IRAK4 degrading agent and use thereof

Abstract

The present invention relates to compounds of formula I, formula II, and to the use of compounds A1 to A52, B1 to B5 and C1 to C36 in the manufacture of a medicament for treating and / or preventing inflammatory and / or immune diseases, and to the use of compounds A1 to A52, B1 to B5 or C1 to C36 in a medicament for treating and / or preventing rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

Description

[Technical Field]

[0001] The present invention relates to compounds that modulate one or more interleukin-1 receptor-associated kinases 4 (IRAK4) by ubiquitination and / or degradation, and their use in medicine for immune and / or inflammatory diseases. [Background technology]

[0002] Interleukin-1 receptor kinase 4 (IRAK4) is a serine / threonine-specific protein kinase with biologically significant kinase activity and plays a key role in immune system activation. Studies have shown that IRAK4 is a key downstream factor in the interleukin (IL)-1β family receptor (including IL-1R, IL-18R, IL-33R, and IL-36R) and Toll-like receptor (TLR) signaling pathways, and that both IRAK4-deficient mice and IRAK4-deficient patients are unresponsive to stimulation by TLRs (except TLR3) and the IL-1β family (Suzuki, Suzuki et al., Nature, 2002; Davidson, Currie et al., The Journal of Immunology, 2006; Kuvon Bernuth et al., JEM, 2007; Kim, Staschke et al., JEM, 2007).

[0003] Depending on the involvement of MyD88, TLR / IL-1β-mediated signaling pathways can be divided into MyD88-dependent and MyD88-independent pathways, in which the signaling pathways mediated by IL-1R and TLR2, TLR4, TLR7 / 8, and TLR9 all depend on MyD88 as a regulator to activate downstream inflammatory signaling pathways. After TLR / IL-1β ligand binding, it recruits the MyD88 molecule, which further recruits IRAK4 to the TLR / IL-1β complex via its N-terminal death domain, interacts with and activates IRAK1 or IRAK2 (Kollewe, Mackensen et al., Journal of Biological Chemistry, 2004; Precious et al., J. Biol. Chem., 2009), thereby transmitting signals downstream to the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6), which activates the serine / threonine kinase TAK1, and further activates the NF-κB and MAPK signaling pathways (Wang, Deng et al., Nature, 2001), resulting in the release of various inflammatory cytokines and anti-apoptotic molecules. The IRAK4-dependent TLR / IL-1β signaling pathway has been implicated in various diseases. For example, multiple sclerosis, atherosclerosis, myocardial infarction, myocarditis (Valaperti, Nishii et al., Circulation, 2013), Vogt-Koyanagi-Harada syndrome, systemic lupus erythematosus (SLE), obesity (Ahmad, R., P. Shihab et al., Diabetology & Metabolic Syndrome, 2015), type 1 diabetes, rheumatoid arthritis, spondyloarthritis (especially psoriatic spondyloarthritis and Bekhterev's disease), lupus erythematosus, psoriasis, vitiligo, giant cell arteritis, chronic inflammatory bowel disease, and viral diseases such as HIV (human immunodeficiency virus), hepatitis virus (Staschke et al., The Journal of Immunology, 2009; Marquez et al., Ann Rheum Dis, 2014; Zambrano-Zaragoza et al., International Journal of Inflammation, 2014; Wang et al., Experimental and Therapeutic Medicine, 2015; Ciccia et al., Rheumatology, 2015), skin diseases such as psoriasis, atopic dermatitis, Kindler's syndrome, bullous pemphigoid, allergic contact dermatitis, alopecia areata, acne inversa and acne vulgaris, other inflammatory diseases such as allergies, Behçet's disease, gout, adult-onset Still's disease, pericarditis, and chronic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. disease), transplant rejection and graft-versus-host reaction, gynecological disorders such as adenomyosis, dysmenorrhea, dyspareunia, and endometriosis, particularly pain associated with adenomyosis, and other endometriosis-related symptoms such as dysmenorrhea, dyspareunia, dysuria, and difficulty defecating (Akoum, Lawson et al., Human Reproduction, 2007; Allhorn, Boing et al., Reproductive Biology and Endocrinology, 2008; Lawson, Bourcier et al., Journal of Reproductive Immunology, 2008; Sikora, Mielczarek-Palacz et al., American Journal of Reproductive Immunology, 2012; Khan, Kitajima et al., Journal of Obstetrics and Gynaecology Research, 2013; Santulli, Borghese et al., Human Reproduction, 2013), eye diseases, such as retinal anemia, keratitis, keratoconjunctivitis dryness, keratoconjunctivitis sicca, macular degeneration and ocular disease (Kaarniranta and Salminen, J Mol Med (Berl), 2009;Sun and Pearlman, Investigative Ophthalmology & Visual Science, 2009;Redfern and McDermott, Experimental EyeResearch, 2010;Kezic, Taylor et al., J Leukoc Biol, 2011;Chang, McCluskey et al., Clinical & Experimental Ophthalmology, 2012;Guo, Gao et al., Immunol Cell Biol, 2012;Lee, Hattori et al., Investigative Ophthalmology & Visual Science, 2012; Qi, Zhao et al., Investigative Ophthalmology & Visual Science, 2014), fibrotic diseases, such as hepatic fibrosis, cardiomyopathy, primary biliary hepatic enhancement syndrome, cystic fibrosis (Zhao, Zhao et al., Scand J Gastroenterol, 2011; Benias, Gopal et al., Clin Res Hepatol Gastroenterol, 2012; Yang, L. and E. Seki, Front Physiol, 2012; Liu, Hu et al., Biochim Biophys Acta., 2015), chronic liver disease, fatty liver hepatitis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic fatty liver disease (NASH), fatty liver disease (ASH) (Nozaki, Saibara et al., Alcohol Clin EXp Res, 2004; Csak, T., A. Velayaudham et al., Am J Physiol Gastrointest Liver Physiol, 2011; Miura, Kodama et al., Gastroenterology, 2010; Kamari, Shaish et al., J Hepatol, 2011; Ye, Li et al., Gut, 2012; Roh, Seki, J Gastroenterol Hepatol, 2013; Ceccarelli, S., V. Nobili et al., World J Gastroenterol, 2014; Miura, Ohnishi, World J Gastroenterol, 2014; Stojsavljevic, Palcic et al., World J Gastroenterol, 2014), cardiovascular diseases and neurological disorders such as myocardial reperfusion injury, myocardial infarction, and hypertension (Oyama, Blais et al., Circulation, 2004; Timmers, Sluijter et al., Circulation Research, 2008; Fang and Hu, MedSci Monit, 2011; Bijani, International Reviews of Immunology, 2012; Bomfim, DosSantos et al., Clin Sci(Lond), 2012; Christia and Frangogiannis, European Journal of Clinical Investigation, 2013; Thompson and Webb, Clin Sci(Lond), 2013; Hernanz, Martinez-Revelles et al., British Journal of Pharmacology, 2015; Frangogiannis, Curr Opin Cardiol, 2015; Bomfim, Echem et al., Life Sciences, 2015), as well as Alzheimer's disease, stroke, traumatic brain injury, amyotrophic lateral sclerosis (ALS), and Parkinson's disease (Brough, Tyrrell et al., Trends in Pharmacological Sciences, 2011; Carty and Bowie, Biochemical Pharmacology, 2011; Denes, Kitazawa, Cheng et al. al., The Journal of Immunology, 2011;Lim, Kou et al., TheAmerican Journal of Pathology, 2011;Beraud and Maguire-Zeiss, Parkinsonism & Related Disorders, 2012;Denes, Wilkinson et al., Disease Models & Mechanisms, 2013;Noelker, Morel et al., Sci. Rep., 2013;Wang, Wang et al., Stroke, 2013;Xiang, Chao et al., Rev Neurosci, 2015;Lee, Lee et al., J Neuroinflammation, 2015), pruritus, and pain (including acute, chronic, inflammatory, and neuropathic pain), such as hyperalgesia, abnormal pain, premenstrual pain, pain associated with adenomyosis, postoperative pain, interstitial cystitis, complex regional pain syndrome (CRPS), trigeminal neuralgia, prostatitis, pain caused by spinal cord injury, inflammation-induced pain, low back pain, cancer pain, chemotherapy-related pain, HIV treatment-induced neuropathy, pain caused by burns, and chronic pain (Wolf, Livshits et al., Brain, Behavior and Immunity, 2008; Kim, Lee et al., Toll-like Receptors: Roles in Infection and Neuropathology, 2009; del Rey, Apkarian et al., Annals of the New York Academy of Sciences, 2012; Guerrero, Cunha et al., European Journal of Pharmacology, 2012; Kwok, Hutchinson et al., PLoS ONE,2012;Nicotra, Loram et al., Experimental Neurology, 2012;Chopra and Cooper, J Neuroimmune Pharmacol, 2013;David, Ratnayake et al., Neurobiology of Disease, 2013; Han, Zhao et al., Neuroscience, 2013; Liu and Ji, Pflugers Arch., 2013; Stokes, Cheung et al., Journal of Neuroinflammation, 2013; Zhao, Zhang et al., Neuroscience, 2013; Liu, Zhang et al., Cell Research, 2014; Park, Stokes et al., Cancer Chemother Pharmacol, 2014;Van der Watt, Wilkinson et al., BMC Infect Dis, 2014;Won, KA, MJKim et al., J Pain, 2014;Min, Ahmad et al., Photochem Photobiol., 2015;Schrepf, Bradley et al., Brain Behav Immun, 2015;Wong, L., JDDone et al., Prostate, 2015), tumor disease, specific cysts, ABC-DLBCL (activated B-cell type cysts, large cell type B-cell cysts), macrophages Cytozoal leukemia and leukemia, combined with chronic leukemia, leucocytosis, gallbladder ulcer and hepatocellular carcinoma (Ngo, Young et al., Nature, 2011;Puente, Pinyol et al., Nature, 2011;Ochi, Nguyen et al., J EXp Med, 2012;Srivastava, Geng et al., Cancer Research, 2012;Treon, Xu et al., New England Journal of Medicine, 2012;Choi, Kim et al., Human Pathology, 2013;Liang, Chen et al., Clinical Cancer Research, 2013), ras-dependent ulcers, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, lung cancer, prostate cancer.

[0004] Although regulation of IRAK4-mediated signaling pathways is primarily related to its kinase function, some studies have demonstrated that in certain cell types, IRAK4 signaling regulation of downstream processes is related to non-kinase functions of IRAK4. Cushing et al. demonstrated that IRAK4 phosphorylation levels were reduced in IL-1β-stimulated human dermal fibroblasts, but pharmacological inhibition of IRAK4 did not inhibit IL-6 and TNF-α. Supporting these results, IRAK4 scaffolding function is important for IL-1 signaling in IRAK4-deficient fibroblasts compared with wild-type cells. Chiang and colleagues also demonstrated that IRAK4 kinase activity is no longer essential in human B and T cells, dendritic cells, and monocytes, and siRNA gene knockdown also demonstrated that IRAK4 has a scaffolding function in these cells. Currently, many potent selective inhibitors of IRAK4 have been reported, such as CA-4948, BAY-1834845, BMS-986126, and PF-06650833. All of these inhibitors can selectively inhibit the kinase activity of IRAK4 and are mainly used for the prevention and treatment of autoimmune diseases, inflammatory diseases, and tumor diseases. However, on the one hand, IRAK4 acts as a scaffold protein and an active kinase, and on the other hand, traditional small molecule inhibitors are prone to drug resistance, so that the inhibition of IRAK4 kinase activity alone may not have sufficient therapeutic effect.

[0005] Proteolysis targeting chimeras (PROTACs) are a distinct technology from traditional small molecule inhibitors. While traditional small molecule inhibitors typically require target protein activation via the active site, PROTACs utilize a small molecule inhibitor that recognizes the target protein at one end and an E3 ubiquitin ligase ligand at the other end. These bifunctional molecules recognize the target protein and the E3 ubiquitin ligase in vivo, attracting the target protein to the E3 ubiquitin ligase to form a ternary complex, ubiquitinating the target protein, and then degrading the target protein via the ubiquitin-protease pathway in vivo. Compared to traditional small molecule inhibitors, PROTACs only require the target protein to be brought into close proximity with the E3 ubiquitin ligase for substrate degradation. This mechanism of action makes this technology applicable to some undruggable targets. On the other hand, even after the target protein is degraded, the PROTAC molecule can be released and continue to participate in the degradation process of the next protein. This catalytic degradation action allows for efficient degradation with a relatively low PROTAC drug dose. Furthermore, while traditional small molecule inhibitors are prone to drug resistance due to point mutations, which cause the small molecule inhibitor to lose its inhibitory effect on the target, PROTACs can directly degrade the target protein, thereby avoiding drug resistance due to point mutations to some extent. Therefore, the research and development of novel small drug molecules using PROTAC technology has significant advantages and feasibility compared to traditional small molecule inhibitors, and is expected to be a highly promising new generation of drugs. PROTAC technology has also been applied to the improvement of various target drugs, such as male hormone receptors, female hormone protein receptors, and BTK. US2019 / 0151295, US2019 / 0192688, WO2019 / 160915, and WO2020 / 113233 disclose several degrading agents that target IRAK4, and the development of more degrading agents that target IRAK4 is expected. Summary of the Invention

[0006] Many IRAK4 inhibitors have been disclosed in the prior art (see Annual Reports in Medical Chemistry (2014), 49, 117-133).

[0007] WO2016 / 083433A1 Examples disclose the following compound (reference compound (I) of the present invention):

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[0009] WO2015 / 150995A1 Examples disclose the following compound (Comparative Compound (II) of the present invention):

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[0011] As described herein, the present inventors have discovered that an IRAK4 degrading agent is applicable to the treatment and prevention of animal immune diseases characterized by a hyperreactive immune system, and in particular has excellent therapeutic or preventive effects against diseases such as rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis, and / or psoriasis. Compounds A1 to A52 correspond to the following formulae, respectively.

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[0064] The preparation and use of compounds A1 to A52 as drugs is disclosed in WO2022 / 088551A1. Compounds B1 to B5 correspond to the following formulae, respectively.

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[0070] The preparation and use of compounds B1 to B5 as drugs is disclosed in WO2022 / 028547A1. Compounds C1 to C36 correspond to the following formulae, respectively.

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[0108] For the preparation and use of compounds C1 to C36 as drugs, please refer to the part explained in the present invention. The present invention provides the use of IRAK4, and / or its stereoisomers, enantiomers, diastereoisomers, deuterated forms, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts thereof, for treating and / or preventing immune and / or inflammatory diseases.

[0109] The present invention provides use of IRAK4, and / or its stereoisomers, enantiomers, diastereoisomers, deuterated compounds, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts thereof, for treating and / or preventing diseases caused by abnormal expression of IRAK4 and IRAK4-related proteins in the IL-1R / TLR pathway and / or abnormal secretion or proliferation of chemical factors, cytokines or immune cells mediated by IRAK4.

[0110] In some aspects of the invention, the IRAK4 degrader is a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof: [ka]

[0111] where: Ring A is a phenyl group or a pyridyl group; Ring B is C6-C 10 a cycloalkyl group or a 6- to 10-membered heterocycloalkyl group containing 1 to 2 heteroatoms selected from N, O, and S; 10 The cycloalkyl group and the 6- to 10-membered heterocycloalkyl group are optionally substituted with a substituent selected from halogen, oxo, cyano, amino, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, or -O-(C1-C6 alkyl); Ring C is C6-C 12 a cycloalkyl group or a 6- to 12-membered heterocycloalkyl group containing 1 to 2 heteroatoms selected from N, O, and S; 12The cycloalkyl group and the 6- to 12-membered heterocycloalkyl group are optionally substituted with a substituent selected from a halogen, a cyano group, an amino group, a hydroxy group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 hydroxyalkyl group, or —O—(C1-C6 alkyl); Ring D is C6-C 10 An aryl group, C6-C 10 the aryl group is optionally substituted with a halogen, cyano, amino, hydroxy, C-C alkyl, C-C haloalkyl, C-C hydroxyalkyl or -O-(C-C alkyl) substituent; Ring Y is a 5- to 6-membered heteroaryl group; X is a bond, —O—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NHC(O)—, or —C(O)NH—; L is -(CH2) j -, and the above -(CH2) j One or more methylene groups in - may optionally be -NR 3' -, -O-, -S-, -S(O)-, -S(O)2-, -S(O)2NR 3' -, -CR 1' R 2' -, -C(O)-, -C(O)O-, -OC(O)-, -NR 3' C(O)O-, -OC(O)NR 3' -, -C(O)NR 3' -, -NR 3' C(O)-, -NR 4' C(O)NR 3' -, vinylidene group, or ethynylene group; R 1' , R 2' are each independently halogen, —OH, —NH, a C1-C4 alkyl group, a C1-C4 haloalkyl group, a C1-C4 hydroxyalkyl group, —O(C1-C4 alkyl) or —NH(C1-C4 alkyl); R 3' , R 4' are each independently hydrogen or a C1-C6 alkyl group; R dare each independently hydrogen, deuterium, halogen, cyano, or C1-C6 alkyl, and the alkyl group is optionally substituted with one or more groups selected from halogen, hydroxy, and amino; R c -O(C1-C3 alkyl), -N(C1-C3 alkyl) 1-2 or a C1-C6 alkyl group, wherein the alkyl group is optionally substituted with one or more groups independently selected from a hydroxy group, an amino group, a halogen atom, a cyano group, or —O—(C1-C3 alkyl); R b is hydrogen or a C1-C6 alkyl group, said alkyl group being optionally substituted with one or more groups independently selected from a hydroxy group, an amino group, a halogen, and a cyano group; R a is hydrogen, halogen, a C1-C6 alkyl group, or —O—(C1-C6 alkyl), wherein the alkyl group is optionally substituted with a halogen or hydroxy group; Each R1 independently represents a C1-C4 alkyl group, -O(C1-C4 alkyl), -N(C1-C4 alkyl) 1-2 , CN, halogen, —OH, —NH2, wherein the alkyl group is optionally substituted with a group selected from halogen, cyano group, —OH, C1-C4 alkyl group, —O(C1-C4 alkyl); each R2 independently represents hydrogen, a C1-C4 alkyl group, -O(C1-C4 alkyl), a C3-C8 cycloalkyl group, a 3- to 8-membered heterocycloalkyl group, a 6- to 10-membered aryl group, a 5- to 6-membered heteroaryl group, CN, halogen, or -OH, wherein the alkyl group, cycloalkyl group, heterocycloalkyl group, aryl group, or heteroaryl group is optionally substituted with a group selected from halogen, cyano group, -OH, -NH2, a C1-C4 alkyl group, and -O(C1-C4 alkyl); X' is CH or N; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; p is 1 or 2; j is 0, 1, 2, 3, 4 or 5.

[0112] In some preferred embodiments of the present invention, the IRAK4 degrader is a compound of formula I-1, I-2, or II-1, or a pharmaceutically acceptable salt thereof: [ka]

[0113] where: R3 is H, halogen, a C1-C6 alkyl group or —O—(C1-C6 alkyl); R c , R d , Ring B, L, Ring C, X, X', p, R2 and m are defined by the present invention.

[0114] In some preferred embodiments of the present invention, the disease is characterized in that an IRAK4 degrading agent is used to treat and / or prevent an immune-mediated disease.

[0115] In some preferred embodiments of the present invention, the IRAK4 degrading agent is used to treat immune diseases, including adult Still's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behçet's disease, benign mucous membrane pemphigoid (mucous membrane pemphigoid), bullous pemphigoid, Castleman's disease (CD), celiac disease, Coxsackie myocarditis, Crohn's disease, dermatitis herpetiformis, atopic dermatitis, dermatomyositis, discoid lupus erythematosus, adenomyosis, eosinophilic fasciitis, erythema nodosum, fibrosing alveolitis, and primary glomerulonephritis. , Goodpasture syndrome, autoimmune hemolytic anemia, Henoch-Schönlein purpura (HSP), hidradenitis suppurativa (HS), IgG4-related sclerosing disease, inclusion body myositis (IBM), interstitial cystitis (IC), Lambert-Eaton syndrome, linear IgA disease (LAD), systemic lupus erythematosus, chronic Lyme disease, multiple sclerosis, systemic sclerosis, idiopathic inflammatory myopathy (IIM), ocular cicatricial pemphigoid optic neuritis, relapsing rheumatoid arthritis (PR), pemphigus, autoimmune encephalomyelitis, POEMS syndrome, polyarteritis nodosa, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriasis ectopic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, autoimmune scleritis, scleroderma, Sjögren's syndrome, aortitis, temporal arteritis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thyroid eye disease (Ted), Tolosa-Hunt syndrome (THS), transverse myelitis, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, systemic vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, myasthenia gravis, gout, spondyloarthropathy, crystal arthropathy, osteoarthritis, rheumatoid arthritis , polymyositis, interstitial lung disease, giant cell arteritis, polymyalgia rheumatica, granulomatous vasculitis, eosinophilic granulomatous vasculitis, eosinophilic vasculitis, microscopic polyangiitis, cryoglobulinemia, cutaneous leukocytoclastic vasculitis, mixed connective tissue disease, Stevens-Johnson syndrome, pulmonary hypertension, endocarditis, atherosclerosis, erythema multiforme, acute coronary syndrome, idiopathic pulmonary fibrosis, non-alcoholic fatty liver disease, renal fibrosis, type 1 diabetes, primary xerosis, Kawasaki disease, pustular psoriasis, chronic granulomatous disease, neuromyelitis optica, urticaria, palmoplantar pustulosis, sepsis, bullous skin diseases, Alzheimer's disease,Selected from chronic idiopathic urticaria, chronic psoriasis, juvenile idiopathic arthritis, axial spondyloarthritis, and Graves' disease.

[0116] In some preferred embodiments of the present invention, the IRAK4 degrading agent is used to treat an immune disease, wherein the immune disease is selected from rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis, and / or psoriasis.

[0117] In some preferred forms of the invention, the disease is characterized in that an IRAK4 degrading agent is used to treat and / or prevent the inflammatory disease.

[0118] In some preferred embodiments of the present invention, the IRAK4 degrading agent is used to treat immune disorders, including inflammatory diseases such as familial Mediterranean fever, tumor necrosis factor-associated periodic syndrome, mevalonate kinase deficiency, pyridine-associated autoinflammatory neutrophilic dermatosis (PAAND), suppurative sterile arthritis, pyoderma gangrenosum, and acne (PAPA), familial cold autoinflammatory syndrome (FCAS), familial chronic lichenoid keratosis (FKLC), and NLRP1-associated autoinflammatory The disease is selected from non-alcoholic idiopathic arthritis and dyskeratosis (NAIAD), IL-1 receptor antagonist (DIRA) deficiency, IL-36 receptor antagonist (DITRA) deficiency, allergic contact dermatitis, CAR-T cell-induced cytokine release syndrome, Crohn's disease, chronic bronchitis, COPD, active ankylosing spondylitis, axial spondyloarthritis, pityriasis rubra pilaris, inflammatory bowel disease, spondyloarthritis, acute lung injury, generalized pustular psoriasis, acne vulgaris, or colitis.

[0119] In some preferred embodiments of the present invention, the IRAK4 degrading agent is used to treat and / or prevent diseases mediated by IL-2R alpha, IL-6, IFA-alpha2, IFN-gamma, IL-1ra, MCP-3, IL-16, IL-12(p40), LIF, IL-5, GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, Eotaxin, basic FGF, beta-NGF, PDGF-BB, IL-4, MCP-1, IL-8, IL-10, GRO-alpha, HGF, IL-1alpha, IL-1beta, IL-3, SCF, TRAIL, M-CSF, CTACK, IL-15, IL-12(P70), IL-17, IL-23, IL-33, and / or IL-36 cytokines.

[0120] In some preferred forms of the invention, IRAK4 degrading agents are used to treat and / or prevent diseases mediated by IL-4, IL-6, IL-12(p40), GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, IL-8, IL-10, IL-17, IL-23, IL-33 and / or IL-36 cytokines.

[0121] In some preferred forms of the invention, the sample is a spleen, skin and / or blood sample.

[0122] In some preferred embodiments of the invention, the blood sample is normal human whole blood and / or patient whole blood.

[0123] In some preferred embodiments of the present invention, the method for treating the above diseases comprises administering to the subject an effective amount of an IRAK4 degrading agent.

[0124] In some preferred embodiments of the present invention, the method of treating the above diseases includes the IRAK4 degrading agent being the above compound alone or in combination with other drugs.

[0125] In the present invention, IRAK4 kinase activity measurement experiments demonstrated that compounds A1-A52, B1-B5, or C1-C36 described in the present invention can effectively bind to the IRAK4 target protein and produce degradation and / or inhibition effects. Western blot analysis demonstrated that compounds A1-A52, B1-B5, or C1-C36 described in the present invention can effectively and specifically degrade IRAK4 protein in THP-1 cells. Compounds A1-A52, B1-B5, or C1-C36 described in the present invention, and / or their stereoisomers, enantiomers, diastereoisomers, deuterated salts, hydrates, solvates, metabolites, prodrugs, and / or pharmaceutically acceptable salts, can effectively degrade IRAK4 protein, thereby achieving the effects of preventing or treating IRAK4-related diseases or conditions.

[0126] The present invention has experimentally demonstrated that the compounds described herein can significantly reduce the expression of IRAK4 in whole blood, skin, or liver, indicating that IRAK4 degraders can be used to treat immune-mediated diseases.

[0127] In the present invention, the above-mentioned drug comprises an IRAK4 degrading agent as an active ingredient, and the drug according to the present invention may optionally further comprise a pharmaceutically acceptable carrier, diluent or excipient.

[0128] In the present invention, the drug IRAK4 degrader can be used alone or in combination with other drugs to achieve better therapeutic effects on inflammatory or immune diseases.

[0129] In the present invention, the dosage form of the above drug may be tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, syrups, elixirs, ointments, drops, suppositories, inhalants, or sprays. The drug containing an IRAK4 degrader as an active ingredient can be formulated into any one of the above dosage forms according to actual needs, and each dosage form of the drug can be prepared according to conventional methods in the pharmaceutical field.

[0130] In the present invention, the administration route of the above-mentioned drug can be selected according to actual needs from any one of oral administration, sublingual administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intranasal administration, transdermal administration, parenteral administration, inhalation administration, intratracheal administration, intrapulmonary administration, and bronchial administration.

[0131] The present invention further provides pharmaceutically acceptable salts of compounds A1-A52, B1-B5, or C1-C36. The term "pharmaceutically acceptable salt" refers to acid or base addition salts of the relatively non-toxic compounds of the present invention. The acid addition salts are salts formed by combining compounds A1-A52, B1-B5, or C1-C36 of the present invention with a suitable inorganic or organic acid. These salts may be prepared during the final isolation and purification process of the compounds, or by reacting purified compounds A1-A52, B1-B5, or C1-C36 in their free base form with a suitable organic or inorganic acid. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleate, fumarate, succinate, tartrate, methanesulfonate, p-toluenesulfonate, gluconate, lactobionate, laurylsulfonate, and the like. The base addition salts are salts formed by combining a compound of A1 to A52, B1 to B5, or C1 to C36 with an appropriate inorganic or organic base, and include salts formed with basic metals, basic earth metals, and quaternary ammonium cations, such as sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt, tetramethyl quaternary ammonium salt, and tetraethyl quaternary ammonium salt. The amine salts include salts formed with ammonia (NH), primary amines, secondary amines, or tertiary amines, such as methylamine salt, dimethylamine salt, trimethylamine salt, triethylamine salt, and ethylamine salt.

[0132] The compounds of the present invention, or pharmaceutically acceptable salts thereof, can be administered to mammals, including humans, orally, rectally, topically (intravenously, intramuscularly, or subcutaneously), locally (as powders, ointments, or drops), or intratumorally.

[0133] The dosage of the compound of the present invention may be about 0.05 to 300 mg / kg body weight / day, preferably 10 to 300 mg / kg body weight / day, and more preferably 10 to 150 mg / kg body weight / day.

[0134] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into solid dosage forms for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, etc. In these solid dosage forms, the compounds of A1 to A52, B1 to B5, or C1 to C36 of the present invention are mixed as an active ingredient with at least one conventional inert excipient (or carrier), for example, sodium citrate or dicalcium phosphate, or the following ingredients: (1) a filler or solubilizer such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, (2) a binder such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic, and (3) a moisturizer such as glycerin. The formulation may be mixed with (4) disintegrating agents such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (5) solubilizing agents such as paraffin, (6) absorption enhancers such as quaternary ammonium compounds, (7) wetting agents such as cetyl alcohol and glyceryl monostearate, (8) adsorbents such as kaolin, and (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, or mixtures thereof. Capsules, tablets, and pills may also contain buffering agents. The above solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be coated or microencapsulated with coating and shell materials, such as enteric coatings, and other materials well known in the art. They may also contain opacifying agents, so that the release of the active ingredient in such compositions can be delayed in a specific part of the digestive tract. Examples of embedding components that can be used include polymeric and wax-based materials. If desired, the active ingredient can also be in micro-encapsulated form with one or more of the above-mentioned excipients.

[0135] The compounds of the present invention or their pharmaceutically acceptable salts can be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and tinctures. In addition to the compounds of A1-A52, B1-B5, or C1-C36 or their pharmaceutically acceptable salts as the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water, and other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances. In addition to these inert diluents, the liquid dosage forms of the present invention may contain conventional auxiliary agents, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and fragrances. Such suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, agar, and the like, or mixtures of these substances.

[0136] The compounds of the present invention or their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable carriers, diluents, solvents or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0137] The compounds of the present invention or pharmaceutically acceptable salts thereof can also be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, inhalants, etc. The compounds of A1 to A52, B1 to B5, or C1 to C36 of the present invention or pharmaceutically acceptable salts thereof as active ingredients are mixed under sterile conditions with a physiologically acceptable carrier and optional preservatives, buffers, or propellants that may be required as needed.

[0138] The present invention also provides a pharmaceutical composition comprising a compound of the present invention represented by formula A1-A52, B1-B5, or C1-C36, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier, excipient, or diluent. To prepare the pharmaceutical composition, the compound of the present invention represented by formula A1-A52, B1-B5, or C1-C36, or a pharmaceutically acceptable salt thereof, is generally mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The composition of the present invention can be prepared into conventional pharmaceutical formulations, such as tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, syrups, elixirs, ointments, drops, suppositories, sprays, and propellants.

[0139] The compounds according to the present invention or their pharmaceutically acceptable salts may be administered alone or (if necessary) in combination with other pharmaceutically acceptable therapeutic agents, such as other anti-tumor drugs, anti-inflammatory drugs, or autoimmune drugs. The combined components may be administered simultaneously or sequentially in a single formulation or in different formulations. The above combinations may include combinations of the compounds of the present invention with one other active agent, or may include combinations of the compounds of the present invention with two or more other active agents.

[0140] Pharmaceutically acceptable according to the present invention means that the substance or composition must be compatible with the other ingredients of the formulation and not harmful to the patient.

[0141] Treatment according to the present invention includes preventative and palliative treatment.

[0142] In some embodiments of the invention, the sample is a patient or normal human blood sample, in some embodiments the sample is a patient or normal human plasma sample, and in some embodiments the sample is a patient or normal human peripheral blood mononuclear cell sample.

[0143] In some embodiments of the invention, measuring the level of an immune biomarker in a sample comprises using an AlphaLISA method. In some embodiments, measuring the level of an immune biomarker in a sample comprises using a method selected from the Examples.

[0144] DETAILED DESCRIPTION: Unless stated to the contrary, the following terms used in the specification and claims have the following meanings.

[0145] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group including a straight-chain or branched-chain alkyl group, and C1-C8 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylbutyl, 2-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylpent ... The alkyl group refers to an alkyl group having 1 to 8 carbon atoms, such as n-octyl, 2,3-dimethylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, or various branched chain isomers thereof, preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group. The alkyl group may be substituted or unsubstituted. In some embodiments, the alkyl group is a C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group.

[0146] "Cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, including "C3-C 11 "Cycloalkyl group" refers to a cycloalkyl group containing 3 to 11 carbon atoms, "C3-C8 membered cycloalkyl group" refers to a cycloalkyl group containing 3 to 8 carbon atoms, and "C5-C 10"membered cycloalkyl group" refers to a cycloalkyl group containing 5 to 10 carbon atoms; Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentene, cyclohexyl, cyclohexene, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl groups, etc., preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexa groups, and more preferably C3-C8 membered cycloalkyl groups, more preferably C3-C6 membered cycloalkyl groups.

[0147] Polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. "Spirocycloalkyl groups" refer to polycyclic groups in which single rings share one carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. Depending on the number of spiro atoms shared between the rings, spirocycloalkyl groups are classified as monospirocycloalkyl groups, bisspirocycloalkyl groups, or polyspirocycloalkyl groups, with 7- to 12-membered bisspirocycloalkyl groups being preferred. Non-limiting examples of spirocycloalkyl groups include: [ka] Includes:

[0148] "Fused cycloalkyl group" refers to an all-carbon polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with another ring in the system, where one or more rings may contain one or more double bonds, but no ring has a completely conjugated π-electron system. Depending on the number of rings, fused cycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, with bicyclic fused cycloalkyl groups being preferred. Non-limiting examples of fused cycloalkyl groups include: [ka] Includes:

[0149] "Bridged-ring alkyl group" refers to an all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, and may contain one or more double bonds, but no ring has a completely conjugated π-electron system, and can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged-ring alkyl group depending on the number of rings it comprises. Non-limiting examples of bridged-ring alkyl groups include: [ka] Includes:

[0150] The cycloalkyl ring may be fused to an aryl group, a heteroaryl group, or a heterocycloalkyl ring, where the ring connected to the parent structure is a cycloalkyl group ring, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted.

[0151] In some embodiments, the cycloalkyl group is selected from the group consisting of C, ... 10 , C 11 , C 12 Mono- or polycyclic (eg, spirocyclic, fused or bridged) cycloalkyl groups.

[0152] "Heterocycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent in which one or more (e.g., 2, 3, 4, or 5) ring atoms are nitrogen, oxygen, or S(O). r(where r is an integer of 0, 1, or 2), but does not contain a ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. A "3- to 11-membered heterocycloalkyl group" refers to a cyclyl group containing 3 to 11 ring atoms, a "5- to 10-membered heterocycloalkyl group" refers to a cyclyl group containing 5 to 10 ring atoms, and a "3- to 8-membered heterocycloalkyl group" refers to a cyclyl group containing 3 to 8 ring atoms, and is preferably a "3- to 11-membered heterocycloalkyl group" containing 1 to 2 heteroatoms selected from N, O, or S, and more preferably a 3- to 11-membered heterocycloalkyl group containing 1 or 2 N atoms.

[0153] The monocyclic heterocycloalkyl group is preferably a 3- to 8-membered monocyclic heterocycloalkyl group containing 1 to 2 N heteroatoms, and non-limiting examples of the monocyclic heterocycloalkyl group include a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a morpholinyl group, a thiomorpholinyl group, a homopiperazinyl group, etc., and preferably a piperidinyl group or a piperazinyl group.

[0154] Polycyclic heterocycloalkyl groups include spirocyclic, fused-ring, and bridged-ring heterocycloalkyl groups. A "spiroheterocycloalkyl group" refers to a polycyclic heterocycloalkyl group that shares one atom (referred to as a spiroatom) between the rings, where one or more of the ring atoms is nitrogen, oxygen, or S(O). r (where r is an integer selected from 0, 1, and 2), and the remaining ring atoms are carbon. They may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. Spirocycloalkyl groups are classified as monospiroheterocycloalkyl groups, bisspiroheterocycloalkyl groups, or polyspiroheterocycloalkyl groups depending on the number of spiro atoms shared by the rings. Preferred are saturated "3- to 11-membered bisspiroheterocycloalkyl groups" containing 1 to 2 heteroatoms selected from N, O, or S, and more preferred are saturated "7- to 11-membered bisspiroheterocycloalkyl groups" containing 1 or 2 N atoms. Non-limiting examples of spiroheterocycloalkyl groups include: [ka] Includes:

[0155] "Fused heterocycloalkyl group" refers to a polycyclic heterocycloalkyl group in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated pi-electron system, and wherein one or more ring atoms is nitrogen, oxygen, or S(O) r (where r is an integer selected from 0, 1, and 2), and the remaining ring atoms are carbon. Depending on the number of rings constituting the heterocycloalkyl group, it can be divided into bicyclic, tricyclic, tetracyclic, and polycyclic fused heterocycloalkyl groups, and is preferably a "3- to 11-membered bicyclic fused heterocycloalkyl group" containing 1 to 3 heteroatoms selected from N, O, and S, and more preferably a saturated "3- to 11-membered bicyclic fused heterocycloalkyl group" containing 1 or 2 N atoms. Non-limiting examples of fused heterocycloalkyl groups include: [ka] Includes:

[0156] A "bridged heterocycloalkyl group" refers to a polycyclic heterocycloalkyl group in which any two rings share two atoms that are not directly connected, and may contain one or more double bonds, but no ring has a completely conjugated pi-electron system, and in which one or more ring atoms is not nitrogen, oxygen, or S(O). r (wherein r is an integer selected from 0, 1, and 2), and the remaining ring atoms are carbon. Depending on the number of rings constituting the group, the group can be classified into bicyclic, tricyclic, tetracyclic, or polycyclic bridged ring alkyl groups. Non-limiting examples of bridged heterocycloalkyl groups are: [ka] Includes:

[0157] The heterocycloalkyl group ring may be fused to an aryl group, heteroaryl group, or cycloalkyl group ring, where the ring connected to the parent structure is a heterocycloalkyl group ring, non-limiting examples include: [ka] wherein the heterocycloalkyl group may be optionally substituted or unsubstituted.

[0158] In some embodiments, the heterocycloalkyl group is a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered monocyclic or polycyclic (e.g., spirocyclic, fused, or bridged) heterocycloalkyl group, wherein the number of heteroatoms can be 1, 2, 3, 4, or 5, and each heteroatom is independently nitrogen, oxygen, or S(O). r (where r is an integer 0, 1, or 2).

[0159] The term "aryl group" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) polycyclic group having a conjugated pi-electron system, and includes "C-C 10 "Aryl" refers to an all-carbon aryl group containing 6 to 10 carbons, such as, for example, phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, where the ring connected to the parent structure is an aryl ring; non-limiting examples include: [ka] wherein the aryl group may be optionally substituted or unsubstituted. In some embodiments, the aryl group is a 6- to 10-membered aryl group.

[0160] "Heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms, where the heteroatoms are nitrogen, oxygen, or S(O). r(where r is an integer 0, 1, or 2), and the 5- to 6-membered heteroaryl group refers to a heteroaromatic system containing 5 to 6 ring atoms, and the 5- to 10-membered heteroaryl group refers to a heteroaromatic system containing 5 to 10 ring atoms, preferably a 5- to 6-membered heteroaryl group, more preferably a 5- to 6-membered heteroaryl group containing one or two N atoms. Non-limiting examples include furan, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidine, pyrazinyl, pyrazole, imidazolyl, triazolyl, tetrazolyl, etc., preferably a pyridyl group. The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, where the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include [ka] In some embodiments, the heteroaryl group is a 5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl group, where the number of heteroatoms can be 1, 2, 3, 4, or 5, and each heteroatom is independently nitrogen, oxygen, or sulfur.

[0161] Unless otherwise stated, structures depicted herein also refer to all isomers of the structure (e.g., enantiomers, diastereoisomers, and geometric (or conformational) forms, R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Thus, single chemical isomers and enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Unless otherwise stated, structures depicted herein also refer to compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure may have different structures such as those having the replacement of a hydrogen by deuterium or tritium, or the replacement of a carbon atom by another isotopically enriched atom. 13 C- or 14Substitution of C isotopes is within the scope of the present invention. These compounds can be used as analytical tools, probes in biological assays, or therapeutic agents of the present invention. "Stereoisomers" include all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes enantiomers of a compound (enantiomers containing the (R-) or (S-) configuration of a compound), mixtures of enantiomers (physical mixtures of enantiomers, and racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of a compound, and isomers of a compound that have multiple chiral centers but are not mirror images of one another (diastereoisomers). Because the chiral centers of a compound can racemize in the body, for these compounds, administration of a compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Thus, the compounds of the present invention can be prepared and used in the form of a single isomer and essentially free of other isomers, or in the form of a mixture of various isomers, such as a racemic mixture of stereoisomers. In some embodiments, the bifunctional compounds of the present invention are isotopic derivatives having a desired isotopic substitution of at least one atom, resulting in an amount greater than the natural abundance of the isotope, i.e., enriched. In one embodiment, the compounds contain deuterium or multiple deuterium atoms. Substitution with a heavier isotope (e.g., deuterium), i.e., 2H, can be advantageous in certain situations because it can provide certain therapeutic advantages due to greater metabolic stability, such as increased half-life in the body or reduced dosage requirements. Furthermore, the bifunctional compounds of the present invention include N-oxides, crystalline forms (also called crystalline polymorphs), active metabolites of compounds with the same type of activity, tautomers, and unsolvated and solvated forms with pharmaceutically acceptable solvents, such as water, ethanol, and the like, of the compounds. Solvated forms of the complexes proposed herein are also considered to be hereby disclosed.

[0162] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0163] "Control sample" or "control sample" refers to an individual or sample from a population not affected by a disease or condition (e.g., arthritis, rheumatoid arthritis, myositis, lupus erythematosus, and / or systemic sclerosis), respectively, or an internal control determined by techniques known in the art. In some embodiments, a control or baseline level is first determined, or measured prior to measurement in a sample, or obtained from a database of such control samples.

[0164] "Control compound (I)" and "Control (I)" are used interchangeably in the present invention. "Control compound (II)" and "Control (II)" are used interchangeably in the present invention. "Induction" and "stimulation" are used interchangeably in the present invention. An "effective amount" refers to an amount that, when administered to a subject, produces beneficial or desired results, including clinical results, e.g., an amount that resolves, inhibits, or alleviates the symptoms of the condition being treated in a subject compared to a control.

[0165] "Pharmaceutically acceptable carrier, adjuvant or carrier" refers to a non-toxic carrier, adjuvant or carrier that does not impair the pharmacological activity of the compound in which it is formulated. Pharmaceutically acceptable carrier, adjuvant or carrier that can be used in the compositions of the present invention includes, but is not limited to, ion exchangers, alumina, stearates, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, glycerol mixtures with some salts or electrolytes such as guanidine sulfate, disodium phosphate, sodium chloride, zinc salts, colloidal silica, magnesium gluconate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0166] "Patient" refers to an animal, preferably a mammal, and most preferably a human.

[0167] "Cytokines" include IL-2Ralpha, MIG, MIP-1beta, IL-6, and IFN-al pha2, IFN-gamma, SDF-1alpha, IL-1ra, MCP-3, IL-16, IL-12(p40), LIF, TNF-beta, IL-5, GM-CSF, MIF, TNF-alpha, RANTES, IL-2, IL-1beta, IL-18, Eotaxin, BasicFGF, VEGF, beta-NGF, PDGF-BB, IP-10, I L-13, IL-4, MCP-1, IL-8, MIP-1alpha, IL-10, G-CSF, GRO-alpha, HGF, IL-1alpha, IL-3, SCF, TRAIL, M - Refers to one selected from CSF, CTACK, IL-15, IL-7, IL-12(p70), IL-17, IL-9, SCGF-beta, KC, IL-17A and MCP-17A. In some embodiments, measuring cytokine levels in normal human or mouse samples comprises measuring using cultured peripheral blood mononuclear cells (PBMCs). In some embodiments, measuring cytokine levels in normal human or mouse samples comprises measuring using Luminex methods.

[0168] The following exemplary embodiments are used to illustrate the present invention, but the present invention is not limited to these examples.

[0169] The present invention will be described in more detail and comprehensively with reference to the following examples, but the present invention is not limited thereto. The present invention is also not limited to the contents of the examples. The raw materials in the examples of the present invention are known and commercially available, or can be synthesized by methods known in the art. Unless otherwise specified, experimental methods for which specific conditions are not specified in the examples of the present invention generally follow conventional conditions or conditions recommended by the manufacturers of raw materials or products.

[0170] Abbreviations "DMSO" refers to N,N-dimethylformamide. "Glucose" refers to glucose. "Solutol" refers to polyethylene glycol-15 hydroxystearate. "PO" refers to oral administration. "LPS" refers to lipopolysaccharide. "R848" refers to lesinurad. "PBMC" refers to human peripheral blood mononuclear cells. "IRAK4" refers to interleukin-1 receptor-associated kinase 4. "IL" refers to interleukin. "IMQ" refers to imiquimod. "Topical" refers to local administration. "BID" refers to twice daily. "QD" refers to once daily. [Brief explanation of the drawings]

[0171] [Figure 1A] FIG. 1A shows the degradation of IRAK4 by Compound A. [Figure 1B] FIG. 1B shows the degradation of IRAK4 by Compound A. [Figure 2] FIG. 2 shows the intracellular IRAK4 protein expression levels in PBMCs from each patient. [Figure 3] FIG. 3 is a diagram illustrating the inhibitory effect of Compound A on IL-6 production in normal human PMBCs induced by LPS. [Figure 4] FIG. 4 is a graph illustrating the inhibitory effect of Compound A on LPS-stimulated cytokine secretion from human peripheral blood mononuclear cells. [Figure 5] FIG. 5 is a graph illustrating the inhibitory effect of Compound A on R848-stimulated cytokine secretion from human peripheral blood mononuclear cells. [Figure 6A] FIG. 6A is a diagram illustrating the inhibitory effect of compound A on IL-6 in normal human whole blood and patient whole blood. [Figure 6B] FIG. 6B is a graph illustrating the inhibitory effect of compound A on IL-6 in normal human whole blood and patient whole blood. [Figure 6C]FIG. 6C is a graph illustrating the inhibitory effect of compound A on IL-6 in normal human whole blood and patient whole blood. [Figure 6D] FIG. 6D is a graph illustrating the inhibitory effect of compound A on IL-6 in normal human whole blood and patient whole blood. [Figure 7A] FIG. 7A is a diagram illustrating the degradation of IRAK4 in C57 mouse PBMC, spleen, and skin by compound A. [Figure 7B] FIG. 7B is a diagram illustrating the degradation of IRAK4 in C57 mouse PBMC, spleen, and skin by compound A. [Figure 7C] FIG. 7C is a diagram illustrating the degradation of IRAK4 in C57 mouse PBMC, spleen, and skin by compound A. [Figure 8A] FIG. 8A is a diagram illustrating the efficacy of Compound A in an imiquimod-induced C57 mouse psoriasis model. [Figure 8B] FIG. 8B is a diagram illustrating the efficacy of Compound A in an imiquimod-induced C57 mouse psoriasis model. [Figure 8C] FIG. 8C is a diagram illustrating the efficacy of Compound A in an imiquimod-induced C57 mouse psoriasis model. DETAILED DESCRIPTION OF THE INVENTION

[0172] I. Example of compound production Example 1: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0173] A solution of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (150 mg, crude, 0.18 mmol), 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (78 mg, 0.18 mmol), and N,N-diisopropylethylamine (116 mg, 0.9 mmol) in dimethyl sulfoxide (5 mL) was stirred overnight at room temperature. The resulting mixture was poured into water (50 mL) and stirred for 10 min. After filtration, the filter cake was purified by preparative HPLC to give the target product, 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0174] LC-MS: (ESI, m / z): [M+H] + =874.5. 1H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 9.50 (d, J = 5.9 Hz, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.39 (d, J = 4.0 Hz, 1H), 8.26 (d, J = 5.7 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.39 (dd, J = 8.3, 2.0 Hz, 1H), 7.27-6.96 (m, 1H), 6.90-6.41 (m, 1H), 5.30-5.05 (m, 1H), 4.77 (d, J = 17.4 Hz, 1H), 4.26-4.14 (m, 1H), 3.86-3.70 (m, 3H), 3.66-3.41 (m, 5H), 3.30-3.25 (m, 2H), 2.96-2.84 (m, 2H), 2.79-2.70 (m, 2H), 2.22-2.08 (m, 2H), 2.06-1.83 (m, 8H), 1.75-1.65 (m, 2H), 1.63-1.24 (m, 7H), 1.19-0.91 (m, 4H).

[0175] Example 2: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-((1r,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0176] The production method was the same as in Example 1.

[0177] LC-MS: (ESI, m / z): [M+H] + =874.4 1H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 9.50 (d, J = 6.3 Hz, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.38 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 5.7 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.39 (dd, J = 8.2, 1.9 Hz, 1H), 7.26-6.95 (m, 1H), 6.91-6.41 (m, 1H), 5.32-5.03 (m, 1H), 4.77 (d, J = 17.4 Hz, 1H), 4.17 (t, J= 12.9 Hz, 1H), 3.84-3.71 (m, 3H), 3.67-3.57 (m, 4H), 3.45 (d, J = 9.8 Hz, 1H), 3.30-3.25 (m, 2H), 2.79-2.71 (m, 2H), 2.38-2.24 (m, 4H), 2.13-1.77 (m, 8H), , 1.79-1.66 (m, 2H), 1.60-1.35 (m, 9H), 1.12-0.92 (m, 2H).

[0178] Example 3: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0179] A mixture of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (160 mg, 0.26 mmol), 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid pentafluorophenyl ester (110 mg, 0.26 mmol), and DIEA (168 mg, 1.3 mmol) in DMSO (5 mL) was stirred at room temperature overnight. The reaction mixture was poured into water (50 mL) and stirred for 10 min. After filtration, the filter cake was purified by Prep-HPLC to give 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0180] LC-MS: (ESI, m / z): [M+H] + =870.3. 1H NMR (400 MHz, DMSO-d6) δ10.33 (s, 1H), 9.50 (d, J = 5.7 Hz, 1H), 8.79 (d, J = 7.7 Hz, 1H), 8.39 (d, J = 4.0 Hz, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.41-7.29 (m, 2H), 7.27-6.95 (m, 2H), 6.91-6.40 (m, 1H), 5.31-5.00 (m, 1H), 4.77 (d, J = 17.5 Hz, 1H), 4.27-4.15 (m, 1H), 3.86-3.71 (m, 5H), 3.65-3.39 (m, 8H), 2.89 (d, J = 10.4 Hz, 2H), 2.68 (t, J = 6.5 Hz, 2H), 2.15 (d, J = 6.1 Hz, 2H), 2.08-1.87 (m, 8H), 1.76-1.65 (m, 2H), 1.63-1.38 (m, 5H), 1.36-1.21 (m, 2H), 1.19-0.95 (m, 4H).

[0181] Example 4: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide

[0182] Step 1: Preparation of tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0183] To a solution of 5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxylic acid (470 mg, 1.9 mmol) and DIEA (1 mL, 4.7 mmol) in DMF (20 mL) was added HATU (900 mg, 2.4 mmol) at 25° C. The mixture was stirred at 25° C. for 2 h, and then tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.5 g, 1.5 mmol) was added, and the reaction mixture was further stirred overnight at 25° C. Water (30 mL) was added to the reaction mixture, which was then extracted with EA (50 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (EA) to give tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0184] LC-MS: (ESI, m / z): [M+H] + = 547.4.

[0185] Step 2: Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0186] To a solution of tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.2 g, 0.366 mmol) in DCM (3 mL) was added TFA (1 mL) at 25° C. The reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0187] LC-MS: (ESI, m / z): [M+H] + = 447.2.

[0188] Step 3: Preparation of tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate [ka]

[0189] To a solution of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (200 mg, 0.45 mmol) and DIEA (0.5 mL, 1.35 mmol) in MeOH (5 mL) was added tert-butyl 9-aldehyde-3-azaspiro[5.5]undecane-3-carboxylate (150 mg, 0.45 mmol) and NaBHCN (100 mg, 2.25 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. After filtration and concentration under reduced pressure, the resulting crude product was purified by flash column (MeOH:DCM=3:97) to give tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0190] LC-MS: (ESI, m / z): [M+H] + = 712.4.

[0191] Step 4: Preparation of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0192] To a solution of tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate (0.14 g, 0.2 mmol) in DCM (3 mL) was added TFA (1 mL) at 25° C. The reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide. The resulting product was used directly in the next reaction.

[0193] LC-MS: (ESI, m / z): [M+H] + = 612.5.

[0194] Step 5: Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0195] To a solution of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.18 mmol) and 1-(2-chloro-5-pentafluorobenzoyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (90 mg, 0.19 mmol) in DMSO (2 mL) was added DIEA (0.1 mL, 0.54 mmol) at 25°C. The reaction mixture was stirred at 25°C for 16 h. The reaction mixture was poured into water (10 mL), filtered, and the filter cake was purified by Prep-HPLC.

[0196] LC-MS: (ESI, m / z): [M+H] + = 862.4. 1 H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 9.40 (s, 1H), 8.83 (d, J= 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (d, J= 1.9 Hz, 1H), 7.39 (dd, J = 8.2, 1.8 Hz, 1H), 7.25-6.95 (m, 1H), 6.91 (d, J= 8.0 Hz, 1H), 4.26-4.14 (m, 1H), 3.87-3.68 (m, 9H), 3.66-3.50 (m, 3H), 3.31-3.20 (m, 2H), 2.95-2.83 (m, 2H), 2.80-2.68 (m, 2H), 2.21-2.08 (m, 2H), 2.07-1.87 (m, 6H), 1.70 (d, J = 8.7 Hz, 2H), 1.63-1.41 (m, 5H), 1.38-1.25 (m, 2H), 1.16-1.02 (m, 4H).

[0197] Example 5: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0198] Step 1: Preparation of ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate [ka]

[0199] Ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (3.0 g, 13.3 mmol) and benzyl piperazine-1-carboxylate (2.9 g, 13.3 mmol) were dissolved in ACN (30 mL), DIEA (5.2 g, 40 mmol) was added, and the reaction mixture was heated to 60°C and stirred for 2 h. The mixture was then concentrated under reduced pressure. The crude product was dissolved in ethyl acetate (100 mL), washed with saturated brine, and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 2:1) to give ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate.

[0200] LC-MS: (ESI, m / z): [M+H] + =410.2. 1H NMR (400 MHz, DMSO-d6) δ8.76 (d, J = 7.9 Hz, 1H), 8.22 (s, 1H), 7.41-7.33 (m, 5H), 6.86 (d, J = 7.9 Hz, 1H), 5.13 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.88-3.74 (m, 4H), 3.62-3.49 (m, 4H), 1.28 (t, J = 7.1 Hz, 3H).

[0201] Step 2: Preparation of 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid [ka]

[0202] Ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2.6 g, 6.3 mmol) was dissolved in methanol (26 mL), lithium hydroxide (1.0 g, 25.2 mmol) was added, and the reaction mixture was stirred at 60°C for 2 h. After the reaction was completed, dilute hydrochloric acid (2 mol / L) was added to the reaction mixture until the pH of the reaction mixture reached 2.0, and then the mixture was filtered. The filter cake was washed with water and dried in vacuo to give 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.

[0203] LC-MS: (ESI, m / z): [M+H] + =382.2.

[0204] Step 3: Preparation of 4-(3-((1-(1-(tert-butoxycarbonyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate [ka]

[0205] To a solution of 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (270 mg, 0.4 mmol) and DIEA (0.5 mL, 1.0 mmol) in DMF (5 mL) was added HATU (380 mg, 0.50 mmol) at 25°C, followed by stirring at room temperature for 2 h. Then, tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.21 g, 0.3 mmol) was added, and the reaction mixture was stirred at 25°C overnight. The reaction was quenched by adding water (30 mL) and extracted with EA (50 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was purified by chromatography column (MeOH:DCM = 0 to 3:97) to give 4-(3-((1-(1-(tert-butoxycarbonyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0206] LC-MS: (ESI, m / z): [M+H] + = 680.4.

[0207] Step 4: Preparation of 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate [ka]

[0208] To a solution of 4-(3-((1-(1-(tert-butoxycarbonyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (0.29 g, 0.42 mmol) in DCM (5 mL) was added TFA (1 mL), and the reaction was stirred at room temperature for 2 h. The reaction was directly concentrated under reduced pressure to give 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0209] LC-MS: (ESI, m / z): [M+H] + = 580.5.

[0210] Step 5: Preparation of tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate [ka]

[0211] To a solution of 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (230 mg, 0.4 mmol) and DIEA (0.35 mL, 2.0 mmol) in MeOH (10 mL) was added tert-butyl 9-aldehyde-3-azaspiro[5.5]undecane-3-carboxylate (144 mg, 0.45 mmol) and NaBHCN (138 mg, 1.2 mmol). The reaction was stirred at 25 °C overnight. The reaction was quenched by adding water (30 mL) and then extracted with EA (50 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column (MeOH:DCM = 0 to 3:97) to give tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0212] LC-MS: (ESI, m / z): [M+H] + = 845.5.

[0213] Step 6: Preparation of 4-(3-((1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate [ka]

[0214] To a solution of tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate (0.29 g, 0.34 mmol) in DCM (3 mL) was added TFA (1 mL) and stirred at 25° C. for 1 h. The reaction mixture was directly concentrated under reduced pressure to give 4-(3-((1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0215] LC-MS: (ESI, m / z): [M+H] + = 745.4.

[0216] Step 7: Preparation of 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate [ka]

[0217] To a solution of 4-(3-((1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (240 mg, 0.32 mmol) and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (144 mg, 0.33 mmol) in DMSO (5 mL) was added DIEA (0.1 mL, 0.6 mmol). The reaction mixture was stirred at 25°C for 16 h. Water (30 mL) was added to the reaction mixture, which was then extracted with EA (50 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (MeOH:DCM=0 to 3:97) to give 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0218] LC-MS: (ESI, m / z): [M+H] + = 995.0.

[0219] Step 8: Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0220] To a solution of 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (290 mg, 0.29 mmol) in DCM (15 mL) was added trimethylsilane iodide (0.85 mL, 0.6 mmol) at 0° C., and the reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was poured into water, extracted with DCM (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The resulting crude product was purified by prep-HPLC to give N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0221] LC-MS: (ESI, m / z): [M+H] + = 861.1. 1H NMR (400 MHz, MeOD) δ 8.62 (d, J = 7.9 Hz, 1H), 8.43 (brs, 2.67H, FA), 8.35 (s, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.08-6.77 (m, 2H), 4.57-4.43 (m, 1H), 4.15-4.03 (m, 4H), 3.79 (t, J = 6.4 Hz, 2H), 3.75-3.65 (m, 2H), 3.63-3.48 (m, 2H), 3.47-3.37 (m, 2H), 3.37-3.30 (m, 2H), 3.05-2.95 (m, 2H), 2.95-2.77 (m, 4H), 2.43-2.27 (m, 4H), 1.92-1.75 (m, 3H), 1.74-1.65 (m, 3H), 1.65-1.45 (m, 2H), 1.39-1.12 (m, 5H).

[0222] Example 6: 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0223] Step 1: Preparation of ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate [ka]

[0224] To a solution of ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (2.0 g, 8.9 mmol) in ACN (20 mL) was added 8-oxa-3-azabicyclo[3.2.1]octane (1.3 g, 8.9 mmol) and DIEA (3.4 g, 26 mmol). The reaction mixture was then heated to 60 °C for 2 h. The reaction was quenched with water (200 mL) and extracted with EA (200 mL × 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by chromatography (PE:EA = 1 to 5:1) to give ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate.

[0225] LC-MS: (ESI, m / z): [M+H] + = 303.2.

[0226] Step 2: Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid [ka]

[0227] Ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2.6 g, 8.6 mmol) in a HO / MeOH (3 mL / 26 mL) mixture was added with LiOH (1.4 g, 34 mmol), and the reaction mixture was heated to 60 °C for 12 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by prep-HPLC (acetonitrile / 0.05% NH4OH aqueous solution, 5%-95%) to give 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.

[0228] LC-MS: (ESI, m / z): [M+H] += 275.3.

[0229] Step 3: Preparation of tert-butyl 4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0230] To a solution of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (274 mg, 1 mmol) in DMF (5 mL) was added tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (316 mg, 1 mmol), HATU (456 mg, 1.2 mmol), and DIEA (387 mg, 3 mmol), and the reaction mixture was reacted at 25 °C for 12 h. The reaction mixture was poured into water and extracted with EA (50 mL × 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by chromatography column (MeOH:DCM=0-1:9) to give tert-butyl 4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0231] LC-MS: (ESI, m / z): [M+H] + = 573.4.

[0232] Step 4: Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0233] A solution of tert-butyl 4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (240 mg, 0.42 mmol) in HCl / 1,4-dioxane (5 mL) was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0234] LC-MS: (ESI, m / z): [M+H] + = 473.4.

[0235] Step 5: Preparation of tert-butyl 9-((4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate [ka]

[0236] To a solution of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (130 mg, 0.27 mmol) in THF (10 mL) was added tert-butyl 9-aldehyde-3-azaspiro[5.5]undecane-3-carboxylate (77 mg, 0.27 mmol) and STAB (175 mg, 0.8 mmol). The reaction mixture was stirred at 25°C for 12 h. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by column chromatography (MeOH:DCM = 0 to 1:9) to give tert-butyl 9-((4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0237] LC-MS: (ESI, m / z): [M+H] + = 738.3.

[0238] Step 6: Preparation of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0239] A solution of tert-butyl 9-((4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate (100 mg, 0.14 mmol) in HCl / 1,4-dioxane (4 mL) was stirred at 25° C. for 1 h. The reaction mixture was then directly concentrated under reduced pressure to give N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0240] LC-MS: (ESI, m / z): [M+H] + = 638.3.

[0241] Step 7: Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0242] To a solution of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (150.0 mg, 0.24 mmol) in DMSO (2 mL) was added pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (123 mg, 0.28 mmol) and DIEA (93 mg, 0.72 mmol), and the reaction was stirred at room temperature for 12 h. The reaction mixture was poured into water (20 mL) and extracted with EA (30 mL × 2). The organic phase was dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by prep-HPLC (acetonitrile / 0.05% NHHCO aqueous solution, 5% to 95%) to give 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0243] LC-MS: (ESI, m / z): [M+H] + = 884.5. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.41 (s, 1H), 8.81 (d, J = 7.8 Hz, 1H), 8.39 (s, 1H), 8.28 (d, J = 1.3 Hz, 1H), 7.37 (d, J= 8.4 Hz, 1H), 7.32 (s, 1H), 7.27-6.95 (m, 2H), 6.82 (d, J = 7.9 Hz, 1H), 4.50-4.37 (m, 2H), 4.30-4.00 (m, 2H), 3.84 (s, 3H), 3.68-3.34 (m, 6H), 3.28-3.11 (m, 2H), 2.98-2.78 (m, 2H), 2.73-2.60 (m, 2H), 2.22-1.63 (m, 14H), 1.61-1.17 (m, 8H), 1.18-0.93 (m, 4H).

[0244] Example 7: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)butyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0245] Step 1: Preparation of tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate [ka]

[0246] To a solution of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.24 mmol) in THF (5 mL) was added tert-butyl 4-(4-oxobutyl)piperidine-1-carboxylate (100 mg, 0.36 mmol) and STAB (170 mg, 0.71 mmol). The reaction mixture was stirred at 25 °C for 2 h. The reaction was quenched by the addition of water (30 mL) and extracted with DCM (30 mL x 3). The organic phase was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (MeOH:DCM=0 to 3:97) to give tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate.

[0247] LC-MS: (ESI, m / z): [M+H] + = 698.0.

[0248] Step 2: Preparation of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-(4-(piperidin-4-yl)butyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0249] To a solution of tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate (0.2 g, 0.26 mmol) in DCM (5 mL) was added TFA (1 mL). The reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was directly concentrated under reduced pressure to give 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-(4-(piperidin-4-yl)butyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0250] LC-MS: (ESI, m / z): [M+H] + = 598.1.

[0251] Step 3: Preparation of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)butyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0252] To a solution of 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-(4-(piperidin-4-yl)butyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (160 mg, 0.26 mmol) and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (130 mg, 0.26 mmol) in DMSO (4 mL) was added DIEA (0.14 mL, 0.78 mmol). The reaction mixture was stirred at 25° C. for 2 h. The reaction was quenched by adding water (10 mL), extracted with DCM (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The resulting crude product was purified by Prep-HPLC to give 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(4-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)butyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0253] LC-MS: (ESI, m / z): [M+H] + = 848.3.

[0254] 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.50 (d, J = 5.3 Hz, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.40 (s, 1H), 8.25 (d, J = 5.0 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.54 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.20-7.00 (m, 1H), 6.98-6.45 (m, 1H), 5.40-5.05 (m, 1H), 4.77 (d, J = 17.3Hz, 1H), 4.54-4.34 (m, 1H), 4.28-4.12 (m, 1H), 3.83-3.72 (m, 3H), 3.67-3.57 (m, 3H), 3.45 (d, J = 9.6 Hz, 1H), 3.02-2.85 (m, 3H), 2.80-2.70 (m, 2H), 2.36-2.26 (m, 2H), 2.08-1.88 (m, 8H), 1.76-1.47 (m, 4H), 1.47-1.22 (m, 6H), 1.18-1.02 (m, 2H).

[0255] Example 8: N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide

[0256] Step 1: Preparation of tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate [ka]

[0257] N-(3-(Difluoromethyl)-1-((1R,4R)-4-(aldehyde)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (50 mg, 0.10 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (24 mg, 0.10 mmol) were dissolved in THF (3 mL). Sodium triacetoxyborohydride (67 mg, 0.32 mmol) was added to the stirred reaction mixture, and the mixture was stirred at room temperature for 2 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by normal phase column chromatography to give tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0258] LC-MS: (ESI, m / z): [M+H] + = 712.3.

[0259] Step 2: Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0260] Tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (95 mg, 0.13 mmol) was dissolved in DCM (3 mL), TFA (1 mL) was added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (crude product), which was used directly in the next reaction.

[0261] LC-MS: (ESI, m / z): [M+H] + =612.3.

[0262] Step 3: Preparation of N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0263] N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (130 mg, 0.13 mmol) and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (57 mg, 0.13 mmol) were dissolved in DMSO (5 mL). DIEA (84 mg, 0.65 mmol) was added to the reaction mixture with stirring, and the mixture was stirred at room temperature for 1 h. After completion of the reaction, the reaction mixture was poured into water (50 mL) and extracted with EA (30 mL × 3). The combined organic phase was washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and then purified by prep-HPLC separation to give N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0264] LC-MS: (ESI, m / z): [M+H] + = 862.3. 1H NMR (400 MHz, DMSO-d6) δ10.50 (s, 1H), 9.39 (s, 1H), 8.82 (d, J= 6.3 Hz, 1H), 8.37 (s, 1H), 8.28 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.25-6.95 (m, 1H), 6.90 (d, J = 7.6 Hz, 1H), 4.27-4.07 (m, 1H), 3.90-3.50 (m, 12H), 3.40-3.30 (m, 2H), 2.79-2.70 (m, 2H), 2.43-2.23 (m, 4H), 2.20-1.95 (m, 4H), 1.93-1.83 (m, 2H), 1.82-1.30 (m, 11H), 1.09-0.94 (m, 2H).

[0265] Example 9: N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide

[0266] Step 1: Preparation of tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate [ka]

[0267] N-(3-(difluoromethyl)-1-((1R,4R)-4-(aldehyde)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.23 mmol) was dissolved in THF (10 mL), and tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (56 mg, 0.23 mmol) and STAB (146 mg, 0.69 mmol) were added. The mixture was allowed to react at room temperature for 2 hours, diluted with water (50 mL), extracted with ethyl acetate (50 mL × 3), and then saturated brine (100 mL) was added. The organic phase was washed with 1 mL of ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the crude product was purified by column chromatography (MeOH:DCM=1:11) to give tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0268] LC-MS: (ESI, m / z): [M+H] + =700.4.

[0269] Step 2: Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0270] Tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate (95 mg, 0.14 mmol) was dissolved in DCM (3 mL), and TFA (1 mL) was added. The reaction mixture was reacted at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step.

[0271] LC-MS: (ESI, m / z): [M+H] + =600.3.

[0272] Step 3: Preparation of N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0273] N-(3-(Difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (95 mg, 0.14 mmol) was dissolved in DMSO (5 mL), DIEA (77 mg, 0.60 mmol) and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (61 mg, 0.14 mmol) were added, and the mixture was allowed to react at room temperature for 2 h. Water (50 mL) was added, and the mixture was stirred for 5 minutes. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to reverse-phase preparative separation to give N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0274] LC-MS: (ESI, m / z): [M+H] + =850.4. 1H NMR (400 MHz, DMSO-d6) δ10.50 (s, 1H), 9.39 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.38 (s, 1H), 8.29 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.54 (d, J= 1.8 Hz, 1H), 7.38 (dd, J = 8.2, 1.8 Hz, 1H), 7.24-6.97 (m, 1H), 6.91 (d, J= 8.0 Hz, 1H), 4.49-4.38 (m, 1H), 4.23-4.13 (m, 1H), 3.83-3.76 (m, 4H), 3.75-3.70 (m, 4H), 3.66-3.52 (m, 2H), 3.10-2.95 (m, 1H), 2.84-2.67 (m, 3H), 2.29 (t, J = 7.0 Hz, 2H), 2.14-2.01 (m, 7H), 1.93-1.84 (m, 2H), 1.82-1.46 (m, 7H), 1.41-1.32 (m, 2H), 1.17-0.95 (m, 4H).

[0275] Example 10: 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0276] Step 1: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0277] ((1R,4R)-4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methanol (410 mg, 1.67 mmol) was dissolved in DMF (10 mL), and 5-(8-oxo-3-azaspirobicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (459 mg, 1.67 mmol), HATU (825 mg, 2.17 mmol), and DIEA (646 mg, 5.01 mmol) were added. The mixture was allowed to react at room temperature for 2 h. Water (50 mL) was added and stirred for 5 min, followed by extraction with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0278] LC-MS: (ESI, m / z): [M+H] + =502.3.

[0279] Step 2: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0280] 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (290 mg, 0.58 mmol) was dissolved in ACN (10 mL), IBX (324 mg, 1.16 mmol) was added, and the mixture was reacted at 85°C for 1.5 h. The mixture was concentrated under reduced pressure to remove ACN, water (30 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 3), and saturated brine (50 mL) was added. The organic phase was washed with 1 mL of HCl, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0281] LC-MS: (ESI, m / z): [M+H] + =500.3.

[0282] Step 3: Preparation of tert-butyl 4-(2-((((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate [ka]

[0283] 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.20 mmol) was dissolved in THF (10 mL), and tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (48 mg, 0.20 mmol) and STAB (127 mg, 0.6 mmol) were added. The mixture was allowed to react at room temperature for 2 hours, diluted with water (50 mL), extracted with ethyl acetate (50 mL × 3), and saturated brine (100 The organic phase was washed with 1 mL of tert-butyl 4-(2-((((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0284] LC-MS: (ESI, m / z): [M+H] + =726.4.

[0285] Step 4: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0286] tert-Butyl 4-(2-((((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate (110 mg, 0.15 mmol) was dissolved in DCM (3 mL), TFA (1 mL) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was allowed to proceed for 1 hour and concentrated under reduced pressure to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step.

[0287] LC-MS: (ESI, m / z): [M+H] + =626.3.

[0288] Step 5: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0289] 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.15 mmol) was dissolved in DMSO (5 mL), DIEA (97 mg, 0.75 mmol) and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (65 mg, 0.15 mmol) were added, and the mixture was allowed to react at room temperature for 2 hours. Water (50 mL) was added and the mixture was stirred for 5 minutes, extracted with ethyl acetate (50 mL × 3), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to reverse-phase preparative separation to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0290] LC-MS: (ESI, m / z): [M+H] + =876.2. 1H NMR (400 MHz, DMSO-d6) δ10.50 (s, 1H), 9.41 (s, 1H), 8.82 (d, J= 7.9 Hz, 1H), 8.38 (s, 1H), 8.28 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.54 (d, J= 1.9 Hz, 1H), 7.38 (dd, J = 8.2, 2.0 Hz, 1H), 7.30-6.95 (m, 1H), 6.82 (d, J= 8.0 Hz, 1H), 4.48-4.42 (m, 2H), 4.26-4.09 (m, 2H), 3.79-3.50 (m, 4H), 3.27-3.17 (m, 2H), 3.10-2.90 (m, 1H), 2.85-2.70 (m, 3H), 2.29 (t, J = 6.8 Hz, 2H), 2.15-2.07 (m, 5H), 2.07-2.01 (m, 2H), 1.95-1.83 (m, 4H), 1.82-1.47 (m, 9H), 1.41-1.31 (m, 2H), 1.17-0.96 (m, 4H).

[0291] Example 11: 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0292] Step 1: Preparation of tert-butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate [ka]

[0293] 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.20 mmol) was dissolved in THF (10 mL), and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (48 mg, 0.20 mmol) and STAB (127 mg, 0.6 mmol) were added. The mixture was reacted at room temperature for 2 hours, diluted with water (50 mL), extracted with ethyl acetate (50 mL × 3), and saturated brine (100 The organic phase was washed with 1 mL of ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM=1:11) to give tert-butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0294] LC-MS: (ESI, m / z): [M+H] + =738.5.

[0295] Step 2: Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0296] tert-Butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (105 mg, 0.14 mmol) was dissolved in DCM (3 mL), TFA (1 mL) was added, and the mixture was allowed to react at room temperature for 1 h. Concentration under reduced pressure gave N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step.

[0297] Step 3: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0298] N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.14 mmol) was dissolved in DMSO (5 mL), DIEA (90 mg, 0.70 mmol) and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (61 mg, 0.14 mmol) were added, and the mixture was allowed to react at room temperature for 2 h. Water (50 mL) was added and the mixture was stirred for 5 min, extracted with ethyl acetate (50 mL × 3), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to reverse-phase preparative separation to give 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0299] LC-MS: (ESI, m / z): [M+H] + =888.2. 1H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 9.41 (s, 1H), 8.81 (d, J= 7.9 Hz, 1H), 8.37 (s, 1H), 8.28 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.55 (d, J= 1.8 Hz, 1H), 7.39 (dd, J = 8.2, 1.9 Hz, 1H), 7.24-6.97 (m, 1H), 6.82 (d, J= 8.0 Hz, 1H), 4.47-4.41 (m, 2H), 4.31-3.93 (m, 3H), 3.81-3.72 (m, 1H), 3.67-3.51 (m, 3H), 3.25-3.15 (m, 2H), 2.77-2.72 (m, 2H), 2.35-2.25 (m, 4H), 2.15-2.07 (m, 2H), 2.16-2.98 (m, 2H), 1.96-1.29 (m, 19H), 1.10-0.92 (m, 2H).

[0300] Example 12: N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0301] N-(1-(1-((3-Azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.14 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoic acid pentafluorophenyl ester (58 mg, 0.14 mmol) and N,N-diisopropylethylamine (54 mg, 0.42 mmol) were added with stirring. After stirring at room temperature for 2 h, water (50 mL) was added, filtered, and the filter cake was purified by pre-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5% to 95%) to give N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0302] LC-MS: (ESI, m / z): [M+H] + =846.4. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.40 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.36 (d, J = 4.0 Hz, 2H), 7.23-6.97 (m, 1H), 6.91 (d, J = 8.0 Hz, 1H), 4.25-4.15 (m, 1H), 3.84-3.69 (m, 10H), 3.65-3.49 (m, 2H), 3.31-3.24 (m, 2H), 2.89 (d, J = 10.3 Hz, 2H), 2.73 (t, J = 6.6 Hz, 2H), 2.20-2.04 (m, 2H), 2.04-1.80 (m, 6H), 1.70-1.43 (m, 7H), 1.43-1.20 (m, 2H), 1.20-0.96 (m, 4H).

[0303] Example 13: N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0304] N-(1-(1-((3-Azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.12 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoic acid pentafluorophenyl ester (58 mg, 0.14 mmol) and N,N-diisopropylethylamine (46 mg, 0.36 mmol) were added with stirring. After stirring at room temperature for 2 h, water (50 mL) was added, filtered, and the filter cake was purified by pre-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5% to 95%) to give N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0305] LC-MS: (ESI, m / z): [M+H] + =842.4. 1 H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.40 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 7.37-7.29 (m, 2H), 7.28-6.96 (m, 2H), 6.91 (d, J = 7.9 Hz, 1H), 4.25-4.15 (m, 1H), 3.83-3.63 (m, 9H), 3.61-3.47 (m, 3H), 3.32-3.24 (m, 2H), 2.95-2.65 (m, 4H), 2.25-1.85 (m, 11H), 1.70 (d, J = 9.3 Hz, 2H), 1.58-1.15 (m, 7H), 1.12-0.91 (m, 4H).

[0306] Example 14: N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0307] Step 1: Preparation of tert-butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate [ka]

[0308] N-(3-(difluoromethyl)-1-((1R,4S)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)-5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (120 mg, 0.21 mmol) and tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (51 mg, 0.21 mmol) were dissolved in tetrahydrofuran (10 mL), and sodium triacetoxyborohydride (222 mg, 1.05 mmol) was added with stirring. After stirring overnight at room temperature, water (50 mL) was added, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (methanol:dichloromethane = 1:9) to give tert-butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0309] LC-MS: (ESI, m / z): [M+H] + =798.5.

[0310] Step 2: Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0311] tert-Butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate (80 mg, 0.10 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 2 hours. The mixture was stirred for 1 h, and the reaction mixture was directly concentrated under reduced pressure to give N-(3-(difluoromethyl)-1-((1R,4S)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next reaction without purification.

[0312] LC-MS: (ESI, m / z): [M+H] + =614.3.

[0313] Step 3: Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0314] N-(3-(difluoromethyl)-1-((1R,4S)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, 0.10 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid pentafluorophenyl ester (47 mg, 0.11 mmol) and N,N-diisopropylethylamine (38 mg, 0.30 mmol) were added with stirring. After stirring overnight at room temperature, water (50 mL) was added and filtered. The filter cake was purified by pre-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5% to 95%) to give N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0315] LC-MS: (ESI, m / z): [M+H] + =860.4. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.37 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.37 (s, 1H), 8.24 (d, J = 5.1 Hz, 1H), 7.36 (dd, J= 8.5, 2.1 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H), 7.24-6.94 (m, 2H), 6.86 (d, J= 8.1 Hz, 1H), 4.89 (d, J = 4.1 Hz, 1H), 4.50-4.15 (m, 2H), 4.03-3.80 (m, 5H), 3.72-3.53 (m, 4H), 3.51-3.40 (m, 1H), 3.10-2.65 (m, 4H), 2.30 (t, J = 7.0 Hz, 2H), 2.15-1.95 (m, 7H), 1.92-1.25 (m, 15H), 1.17-0.95 (m, 4H).

[0316] Example 15: 5-((S)-3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0317] Step 1: Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate [ka]

[0318] (S)-5-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (150 mg, 0.42 mmol) was dissolved in DMF (10 mL), and ((1R,4R)-4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methanol (112 mg, 0.46 mmol), DIEA (163 mg, 1.26 mmol), and py-BOP (284 mg, 0.55 mmol) were added. The mixture was allowed to react at room temperature for 2 h. Water (50 mL) was added and stirred for 5 min, followed by extraction with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA: PE = 0-1) to give tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0319] LC-MS: (ESI, m / z): [M+H] + =589.3.

[0320] Step 2: Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate [ka]

[0321] Tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(hydroxymethyl)cyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate (190 mg, 0.32 mmol) was dissolved in ACN (10 mL), and IBX (181 mg, 0.65 mmol) was added. The mixture was reacted at 85°C for 1.5 hours. Water (30 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA: PE = 0-1) to give tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0322] LC-MS: (ESI, m / z): [M+H] + =586.6.

[0323] Step 3: Preparation of N-benzyl-N-methyl-2-(piperidin-4-yl)ethan-1-amine [ka]

[0324] tert-Butyl 4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carboxylate (345 mg, 1.04 mmol) was dissolved in DCM (5 mL), TFA (1 mL) was added, and the mixture was reacted at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-benzyl-N-methyl-2-(piperidin-4-yl)ethan-1-amine, which was used directly in the next step.

[0325] LC-MS: (ESI, m / z): [M+H] + =233.2.

[0326] Step 5: Preparation of 1-(5-(4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0327] N-Benzyl-N-methyl-2-(piperidin-4-yl)ethan-1-amine (250 mg, 1.04 mmol) was dissolved in DMSO (10 mL), and DIEA (671 mg, 5.2 mmol) and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (451 mg, 1.04 mmol) were added, followed by reaction at room temperature for 2 h. Water (100 mL) was added to the reaction mixture, and the mixture was stirred for 5 min. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0 to 1:9) to give 1-(5-(4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0328] LC-MS: (ESI, m / z): [M+H] + =479.2.

[0329] Step 6: Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0330] 1-(5-(4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (240 mg, 0.5 mmol) was dissolved in MeOH (10 mL), and Pd / C (72 mg, 30%) was added. The mixture was reacted at room temperature for 2 h. After filtration, the mixture was concentrated under reduced pressure to give 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was used directly in the next step.

[0331] LC-MS: (ESI, m / z): [M+Na] + =411.1.

[0332] Step 7: Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate [ka]

[0333] 1-(2-Methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (95 mg, 0.24 mmol) was dissolved in THF (10 mL), and tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate (120 mg, 0.20 mmol) and STAB (127 mg, 0.60 mmol) were added, followed by a reaction at room temperature for 2 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0 to 1:9) to give tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0334] LC-MS: (ESI, m / z): [M+H] + =959.4.

[0335] Step 8: Preparation of 5-((S)-3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0336] Tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate (100 mg, 0.10 mmol) was dissolved in DCM (5 mL), TFA (1 mL) was added, and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was directly concentrated under reduced pressure, and the crude product was purified by reverse-phase preparative separation to give 5-((S)-3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-((1R,4S)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0337] LC-MS: (ESI, m / z): [M+H] + =859.4. 1H NMR (400 MHz, DMSO-d6) δ10.33 (s, 1H), 9.36 (s, 1H), 8.74 (d, J= 8.0 Hz, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 7.36 (dd, J = 8.4, 2.1 Hz, 1H), 7.32 (d, J= 2.1 Hz, 1H), 7.27-6.97 (m, 2H), 6.88 (d, J= 8.1 Hz, 1H), 4.52-4.15 (m, 4H), 3.84 (s, 3H), 3.59 (t, J = 6.6 Hz, 2H), 3.24-2.93 (m, 3H), 2.80-2.60 (m, 4H), 2.40-2.25 (m, 2H), 2.16-1.99 (m, 8H), 1.95-1.80 (m, 3H), 1.80-1.29 (m, 13H), 1.17-0.96 (m, 4H).

[0338] Example 16: N-(3-(difluoromethyl)-1-(1-(2-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka] N-(1-(1-(2-(3-Azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (103 mg, 0.165 mmol) and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid pentafluorophenyl ester (85 mg, 0.198 mmol) were added to DMSO (3 mL), followed by the addition of DIEA (63.8 mg, 0.495 mmol). The mixture was stirred at room temperature for 16 h, and then water (20 mL) was added. The mixture was extracted with ethyl acetate (25 mL × 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (acetonitrile / 0.5% FA aqueous solution, Purification by 5% to 95% gave the product N-(3-(difluoromethyl)-1-(1-(2-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0339] LC-MS: (ESI, m / z): [M+H] + = 872.5. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.40 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 8.27 (s, 0.62H, HCOOH), 7.42-7.28 (m, 2H), 7.26-6.94 (m, 2H), 6.91 (d, J = 7.9 Hz, 1H), 4.30-4.10 (m, 1H), 3.84 (s, 3H), 3.79-3.61(m, 8H), 3.62-3.54 (m, 6H), 2.97-2.91 (m, 2H), 2.71-2.65 (m, 2H), 2.37-2.17 (m, 2H), 2.05-1.85 (m, 6H), 1.75-1.62 (m, 2H), 1.55-1.17 (m, 9H), 1.17-0.93 (m, 4H).

[0340] Example 17: N-(3-(difluoromethyl)-1-((1R,4R)-4-((2-(1-(3-(2,6-dioxopyridin-3-yl)-4-fluorobenzoyl)piperidin-4-yl)ethyl)(methyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide

[0341] Step 1: Preparation of methyl (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylate [ka]

[0342] 5-Morpholinepyrazolo[1,5-a]pyrimidine-3-carboxylic acid (200 mg, 0.73 mmol) was dissolved in DMF (4 mL), and methyl (1R,4R)-4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexane-1-carboxylate (182 mg, 0.73 mmol), HATU (139 mg, 0.95 mmol), and DIEA (377 mg, 2.92 mmol) were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (40 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA:PE = 9:1) to give methyl (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylate.

[0343] LC-MS: (ESI, m / z): [M+H] + =504.1.

[0344] Step 2: Preparation of (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazole[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid [ka]

[0345] Methyl (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylate (230 mg, 0.46 mmol) was dissolved in a mixed solution of methanol / water (5 mL, V / V=5 / 1), and lithium hydroxide (44 mg, 1.84 mmol) was added. The reaction mixture was heated to 60°C and stirred for 2 h. The reaction mixture was adjusted to pH 4 with dilute hydrochloric acid (2 mol / L) and extracted with ethyl acetate (40 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazole[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid, which was used directly in the next reaction.

[0346] LC-MS: (ESI, m / z): [M+H] + =490.6.

[0347] Step 3: Preparation of tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxyacylamino)ethyl)piperidine-1-carboxylate [ka]

[0348] (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazole[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid (140 mg, 0.29 mmol) and tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (70 mg, 0.29 mmol) were dissolved in DMF (4 mL), and HATU (144 mg, 0.38 mmol) and DIEA (155 mg, 1.2 mmol) were added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (40 mL × 4). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA) to give tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxyacylamino)ethyl)piperidine-1-carboxylate.

[0349] LC-MS: (ESI, m / z): [M+H] + =714.8.

[0350] Step 4: Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0351] Tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxyacylamino)ethyl)piperidine-1-carboxylate (130 mg, 0.18 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.3 mL) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step without purification.

[0352] LC-MS: (ESI, m / z): [M+H] + =614.2.

[0353] Step 5: Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-((2-(1-(3-(2,6-dioxopyridin-3-yl)-4-fluorobenzoyl)piperidin-4-yl)ethyl)(methyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0354] N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (130 mg, 0.18 mmol) and 3-(2,6-dioxopiperidin-3-yl)-4-fluorobenzoic acid pentafluorophenyl ester (75.3 mg, 0.18 mmol) were dissolved in DMSO (2 mL), DIEA (93 mg, 0.72 mmol) was added, and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give N-(3-(difluoromethyl)-1-((1R,4R)-4-((2-(1-(3-(2,6-dioxopyridin-3-yl)-4-fluorobenzoyl)piperidin-4-yl)ethyl)(methyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0355] LC-MS: (ESI, m / z): [M+H] + =847.4. 1H NMR (400 MHz, DMSO-d6) δ10.90 (s, 1H), 9.40 (s, 1H), 8.83 (d, J= 7.9 Hz, 1H), 8.40 (d, J = 5.5 Hz, 1H), 8.29 (s, 1H), 7.40-7.33 (m, 2H), 7.31-7.23 (m, 1H), 7.23-6.96 (m, 1H), 6.91 (d, J = 8.0 Hz, 1H), 4.52-4.20 (m, 2H), 4.12 (dd, J = 12.7, 4.9 Hz, 1H), 3.88-3.66 (m, 8H), 3.62-3.48 (m, 1H), 3.433.34 (m, 1H), 3.01 (s, 3H), 2.82-2.62 (m, 4H), 2.60-2.54 (m, 1H), 2.29-2.17 (m, 1H), 2.08-1.98 (m, 3H), 1.95-1.67 (m, 6H), 1.66-1.35 (m, 6H), 1.21-1.02 (m, 2H).

[0356] Example 18: 5-((S)-3-aminopiperidin-1-yl)-N-(1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0357] Step 1: Preparation of tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate [ka]

[0358] tert-Butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (240 mg, 1.0 mmol) was dissolved in DCM (20 mL), and FmocCl (311 mg, 1.2 mmol) and pyridine (237 mg, 3.0 mmol) were added. The mixture was allowed to react at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with DCM (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA:PE = 0 to 4:1) to give tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0359] LC-MS: (ESI, m / z): [M+Na] + =487.3.

[0360] Step 2: Preparation of (9H-fluoren-9-yl)methyl methyl(2-(piperidin-4-yl)ethyl)carbamate [ka]

[0361] tert-Butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate (210 mg, 0.45 mmol) was dissolved in DCM (4 mL), and TFA (1 mL) was added and the reaction was allowed to proceed at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give (9H-fluoren-9-yl)methylmethyl(2-(piperidin-4-yl)ethyl)carbamate, which was used directly in the next step.

[0362] LC-MS: (ESI, m / z): [M+H] + =365.2.

[0363] Step 3: Preparation of 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0364] (9H-Fluoren-9-yl)methyl methyl(2-(piperidin-4-yl)ethyl)carbamate (210 mg, 0.45 mmol) was dissolved in DMSO (5 mL), DIEA (264 mg, 2.05 mmol) and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (178 mg, 0.41 mmol) were added, and the mixture was allowed to react at room temperature overnight. The reaction solution was purified by reverse-phase chromatography (45% ACN / 0.1% NH4HCO3 aqueous solution) to give 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0365] LC-MS: (ESI, m / z): [M+H] + =393.1.

[0366] Step 4: Preparation of tert-butyl ((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate [ka]

[0367] 1-(2-Chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (50 mg, 0.13 mmol) was dissolved in THF (10 mL), and tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-formylcyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate (30 mg, 0.05 mmol) and STAB (83 mg, 0.39 mmol) were added. After reacting for 1 h, tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-formylcyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate (60 mg, 0.10 mmol) was added in two portions within 1 h, and the reaction was continued at room temperature for 2 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0 to 1:9) to give tert-butyl ((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate.

[0368] LC-MS: (ESI, m / z): [M+H] + =963.3.

[0369] Step 5: Preparation of 5-((S)-3-aminopiperidin-1-yl)-N-(1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0370] Tert-butyl ((S)-1-(3-((1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate (100 mg, 0.10 mmol) was dissolved in DCM (5 mL), TFA (1 mL) was added, and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, then saturated NaHCO3 solution was added to adjust the pH to 8-9, followed by extraction with MeOH / DCM (10%, 20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by reverse-phase preparative separation to give 5-((S)-3-aminopiperidin-1-yl)-N-(1-((1R,4S)-4-(((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0371] LC-MS: (ESI, m / z): [M+H] + =863.1. 1H NMR (400 MHz, DMSO-d6) δ11.18-9.87 (s, 1H), 9.36 (s, 1H), 8.74 (d, J= 8.0 Hz, 1H), 8.37 (s, 1H), 8.24 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.54 (d, J= 2.0 Hz, 1H), 7.38 (dd, J = 8.2, 2.0 Hz, 1H), 7.26-6.97 (m, 1H), 6.87 (d, J= 8.0 Hz, 1H), 4.50-4.25 (m, 2H), 4.18 (t, J= 11.9 Hz, 2H), 3.80-3.70 (m, 1H), 3.70-3.51 (m, 2H), 3.26-3.18 (m, 1H), 3.08-2.97 (m, 2H), 2.85-2.64 (m, 5H), 2.30 (t, J = 7.2 Hz, 2H), 2.14-2.07 (m, 5H), 2.07-1.97 (m, 2H), 1.95-1.83 (m, 3H), 1.80-1.30 (m, 12H), 1.18-0.95 (m, 4H).

[0372] Example 19: N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0373] Step 1: Preparation of tert-butyl 9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate [ka]

[0374] Tert-Butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (100 mg, 0.39 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (205 mg, 0.47 mmol) and N,N-diisopropylethylamine (153 mg, 1.18 mmol) were added with stirring at room temperature overnight. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate) to give tert-butyl 9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0375] LC-MS: (ESI, m / z): [M-Boc] + =405.1.

[0376] Step 2: Preparation of 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0377] tert-Butyl 9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (170 mg, 0.34 mmol) was dissolved in dichloromethane (2.5 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was directly concentrated under reduced pressure to give 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was used directly in the next reaction without purification.

[0378] LC-MS: (ESI, m / z): [M+H] + =405.1.

[0379] Step 3: Preparation of N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0380] N-(3-(Difluoromethyl)-1-((1R,4R)-4-aldehydecyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (50 mg, 0.10 mmol) and 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (42 mg, 0.10 mmol) were dissolved in dichloroethane (10 mL), and sodium triacetoxyborohydride (44 mg, 0.20 mmol) was added. The mixture was heated to 70°C and stirred overnight. Water (50 mL) was added to the reaction mixture, which was then extracted with dichloromethane (20 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5% to 95%) to give N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0381] LC-MS: (ESI, m / z): [M+H] + =874.2. 1H NMR (400 MHz, CDCl3) δ9.76 (s, 1H), 8.44 (s, 1H), 8.39 (d, J= 5.3 Hz, 2H), 8.30 (d, J = 7.6 Hz, 1H), 7.58-7.48 (m, 2H), 7.43 (d, J = 1.8 Hz, 1H), 7.38-7.34 (m, 1H), 6.97-6.65 (m, 1H), 6.02 (d, J = 7.5 Hz, 1H), 4.98-4.85 (m, 4H), 4.47-4.37 (m, 4H), 4.13-4.06 (m, 1H), 3.86-3.74 (m, 4H), 3.46-3.38 (m, 2H), 3.00-2.74 (m, 3H), 2.39-2.29 (m, 3H), 2.29-2.10 (m, 3H), 2.10-2.00 (m, 2H), 1.92-1.76 (m, 3H), 1.40-1.10 (m, 10H), 0.95-0.85 (m, 1H).

[0382] Example 20: N-(1-(1-(2-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0383] N-(1-(1-(2-(3-Azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (23 mg, 0.03 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (14 mg, 0.03 mmol) and N,N-diisopropylethylamine (38 mg, 0.30 mmol) were added with stirring at room temperature for 2 h. Water (20 mL) was added to the reaction mixture, which was then filtered. The filter cake was purified by p-TLC (methanol:dichloromethane=1:9) to give N-(1-(1-(2-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0384] LC-MS: (ESI, m / z): [M+H] + =876.3. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.40 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.42-7.35 (m, 1H), 7.26-6.97 (m, 1H), 6.91 (d, J = 8.0 Hz, 1H), 4.31-4.11 (m, 1H), 3.86-3.66 (m, 9H), 3.61-3.50 (m, 3H), 3.29-3.24 (m, 1H), 2.99-2.90 (m, 2H), 2.80-2.71 (m, 2H), 2.43-2.30 (m, 2H), 2.15-1.84 (m, 6H), 1.73-1.60 (m, 2H), 1.55-1.23 (m, 10H), 1.16-1.01 (m, 4H).

[0385] Example 21: 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0386] Step 1: Preparation of tert-butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate [ka]

[0387] 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, crude product) was dissolved in THF (10 mL), and tert-butyl 9-formyl-3-azaspiro[5.5]undecane-3-carboxylate (50 mg, 0.18 mmol) and STAB (115 mg, 0.54 mmol) were added. The mixture was allowed to react at room temperature for 2 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM = 0 to 1:9) to give tert-butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0388] LC-MS: (ESI, m / z): [M+H] + =724.3.

[0389] Step 2: Preparation of N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0390] tert-Butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate (100 mg, 0.14 mmol) was dissolved in DCM (4 mL), TFA (1 mL) was added, and the mixture was allowed to react at room temperature for 1 h. The reaction was directly concentrated under reduced pressure to give N-(1-(1-((3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step.

[0391] LC-MS: (ESI, m / z): [M+H] + =624.4.

[0392] Step 3: Preparation of 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0393] N-(1-(1-((3-Azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.14 mmol) was dissolved in DMSO (10 mL), DIEA (90 mg, 0.70 mmol) and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoate (58 mg, 0.14 mmol) were added, and the mixture was reacted at room temperature for 2 h. Water (100 mL) was added to the reaction mixture, and the mixture was stirred for 5 min. The mixture was then extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by reverse-phase preparative separation to give 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0394] LC-MS: (ESI, m / z): [M+H] + =854.4. 1H NMR (400 MHz, DMSO-d6) δ10.37 (s, 1H), 9.50 (d, J = 5.7 Hz, 1H), 8.78 (d, J = 7.6 Hz, 1H), 8.39 (d, J = 3.6 Hz, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.36-7.29 (m, 2H), 7.26-6.96 (m, 2H), 6.88-6.44 (m, 1H), 5.28-5.07 (m, 1H), 4.77 (d, J = 17.6 Hz, 1H), 4.31-4.11 (m, 1H), 3.88-3.71 (m, 3H), 3.63-3.45 (m, 5H), 2.95-2.85 (m, 2H), 2.81-2.65 (m, 2H), 2.25 -2.10 (m, 5H), 2.07-1.87 (m, 8H).

[0395] Example 22 Preparation of N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0396] N-(3-(Difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (60 mg, 0.1 mmol) and 3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecane-9-formaldehyde (41 mg, 0.1 mmol) were dissolved in tetrahydrofuran (2 mL), and sodium triacetoxyborohydride (84 mg, 0.4 mmol) was added with stirring. The mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0397] LC-MS: (ESI, m / z): [M+H] + =890.1 1H NMR (400 MHz, CD3OD-d4) δ 8.64 (d, J = 7.9 Hz, 1H), 8.37 (d, J= 5.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H), 7.36-7.29 (m, 2H), 7.06-6.77 (m, 2H), 4.60-4.52 (m, 2H), 4.42-4.34 (m, 4H), 4.25-4.14 (m, 1H), 3.92-3.82 (m, 1H), 3.77-3.62 (m, 3H), 3.49-3.40 (m, 2H), 3.30-3.22 (m, 4H), 3.06-3.00 (m, 2H), 2.92-2.82 (m, 2H), 2.31 (s, 3H), 2.28-2.22 (m, 2H), 2.20-2.06 (m, 6H), 1.82-1.72 (m, 2H), 1.70-1.63 (m, 2H), 1.55-1.39 (m, 2H), 1.33-1.25 (m, 2H), 1.24-1.12 (m, 3H).

[0398] Example 23: (S)-5-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0399] Step 1: Preparation of tert-butyl (S)-(1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate [ka]

[0400] (S)-tert-Butyl (1-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate (45 mg, 0.08 mmol) and 3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecane-9-formaldehyde (33 mg, 0.08 mmol) were dissolved in DMF (2 mL), and NaBH(OAc) (51 mg, 0.24 mmol) was added. The mixture was stirred at room temperature for 2 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The combined upper organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by normal phase column chromatography to give tert-butyl (S)-(1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0401] LC-MS: (ESI, m / z): [M+H] + = 955.5.

[0402] Step 2: Preparation of (S)-5-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0403] (S)-tert-Butyl (1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate (30 mg, 0.03 mmol) was dissolved in DCM (0.5 mL), followed by addition of TFA (0.1 mL) and stirring at room temperature for 1 h. Saturated aqueous NaHCO3 was added to the reaction mixture until the pH of the solution became slightly basic, and the mixture was extracted with a mixed organic solvent (MeOH:DCM = 1:10, 30 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by p-HPLC to give (S)-5-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0404] LC-MS: (ESI, m / z): [M+H] + =855.7. 1H NMR (400 MHz, MeOD) δ 8.49 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 7.40 (d, J= 7.9 Hz, 1H), 7.35-7.29 (m, 2H), 7.04-6.74 (m, 2H), 4.60-4.50 (m, 1H), 4.50-4.25 (m, 2H), 4.25-4.15 (m, 1H), 3.97-3.79 (m, 1H), 3.80-3.63 (m, 3H), 3.50-3.38 (m, 3H), 3.15-3.08 (m, 1H), 3.06-3.01 (m, 2H), 2.94-2.79 (m, 3H), 2.31 (s, 3H), 2.27-2.21 (m, 2H), 2.21-2.04 (m, 7H), 1.93-1.83 (m, 1H), 1.83-1.76 (m, 2H), 1.70-1.44 (m, 8H), 1.22-1.02 (m, 4H).

[0405] Example 24: N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0406] Step 1: Preparation of tert-butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate [ka]

[0407] 5-(2-Oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (130 mg, 0.5 mmol), tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (158 mg, 0.5 mmol), and Py-BOP (780 mg, 1.5 mmol) were dissolved in DMF (6 mL). DIEA (780 mg, 1.5 mmol) was added and the mixture was stirred at room temperature for 16 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The combined upper organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by normal phase column chromatography to give tert-butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0408] LC-MS: (ESI, m / z): [M+H] + = 559.2.

[0409] Step 2: Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0410] tert-Butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (250 mg, 0.45 mmol) was dissolved in DCM (4 mL), followed by the addition of TFA (1 mL) and stirring at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give crude N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0411] LC-MS: (ESI, m / z): [M+H] + = 459.3.

[0412] Step 3: Preparation of N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0413] N-(3-(Difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (114 mg, 0.25 mmol) and 3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecane-9-formaldehyde (102 mg, 0.25 mmol) were dissolved in THF (5 mL), and sodium triacetoxyborohydride (160 mg, 0.75 mmol) was added and the mixture was stirred at room temperature for 16 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The combined upper organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by prep-HPLC to give a white solid, N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0414] LC-MS: (ESI, m / z): [M+H] + =854.6. 1H NMR (400 MHz, MeOD) δ 8.47 (d, J = 7.8 Hz, 1H), 8.35 (s, 1H), 8.27 (s, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.36-7.27 (m, 2H), 7.05-6.95 (m, 1H), 6.31 (d, J = 8.0 Hz, 1H), 4.62-4.50 (m, 2H), 4.50-4.39 (m, 4H), 4.26-4.11 (m, 1H), 3.95-3.82 (m, 1H), 3.77-3.60 (m, 3H), 3.49-3.40 (m, 2H), 3.05-2.97 (m, 2H), 2.89-2.79 (m, 2H), 2.37-2.20 (m, 5H), 2.20-2.09 (m, 6H), 1.83-1.75 (m, 2H), 1.71-1.41 (m, 7H), 1.37-1.27 (m, 2H), 1.24-1.08 (m, 4H).

[0415] Example 25: (S)—N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0416] (S)—N-(1-(1-((3-Azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, crude product, 0.11 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoic acid pentafluorophenyl ester (46 mg, 0.11 mmol) and N,N-diisopropylethylamine (142 mg, 1.1 mmol) were added with stirring at room temperature overnight. Water (50 mL) was added to the reaction mixture, which was then filtered. The filter cake was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give (S)—N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0417] LC-MS: (ESI, m / z): [M+H] + =856.3. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.38 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 7.37-7.29 (m, 2H), 7.26-6.96 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 4.89 (d, J = 2.0 Hz, 1H), 4.26-4.14 (m, 1H), 4.00-3.75 (m, 3H), 3.70-3.39 (m, 6H), 3.30-3.24 (m, 2H), 2.93-2.64 (m, 4H), 2.21 (s, 3H), 2.18-2.10 (m, 2H), 2.06-1.65 (m, 10H), 1.61-1.22 (m, 9H), 1.16-0.95 (m, 4H).

[0418] Example 26: (S)—N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0419] (S)—N-(1-(1-((3-Azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, crude product, 0.11 mmol) was dissolved in dimethyl sulfoxide (3 mL), and perfluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (48 mg, 0.11 mmol) and N,N-diisopropylethylamine (142 mg, 1.1 mmol) were added with stirring and the mixture was stirred at room temperature overnight. Water (30 mL) was added to the reaction mixture, which was then filtered. The filter cake was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give (S)—N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0420] LC-MS: (ESI, m / z): [M+H] + =876.3. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.38 (s, 1H), 8.72 (d, J = 7.9 Hz, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.26-6.97 (m, 1H), 6.86 (d, J = 8.0 Hz, 1H), 4.88 (d, J = 2.0 Hz, 1H), 4.25-4.15 (m, 1H), 3.97-3.40 (m, 9H), 3.30-3.18 (m, 2H), 2.93-2.85 (m, 2H), 2.78-2.72 (m, 2H), 2.20-2.10 (m, 2H), 2.05-1.65 (m, 10H), 1.60-1.31 (m, 9H), 1.20-0.94 (m, 4H).

[0421] Example 27: N-(3-(difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0422] Step 1: Preparation of tert-butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate [ka]

[0423] N-(3-(difluoromethyl)-1-((1R,4S)-4-formaldehydecyclohexyl)-1H-pyrazol-4-yl)-5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (90 mg, 0.15 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (40 mg, 0.15 mmol) were dissolved in tetrahydrofuran (10 mL), sodium triacetoxyborohydride (95 mg, 0.45 mmol) was added, and the mixture was stirred at room temperature for 2 h. Water (50 mL) was added, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was then washed with saturated brine (60 mL). The mixture was washed with 1 mL of tert-butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0424] LC-MS: (ESI, m / z): [M+H] + =810.4.

[0425] Step 2: Preparation of N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0426] tert-Butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-(tetrahydro-2H-pyran-2-yl)oxo)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxyacylamino)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (100 mg, 0.12 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next reaction without purification.

[0427] LC-MS: (ESI, m / z): [M+H] + =626.3.

[0428] Step 3: Preparation of N-(3-(difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0429] N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.12 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoic acid pentafluorophenyl ester (55 mg, 0.13 mmol) and N,N-diisopropylethylamine (155 mg, 1.2 mmol) were added and the mixture was stirred at room temperature overnight. Water (30 mL) was added to the reaction mixture, which was then filtered. The filter cake was purified by pre-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5% to 95%) to give N-(3-(difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0430] LC-MS: (ESI, m / z): [M+H] + =856.4. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.38 (s, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.36 (s, 1H), 8.24 (s, 1H), 7.36-7.29 (m, 2H), 7.27-6.94 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 4.93-4.88 (m, 1H), 4.20-4.10 (m, 1H), 4.01-3.74 (m, 3H), 3.70-3.44 (m, 6H), 3.33-3.25 (m, 2H), 2.84-2.64 (m, 2H), 2.40-2.21 (m, 4H), 2.21 (s, 3H), 2.15-1.96 (m, 4H), 1.87-1.70 (m, 6H), 1.65-1.26 (m, 11H), 1.10-0.95 (m, 2H).

[0431] Example 28: N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide

[0432] Step 1: Preparation of tert-butyl 9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate [ka]

[0433] Tert-Butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (200 mg, 0.79 mmol) was dissolved in dimethyl sulfoxide (4 mL). Pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (340 mg, 0.79 mmol) and N,N-diisopropylethylamine (413 mg, 3.24 mmol) were added with stirring, and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:petroleum ether = 9:1) to give tert-butyl 9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0434] LC-MS: (ESI, m / z): [M+H] + =501.2.

[0435] Step 2: Preparation of 1-(2-methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0436] Tert-butyl 9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (230 mg, 0.46 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.3 mL) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give 1-(2-methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione, which was used directly in the next reaction without purification.

[0437] LC-MS: (ESI, m / z): [M+H] + =401.1.

[0438] Step 3: Preparation of 1-(5-(9-(2-(1,3-dioxopentan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0439] 1-(2-Methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (180 mg, 0.45 mmol) and 2-(2-bromoethyl)-1,3-dioxopentane (121 mg, 0.67 mmol) were dissolved in DMF (5 mL), and KCO (186 mg, 1.3 mmol) was added with stirring. The reaction mixture was heated to 80 °C and stirred for 12 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (methanol:dichloromethane = 1:7) to give 1-(5-(9-(2-(1,3-dioxopentan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0440] LC-MS: (ESI, m / z): [M+H] + =501.7.

[0441] Step 4: Preparation of 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propionaldehyde [ka]

[0442] 1-(5-(9-(2-(1,3-dioxopentan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (200 mg, 0.5 mmol) was dissolved in acetone (5 mL), and hydrochloric acid (2 mol / L, 10 mL) was added and stirred at room temperature for 6 h. The reaction mixture was adjusted to pH 8 with sodium bicarbonate and extracted with ethyl acetate (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propionaldehyde, which was used directly in the next reaction without further purification.

[0443] LC-MS: (ESI, m / z): [M+H] + =457.7.

[0444] Step 5: Preparation of N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0445] 3-(9-(3-(2,4-Dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propionaldehyde (70 mg, 0.15 mmol) and N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide (67 mg, 0.15 mmol) were dissolved in dichloroethane (5 mL), and sodium triacetoxyborohydride (126 mg, 0.6 mmol) was added with stirring. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (50 mL) and extracted with dichloromethane (60 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give N-(3-(difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinepyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0446] LC-MS: (ESI, m / z): [M+H] + =887.2. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.40 (s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.29 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H), 7.26-6.97 (m, 2H), 6.91 (d, J = 7.9 Hz, 1H), 4.25-4.15 (m, 1H), 3.84 (s, 3H), 3.82-3.70 (m, 8H), 3.62-3.33 (m, 6H), 2.95-2.89 (m, 2H), 2.72-2.62 (m, 2H), 2.36-2.23 (m, 8H), 2.05-1.80 (m, 6H), 1.60-1.24 (m, 10H).

[0447] Example 29: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0448] N-(3-(Difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, 0.16 mmol) and 3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecane-9-formaldehyde (70 mg, 0.16 mmol) were dissolved in THF (3 mL), and sodium triacetoxyborohydride (102 mg, 0.48 mmol) was added. The mixture was stirred at room temperature for 16 h. Water (50 mL) was added to the reaction mixture, which was then extracted with EA (30 mL × 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by prep-HPLC separation to give N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0449] LC-MS: (ESI, m / z): [M+H] + =910.4. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 9.25 (s, 1H), 8.92 (d, J = 7.9 Hz, 1H), 8.39 (s, 1H), 8.34 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.25-6.97 (m, 2H), 4.31-4.20 (m, 5H), 3.77-3.70 (m, 1H), 3.67-3.57 (m, 3H), 3.33-3.23 (m, 5H), 2.90 (d, J = 9.3 Hz, 2H), 2.75-2.67 (m, 3H), 2.18-2.11 (m, 2H), 2.07-1.87 (m, 6H), 1.73-1.65 (m, 2H), 1.62-1.23 (m, 7H), 1.15-0.92 (m, 4H).

[0450] Example 30: (S)-5-(3-aminopiperidin-1-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0451] Step 1: Preparation of tert-butyl (S)-(1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate [ka]

[0452] (S)-tert-Butyl (1-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate (70 mg, 0.12 mmol) and 3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecane-9-formaldehyde (70 mg, 0.15 mmol) were dissolved in THF (2 mL). NaBH(OAc) (56 mg, 0.37 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (methanol:dichloromethane = 1:24) to give tert-butyl (S)-(1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0453] LC-MS: (ESI, m / z): [M+H] + =975.4.

[0454] Step 2: Preparation of (S)-5-(3-aminopiperidin-1-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0455] (S)-(1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl) tert-Butyl carboxylate (30 mg, 0.03 mmol) was dissolved in DCM (5 mL), TFA (1 mL) was added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure, and the crude product was purified by Prep-HPLC to give (S)-5-(3-aminopiperidin-1-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0456] LC-MS: (ESI, m / z): [M+H] + =875.3. 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 7.9 Hz, 1H), 8.37 (s, 1H), 8.34-8.30 (m, 2H), 7.65 (d, J = 8.2 Hz, 1H), 7.53 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.26-6.95 (m, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.23-4.13 (m, 2H), 3.82-3.74 (m, 1H), 3.70-3.40 (m, 7H), 3.30-3.10 (m, 3H), 2.97-2.92 (m, 2H), 2.77-2.73 (m, 2H), 2.25-1.75 (m, 11H), 1.75- 1.15 (m, 12H), 1.15-0.96 (m, 4H).

[0457] Example 31: 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0458] Step 1: Preparation of 4-(3-hydroxypropoxy)piperidine-1-carboxylate [ka]

[0459] 4-(3-Methoxy-3-oxypropyl)piperidine-1-carboxylate (500 mg, 1.49 mmol) was dissolved in anhydrous THF (10 mL), and a BH3 / THF solution (15 mL, 14.9 mmol) was added at 0 °C. The mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with methanol and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (ethyl acetate:petroleum ether = 9:1) to give 4-(3-hydroxypropoxy)piperidine-1-carboxylate.

[0460] LC-MS: (ESI, m / z): [M+H] + =294.2.

[0461] Step 2: Preparation of 3-(piperidin-4-yloxy)propan-1-ol [ka]

[0462] 4-(3-Hydroxypropoxy)piperidine-1-carboxylate (200 mg, 0.68 mmol) was dissolved in ethyl acetate (10 mL), Pd(OH) / C (80 mg) was added, and the mixture was heated to 70 °C under a hydrogen gas atmosphere and stirred overnight. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to give 3-(piperidin-4-yloxy)propan-1-ol, which was used directly in the next reaction without further purification.

[0463] LC-MS: (ESI, m / z): [M+H] + =160.2.

[0464] Step 3: Preparation of 1-(2-chloro-5-(4-(3-hydroxypropoxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0465] 3-(Piperidin-4-yloxy)propan-1-ol (100 mg, 0.62 mmol) was dissolved in DMSO (10 mL), and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (300 mg, 0.69 mmol) and DIEA (240 mg, 1.86 mmol) were added. The mixture was stirred at room temperature for 2 h. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (methanol:dichloromethane = 1:9) to give 1-(2-chloro-5-(4-(3-hydroxypropoxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0466] LC-MS: (ESI, m / z): [M+H] + =410.1.

[0467] Step 4: Preparation of 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propionaldehyde [ka]

[0468] 1-(2-chloro-5-(4-(3-hydroxypropoxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (60 mg, 0.147 mmol) was dissolved in DCM (5 mL), PCC (63 mg, 0.294 mmol) was added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (methanol:dichloromethane = 1:9) to give 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propionaldehyde.

[0469] LC-MS: (ESI, m / z): [M+H] + =408.1.

[0470] Step 5: Preparation of 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0471] 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propionaldehyde (55 mg, 0.135 mmol) was dissolved in THF (5 mL), and 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (crude product 70 mg, 0.135 mmol) and STAB (86 mg, 0.405 mmol) were added and the mixture was stirred at room temperature for 2 h. Water (100 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated aqueous sodium chloride (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptan-5-yl)-N-(1-(1-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxo)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0472] LC-MS: (ESI, m / z): [M+H] + =850.4. 1H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 9.50 (d, J = 5.7 Hz, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 3.2 Hz, 1H), 8.26 (d, J = 5.5 Hz, 1H), 7.67-7.55 (m, 2H), 7.40 (d, J = 8.7 Hz, 1H), 7.30-6.92 (m, 1H), 6.88-6.45 (m, 1H), 5.28-5.08 (m, 1H), 4.77 (d, J = 17.7 Hz, 1H), 4.30-4.10 (m, 1H), 4.02-3.68 (m, 4H), 3.62-3.47 (m, 7H), 3.30-3.10 (m, 2H), 3.00-2.85 (m, 2H), 2.78-2.69 (m, 2H), 2.45-2.31 (m, 2H), 2.10-1.55 (m, 12H), 1.52-1.37 (m, 2H).

[0473] Example 32: N-(3-(difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0474] Step 1: Preparation of tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate [ka]

[0475] tert-Butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (200 mg, 0.82 mmol) was dissolved in dichloromethane (4 mL), triethylamine (249, 2.46 mmol) and CbzCl (139 mg, 0.82 mmol) were added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with water (50 mL), extracted with dichloromethane (80 mL × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 3:7) to give tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0476] LC-MS: (ESI, m / z): [M+H-100] + =277.2.

[0477] Step 2: Preparation of benzyl methyl (2-(piperidin-4-yl)ethyl)carbamate [ka]

[0478] Compound tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate (200 mg, 0.53 mmol) was dissolved in hydrogen chloride / 1-4, dioxane (4 mL), and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated under reduced pressure to give benzylmethyl(2-(piperidin-4-yl)ethyl)carbamate.

[0479] LC-MS: (ESI, m / z): [M+H] + =277.2.

[0480] Step 3: Preparation of benzyl (2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate [ka]

[0481] The compound benzyl methyl (2-(piperidin-4-yl)ethyl)carbamate (200 mg, 0.72 mmol) and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (309 mg, 0.72 mmol) were added to a solution of DMSO (4 mL), followed by DIEA (278 mg, 2.1 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (70 mL × 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 1:3) to give compound benzyl (2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate.

[0482] LC-MS: (ESI, m / z): [M+H] + =523.2.

[0483] Step 4: Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione [ka]

[0484] Compound benzyl (2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate (120 mg, 0.23 mmol) was dissolved in methanol (3 mL), palladium on carbon (10%, 24 mg) was added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was suction filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0485] LC-MS: (ESI, m / z): [M+H] + =389.2.

[0486] Step 5: Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide [ka]

[0487] The compound N-(3-(difluoromethyl)-1-((1R,4R)-4-aldehydecyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, 0.165 mmol) and 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (64 mg, 0.165 mmol) were dissolved in DCE (3 mL), STAB (70 mg, 0.330 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (25 mL × 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was subjected to reverse phase separation to obtain compound N-(3-(difluoromethyl)-1-((1R,4R)-4-(((2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptan-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0488] LC-MS: (ESI, m / z): [M+H] + =858.4. 1H NMR (400 MHz, DMSO-d6) δ10.34 (s, 1H), 9.69 (s, 1H), 8.76 (d, J= 7.6 Hz, 1H), 8.37 (s, 1H), 8.25 (s, 1H), 7.35 (s, 1H), 7.42-7.00 (m, 3H), 6.38 (d, J = 7.6 Hz, 1H), 4.78-4.70 (m, 4H), 4.48-4.35 (m, 4H), 4.25-4.10 (m, 1H), 3.84 (s, 3H), 3.59 (t, J = 5.0 Hz, 2H), 3.30-3.10 (m, 3H), 2.75-2.63 (m, 2H), 2.36-2.25 (m, 2H), 2.20-1.99 (m, 7H), 1.93-1.83 (m, 2H), 1.77-1.50 (m, 7H), 1.44-1.30 (m, 2H), 1.20-0.96 (m, 4H).

[0489] Example 33: N-(2-((1S,4S)-4-(((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide

[0490] Step 1: Preparation of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate [ka]

[0491] At 0°C, Me3OS was added with stirring. + I -To a solution of (6.2 g, 28.2 mmol) in DMSO (50 mL) was added NaH (1.17 g, 29.3 mmol), and the reaction mixture was stirred at room temperature for 1 h. Next, a solution of tert-butyl (4-oxocyclohexyl)carbamate (5.0 g, 23.5 mmol) in DMSO (20 mL) was added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h. Water (700 mL) was added to the reaction mixture, and the mixture was extracted with EtOAc (100 mL × 3). The organic phase was washed with saturated brine (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (EA:PE = 0 to 1:1) to give tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate.

[0492] 1 H NMR (400 MHz, DMSO-d6) δ6.79 (d, J = 7.2 Hz, 1H), 3.37 (m, 1H), 2.57 (s, 2H), 1.83 (td, J = 13.3, 4.1 Hz, 2H), 1.77-1.64 (m, 2H), 1.48-1.40 (m, 2H), 1.38 (s, 9H), 1.20 (d, J = 13.1 Hz, 2H).

[0493] Step 2: Preparation of tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate [ka]

[0494] To a stirred solution of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate (4.7 g, 20.7 mmol) in NMP (50 mL) was added 2N NaOH (47 mL), and the reaction mixture was stirred at 100 °C for 2 h. Water (500 mL) was added to the reaction mixture, and the mixture was extracted with EA (100 mL × 3). The organic phase was washed with saturated brine (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (MeOH:DCM = 0 to 1:9) to give tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate.

[0495] 1 H NMR (400 MHz, DMSO-d6) δ 6.67 (d, J = 7.9 Hz, 1H), 4.46 (t, J = 5.8 Hz, 1H), 3.81 (s, 1H), 3.11 (t, J = 6.8 Hz, 3H), 1.56-1.45 (m, 4H), 1.44-1.34 (m, 13H).

[0496] Step 3: Preparation of tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate [ka]

[0497] To a solution of tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate (600 mg, 2.4 mmol) in THF (20 mL) was added NaH (292 mg, 7.3 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 20 min. Stirring was continued at room temperature for an additional 30 min, followed by the addition of 4-(bromomethyl)pyridine hydrobromide (617 mg, 2.4 mmol). The reaction mixture was stirred overnight at room temperature. Water (30 mL) was added to the reaction mixture, followed by extraction with EA (20 mL × 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA:PE = 0 to 4:1) to give tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate.

[0498] LC-MS: (ESI, m / z): [M+H] + =337.1. 1 H NMR (400 MHz, DMSO-d6) δ8.53 (d, J = 5.8 Hz, 2H), 7.34 (d, J = 5.6 Hz, 2H), 6.69 (d, J = 7.6 Hz, 1H), 4.55 (s, 2H), 4.19 (s, 1H), 3.23 (s, 2H), 3.18-3.07 (m, 1H), 1.60-1.48 (m, 6H), 1.42-1.35 (m, J = 9.2 Hz, 11H).

[0499] Step 4: Preparation of tert-butyl (4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)carbamate [ka]

[0500] A mixture of tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate (700 mg, 2.07 mmol) and Pd / C (400 mg) in i-PrOH / HO (18 mL / 21 mL) was stirred under a H atmosphere at 75 °C overnight. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give tert-butyl (4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)carbamate. The crude product was used directly in the next reaction.

[0501] LC-MS: (ESI, m / z): [M+H] + =343.2.

[0502] Step 5: Preparation of 4-(((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate [ka]

[0503] A solution of tert-butyl (4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)carbamate (630 mg, 1.83 mmol), CbzCl (376 mg, 2.20 mmol) in ACN (10 mL), and saturated aqueous NaHCO3 (10 mL) was stirred at room temperature overnight. The reaction mixture was first concentrated under reduced pressure and then extracted with DCM (50 mL × 3). The organic phase was washed with saturated brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA:PE = 0 to 7:3) to give 4-(((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0504] LC-MS: (ESI, m / z): [M+Na] + =499.3.

[0505] Step 6: Preparation of 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate [ka]

[0506] To a stirred solution of 4-(((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate (300 mg, 0.63 mmol) in DCM (12 mL) was added TFA (3 mL) at −10 to 0° C., and the reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was adjusted to pH 9.0 with DIEA and then directly concentrated under reduced pressure to give 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate. The resulting crude product was used directly in the next reaction without purification.

[0507] LC-MS: (ESI, m / z): [M+H] + =377.2.

[0508] Step 7: Preparation of 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate [ka]

[0509] To a stirred solution of 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate (237 mg, 0.63 mmol) in toluene (10 mL) was added 2-azido-4-methoxy-5-nitrobenzeneformaldehyde (170 mg, 0.76 mmol). The reaction mixture was stirred at 100 °C for 3 h. The reaction mixture was then directly concentrated under reduced pressure. The crude product was first purified by flash column chromatography (EA:PE = 0-4:1) and further purified by prep-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5%-95%) to give 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0510] LC-MS: (ESI, m / z): [M+H] + =553.2. 1 H NMR (400 MHz, DMSO-d6) δ8.58 (s, 1H), 8.37 (s, 1H), 7.40-7.29 (m, 5H), 7.26 (s, 1H), 5.07 (s, 2H), 4.45 (t, J = 12.0 Hz, 1H), 4.36 (s, 1H), 4.06-3.99 (m, 2H), 3.90 (s, 3H), 3.29 (d, J= 6.2 Hz, 2H), 3.20 (s, 2H), 2.85-2.75 (m, 2H), 2.35-2.20 (m, 2H), 1.89 (d, J = 11.8 Hz, 2H), 1.76-1.61 (m, 7H), 1.15-1.02 (m, 2H).

[0511] Step 8: Preparation of tert-butyl 4-((((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate [ka]

[0512] To a solution of 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate (200 mg, 0.36 mmol) in EtOH (20 mL) was added Raney-Ni (0.3 mL) and N2H4H2O (0.3 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was directly concentrated under reduced pressure to give tert-butyl 4-((((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate. The crude product obtained was used directly in the next reaction.

[0513] LC-MS: (ESI, m / z): [M+H] + =523.2.

[0514] Step 9: Preparation of 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate [ka]

[0515] To a stirred solution of tert-butyl 4-((((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate (190 mg, 0.36 mmol) in DMF (5 mL) were added 6-(trifluoromethyl)pyridineformate (90 mg, 0.47 mmol), HATU (178 mg, 0.47 mmol), and DIEA (140 mg, 1.08 mmol). The reaction mixture was stirred at room temperature for 2 h. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with EA (20 mL × 3). The organic layer was washed with saturated brine (60 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (EA:PE=0-4:1) to give 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0516] LC-MS: (ESI, m / z): [M+H] + =696.3. 1 H NMR (400 MHz, DMSO-d6) δ10.51 (s, 1H), 8.69 (s, 1H), 8.47 (d, J= 7.7 Hz, 1H), 8.41 (t, J = 7.8 Hz, 1H), 8.32 (s, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.41-7.29 (m, 5H), 7.16 (s, 1H), 5.08 (s, 2H), 4.40-4.29 (m, 2H), 4.15-3.95 (m 5H), 3.29 (d, J = 6.2 Hz, 2H), 3.21 (s, 2H), 2.91-2.70 (m, 2H), 2.35-2.15 (m, 2H), 1.90 (d, J = 9.5 Hz, 2H), 1.85-1.60 (m, 7H), 1.12-.1.02 (m, 2H).

[0517] Step 10: Preparation of N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0518] To a solution of 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamide)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate (170 mg, 0.24 mmol) in EA (20 mL) was added Pd(OH)2 / C (80 mg). The reaction mixture was then stirred overnight at 70°C under a H2 atmosphere. The reaction mixture was filtered through diatomaceous earth, and the filtrate was directly concentrated under reduced pressure to give N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide.

[0519] LC-MS: (ESI, m / z): [M+H] + =562.3.

[0520] Step 11: Preparation of N-(2-((1S,4S)-4-(((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0521] A mixture of N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide (130 mg, 0.23 mmol), 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (121 mg, 0.28 mmol), and DIEA (89 mg, 0.69 mmol) in DMSO (6 mL) was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with DCM (20 mL × 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was purified by prep-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5%-95%) to give N-(2-((1S,4S)-4-(((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)methoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide.

[0522] LC-MS: (ESI, m / z): [M+H] + =812.1. 1H NMR (400 MHz, DMSO-d6) δ10.51 (d, J = 7.5 Hz, 2H), 8.69 (s, 1H), 8.46 (d, J = 7.7 Hz, 1H), 8.40 (t, J = 7.8 Hz, 1H), 8.32 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 1.4 Hz, 1H), 7.40 (dd, J = 8.2, 1.5 Hz, 1H), 7.16 (s, 1H), 4.58-4.29 (m, 3H), 3.99 (s, 3H), 3.84-3.55 (m, 3H), 3.33-3.28 (m, 2H), 3.22 (s, 2H), 3.13-3.03 (m, 1H), 2.88-2.70 (m, 3H), 2.35-2.20 (m, 2H), 1.94-1.59 (m, 9H), 1.21-1.15 (m, 2H).

[0523] Example 34: N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide

[0524] Step 1: Preparation of 4-(2-((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0525] To a solution of tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate (770 mg, 3.13 mmol) in THF (20 mL) was added NaH (376 mg, 9.4 mmol) at 0 °C and stirred at 0 °C for 2 h. Then, 4-(2-(p-toluenesulfonyl)ethyl)piperidine-1-carboxylate (2.62 g, 6.26 mmol) and KI (1.04 g, 6.26 mmol) were added. The reaction mixture was stirred at 70 °C overnight. The reaction mixture was diluted with water (50 mL) and extracted with EA (30 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (EA:PE=0 to 3:2) to give 4-(2-((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0526] LC-MS: (ESI, m / z): [M+Na] + =513.1. 1 H NMR (400 MHz, DMSO-d6) δ7.41-7.29 (m, 5H), 6.67 (d, J = 7.5 Hz, 1H), 5.06 (s, 2H), 4.05-3.90 (m, 3H), 3.42 (t, J = 6.4 Hz, 2H), 3.10 (s, 3H), 2.88-2.68 (m, 2H), 1.65 (d, J = 12.6 Hz, 2H), 1.55-1.32 (m, 20H), 1.13-0.92 (m, 2H).

[0527] Step 2: Preparation of 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0528] To a stirred solution of 4-(2-((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (270 mg, 0.55 mmol) in DCM (3 mL) was added TFA (1.2 mL) at −10 to 0° C., and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was adjusted to pH 9.0 with DIEA and concentrated under reduced pressure to give 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0529] LC-MS: (ESI, m / z): [M+H] + =391.2.

[0530] Step 3: Preparation of 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0531] To a stirred solution of 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (400 mg, 0.55 mmol) in toluene (15 mL) was added 2-azido-4-methoxy-5-nitrobenzeneformaldehyde (150 mg, 0.66 mmol). The reaction mixture was stirred at 100°C for 3 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by flash column chromatography (EA:PE = 0 to 9:1) to give 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0532] LC-MS: (ESI, m / z): [M+H] + =567.3. 1H NMR (400 MHz, DMSO-d6) δ8.59 (s, 1H), 8.38 (s, 1H), 7.41-7.29 (m, 5H), 7.27 (s, 1H), 5.07 (s, 2H), 4.50-4.40 (m, 1H), 4.34 (s, 1H), 4.05-3.95 (m, 2H), 3.91 (s, 3H), 3.47 (t, J = 6.4 Hz, 2H), 3.21 (s, 2H), 2.90-2.70 (m, 2H), 2.35-2.19 (m, 2H), 1.90 (d, J= 9.7Hz, 2H), 1.75-1.50 (m, 7H), 1.50-1.40 (m, 2H), 1.11-0.98 (m, 2H).

[0533] Step 4: Preparation of 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0534] To a solution of 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (250 mg, 0.44 mmol) in EtOH (20 mL) was added Raney-Ni (0.5 mL) and N2H4H2O (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate. The resulting product was used directly in the next reaction.

[0535] LC-MS: (ESI, m / z): [M+H] + =537.3

[0536] Step 5: Preparation of 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0537] To a stirred solution of 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (236 mg, 0.44 mmol) in DMF (5 mL) were added 6-(trifluoromethyl)pyridineformic acid (101 mg, 0.53 mmol), HATU (217 mg, 0.57 mmol), and DIEA (170 mg, 1.32 mmol). The reaction mixture was stirred at room temperature for 2 h. The mixture was diluted with water (50 mL) and extracted with EA (20 mL × 3). The organic layer was washed with saturated brine (60 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (EA:PE=0-4:1) to give 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0538] LC-MS: (ESI, m / z): [M+H] + =710.2. 1H NMR (400 MHz, DMSO-d6) δ10.50 (s, 1H), 8.69 (s, 1H), 8.46 (d, J= 7.7 Hz, 1H), 8.40 (t, J = 7.8 Hz, 1H), 8.32 (s, 1H), 8.21 (d, J = 7.7 Hz, 1H), 7.41-7.28 (m, 5H), 7.16 (s, 1H), 5.06 (s, 2H), 4.40-4.20 (m, 2H), 4.10-3.90 (m, 5H), 3.47 (t, J = 6.4 Hz, 2H), 3.21 (s, 2H), 2.90-2.70 (m, 2H), 2.32-2.18 (m, 2H), 1.90 (d, J = 10.4 Hz, 2H), 1.72-1.53 ​​(m, 7H), 1.52-1.40 (m, 2H), 1.15-1.00 (m, 2H).

[0539] Step 6: Preparation of N-(2-((1S,4S)-4-hydroxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0540] To a solution of 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamide)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (200 mg, 0.28 mmol) in EA (20 mL) was added Pd(OH)2 / C (100 mg). The reaction mixture was then stirred overnight at 70°C under a H2 atmosphere. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated to give N-(2-((1S,4S)-4-hydroxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide, which was used directly in the next reaction.

[0541] LC-MS: (ESI, m / z): [M+H] + =576.2.

[0542] Step 7: Preparation of N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0543] A solution of N-(2-((1S,4S)-4-hydroxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide (130 mg, 0.22 mmol), 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (118 mg, 0.27 mmol), and DIEA (87 mg, 0.68 mmol) in DMSO (3 mL) was stirred overnight at room temperature. The reaction mixture was diluted with water (30 mL) and extracted with DCM (10 mL × 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (MeOH:DCM=0 to 1:9) to give N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide.

[0544] LC-MS: (ESI, m / z): [M+H] + =826.1. 1 H NMR (400 MHz, DMSO-d6) δ10.51 (d, J = 5.7 Hz, 2H), 8.69 (s, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.41 (t, J = 7.8 Hz, 1H), 8.33 (s, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.39 (dd, J = 8.2, 1.8 Hz, 1H), 7.16 (s, 1H), 4.50-4.26 (m, 3H), 3.98 (s, 3H), 3.83-3.53 (m, 3H), 3.48 (t, J = 6.3 Hz, 2H), 3.21 (s, 2H), 3.13-2.97 (m, 1H), 2.86-2.71 (m, 3H), 2.32-2.18 (m, 2H), 1.90 (d, J = 9.8 Hz, 2H), 1.85-1.45 (m, 9H), 1.15-1.05 (m, 2H).

[0545] Example 35: N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide

[0546] Step 1: Preparation of tert-butyl (4-hydroxy-4-((methylamino)methyl)cyclohexyl)carbamate [ka]

[0547] To a solution of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate (4.2 g, 18.42 mmol) in EtOH / HO (90 mL / 15 mL) was added methylamine (40 mL, 25%-30% aqueous solution). The reaction mixture was then stirred at 25°C overnight. The reaction mixture was directly concentrated under reduced pressure, and the resulting crude product was purified by flash (MeOH:DCM = 0-1:1) to give tert-butyl (4-hydroxy-4-((methylamino)methyl)cyclohexyl)carbamate.

[0548] LC-MS: (ESI, m / z): [M+H] + =259.2.

[0549] Step 2: Preparation of benzyl ((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate [ka]

[0550] A mixture of tert-butyl (4-hydroxy-4-((methylamino)methyl)cyclohexyl)carbamate (1.2 g, 4.61 mmol) and benzyl chloroformate (941 mg, 5.53 mmol) in ACN / sat. NaHCO3 (20 mL / 20 mL) was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and then extracted with EA (50 mL × 3). The organic phase was washed with saturated brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash elution (EA:PE = 0 to 7:3) to give benzyl ((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate.

[0551] LC-MS: (ESI, m / z): [M+Na] + =415.2.

[0552] Step 3: Preparation of benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate [ka]

[0553] To a stirred solution of benzyl ((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate (1.5 g, 3.81 mmol) in DCM (15 mL) was added TFA (5 mL) at 0° C., and the reaction was stirred at 0° C. for 2 h. The reaction was adjusted to pH=9.0 with DIEA and then directly concentrated under reduced pressure to give benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate, and the resulting crude product was used directly in the next reaction.

[0554] LC-MS: (ESI, m / z): [M+H] + =293.1.

[0555] Step 4: Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate [ka]

[0556] To a stirred solution of benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate (1.5 g, 3.8 mmol) in toluene (40 mL) was added 2-azido-4-methoxy-5-nitrobenzeneformaldehyde (1.2 g, 5.7 mmol). The reaction mixture was stirred at 100 °C for 3 h. The reaction mixture was then directly concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA: PE = 0-1) to give benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate.

[0557] LC-MS: (ESI, m / z): [M+H] + =469.2 1 H NMR (400 MHz, DMSO-d6) δ8.58 (s, 1H), 8.38 (s, 1H), 7.45-7.23 (m, 6H), 5.09 (s, 2H), 4.66-4.36 (m, 2H), 3.91 (s, 3H), 3.28 (s, 2H), 3.10-2.95 (m, 3H), 2.30-2.20 (m, 2H), 1.95-1.60 (m, 2H), 1.78-1.63 (m, 2H), 1.59-1.41 (m, 2H).

[0558] Step 5: Preparation of benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate [ka]

[0559] To a solution of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate (1.1 g, 2.34 mmol) in EtOH (20 mL) was added N2H4H2O (1.2 mL) and Raney-Ni (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate, which was used directly in the next reaction.

[0560] LC-MS: (ESI, m / z): [M+H] + =439.2

[0561] Step 6: Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridylcarbonyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate [ka]

[0562] To a stirred solution of (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)benzylcarbamate (940 mg, 2.14 mmol) in DMF (10 mL) were added 6-(trifluoromethyl)pyridineformic acid (538 mg, 2.78 mmol), HATU (1.06 g, 2.78 mmol), and DIEA (828 mg, 6.42 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (100 mL) and extracted with EA (30 mL × 3). The organic phase was diluted with saturated brine (100 mL). The crude product was washed with 1 mL of ethyl acetate, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography (EA:PE = 0-9:1) to give benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridylcarbonyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate.

[0563] LC-MS: (ESI, m / z): [M+H] + =612.2. 1H NMR (400 MHz, DMSO-d6) δ10.50 (s, 1H), 8.69 (s, 1H), 8.46 (d, J= 7.5 Hz, 1H), 8.40 (t, J = 7.8 Hz, 1H), 8.31 (s, 1H), 8.21 (dd, J = 7.7, 1.0 Hz, 1H), 7.42-7.28 (m, 5H), 7.16 (s, 1H), 5.09 (s, 2H), 4.60-4.31 (m, 2H), 3.98 (s, 3H), 3.28 (s, 2H), 3.10-2.95 (m, 3H), 2.30-2.15 (m, 2H), 1.95-1.60 (m, 2H), 1.67 (t, J = 14.9 Hz, 2H), 1.57-1.40 (m, 2H).

[0564] Step 7: Preparation of N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0565] To a solution of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridylcarbonyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate (500 mg, 0.81 mmol) in EA (20 mL) was added Pd(OH)2 / C (200 mg). The reaction was then stirred under a H2 atmosphere at 70 °C overnight. The reaction was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide, which was used crude directly in the next reaction.

[0566] LC-MS: (ESI, m / z): [M+H] + =478.1.

[0567] Step 8: Preparation of tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate [ka]

[0568] A mixture of N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide (200 mg, 0.41 mmol), tert-butyl 9-aldehyde-3-azaspiro[5.5]undecane-3-carboxylate (141 mg, 0.50 mmol), and STAB (434 mg, 2.05 mmol) in THF (10 mL) was stirred at room temperature overnight. The reaction mixture was diluted with water (30 mL) and then extracted with DCM (10 mL × 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by flash column (MeOH:DCM = 0 to 1:9) to give tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0569] LC-MS: (ESI, m / z): [M+H] + =743.3.

[0570] Step 9: Preparation of N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0571] To a solution of tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate (200 mg, 0.27 mmol) in DCM (5 mL) was added TFA (2 mL) at 0° C. The reaction mixture was then stirred at room temperature for 2 h. The pH of the reaction mixture was adjusted to 9.0 with DIE. The organic phase was washed with water (100 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide, which was used crude directly in the next reaction.

[0572] LC-MS: (ESI, m / z): [M+H] + =643.3.

[0573] Step 10: Preparation of N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0574] A mixture of N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide (130 mg, 0.20 mmol), 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (105 mg, 0.24 mmol), and DIEA (77 mg, 0.60 mmol) in DMSO (5 mL) was stirred overnight at room temperature. The reaction mixture was diluted with water (50 mL) and then extracted with DCM (20 mL × 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was purified by prep-HPLC (CHCN / 0.08% NHHCO aqueous solution, 5%-95%) to give N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undecan-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide.

[0575] LC-MS: (ESI, m / z): [M+H] + =893.1. 1H NMR (400 MHz, DMSO-d6) δ10.51 (s, 2H), 8.69 (s, 1H), 8.47 (d, J= 7.8 Hz, 1H), 8.41 (t, J = 7.8 Hz, 1H), 8.32 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 4.37-4.27 (m, 1H), 4.10-3.90 (m, 4H), 3.82-3.49 (m, 4H), 3.35-3.25 (m, 1H), 2.80-2.70 (m, 2H), 2.36-2.15 (m, 9H), 1.95-1.85 (m, 2H), 1.78-1.22 (m, 14H), 1.18-0.98 (m, 4H).

[0576] Example 36: N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-methoxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)pyridineamide

[0577] Step 1: Preparation of 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0578] The compound 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (700 mg, 1.24 mmol) and AcO (151 mg, 1.48 mmol) were dissolved in anhydrous DMSO (20 mL) and stirred at room temperature under nitrogen gas protection for 16 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (50 mL × 3). The organic phase was washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:petroleum ether=1:9) to give 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0579] LC-MS: (ESI, m / z): [M+H] + =627.2.

[0580] Step 2: Preparation of 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0581] The compound 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (600 mg, 0.96 mmol) and pretreated Raney-Ni (200 mg) (pretreatment method: first washed three times with water, then three times with acetone, and finally three times with ethanol) were dissolved in absolute ethanol (50 mL), stirred under a hydrogen gas atmosphere at room temperature for 3 h, filtered, and the filtrate was directly concentrated under reduced pressure. The crude product was purified using a reverse-phase silica gel column (ACN:HO = 1:1, flow rate 40 mL / min) to give 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0582] LC-MS: (ESI, m / z): [M+H] + =551.3.

[0583] Step 3: Preparation of 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate [ka]

[0584] Compound 6-(trifluoromethyl)pyridineformic acid (60 mg, 0.3 mmol), HATU (114 mg, 0.3 mmol), and DIEA (100 mg, 0.78 mmol) were dissolved in DMF (10 mL) and stirred at room temperature for 20 min. After that, 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (150 mg, 0.26 mmol) was added and the mixture was allowed to react at room temperature for 1 h. Water (100 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:petroleum ether=1:1) to give 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamido)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0585] LC-MS: (ESI, m / z): [M+H] + =724.4.

[0586] Step 4: Preparation of N-(6-methoxy-2-((1S,4S)-4-methoxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-2H-indazol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0587] 4-(2-(((1S,4S)-1-Methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)pyridinecarboxamide)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate (70 mg, 0.097 mmol) was dissolved in trifluoroacetic acid (3 mL), heated to 90° C., and stirred for 1 h. The reaction mixture was directly concentrated under reduced pressure to give N-(6-methoxy-2-((1S,4S)-4-methoxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-2H-indazol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide, which was used directly in the next reaction without purification.

[0588] LC-MS: (ESI, m / z): [M+H] + =590.4.

[0589] Step 5: Preparation of N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-methoxycyclohexyl)-6-methoxy-2H-indazol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide [ka]

[0590] N-(6-Methoxy-2-((1S,4S)-4-methoxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-2H-indazol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide (70 mg, 0.097 mmol) was dissolved in dimethyl sulfoxide (3 mL), and 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)pentafluorophenyl benzoate (48 mg, 0.11 mmol) and N,N-diisopropylethylamine (37 mg, 0.29 mmol) were added with stirring. After stirring at room temperature for 2 h, water (50 mL) was added, extracted with dichloromethane (20 mL × 3), and the organic phase was washed with saturated brine (3 × 60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by pre-HPLC (CHCN / 0.08% aqueous NHHCO, 5% to 95%) to give N-(2-((1S,4S)-4-((2-(1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)ethoxy)methyl)-4-methoxycyclohexyl)-6-methoxy-2H-indazol-5-yl)-6-(trifluoromethyl)pyridinecarboxamide.

[0591] LC-MS: (ESI, m / z): [M+H] + =840.3. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (d, J = 4.5 Hz, 2H), 8.68 (s, 1H), 8.49-8.37 (m, 2H), 8.32 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.64 (d, J= 8.2 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.39 (dd, J = 8.2, 2.0 Hz, 1H), 7.18 (s, 1H), 4.52-4.30 (m, 2H), 3.98 (s, 3H), 3.80-3.52 (m, 3H), 3.47 (t, J = 6.2 Hz, 2H), 3.34 (s, 2H), 3.19 (s, 3H), 3.15-2.95 (m, 1H), 2.85-2.70 (m, 3H), 2.19-1.97 (m, 2H), 1.96-1.85 (m, 4H), 1.85-1.55 (m, 3H), 1.55-1.45 (m, 4H), 1.25-1.05 (m, 2H).

[0592] II Bioactivity Test Examples Test Example 1: IRAK4 キナーゼActivity Test The inhibitory effect of compounds on IRAK4 kinase activity was detected using the KinEASE-STK S1 Serine / Threonine Kinase Reagent Kit (Cisbio). The specific method is as follows: Compounds were dissolved in dimethyl sulfoxide and diluted isocratically with the reagent kit's buffer to achieve final concentrations ranging from 10,000 nM to 0.038 nM in the reaction system. 2.5 nM IRAK4 kinase (Carna), 1 μM biotinylated polypeptide substrate (Cisbio), and 7 μM adenosine triphosphate (Sigma-Aldrich) were then added, in that order, and the mixture was incubated at 37°C for 120 min. Next, an anti-phosphorylated serine / threonine antibody (Cisbio) coupled to a europium-based element compound and modified XL665 streptavidin (Cisbio) were added to the reaction system to stop the reaction. After incubation at room temperature for 1 hour, each well was measured using a microplate reader EnVision (PerkinElmer) in HTRF mode at an excitation wavelength of 337 nm. The fluorescence intensity of each well at emission wavelengths of 620 nm and 665 nm was read, and the ratio was calculated using the formula: Ratio = (665 nm / 620 nm) × 10. 4 The ratio was calculated using the formula: The inhibition rate of the compound at each concentration was calculated by aligning the fluorescence intensity ratio with the control group, and then nonlinear curve fitting was performed using GraphPad Prism 7 with the logarithmic concentration-inhibition rate to obtain the IC value of the compound. 50 values ​​were obtained.

[0593] [Table 1]

[0594] Experimental Results: The compounds described in this invention can effectively bind to the target protein and inhibit IRAK4 kinase activity.

[0595] Test Example 2: IRAK4 Expression in THP-1 Cells by Compounds 0.95 mL of THP-1 cells (Stem Cell Bank, Chinese Academy of Sciences) was added to each well of a 24-well cell culture plate at a concentration of 5 × 10 5Cells were seeded at a density of 100 cells / well and incubated overnight at 37°C in a 5% CO2 incubator. 50 μL of compound dimethyl sulfoxide solution was added to adjust the final compound concentrations to 0.03–3000 nM. After 24 h of incubation, the cells were collected in a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 min at 4°C. The cell pellet was washed twice with 1x DPBS and resuspended in 200 μL of Western and IP cell lysis solution (Biyuntian) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Biyuntian). The cells were incubated on ice for 30 min and then centrifuged at 14000 g for 10 min at 4°C. The supernatant was analyzed by Western blotting to detect IRAK4 protein levels.

[0596] Human IRAK4 AlphaLISA detection The cell lysates were simultaneously assayed for IRAK4 degradation using a human IRAK4 AlphaLISA detection reagent kit (PerkinElmer, AL3117C). The specific procedure was as follows: 2 μL of the supernatant sample was added to a white 384-well plate (PerkinElmer, 6007299). Then, 4 μL of 5× anti-IRAK4 receptor microbeads (PerkinElmer, final concentration 10 μg / mL) were added and incubated at 23°C for 30 min. Next, 4 μL of 5× biotin-labeled anti-IRAK4 antibody (PerkinElmer, final concentration 1 nM) was added and incubated at 23°C for 60 min. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C for 30 min in the dark. After incubation, AlphaLISA signal values ​​were measured using the microplate reader EnVision (PerkinElmer, 2105) in AlphaScreen standard setting mode, and IRAK4 concentrations were calculated based on the measured values. Furthermore, nonlinear curve fitting was performed using GraphPad Prism 7 with logarithmic concentration versus inhibition rate to obtain the DC50 and D max values ​​were obtained.

[0597] [Table 2]

[0598] Experimental results: The compounds described in the present invention can effectively decompose IRAK4 kinase protein in cells and have excellent decomposition activity.

[0599] Test Example 3: Measurement of cytokine concentrations secreted from normal human whole blood and PBMCs following LPS induction (1) Measurement of cytokine concentrations secreted from normal human whole blood following LPS induction 190 μL of fresh normal human whole blood containing heparin sodium anticoagulant (from healthy volunteers aged 22–50 years, collected live) was seeded into each well of a 96-well clear cell plate (Labserv, 310109008). Then, 10 μL of diluted compound solution was added to the corresponding well to achieve final compound concentrations in the range of 10–20,000 nM. The final concentration of dimethyl sulfoxide was 0.1%. The compound-treated cell plate was covered with a sealing film and incubated in a 5% CO2 incubator at 37°C for 20 h. Then, 10 μL of LPS (0111:B4) (Sigma, L4391) was added to a final concentration of 100 ng / mL. A sealing film was attached to the cell plate, which was then placed in a 5% carbon dioxide incubator and incubated at 37°C for 5 hours. After centrifugation at 2000 rpm for 10 minutes, 30 μL of supernatant plasma was removed from each well and transferred to a new 96-well clear cell plate. The plate was then frozen at -80°C and stored in preparation for cytokine concentration testing.

[0600] Detection of interleukin-6 (IL-6) AlphaLISA. Interleukin-6 (IL-6) AlphaLISA detection reagent kit (PerkinElmer, AL223 C) was used to measure IL-6 concentrations in supernatant plasma samples of LPS-induced human whole blood (blood collected on-site from healthy individuals aged 22–50).

[0601] IL-6 standard solutions were prepared at various concentrations ranging from 0 to 100,000 pg / mL according to the product manual. 2 μL of each IL-6 standard solution and the cell supernatant samples to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (PerminElmer, final concentration 10 μg / mL) and biotin-labeled anti-IL-6 antibody (PerminElmer, final concentration 1 nM) was added to each well and incubated for 60 min at 23°C. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration 40 μg / mL) was added to each well and incubated for a further 30 min at 23°C in the dark. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IL-6 standard solution at each concentration, and the IL-6 concentration of each cell lysate supernatant was determined based on the corresponding concentration on the standard curve of the AlphaLISA signal values ​​of the measured samples. The inhibitory rate of the compound at each concentration was calculated compared with the control IL-6 concentration, and nonlinear curve fitting was performed using GraphPad Prism 7 based on the logarithmic concentration-inhibitory rate to determine the IC of the compound. 50 values ​​were calculated.

[0602] (2) Measurement of cytokine levels secreted by human PBMCs following LPS induction Cryopreserved human PBMCs (Myoshun, PB010C) were resuscitated and resuspended in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% fetal bovine serum (Gibco, 10099141), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, 15140122). On the same day, the cell density was adjusted to 2 × 10 5 150 μL of PBMCs were seeded into each well of a 96-well clear cell plate (Labserv, 310109008) at 1 cell / well. Next, 50 μL of diluted compound solution was added to the corresponding well to adjust the final compound concentration to a range of 0.026–10,000 nM. The final DMSO concentration was 0.25%. The treated cell plates were incubated in a 5% CO2 incubator at 37°C for 1 or 20 h, after which 10 μL of LPS (0111:B4) (Sigma, L4391) was added to adjust the final concentration to 100 ng / mL. The cell plate was placed back into a 5% CO2 incubator and incubated at 37°C for 5 hours, then centrifuged at 2000 rpm for 10 minutes. 100 μL of cell supernatant was taken from each well and transferred to a new 96-well clear cell plate. The plate was then frozen at -80°C for cytokine concentration testing.

[0603] Interleukin-6 (IL-6) AlphaLISA detection The interleukin-6 (IL-6) AlphaLISA detection reagent kit (PerkinElmer, AL223 C) was used to measure the IL-6 concentration in the supernatant of LPS-induced human PBMC cells. IL-6 standard solutions were prepared at various concentrations ranging from 0 to 100,000 pg / mL according to the product manual. 2 μL of each IL-6 standard solution and the cell supernatant samples to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-conjugated anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 min. Finally, 10 μL of 2× streptavidin-conjugated donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C for 30 min in the dark. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IL-6 standard solution at each concentration, and the IL-6 concentration of each cell lysate supernatant was determined based on the corresponding concentration on the standard curve of the AlphaLISA signal values ​​of the measured samples. The inhibitory rate of the compound at each concentration was calculated compared with the control IL-6 concentration, and nonlinear curve fitting was performed using GraphPad Prism 7 based on the logarithmic concentration-inhibitory rate to determine the IC of the compound. 50 values ​​were calculated.

[0604] Test Example 4: Degradation of IRAK4 by Compounds (1) Degradation of IRAK4 protein by compounds in normal human PBMCs Cryopreserved normal human PBMCs (Myojun, PB010C) were resuscitated and resuspended in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% fetal bovine serum (Gibco, 10099141), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, 15140122). 0.95 mL of cells were seeded into each well of a 24-well cell culture plate. 50 μL of diluted compound solution was then added to the corresponding well to achieve final compound concentrations ranging from 0.01 to 3000 nM. The final DMSO concentration was 0.25%. After treatment, the cell plates were placed in a 5% CO2 incubator at 37°C for 24 h. The cells were then collected into a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 min at 4°C. The cell pellet was washed twice with 1x DPBS, and the resuspended cells were lysed in 100 μL of lysis buffer. The cells were then incubated on ice for 30 min and centrifuged at 14,000 g for 10 min at 4°C. The supernatant samples were frozen at -80°C and stored for further analysis. The cell lysis buffer was Western and IP cell lysis buffer (Biyuntian, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Biyuntian, P1008).

[0605] The concentration of total protein in the cell lysate samples was measured using a BCA protein quantification reagent kit (Amane, PA115-02).

[0606] The prepared PBMC lysate samples were subjected to measurement of IRAK4 concentration using a human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C), thereby determining the IRAK4 degradation activity of the compounds. Five microliters of each IRAK4 standard solution and the cell lysate supernatant sample to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 10 μL of 5x anti-IRAK4 receptor microbeads (final concentration: 10 μg / mL) were added to each well and incubated at room temperature for 30 min. Next, 10 μL of 5x biotin-conjugated anti-IRAK4 antibody (final concentration: 1 nM) was added to each well and incubated at room temperature for a further 60 min. Finally, 25 μL of 2x streptavidin-conjugated donor microbeads (final concentration: 40 μg / mL) were added to each well and incubated at room temperature for a further 30 min. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IRAK4 standard solution at each concentration. The AlphaLISA signal values ​​of the samples to be measured were then correspondingly corrected using the total protein concentration of each sample obtained by BCA quantification. The IRAK4 concentration in each cell lysis supernatant was then determined. The inhibitory rate of the compound at each concentration was calculated compared with the control IRAK4 concentration. A nonlinear curve fitting was performed using GraphPad Prism 8 based on the logarithmic concentration-inhibitory rate to determine the DC concentration of the compound. 50 and D max values ​​were calculated.

[0607] [Table 3]

[0608] (2) Degradation of IRAK4 protein in patient whole blood by the compound Fresh whole blood was collected from normal subjects or volunteers with rheumatoid arthritis or dermatomyositis and anticoagulated with sodium heparin. 0.95 mL of whole blood was seeded into each well of a 24-well cell culture plate, and 50 μL of diluted compound solution was added to the corresponding well to achieve a final compound concentration in the range of 10–10,000 nM. The final DMSO concentration was 0.1%. After administration, the cell plates were placed in a 5% CO2 incubator and incubated at 37°C for 24 h. Afterward, whole blood samples were collected from each well. PBMCs were isolated from the whole blood using FICOLL Separation Solution (GE, 17-1440-02) according to the product manual. The isolated cells were washed once with 1x DPBS, then erythrocytes were lysed in erythrocyte lysis solution (Nanjing Senbei Ka, BL-O51-100 mL) for 5 min, and the cells were washed once with 1x DPBS. Finally, after centrifugation at 3000 rpm / min for 5 min, the collected white pellet cells were PBMCs.

[0609] The resulting PBMCs were separated and the supernatant removed. The cells were then lysed in a cell lysis solution, incubated on ice for 30 minutes, and centrifuged at 14,000 g for 10 minutes at 4°C. The cell lysis solution was Western and IP cell lysis solution (Biyuntian, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Biyuntian, P1008). Protein samples from the lysed supernatant were frozen at -80°C and stored for further analysis.

[0610] The total protein concentration in the prepared whole blood lysate samples from normal humans or patients (rheumatoid arthritis, dermatomyositis) was measured using a BCA protein assay reagent kit (Amane, PA115-02).

[0611] The whole blood lysate samples prepared from normal subjects or patients (rheumatoid arthritis, dermatomyositis) were used to measure the IRAK4 concentration using a human IRAK4 AlphaLISA detection reagent kit (PerkinElmer, AL3117C), thereby determining the IRAK4 degradation activity of compounds. Five microliters of each IRAK4 standard solution and the cell lysate supernatant sample to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 10 μL of 5x anti-IRAK4 receptor microbeads (final concentration: 10 μg / mL) were added to each well and incubated at room temperature for 30 min. Next, 10 μL of 5x biotin-conjugated anti-IRAK4 antibody (final concentration: 1 nM) was added to each well and incubated at room temperature for a further 60 min. Finally, 25 μL of 2x streptavidin-conjugated donor microbeads (final concentration: 40 μg / mL) were added to each well and incubated at room temperature for a further 30 min. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IRAK4 standard solution at each concentration. The AlphaLISA signal values ​​of the samples to be measured were then correspondingly corrected using the total protein concentration of each sample obtained by BCA quantification. The IRAK4 concentration in each cell lysis supernatant was then determined. The inhibitory rate of the compound at each concentration was calculated compared with the control IRAK4 concentration. A nonlinear curve fitting was performed using GraphPad Prism 8 based on the logarithmic concentration-inhibitory rate to determine the DC concentration of the compound. 50 and D max values ​​were calculated.

[0612] 1A and 1B show that compound A of the present invention has a good decomposition activity against IRAK4 in normal human peripheral blood mononuclear cells, dermatomyositis patients, and rheumatoid arthritis patients.

[0613] Test Example 5: Measurement of IRAK4 protein expression levels in PBMCs from individual patients Fresh whole blood was collected from volunteers and anticoagulated with sodium heparin. PBMCs were then isolated from the whole blood using FICOLL Separation Solution (GE, 17-1440-02) according to the instructions in the product manual. The isolated cells were washed once with 1x DPBS. Red blood cells were then lysed with red blood cell lysis solution (Nanjing Senbei Ka, BL-O51-100 mL) and washed once with 1x DPBS. Finally, after 5 min of centrifugation at 3000 rpm / min, the collected white pellet contained PBMCs. This method is suitable for isolating PBMCs from whole blood collected from patients with osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, and other conditions, as well as from normal human whole blood.

[0614] The resulting PBMCs were separated and the supernatant removed. The cells were then lysed in a cell lysis solution, incubated on ice for 30 minutes, and centrifuged at 14,000 g for 10 minutes at 4°C. The cell lysis solution was Western and IP cell lysis solution (Biyuntian, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Biyuntian, P1008). Protein samples from the lysed supernatant were frozen at -80°C and stored for further analysis.

[0615] The concentration of total protein in the cell protein samples was measured using a BCA protein quantification reagent kit (Amane, PA115-02).

[0616] The prepared cell protein samples were used to measure the expression level of IRAK4 protein in each patient or normal human PBMC using a human IRAK4 AlphaLISA detection reagent kit (PerkinElmer, AL3117C). The method was as follows: Five microliters of each IRAK4 standard solution and the cell lysate supernatant sample to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 10 μL of 5x anti-IRAK4 receptor microbeads (final concentration: 10 μg / mL) were added to each well and incubated at room temperature for 30 min. Next, 10 μL of 5x biotin-conjugated anti-IRAK4 antibody (final concentration: 1 nM) was added to each well and incubated at room temperature for a further 60 min. Finally, 25 μL of 2x streptavidin-conjugated donor microbeads (final concentration: 40 μg / mL) were added to each well and incubated at room temperature for a further 30 min. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IRAK4 standard solutions at various concentrations. The AlphaLISA signal values ​​of the samples to be measured were then correspondingly corrected using the total protein concentration of each sample obtained by BCA quantification. A bar graph was plotted using GraphPad Prism 8 software, and the results are shown in Figure 2.

[0617] FIG. 2 shows that IRAK4 protein is expressed at high levels in PBMC cells from patients with rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus, and systemic sclerosis.

[0618] Test Example 6: Inhibitory effect of compounds on IL-6 levels in normal human PMBCs induced by LPS Cryopreserved normal human PBMCs (Myoshun, PB010C) were resuscitated and resuspended in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% fetal bovine serum (Gibco, 10099141), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, 15140122). 150 μL of PBMCs were seeded into each well of a 96-well clear cell plate (Labserv, 310109008) at a cell density of 2 × 10 cells / well. Next, 50 μL of diluted compound solution was added to the corresponding wells to adjust the final compound concentration to a range of 0.026–10,000 nM. The final concentration of DMSO was 0.25%. The treated cell plates were placed in a 5% CO2 incubator and incubated at 37°C for 20 hours, after which 10 μL of LPS (0111:B4) (Sigma, L4391) was added. The cell plates were then placed in a 5% CO2 incubator again and incubated at 37°C for 5 hours, after which they were centrifuged at 2000 rpm for 10 minutes. 100 μL of cell supernatant was removed from each well and transferred to a new 96-well clear cell plate. The plates were then frozen and stored at -80°C for cytokine concentration testing.

[0619] The IL-6 concentration in normal human PBMC cell supernatant was measured using a human IL-6 AlphaLISA detection reagent kit (PerkinElmer, AL223 C). The method was as follows: IL-6 standard solutions were prepared at various concentrations ranging from 0 to 100,000 pg / mL according to the product manual. 2 μL of each IL-6 standard solution and the cell supernatant samples to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-conjugated anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 min. Finally, 10 μL of 2× streptavidin-conjugated donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C for 30 min in the dark. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IL-6 standard solution at each concentration, and the IL-6 concentration of each cell lysate supernatant was determined based on the corresponding concentration in the standard curve of the AlphaLISA signal values ​​of the measured samples. The inhibitory rate of the compound at each concentration was calculated compared with the control IL-6 concentration, and nonlinear curve fitting was performed using GraphPad Prism 8 based on the logarithmic concentration-inhibitory rate to determine the IC of the compound. 50 values ​​were calculated.

[0620] FIG. 3 shows that compound A of the present invention has a good inhibitory effect on IL-6 in human peripheral blood mononuclear cells stimulated with lipopolysaccharide.

[0621] Test Example 7: Human PBMC cytokine test Test equipment and reagents: Normal human peripheral blood mononuclear cells (PBMC) (TPCS, PB010C) RPMI 1640 medium (Gibco, A1049101) Fetal bovine serum (Gibco, 10099141) Double antibody (Gibco, 15140122) Lipopolysaccharide (LPS) (0111:B4) (Sigma-Aldrich, L4391) Lesinurad (R848) (MCE, HY-13740) DMSO (Sigma-Aldrich, D8418-500ML) Ficoll separation liquid (Cytiva, 17544602) ACK red blood cell lysate (sterile) (Sbjbio, BL-O51) 96-well cell culture plate (Corning, 3596) Vacuum blood collection tube (lithium heparin anticoagulant) (BD, 367880) Centrifuge (Thermo Scientific, ST16R) Pipette (Eppendorf)

[0622] (1) Lipopolysaccharide- or lesinurad-induced cytokine test in normal human PMBC Cryopreserved normal human PBMCs were resuscitated and resuspended in RPMI 1640 medium (supplemented with 10% fetal bovine blood, 100 U / mL penicillin, and 100 μg / mL streptomycin) at a cell density of 2 × 10 5 PBMCs were seeded into a 96-well clear cell plate at 1000 cells / well. Compounds diluted in medium were added to the compound wells to a final concentration of 500 nM (containing 0.25% DMSO). At the same time, medium containing 0.25% DMSO was added to the positive and negative control wells. After sample addition, the cell plate was placed in a 5% CO2 incubator and incubated at 37°C for 20 h. LPS or R848 was then added to the compound and positive control wells. After sample addition, the cell plate was again placed in a 5% CO2 incubator and incubated at 37°C for 5 h. Then, the plate was centrifuged at 2000 rpm for 10 min. Finally, the cell supernatant was collected from each well and frozen at -80°C for further use.

[0623] The detection of 48 human cytokines (Shanghai Youningwei Biotechnology Co., Ltd.) was performed according to the Bio-Plex Pro Human Cytokine Screening Panel Reagent Kit (Bio-Rad, 12007283) product manual, and detection was performed using Luminex (X-200) in the machine. The expression detection results for each factor in the samples are shown in Figures 4 and 5.

[0624] Figures 4 and 5 show that at a concentration of 500 nM, the decomposing agent compound A of the present invention has a general inhibitory effect on the cytokines secreted by human peripheral blood mononuclear cells stimulated with lipopolysaccharide or racemate, and is superior to the inhibitor compounds (Control I and Control II) at the same concentration.

[0625] Test Example 8: Inhibitory effect of compounds on IL-6 in whole blood of normal humans or patients Fresh whole blood was collected from normal volunteers or patients with rheumatoid arthritis, systemic lupus erythematosus, or dermatomyositis and anticoagulated with sodium heparin. 190 μL of whole blood was seeded into a 96-well clear cell plate (Labserv, 310109008). 10 μL of diluted compound solution was added to the corresponding well to achieve final compound concentrations in the 10–10,000 nM range. The final DMSO concentration was 0.25%. The treated cell plate was placed in a 5% CO2 incubator and incubated at 37°C for 20 h, after which 10 μL of LPS was added. The cell plate was then placed in a 5% CO2 incubator and incubated at 37°C for 5 h. The plate was then centrifuged at 2000 rpm for 10 min. 50 μL of supernatant was removed from each well and transferred to a new 96-well clear cell plate. The plates were then frozen and stored at -80°C for cytokine concentration testing.

[0626] The IL-6 concentration in whole blood supernatants from normal subjects or patients (with dermatomyositis, rheumatoid arthritis, or systemic lupus erythematosus) was measured using a human IL-6 AlphaLISA detection reagent kit (PerkinElmer, AL223 C). The method was as follows: IL-6 standard solutions were prepared at various concentrations ranging from 0 to 100,000 pg / mL according to the product manual. 2 μL of each IL-6 standard solution and the cell supernatant samples to be measured were added to a white 384-well plate (PerkinElmer, 6007299). Next, 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-conjugated anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 min. Finally, 10 μL of 2× streptavidin-conjugated donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C for 30 min in the dark. After incubation, AlphaLISA signal values ​​were measured using the EnVision microplate reader (PerkinElmer, 2105) in AlphaScreen standard mode. A standard curve was plotted based on the AlphaLISA signal values ​​of the IL-6 standard solution at each concentration, and the IL-6 concentration of each cell lysate supernatant was determined based on the corresponding concentration on the standard curve of the AlphaLISA signal values ​​of the measured samples. The inhibitory rate of each compound at each concentration was calculated compared with the control IL-6 concentration, and nonlinear curve fitting was performed using GraphPad Prism 7 based on the logarithmic concentration-inhibitory rate to determine the IC of the compound. 50 values ​​were calculated.

[0627] Figures 6A, 6B, 6C, and 6D show that compound A of the present invention has a good inhibitory effect on lipopolysaccharide-induced IL-6 in normal human whole blood, IL-6 in whole blood of dermatomyositis patients, IL-6 in whole blood of rheumatoid arthritis patients, and IL-6 in whole blood of systemic lupus erythematosus patients.

[0628] Test Example 9: Test of the effect of compound A on tissue IRAK4 protein expression after multiple oral administration to male BALB / C mice Target information Male BALB / C mice, 6-8 weeks old Test Reagents DMSO, Solutol, Glucose (Lot No. 20181102, AR, Sinopharm Group Chemical Reagents Co., Ltd.).

[0629] Preparation of dosage formulations A predetermined amount of compound A was weighed, and 5% DMSO was added to dissolve the compound in a clear solution. 15% Solutol was then added and the mixture was shaken to mix uniformly. Finally, 80% saline was added and the mixture was mixed uniformly.

[0630] Dose setting and grouping The doses of Compound A were set at 30 mpk, 60 mpk, and 90 mpk, and were orally administered twice daily. The vehicle was 5% DMSO + 15% Solutol + 80% saline. The grouping information is shown in Table 5.

[0631] [Table 4]

[0632] Experimental procedure The animals were divided into groups according to body weight (see Table 5) and then administered the drug. After multiple intragastric administration, the animals were euthanized with CO2 euthanasia. After CO2 euthanasia, blood was collected from the heart and anticoagulated with heparin. PBMCs were isolated and prepared to detect IRAK4 degradation. Spleen and skin tissues were collected and used to detect IRAK4 protein levels.

[0633] Detection indicators Protein levels of IRAK4 in PBMCs, spleen, and skin.

[0634] Test results Figures 7A, 7B, and 7C show that IARK4 was successfully degraded in the animal's PBMCs, spleen, and skin after oral administration of Compound A. With the prolonged administration time, the degradation rates of IRAK4 in PBMCs and spleen tissues increased, reaching over 80%.

[0635] Test Example 10: Beneficial effect of Compound A in imiquimod-induced C57 mouse psoriasis (psoriasis) model reagent 5% Imiquimod Cream, Aldara, 3M Pharmaceuticals, LOT: GVJ005C VecticalTM (calcitriol) Ointment,Galderma,LOT:321449 Dexamethasone acetate tablets, Chenxin Pharmaceutical, National Approval: H37021898 Target information Female C57BL / 6 mice (weight 19–21 g), 9 weeks old, 10 per group.

[0636] Experimental Method A. Reagent Preparation i. Imiquimod cream: For each mouse, 62.5 mg of imiquimod cream was weighed and applied directly to the back, and 20 mg of imiquimod cream was weighed and applied directly to the right ear. ii. Calcitriol: For each mouse, 75 mg of calcitriol was weighed and applied directly to the back, and 24 mg of calcitriol was weighed and applied directly to the right ear. iii. Test compound Dex is prepared at the corresponding concentration for oral administration; Five 5 mg / kg Dex tablets were taken, 7.5 mL of saline was added, and the mixture was vortexed and sonicated until uniformly suspended. The mixture was then stored at 4°C for use. It was noted that the mixture was administered once every three days and that it was vortexed and sonicated beforehand until uniformly suspended. iv. Test compound A was prepared to the corresponding concentration for oral administration. Vehicle: 1% DMSO + 10% Solutol + 89% (5% Dextrose), prepared immediately before use for each administration. Test compound A 10, 100 mg / kg group: Prepared in EP tube. Compound A was weighed, DMSO was added to a total volume of 1%, and the mixture was vortexed and sonicated until completely dissolved. Solutol was further added to a total volume of 10%, and the mixture was vortexed and sonicated until clear. 5% glucose solution to a total volume of 89% was added in several portions, vortexing while adding, and the mixture was constantly stirred with a magnetic stirrer before administration. The compound was prepared immediately before use.

[0637] B. Imiquimod was sensitized and administered to C57BL / 6J mice. The specific administration times are shown in Table 6. i. Day 1: Shave the backs of the mice, and use a mold to secure an area of ​​approximately 2 cm x 3 cm. Select 60 mice. ii. Mice were randomly divided into 6 groups of 10 mice each according to body weight and right ear thickness. iii. To induce psoriasis symptoms in the model mice, imiquimod was applied to the dorsal skin and right ear of the mice in groups G2 to G6 at noon for seven consecutive days starting from day 0 to sensitize them. iv. From Day 0, the dorsal skin of the mice was clinically scored every morning before administration, and the ear thickness was measured every other day. Compound A was administered at 9:00 AM and 5:00 PM, and imiquimod was applied at 1:30 PM for sensitization. The detailed group administration schedule is shown in Table 6. v. Mice were collected at endpoint on Day 5 or Day 7.

[0638] [Table 5]

[0639] Detection indicators Clinical scores and right ear thickness. Test results Clinical score

[0640] This study evaluated the compound's effect on improving clinical scores in an imiquimod-induced mouse psoriasis model. The mean clinical score in the vehicle control group gradually increased, reaching 8.00 points by day 5, suggesting that the imiquimod-induced mouse psoriasis model was successfully established. Compound A had a dose-dependent inhibitory effect on the clinical score of psoriasis in mice at both doses of 10 mg / kg and 100 mg / kg. Compound A showed significant differences compared to the vehicle control group from Day 3, and these differences continued until the end of the experiment (Day 5). The area under the curve (AUC) was calculated by analyzing the clinical score curves for each animal in each group, and the inhibition rate of each treatment group relative to the vehicle control group was calculated based on the mean AUC values ​​between groups. The results are shown in Figures 8A and 8B. The inhibition rates of Compound A at doses of 10 mg / kg and 100 mg / kg were 19.2% and 26.9%, respectively (p<0.0001). The inhibition rates of the positive drug groups, calcitriol and dexamethasone, were 44.5% and 20.7%, respectively (p<0.0001).

[0641] Right ear thickness Figure 8C shows that on day 5, compound A had an inhibitory effect on the ear thickness difference Δ ear thickness (ear thickness value on the day of administration - ear thickness value before group administration) at both 10 mg / kg and 100 mg / kg doses, and the inhibitory effect of compound A on the ear thickness difference Δ ear thickness at 100 mg / kg was statistically significant (p<0.0001).

[0642] Test Conclusion As can be seen from the above biological activity test data, the compounds of the present invention, as IRAK4 degraders, can treat rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

Claims

1. an IRAK4 degrader, and / or its stereoisomer, enantiomer, diastereoisomer, deuterated form, hydrate, solvate, prodrug and / or pharmaceutically acceptable salt thereof, for treating and / or preventing immune and / or inflammatory diseases; use.

2. An IRAK4 degrader, and / or a stereoisomer, enantiomer, diastereoisomer, deuterated product, hydrate, solvate, prodrug and / or pharmaceutically acceptable salt thereof, for treating and / or preventing a disease caused by the abnormal expression of IRAK4 and IRAK4-related proteins in the IL-1R / TLR pathway and / or the abnormal secretion or proliferation of chemical factors, cytokines or immune cells mediated by IRAK4, use.

3. The use according to claim 1 or 2, wherein the IRAK4 degrader is a compound represented by formula I or formula II or a pharmaceutically acceptable salt thereof: 【Chemistry 240】 【Chemistry 241】 where: Ring A is a phenyl group or a pyridyl group; Ring B is C 6 -C 10 a cycloalkyl group or a 6- to 10-membered heterocycloalkyl group containing 1 to 2 heteroatoms selected from N, O, and S, 6 -C 10 The cycloalkyl group and the 6- to 10-membered heterocycloalkyl group may optionally be selected from the group consisting of halogen, oxo, cyano, amino, hydroxy, C 1 -C 6 Alkyl group, C 1 -C 6 Haloalkyl group, C 1 -C 6 Hydroxyalkyl group or -O-(C 1 -C 6 alkyl), Ring C is C 6 -C 12 a cycloalkyl group or a 6- to 12-membered heterocycloalkyl group containing 1 to 2 heteroatoms selected from N, O, and S, 6 -C 12 The cycloalkyl group and the 6- to 12-membered heterocycloalkyl group may optionally be selected from the group consisting of halogen, cyano, amino, hydroxy, C 1 -C 6 Alkyl group, C 1 -C 6 Haloalkyl group, C 1 -C 6 Hydroxyalkyl group or -O-(C 1 -C 6 alkyl), Ring D is C 6 -C 10 is an aryl group, 6 -C 10 The aryl group may optionally be a halogen, a cyano group, an amino group, a hydroxy group, a C 1 -C 6 Alkyl group, C 1 -C 6 Haloalkyl group, C 1 -C 6 Hydroxyalkyl group or -O-(C 1 -C 6 alkyl) substituents, Ring Y is a 5- to 6-membered heteroaryl group; X is a bond, -O-, -NH-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-; L is -(CH 2 ) j -, wherein -(CH 2 ) j One or more methylene groups in - may optionally be -NR 3' -, -O-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR 3' -, -CR 1' R 2' -, -C(O)-, -C(O)O-, -OC(O)-, -NR 3' C(O)O-, -OC(O)NR 3' -, -C(O)NR 3' -, -NR 3' C(O)-, -NR 4' C(O)NR 3' -, vinylidene group, or ethynylene group; R 1' , R 2' are each independently a halogen, -OH, or -NH 2 , C 1 -C 4 Alkyl group, C 1 -C 4 Haloalkyl group, C 1 -C 4 Hydroxyalkyl groups, -O(C 1 -C 4 alkyl) or -NH(C 1 -C 4 alkyl), R 3' , R 4' are each independently hydrogen or C 1 -C 6 is an alkyl group, R d are independently hydrogen, deuterium, halogen, cyano group, C 1 -C 6 an alkyl group, optionally substituted with one or more groups selected from halogen, hydroxyl, and amino; R c is -O(C 1 -C 3 alkyl), -N(C 1 -C 3 alkyl) 1-2 or C 1 -C 6 an alkyl group, the alkyl group optionally being a hydroxy group, an amino group, a halogen, a cyano group or -O-(C 1 -C 3 alkyl), R b is hydrogen or C 1 -C 6 an alkyl group, optionally substituted with one or more groups independently selected from a hydroxy group, an amino group, a halogen atom, and a cyano group; R a is hydrogen, halogen, C 1 -C 6 Alkyl group or -O-(C 1 -C 6 alkyl), wherein the alkyl group is optionally substituted with a halogen or hydroxy group; Each R 1 is independently C 1 -C 4 Alkyl group, -O(C 1 -C 4 alkyl), -N(C 1 -C 4 alkyl) 1-2 , CN, halogen, -OH, -NH 2 and C 1 -C 4 The alkyl group may optionally be a halogen, a cyano group, —OH, C 1 -C 4 Alkyl group, -O(C 1 -C 4 alkyl), Each R 2 are independently hydrogen, C 1 -C 4 Alkyl group, -O(C 1 -C 4 alkyl), C 3 -C 8 Cycloalkyl groups, 3- to 8-membered heterocycloalkyl groups, C 6 -C 10 an aryl group, a 5- to 6-membered heteroaryl group, CN, halogen, or —OH; 3 -C 8 Cycloalkyl groups, 3- to 8-membered heterocycloalkyl groups, C 6 -C 10 The aryl group and the 5- to 6-membered heteroaryl group may optionally contain halogen, cyano, —OH, —NH 2 , C 1 -C 4 Alkyl groups and -O(C 1 -C 4 alkyl), X' is CH or N; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; p is 1 or 2; j is 0, 1, 2, 3, 4 or 5; 3. Use according to claim 1 or 2.

4. The IRAK4 decomposing agent is a compound represented by formula I-1, I-2 or II-1 or a pharmaceutically acceptable salt thereof, 【Chemistry 242】 【Chemistry 243】 【Chemistry 244】 where: R 3 H, halogen, C 1 -C 6 Alkyl group or -O-(C 1 -C 6 alkyl), R c , R d , ring B, L, ring C, X, X', p, R 2 and m is defined and described by claim 3, 4. The use according to claim 3.

5. The IRAK4 decomposing agent is a compound selected from A1 to A52, B1 to B5, and C1 to C36; 【Chemistry 245】 【Chemistry 246】 【Chemistry 247】 【Chemistry 248】 【Chemistry 249】 [Chemical 250] 【Chemistry 251】 【Chemistry 252】 【Chemistry 253】 【Chemistry 254】 【Chemistry 255】 【Chemistry 256】 【Chemistry 257】 【Chemistry 258】 【Chemistry 259】 【Chemical 260】 【Chemistry 261】 【Chemistry 262】 【Chemical 263】 【Chemistry 264】 【Chemical 265】 【Chemical 266】 【Chemistry 267】 【Chemical 268】 【Chemistry 269】 【Chemistry 270】 【Chemistry 271】 【Chemistry 272】 【Chemistry 273】 【Chemistry 274】 【Chemistry 275】 【Chemistry 276】 【Chemistry 277】 【Chemistry 278】 【Chemistry 279】 【Chemistry 280】 【Chemistry 281】 【Chemistry 282】 【Chemistry 283】 【Chemistry 284】 【Chemical 285】 【Chemistry 286】 【Chemistry 287】 【Chemical 288】 【Chemistry 289】 【Chemistry 290】 【Chemistry 291】 【Chemistry 292】 【Chemistry 293】 【Chemistry 294】 【Chemistry 295】 【Chemistry 296】 【Chemistry 297】 【Chemistry 298】 【Chemistry 299】 [Chemical 300] 【Chemical 301】 【Chemical 302】 【Chemical 303】 【Chemical 304】 【Chemical 305】 【Chemical 306】 【Chemical 307】 【Chemical 308】 【Chemical 309】 【Chemical 310】 【Chemical 311】 【Chemical 312】 【Chemistry 313】 【Chemical 314】 【Chemical Industry 315】 【Chemical 316】 【Chemical 317】 【Chemical 318】 【Chemical 319】 【Chem.320】 【Chemistry 321】 【Chemistry 322】 【Chemical 323】 【Chemical 324】 【Chemical 325】 【Chemistry 326】 【Chemistry 327】 【Chemical 328】 【Chemistry 329】 【Chemistry 330】 【Chemistry 331】 【Chemistry 332】 【Chemical 333】 【Chemistry 334】 【Chemistry 335】 【Chemistry 336】 【Chemistry 337】 or a pharmaceutically acceptable salt, deuterated salt, solvate or prodrug thereof; 5. Use according to claim 3 or 4.

6. The IRAK4 degrading agent is used to treat and / or prevent an immune disease.

2. The use according to claim 1 .

7. The immune diseases include adult Still's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behçet's disease, benign mucous membrane pemphigoid (mucous membrane pemphigoid), bullous pemphigoid, Castleman's disease (CD), celiac disease, Coxsackie myocarditis, Crohn's disease, dermatitis herpetiformis, atopic dermatitis, dermatomyositis, discoid lupus erythematosus, adenomyosis, eosinophilic fasciitis, erythema nodosum, fibrosing alveolitis, primary glomerulonephritis, Goodpasture's syndrome, autoimmune hemolytic anemia, Henoch-Schön's syndrome, and the like. Hidradenitis suppurativa (HSP), hidradenitis suppurativa (HS), IgG4-related sclerosing disease, inclusion body myositis (IBM), interstitial cystitis (IC), Lambert-Eaton syndrome, linear IgA disease (LAD), systemic lupus erythematosus, chronic Lyme disease, multiple sclerosis, systemic sclerosis, idiopathic inflammatory myopathy (IIM), ocular cicatricial pemphigoid optic neuritis, relapsing rheumatoid arthritis (PR), pemphigus, autoimmune encephalomyelitis, POEMS syndrome, polyarteritis nodosa, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatoid arthritis rheumatoid arthritis, sarcoidosis, autoimmune scleritis, scleroderma, Sjögren's syndrome, aortitis, temporal arteritis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, systemic vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, myasthenia gravis, gout, spondyloarthropathy, crystalline arthropathy, osteoarthritis, rheumatoid arthritis, polymyositis, interstitial lung disease, giant cell arteritis, rheumatic Polymyalgia, granulomatous vasculitis, eosinophilic granulomatous vasculitis, eosinophilic vasculitis, microscopic polyangiitis, cryoglobulinemia, cutaneous leukocytoclastic vasculitis, mixed connective tissue disease, Stevens-Johnson syndrome, pulmonary hypertension, endocarditis, atherosclerosis, erythema multiforme, acute coronary syndrome, idiopathic pulmonary fibrosis, non-alcoholic fatty liver disease, renal fibrosis, type 1 diabetes, primary xerosis, Kawasaki disease, pustular psoriasis, chronic granulomatous disease, neuromyelitis optica, urticaria, palmoplantar pustulosis, sepsis, bullous skin diseases, Alzheimer's disease, chronic idiopathic urticaria, chronic psoriasis, juvenile idiopathic arthritis,selected from axial spondyloarthritis or Graves' disease, 7. Use according to claim 6.

8. The immune disease is selected from rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and / or psoriasis; 8. Use according to claim 7.

9. The IRAK4 degrading agent is used to treat and / or prevent an inflammatory disease.

2. The use according to claim 1 .

10. The inflammatory disease is selected from familial Mediterranean fever, tumor necrosis factor-associated periodic syndrome, mevalonate kinase deficiency, pyridine-associated autoinflammatory neutrophilic dermatosis (PAAND), pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA), familial cold autoinflammatory syndrome (FCAS), familial chronic lichenoid keratosis (FKLC), NLRP1-associated autoinflammatory arthritis and dyskeratosis (NAIAD), IL-1 receptor antagonist (DIRA) deficiency, IL-36 receptor antagonist (DITRA) deficiency, allergic contact dermatitis, CAR-T cell-induced cytokine release syndrome, Crohn's disease, chronic bronchitis, COPD, active ankylosing spondylitis, axial spondyloarthritis, pityriasis rubra pilaris, inflammatory bowel disease, spondyloarthritis, acute lung injury, generalized pustular psoriasis, acne vulgaris, or colitis; 10. The use according to claim 9.

11. the IRAK4 degrading agent is used for treating and / or preventing a disease mediated by IL-2R alpha, IL-6, IFA-alpha2, IFN-gamma, IL-1ra, MCP-3, IL-16, IL-12(p40), LIF, IL-5, GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, Eotaxin, Basic FGF, beta-NGF, PDGF-BB, IL-4, MCP-1, IL-8, IL-10, GRO-alpha, HGF, IL-1alpha, IL-1beta, IL-3, SCF, TRAIL, M-CSF, CTACK, IL-15, IL-12(p70), IL-17, IL-23, IL-33 and / or IL-36 cytokines; 3. The use according to claim 2.

12. The IRAK4 degrading agent is used to treat and / or prevent diseases mediated by IL-4, IL-6, IL-12(p40), GM-CSF, TNF-alpha, IL-2, IL-1alpha, IL-1beta, IL-18, IL-8, IL-10, IL-17, IL-23, IL-33 and / or IL-36 cytokines; 12. Use according to claim 11.

13. The disease sample is a spleen, skin and / or blood sample.

13. Use according to any one of claims 1 to 12.

14. The blood sample is normal human whole blood and / or patient whole blood.

14. The use according to claim 13.

15. 13. A method for treating a disease according to any one of claims 1 to 12, comprising: A method comprising administering to the subject an effective amount of an IRAK4 degrading agent.

16. The IRAK4 degrading agent is the compound of claim 5, either alone or in combination with other drugs. The method of treatment according to claim 15.

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