IRAK4 degrading agent and application thereof

Abstract

Relates to application of compounds shown in a formula I and a formula II, in particular to application of compounds A1-A52, B1-B5 or C1-C36 in preparation of drugs for treating and / or preventing inflammatory or immune diseases. Relates to application of compounds A1-A52, B1-B5 or C1-C36 in preparation of medicines for treating and / or preventing diseases such as rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa, vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung diseases, systemic lupus erythematosus, systemic sclerosis and psoriasis.

Description

IRAK4 degraders and uses thereof

[0001] Technical Field: The present invention relates to compounds that regulate one or more interleukin-1 receptor-associated kinases 4 (IRAK4) by ubiquitination and / or degradation, and their use as drugs for immune and / or inflammatory diseases.

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

[0003] Depending on the presence or absence of MyD88, TLR / IL-1β-mediated signaling pathways can be divided into MyD88-dependent signaling pathways and MyD88-independent pathways. Among them, the signal transduction pathways mediated by IL-1R and TLR2, TLR4, TLR7 / 8, and TLR9 all rely on MyD88 as a regulatory factor to activate downstream inflammatory signaling pathways. After TLR / IL-1β binds to the ligand, it recruits MyD88 molecules. MyD88 further recruits IRAK4 to the TLR / IL-1β complex through 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 (TRAF6), activating the serine / threonine kinase TAK1, and further activating the NF-κB and MAPK signaling pathways (Wang, Deng et al., Nature, 2001), leading to the release of multiple inflammatory cytokines and anti-apoptotic molecules. IRAK4-dependent TLR / IL-1β signaling has been shown to be associated with a variety of diseases, such as 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) and hepatitis viruses (Staschke et al., The Journal of Immunology, 2009; Marquez et al., Ann Rheum Dis, 2014; Zambrano-Zaragoza et al., International Journal of Inflammation, 2014; Wang et al., Experimental and Therapeutic Medicine, 2015; Ciccia et al., Rheumatology, 2015);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, Behcet's disease, gout, adult-onset Still's disease, pericarditis and chronic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, transplant rejection and graft-versus-host reaction; gynecological diseases such as endometriosis, dysmenorrhea, dyspareunia and endometriosis, in particular pain associated with endometriosis and other symptoms associated with endometriosis such as dysmenorrhea, dyspareunia, dysuria and dysfecation (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); ocular diseases such as retinal ischemia, keratitis, allergic conjunctivitis, keratoconjunctivitis sicca, macular degeneration, and uveitis (Kaarniranta and Salminen, J Mol Med (Berl), 2009; Sun and Pearlman, Investigative Ophthalmology & Visual Science, 2009; Redfern and McDermott, Experimental Eye Research, 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 liver fibrosis, myocarditis, primary biliary cirrhosis, 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 diseases such as fatty liver hepatitis, in particular non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH) (Nozaki, Saibara et al., Alcohol Clin Exp Res, 2004; Csak, T., A. Velayudham, 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, 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, Martínez-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 trauma, 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., The Journal of Immunology, 2011; Lim, Kou et al., The American Journal of Pathology, 2011; Béraud 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); itch and pain (including acute, chronic, inflammatory and neuropathic pain) such as hyperalgesia, allodynia, premenstrual pain, pain associated with endometriosis, postoperative pain, interstitial cystitis, CRPS (complex regional pain syndrome), trigeminal neuralgia, prostatitis, pain caused by spinal cord injury, pain caused by inflammation, low back pain, cancer pain, chemotherapy-related pain, HIV treatment-induced neuropathy, pain caused by burns and chronic pain (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, MJ Kim, et al., J Pain, 2014; Min, Ahmad, et al., Photochem Photobiol., 2015; Schrepf, Bradley, et al., Brain Behav Immun, 2015; Wong, L., JD One, et al., Prostate, 2015); neoplastic diseases such as certain lymphomas: ABC-DLBCL (activated B-cell diffuse large B-cell lymphoma), mantle cell lymphoma, and Waldenström disease, as well as chronic lymphocytic leukemia, melanoma, pancreatic tumors, 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 tumors, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, lung cancer, and prostate cancer.

[0004] The regulation of IRAK4-mediated signal transduction pathways is mainly related to its kinase function. However, there are also some reports indicating that in some cell types, the signal regulation of IRAK4 on downstream processes is related to the non-kinase function of IRAK4. Cushing et al. showed that although the phosphorylation level of IRAK4 was reduced in human skin fibroblasts stimulated by IL-1β, pharmacological inhibition of IRAK4 did not lead to the inhibition of IL-6 and TNF-α. Supporting these results, the scaffold function of IRAK4 in IRAK4-deficient fibroblasts was important for IL1 signaling compared with wild-type cells. At the same time, Chiang and his colleagues also showed that IRAK4 kinase activity was not necessary in human B cells and T cells, dendritic cells and monocytes, and siRNA gene ablation also showed that IRAK4 has a scaffold function in these cells. Several potent and selective inhibitors targeting IRAK4 have been reported, such as CA-4948, BAY-1834845, BMS-986126, and PF-06650833. These inhibitors selectively inhibit the kinase activity of IRAK4 and are primarily used for the prevention and treatment of autoimmune, inflammatory, and neoplastic diseases. However, because IRAK4 functions as both a scaffold protein and an active kinase, and because traditional small molecule inhibitors are prone to drug resistance, inhibiting IRAK4 kinase activity alone may not be sufficient to produce a therapeutic effect.

[0005] Proteolysis Targeting Chimeria (PROTAC) is a technology different from traditional small molecule inhibitors. Traditional small molecule inhibitors usually need to act on the active site of the target protein to inhibit its activity. PROTAC is a heterogeneous bifunctional molecule, one end of which is a small molecule inhibitor that can recognize the target protein through a connecting chain, and the other end is an E3 ubiquitin ligase ligand that can recognize E3 ubiquitin ligase. This bifunctional molecule recognizes the target protein and E3 ubiquitin ligase in vivo, brings the target protein and E3 ubiquitin ligase closer to form a ternary complex, ubiquitinates the target protein, and then degrades the target protein through the ubiquitin-proteasome pathway in vivo. Compared to traditional small molecule inhibitors, PROTACs only need to bring the target protein into close proximity with the E3 ubiquitin ligase to degrade the substrate. This mode of action allows this technology to be applied to some undruggable targets. On the other hand, since the PROTAC molecule can be released after the target protein is degraded and continues to participate in the degradation process of the next protein, this catalytic degradation effect allows a smaller PROTAC drug dose to achieve efficient degradation. On the other hand, traditional small molecule inhibitors are prone to drug resistance, which is often due to point mutations that cause the small molecule inhibitors to lose their inhibitory effect on the target. PROTACs can directly degrade the target protein, which can, to a certain extent, avoid the drug resistance caused by point mutations. Therefore, compared with traditional small molecule inhibitors, the use of PROTAC technology for the development of new drug small molecules has great advantages and feasibility, and is expected to become the next generation of promising new drugs. PROTAC technology has also been applied to the modification of drugs for various targets, such as androgen receptors, estrogen protein receptors, BTK, etc. Several types of degraders targeting IRAK4 are disclosed in US2019 / 0151295, US2019 / 0192688, WO2019 / 160915 and WO2020 / 113233, and more degraders targeting IRAK4 are awaiting development.

[0006] SUMMARY OF THE INVENTION

[0007] The prior art discloses many IRAK4 inhibitors (see: Annual Reports in Medical Chemistry (2014), 49, 117-133).

[0008] WO2016 / 083433A1 Examples disclose the following compounds (reference compounds (I) of the present invention):

[0009] WO2015 / 150995A1 Examples disclose the following compounds (reference compounds (II) of the present invention):

[0010] As described herein, the present inventors have discovered that IRAK4 degraders are suitable for treating and preventing animal immune diseases characterized by an overreactive immune system, and particularly have excellent therapeutic or preventive effects on the following diseases: rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

[0011] Summary of the Invention

[0012] Compounds A1-A52 correspond to the following formulas:

[0013] The preparation and use of compounds A1-A52 as medicines have been disclosed in WO2022 / 088551A1.

[0014] Compounds B1-B5 correspond to the following formulas:

[0015] The preparation and use of compounds B1-B5 as drugs have been disclosed in WO2022 / 028547A1.

[0016] Compounds C1-C36 correspond to the following formulas:

[0017] The preparation and use of compounds C1-C36 as medicines are described in the description of the present invention.

[0018] The present invention provides IRAK4, and / or its stereoisomers, enantiomers, diastereomers, deuterated forms, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts, for use in treating and / or preventing immune and / or inflammatory diseases.

[0019] The present invention provides IRAK4, and / or its stereoisomers, enantiomers, diastereomers, deuterated forms, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts, for use in 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.

[0020] In some embodiments of the present invention, the IRAK4 degrading agent is a compound represented by Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof:

[0021] in:

[0022] Ring A is phenyl or pyridyl;

[0023] Ring B is C6-C 10 Cycloalkyl or 6-10 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, the C6-C 10 Cycloalkyl and 6-10 membered heterocycloalkyl 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);

[0024] Ring C is C6-C 12 Cycloalkyl or 6-12 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, the C6-C 12 Cycloalkyl and 6-12 membered heterocycloalkyl are optionally substituted with a substituent selected from halogen, cyano, amino, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl or -O-(C1-C6 alkyl);

[0025] Ring D is C6-C 10 Aryl, the C6-C 10 The aryl group is optionally substituted with a halogen, cyano, amino, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, or -O-(C1-C6 alkyl) substituent;

[0026] Ring Y is a 5-6 membered heteroaryl group;

[0027] X is a bond, -O-, -NH-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-;

[0028] L is -(CH2) j -, the -(CH2) j -One or more methylene groups are optionally selected from -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’ -, vinylene or ethynylene group replacement;

[0029] R 1’ 、R 2’ Each is independently halogen, -OH, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, -O(C1-C4 alkyl) or -NH(C1-C4 alkyl);

[0030] R 3’ 、R 4’ Each is independently hydrogen or C1-C6 alkyl;

[0031] R d Each is independently hydrogen, deuterium, halogen, cyano, C1-C6 alkyl, wherein the alkyl is optionally substituted by one or more groups selected from halogen, hydroxyl, and amino;

[0032] R c -O(C1-C3 alkyl), -N(C1-C3 alkyl) 1-2 or C1-C6 alkyl, which is optionally substituted by one or more groups independently selected from hydroxy, amino, halogen, cyano or -O-(C1-C3 alkyl);

[0033] R b is hydrogen or C1-C6 alkyl, which is optionally substituted by one or more groups independently selected from hydroxy, amino, halogen, and cyano;

[0034] R a is hydrogen, halogen, C1-C6 alkyl or -O-(C1-C6 alkyl), wherein the alkyl group is optionally substituted with halogen or hydroxy;

[0035] Each R1 is independently selected from: C1-C4 alkyl, -O(C1-C4 alkyl), -N(C1-C4 alkyl) 1-2 , CN, halogen, -OH, -NH2, the alkyl group is optionally substituted by a group selected from halogen, cyano, -OH, C1-C4 alkyl, -O(C1-C4 alkyl)

[0036] each R2 is independently hydrogen, C1-C4 alkyl, -O(C1-C4 alkyl), C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, 6-10 membered aryl, 5-6 membered heteroaryl, CN, halogen, -OH, wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally selected from halogen, cyano, -OH, -NH2, C1-C4 alkyl and -O(C1-C4 alkyl);

[0037] X' is CH or N;

[0038] m is 0, 1, 2, 3, or 4;

[0039] n is 0, 1, 2, 3, or 4;

[0040] p is 1 or 2;

[0041] j is 0, 1, 2, 3, 4 or 5.

[0042] In some preferred embodiments of the present invention, the IRAK4 degrading agent is a compound represented by Formula I-1, I-2 or Formula II-1, or a pharmaceutically acceptable salt thereof:

[0043] in:

[0044] R3 is H, halogen, C1-C6 alkyl or -O-(C1-C6 alkyl);

[0045] R c 、R d , Ring B, L, Ring C, X, X', p, R2 and m are as defined in the present invention.

[0046] In some preferred embodiments of the present invention, the disease is characterized by the use of an IRAK4 degrader for treating and / or preventing an immune disease.

[0047] In some preferred embodiments of the present invention, the IRAK4 degrader is used for an immune disease selected from the group consisting of: adult-onset Still's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet'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, endometriosis, eosinophilic fasciitis, erythema nodosum, fibrosing alveolitis, primary glomerulonephritis, Goodpasture syndrome, autoimmune Hemolytic anemia, Henoch-Schonlein 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, palliative rheumatism (PR), pemphigus, autoimmune encephalomyelitis, POEMS syndrome, polyarteritis nodosa, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, autoimmune scleritis, morphea, Sjögren's syndrome, Takayasu arteritis, 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, spondyloarthropathies, crystalline arthropathy, osteoarthritis, rheumatoid arthritis, polymyositis, interstitial lung disease, giant cell arteritis, polymyositis rheumatica pain, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, 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, nonalcoholic fatty liver disease, renal fibrosis, type 1 diabetes mellitus, primary sicca, Kawasaki disease, pustular psoriasis, chronic granulomatous disease, neuromyelitis optica urticaria, palmoplantar pustulosis, sepsis, bullous dermatoses, Alzheimer's disease, chronic spontaneous urticaria, chronic psoriasis, juvenile atopic arthritis, axial spondyloarthritis, or Graves' disease.

[0048] In some preferred embodiments of the present invention, the IRAK4 degrader is used for an immune disease selected from the group consisting of rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

[0049] In some preferred embodiments of the present invention, the disease is characterized by the use of an IRAK4 degrader for treating and / or preventing an inflammatory disease.

[0050] In some preferred embodiments of the present invention, the IRAK4 degrader is used for an immune disease, and the inflammatory disease is selected from: familial Mediterranean fever, tumor necrosis factor-associated periodic syndrome, mevalonate kinase deficiency, pyridine-associated autoinflammatory with neutrophilic dermatosis (PAAND), suppurative sterile arthritis, pyoderma gangrenosum and acne (PAPA), familial cold autoinflammatory syndrome (FCAS), familial chronic lichenoid keratosis (FKLC), NLRP1-associated autoinflammatory with 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.

[0051] In some preferred embodiments of the present invention, IRAK4 degraders are 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.

[0052] In some preferred embodiments of the present invention, IRAK4 degraders 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.

[0053] In some preferred embodiments of the present invention, the sample is a spleen, skin and / or blood sample.

[0054] In some preferred embodiments of the present invention, the blood sample is normal human whole blood and / or patient whole blood.

[0055] In some preferred embodiments of the present invention, the method for treating the disease comprises administering an effective amount of an IRAK4 degrading agent to the subject.

[0056] In some preferred embodiments of the present invention, the method for treating the disease comprises the use of the IRAK4 degrader as a single compound or in combination with other drugs.

[0057] The present invention demonstrates, through IRAK4 kinase activity assays, that the compounds A1-A52, B1-B5, or C1-C36 of the present invention can effectively bind to the IRAK4 target protein to produce degradation and / or inhibitory effects. Western-blotting demonstrates that the compounds A1-A52, B1-B5, or C1-C36 of the present invention can effectively and specifically degrade IRAK4 protein in THP-1 cells. The compounds A1-A52, B1-B5, or C1-C36 of the present invention, and / or their stereoisomers, enantiomers, diastereomers, deuterated forms, hydrates, solvates, metabolites, prodrugs, and / or pharmaceutically acceptable salts can effectively degrade IRAK4 protein, thereby preventing or treating diseases or conditions associated with IRAK4.

[0058] The present invention demonstrates through experiments 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 diseases.

[0059] In the present invention, the drug comprises an IRAK4 degrader as an active ingredient; the drug of the present invention may optionally further comprise a pharmaceutically acceptable carrier, diluent or excipient.

[0060] In the present invention, the IRAK4 degrader can be used alone or in combination with other drugs to achieve a better therapeutic effect on inflammatory or immune diseases.

[0061] In the present invention, the drug can be in the form of 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 prepared into any of the above-mentioned dosage forms as needed, and each dosage form can be prepared according to conventional methods in the pharmaceutical field.

[0062] In the present invention, the route of administration of the drug can be selected according to actual needs, including oral administration, sublingual administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal administration, transdermal administration, parenteral administration, inhalation administration, intratracheal administration, intrapulmonary administration, and bronchial administration.

[0063] The present invention also provides pharmaceutically acceptable salts of compounds A1-A52, B1-B5, or C1-C36. The term "pharmaceutically acceptable salt" refers to relatively nontoxic acid addition salts or base addition salts of the compounds of the present invention. Such acid addition salts are salts of compounds A1-A52, B1-B5, or C1-C36 of the present invention formed with suitable inorganic or organic acids. These salts can be prepared during the final isolation and purification of the compounds, or by reacting the 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, hydrogenphosphate, carbonate, bicarbonate, toluate, citrate, maleate, fumarate, succinate, tartrate, benzoate, methanesulfonate, p-toluenesulfonate, gluconate, lactobionate, and laurylsulfonate, among others. The base addition salt is a salt formed by the A1-A52, B1-B5 or C1-C36 compound with a suitable inorganic base or organic base, including, for example, salts formed with alkali metals, alkaline earth metals, and quaternary ammonium cations, such as sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, tetramethylammonium salts, tetraethylammonium salts, etc.; amine salts include salts formed with ammonia (NH3), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.

[0064] The compounds of the present invention or their pharmaceutically acceptable salts can be administered to mammals, including humans, orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments or drops), or intratumorally.

[0065] The compound of the present invention can be administered at a dosage of approximately 0.05-300 mg / kg body weight / day, preferably 10-300 mg / kg body weight / day, more preferably 10-150 mg / kg body weight / day.

[0066] The compounds of the present invention or their pharmaceutically acceptable salts can be formulated into solid dosage forms for oral administration, including but not limited to capsules, tablets, pills, powders and granules. In these solid dosage forms, the compounds A1-A52, B1-B5 or C1-C36 of the present invention are mixed as active ingredients with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (2) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (3) Humectants such as glycerol, etc.; (4) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, etc.; (5) solubilizing agents such as paraffin, etc.; (6) absorption accelerators such as quaternary ammonium compounds, etc.; (7) wetting agents such as cetyl alcohol and glyceryl monostearate, etc.; (8) adsorbents such as kaolin, etc.; and (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, etc., or mixtures thereof. Capsules, tablets and pills may also contain buffering agents. The solid dosage forms such as tablets, pills, capsules, pills and granules may be coated or microencapsulated with coatings and shell materials such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If desired, the active ingredient can also be in microencapsulated form with one or more of the above-mentioned excipients.

[0067] 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 active ingredient, compound A1-A52, B1-B5, or C1-C36, or its pharmaceutically acceptable salt, the liquid dosage form may contain inert diluents commonly used in the art, such as water and other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the liquid dosage form of the present invention may also contain conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. Suspending agents include, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0068] The compounds of the present invention or pharmaceutically acceptable salts thereof 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.

[0069] The compounds of the present invention or their pharmaceutically acceptable salts can also be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, and inhalants. The active ingredient, A1-A52, B1-B5, or C1-C36 compounds of the present invention or their pharmaceutically acceptable salts, are mixed under sterile conditions with a physiologically acceptable carrier and optionally a preservative, buffer, or propellant as needed.

[0070] The present invention also provides pharmaceutical compositions comprising the compounds A1-A52, B1-B5, or C1-C36 of the present invention, or pharmaceutically acceptable salts thereof, as active ingredients, and a pharmaceutically acceptable carrier, excipient, or diluent. To prepare the pharmaceutical compositions, the compounds A1-A52, B1-B5, or C1-C36 of the present invention, or pharmaceutically acceptable salts thereof, are typically mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The compositions of the present invention can be formulated into conventional pharmaceutical preparations according to conventional preparation methods, such as tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, syrups, elixirs, ointments, drops, suppositories, inhalants, sprays, and the like.

[0071] The compounds of the present invention or their pharmaceutically acceptable salts can be administered alone or, if necessary, in combination with other pharmaceutically acceptable therapeutic agents, such as with other anti-tumor drugs, anti-inflammatory drugs or autoimmune drugs. The components to be combined can be administered simultaneously or sequentially, in the form of a single formulation or in the form of different formulations. The combination can include not only a combination of a compound of the present invention and one other active agent, but also a combination of a compound of the present invention and two or more other active agents.

[0072] Pharmaceutically acceptable as used herein means that the substance or composition must be compatible with other ingredients of the formulation and not harmful to patients.

[0073] The treatments described herein include preventive and palliative treatments.

[0074] In some embodiments of the present invention, the sample is a blood sample from a patient or a normal person, in some embodiments, the sample is a plasma sample from a patient or a normal person, and in some embodiments, the sample is a peripheral blood mononuclear cell sample from a patient or a normal person.

[0075] In some embodiments of the present invention, measuring the level of immune biomarkers in a sample comprises using an AlphaLISA method. In some embodiments, measuring the level of immune biomarkers in a sample comprises using a method selected from the group consisting of those described in the Examples.

[0076] DETAILED DESCRIPTION: Unless otherwise stated, the following terms used in the specification and claims have the following meanings.

[0077] “Alkyl” refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched alkyl groups; C1-C8 alkyl refers to an alkyl group containing 1 to 8 carbon atoms, such as 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-methylhexyl, 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 isomers thereof; preferably C1-C6 alkyl; more preferably C1-C4 alkyl. The alkyl group may be substituted or unsubstituted. In some embodiments, the alkyl group is C1, C2, C3, C4, C5, C6, C7 or C8 alkyl.

[0078] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent; "C3-C 11 "Cycloalkyl" refers to a cycloalkyl group containing 3 to 11 carbon atoms; "C3-C8 cycloalkyl" refers to a cycloalkyl group containing 3 to 8 carbon atoms; "C5-C 10 "Membered cycloalkyl" refers to a cycloalkyl group comprising 5 to 10 carbon atoms;

[0079] Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; preferably C3-C8 membered cycloalkyl; more preferably C3-C6 membered cycloalkyl.

[0080] Polycyclic cycloalkyl includes cycloalkyl of spiro ring, fused ring and bridged ring. "Spiroalkyl" refers to a polycyclic group in which a carbon atom (called spiro atom) is shared between the monocyclic rings. They may contain one or more double bonds, but no ring has a completely conjugated π electron system. Spiroalkyl is divided into monospiroalkyl, bispiroalkyl or polyspiroalkyl according to the number of spiro atoms shared between the rings, preferably 7-12 membered bispiroalkyl. Non-limiting examples of spiroalkyl include:

[0081] wait.

[0082] "Fused cycloalkyl" refers to an all-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with other rings in the system, wherein 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 constituent rings, fused cycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic. Non-limiting examples of fused cycloalkyl groups include:

[0083] wait.

[0084] "Bridged cycloalkyl" refers to a fully carbon-containing polycyclic group in which any two rings share two carbon atoms that are not directly connected. They may contain one or more double bonds, but no ring has a completely conjugated π electron system. Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl. Non-limiting examples of bridged cycloalkyl groups include:

[0085] wait.

[0086] The cycloalkyl ring can be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl can be optionally substituted or unsubstituted.

[0087] In some embodiments, the cycloalkyl group is C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 、C 12 Monocyclic or polycyclic (eg, spirocyclic, fused or bridged) cycloalkyl groups.

[0088] "Heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent in which one or more (e.g., 2, 3, 4, or 5) ring atoms are selected from nitrogen, oxygen, or S(O) r(wherein 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. "3-11-membered heterocycloalkyl" refers to a ring group containing 3 to 11 ring atoms, "5-10-membered heterocycloalkyl" refers to a ring group containing 5 to 10 ring atoms, and "3-8-membered heterocycloalkyl" refers to a ring group containing 3 to 8 ring atoms, preferably a "3-11-membered heterocycloalkyl" containing 1-2 heteroatoms selected from N, O or S, and more preferably a 3-11-membered heterocycloalkyl containing 1 or 2 N atoms.

[0089] Monocyclic heterocycloalkyl is preferably a 3-8 membered monocyclic heterocycloalkyl containing 1-2 N heteroatoms; non-limiting examples of monocyclic heterocycloalkyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and piperazinyl.

[0090] Polycyclic heterocycloalkyl groups include spiro, fused and bridged heterocycloalkyl groups. "Spiroheterocycloalkyl" refers to a polycyclic heterocycloalkyl group in which the single rings share an atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O). r (wherein r is an integer 0, 1, 2), and the remaining ring atoms are carbon. They may contain one or more double bonds, but no ring has a completely conjugated π electron system. Spirocycloalkyl groups are divided into monospiroheterocycloalkyl, bispiroheterocycloalkyl or polyspiroheterocycloalkyl groups according to the number of spiro atoms shared between the rings, preferably saturated "3-11 membered bispiroheterocycloalkyl groups" containing 1-2 heteroatoms selected from N, O or S; more preferably saturated "7-11 membered bispiroheterocycloalkyl groups" containing 1 or 2 N atoms. Non-limiting examples of spiroheterocycloalkyl groups include: wait.

[0091] "Fused heterocycloalkyl" refers to a polycyclic heterocycloalkyl group in which each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, and one or more ring atoms are selected from nitrogen, oxygen or S(O) r (where r is an integer of 0, 1, or 2), and the remaining ring atoms are carbon. Depending on the number of constituent rings, fused heterocycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably "3-11-membered bicyclic fused heterocycloalkyl groups" containing 1-3 heteroatoms selected from N, O, or S; and more preferably "3-11-membered bicyclic fused heterocycloalkyl groups" containing 1 or 2 saturated N atoms. Non-limiting examples of fused heterocycloalkyl groups include: wait.

[0092] "Bridged heterocycloalkyl" refers to a polycyclic heterocycloalkyl group in which any two rings share two atoms that are not directly connected, they may contain one or more double bonds, but no ring has a completely conjugated π electron system, and one or more ring atoms are selected from nitrogen, oxygen or S(O) r (wherein r is an integer of 0, 1, or 2), and the remaining ring atoms are carbon. Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl groups. Non-limiting examples of bridged heterocycloalkyl groups include:

[0093] wait.

[0094] The heterocycloalkyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

[0095] The heterocycloalkyl group may be optionally substituted or unsubstituted.

[0096] 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, each heteroatom being independently nitrogen, oxygen, or S(O). r (where r is an integer 0, 1, or 2).

[0097] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) polycyclic group having a conjugated π electron system, "C6-C 10 "Aryl" refers to an all-carbon aromatic group containing 6-10 carbon atoms, such as phenyl and naphthyl; preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include: The aryl group may be optionally substituted or unsubstituted. In some embodiments, the aryl group is a 6-10 membered aryl group.

[0098] "Heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms including nitrogen, oxygen or S(O) r(wherein r is an integer of 0, 1, or 2), a 5-6 membered heteroaryl group refers to a heteroaromatic system containing 5-6 ring atoms, and a 5-10 membered heteroaryl group refers to a heteroaromatic system containing 5-10 ring atoms, preferably a 5-6 membered heteroaryl group; more preferably a 5-6 membered heteroaryl group containing 1 or 2 N atoms; non-limiting examples include furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyrazole, imidazolyl, triazolyl, tetrazolyl, etc.; preferably pyridinyl. The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples include: The heteroaryl group may be optionally substituted or unsubstituted. In some embodiments, the heteroaryl group is a 5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl group, wherein the number of heteroatoms may be 1, 2, 3, 4, or 5, each heteroatom being independently nitrogen, oxygen, or S.

[0099] Unless otherwise indicated, structures depicted herein are also intended to encompass all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformers. Therefore, single chemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the present invention. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention. Additionally, unless otherwise indicated, structures depicted herein are also intended to encompass compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure include replacement of a hydrogen by deuterium or tritium, or replacement of a carbon by a 13C- or 14-C oxygen-rich carbons are within the scope of the present invention. These compounds are useful as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention. "Stereoisomers" include all isomers of a single compound that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers of a compound (including enantiomers in the (R-) or (S-) configuration of a compound), mixtures of mirror image isomers (physical mixtures of enantiomers, as well as racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of a compound, and isomers of a compound with multiple chiral centers that are not mirror images of one another (diastereomers). The chiral centers of a compound can undergo epimerization in vivo; therefore, for these compounds, administration of the compound in the (R-) form is considered equivalent to administration of the compound in the (S-) form. Therefore, the compounds of the present invention can be prepared and used as an individual isomer substantially free of other isomers, or as 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 because they have at least one desired isotopic substitution of an atom in an amount greater than the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium or multiple deuterium atoms. Substitution with heavier isotopes, such as deuterium, i.e., 2H, can provide certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and thus may be advantageous in certain circumstances. Furthermore, the bifunctional compounds of the present invention include N-oxides, crystalline forms (also known as polymorphs), active metabolites of the compound having the same type of activity, tautomers, and unsolvated as well as solvated and hydrated forms with pharmaceutically acceptable solvents, such as water, ethanol, etc., in the compound. Solvated forms of the conjugates presented herein are also considered disclosed herein.

[0100] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0101] A "control sample" or "control sample" refers to an individual or sample from a group of individuals not suffering from a disease or condition (e.g., arthritis, rheumatoid arthritis, myositis, lupus erythematosus, and / or systemic sclerosis), or an internal control, as determined by techniques known in the art. In some embodiments, a control or baseline level is determined, either prior to measurement in a sample, or obtained from a database of such control samples.

[0102] "Control compound (I)" and "control (I)" are used interchangeably in the present invention.

[0103] "Control compound (II)" and "control (II)" are used interchangeably in the present invention.

[0104] "Induce" and "stimulate" are used interchangeably in the present invention.

[0105] An "effective amount" refers to an amount that, when administered to a subject, produces beneficial or desired results, including clinical results, eg, degradation, inhibition, or alleviation of symptoms of the disorder being treated in the subject as compared to a control.

[0106] "Pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound in which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are 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, salts or partial glycerol mixtures of electrolytes such as presulfated guanidine, disodium phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium gluconate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0107] "Patient" refers to an animal, preferably a mammal, most preferably a human.

[0108] "Cytokines" are selected from IL-2Ralpha, MIG, MIP-1beta, IL-6, IFN-alpha 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,I P-10,IL-13,IL-4,MCP-1,IL-8,MIP-1alpha,IL-10,G-CSF,GRO-alpha,HGF,IL-1alpha,IL-3,SC F, TRAIL, M-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 the cytokine level in a normal human or mouse sample comprises measuring using cultured peripheral blood mononuclear cells (PBMC). In some embodiments, measuring the cytokine level in a normal human or mouse sample comprises measuring using a Luminex method.

[0109] The following exemplary embodiments are provided to illustrate the present invention, but the present invention is not limited to these examples.

[0110] The present invention will be further described in detail and completely below in conjunction with the examples, but the present invention is by no means limited thereto, and the present invention is not limited thereto. The raw materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using methods known in the art. Unless otherwise specified, the experimental methods in the examples of the present invention that do not specify specific conditions are usually prepared under conventional conditions or under the conditions recommended by the raw materials or commodity manufacturers.

[0111] Abbreviation Definition

[0112] "DMSO" refers to N,N-dimethylformamide.

[0113] "Glucose" refers to glucose.

[0114] "Solutol" refers to polyethylene glycol-15 hydroxystearate.

[0115] "PO" means oral.

[0116] "LPS" refers to lipopolysaccharide.

[0117] "R848" refers to Reximod.

[0118] "PBMC" refers to human peripheral blood mononuclear cells.

[0119] "IRAK4" refers to interleukin 1 receptor-associated kinase 4.

[0120] "IL" refers to interleukin.

[0121] "IMQ" refers to imiquimod.

[0122] "Topical" refers to local administration.

[0123] "BID" means twice daily.

[0124] “QD” means once daily. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] FIG1 shows the degradation effect of compound A on IRAK4 in FIG1A and FIG1B respectively.

[0126] Figure 2. Intracellular IRAK4 protein expression levels in PBMCs of various patients.

[0127] FIG3 shows the inhibitory effect of compound A on LPS-induced IL-6 production in normal human PMBCs.

[0128] FIG4 shows the inhibitory effect of compound A on cytokine secretion by human peripheral blood mononuclear cells stimulated by LPS.

[0129] FIG5 shows the inhibitory effect of compound A on cytokine secretion by human peripheral blood mononuclear cells stimulated by R848.

[0130] FIG6 shows the inhibitory effects of compound A on IL-6 in normal human whole blood and patient whole blood in FIG6A, FIG6B, FIG6C, and FIG6D, respectively.

[0131] FIG7 shows the degradation of IRAK4 by compound A in PBMC, spleen, and skin of C57 mice ( FIG7A , FIG7B , and FIG7C ), respectively.

[0132] FIG8 shows the efficacy of compound A in the imiquimod-induced psoriasis model in C57 mice ( FIG8A , FIG8B , and FIG8C ), respectively.

[0133] Specific implementation methods:

[0134] Ⅰ Compound Preparation Example:

[0135] Example 1: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0136] A solution of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (150 mg, crude, 0.18 mmol), pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (78 mg, 0.18 mmol) and N,N-diisopropylethylamine (116 mg, 0.9 mmol) in dimethyl sulfoxide (5 mL) was stirred at room temperature overnight. The resulting mixture was poured into water (50 mL) and stirred for 10 minutes. The mixture was filtered and the filter cake was purified by preparative HPLC to give the target product 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0137] LC-MS:(ESI,m / z):[M+H] + =874.5.

[0138] 1H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.50(d,J=5.9Hz,1H),8.78(d,J=7.7Hz,1H),8.39(d,J=4.0Hz,1H),8.26(d,J=5.7Hz ,1H),7.63(d,J=8.2Hz,1H),7.55(d,J=1.9Hz,1H),7.39(dd,J=8.3,2.0Hz,1H),7.27-6.96(m,1H),6.90-6.41(m,1H),5.30- 5.05(m,1H),4.77(d,J=17.4Hz,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),2.96-2.8 4(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).

[0139] Example 2: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-((1r,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0140] The preparation method is as in Example 1.

[0141] LC-MS:(ESI,m / z):[M+H] + =874.4

[0142] 1H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.50(d,J=6.3Hz,1H),8.78(d,J=7.7Hz,1H),8.38(d,J=4.4Hz,1H),8.25(d,J=5.7Hz,1 H),7.64(d,J=8.2Hz,1H),7.55(d,J=1.9Hz,1H),7.39(dd,J=8.2,1.9Hz,1H),7.26-6.95(m,1H),6.91-6.41(m,1H),5.32-5.03 (m,1H),4.77(d,J=17.4Hz,1H),4.17(t,J=12.9Hz,1H),3.84-3.71(m,3H),3.67-3.57(m,4H),3.45(d,J=9.8Hz,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).

[0143] Example 3: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0144] A mixture of N-(1-(1-((3-azaspiro[5.5]undec-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), pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (110 mg, 0.26 mmol), and DIEA (168 mg, 1.3 mmol) in DMSO (5 mL) was stirred at room temperature overnight. The reaction was poured into water (50 mL) and stirred for 10 min. The mixture was filtered and the filter cake was purified by Prep-HPLC to give 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0145] LC-MS:(ESI,m / z):[M+H] + =870.3.

[0146] 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),9.50(d,J=5.7Hz,1H),8.79(d,J=7.7Hz,1H),8.39(d,J=4.0Hz,1H),8.26( d,J=5.6Hz,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=17.5Hz,1 H),4.27-4.15(m,1H),3.86-3.71(m,5H),3.65-3.39(m,8H),2.89(d,J=10.4Hz,2H),2.68(t,J=6.5Hz,2H),2.15 (d,J=6.1Hz,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).

[0147] Example 4: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0148] Step 1: Preparation of tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidine-1-carboxylate

[0149] To a solution of 5-morpholinopyrazolo[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, followed by the addition of tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.5 g, 1.5 mmol). The reaction mixture was stirred at 25°C overnight. Water (30 mL) was added to the reaction mixture, which was then extracted with EA (50 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column (EA) to give tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0150] LC-MS:(ESI,m / z):[M+H] + =547.4.

[0151] Step 2: Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0152] To a solution of tert-butyl 4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[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 then concentrated under reduced pressure to afford N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0153] LC-MS:(ESI,m / z):[M+H] + =447.2.

[0154] Step 3: Preparation of tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate

[0155] To a solution of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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-formyl-3-azaspiro[5.5]undecane-3-carboxylate (150 mg, 0.45 mmol) and NaBH3CN (100 mg, 2.25 mmol) at 25°C. The reaction was stirred at 25°C for 16 h. The mixture was filtered and concentrated under reduced pressure. The crude product was purified by flash column (MeOH:DCM=3:97) to give tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0156] LC-MS:(ESI,m / z):[M+H] + =712.4.

[0157] Step 4: Preparation of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0158] To a solution of tert-butyl 9-((4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[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 then concentrated under reduced pressure to afford N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide. The resulting product was used directly in the next reaction.

[0159] LC-MS:(ESI,m / z):[M+H] + =612.5.

[0160] Step 5: Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0161] To a solution of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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 to obtain the product.

[0162] LC-MS:(ESI,m / z):[M+H] + =862.4.

[0163] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.63(d,J=8 .2Hz,1H),7.55(d,J=1.9Hz,1H),7.39(dd,J=8.2,1.8Hz,1H),7.25-6.95(m,1H),6.91(d,J=8.0Hz,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=8.7Hz,2H),1.63-1.41(m,5H),1.38-1.25(m,2H),1.16-1.02(m,4H).

[0164] Example 5: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0165] Step 1: Preparation of ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0166] Ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (3.0 g, 13.3 mmol) and benzylpiperazine-1-carboxylate (2.9 g, 13.3 mmol) were dissolved in ACN (30 mL) solution, and DIEA (5.2 g, 40 mmol) was added. The reaction solution was heated to 60 ° C. and stirred for 2 hours, and 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 obtain ethyl 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate.

[0167] LC-MS:(ESI,m / z):[M+H] + =410.2.

[0168] 1 H NMR(400MHz, DMSO-d6)δ8.76(d,J=7.9Hz,1H),8.22(s,1H),7.41-7.33(m,5H),6.86(d,J=7.9Hz,1H ),5.13(s,2H),4.19(q,J=7.1Hz,2H),3.88-3.74(m,4H),3.62-3.49(m,4H),1.28(t,J=7.1Hz,3H).

[0169] Step 2: Preparation of 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

[0170] 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), and lithium hydroxide (1.0 g, 25.2 mmol) was added. The reaction solution was stirred at 60°C for 2 hours. After the reaction, dilute hydrochloric acid (2 mol / L) was added to the reaction solution to adjust the pH to 2, and then the solution was filtered. The filter cake was washed with water and dried in vacuo to obtain 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.

[0171] LC-MS:(ESI,m / z):[M+H] + =382.2.

[0172] Step 3: Preparation of benzyl 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

[0173] At 25 ° C, 5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (270 mg, 0.4 mmol and DIEA) was added. HATU (380 mg, 0.50 mmol) was added to a solution of 1,4-difluoromethyl-1H-pyrazol-1-yl)piperidine-1-carboxylic acid (0.5 ml, 1.0 mmol) in DMF (5 ml). The mixture was stirred at room temperature for 2 h, followed by the addition of tert-butyl 4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.21 g, 0.3 mmol). The reaction mixture was stirred at 25°C overnight. The reaction was quenched with 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 crude product was purified by column chromatography (MeOH:DCM = 0-3:97) to afford benzyl 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.

[0174] LC-MS:(ESI,m / z):[M+H] + =680.4.

[0175] Step 4: Preparation of benzyl 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate

[0176] To a solution of benzyl 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 mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure to yield benzyl 4-(3-((3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0177] LC-MS:(ESI,m / z):[M+H] + =580.5.

[0178] Step 5: Preparation of tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate

[0179] To a solution of benzyl 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-formyl-3-azaspiro[5.5]undecane-3-carboxylate (144 mg, 0.45 mmol) and NaBH3CN (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 crude product was purified by flash column (MeOH:DCM=0~3:97) to give 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester.

[0180] LC-MS:(ESI,m / z):[M+H] + =845.5.

[0181] Step 6: Preparation of benzyl 4-(3-((1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate

[0182] To a solution of tert-butyl 9-((4-(4-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-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), followed by stirring at 25°C for 1 h. The reaction mixture was concentrated under reduced pressure to afford benzyl 4-(3-((1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0183] LC-MS:(ESI,m / z):[M+H] + =745.4.

[0184] Step 7: Preparation of benzyl 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate

[0185] To a solution of benzyl 4-(3-((1-(1-((3-azaspiro[5.5]undec-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 pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (144 mg, 0.33 mmol) in DMSO (5 mL) was added DIEA (0.1 mL, 0.6 mmol). The reaction mixture was then 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 crude product was purified by flash column (MeOH:DCM=0-3:97) to give benzyl 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.

[0186] LC-MS:(ESI,m / z):[M+H] + =995.0.

[0187] Step 8: Preparation of N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0188] To a solution of benzyl 4-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-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 iodotrimethylsilane (0.85 mL, 0.6 mmol) at 0°C, and the reaction was stirred at 25°C for 16 h. The reaction solution was poured into water, extracted with DCM (20 mL×3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The 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]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0189] LC-MS:(ESI,m / z):[M+H] + =861.1.

[0190] 1 H NMR (400MHz, MeOD) δ8.62(d,J=7.9Hz,1H),8.43(brs,2.67H,FA),8.35(s,1H),7.64(d,J=8.3Hz,1H),7.54(d,J=1.6Hz,1H),7.43(d,J=8.2Hz,1H),7.08 6.77(m,2H),4.57-4.43(m,1H),4.15-4.03(m,4H),3.79(t,J=6.4Hz,2 H),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).

[0191] 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0192] Step 1: Preparation of ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0193] 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 x 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 1 to 5:1) to afford ethyl 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate.

[0194] LC-MS:(ESI,m / z):[M+H] + =303.2.

[0195] Step 2: Preparation of 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

[0196] To a mixture of 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 H₂O / MeOH (3 ml / 26 mL) was added LiOH (1.4 g, 34 mmol). The reaction mixture was then heated to 60°C for 12 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The crude product was purified by Prep-HPLC (acetonitrile / 0.05% aqueous NH₄OH, 5% to 95%) to afford 5-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.

[0197] LC-MS:(ESI,m / z):[M+H] + =275.3.

[0198] 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-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate

[0199] 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). The reaction mixture was then reacted at 25°C for 12 h. The reaction mixture was poured into water and extracted with EA (50 mL x 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude product was purified by column chromatography (MeOH:DCM=0-1:9) to give tert-butyl 4-(4-(5-(8-oxa-3-azabicyclo[3.2.1]octane-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0200] LC-MS:(ESI,m / z):[M+H] + =573.4.

[0201] 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

[0202] 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 hour. The reaction solution was directly concentrated under reduced pressure to obtain 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.

[0203] LC-MS:(ESI,m / z):[M+H] + =473.4.

[0204] 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-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylate

[0205] 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-formyl-3-azaspiro[5.5]undecane-3-carboxylate (77 mg, 0.27 mmol) and STAB (175 mg, 0.8 mmol). . The reaction solution was then stirred at 25°C for 12 hours. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM=0-1:9) to give 9-((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)piperidin-1-yl)methyl)-3-azaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester.

[0206] LC-MS:(ESI,m / z):[M+H] + =738.3.

[0207] Step 6: Preparation of N-(1-(1-((3-azaspiro[5.5]undec-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

[0208] 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-carboxamide)-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 solution was then directly concentrated under reduced pressure to obtain N-(1-(1-((3-azaspiro[5.5]undec-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.

[0209] LC-MS:(ESI,m / z):[M+H] + =638.3.

[0210] 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0211] To a solution of N-(1-(1-((3-azaspiro[5.5]undec-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). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was poured into water (20 mL) and extracted with EA (30 mL x 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% NH4HCO3 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0212] LC-MS:(ESI,m / z):[M+H] + =884.5.

[0213] 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),9.41(s,1H),8.81(d,J=7.8Hz,1H),8.39(s,1H),8.28 (d,J=1.3Hz,1H),7.37(d,J=8.4Hz,1H),7.32(s,1H),7.27-6.95(m,2H),6.82(d,J=7.9Hz,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).

[0214] Example 7: 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-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

[0215] Step 1: Preparation of tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate

[0216] 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 mixture was quenched with 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 crude product was purified by flash column (MeOH:DCM=0~3:97) to give tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidin-1-yl)butyl)piperidine-1-carboxylate.

[0217] LC-MS:(ESI,m / z):[M+H] + =698.0.

[0218] 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

[0219] To a solution of tert-butyl 4-(4-(4-(4-(5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-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 solution was directly concentrated under reduced pressure to obtain 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-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.

[0220] LC-MS:(ESI,m / z):[M+H] + =598.1.

[0221] 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

[0222] 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 pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (130 mg, 0.26 mmol) in DMSO (4 mL) was added DIEA (0.14 mL, 0.78 mmol). The reaction 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 Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC to give 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-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.

[0223] LC-MS:(ESI,m / z):[M+H] + =848.3.

[0224] 1 H NMR (400MHz, DMSO-d6) δ10.49(s,1H),9.50(d,J=5.3Hz,1H),8.78(d,J=7.7Hz,1H),8.40(s,1H),8.25(d,J=5.0Hz,1H),7.6 3(d,J=8.3Hz,1H),7.54(s,1H),7.38(d,J=8.1Hz,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=9.6Hz,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).

[0225] Example 8: N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0226] Step 1: Preparation of tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate

[0227] N-(3-(Difluoromethyl)-1-((1R,4R)-4-(aldehyde)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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 reaction solution with stirring, and the reaction solution was stirred at room temperature for 2 h. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by normal column chromatography to obtain tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0228] LC-MS:(ESI,m / z):[M+H] + =712.3.

[0229] Step 2: Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0230] Tert-butyl 9-(((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-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 solution was stirred at room temperature for 1 h. The reaction solution was directly concentrated under reduced pressure to afford N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (crude product), which was used directly in the next step.

[0231] LC-MS:(ESI,m / z):[M+H] + =612.3.

[0232] 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0233] N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (130 mg, 0.13 mmol) and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (57 mg, 0.13 mmol) were dissolved in DMSO (5 mL). DIEA (84 mg, 0.65 mmol) was added to the reaction solution with stirring, and the reaction solution was stirred at room temperature for 1 h. After the reaction, the reaction solution was poured into water (50 mL), extracted with EA (30 mL x 3), the organic phases were combined and washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by prep-HPLC to obtain N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0234] LC-MS:(ESI,m / z):[M+H] + =862.3.

[0235] 1H NMR (400MHz, DMSO-d6) δ10.50(s,1H),9.39(s,1H),8.82(d,J=6.3Hz,1H),8.37(s,1H),8.28( s,1H),7.63(d,J=7.9Hz,1H),7.55(s,1H),7.39(d,J=7.9Hz,1H),7.25-6.95(m,1H),6.90(d,J =7.6Hz,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).

[0236] 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0237] Step 1: Preparation of tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate

[0238] N-(3-(difluoromethyl)-1-((1R,4R)-4-(aldehyde)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (110 mg, 0.23 mmol) was dissolved in THF (10 mL), tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (56 mg, 0.23 mmol) and STAB (146 mg, 0.69 mmol) were added, and the mixture was reacted at room temperature for 2 h, diluted with water (50 mL), and washed with ethyl acetate (50 mL x 40 mL). 3) extraction, 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=1:11) to give tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0239] LC-MS:(ESI,m / z):[M+H] + =700.4.

[0240] 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0241] Tert-butyl 4-(2-((((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[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), TFA (1 mL) was added, and the mixture was allowed to react at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to afford N-(3-(difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step.

[0242] LC-MS:(ESI,m / z):[M+H] + =600.3.

[0243] 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0244] N-(3-(Difluoromethyl)-1-((1R,4R)-4-((methyl(2-(piperidin-4-yl)ethyl)amino)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (95 mg, 0.14 mmol) was dissolved in DMSO (5 mL), and DIEA (77 mg, 0.60 mmol) and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (61 mg, 0.14 mmol) were added, and the reaction was carried out at room temperature for 2 h. Water (50 mL) was added and stirred for 5 minutes, extracted with ethyl acetate (50 mL x 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 prepared by reverse phase 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0245] LC-MS:(ESI,m / z):[M+H] + =850.4.

[0246] 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),9.39(s,1H),8.83(d,J=7.9Hz,1H),8.38(s,1H),8.29(s,1H),7.63(d,J=8.2Hz ,1H),7.54(d,J=1.8Hz,1H),7.38(dd,J=8.2,1.8Hz,1H),7.24-6.97(m,1H),6.91(d,J=8.0Hz,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=7.0Hz,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).

[0247] 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

[0248] 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

[0249] ((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 and reacted at room temperature for 2 h. Water (50 mL) was added and stirred for 5 minutes, extracted with ethyl acetate (50 mL x 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 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.

[0250] LC-MS:(ESI,m / z):[M+H] + =502.3.

[0251] Step 2: Preparation of 5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0252] 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), and IBX (324 mg, 1.16 mmol) was added. The mixture was reacted at 85° C. for 1.5 h, and the ACN was removed by concentration under reduced pressure. Water (30 mL) was added, and the mixture was washed with ethyl acetate (30 mL x 40 mL). 3) extraction, 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 to give 5-(8-oxo-3-azabicyclo[3.2.1]octane-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0253] LC-MS:(ESI,m / z):[M+H] + =500.3.

[0254] 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-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate

[0255] 5-(8-Oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.20 mmol) was dissolved in THF (10 mL), tert-butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (48 mg, 0.20 mmol) and STAB (127 mg, 0.6 mmol) were added, and the mixture was reacted at room temperature for 2 h. The mixture was diluted with water (50 mL) and washed with ethyl acetate (50 mL x 40 mL). 3) extraction, 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=1:11) to give tert-butyl 4-(2-((((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octane-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0256] LC-MS:(ESI,m / z):[M+H] + =726.4.

[0257] 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

[0258] 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-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate (110 mg, 0.15 mmol) was dissolved in DCM (3 To the precipitate (1 mL), TFA (1 mL) was added, the mixture was reacted at room temperature for 1 h, and the mixture was 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.

[0259] LC-MS:(ESI,m / z):[M+H] + =626.3.

[0260] 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

[0261] 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), and 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 reaction was carried out at room temperature for 2 h. Water (50 mL) was added and stirred for 5 minutes, extracted with ethyl acetate (50 mL x 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 prepared by reverse phase 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.

[0262] LC-MS:(ESI,m / z):[M+H] + =876.2.

[0263] 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),9.41(s,1H),8.82(d,J=7.9Hz,1H),8.38(s,1H),8.28(s,1H),7.63(d,J=8.2Hz ,1H),7.54(d,J=1.9Hz,1H),7.38(dd,J=8.2,2.0Hz,1H),7.30-6.95(m,1H),6.82(d,J=8.0Hz,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=6.8Hz,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).

[0264] 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0265] 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-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate

[0266] 5-(8-Oxo-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-(difluoromethyl)-1-((1R,4R)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.20 mmol) was dissolved in THF (10 mL), tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (48 mg, 0.20 mmol) and STAB (127 mg, 0.6 mmol) were added, and the mixture was reacted at room temperature for 2 h, diluted with water (50 mL), and washed with ethyl acetate (50 mL x 40 mL). 3) extraction, 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=1:11) to give tert-butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octane-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0267] LC-MS:(ESI,m / z):[M+H] + =738.5.

[0268] Step 2: Preparation of N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-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

[0269] Tert-butyl 9-(((1R,4R)-4-(4-(5-(8-oxo-3-azabicyclo[3.2.1]octan-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-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 reaction was carried out at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-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.

[0270] 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0271] N-(1-((1R,4R)-4-((3,9-diazaspiro[5.5]undec-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), and 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 reaction was carried out at room temperature for 2 h. Water (50 mL) was added and stirred for 5 minutes, extracted with ethyl acetate (50 mL x 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 prepared by reverse phase 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0272] LC-MS:(ESI,m / z):[M+H] + =888.2.

[0273] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.41(s,1H),8.81(d,J=7.9Hz,1H),8.37(s,1H),8.28(s,1H),7.64( d,J=8.2Hz,1H),7.55(d,J=1.8Hz,1H),7.39(dd,J=8.2,1.9Hz,1H),7.24-6.97(m,1H),6.82(d,J=8.0Hz,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).

[0274] Example 12: N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0275] N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.14 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-fluorobenzoate (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 hours, water (50 mL) was added and the mixture was filtered. The filter cake was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0276] LC-MS:(ESI,m / z):[M+H] + =846.4.

[0277] 1 H NMR (400MHz, DMSO-d6) δ10.53(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1H),8.29(s,1H),7 .50(d,J=7.7Hz,1H),7.36(d,J=4.0Hz,2H),7.23-6.97(m,1H),6.91(d,J=8.0Hz,1H),4.25-4.15(m,1 H),3.84-3.69(m,10H),3.65-3.49(m,2H),3.31-3.24(m,2H),2.89(d,J=10.3Hz,2H),2.73(t,J=6.6H z,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).

[0278] Example 13: N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0279] N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.12 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoate (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 hours, water (50 mL) was added and the mixture was filtered. The filter cake was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0280] LC-MS:(ESI,m / z):[M+H] + =842.4.

[0281] 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1 H),8.29(s,1H),7.37-7.29(m,2H),7.28-6.96(m,2H),6.91(d,J=7.9Hz,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,4 H),2.25-1.85(m,11H),1.70(d,J=9.3Hz,2H),1.58-1.15(m,7H),1.12-0.91(m,4H).

[0282] 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

[0283] Step 1: Preparation of tert-butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate

[0284] N-(3-(Difluoromethyl)-1-((1R,4S)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)-5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)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. The mixture was stirred at room temperature overnight, water (50 mL) was added, and the mixture was extracted with dichloromethane (20 mL x 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)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0285] LC-MS:(ESI,m / z):[M+H] + =798.5.

[0286] 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

[0287] Tert-butyl 4-(2-((((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)cyclohexyl)methyl)(methyl)amino)ethyl)piperidine-1-carboxylate (80 mg, 0.10 mmol) was dissolved in dichloromethane (3 mL) and the mixture was stirred for 2 h. The reaction mixture was dissolved in 1% paraffin wax, trifluoroacetic acid (1 mL) was added and stirred at room temperature for 2 h. The reaction solution was 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 in the next step without purification.

[0288] LC-MS:(ESI,m / z):[M+H] + =614.3.

[0289] 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

[0290] 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 pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoate (47 mg, 0.11 mmol) and N,N-diisopropylethylamine (38 mg, 0.30 mmol) were added with stirring. The mixture was stirred overnight at room temperature, water (50 mL) was added, and the mixture was filtered. The filter cake was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 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.

[0291] LC-MS:(ESI,m / z):[M+H] + =860.4.

[0292] 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.37(s,1H),8.72(d,J=8.0Hz,1H),8.37(s,1H),8.24(d,J=5. 1Hz,1H),7.36(dd,J=8.5,2.1Hz,1H),7.32(d,J=2.1Hz,1H),7.24-6.94(m,2H),6.86(d,J=8.1Hz,1H ),4.89(d,J=4.1Hz,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=7.0Hz,2H),2.15-1.95(m,7H),1.92-1.25(m,15H),1.17-0.95(m,4H).

[0293] 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

[0294] 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

[0295] (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 and reacted at room temperature for 2 h. Water (50 mL) was added and stirred for 5 minutes, and the mixture was extracted with ethyl acetate (50 mL x 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.

[0296] LC-MS:(ESI,m / z):[M+H] + =589.3.

[0297] Step 2: Preparation of tert-butyl ((S)-1-(3-((3-(difluoromethyl)-1-((1R,4S)-4-carbaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate

[0298] 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 and reacted at 85°C for 1.5 h. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 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-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0299] LC-MS:(ESI,m / z):[M+H] + =586.6.

[0300] Step 3: Preparation of N-benzyl-N-methyl-2-(piperidin-4-yl)ethan-1-amine

[0301] Dissolve tert-butyl 4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carboxylate (345 mg, 1.04 mmol) in DCM (5 mL) and add TFA (1 mL). Allow to react at room temperature for 1 h. The reaction mixture is then concentrated under reduced pressure to yield N-benzyl-N-methyl-2-(piperidin-4-yl)ethan-1-amine, which is used directly in the next step.

[0302] LC-MS:(ESI,m / z):[M+H] + =233.2.

[0303] Step 5: Preparation of 1-(5-(4-(2-(benzyl(methyl)amino)ethyl)piperidine-1-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0304] 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, and the mixture was reacted at room temperature for 2 h. Water (100 mL) was added to the reaction solution and stirred for 5 minutes. The mixture was extracted with ethyl acetate (100 mL x 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.

[0305] LC-MS:(ESI,m / z):[M+H] + =479.2.

[0306] Step 6: Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0307] 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 allowed to react at room temperature for 2 h. After filtration and concentration under reduced pressure, 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione was obtained, which was used directly in the next step.

[0308] LC-MS:(ESI,m / z):[M+Na] + =411.1.

[0309] 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

[0310] 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-carbaldehydecyclohexyl)-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, and the reaction was carried out at room temperature for 2 h. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 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=0-1;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.

[0311] LC-MS:(ESI,m / z):[M+H] + =959.4.

[0312] 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

[0313] 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 reaction was carried out at room temperature for 1 h. The reaction solution was directly concentrated under reduced pressure, and the crude product was purified by reverse phase preparative method 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.

[0314] LC-MS:(ESI,m / z):[M+H] + =859.4.

[0315] 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),9.36(s,1H),8.74(d,J=8.0Hz,1H),8.38(s,1H),8.24(s, 1H),7.36(dd,J=8.4,2.1Hz,1H),7.32(d,J=2.1Hz,1H),7.27-6.97(m,2H),6.88(d,J=8.1Hz,1H ),4.52-4.15(m,4H),3.84(s,3H),3.59(t,J=6.6Hz,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).

[0316] Example 16: N-(3-(difluoromethyl)-1-(1-(2-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0317] N-(1-(1-(2-(3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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), and then DIEA (63.8 mg, 0.495 mmol) was added. The mixture was stirred at room temperature for 16 h, and then water (20 mL) was added. The mixture was washed with ethyl acetate (25 mL x 40 mL). 4) extraction, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by Prep-HPLC (acetonitrile / 0.5% FA aqueous solution, 5% to 95%) to obtain the product N-(3-(difluoromethyl)-1-(1-(2-(3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0318] LC-MS:(ESI,m / z):[M+H] + =872.5.

[0319] 1H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1H),8.29(s,1 H),8.27(s,0.62H,HCOOH),7.42-7.28(m,2H),7.26-6.94(m,2H),6.91(d,J=7.9Hz,1H),4.30-4.1 0(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).

[0320] 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0321] Step 1: Preparation of (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid methyl ester

[0322] 5-Morpholinopyrazolo[1,5-a]pyrimidine-3-carboxylic acid (200 mg, 0.73 mmol) was dissolved in DMF (4 mL), and (1R,4R)-4-(4-amino-3-(difluoromethyl)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid methyl ester (182 mg, 0.73 mmol), HATU (139 mg, 0.95 mmol) and DIEA (377 mg, 2.92 mmol) were added, and the reaction solution was stirred at room temperature overnight. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (40 mL x 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 (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid methyl ester.

[0323] LC-MS:(ESI,m / z):[M+H] +=504.1.

[0324] Step 2: Preparation of (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid

[0325] Methyl (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylate (230 mg, 0.46 mmol) was dissolved in a methanol / water mixture (5 mL, V / V = 5 / 1). Lithium hydroxide (44 mg, 1.84 mmol) was added, and the reaction mixture was heated to 60°C and stirred for 2 hours. The reaction mixture was adjusted to pH 4 with dilute hydrochloric acid (2 mol / L) and extracted with ethyl acetate (40 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford (1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexane-1-carboxylic acid, which was used directly in the next reaction.

[0326] LC-MS:(ESI,m / z):[M+H] + =490.6.

[0327] Step 3: Preparation of tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxamido)ethyl)piperidine-1-carboxylate

[0328] (1R,4R)-4-(3-(Difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-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). HATU (144 mg, 0.38 mmol) and DIEA (155 mg, 1.2 mmol) were added, and the reaction solution was stirred at room temperature overnight. The reaction solution was diluted with water (40 mL) and extracted with ethyl acetate (40 mL x 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxamido)ethyl)piperidine-1-carboxylate.

[0329] LC-MS:(ESI,m / z):[M+H] + =714.8.

[0330] Step 4: Preparation of N-(3-(difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0331] Tert-butyl 4-(2-((1R,4R)-4-(3-(difluoromethyl)-4-(5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)-N-methylcyclohexane-1-carboxamido)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 hour. The reaction solution 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide, which was used directly in the next step without purification.

[0332] LC-MS:(ESI,m / z):[M+H] + =614.2.

[0333] 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0334] N-(3-(Difluoromethyl)-1-((1R,4R)-4-(methyl(2-(piperidin-4-yl)ethyl)carbamoyl)cyclohexyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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 solution was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 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 (CH3CN / 0.08% NH4HCO3 aqueous solution, 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-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide).

[0335] LC-MS:(ESI,m / z):[M+H] + =847.4.

[0336] 1H NMR (400MHz, DMSO-d6) δ10.90(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.40(d,J=5.5Hz,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=8.0Hz,1H),4.52-4.20(m,2H),4.12(dd,J= 12.7,4.9Hz,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),2.6 0-2.54(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).

[0337] 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

[0338] Step 1: Preparation of tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate

[0339] 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 solution 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 obtain tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0340] LC-MS:(ESI,m / z):[M+Na] + =487.3.

[0341] Step 2: Preparation of (9H-fluoren-9-yl)methylmethyl(2-(piperidin-4-yl)ethyl)carbamic acid

[0342] Dissolve tert-Butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate (210 mg, 0.45 mmol) in DCM (4 mL), add TFA (1 mL), and react at room temperature for 1 h. The reaction solution is directly concentrated under reduced pressure to obtain (9H-fluoren-9-yl)methylmethyl(2-(piperidin-4-yl)ethyl)carbamic acid, which is used directly in the next step.

[0343] LC-MS:(ESI,m / z):[M+H] + =365.2.

[0344] Step 3: Preparation of 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0345] (9H-fluoren-9-yl)methylmethyl(2-(piperidin-4-yl)ethyl)carbamic acid (210 mg, 0.45 mmol) was dissolved in DMSO (5 mL), and DIEA (264 mg, 2.05 mmol) and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (178 mg, 0.41 mmol) were added. 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 obtain 1-(2-chloro-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0346] LC-MS:(ESI,m / z):[M+H] + =393.1.

[0347] 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

[0348] 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-((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)carbamic acid was added. Butyl ester (30 mg, 0.05 mmol) and STAB (83 mg, 0.39 mmol) were reacted for 0.5 hours, and then 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 twice within 1 hour, and then reacted at room temperature for 2 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 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=0-1:9) 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.

[0349] LC-MS:(ESI,m / z):[M+H] + =963.3.

[0350] 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

[0351] 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 reaction was carried out at room temperature for 1 h. The reaction solution was directly concentrated under reduced pressure, and then a saturated solution of NaHCO3 was added to adjust the pH to 8-9. The product was extracted 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 method 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.

[0352] LC-MS:(ESI,m / z):[M+H] + =863.1.

[0353] 1 H NMR (400MHz, DMSO-d6) δ11.18-9.87(s,1H),9.36(s,1H),8.74(d,J=8.0Hz,1H),8.37(s,1H),8.24(s,1H),7.63(d,J=8. 2Hz,1H),7.54(d,J=2.0Hz,1H),7.38(dd,J=8.2,2.0Hz,1H),7.26-6.97(m,1H),6.87(d,J=8.0Hz,1H),4.50-4.25(m,2H) ,4.18(t,J=11.9Hz,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=7.2Hz,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).

[0354] Example 19: N-(1-((1R,4R)-4-((9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0355] 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

[0356] Tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (100 mg, 0.39 mmol) was dissolved in dimethyl sulfoxide (3 mL). 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 and stirred at room temperature overnight. Water (50 mL) was added to the reaction solution, which was then extracted with ethyl acetate (30 mL x 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 obtain tert-butyl 9-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.

[0357] LC-MS:(ESI,m / z):[M-Boc] + =405.1.

[0358] Step 2: Preparation of 1-(2-chloro-5-(3,9-diazaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0359] 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 hours. The reaction solution was directly concentrated under reduced pressure to afford 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.

[0360] LC-MS:(ESI,m / z):[M+H] + =405.1.

[0361] 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0362] N-(3-(Difluoromethyl)-1-((1R,4R)-4-formylcyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-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), sodium triacetoxyborohydride (44 mg, 0.20 mmol) was added, and the temperature was raised to 70°C and stirred overnight. Water (50 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 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]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0363] LC-MS:(ESI,m / z):[M+H] + =874.2.

[0364] 1H NMR (400MHz, CDCl3) δ9.76 (s, 1H), 8.44 (s, 1H), 8.39 (d, J = 5.3Hz, 2H), 8.30 (d, J = 7.6Hz, 1H), 7.58-7.48 (m, 2H), 7.43 (d, J = 1.8Hz, 1H), 7.38-7.34 (m,1H),6.97-6.65(m,1H),6.02(d,J=7.5Hz,1H),4.98-4.85(m,4H),4.4 7-4.37(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).

[0365] Example 20: N-(1-(1-(2-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0366] N-(1-(1-(2-(3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide (23 mg, 0.03 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (14 mg, 0.03 mmol) and N,N-diisopropylethylamine (38 mg, 0.30 mmol) were added with stirring, and the mixture was stirred at room temperature for 2 hours. Water (20 mL) was added to the reaction solution, and the mixture was filtered. The filter cake was purified by p-TLC (methanol:dichloromethane = 1:9) to obtain N-(1-(1-(2-(3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)ethyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0367] LC-MS:(ESI,m / z):[M+H] + =876.3.

[0368] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1H),8.29(s,1H),7.6 3(d,J=8.2Hz,1H),7.55(d,J=1.9Hz,1H),7.42-7.35(m,1H),7.26-6.97(m,1H),6.91(d,J=8.0Hz,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).

[0369] Example 21: 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0370] Step 1: Preparation of tert-butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-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

[0371] 5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, crude) 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, and the reaction was carried out at room temperature for 2 h. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 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]heptane-5-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.

[0372] LC-MS:(ESI,m / z):[M+H] + =724.3.

[0373] Step 2: Preparation of N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0374] Tert-butyl 9-((4-(4-(5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-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) was dissolved in DCM (4 mL), TFA (1 mL) was added, and the reaction was carried out at room temperature for 1 h. The reaction solution was directly concentrated under reduced pressure to obtain N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, which was directly used in the next step.

[0375] LC-MS:(ESI,m / z):[M+H]+ =624.4.

[0376] 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0377] N-(1-(1-((3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.14 mmol) was dissolved in DMSO (10 mL), and 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 reaction was carried out at room temperature for 2 h. Water (100 mL) was added to the reaction solution and stirred for 5 minutes. The mixture was extracted with ethyl acetate (100 mL x 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 chromatography to give 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0378] LC-MS:(ESI,m / z):[M+H] + =854.4.

[0379] 1H NMR (400MHz, DMSO-d6) δ10.37(s,1H),9.50(d,J=5.7Hz,1H),8.78(d,J=7.6Hz,1H),8.39(d ,J=3.6Hz,1H),8.26(d,J=5.6Hz,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=17.6Hz,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).

[0380] Example 22: Preparation of N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazole-4-)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0381] 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-carbaldehyde (41 mg, 0.1 mmol) were dissolved in tetrahydrofuran (2 mL), sodium triacetoxyborohydride (84 mg, 0.4 mmol) was added with stirring, and the mixture was stirred at room temperature for 3 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 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 (CH3CN / 0.08% NH4HCO3 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazole-4-)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0382] LC-MS:(ESI,m / z):[M+H] + =890.1

[0383] 1 H NMR (400MHz, CD3OD-d4) δ8.64(d,J=7.9Hz,1H),8.37(d,J=5.8Hz,2H),7.40(d,J=7.8Hz,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).

[0384] 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0385] Step 1: Preparation of (S)-tert-butyl (1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate

[0386] Tert-butyl (S)-(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-carbaldehyde (33 mg, 0.08 mmol) were dissolved in DMF (2 mL), and NaBH(OAc)3 (51 mg, 0.24 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 3). The upper organic phases were combined and 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-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate.

[0387] LC-MS:(ESI,m / z):[M+H] + =955.5.

[0388] 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0389] (S)-tert-Butyl (1-(3-((3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-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), and then TFA (0.1 mL) was added and stirred at room temperature for 1 hour. Saturated NaHCO3 aqueous solution was added to the reaction solution until the pH value of the solution became weakly alkaline, and the solution was extracted with a mixed organic solvent (MeOH:DCM=1:10, 30 mL x 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0390] LC-MS:(ESI,m / z):[M+H] + =855.7.

[0391] 1 H NMR(400MHz,MeOD)δ8.49(d,J=8.0Hz,1H),8.35(s,1H),8.29(s,1H),7.40(d,J=7.9Hz,1H),7.35-7.29(m,2 H),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).

[0392] Example 24: N-(3-(Difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0393] Step 1: Preparation of tert-butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate

[0394] 5-(2-Oxo-6-azaspiro[3.3]heptane-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), and DIEA (780 mg, 1.5 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 3). The upper organic phases were combined and 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]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate.

[0395] LC-MS:(ESI,m / z):[M+H] + =559.2.

[0396] Step 2: Preparation of N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0397] tert-Butyl 4-(4-(5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-3-(difluoromethyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (250 mg, 0.45 mmol) was dissolved in DCM (4 mL), and then TFA (1 mL) was added and stirred at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain crude N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0398] LC-MS:(ESI,m / z):[M+H] + =459.3.

[0399] Step 3: Preparation of N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0400] N-(3-(Difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-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-carbaldehyde (102 mg, 0.25 mmol) were dissolved in THF (5 mL), sodium triacetoxyborohydride (160 mg, 0.75 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 3). The upper organic phases were combined and 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 N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a white solid.

[0401] LC-MS:(ESI,m / z):[M+H] +=854.6.

[0402] 1 H NMR (400MHz, MeOD) δ8.47(d,J=7.8Hz,1H),8.35(s,1H),8.27(s,1H),7.40(d,J=7.4Hz,1H),7.36-7.27(m ,2H),7.05-6.95(m,1H),6.31(d,J=8.0Hz,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),2.37-2.2 0(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).

[0403] Example 25: (S)-N-(3-(difluoromethyl)-1-(1-((3-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0404] (S)-N-(1-(1-((3-azaspiro[5.5]undec-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, 0.11 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoate (46 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 (50 mL) was added to the reaction solution, which was filtered. The filter cake was purified by Pre-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 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]undec-9-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0405] LC-MS:(ESI,m / z):[M+H] + =856.3.

[0406] 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),9.38(s,1H),8.72(d,J=8.0Hz,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=8.0Hz,1H),4.89(d,J=2.0Hz,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).

[0407] Example 26: (S)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0408] (S)-N-(1-(1-((3-azaspiro[5.5]undec-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, 0.11 mmol) was dissolved in dimethyl sulfoxide (3 mL). Pentafluorophenyl perfluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)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 solution, and the mixture was filtered. The filter cake was purified by Pre-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 5% to 95%) to obtain (S)-N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0409] LC-MS:(ESI,m / z):[M+H] + =876.3.

[0410] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.38(s,1H),8.72(d,J=7.9Hz,1H),8.38(s,1H),8.24(s,1H) ,7.63(d,J=8.2Hz,1H),7.55(s,1H),7.39(d,J=8.5Hz,1H),7.26-6.97(m,1H),6.86(d,J=8.0Hz,1H ),4.88(d,J=2.0Hz,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).

[0411] Example 27: N-(3-(Difluoromethyl)-1-((1R,4S)-4-((9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0412] Step 1: Preparation of tert-butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate

[0413] N-(3-(Difluoromethyl)-1-((1R,4S)-4-carboxaldehydecyclohexyl)-1H-pyrazol-4-yl)-5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)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 hours. Water (50 mL) was added and the mixture was washed with dichloromethane (20 mL x 4 ). 3) extraction, 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 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)cyclohexyl)methyl)-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester.

[0414] LC-MS:(ESI,m / z):[M+H] + =810.4.

[0415] Step 2: Preparation of N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undec-3-yl)methyl)cyclohexyl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0416] Tert-butyl 9-(((1S,4R)-4-(3-(difluoromethyl)-4-(5-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-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 stirred at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undec-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.

[0417] LC-MS:(ESI,m / z):[M+H] + =626.3.

[0418] 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]undec-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0419] N-(1-((1R,4S)-4-((3,9-diazaspiro[5.5]undec-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 pentafluorophenyl 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methylbenzoate (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 filtered. The filter cake was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 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]undec-3-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)-5-((S)-3-hydroxypiperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0420] LC-MS:(ESI,m / z):[M+H] + =856.4.

[0421] 1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.38(s,1H),8.72(d,J=8.0Hz,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=8.0Hz,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).

[0422] Example 28: N-(3-(Difluoromethyl)-1-(1-(3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0423] Step 1: Preparation of tert-butyl 9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate

[0424] Tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (200 mg, 0.79 mmol) was dissolved in dimethyl sulfoxide (4 mL), and 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. The reaction solution was stirred at room temperature for 3 hours. The reaction solution was diluted with water (40 mL) and extracted with ethyl acetate (50 mL x 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.

[0425] LC-MS:(ESI,m / z):[M+H] + =501.2.

[0426] Step 2: Preparation of 1-(2-methoxy-5-(3,9-azaspiro[5.5]undecane-3-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0427] Dissolve 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) in dichloromethane (3 mL), add trifluoroacetic acid (0.3 mL), and stir at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain 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.

[0428] LC-MS:(ESI,m / z):[M+H] + =401.1.

[0429] Step 3: Preparation of 1-(5-(9-(2-(1,3-dioxolan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0430] 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-dioxolane (121 mg, 0.67 mmol) were dissolved in DMF (5 mL), and K2CO3 (186 mg, 1.3 mmol) was added with stirring. The reaction solution was heated to 80°C and stirred for 12 hours. The reaction solution was diluted with water (80 mL) and extracted with ethyl acetate (50 mL x 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-dioxolan-2-yl)ethyl)-3,9-diazaspiro[5.5]undecane-3-carbonyl)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0431] LC-MS:(ESI,m / z):[M+H] + =501.7.

[0432] Step 4: Preparation of 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)propanal

[0433] 1-(5-(9-(2-(1,3-dioxolan-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. The mixture was stirred at room temperature for 6 hours. The pH of the reaction solution was adjusted to 8 with sodium bicarbonate, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undecane-3-yl)propanal, which was used directly in the next reaction without purification.

[0434] LC-MS:(ESI,m / z):[M+H] + =457.7.

[0435] 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]undec-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide

[0436] 3-(9-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)-3,9-diazaspiro[5.5]undec-3-yl)propanal (70 mg, 0.15 mmol) and N-(3-(difluoromethyl)-1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[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 solution was stirred at room temperature for 3 hours. The reaction solution was diluted with water (50 mL) and extracted with dichloromethane (60 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 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]undec-3-yl)propyl)piperidin-4-yl)-1H-pyrazol-4-yl)-5-morpholinopyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0437] LC-MS:(ESI,m / z):[M+H] + =887.2.

[0438] 1H NMR(400MHz,DMSO-d6)δ10.32(s,1H),9.40(s,1H),8.83(d,J=7.9Hz,1H),8.39(s,1H),8 .29(s,1H),7.37(d,J=8.4Hz,1H),7.32(d,J=2.1Hz,1H),7.26-6.97(m,2H),6.91(d,J=7 .9Hz,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).

[0439] Example 29: N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0440] 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-carbaldehyde (70 mg, 0.16 mmol) were dissolved in THF (3 mL), sodium triacetoxyborohydride (102 mg, 0.48 mmol) was added and stirred at room temperature for 16 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with EA (30 mL x 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 separated and purified by Prep-HPLC to give N-(1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)-5-(1,1-dioxothiomorpholinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0441] LC-MS:(ESI,m / z):[M+H] + =910.4.

[0442] 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),9.25(s,1H),8.92(d,J=7.9Hz,1H),8.39(s,1H),8.34(s,1H), 7.63(d,J=8.2Hz,1H),7.55(d,J=1.8Hz,1H),7.39(d,J=8.2Hz,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=9.3Hz,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).

[0443] 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]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0444] Step 1: Preparation of (S)-tert-butyl (1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylate

[0445] Tert-butyl (S)-(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-carbaldehyde (70 mg, 0.15 mmol) were dissolved in THF (2 mL), and NaBH(OAc)3 (56 mg, 0.37 mmol) was added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was diluted with water (20 mL) and ethyl acetate (20 mL x 3). The organic phases were combined, 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 (S)-(1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carboxylic acid tert-butyl ester.

[0446] LC-MS:(ESI,m / z):[M+H] + =975.4.

[0447] 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]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0448] (S)-tert-Butyl (1-(3-((1-(1-((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-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 (5 mL), TFA (1 mL) was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution 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]undec-9-yl)methyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0449] LC-MS:(ESI,m / z):[M+H] + =875.3.

[0450] 1 H NMR (400MHz, DMSO-d6) δ8.77(d,J=7.9Hz,1H),8.37(s,1H),8.34-8.30(m,2H),7.65(d,J =8.2Hz,1H),7.53(s,1H),7.40(d,J=8.4Hz,1H),7.26-6.95(m,1H),6.85(d,J=7.9Hz,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),2.97-2.9 2(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).

[0451] Example 31: 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl)-N-(1-(1-(3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0452] Step 1: Preparation of benzyl 4-(3-hydroxypropyloxy)piperidine-1-carboxylate

[0453] Benzyl 4-(3-methoxy-3-oxopropyl)piperidine-1-carboxylate (500 mg, 1.49 mmol) was dissolved in anhydrous THF (10 mL). A BH3 / THF solution (15 mL, 14.9 mmol) was added at 0°C and stirred at room temperature for 16 hours. The reaction mixture was quenched with methanol and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate:petroleum ether = 9:1) to obtain benzyl 4-(3-hydroxypropyloxy)piperidine-1-carboxylate.

[0454] LC-MS:(ESI,m / z):[M+H] + =294.2.

[0455] Step 2: Preparation of 3-(piperidin-4-yloxy)propan-1-ol

[0456] Benzyl 4-(3-hydroxypropoxy)piperidine-1-carboxylate (200 mg, 0.68 mmol) was dissolved in ethyl acetate (10 mL), and Pd(OH)2 / C (80 mg) was added. The mixture was heated to 70°C under a hydrogen atmosphere and stirred overnight. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to afford 3-(piperidin-4-yloxy)propan-1-ol. The crude product was used directly in the next reaction without purification.

[0457] LC-MS:(ESI,m / z):[M+H] + =160.2.

[0458] Step 3: Preparation of 1-(2-chloro-5-(4-(3-hydroxypropyloxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0459] 3-(Piperidin-4-yloxy)propan-1-ol (100 mg, 0.62 mmol) was dissolved in DMSO (10 mL), and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (300 mg, 0.69 mmol) and DIEA (240 mg, 1.86 mmol) were added. The mixture was stirred at room temperature for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (methanol:dichloromethane = 1:9) to obtain 1-(2-chloro-5-(4-(3-hydroxypropyloxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0460] LC-MS:(ESI,m / z):[M+H] + =410.1.

[0461] Step 4: Preparation of 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propanal

[0462] 1-(2-Chloro-5-(4-(3-hydroxypropyloxy)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (60 mg, 0.147 mmol) was dissolved in DCM (5 mL), and PCC (63 mg, 0.294 mmol) was added. The mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (methanol:dichloromethane = 1:9) to provide 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propanal.

[0463] LC-MS:(ESI,m / z):[M+H] + =408.1.

[0464] 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)oxy)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0465] 3-((1-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)piperidin-4-yl)oxy)propanal (55 mg, 0.135 mmol) was dissolved in THF (5 mL), and 5-((1R,4R)-2-oxo-5-azabicyclo[2.2.1]heptane-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 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL x 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 obtain the crude product. The crude product was purified by pre-HPLC (CH3CN / 0.08% aqueous NH4HCO3, 5% to 95%) to obtain 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)oxy)propyl)piperidin-4-yl)-3-(difluoromethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0466] LC-MS:(ESI,m / z):[M+H] + =850.4.

[0467] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.50(d,J=5.7Hz,1H),8.79(d,J=7.8Hz,1H),8.40(d,J=3.2Hz,1H), 8.26(d,J=5.5Hz,1H),7.67-7.55(m,2H),7.40(d,J=8.7Hz,1H),7.30-6.92(m,1H),6.88-6.45(m,1H),5.2 8-5.08(m,1H),4.77(d,J=17.7Hz,1H),4.30-4.10(m,1H),4.02-3.68(m,4H),3.62-3.47(m,7H),3.30-3.1 0(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).

[0468] 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]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0469] Step 1: Preparation of tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate

[0470] tert-Butyl 4-(2-(methylamino)ethyl)piperidine-1-carboxylate (200 mg, 0.82 mmol) was dissolved in dichloromethane (4 mL), and triethylamine (249 mg, 2.46 mmol) and CbzCl (139 mg, 0.82 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with water (50 mL), extracted with dichloromethane (80 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 3:7) to obtain tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate.

[0471] LC-MS:(ESI,m / z):[M+H-100] + =277.2.

[0472] Step 2: Preparation of benzylmethyl (2-(piperidin-4-yl)ethyl)carbamate

[0473] Dissolve tert-butyl 4-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)piperidine-1-carboxylate (200 mg, 0.53 mmol) in a hydrogen chloride / 1-4,4-dioxane solution (4 mL). Stir the reaction mixture at room temperature for 1 hour. The reaction mixture is then concentrated under reduced pressure to yield benzylmethyl(2-(piperidin-4-yl)ethyl)carbamate.

[0474] LC-MS:(ESI,m / z):[M+H] + =277.2.

[0475] Step 3: Preparation of benzyl (2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate

[0476] Benzylmethyl(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 DMSO (4 mL) solution, followed by DIEA (278 mg, 2.1 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (70 mL x 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 obtain benzyl(2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperidin-4-yl)ethyl)(methyl)carbamate.

[0477] LC-MS:(ESI,m / z):[M+H] + =523.2.

[0478] Step 4: Preparation of 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0479] 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 hour. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain 1-(2-methoxy-5-(4-(2-(methylamino)ethyl)piperidine-1-carbonyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione.

[0480] LC-MS:(ESI,m / z):[M+H] + =389.2.

[0481] 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]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0482] Compound N-(3-(difluoromethyl)-1-((1R,4R)-4-formylcyclohexyl)-1H-pyrazol-4-yl)-5-(2-oxo-6-azaspiro[3.3]heptane-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 solution was stirred at room temperature for 2 hours. The reaction solution was diluted with water (20 mL), extracted with ethyl acetate (25 mL x 4), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was prepared by reverse phase to give the 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]heptane-6-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0483] LC-MS:(ESI,m / z):[M+H] + =858.4.

[0484] 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.69(s,1H),8.76(d,J=7.6Hz,1H),8.37(s,1H),8.25(s,1H), 7.35(s,1H),7.42-7.00(m,3H),6.38(d,J=7.6Hz,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=5.0Hz,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).

[0485] 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)picolinamide

[0486] Step 1: Preparation of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate

[0487] At 0℃, add Me3OS + I - To a solution of tert-butyl (6.2 g, 28.2 mmol) in DMSO (50 mL) was added NaH (1.17 g, 29.3 mmol). The reaction mixture was stirred at room temperature for 1 hour. Then, 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 another 1 hour. Water (700 mL) was added to the reaction mixture, and the mixture was extracted with EtOAc (100 mL x 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 afford tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate.

[0488] 1 H NMR (400MHz, DMSO-d6) δ6.79 (d, J = 7.2Hz, 1H), 3.37 (m, 1H), 2.57 (s, 2H), 1.83 (td, J = 13. 3,4.1Hz,2H),1.77-1.64(m,2H),1.48-1.40(m,2H),1.38(s,9H),1.20(d,J=13.1Hz,2H).

[0489] Step 2: Preparation of tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate

[0490] To a 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) with stirring, and the reaction solution was stirred at 100°C for 2 h. Water (500 mL) was added to the reaction solution, 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 (MeOH:DCM=0~1:9) to obtain tert-butyl (4-hydroxy-4-(hydroxymethyl)cyclohexyl)carbamate.

[0491] 1 H NMR (400MHz, DMSO-d6) δ6.67(d,J=7.9Hz,1H),4.46(t,J=5.8Hz,1H),3.81(s,1H),3.11(t,J=6.8Hz,3H),1.56-1.45(m,4H),1.44-1.34(m,13H).

[0492] Step 3: Preparation of tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate

[0493] 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 minutes, then stirred at room temperature for an additional 30 minutes before the addition of 4-(bromomethyl)pyridinium hydrobromide (617 mg, 2.4 mmol). The reaction mixture was stirred at room temperature overnight. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with EA (20 mL x 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 afford tert-butyl (4-hydroxy-4-((pyridin-4-ylmethoxy)methyl)cyclohexyl)carbamate.

[0494] LC-MS:(ESI,m / z):[M+H] + =337.1.

[0495] 1 H NMR (400MHz, DMSO-d6) δ8.53(d,J=5.8Hz,2H),7.34(d,J=5.6Hz,2H),6.69(d,J=7.6Hz,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=9.2Hz,11H).

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

[0497] 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 / H₂O (18 mL / 21 mL) was stirred overnight at 75°C under an H₂ atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford tert-butyl (4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)carbamate. The crude product was used directly in the next reaction.

[0498] LC-MS:(ESI,m / z):[M+H] + =343.2.

[0499] Step 5: Preparation of benzyl 4-(((4-((tert-Butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate

[0500] 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 concentrated under reduced pressure and then extracted with DCM (50 mL x 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 afford benzyl 4-(((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0501] LC-MS:(ESI,m / z):[M+Na] + =499.3.

[0502] Step 6: Preparation of benzyl 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate

[0503] To a solution of benzyl 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) with stirring 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 then concentrated under reduced pressure to provide benzyl 4-(((4-amino-1-hydroxycyclohexyl)methoxy)methyl)piperidine-1-carboxylate. The crude product was used directly in the next reaction without purification.

[0504] LC-MS:(ESI,m / z):[M+H] + =377.2.

[0505] Step 7: Preparation of benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate

[0506] To a stirred solution of benzyl 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-nitrobenzaldehyde (170 mg, 0.76 mmol). The reaction mixture was stirred at 100°C for 3 h. The reaction mixture was then concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA:PE = 0 to 4:1) and then by Prep-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 5% to 95%) to afford benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0507] LC-MS:(ESI,m / z):[M+H] + =553.2.

[0508] 1 H NMR(400MHz,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=12.0Hz,1H),4.36(s,1H),4.06-3.99(m,2H) ,3.90(s,3H),3.29(d,J=6.2Hz,2H),3.20(s,2H),2.85-2.75(m,2H),2.35- 2.20(m,2H),1.89(d,J=11.8Hz,2H),1.76-1.61(m,7H),1.15-1.02(m,2H).

[0509] 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

[0510] To a solution of benzyl 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 solution was stirred at room temperature for 2 h. The reaction solution was filtered through celite, 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 was used directly in the next reaction.

[0511] LC-MS:(ESI,m / z):[M+H] + =523.2.

[0512] Step 9: Preparation of benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate

[0513] 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) was added 6-(trifluoromethyl)picolinic acid (90 mg, 0.47 mmol), HATU (178 mg, 0.47 mmol), and DIEA (140 mg, 1.08 mmol). The reaction mixture was then stirred at room temperature for 2 h. Water (30 mL) was added to the reaction mixture, and EA (20 mL x 3) was added. 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 (EA:PE=0-4:1) to give benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)methyl)piperidine-1-carboxylate.

[0514] LC-MS:(ESI,m / z):[M+H] + =696.3.

[0515] 1H NMR (400MHz, DMSO-d6) δ10.51(s,1H),8.69(s,1H),8.47(d,J=7.7Hz,1H),8.41(t,J=7.8Hz,1H),8.32(s, 1H),8.22(d,J=7.7Hz,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=6.2Hz,2H),3.21(s,2H),2.91-2.70(m,2H),2.35-2.15(m,2H),1.90(d,J=9.5Hz,2H),1.85-1.60(m,7H),1.12-.1.02(m,2H).

[0516] Step 10: Preparation of N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide

[0517] To a solution of benzyl 4-((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-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 was then stirred at 70°C overnight under a H2 atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide.

[0518] LC-MS:(ESI,m / z):[M+H] + =562.3.

[0519] 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)picolinamide

[0520] A mixture of N-(2-((1S,4S)-4-hydroxy-4-((piperidin-4-ylmethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide (130 mg, 0.23 mmol), pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)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 washed with DCM (20 mL x 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 5% to 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)picolinamide.

[0521] LC-MS:(ESI,m / z):[M+H] + =812.1.

[0522] 1 H NMR (400MHz, DMSO-d6) δ10.51(d,J=7.5Hz,2H),8.69(s,1H),8.46(d,J=7.7Hz,1H),8.40(t,J=7.8Hz, 1H),8.32(s,1H),8.21(d,J=7.6Hz,1H),7.65(d,J=8.2Hz,1H),7.57(d,J=1.4Hz,1H),7.40(dd,J=8.2, 1.5Hz,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)picolinamide

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

[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, benzyl 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 x 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA:PE = 0 to 3:2) to afford benzyl 4-(2-((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0526] LC-MS:(ESI,m / z):[M+Na] + =513.1.

[0527] 1 H NMR (400MHz, DMSO-d6) δ7.41-7.29(m,5H),6.67(d,J=7.5Hz,1H),5.06(s,2H),4.05-3.90(m,3H ),3.42(t,J=6.4Hz,2H),3.10(s,3H),2.88-2.68(m,2H),1.65(d,J=12.6Hz,2H),1.55-1.32(m, 20H),1.13-0.92(m,2H).

[0528] Step 2: Preparation of benzyl 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0529] To a solution of benzyl 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) with stirring at -10 to 0°C. The reaction mixture was then stirred at room temperature for 2 h. DIEA was added to adjust the pH to 9.0, and the mixture was concentrated under reduced pressure to yield benzyl 4-(2-((4-amino-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0530] LC-MS:(ESI,m / z):[M+H] + =391.2.

[0531] Step 3: Preparation of benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0532] To a stirred solution of benzyl 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-nitrobenzaldehyde (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 afford benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0533] LC-MS:(ESI,m / z):[M+H] + =567.3.

[0534] 1 H NMR(400MHz,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,3 H),3.47(t,J=6.4Hz,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).

[0535] Step 4: Preparation of benzyl 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0536] To a solution of benzyl 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 N2H 4. H2O (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain benzyl 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.

[0537] LC-MS:(ESI,m / z):[M+H] + =537.3

[0538] Step 5: Preparation of benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0539] To a stirred solution of benzyl 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) was added 6-(trifluoromethyl)picolinic acid (101 mg, 0.53 mmol), HATU (217 mg, 0.57 mmol), and DIEA (170 mg, 1.32 mmol). The reaction mixture was then stirred at room temperature for 2 h. The reaction mixture was diluted with water (50 mL) and extracted with EA (20 mL x 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 (EA:PE=0-4:1) to give benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0540] LC-MS:(ESI,m / z):[M+H] + =710.2.

[0541] 1H NMR (400MHz, DMSO-d6) δ10.50(s,1H),8.69(s,1H),8.46(d,J=7.7Hz,1H),8.40(t,J=7.8Hz, 1H),8.32(s,1H),8.21(d,J=7.7Hz,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=6.4Hz,2H),3.21(s,2H),2.90-2.70(m,2H),2.32-2 .18(m,2H),1.90(d,J=10.4Hz,2H),1.72-1.53(m,7H),1.52-1.40(m,2H),1.15-1.00(m,2H).

[0542] 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)picolinamide

[0543] To a solution of benzyl 4-(2-(((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-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 was then stirred at 70°C overnight under an H2 atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated to afford N-(2-((1S,4S)-4-hydroxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide, which was used directly in the next reaction.

[0544] LC-MS:(ESI,m / z):[M+H] + =576.2.

[0545] 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)picolinamide

[0546] 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)picolinamide (130 mg, 0.22 mmol), pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (118 mg, 0.27 mmol) and DIEA (87 mg, 0.68 mmol) in DMSO (3 mL) was stirred at room temperature overnight. The reaction solution was diluted with water (30 mL). The mixture was 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 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)picolinamide.

[0547] LC-MS:(ESI,m / z):[M+H] + =826.1.

[0548] 1 H NMR (400MHz, DMSO-d6) δ10.51(d,J=5.7Hz,2H),8.69(s,1H),8.46(d,J=7.8Hz,1H),8.41(t,J=7.8Hz,1H),8. 33(s,1H),8.22(d,J=7.7Hz,1H),7.64(d,J=8.2Hz,1H),7.55(d,J=1.8Hz,1H),7.39(dd,J=8.2,1.8Hz,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=6.3Hz,2H),3.21(s,2H),3.13-2.9 7(m,1H),2.86-2.71(m,3H),2.32-2.18(m,2H),1.90(d,J=9.8Hz,2H),1.85-1.45(m,9H),1.15-1.05(m,2H).

[0549] Example 35: N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide

[0550] Step 1: Preparation of tert-butyl (4-hydroxy-4-(methylamino)methyl)cyclohexyl)carbamate

[0551] To a mixture of tert-butyl (1-oxaspiro[2.5]octan-6-yl)carbamate (4.2 g, 18.42 mmol) in EtOH / H₂O (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 chromatography (MeOH:DCM = 0-1:1) to afford tert-butyl (4-hydroxy-4-(methylamino)methyl)cyclohexyl)carbamate.

[0552] LC-MS:(ESI,m / z):[M+H] + =259.2.

[0553] Step 2: Preparation of benzyl ((4-((tert-Butoxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate

[0554] 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 extracted with EA (50 mL x 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 chromatography (EA:PE = 0 to 7:3) to yield benzyl ((4-((tert-butoxycarbonyl)amino)-1-hydroxycyclohexyl)methyl)(methyl)carbamate.

[0555] LC-MS:(ESI,m / z):[M+Na] + =415.2.

[0556] Step 3: Preparation of benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate

[0557] To a 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) with stirring at 0°C, and the reaction mixture was stirred at 0°C for 2 h. The pH of the reaction mixture was adjusted to 9.0 with DIEA and then concentrated under reduced pressure to afford benzyl ((4-amino-1-hydroxycyclohexyl)methyl)(methyl)carbamate, which was used directly in the next reaction.

[0558] LC-MS:(ESI,m / z):[M+H] + =293.1.

[0559] Step 4: Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate

[0560] 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-nitrobenzaldehyde (1.2 g, 5.7 mmol). The reaction mixture was stirred at 100°C for 3 h. The reaction mixture was then concentrated under reduced pressure. The crude product was purified by flash column chromatography (EA:PE = 0-1) to afford benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-nitro-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate.

[0561] LC-MS:(ESI,m / z):[M+H] + =469.2

[0562] 1 H NMR(400MHz,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,3 H),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).

[0563] Step 5: Preparation of benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate

[0564] 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 celite, and the filtrate was concentrated under reduced pressure to afford benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate, which was used directly in the next reaction.

[0565] LC-MS:(ESI,m / z):[M+H] + =439.2

[0566] Step 6: Preparation of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate

[0567] To a stirred solution of benzyl (((1S,4S)-4-(5-amino-6-methoxy-2H-indol-2-yl)-1-hydroxycyclohexylmethyl)(methyl)carbamate (940 mg, 2.14 mmol) in DMF (10 mL) were added 6-(trifluoromethyl)picolinic acid (538 mg, 2.78 mmol), HATU (1.06 g, 2.78 mmol) and DIEA (828 mg, 6.42 mmol). The reaction mixture was then heated to room temperature. Stir overnight. The reaction mixture was diluted with water (100 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 crude product was purified by flash column (EA:PE = 0 to 9:1) to obtain benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinyl)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)carbamate.

[0568] LC-MS:(ESI,m / z):[M+H] + =612.2.

[0569] 1H NMR (400MHz, DMSO-d6) δ10.50 (s, 1H), 8.69 (s, 1H), 8.46 (d, J = 7.5Hz, 1H), 8.40 (t, J=7.8Hz,1H),8.31(s,1H),8.21(dd,J=7.7,1.0Hz,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=14.9Hz,2H),1.57-1.40(m,2H).

[0570] Step 7: Preparation of N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide

[0571] To a solution of benzyl (((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinoyl)-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 at 70°C overnight under an H2 atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide. The crude product was used directly in the next reaction.

[0572] LC-MS:(ESI,m / z):[M+H] + =478.1.

[0573] Step 8: Preparation of tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate

[0574] A mixture of N-(2-((1S,4S)-4-hydroxy-4-((methylamino)methyl)cyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide (200 mg, 0.41 mmol), tert-butyl 9-formyl-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 overnight at room temperature. The reaction mixture was diluted with water (30 mL) and then extracted with DCM (10 mL x 3). The organic phase was washed with saturated brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by flash column (MeOH:DCM=0-1:9) to give tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indol-2-yl)cyclohexyl)methyl)(methyl)amino)methyl)-3-azaspiro[5.5]undecane-3-carboxylate.

[0575] LC-MS:(ESI,m / z):[M+H] + =743.3.

[0576] Step 9: Preparation of N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide

[0577] To a solution of tert-butyl 9-(((((1S,4S)-1-hydroxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamido)-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) at 0°C was added TFA (2 mL). 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 Na2SO4, filtered, and concentrated under reduced pressure to give N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide, which was used directly in the next reaction.

[0578] LC-MS:(ESI,m / z):[M+H] + =643.3.

[0579] Step 10: Preparation of N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide

[0580] A mixture of N-(2-((1S,4S)-4-((((3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide (130 mg, 0.20 mmol), pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoate (105 mg, 0.24 mmol), and DIEA (77 mg, 0.60 mmol) in DMSO (5 mL) was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with DCM (20 mL x 3). The organic phase was washed with saturated brine (60 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 5% to 95%) to give N-(2-((1S,4S)-4-((((3-(4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)benzoyl)-3-azaspiro[5.5]undec-9-yl)methyl)(methyl)amino)methyl)-4-hydroxycyclohexyl)-6-methoxy-2H-indol-5-yl)-6-(trifluoromethyl)picolinamide.

[0581] LC-MS:(ESI,m / z):[M+H] + =893.1.

[0582] 1H NMR (400MHz, DMSO-d6) δ10.51(s,2H),8.69(s,1H),8.47(d,J=7.8Hz,1H),8.41(t,J=7.8Hz,1H), 8.32(s,1H),8.22(d,J=7.8Hz,1H),7.63(d,J=8.2Hz,1H),7.55(d,J=1.7Hz,1H),7.39(d,J=8.3H z,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).

[0583] 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)picolinamide

[0584] Step 1: Preparation of benzyl 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0585] Benzyl 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 for 16 h. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL x 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 benzyl 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0586] LC-MS:(ESI,m / z):[M+H] + =627.2.

[0587] Step 2: Preparation of benzyl 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0588] The compound 4-(2-(((1S,4S)-4-(6-methoxy-5-nitro-2H-indazol-2-yl)-1-((methylthio)methoxy)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylic acid benzyl ester (600 mg, 0.96 mmol) and pretreated Raney-Ni (200 mg) (pretreatment method: first washing with water 3 times, then washing with acetone 3 times, and finally washing with ethanol 3 times) were dissolved in anhydrous ethanol (50 mL) and stirred at room temperature for 3 h under a hydrogen atmosphere. The mixture was filtered and the filtrate was directly concentrated under reduced pressure. The crude product was purified using a reverse phase silica gel column (ACN:H2O=1:1, flow rate 40 mL / min) to obtain 4-(2-(((1S,4S)-4-(5-amino-6-methoxy-2H-indazol-2-yl)-1-methoxycyclohexyl)methoxy)ethyl)piperidine-1-carboxylic acid benzyl ester.

[0589] LC-MS:(ESI,m / z):[M+H] + =551.3.

[0590] Step 3: Preparation of benzyl 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate

[0591] 6-(Trifluoromethyl)picolinic 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. Then, benzyl 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 allowed to react at room temperature for 1 h. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 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 benzyl 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-2H-indazol-2-yl)cyclohexyl)methoxy)ethyl)piperidine-1-carboxylate.

[0592] LC-MS:(ESI,m / z):[M+H] + =724.4.

[0593] 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)picolinamide

[0594] Benzyl 4-(2-(((1S,4S)-1-methoxy-4-(6-methoxy-5-(6-(trifluoromethyl)picolinamide)-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 hour. The reaction solution 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)picolinamide, which was used directly in the next reaction without purification.

[0595] LC-MS:(ESI,m / z):[M+H] + =590.4.

[0596] 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)picolinamide

[0597] N-(6-methoxy-2-((1S,4S)-4-methoxy-4-((2-(piperidin-4-yl)ethoxy)methyl)cyclohexyl)-2H-indazol-5-yl)-6-(trifluoromethyl)picolinamide (70 mg, 0.097 mmol) was dissolved in dimethyl sulfoxide (3 mL), and pentafluorophenyl 4-chloro-3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)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 hours, water (50 mL) was added and the mixture was extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine (3 x 60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by pre-HPLC (CH3CN / 0.08% NH4HCO3 aqueous solution, 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)picolinamide.

[0598] LC-MS:(ESI,m / z):[M+H] + =840.3.

[0599] 1 H NMR (400MHz, DMSO-d6) δ10.51(d,J=4.5Hz,2H),8.68(s,1H),8.49-8.37(m,2H),8.32(s,1H),8.22(d,J=7.6H z,1H),7.64(d,J=8.2Hz,1H),7.55(d,J=1.9Hz,1H),7.39(dd,J=8.2,2.0Hz,1H),7.18(s,1H),4.52-4.30(m,2 H),3.98(s,3H),3.80-3.52(m,3H),3.47(t,J=6.2Hz,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).

[0600] II. Biological Activity Test Example

[0601] Test Example 1: IRAK4 kinase activity test

[0602] The KinEASE-STK S1 serine / threonine kinase kit (Cisbio) was used to detect the inhibitory effect of compounds on IRAK4 kinase activity. The specific method was as follows: the compound was dissolved in dimethyl sulfoxide and then diluted isocratically with the kit buffer to adjust the final concentration of the test compound in the reaction system to 10,000 nM to 0.038 nM. Then, 2.5 nM IRAK4 kinase (Carna), 1 μM biotinylated peptide substrate (Cisbio), and 7 μM adenosine triphosphate (Sigma-Aldrich) were added in sequence and incubated at 37°C for 120 min. Subsequently, an anti-phosphorylated serine / threonine antibody conjugated with a europium compound (Cisbio) and a modified XL665 streptavidin conjugated with a phospho-Ser (Cisbio) were added to the reaction system to terminate the reaction. After incubation at room temperature for 1 h, the fluorescence intensity of each well was measured on an EnVision microplate reader (PerkinElmer) in HTRF mode at an excitation wavelength of 337 nm and at emission wavelengths of 620 nm and 665 nm, using the formula Ratio = (665 nm / 620 nm) × 10 4 Calculate the Ratio value. By comparing the fluorescence intensity ratio with the control group, calculate the inhibition rate of the compound at each concentration, and then use GraphPad Prism 7 to perform nonlinear curve fitting based on the logarithmic concentration-inhibition rate to obtain the IC value of the compound. 50 value.

[0603] Table 1:

[0604] Experimental results: The compounds of the present invention can effectively bind to the target protein and inhibit the activity of IRAK4 kinase.

[0605] Test Example 2: Degradation of IRAK4 in THP-1 cells by compounds

[0606] 0.95 mL THP-1 cells (Stem Cell Bank, Chinese Academy of Sciences) were seeded into each well of a 24-well cell culture plate at a cell density of 5 × 10 5Cells were plated at 37°C in a 5% CO2 incubator overnight, then 50 μL of compound dimethyl sulfoxide solution was added to a final concentration ranging from 0.03 to 3000 nM. After 24 hours of incubation, cells were harvested into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm at 4°C for 5 minutes. The cell pellet was washed twice with 1× DPBS, and the resuspended cells were lysed with 200 μL of lysis buffer (Western and IP cell lysis buffer (Beyotime) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Beyotime)). After standing on ice for 30 minutes, the cells were centrifuged at 14,000 g at 4°C for 10 minutes. The supernatant was then analyzed for IRAK4 protein levels by Western blotting.

[0607] Human IRAK4 AlphaLISA Assay

[0608] The above cell lysates were also used to measure IRAK4 degradation using the Human IRAK4 AlphaLISA Detection Kit (PerkinElmer, AL3117C). The specific method was to take 2 μL of the cell lysate supernatant sample to be tested and add it to a white 384-well plate (PerkinElmer, 6007299). Then, 4 μL of 5× anti-IRAK4 acceptor microbeads (PerkinElmer, final concentration of 10 μg / mL) were added and incubated at 23°C for 30 minutes. Then, 4 μL of 5× biotin-labeled anti-IRAK4 antibody (PerkinElmer, final concentration of 1 nM) was added and incubated at 23°C for 60 minutes. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration of 40 μg / mL) was added to each well and incubated at 23°C in the dark for 30 minutes. After incubation, the AlphaLISA signal value was measured on the microplate reader EnVision (PerkinElmer, 2105) in the AlphaScreen standard setting mode, and the IRAK4 concentration was calculated based on this. Then, GraphPad Prism 7 was used to perform nonlinear curve fitting based on the logarithmic concentration-inhibition rate to obtain the DC of the compound. 50 and D max value.

[0609] Table 2:

[0610] Experimental results: The compound of the present invention can effectively degrade IRAK4 kinase protein in cells and has excellent degradation activity.

[0611] Test Example 3: Determination of cytokine concentrations induced by LPS in normal human whole blood and PBMC

[0612] (1) Determination of cytokine concentrations induced by LPS in normal human whole blood

[0613] 190 μL of fresh normal human whole blood (blood collected from healthy individuals aged 22-50 years) anticoagulated with sodium heparin was plated into each well of a 96-well clear cell plate (Labserv, 310109008). Then, 10 μL of the diluted compound solution was added to the corresponding well to achieve a final compound concentration ranging from 10 to 20,000 nM. The final dimethyl sulfoxide concentration was 0.1%. The cell plate treated with compound was sealed with film and incubated at 37°C in a 5% CO2 incubator for 20 hours. Then, 10 μL of LPS (0111:B4) (Sigma, L4391) was added to a final concentration of 100 ng / mL. The cell plate was sealed with film and incubated again at 37°C in a 5% CO2 incubator for 5 hours. After centrifugation at 2000 rpm for 10 minutes, 30 μL of supernatant plasma from each well was transferred to a new 96-well clear cell plate for cytokine concentration measurement and stored frozen at -80°C until assayed.

[0614] Interleukin-6 (IL-6) AlphaLISA detection.

[0615] The interleukin-6 (IL-6) AlphaLISA assay kit (PerkinElmer, AL223C) was used to measure the IL-6 concentration in the supernatant plasma samples of LPS-induced human whole blood (blood collected from healthy individuals aged 22-50 years).

[0616] IL-6 standard solutions of various concentrations were prepared according to the product instructions, ranging from 0 to 100,000 pg / mL. 2 μL of each IL-6 standard solution and the cell supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (PerkinElmer, final concentration of 10 μg / mL) and biotin-labeled anti-IL-6 antibody (PerkinElmer, final concentration of 1 nM) was added to each well and incubated at 23°C for 60 minutes. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration of 40 μg / mL) was added to each well and incubated at 23°C in the dark for another 30 minutes. After incubation, AlphaLISA signals were measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn using the AlphaLISA signal values ​​of the IL-6 standard solutions at various concentrations. The IL-6 concentration of each cell lysate supernatant was then determined based on the AlphaLISA signal values ​​of the test samples at the corresponding concentrations on the standard curve. The IL-6 concentrations were then compared with the IL-6 concentrations of the control group, and the inhibition rate of the compound at each concentration was calculated. The IC value of the compound was then calculated using a nonlinear curve fit using the logarithmic concentration-inhibition rate method using GraphPad Prism 7. 50 value.

[0617] (2) Determination of cytokine concentrations secreted by human PBMC induced by LPS

[0618] Cryopreserved human PBMCs (Miaoshun, PB010C) were 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, 150 μL of PBMCs were plated per well in a 96-well clear cell plate (Labserv, 310109008) to a cell density of 2 × 10 5 / well. Then, 50 μL of the diluted compound solution was added to the corresponding wells with cells to make the final concentration of the compound in the range of 0.026 to 10000 nM. The final DMSO concentration was 0.25%. After the drug-treated cell plate was incubated at 37°C in a 5% CO2 incubator for 1 hour or 20 hours, 10 μL of LPS (0111:B4) (Sigma, L4391) was added to a final concentration of 100 ng / mL. The cell plate was again incubated at 37°C in a 5% CO2 incubator for 5 hours, centrifuged at 2000 rpm for 10 minutes, and 100 μL of cell supernatant was transferred from each well to a new 96-well clear cell plate for cytokine concentration testing and frozen at -80°C for testing.

[0619] Interleukin-6 (IL-6) AlphaLISA Detection

[0620] The interleukin-6 (IL-6) AlphaLISA detection kit (PerkinElmer, AL223C) was used to determine the IL-6 concentration in the supernatant of LPS-induced human PBMC cells.

[0621] IL-6 standard solutions of varying concentrations were prepared according to the product instructions, ranging from 0 to 100,000 pg / mL. 2 μL of each IL-6 standard solution and the cell supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-labeled anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 minutes. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C in the dark for another 30 minutes. After incubation, AlphaLISA signals were measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn using the AlphaLISA signal values ​​of the IL-6 standard solutions at various concentrations. The IL-6 concentration of each cell lysate supernatant was then determined based on the AlphaLISA signal values ​​of the test samples at the corresponding concentrations on the standard curve. The IL-6 concentrations were then compared with the IL-6 concentrations of the control group, and the inhibition rate of the compound at each concentration was calculated. The IC value of the compound was then calculated using a nonlinear curve fit using the logarithmic concentration-inhibition rate method using GraphPad Prism 7. 50 value.

[0622] Test Example 4: Degradation effect of compounds on IRAK4

[0623] (1) Effect of compounds on IRAK4 protein degradation in normal human PBMCs

[0624] Cryopreserved normal human PBMCs (Miaoshun, PB010C) were 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 plated per well of a 24-well cell culture plate. Then, 50 μL of the diluted compound solution was added to the corresponding wells containing cells, resulting in a final compound concentration ranging from 0.01 to 3000 nM. The final DMSO concentration was 0.25%. After dosing, the cell plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The cells were then harvested into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm at 4°C for 5 minutes. The cell pellet was washed twice with 1× DPBS, and the resuspended cells were lysed with 100 μL of lysis buffer. After standing on ice for 30 minutes, the cells were centrifuged at 14,000 g at 4°C for 10 minutes. The supernatant samples were frozen at -80°C until analysis. The cell lysis buffer used was Western elution and IP cell lysis buffer (Beyotime, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Beyotime, P1008).

[0625] The total protein concentration in the cell lysate samples was determined using a BCA protein quantification kit (Tiangen, PA115-02).

[0626] The prepared PBMC lysate samples were used to measure the IRAK4 concentration using the Human IRAK4 AlphaLISA Detection Kit (PerkinElmer, AL3117C) to determine the degradation effect of the compound on IRAK4. The method is as follows:

[0627] 5 μL of each IRAK4 standard solution and the cell lysate supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). Then, 10 μL of 5× anti-IRAK4 acceptor beads (final concentration 10 μg / mL) was added to each well and incubated at room temperature for 30 minutes. Next, 10 μL of 5× biotin-labeled anti-IRAK4 antibody (final concentration 1 nM) was added to each well and incubated at room temperature for another 60 minutes. Finally, 25 μL of 2× streptavidin-labeled donor beads (final concentration 40 μg / mL) was added to each well and incubated at room temperature for another 30 minutes. After incubation, the AlphaLISA signal was measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn by the AlphaLISA signal value of each concentration of IRAK4 standard solution. According to the corresponding concentration of the AlphaLISA signal value of the test sample on the standard curve, the total protein concentration of each sample obtained by BCA quantification was corrected accordingly to determine the IRAK4 concentration of each cell lysate supernatant. The IRAK4 concentration was then compared with the IRAK4 concentration of the control group, and the degradation rate of the compound at each concentration was calculated. Then, a nonlinear curve fitting was performed using GraphPad Prism 8 using the logarithmic concentration-inhibition rate to obtain the DC of the compound. 50 and D max value.

[0628] Table 4:

[0629] (2) Effect of compounds on IRAK4 protein degradation in patient whole blood

[0630] Fresh whole blood was collected from healthy volunteers or patients (rheumatoid arthritis, dermatomyositis) and anticoagulated with sodium heparin. 0.95 mL of whole blood was plated in each well of a 24-well cell culture plate. 50 μL of the diluted compound solution was added to the corresponding well containing whole blood, resulting in a final compound concentration ranging from 10 to 10,000 nM. The final DMSO concentration was 0.1%. After dosing, the cell plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. Whole blood samples were collected from each well and PBMCs were isolated from the whole blood using FICOLL separation buffer (GE, 17-1440-02) according to the product instructions. The isolated cells were washed once with 1× DPBS, then lysed with erythrocyte lysis buffer (Nanjing Senbeijia, BL-O51-100 ml) for 5 minutes and washed once with 1× DPBS. Finally, the white pellet of cells collected after centrifugation at 3000 rpm for 5 minutes was PBMCs.

[0631] After the isolated PBMCs were separated, the supernatant was removed and lysed with cell lysis buffer. The cells were allowed to stand on ice for 30 minutes, then centrifuged at 14,000 g at 4°C for 10 minutes. The cell lysis buffer used was Western and IP cell lysis buffer (Beyotime, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Beyotime, P1008). Protein samples from the centrifuged cell lysate supernatant were frozen at -80°C until analysis.

[0632] The total protein concentration in the whole blood cell lysate samples prepared from normal subjects or patients (rheumatoid arthritis, dermatomyositis) was determined using a BCA protein quantification kit (Tian Gen, PA115-02).

[0633] Whole blood cell lysate samples from normal subjects or patients (rheumatoid arthritis, dermatomyositis) were prepared and the IRAK4 concentration was measured using the Human IRAK4 AlphaLISA Detection Kit (PerkinElmer, AL3117C) to determine the degradation effect of the compound on IRAK4. The method is as follows:

[0634] 5 μL of each IRAK4 standard solution and the cell lysate supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). Then, 10 μL of 5× anti-IRAK4 acceptor beads (final concentration 10 μg / mL) was added to each well and incubated at room temperature for 30 minutes. Next, 10 μL of 5× biotin-labeled anti-IRAK4 antibody (final concentration 1 nM) was added to each well and incubated at room temperature for another 60 minutes. Finally, 25 μL of 2× streptavidin-labeled donor beads (final concentration 40 μg / mL) was added to each well and incubated at room temperature for another 30 minutes. After incubation, the AlphaLISA signal was measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn by the AlphaLISA signal value of each concentration of IRAK4 standard solution. According to the corresponding concentration of the AlphaLISA signal value of the test sample on the standard curve, the total protein concentration of each sample obtained by BCA quantification was corrected accordingly to determine the IRAK4 concentration of each cell lysate supernatant. The IRAK4 concentration was then compared with the IRAK4 concentration of the control group, and the degradation rate of the compound at each concentration was calculated. Then, a nonlinear curve fitting was performed using GraphPad Prism 8 using the logarithmic concentration-inhibition rate to obtain the DC of the compound. 50 and D max value.

[0635] FIG1A and FIG1B show that compound A of the present invention has a good degradation effect on IRAK4 in normal human peripheral blood mononuclear cells, dermatomyositis patients, and rheumatoid arthritis patients.

[0636] Test Example 5: Determination of IRAK4 protein expression levels in PBMCs of various patients

[0637] Fresh whole blood was collected from volunteers and anticoagulated with sodium heparin. PBMCs were then isolated from the whole blood using FICOLL separation buffer (GE, 17-1440-02) according to the product instructions. The isolated cells were washed once with 1× DPBS, then lysed with erythrocyte lysis buffer (Nanjing Senbeijia, BL-O51-100ml), and washed once with 1× DPBS. Finally, the white precipitate collected after centrifugation at 3000 rpm for 5 minutes was PBMCs. This method is suitable for isolating PBMCs from whole blood of patients with osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, and other diseases, as well as from healthy subjects.

[0638] After the isolated PBMCs were separated, the supernatant was removed and lysed with cell lysis buffer. The cells were allowed to stand on ice for 30 minutes, then centrifuged at 14,000 g at 4°C for 10 minutes. The cell lysis buffer used was Western and IP cell lysis buffer (Beyotime, P0013) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Beyotime, P1008). Protein samples from the centrifuged cell lysate supernatant were frozen at -80°C until analysis.

[0639] The total protein concentration in the cell protein samples was determined using the BCA protein quantification kit (Tiangen, PA115-02).

[0640] The prepared cell protein samples were used to measure the expression level of IRAK4 protein in PBMCs of various patients or normal subjects using the human IRAK4 AlphaLISA detection kit (PerkinElmer, AL3117C). The method is as follows:

[0641] 5 μL of each IRAK4 standard solution and the cell lysate supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). Then, 10 μL of 5× anti-IRAK4 acceptor beads (final concentration 10 μg / mL) was added to each well and incubated at room temperature for 30 minutes. Next, 10 μL of 5× biotin-labeled anti-IRAK4 antibody (final concentration 1 nM) was added to each well and incubated at room temperature for another 60 minutes. Finally, 25 μL of 2× streptavidin-labeled donor beads (final concentration 40 μg / mL) was added to each well and incubated at room temperature for another 30 minutes. After incubation, the AlphaLISA signal was measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. A standard curve was constructed using the AlphaLISA signal values ​​of various IRAK4 standard solution concentrations. Based on the AlphaLISA signal values ​​of the test samples corresponding to the standard curve, the total protein concentration of each sample, as determined by BCA quantification, was then corrected to determine the IRAK4 concentration in each cell lysate sample. A histogram was created using GraphPad Prism 8 software, and the results are shown in Figure 2.

[0642] FIG2 shows that PBMCs from patients with rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus, and systemic sclerosis express high levels of IRAK4 protein.

[0643] Test Example 6: Inhibitory effect of compounds on LPS-induced IL-6 in normal human PMBC

[0644] Cryopreserved normal human PBMCs (Miaoshun, PB010C) were 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 plated per well of a 96-well clear cell plate (Labserv, 310109008) to a cell density of 2 × 105 cells / well. 50 μL of the diluted compound solution was then added to the corresponding wells containing cells, resulting in a final compound concentration range of 0.026 to 10,000 nM. The final DMSO concentration was 0.25%. After incubation at 37°C in a 5% CO2 incubator for 20 hours, 10 μL of LPS (0111:B4) (Sigma, L4391) was added. The cell plate was again placed in a 5% CO2 incubator and cultured at 37°C for 5 hours. The cells were 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 for cytokine concentration testing and frozen at -80°C for testing.

[0645] The IL-6 concentration in the supernatant of normal human PBMC cells was determined using a human IL-6 AlphaLISA assay kit (PerkinElmer, AL223C). The method is as follows:

[0646] IL-6 standard solutions of varying concentrations were prepared according to the product instructions, ranging from 0 to 100,000 pg / mL. 2 μL of each IL-6 standard solution and the cell supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-labeled anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 minutes. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C in the dark for another 30 minutes. After incubation, AlphaLISA signals were measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn using the AlphaLISA signal values ​​of the IL-6 standard solutions at various concentrations. The IL-6 concentration of each cell lysate supernatant was then determined based on the AlphaLISA signal values ​​of the test samples at the corresponding concentrations on the standard curve. The IL-6 concentrations were then compared with the IL-6 concentrations of the control group, and the inhibition rate of the compound at each concentration was calculated. The IC value of the compound was then calculated using a nonlinear curve fit using the logarithmic concentration-inhibition rate method using GraphPad Prism 8. 50 value.

[0647] FIG3 illustrates that compound A of the present invention has a good inhibitory effect on IL-6 in human peripheral blood mononuclear cells stimulated by lipopolysaccharide.

[0648] Test Example 7: Human PBMC cytokine test

[0649] Test instruments and reagents:

[0650] Normal human peripheral blood mononuclear cells (PBMC) (TPCS, PB010C)

[0651] RPMI 1640 medium (Gibco, A1049101)

[0652] Fetal bovine serum (Gibco, 10099141)

[0653] Bispecific antibody (Gibco, 15140122)

[0654] Lipopolysaccharide (LPS) (0111:B4) (Sigma-Aldrich, L4391)

[0655] Reximol (R848) (MCE, HY-13740)

[0656] DMSO (Sigma-Aldrich, D8418-500ML)

[0657] Ficoll separation solution (Cytiva, 17544602)

[0658] ACK red blood cell lysis buffer (sterile) (Sbjbio, BL-O51)

[0659] 96-well cell culture plate (Corning, 3596)

[0660] Vacuum blood collection tube (lithium heparin anticoagulation) (BD, 367880)

[0661] Centrifuge (Thermo Scientific, ST16R)

[0662] Pipette (Eppendorf)

[0663] (1) Cytokine test of normal human PMBC induced by lipopolysaccharide or reximod

[0664] After thawing, normal human PBMCs were resuspended in RPMI 1640 medium (supplemented with 10% fetal bovine blood, 100 U / mL penicillin, and 100 μg / mL streptomycin). PBMCs were plated in 96-well clear cell plates at a cell density of 2×10 5 The test compound, diluted in culture medium, was then added to the compound wells to a final concentration of 500 nM (containing 0.25% DMSO). Culture medium containing 0.25% DMSO was also added to the positive and negative control wells. After the sample was added, the cell plate was incubated at 37°C in a 5% CO2 incubator for 20 hours. LPS or R848 was then added to the compound wells and positive control wells. After the sample was added, the cell plate was again incubated at 37°C in a 5% CO2 incubator for 5 hours. The plate was then centrifuged at 2000 rpm for 10 minutes. Finally, the cell supernatant was collected from each well and stored frozen at -80°C until further use.

[0665] Detection of 48 human cytokines (Shanghai Union Biotech Co., Ltd.) was performed using a Luminex (X-200) instrument according to the Bio-Plex Pro Human Cytokine Screening Panel kit (Bio-Rad, 12007283) product instructions. The expression results of each cytokine in the samples are shown in Figures 4 and 5.

[0666] Figures 4 and 5 show that at a concentration of 500 nM, the degrader compound A of the present invention generally has an inhibitory effect on cytokine secretion by human peripheral blood mononuclear cells stimulated by lipopolysaccharide or reximod, and is superior to the inhibitor compounds of the same concentration (control I and control II).

[0667] Test Example 8: Inhibitory effect of compounds on whole blood IL-6 in normal subjects or patients

[0668] Fresh whole blood was collected from healthy volunteers or patients (rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis) and anticoagulated with sodium heparin. 190 μL of whole blood was plated in a 96-well clear cell plate (Labserv, 310109008). 10 μL of diluted compound solution was added to the corresponding wells containing whole blood, resulting in a final compound concentration ranging from 10 to 10,000 nM. The final DMSO concentration was 0.25%. After incubating the treated cell plates at 37°C in a 5% CO2 incubator for 20 hours, 10 μL of LPS was added. The cell plates were again incubated at 37°C in a 5% CO2 incubator for 5 hours. The plates were centrifuged at 2000 rpm for 10 minutes, and 50 μL of supernatant from each well was transferred to a new 96-well clear cell plate for cytokine concentration analysis and stored frozen at -80°C until further analysis.

[0669] The IL-6 concentration in the whole blood supernatant of healthy subjects or patients (dermatomyositis, rheumatoid arthritis, systemic lupus erythematosus) was measured using the human IL-6 AlphaLISA detection kit (PerkinElmer, AL223C). The method is as follows:

[0670] IL-6 standard solutions of varying concentrations were prepared according to the product instructions, ranging from 0 to 100,000 pg / mL. 2 μL of each IL-6 standard solution and the cell supernatant sample to be tested were added to a white 384-well plate (PerkinElmer, 6007299). 8 μL of a mixture of 5× anti-IL-6 receptor microbeads (final concentration 10 μg / mL) and biotin-labeled anti-IL-6 antibody (final concentration 1 nM) was added to each well and incubated at 23°C for 60 minutes. Finally, 10 μL of 2× streptavidin-labeled donor microbeads (final concentration 40 μg / mL) was added to each well and incubated at 23°C in the dark for another 30 minutes. After incubation, AlphaLISA signals were measured on an EnVision microplate reader (PerkinElmer, 2105) using the AlphaScreen standard setting. The standard curve was drawn by the AlphaLISA signal value of each concentration of IL-6 standard solution, and the IL-6 concentration of each cell lysate supernatant was determined based on the corresponding concentration of the α-LISA signal value of the test sample on the standard curve. The IL-6 concentration was then compared with the IL-6 concentration of the control group, and the inhibition rate of the compound at each concentration was calculated. GraphPad Prism 7 was then used to perform nonlinear curve fitting using the logarithmic concentration-inhibition rate to calculate the IC value of the compound. 50 value.

[0671] Figures 6A, 6B, 6C, and 6D show that compound A of the present invention has a good inhibitory effect on IL-6 in the whole blood of normal people induced by lipopolysaccharide, IL-6 in the whole blood of dermatomyositis patients, IL-6 in the whole blood of rheumatoid arthritis patients, and IL-6 in the whole blood of systemic lupus erythematosus patients.

[0672] Test Example 9: Effect of Compound A on Tissue IRAK4 Protein Expression After Multiple Oral Administration in Male BALB / C Mice

[0673] Subject information

[0674] Male BALB / C mice, 6-8 weeks old.

[0675] Test reagents

[0676] DMSO, Solutol, Glucose (Batch No. 20181102, AR, Sinopharm Chemical Reagent Co., Ltd.).

[0677] Preparation of drug delivery preparations

[0678] A certain amount of compound A was weighed, and 5% DMSO was first added to dissolve the compound into a clear solution, and then 15% Solutol was added and shaken to mix, and finally 80% physiological saline was added and mixed.

[0679] Dose setting and grouping

[0680] The dose of Compound A was set at 30 mpk, 60 mpk, and 90 mpk; it was orally administered twice a day; the vehicle was 5% DMSO + 15% Solutol + 80% normal saline. The grouping information is shown in Table 5:

[0681] Table 5 Compound dosage setting and grouping information

[0682] Experimental Procedure

[0683] Animals were grouped according to body weight (see Table 5 for group information) and then dosed. Multiple oral administrations were performed, and animals were euthanized with CO2 three days later. Cardiac blood was collected after CO2 euthanasia, anticoagulated with heparin, and PBMCs were isolated and prepared for analysis of IRAK4 degradation. Spleen and skin tissue were also collected for analysis of IRAK4 protein levels.

[0684] Detection indicators

[0685] IRAK4 protein levels in PBMC, spleen, and skin.

[0686] Test results

[0687] Figures 7A, 7B, and 7C show that after oral administration of Compound A, IARK4 was significantly degraded in the PBMC, spleen, and skin of animals. The degradation rate of IRAK4 in PBMC and spleen tissues also increased with prolonged administration, reaching over 80%.

[0688] Test Example 10: Pharmacodynamic effect of compound A on imiquimod-induced C57 mouse psoriasis model

[0689] Reagents

[0690] 5% Imiquimod Cream, Aldara, 3M Pharmaceuticals, LOT: GVJ005C

[0691] VecticalTM(calcitriol)Ointment, Galderma, LOT: 321449

[0692] Dexamethasone acetate tablets, Chenxin Pharmaceutical, National Medicine Approval Number: H37021898

[0693] Subject information

[0694] Female C57BL / 6 mice (weight 19-21 g), 9 weeks old, 10 mice per group.

[0695] Experimental methods

[0696] A. Reagent Preparation

[0697] i. Imiquimod cream: Weigh 62.5 mg of imiquimod cream for each mouse and apply it directly to the back. Weigh 20 mg of imiquimod cream and apply it directly to the right ear.

[0698] ii. Calcitriol ointment: Weigh 75 mg of calcitriol ointment for each mouse and apply it directly to the back. Weigh 24 mg of calcitriol ointment and apply it directly to the right ear.

[0699] iii. The test compound Dex was formulated into corresponding concentrations for oral administration;

[0700] 5mg / kg: Take 5 Dex tablets, add 7.5ml of normal saline, vortex and sonicate until uniformly suspended, and store at 4°C until needed. Be sure to vortex and sonicate before administration. Prepare every three days.

[0701] iv. Test compound A was prepared into corresponding concentrations and administered orally.

[0702] Vehicle: 1% DMSO + 10% Solutol + 89% (5% Dextrose), prepared and used immediately after each administration.

[0703] Compound A (10 and 100 mg / kg): Prepare in EP tubes. Weigh Compound A, add 1% of the total volume of DMSO, vortex and sonicate until completely dissolved. Then add 10% of the total volume of Solutol, vortex and sonicate until clear. Add 89% of the total volume of 5% glucose solution in steps, vortexing while adding. Stir with a magnetic stir bar until dosing. Prepare the compound freshly before use.

[0704] B. Imiquimod sensitization and drug intervention in C57BL / 6J mice. Specific administration schedules are shown in Table 6.

[0705] i. Day 1: Shave the back of the mice and select 60 mice using a mold to ensure a uniform area of ​​approximately 2 cm x 3 cm.

[0706] ii. Mice were randomly divided into 6 groups based on body weight and right ear thickness, with 10 mice in each group;

[0707] iii. To induce psoriasis symptoms in the model mice, imiquimod was applied to the back skin and right ears of mice in groups G2 to G6 at noon starting on Day 0 for sensitization and continued for 7 days.

[0708] iv. Starting from Day 0, the back skin of the mice was clinically scored before administration every morning, and the ear thickness was measured every other day. Compound A was administered at 9:00 a.m. and 17:00 p.m.; imiquimod was applied at 1:30 p.m. for sensitization. The detailed grouping and dosing schedule is shown in Table 6.

[0709] v. End-point sampling of mice on Day 5 or Day 7

[0710] Table 6 Grouping information

[0711] Detection indicators

[0712] Clinical score and right ear thickness.

[0713] Test results

[0714] Clinical scoring

[0715] This study evaluated the compound's ability to improve clinical scores in an imiquimod-induced mouse psoriasis model. The average clinical score in the solvent control group gradually increased, reaching 8.00 on day 5, indicating the successful establishment of an imiquimod-induced mouse psoriasis model.

[0716] Compound A at both 10 mg / kg and 100 mg / kg doses inhibited the clinical scores of psoriasis in mice in a dose-dependent manner. Significant differences in compound A compared to the solvent control group were observed starting on Day 3 and persisted until the end of the experiment (Day 5).

[0717] The clinical score curves for each animal in each group were analyzed and the area under the curve (AUC) was calculated. The inhibition rate of each treatment group relative to the solvent control group was calculated by averaging the intergroup AUCs. The results are shown in Figures 8A and 8B. Compound A exhibited inhibition rates of 19.2% and 26.9% at doses of 10 mg / kg and 100 mg / kg, respectively (p<0.0001). The inhibition rates of the active drug calcitriol and dexamethasone groups were 44.5% and 20.7%, respectively (p<0.0001).

[0718] Right ear thickness

[0719] Figure 8C shows that on day 5, compound A had an inhibitory effect on the ear thickness difference Δear thickness (ear thickness value on the day of administration - ear thickness value before group administration) at doses of 10 mg / kg and 100 mg / kg, and the inhibitory effect of compound A on the ear thickness difference Δear thickness at 100 mg / kg had a significant statistical difference (p<0.0001).

[0720] Test conclusion

[0721] Based on the above-mentioned biological activity test experimental data, it can be found that the compound of the present invention can be used as an IRAK4 degrader to treat rheumatoid arthritis, dermatomyositis, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

Claims

1. An IRAK4 degrader, and / or its stereoisomers, enantiomers, diastereomers, deuterated forms, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts, for use in treating and / or preventing immune and / or inflammatory diseases.

2. IRAK4 degraders, and / or their stereoisomers, enantiomers, diastereomers, deuterated forms, hydrates, solvates, prodrugs and / or pharmaceutically acceptable salts, for use in 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.

3. The use according to claim 1 or 2, wherein the IRAK4 degrading agent is a compound represented by Formula I or Formula II or a pharmaceutically acceptable salt thereof: in: Ring A is phenyl or pyridyl; Ring B is C6-C 10 Cycloalkyl or 6-10 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, the C6-C 10 Cycloalkyl and 6-10 membered heterocycloalkyl 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 Cycloalkyl or 6-12 membered heterocycloalkyl containing 1-2 heteroatoms selected from N, O or S, the C6-C 12 Cycloalkyl and 6-12 membered heterocycloalkyl are optionally substituted with a substituent selected from halogen, cyano, amino, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl or -O-(C1-C6 alkyl); Ring D is C6-C 10 Aryl, the C6-C 10 The aryl group is optionally substituted with a halogen, cyano, amino, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl or -O-(C1-C6 alkyl) substituent; Ring Y is a 5-6 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 -, the -(CH2) j -One or more methylene groups are optionally selected from -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’ -, vinylene or ethynylene group replacement; R 1’ 、R 2’ Each is independently halogen, -OH, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, -O(C1-C4 alkyl) or -NH(C1-C4 alkyl); R 3’ 、R 4’ Each is independently hydrogen or C1-C6 alkyl; R d Each is independently hydrogen, deuterium, halogen, cyano, C1-C6 alkyl, wherein the alkyl is optionally substituted by one or more groups selected from halogen, hydroxyl, and amino; R c -O(C1-C3 alkyl), -N(C1-C3 alkyl) 1-2 or C1-C6 alkyl, which is optionally substituted by one or more groups independently selected from hydroxy, amino, halogen, cyano or -O-(C1-C3 alkyl); R b is hydrogen or C1-C6 alkyl, which is optionally substituted by one or more groups independently selected from hydroxy, amino, halogen, and cyano; R a is hydrogen, halogen, C1-C6 alkyl or -O-(C1-C6 alkyl), wherein the alkyl group is optionally substituted with halogen or hydroxy; Each R1 is independently selected from: C1-C4 alkyl, -O(C1-C4 alkyl), -N(C1-C4 alkyl) 1-2 , CN, halogen, -OH, -NH2, the C1-C4 alkyl group is optionally substituted by a group selected from halogen, cyano, -OH, C1-C4 alkyl, -O(C1-C4 alkyl); Each R2 is independently hydrogen, C1-C4 alkyl, -O(C1-C4 alkyl), C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, C6-C 10 Aryl, 5-6 membered heteroaryl, CN, halogen, -OH, the C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, C6-C 10 Aryl, 5-6 membered heteroaryl are optionally selected from halogen, cyano, -OH, -NH2, C1-C4 alkyl 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.

4. The use according to claim 3, wherein the IRAK4 degrading agent is a compound represented by Formula I-1, I-2 or II-1 or a pharmaceutically acceptable salt thereof: in: R3 is H, halogen, C1-C6 alkyl or -O-(C1-C6 alkyl); R c 、R d , Ring B, L, Ring C, X, X', p, R2 and m are as defined and described in claim 3.

5. The use according to claim 3 or 4, characterized in that The IRAK4 degrading agent is a compound selected from A1-A52, B1-B5 and C1-C36: or a pharmaceutically acceptable salt, deuterate, solvate or prodrug thereof.

6. The use according to claim 1, wherein Use of the IRAK4 degrader for treating and / or preventing immune diseases.

7. The use according to claim 6, characterized in that The immune disease is selected from the group consisting of: adult Still's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet'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, endometriosis, eosinophilic fasciitis, erythema nodosum, fibrosing alveolitis, primary glomerulonephritis, Goodpasture's syndrome, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), suppurative Hidradenitis (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, palliative rheumatism (PR), pemphigus, autoimmune encephalomyelitis, POEMS syndrome, polyarteritis nodosa, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, Sarcoidosis, autoimmune scleritis, morphea, Sjögren's syndrome, Takayasu arteritis, 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, spondyloarthropathies, crystalline arthropathy, osteoarthritis, rheumatoid arthritis, polymyositis, interstitial lung disease, giant cell arteritis, polymyalgia rheumatica, granulomatous hematoma vasculitis, eosinophilic granulomatosis with polyangiitis, 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, nonalcoholic fatty liver disease, renal fibrosis, type 1 diabetes mellitus, primary sicca, Kawasaki disease, pustular psoriasis, chronic granulomatous disease, neuromyelitis optica urticaria, palmoplantar pustulosis, sepsis, bullous dermatoses, Alzheimer's disease, chronic spontaneous urticaria, chronic psoriasis, juvenile atopic arthritis, axial spondyloarthritis, or Graves' disease.

8. The use according to claim 7, characterized in that The immune disease is selected from the group consisting of rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa (HS), vitiligo, dermatomyositis, alopecia areata, urticaria, polymyositis, interstitial lung disease, systemic lupus erythematosus, systemic sclerosis and / or psoriasis.

9. The use according to claim 1, characterized in that Use of the IRAK4 degrader for treating and / or preventing inflammatory diseases.

10. The use according to claim 9, characterized in that The inflammatory disease is selected from the group consisting of 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), 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.

11. The use according to claim 2, characterized in that The IRAK4 degrader 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.

12. The use according to claim 11, characterized in that The IRAK4 degrader 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.

13. The use according to any one of claims 1 to 12, characterized in that The sample of the disease is a spleen, skin and / or blood sample.

14. The use according to claim 13, characterized in that The blood sample is normal human whole blood and / or patient whole blood.

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

16. The method of claim 15, wherein the IRAK4 degrader is the single compound of claim 5 or is used in combination with other drugs.

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