Compounds for use in the treatment of disease and pharmaceutical compositions thereof

Abstract

This invention discloses compounds of formula I, wherein X1, X2, and R 1 and R 2 As defined herein, this invention relates to compounds for the prevention and / or treatment of the following diseases, methods of their preparation, pharmaceutical compositions comprising them, and methods of treatment using them: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis, wherein the methods are carried out by administering the compounds of the invention.

Description

Technical Field

[0001] This invention relates to compounds, methods for preparing the compounds of this invention, pharmaceutical compositions comprising the compounds of this invention, and uses and methods for preventing and / or treating the following diseases by administering the compounds of this invention: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis. In particular, the compounds of this invention can inhibit salt-inducible kinases (“SIK” kinases). Background Technology

[0002] Protein kinases belong to a large family of structure-associated enzymes responsible for controlling a variety of cellular signal transduction processes. In particular, they have been shown to be key regulators of cellular functions, including, for example, proliferation, metabolism, and apoptosis. Therefore, defective control of protein phosphorylation leading to uncontrolled signal transduction is involved in many diseases, including, for example, inflammation, allergies, cancer, autoimmune diseases, CNS disorders, and angiogenesis.

[0003] In healthy individuals, inflammation is self-limiting and is controlled by the release of anti-inflammatory mediators and cytokines (such as interleukin-10 (IL-10)) produced by “inhibitory” or “regulatory” cells as part of a negative feedback loop.

[0004] In the inflammatory process, the initial pro-inflammatory response is followed by a pro-remission response, which shuts down inflammation after the damage has subsided. This leads to a decrease in pro-inflammatory cytokines such as TNFα and IL-12, accompanied by an increase in the levels of anti-inflammatory cytokines such as IL-10 and TGF-β, resulting in a so-called tolerance-inducing environment.

[0005] Salt-inducible kinases (SIKs) are a family of serine / threonine kinases that are widely expressed in vivo and are particularly involved in cellular energy homeostasis. Three SIK isoforms have been identified and named SIK1 (also known as SNF1-like kinase (SNF1LK) or cardiac SNF1-like kinase (MSK)), SIK2 (SNF1LK2 or KIAA0781), and SIK3 (KIAA0999) (Katoh et al., 2004).

[0006] SIKs play numerous roles in different cell types. They have been found to phosphorylate many substrates, including CREB-responsive transcriptional coactivator (CRTC) and histone deacetylase (HDAC) proteins, thereby regulating the transcription of many different genes. HDAC and CRTC proteins control the phenotype of activated macrophages by reducing the secretion of pro-inflammatory cytokines (TNFα and IL-12) and increasing the production of anti-inflammatory cytokines such as IL-10 (Clark et al., 2012; Ozanne et al., 2015).

[0007] The regulation of ALK5 by SIK1 (Yu et al., 2013), the identification of SIK2 as a risk site in primary sclerosing cholangitis (Liu et al., 2013), and the regulation of SIK gene expression by TGF-β-induced factors involved in fibrotic pathology, such as plasminogen activator inhibitor 1 (PAI-1) (Hutchinson et al., 2020) suggest a role for SIK proteins in fibrotic diseases. Recently, SIK2 and SIK3 have been identified to function in inflammation by secreting high levels of anti-inflammatory cytokines (particularly IL-10) and very low levels of pro-inflammatory cytokines (e.g., TNFα) when their kinase activity is inhibited (Darling et al., 2017; Darling et al., 2021).

[0008] The role of SIK2 in T helper (TH)1 cell differentiation has been described through the regulation of IFNγ and IL-12 signaling, suggesting that SIK2 may be an interesting target for inflammatory diseases (Yao et al., 2013).

[0009] SIK1 has recently been shown to be involved in skeletal muscle sensitivity in obese mice and may be an interesting target for the prevention of type 2 diabetes (Nixon et al., 2016) and diabetic nephropathy (Hsu et al., 2020).

[0010] Recently, it has also been shown that, similar to PTH (parathyroid hormone), small molecule SIK inhibitors induce decreased phosphorylation of HDAC4 / 5 and CRTC2 and increased nuclear translocation. Treatment with the small molecule SIK inhibitor YKL-05-099 increased bone formation and bone quality in mice (Wein et al., 2016), confirming the relevance of SIK inhibition in the treatment of bone turnover diseases (Nishimori et al., 2019; Sato et al., 2022).

[0011] In mouse and human embryonic stem cell-derived kidney organoids, inducible knockout or mutation of SIK isoforms and pharmacological inhibition lead to the induction of Cyp27b1 expression and 1,25-vitamin D synthesis. Therefore, SIK isoforms, particularly SIK2 and SIK3, regulate renal 1,25-vitamin D production downstream of PTH (Yoon et al., 2023), which could be beneficial for the treatment of chronic kidney disease-mineral bone disorder (CKD-MBD).

[0012] Furthermore, inhibition of SIK2 after oxygen-glucose deprivation has been shown to enhance neuronal survival (Sasaki et al., 2011) or promote melanin production in melanoma cells (Kumagai et al., 2011). In this paper, the rapid inhibition or degradation of SIK proteins after various stressors, such as after tissue ischemia and reperfusion, in the chronic phase of cardiac remodeling, in diabetes and neurodegenerative diseases, makes it an interesting target in inflammatory, cardiac or metabolic diseases and neurodegenerative diseases. SIK inhibition could also be applied to cosmetic or pigmentation-related diseases to induce melanin production (Mujahid et al., 2017).

[0013] In addition to their crucial role in cellular energy homeostasis, SIK proteins are also involved in cell cycle regulation. Higher expression of SIK2 is significantly associated with poorer survival in patients with advanced serous ovarian cancer (Ahmed et al., 2010). Furthermore, SIK3 expression is elevated in ovarian cancer, particularly in the serous subtype and at later stages (Charoenfuprasert et al., 2011). Therefore, SIK inhibition could be used for cancer treatment.

[0014] Despite significant progress over the past two decades in antibody therapy targeting pro-inflammatory cytokines (e.g., anti-TNFα) for patients affected by autoimmune diseases, a substantial proportion of patients do not respond to these therapies or experience serious adverse events (e.g., opportunistic infections). Therefore, a substantial unmet medical need remains for the treatment of these diseases, and new agents are required for the prevention and / or treatment of these conditions.

[0015] sequence list

[0016] SEQ ID 1: AMARA peptide (Kaneca Eurogentec SA).

[0017] SEQ ID 2: SIK3 FL Gst-TEV-HsSIK3 (Uniprot# Q9Y2K2-5, amino acid 59-1321)-Thr-6His protein. Summary of the Invention

[0018] This invention is based on the identification of novel compounds and their use in the prevention and / or treatment of the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis. In particular, the compounds of this invention can be SIK inhibitors, more particularly SIK2 and / or SIK3 inhibitors. The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising these compounds, and methods for preventing and / or treating the following diseases by administering the compounds of the present invention: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis.

[0019] Therefore, in a first aspect of the invention, compounds of the invention having Formula I are provided: I in One of X1 and X2 is N and the other is C; R 1 It is H or -P(=O)(OH)2; R 2 yes - H, - Optionally replace C with one or more independently selected halogens. 1-4 alkyl, - -NR 3a R 3b ;and R 3a and R 3b C, each independently selected from H, optionally replacing one or more independently selected halogens. 1-4 Alkyl, or phenyl.

[0020] Therefore, in a first aspect of the invention, compounds of the invention having Formula I are provided: I in One of X1 and X2 is N and the other is C; R 1 It is H or -P(=O)(OH)2; and R 2 Is it H or C? 1-4 alkyl.

[0021] In one specific aspect, compounds of the present invention are provided for the prevention and / or treatment of the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis.

[0022] Furthermore, the compounds of the present invention exhibit potent inhibition of SIKs, particularly SIK2 and SIK3, with a greater emphasis on SIK3, which can lead to tolerance to treatment (i.e., a reduction in pro-inflammatory cytokines such as TNFα and IL-12, accompanied by an increase in the levels of anti-inflammatory cytokines such as IL-10 and TGF-β). In particular, the compounds of the present invention exhibit enhanced inhibition of SIK3 with improved selectivity relative to SIK1 and SIK2.

[0023] In addition, the compounds of the present invention can exhibit low propensity for human pregnane X receptor (PXR, NR1I2) activation, and thus exhibit more stable metabolic characteristics and a reduced risk of harmful drug-drug interactions.

[0024] Certain compounds of the present invention may also exhibit one or more benefits, particularly including favorable levels of bioactivity for the prevention and / or treatment of one or more diseases, improved safety characteristics (e.g., involving hERG inhibition, drug-drug interaction (DDI) or CYP-interaction characteristics, etc.), improved selectivity for one or more disease-related biological targets (e.g., reduced off-target effects, etc.), improved pharmacokinetic properties (e.g., involving administration, solubility, absorption, etc.), improved pharmacodynamic properties (e.g., involving permeability, efflux, etc.), or excellent properties (e.g., stability) for use alone as a pharmaceutical active ingredient or in a pharmaceutical composition, or favorable physicochemical properties for the manufacture of the aforementioned pharmaceutical compositions.

[0025] In another aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutical carrier, excipient, or diluent. In one specific aspect, the pharmaceutical composition may additionally comprise other therapeutically active ingredients suitable for use in combination with the compounds of the present invention. More specifically, the other therapeutically active ingredients are agents for treating the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis.

[0026] Furthermore, the compounds of the present invention that can be used in the pharmaceutical compositions and treatments disclosed herein are pharmaceutically acceptable in their preparation and use.

[0027] In another aspect of the invention, the invention provides a method for treating mammals, particularly humans, suffering from diseases selected from those listed herein, said diseases being particularly: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis, said method comprising administering an effective amount of the pharmaceutical composition or compound of the invention as described herein.

[0028] The present invention also provides pharmaceutical compositions comprising the compounds of the present invention and suitable pharmaceutical carriers, excipients, or diluents for use as a medicament. In one particular aspect, the pharmaceutical compositions are intended for the prevention and / or treatment of the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis.

[0029] In another aspect, the present invention provides a method for synthesizing the compounds of the present invention, having representative synthetic schemes and routes disclosed later herein.

[0030] Other objects and advantages will become apparent to those skilled in the art upon consideration of the following detailed description.

[0031] It should be understood that the compounds of the present invention can be metabolized to produce bioactive metabolites. Detailed Implementation

[0032] definition

[0033] The following terms are intended to have the meanings presented below and are used to understand the description and intended scope of the invention.

[0034] When describing the present invention (which may include compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds and compositions), unless otherwise specified, the following terms (if present) shall have the following meanings. It should also be understood that, as described herein, any portion defined below may be substituted with various substituents, and the respective definitions are intended to include such substituted portions within their scope, as listed below. Unless otherwise stated, the term “substituted” will be defined as described below. It should also be understood that, as used herein, the terms “group” and “radical” may be considered interchangeable.

[0035] The articles “a kind” and “an” can be used in this text to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “analogy” refers to one or more analogies.

[0036] "Alkyl" refers to a straight-chain or branched aliphatic hydrocarbon group having a specified number of carbon atoms. Specific alkyl groups have 1 to 6 carbon atoms or 1 to 4 carbon atoms. Branching refers to one or more alkyl groups, such as methyl, ethyl, or propyl, attached to a straight-chain alkyl chain. Specific alkyl groups are methyl (-CH3), ethyl (-CH2-CH3), n-propyl (-CH2-CH2-CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2-CH2-CH2-CH3), tert-butyl (-C(CH3)3), sec-butyl (-CH(CH3)-CH2CH3), isobutyl (-CH2-CH(CH3)2), n-pentyl (-CH2-CH2-CH2-CH2-CH3), n-hexyl (-CH2-CH2-CH2-CH2-CH2-CH3), and 1,2-dimethylbutyl (-CH(CH3)-CH(CH3)-CH2-CH3). The particular alkyl group has 1 to 4 carbon atoms.

[0037] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents.

[0038] As used herein, the term "substituted by one or more substituents" refers to 1 to 4 substituents. More specifically, it refers to 1 to 3 substituents. More specifically, it refers to 1 or 2 substituents. Most specifically, it refers to 1 substituent.

[0039] "Pharmaceutical acceptable" means approved or permitted by a federal or state government regulatory agency or by the corresponding agency in a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia, for use in animals, and more specifically, for use in humans.

[0040] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and retains the biological activity of the given compound, and is not biologically or otherwise undesirable. In particular, such salts can be inorganic or organic acid addition salts and base addition salts. For example, pharmaceutically acceptable salts are described in the Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Stahl & Wermuth 2011). These salts can be prepared in situ or separately during the final isolation and purification of the compounds described herein, for example, by reacting a free base group with a suitable inorganic or organic acid. The compounds of the present invention can have ionizable groups to enable their preparation as pharmaceutically acceptable salts. These salts can be acid addition salts involving inorganic or organic acids, or, in the case of the acidic form of the compounds of the present invention, the salts can be prepared from inorganic or organic bases. Typically, compounds are prepared or used in the form of pharmaceutically acceptable salts, which are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric acid for forming acid addition salts, and sodium hydroxide for forming basic salts. The term "pharmaceutically acceptable cation" refers to an acceptable cationic counterion with an acidic functional group. Examples of such cations include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, etc.

[0041] "Pharmaceutically acceptable carrier" means a diluent, adjuvant, excipient or carrier that is administered together with the compounds of the present invention.

[0042] "Prodrug" refers to a compound of the present invention that has a cleavable group and is converted into a drug-active compound in vivo by solvent decomposition or under physiological conditions, including derivatives of the present invention. Examples of such compounds include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, etc.

[0043] "Solventrolyte" refers to a compound that typically associates with a solvent via a solvent decomposition reaction. This physical association includes hydrogen bonding. Common solvents include water, EtOH, acetic acid, etc. The compounds of this invention can be prepared, for example, in crystalline form and can be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and also include stoichiometric and non-stoichiometric solvates. In some cases, solvates will be separable, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solventrolyte" includes solution phases and separable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0044] "Subject" includes people. The terms "person," "patient," and "subject" are used interchangeably in this document.

[0045] "Effective amount" refers to the amount of the compound of the present invention sufficient to achieve such treatment of the disease when administered to a subject. "Effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0046] "Prevention" or "avoidance" means reducing the risk of acquiring or developing a disease or condition (i.e., preventing the development of at least one clinical symptom of the disease in a subject who may have been exposed to the causative agent or who was susceptible to the disease before the onset of the disease).

[0047] The term “prophylaxis” is related to “prevention” and refers to measures or procedures whose purpose is to prevent rather than treat or cure a disease. Non-limiting examples of preventive measures may include administering vaccines; administering low molecular weight heparin to hospitalized patients at risk of thrombosis (e.g., due to fixation); and administering antimalarial agents, such as chloroquine, before visiting geographically prevalent or high-risk areas for malaria infection.

[0048] In one embodiment, “treating” or “treatment” for any disease or condition means improving the disease or condition (i.e., stopping the disease or reducing the manifestation, extent, or severity of at least one of its clinical symptoms). In another embodiment, “treatment” means improving at least one bodily parameter that the subject may not be able to discern. In yet another embodiment, “treatment” means physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing bodily parameters), or physically and physiologically modulating the disease or condition. In yet another embodiment, “treatment” involves slowing the progression of the disease.

[0049] As used herein, the term "inflammatory disease" refers to a group of conditions including rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway diseases (e.g., asthma, rhinitis), chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), endotoxin-driven disease states (e.g., complications after bypass surgery, acute kidney injury (AKI), Allport syndrome, or chronic endotoxin states leading to, for example, chronic heart failure), and related diseases involving cartilage, such as joint diseases. Specifically, the term refers to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel disease.

[0050] The term “autoinflammatory disease” as used in this article refers to a group of diseases including cryopyrin-associated periodic syndromes (CAPS), familial Mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic syndromes (TRAPS), Behçet's disease, systemic juvenile idiopathic arthritis (SJIA), or Still's disease.

[0051] As used in this article, the term "autoimmune disease" refers to a group of diseases, including obstructive airway diseases such as COPD, asthma (e.g., intrinsic asthma, extrinsic asthma, dust asthma, infantile asthma), especially chronic or refractory asthma (e.g., advanced asthma and airway hyperresponsiveness), bronchitis including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, and autoimmune liver diseases (e.g., autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis). Sjögren's syndrome, multiple sclerosis, psoriasis, dry eye disease, type 1 diabetes and its associated complications, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto's and autoimmune thyroiditis), pemphigus vulgaris, contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis, type 1 diabetic nephropathy, anti-GBM (Goodpassuia disease), IgA nephropathy (Bergberg's disease), focal segmental glomerulosclerosis or thin basement membrane disease, and amyotrophic lateral sclerosis. Specifically, the term refers to COPD, asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, dry eye disease, and inflammatory bowel disease.

[0052] As used herein, the term "proliferative disorder" refers to conditions such as cancer (e.g., uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (e.g., polycythemia vera, idiopathic thrombocythemia, and myelofibrosis), leukemia (e.g., acute myeloid leukemia, acute and chronic lymphocytic leukemia), multiple myeloma, psoriasis, restenosis, scleroderma, or fibrosis. Specifically, the term refers to psoriasis, scleroderma, and fibrosis.

[0053] As used herein, the term "cancer" refers to the malignant or benign growth of cells in the skin or a body organ, such as, but not limited to, the breast, prostate, lung, kidney, pancreas, stomach, or intestine. Cancer tends to infiltrate adjacent tissues and spread (metastasize) to distant organs, such as bone, liver, lung, or brain. The term cancer as used herein includes metastatic tumor cell types (e.g., but not limited to melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mast cell tumor) and tissue cancer types (e.g., but not limited to colorectal cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, stomach cancer, glioblastoma, primary liver cancer, ovarian cancer, and uterine leiomyosarcoma). Specifically, the term "cancer" refers to acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumors, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brainstem glioma, brain tumors, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, and uterine cancer. Endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma family tumors, ocular cancer, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gastrointestinal stromal cell tumors, germ cell tumors, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, skin Skin T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoma, Waldenström macroglobulinemia, medulloblastoma, medullary epithelioma, melanoma, mesothelioma, oral cancer, myeloid leukemia, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, low-grade malignant potential ovarian tumors, pancreatic cancer, papilloma, parathyroid cancer, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumors, pineal blastoma, and supratentorial primitive neuroectodermal tumors. Pituitary adenoma, plasma cell tumor / multiple myeloma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing sarcoma family tumors, sarcoma, Cezari syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer (stomach cancer), supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0054] As used herein, the term "leukemia" refers to a neoplastic disease of the blood and blood-forming organs. These diseases can cause dysfunction of the bone marrow and immune system, making the host highly susceptible to infection and bleeding. Specifically, the term leukemia refers to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphoblastic leukemia (CLL).

[0055] As used herein, the term "fibrotic disease" refers to a disease characterized by excessive scarring due to the overproduction, deposition, and contraction of the extracellular matrix, and associated with abnormal accumulation of cells and / or fibronectin and / or collagen and / or increased recruitment of fibroblasts, and includes, but is not limited to, fibrosis of individual organs or tissues such as the heart, kidneys, liver, joints, lungs, pleural tissue, peritoneum, skin, cornea, retina, musculoskeletal system, and digestive tract. Specifically, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF); cystic fibrosis; other diffuse solid lung diseases of various etiologies, including iatrogenic drug-induced fibrosis, occupational and / or environmentally induced fibrosis, granulomatous diseases (sarcoidosis, allergic pneumonia), collagen vascular diseases, pulmonary alveolar proteinosis, Langerhans cell granulomatosis, lymphangioleiomyomatosis, and hereditary diseases (Hermansky-Pudlak syndrome). (syndrome), tuberous sclerosis, neurofibromatosis, metabolic storage disease, familial interstitial lung disease; radiation-induced fibrosis; chronic obstructive pulmonary disease (COPD); scleroderma; bleomycin-induced pulmonary fibrosis; chronic asthma; silicosis; asbestos-induced pulmonary fibrosis; acute respiratory distress syndrome (ARDS); renal fibrosis, autosomal dominant polycystic kidney disease, tubulointerstitial fibrosis; glomerulonephritis; diabetic nephropathy, focal segmental glomerulosclerosis; IgA nephropathy; hypertension; Allport syndrome; intestinal fibrosis; liver fibrosis; cirrhosis; ethanol-induced liver fibrosis; toxic / drug-induced liver fibrosis; hemochromatosis; non-alcoholic steatohepatitis (NASH); biliary tract injury; primary biliary cirrhosis; infection-induced liver fibrosis; virus-induced liver fibrosis; and autoimmune hepatitis; corneal scarring; hypertrophic scarring; Dupuytren's disease Diseases including keloids, cutaneous fibrosis; scleroderma; systemic sclerosis, spinal cord injury / fibrosis; myelofibrosis; musculoskeletal fibrosis associated with Duchenne muscular dystrophy, restenosis; atherosclerosis; arteriosclerosis; Wegener's granulomatosis; Peronni's disease, or chronic lymphocytic leukemia. More specifically, the term "fibrotic disease" refers to idiopathic pulmonary fibrosis (IPF), radiation-induced fibrosis, non-alcoholic steatohepatitis (NASH), scleroderma, bleomycin-induced pulmonary fibrosis, cutaneous scleroderma, renal fibrosis, and systemic sclerosis.

[0056] As used herein, the term "transplant rejection" refers to acute or chronic rejection, or graft-versus-host disease (GvHD), of allogeneic or xenografts of cells, tissues, or solid organs, such as islets of Langerhans, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary junction, kidney, liver, intestine, pancreas, trachea, or esophagus. More specifically, the term refers to graft-versus-host disease (GvHD).

[0057] The term “diseases involving impaired cartilage transformation” as used in this article includes conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Tietze syndrome or costochondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis such as endemic degenerative osteoarthritis, Mseleni joint disease, and Handigodu disease; and degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

[0058] As used herein, the term “congenital chondrodysplasia” includes conditions such as hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, and especially, but not limited to, microtia, auria, metaphyseal dysplasia and related conditions.

[0059] The term “diseases involving impaired bone turnover” as used in this article includes conditions such as osteoporosis (including postmenopausal osteoporosis, male osteoporosis, glucocorticoid-induced osteoporosis, and juvenile osteoporosis), osteoporosis caused by neoplastic myelopathy, osteopenia, hormone deficiencies (vitamin D deficiency, male and female hypogonadism), hormone excesses (hyperprolactinemia, excessive glucocorticoids, hyperthyroidism, hyperparathyroidism), Paget's disease, osteoarthritis, renal bone disease, osteogenesis imperfecta, hypophosphodiesterase syndrome, and chronic kidney disease-mineralized bone disease (CKD-MBD).

[0060] As used in this article, the term “diseases associated with excessive IL-6 secretion” includes conditions such as Castleman’s disease, multiple myeloma, psoriasis, Kaposi’s sarcoma, and / or mesangial proliferative glomerulonephritis.

[0061] The term “diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23” as used in this article includes conditions such as systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, spondyloarthritis (especially ankylosing spondylitis) and / or Crohn's disease.

[0062] As used herein, the term "respiratory disease" refers to a disease affecting organs involved in breathing, such as the nose, pharynx, larynx, Eustachian tubes, trachea, bronchi, lungs, related muscles (e.g., diaphragm and intercostal spaces), and nerves. In particular, examples of respiratory diseases include asthma, adult respiratory distress syndrome and allergic (exogenous) asthma, non-allergic (endogenous) asthma, acute severe asthma, chronic asthma, clinical asthma, nocturnal asthma, allergen-induced asthma, aspirin-sensitive asthma, exercise-induced asthma, isovolemic hyperventilation, childhood paroxysmal asthma, adult paroxysmal asthma, cough-variant asthma, occupational asthma, steroid-resistant asthma, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease (COPD), including chronic bronchitis or emphysema, pulmonary hypertension, interstitial pulmonary fibrosis and / or airway inflammation, cystic fibrosis, and hypoxia.

[0063] As used herein, the term "endocrine disorder" refers to a condition of the endocrine system and hormone secretion. In particular, the term refers to hypothyroidism, congenital adrenal hyperplasia, parathyroid disorders, diabetes, adrenal disorders (including Cushing's syndrome and Addison's disease), and ovarian dysfunction (including polycystic ovary syndrome).

[0064] As used herein, the term "metabolic disease" refers to a condition that disrupts normal metabolism, the process of converting food into energy at the cellular level. Metabolic diseases affect the ability to perform key biochemical reactions involving the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Specifically, the term refers to cystic fibrosis, phenylketonuria (PKU), diabetes, hyperlipidemia, gout, type 2 diabetic nephropathy, and rickets. More specifically, the term refers to obesity and / or type 2 diabetes.

[0065] As used herein, the term "cardiovascular disease" refers to a disease affecting the heart or blood vessels, or both. Specifically, cardiovascular disease includes arrhythmias (atrial or ventricular, or both); atherosclerosis and its sequelae; angina; cardiac rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysms; vasculitis (e.g., giant cell arteritis (GCA), retinal vasculitis, rheumatoid vasculitis); stroke; peripheral obstructive arterial disease of the limbs, organs, or tissues; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxic shock, surgical shock, or traumatic shock; hypertension, valvular heart disease, heart failure, blood pressure abnormalities; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, inflammation, or dysfunction limited to a single organ or tissue. More specifically, cardiovascular disease refers to atherosclerosis or giant cell arteritis.

[0066] As used herein, the term "skin disease" refers to a skin condition. Specifically, dermatological conditions include proliferative or inflammatory conditions of the skin, such as atopic dermatitis, bullous conditions, collagen dermatitis, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, vitiligo, pruritus, scleroderma, wound healing, scarring, hypertrophic scarring, keloids, Kawasaki disease, rosacea, Sjögren-Larsson syndrome, or urticaria. More specifically, the term dermatological conditions refers to psoriasis, psoriatic lesions, scleroderma, and vitiligo.

[0067] As used herein, the term "abnormal angiogenesis-related diseases" refers to diseases caused by dysregulation of the processes mediating angiogenesis. Specifically, abnormal angiogenesis-related diseases include atherosclerosis, hypertension, tumor growth, inflammation, rheumatoid arthritis, wet macular degeneration, choroidal neovascularization, retinal neovascularization, and diabetic retinopathy.

[0068] "Compounds of the present invention" and equivalent expressions are intended to include compounds of the general formula described herein, including pharmaceutically acceptable salts and solvates, such as hydrates and solvates of pharmaceutically acceptable salts, where the context permits. Similarly, references to intermediates, whether or not they are claimed in themselves, are intended to include their salts and solvates, where the context permits.

[0069] When referring to scope in this article, such as, but not limited to, C 1-8 Alkyl groups, and the reference to the range should be regarded as representative of each member of the range.

[0070] Other derivatives of the compounds of this invention are active in both their acidic and acid-derived forms, but in their acid-sensitive forms they generally offer advantages such as solubility, tissue compatibility, or delayed release in mammalian organisms (Bundgaard 1985). Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting a parent acid with a suitable alcohol, or amides, anhydrides, or mixed anhydrides prepared by reacting a parent acid compound with a substituted or unsubstituted amine. Simple aliphatic or aromatic esters, amides, and anhydrides derived from the acidic groups side-attached to the compounds of this invention are particularly useful prodrugs. In some cases, diester-type prodrugs are required, such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters. A particular type of such prodrug is the C3 group of the compounds of this invention. 1-8 Alkyl, C 2-8 alkenyl, C 6-10 Optionally substituted aryl and (C 6-10 aryl)-(C 1-4 Alkyl ester.

[0071] This disclosure includes all isotopic forms of the compounds of the invention provided herein, whether in form (i) where all atoms of a given atomic number have a mass number (or a mixture of mass numbers) that is dominant in nature (referred to herein as "natural isotopic form") or (ii) where one or more atoms are replaced by atoms having the same atomic number but a mass number different from that of the dominant atom in nature (referred to herein as "non-natural variant isotopic form"). It should be understood that atoms can exist naturally as a mixture of mass numbers. The term "non-natural variant isotopic form" also includes embodiments in which the proportion of atoms of a given atomic number having a less common mass number found in nature (referred to herein as "uncommon isotopes") has been increased relative to naturally occurring atoms, for example, to a level of >20%, >50%, >75%, >90%, >95%, or >99% based on the number of atoms of that atomic number (the latter embodiment is referred to as "isotope-enriched variant form"). The term "non-natural variant isotopic form" also includes embodiments in which the proportion of uncommon isotopes is reduced relative to naturally occurring proportions. Isotopic forms can include radioactive forms (i.e., they are doped with radioactive isotopes) and non-radioactive forms. Radioactive forms are typically isotope-enriched variants.

[0072] Therefore, the non-natural variant isotopic form of a compound can contain one or more artificial or uncommon isotopes in one or more atoms, such as deuterium ( 2 H or D), carbon-11 ( 11 C), Carbon-13 ( 13 C), Carbon-14 ( 14 C) Nitrogen-13 ( 13 N), nitrogen-15 ( 15 N), Oxygen-15 ( 15 O), Oxygen-17 ( 17 O), Oxygen-18 ( 18 O), Phosphorus-32 ( 32 P), sulfur-35 ( 35 S), Chlorine-36 ( 36 Cl), Chlorine-37 ( 37 Cl), Fluorine-18 ( 18 F), Iodine-123 ( 123 I), iodine-125( 125 I) Or, compared to the proportion of the isotopes that are dominant in nature in one or more atoms, the isotopes may be contained in an increased proportion.

[0073] Non-natural variants of radioactive isotopes can be used, for example, for studies of drug and / or substrate tissue distribution. Radioactive isotope tritium (i.e., 3H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and ease of detection. Incorporation of deuterium (i.e.,...) 2 Non-natural variant isotopic forms of H or D can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and are therefore preferred in some cases. Furthermore, incorporation of positron-emitting isotopes (e.g., H or D) can be prepared. 11 C 18 F, 15 O and 13 The non-natural variant isotope form of N) will be used in positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0074] It should also be understood that compounds with the same molecular formula but different properties, different atomic bonding sequences, or different spatial arrangements of atoms are called "isomers". Isomers with different spatial arrangements of atoms are called "stereoisomers".

[0075] Stereoisomers that are not mirror images of each other are called "diastereomers," and those that are non-overlapping mirror images of each other are called "enantiomers." When a compound has an asymmetric center, for example, when it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric center and described by the R- and S-ordering rules of Cahn, Ingold, and Prelog, or by the way the molecule rotates the plane of polarization, and are designated as dextrorotatory or levorotatory (i.e., (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture."

[0076] "Tautomerism" refers to compounds whose structures are interchangeable, with different substitutions of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they rapidly interconvert upon treatment with an acid or base. Another example of tautomerism is the similar formation of the acid and nitro forms of phenylnitromethane through treatment with an acid or base.

[0077] Tautomerism may be related to the realization of optimal chemical reactivity and biological activity of the compound of interest.

[0078] The compounds of the present invention may have one or more asymmetric centers; therefore, such compounds may be prepared as individual (R)- or (S)- stereoisomers or as mixtures thereof.

[0079] Unless otherwise specified, the description or naming of specific compounds in the specification and claims is intended to include their individual enantiomers and racemic mixtures or other mixtures. Methods for determining stereochemistry and isomerization are well known in the art.

[0080] This invention

[0081] This invention is based on the identification of novel compounds and their use in the prevention and / or treatment of the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis. In particular, the compounds of this invention can be SIK inhibitors, more particularly SIK2 and / or SIK3 inhibitors.

[0082] The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising these compounds, and methods for preventing and / or treating the following diseases by administering the compounds of the present invention: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases, and / or diseases associated with abnormal angiogenesis.

[0083] Therefore, in a first aspect of the invention, compounds of the invention having Formula I are provided: I in One of X1 and X2 is N and the other is C; R 1 It is H or -P(=O)(OH)2; R 2 yes - H, - Optionally replace C with one or more independently selected halogens. 1-4 alkyl, - -NR 3a R 3b ;and R 3a and R 3bEach is independently selected from H, and optionally replaces C with one or more independently selected halogens. 1-4 Alkyl, or phenyl.

[0084] Therefore, in a second aspect of the invention, compounds of the invention having Formula I are provided: I in One of X1 and X2 is N and the other is C; R 1 It is H or -P(=O)(OH)2; and R 2 Is it H or C? 1-4 alkyl.

[0085] In one embodiment, the compound of the present invention is according to Formula II:

[0086] Where X1, X2, R 1 and R 2 As stated above.

[0087] In one embodiment, the compound of the present invention is according to Formula III:

[0088] Where X1, X2, R 1 and R 2 As stated above.

[0089] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein R 2 It is H.

[0090] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein R 2 It is C 1-4 Alkyl group. In one specific embodiment, R 2 It is -CH3, -CH2CH3, or -CH(CH3)2. In a more specific embodiment, R 2 It is -CH3 or -CH2CH3. In another, more specific embodiment, R 2 It is -CH3.

[0091] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein R 2 It is C 1-4 Alkyl, the C 1-4 The alkyl group is substituted with one or more independently selected halogens. In one specific embodiment, R 2It is -CH3, -CH2CH3, or -CH(CH3)2, each substituted by one or more independently selected halogens. In one specific embodiment, R 2 It is -CH3, -CH2CH3, or -CH(CH3)2, each of which is replaced by one or more independently chosen Fs. In a more specific embodiment, R 2 It is -CH3 or -CH2CH3, each of which is replaced by one or more independently chosen Fs. In another, more specific embodiment, R 2 It is -CHF3.

[0092] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein R 2 Yes -NR 3a R 3b , where R 3a and R 3b As previously defined. In one specific implementation, R 2 Yes -NR 3a R 3b And R 3a and R 3b Each is independently selected from H, -CH3, -CH2CH3, and -CH2phenyl. In a more particular embodiment, R 2 It is -NH2, -NHCH3, -NH2, -N(CH3)2, -NH(CH2phenyl) or -NCH3(CH2phenyl).

[0093] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein X1 is N and X2 is C.

[0094] In one embodiment, the compound of the present invention is according to formula I, II or III, wherein X1 is C and X2 is N.

[0095] In one embodiment, the compound of the present invention is according to formula IVa, IVb, IVc or IVd:

[0096] Where R 1 As mentioned above.

[0097] In one embodiment, the compound of the present invention is according to formula Va, Vb, Vc or Vd:

[0098] Where R 1 As mentioned above.

[0099] In one embodiment, the compound of the present invention is any one of formulas I-Vd, wherein R1 It is H.

[0100] In one embodiment, the compound of the present invention is any one of formulas I-Vd, wherein R 1 It is -P(=O)(OH)2.

[0101] In one embodiment, the compounds of the present invention are selected from: 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2S)-3-hydroxy-3-methylbut-2-yl]oxypyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[7-[(2S)-3-hydroxy-3-methylbut-2-yl]oxyimidazo[1,2-b]pyridazin-3-yl]-6-methoxybenzamide, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[7-(3-hydroxy-3-methylbut-2-yl)oxyimidazo[1,2-b]pyridazin-3-yl]-6-methoxybenzamide, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-(3-hydroxy-3-methylbut-2-yl)oxypyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide, [(3S)-3-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxyphenyl]imidazo[1,2-b]pyridazin-7-yl]oxy-2-methylbut-2-yl]dihydrogen phosphate, [(3S)-3-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxyphenyl]pyrazolo[1,5-a]pyrimidin-6-yl]oxy-2-methylbut-2-yl]dihydrogen phosphate, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[7-[(2S)-3-hydroxy-3-methylbut-2-yl]oxy-6-methylimidazo[1,2-b]pyridazin-3-yl]-6-methoxybenzamide, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2R)-3-hydroxy-3-methylbut-2-yl]oxypyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide, [(3S)-3-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxyphenyl]-6-methylimidazo[1,2-b]pyridazin-7-yl]oxy-2-methylbut-2-yl]dihydrogen phosphate, 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2S)-3-hydroxy-3-methylbut-2-yl]oxy-5-methylpyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide, and 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2R)-3-hydroxy-3-methylbut-2-yl]oxy-5-methylpyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide.

[0102] In one embodiment, the compounds of the present invention are selected from:

[0103] Where R or S In This indicates that stereochemistry is arbitrarily specified.

[0104] In one embodiment, the compound of the present invention is 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2S)-3-hydroxy-3-methylbut-2-yl]oxypyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide.

[0105] In one embodiment, the compound of the present invention is not 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[6-[(2S)-3-hydroxy-3-methylbut-2-yl]oxypyrazolo[1,5-a]pyrimidin-3-yl]-6-methoxybenzamide.

[0106] In one embodiment, the compound of the present invention is 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[7-[(2S)-3-hydroxy-3-methylbut-2-yl]oxy-6-methylimidazo[1,2-b]pyridazin-3-yl]-6-methoxybenzamide.

[0107] In one embodiment, the compound of the present invention is not 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-4-[7-[(2S)-3-hydroxy-3-methylbut-2-yl]oxy-6-methylimidazo[1,2-b]pyridazin-3-yl]-6-methoxybenzamide.

[0108] In one embodiment, the compound of the present invention is a compound of formula Va, wherein R 1 It is -P(=O)(OH)2.

[0109] In one embodiment, the compound of the present invention is an amorphous form of a compound according to formula Va, wherein R 1 It is -P(=O)(OH)2.

[0110] In one embodiment, the compound of the present invention is a crystalline compound according to formula Va, wherein R 1 It is -P(=O)(OH)2.

[0111] In one embodiment, the compound of the present invention is a compound of formula Va, wherein R 1 It is -P(=O)(OH)2, wherein the compound is a hydrate. In one specific embodiment, the hydrate is a hemihydrate or a monohydrate. In a more specific embodiment, the hydrate is a crystalline hemihydrate or a monohydrate.

[0112] In one embodiment, the compound of the present invention is a compound according to formula Va, wherein R 1 It is -P(=O)(OH)₂, wherein the compound is a crystalline monohydrate, characterized by an X-ray powder diffraction pattern using Cu Kα radiation containing peaks at 9.1, 10.0, 13.2, 13.6, and 14.9 ± 0.2° 2θ. In one specific embodiment, the X-ray powder diffraction pattern further includes peaks at 15.7, 15.9, 16.6, 17.2, 19.0, 19.3, 20.4, 20.8, 24.2, 24.8, and 26.5 ± 0.2° 2θ.

[0113] In one embodiment, the compound of the present invention is a compound according to formula Va, wherein R 1It is -P(=O)(OH)₂, wherein the compound is a crystalline hemihydrate, characterized by an X-ray powder diffraction pattern using Cu Kα radiation containing peaks at 9.1, 9.6, 15.1, 15.4, and 19.7 ± 0.2° 2θ. In one specific embodiment, the X-ray powder diffraction pattern further includes peaks at 10.6, 12.4, 14.1, 18.1, 18.5, 18.8, 20.0, and 20.3 ± 0.2° 2θ.

[0114] In one embodiment, the compounds of the present invention are provided in the form of natural isotopes.

[0115] In one embodiment, the compounds of the present invention are provided in a non-natural variant isotopic form. In a specific embodiment, the non-natural variant isotopic form is wherein deuterium (i.e., 2 (H or D) is incorporated in the form where hydrogen is specified in the chemical structure of one or more atoms of the compound of the invention. In one embodiment, the atoms of the compound of the invention are in isotopic form, which is not radioactive. In one embodiment, one or more atoms of the compound of the invention are in radioactive isotopic form. Suitable radioactive isotopes are stable isotopes. Suitably, non-natural variant isotopic forms are pharmaceutically acceptable forms.

[0116] In one embodiment, a compound of the present invention is provided, wherein a single atom of said compound is present in a non-natural variant isotopic form. In another embodiment, a compound of the present invention is provided, wherein two or more atoms are present in a non-natural variant isotopic form.

[0117] Non-natural isotopic variants can typically be prepared using conventional techniques known to those skilled in the art or by methods described herein (e.g., methods similar to those described in the appended examples for preparing natural isotopic forms). Therefore, non-natural isotopic variants can be prepared by using appropriate isotopic variant (or labeled) reagents instead of the conventional reagents used in the illustrative examples as examples.

[0118] In one aspect, the compounds of the present invention according to any of the embodiments described herein exist as free bases.

[0119] In one respect, the compounds of the present invention according to any of the embodiments described herein are pharmaceutically acceptable salts.

[0120] In one aspect, the compound of the present invention according to any of the embodiments described herein is a solvate of the compound.

[0121] In one aspect, the compounds of the present invention according to any embodiment described herein are pharmaceutically acceptable salt solvates of compounds.

[0122] While the specified groups in each embodiment are generally listed separately above, the compounds of the present invention include compounds in which several or each of the above formulas and other formulas provided herein are selected from one or more of the respective specified members or groups of the variables. Therefore, the present invention is intended to include all combinations of these embodiments within its scope.

[0123] While specific functional groups for each embodiment are generally listed separately above, the compounds of the present invention may be compounds selected from one or more embodiments according to any of the formulas listed above, including one or more of the variables (e.g., the R group). Therefore, the present invention is intended to include all combinations of variables from any of the disclosed embodiments within its scope.

[0124] Alternatively, the invention also covers excluding one or more specified variables from a group or implementation or a combination thereof.

[0125] In some aspects, the present invention provides prodrugs and derivatives of compounds according to the above formula. A prodrug is a derivative of the compound of the present invention having a metabolically cleavable group and being converted into the compound of the present invention by solvent decomposition or under physiological conditions, having pharmaceutical activity in vivo. Examples of such compounds include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, etc.

[0126] Other derivatives of the compounds of this invention are active in their acidic and acid-derived forms, but acid-sensitive forms generally offer advantages such as solubility, tissue compatibility, or delayed release in mammalian organisms (Bundgaard 1985). Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting a parent acid with a suitable alcohol, or amides, or anhydrides, or mixed anhydrides, prepared by reacting a parent acid compound with a substituted or unsubstituted amine. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups side-attached to the compounds of this invention are preferred prodrugs. In some cases, diester-type prodrugs are required, such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters. Particularly useful are C1 to C8 alkyl, C2 to C8 alkenyl, aryl, C7 to C8 alkenyl groups of the compounds of this invention. 12 Substituted aryl and C7 to C 12 Aryl esters.

[0127] Pharmaceutical Composition

[0128] When used as a medicine, the compounds of the present invention are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical field and comprise at least one active compound of the present invention according to Formula I. Generally, the compounds of the present invention are administered in a pharmaceutically effective amount. The actual amount of the compound of the present invention administered will typically be determined by a physician based on relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound of the present invention administered, the individual patient's age, weight and response, the severity of the patient's symptoms, etc.

[0129] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent.

[0130] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a treatment for the following diseases: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis.

[0131] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being an inflammatory disease treatment agent. Specifically, the term inflammatory disease refers to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel disease.

[0132] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for autoinflammatory diseases. Specifically, the term autoinflammatory disease refers to cryoinflammatory periodic syndrome (CAPS), familial Mediterranean fever (FMF), and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), Behçet's disease, systemic juvenile idiopathic arthritis (SJIA), or Still's disease. More specifically, the term refers to CAPS, FMF, TRAPS, and Still's disease.

[0133] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being an autoimmune disease therapeutic agent. Specifically, the term autoimmune disease refers to COPD, asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, dry eye disease, and inflammatory bowel disease. More specifically, the term refers to COPD, asthma, systemic lupus erythematosus, and inflammatory bowel disease.

[0134] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a proliferative disease therapeutic agent. Specifically, the term proliferative disease refers to cancer, myeloproliferative disorders, leukemia, multiple myeloma, psoriasis, restenosis, scleroderma, or fibrosis. Specifically, the term refers to psoriasis, scleroderma, and fibrosis.

[0135] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a fibrotic disease treatment agent. Specifically, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF), radiation-induced fibrosis, nonalcoholic steatohepatitis (NASH), scleroderma, bleomycin-induced pulmonary fibrosis, cutaneous scleroderma, and systemic sclerosis.

[0136] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a transplant rejection therapeutic agent. Specifically, the term transplant rejection refers to acute or chronic rejection, or graft-versus-host disease, of allogeneic or xenografts of cells, tissues, or solid organs, such as islets of Langerhans, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary junction, kidney, liver, intestine, pancreas, trachea, or esophagus. More specifically, the term refers to graft-versus-host disease.

[0137] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for diseases involving impaired cartilage transformation. Specifically, the term "diseases involving impaired cartilage transformation" refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Tietze syndrome or costochondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis such as endemic degenerative osteoarthritis, Mseleni disease, and Handigodu disease; and degenerative diseases resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. More specifically, the term refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

[0138] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a treatment for congenital chondrodysplasia. Specifically, the term congenital chondrodysplasia refers to hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, microtia, auria, and metaphyseal chondrodysplasia. More specifically, the term refers to microtia, auria, and metaphyseal chondrodysplasia.

[0139] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for a disease involving impaired bone turnover. Specifically, the term disease involving impaired bone turnover refers to osteoporosis, osteopenia, hormone deficiency, hormone excess, Paget's disease, osteoarthritis, renal osteodystrophy, osteogenesis imperfecta, and hypophosphatase syndrome. More specifically, the term refers to osteoporosis.

[0140] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a treatment for a disease associated with excessive IL6 secretion. Specifically, the term "disease associated with excessive IL6 secretion" refers to Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma, and / or mesangial proliferative glomerulonephritis.

[0141] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a treatment for diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23. Specifically, the term "diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23" refers to systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, spondyloarthritis (especially ankylosing spondylitis), and / or Crohn's disease. More specifically, the term refers to Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, ankylosing spondylitis, and / or Crohn's disease.

[0142] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for respiratory diseases. Specifically, the term respiratory disease refers to asthma, adult respiratory distress syndrome, isocapnic hyperventilation, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, emphysema, pulmonary hypertension, interstitial pulmonary fibrosis, cystic fibrosis, or hypoxia. More specifically, the term refers to pulmonary hypertension or interstitial pulmonary fibrosis.

[0143] In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for endocrine disorders. Specifically, the term endocrine disorder refers to hypothyroidism, congenital adrenal hyperplasia, parathyroid disease, diabetes, adrenal disorders, Cushing's syndrome and Addison's disease, ovarian dysfunction, and polycystic ovary syndrome.

[0144] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for metabolic diseases. Specifically, the term metabolic disease refers to cystic fibrosis, phenylketonuria (PKU), diabetes, hyperlipidemia, gout, type II diabetic nephropathy, and rickets. More specifically, the term refers to obesity and / or type II diabetes.

[0145] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for cardiovascular diseases. Specifically, the term cardiovascular disease refers to arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac arrhythmia; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis; stroke; peripheral obstructive arterial disease of a limb, organ, or tissue; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxic shock, surgical shock, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, inflammation, or dysfunction limited to a single organ or tissue. More specifically, cardiovascular disease refers to atherosclerosis or giant cell arteritis.

[0146] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a treatment for a skin disease. Specifically, the term skin disease refers to atopic dermatitis, bullous disease, collagen lesions, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, vitiligo, pruritus, scleroderma, wound healing, scarring, hypertrophic scarring, keloids, Kawasaki disease, rosacea, Sjögren's-Lasson syndrome, or urticaria. More specifically, the term refers to psoriasis, psoriatic lesions, scleroderma, and vitiligo.

[0147] In one embodiment, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and another therapeutic agent, said other therapeutic agent being a therapeutic agent for diseases associated with abnormal angiogenesis. Specifically, the term "diseases associated with abnormal angiogenesis" refers to atherosclerosis, hypertension, tumor growth, inflammation, rheumatoid arthritis, wet macular degeneration, choroidal neovascularization, retinal neovascularization, and diabetic retinopathy. More specifically, the term refers to atherosclerosis, hypertension, or diabetic retinopathy.

[0148] The pharmaceutical compositions of the present invention can be administered via a variety of routes, including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds of the present invention are preferably formulated as injectable or oral compositions, or as ointments, lotions, or patches exclusively for transdermal application.

[0149] Compositions for oral administration may be in the form of bulk liquid solutions or suspensions, or bulk powders. However, more commonly, compositions are present in unit dosage forms to facilitate precise dosing. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, and suitable pharmaceutical excipients, carriers, or agonists. Typical unit dosage forms include pre-filled, pre-quantitative ampoules or syringes of liquid compositions, or, in the case of solid compositions, pills, tablets, capsules, etc. In such compositions, the compound of the invention according to Formula I is typically a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being various carriers or agonists and processing aids that contribute to the formation of the desired form of administration.

[0150] Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers, as well as buffers, suspending agents and dispersants, colorants, flavoring agents, etc. Solid forms may include, for example, any of the following components, or compounds of the present invention having similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as mint or orange flavorings.

[0151] Injectable compositions are typically based on injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. As previously described, the active compound of Formula I of the present invention is typically a minor component in such compositions, usually about 0.05-10% by weight, with the remainder being an injectable carrier, etc.

[0152] Transdermal compositions are typically formulated as topical ointments or creams containing an active ingredient, typically in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, more preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredient is typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with, for example, an oil-in-water emulsion base. Such transdermal formulations are well known in the art and typically include additional ingredients to enhance the skin penetration or stability of the active ingredient or formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.

[0153] The compounds of the present invention can also be administered via transdermal devices. Therefore, transdermal administration can be achieved using reservoir-type, porous membrane-type, or solid matrix-type patches.

[0154] The components described above for use in compositions that can be administered orally, injected, or topically are only representative. Other materials and processing techniques, etc., are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0155] The compounds of the present invention can also be administered in a sustained-release form or from a sustained-release drug delivery system. Descriptions of representative sustained-release materials can be found in Remington's Pharmaceutical Sciences (Remington & Gennaro 1985).

[0156] The following formulation examples illustrate representative pharmaceutical compositions that can be prepared according to the present invention. However, the present invention is not limited to the following pharmaceutical compositions.

[0157] Formulation 1 - Tablets

[0158] The compound of the present invention according to Formula I can be mixed as a dry powder with a dry gelatin binder at a weight ratio of about 1:2. A small amount of magnesium stearate can be added as a lubricant. The mixture can be formed into 240-270 mg tablets (each tablet containing 80-90 mg of the active compound of the present invention of Formula I) in a tableting machine.

[0159] Formulation 2 - Capsules

[0160] The compound of the present invention according to Formula I can be mixed as a dry powder with a starch diluent at a weight ratio of about 1:1. The mixture can be filled into 250 mg capsules (each capsule contains 125 mg of the active compound of the present invention according to Formula I).

[0161] Formulation 3 - Liquid

[0162] The compound of Formula I (125 mg) can be mixed with sucrose (1.75 g) and xanthan gum (4 mg), and the resulting mixture can be blended and passed through a 10-mesh US sieve. Then, it can be mixed with a pre-prepared solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavoring agents, and coloring agents can be diluted with water and added with stirring. Sufficient water can then be added with stirring. Sufficient water can then be added again to produce a total volume of 5 mL.

[0163] Formulation 4 - Tablets

[0164] The compound of the present invention according to Formula I can be mixed as a dry powder with a dry gelatin binder at a weight ratio of about 1:2. A small amount of magnesium stearate may be added as a lubricant. The mixture can be formed into 450-900 mg tablets (150-300 mg of the active compound of the present invention according to Formula I) in a tableting machine.

[0165] Formulation 5 - Injection

[0166] The compounds of the present invention of Formula I can be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of about 5 mg / mL.

[0167] Formulation 6 - Topical

[0168] Stearyl alcohol (250g) and white petrolatum (250g) can be melted at about 75°C, and then a mixture of the compound of the present invention according to formula I (50g), methylparaben (0.25g), propylparaben (0.15g), sodium dodecyl sulfate (10g) and propylene glycol (120g) dissolved in water (about 370g) can be added, and the resulting mixture can be stirred until it solidifies.

[0169] Treatment

[0170] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for use in medicine.

[0171] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis.

[0172] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis.

[0173] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis, said methods comprising administering an effective amount of the compounds of the present invention described herein or one or more pharmaceutical compositions for the treatment or prevention of said conditions.

[0174] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of inflammatory diseases. Specifically, the term inflammatory disease refers to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel disease.

[0175] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of inflammatory diseases. Specifically, the term inflammatory disease refers to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel disease.

[0176] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from inflammatory diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term inflammatory disease refers to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel disease.

[0177] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of autoinflammatory diseases. Specifically, the term autoinflammatory disease refers to cryoinflammatory periodic syndrome (CAPS), familial Mediterranean fever (FMF), and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), Behçet's disease, systemic juvenile idiopathic arthritis (SJIA), or Still's disease. More specifically, the term refers to CAPS, FMF, TRAPS, and Still's disease.

[0178] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of autoinflammatory diseases. Specifically, the term autoinflammatory disease refers to cryoinflammatory periodic syndrome (CAPS), familial Mediterranean fever (FMF), and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), Behçet's disease, systemic juvenile idiopathic arthritis (SJIA), or Still's disease. More specifically, the term refers to CAPS, FMF, TRAPS, and Still's disease.

[0179] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from autoinflammatory diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term autoinflammatory disease refers to cryoinflammatory periodic syndrome (CAPS), familial Mediterranean fever (FMF), and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), Behçet's disease, systemic juvenile idiopathic arthritis (SJIA), or Still's disease. More specifically, the term refers to CAPS, FMF, TRAPS, and Still's disease.

[0180] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of autoimmune diseases. Specifically, the term autoimmune disease refers to COPD, asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, dry eye disease, and inflammatory bowel disease. More specifically, the term refers to COPD, asthma, systemic lupus erythematosus, and inflammatory bowel disease.

[0181] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of autoimmune diseases. Specifically, the term autoimmune disease refers to COPD, asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, dry eye disease, and inflammatory bowel disease. More specifically, the term refers to COPD, asthma, systemic lupus erythematosus, and inflammatory bowel disease.

[0182] In another aspect of treatment, the present invention provides methods for preventing and / or treating mammals suffering from autoimmune diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term autoimmune disease refers to COPD, asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, Sjögren's syndrome, psoriasis, xerophthalmia, and inflammatory bowel disease. More specifically, the term refers to COPD, asthma, systemic lupus erythematosus, and inflammatory bowel disease.

[0183] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of proliferative diseases. Specifically, the term proliferative disease refers to cancer, myeloproliferative disorders, leukemia, multiple myeloma, psoriasis, restenosis, scleroderma, or fibrosis. Specifically, the term refers to psoriasis, scleroderma, and fibrosis.

[0184] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of proliferative diseases. Specifically, the term proliferative disease refers to cancer, myeloproliferative disorders, leukemia, multiple myeloma, psoriasis, restenosis, scleroderma, or fibrosis. Specifically, the term refers to psoriasis, scleroderma, and fibrosis.

[0185] In another aspect of treatment, the present invention provides methods for preventing and / or treating mammals suffering from proliferative diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said disease. Specifically, the term proliferative disease refers to cancer, myeloproliferative disorders, leukemia, multiple myeloma, psoriasis, restenosis, scleroderma, or fibrosis. Specifically, the term refers to psoriasis, scleroderma, and fibrosis.

[0186] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of fibrotic diseases. Specifically, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF), radiation-induced fibrosis, nonalcoholic steatohepatitis (NASH), scleroderma, bleomycin-induced pulmonary fibrosis, cutaneous scleroderma, and systemic sclerosis.

[0187] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of fibrotic diseases. Specifically, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF), radiation-induced fibrosis, nonalcoholic steatohepatitis (NASH), scleroderma, bleomycin-induced pulmonary fibrosis, cutaneous scleroderma, and systemic sclerosis.

[0188] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from fibrotic diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF), radiation-induced fibrosis, nonalcoholic steatohepatitis (NASH), scleroderma, bleomycin-induced pulmonary fibrosis, cutaneous scleroderma, and systemic sclerosis.

[0189] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of transplant rejection. Specifically, the term transplant rejection refers to acute or chronic rejection of allogeneic or xenografts of cells, tissues, or solid organs, such as islets of Langerhans, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary junction, kidney, liver, intestine, pancreas, trachea, or esophagus, or graft-versus-host disease. More specifically, the term refers to graft-versus-host disease.

[0190] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the manufacture of medicaments for the prevention and / or treatment of transplant rejection. Specifically, the term transplant rejection refers to acute or chronic rejection, or graft-versus-host disease, of allogeneic or xenografts of cells, tissues, or solid organs, such as islets of Langerhans, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary junction, kidney, liver, intestine, pancreas, trachea, or esophagus. More specifically, the term refers to graft-versus-host disease.

[0191] In another aspect of treatment, the present invention provides a method for preventing and / or treating mammals suffering from transplant rejection, the method comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said condition. Specifically, the term transplant rejection refers to acute or chronic rejection, or graft-versus-host disease, of allogeneic or xenografts of cells, tissues, or solid organs, such as islets of Langerhans, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, cardiopulmonary junction, kidney, liver, intestine, pancreas, trachea, or esophagus. More specifically, the term refers to graft-versus-host disease.

[0192] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of diseases involving impaired cartilage transformation. Specifically, the term "diseases involving impaired cartilage transformation" refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Titzer syndrome or costochondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis such as endemic degenerative osteoarthritis, M'Selene's disease, and Handigodus disease; and degenerative diseases resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. More specifically, the term refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

[0193] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases involving impaired cartilage transformation. Specifically, the term "diseases involving impaired cartilage transformation" refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Titzer syndrome or costochondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis such as endemic degenerative osteoarthritis, M'Selene's disease, and Handigodou's disease; and degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. More specifically, the term refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

[0194] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from diseases involving impaired cartilage transformation, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term "disease involving impaired cartilage transformation" refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Titzer syndrome or costochondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis such as endemic degenerative osteoarthritis, M'Selene's disease, and Handigodou's disease; and degenerative diseases resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. More specifically, the term refers to osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

[0195] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of congenital chondrodysplasia. Specifically, the term congenital chondrodysplasia refers to hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, microtia, auria, and metaphyseal chondrodysplasia. More specifically, the term refers to microtia, auria, and metaphyseal chondrodysplasia.

[0196] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of congenital chondrodysplasia. Specifically, the term congenital chondrodysplasia refers to hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, microtia, auria, and metaphyseal chondrodysplasia. More specifically, the term refers to microtia, auria, and metaphyseal chondrodysplasia.

[0197] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from congenital chondrodysplasia, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for treating or preventing the condition. Specifically, the term congenital chondrodysplasia refers to hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, microtia, auria, and metaphyseal chondrodysplasia. More specifically, the term refers to microtia, auria, and metaphyseal chondrodysplasia.

[0198] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of diseases involving impaired bone turnover. Specifically, the term diseases involving impaired bone turnover refers to osteoporosis, osteopenia, hormone deficiency, hormone excess, Paget's disease, osteoarthritis, renal osteodystrophy, osteogenesis imperfecta, and hypophosphatase syndrome. More specifically, the term refers to osteoporosis.

[0199] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases involving impaired bone turnover. Specifically, the term diseases involving impaired bone turnover refers to osteoporosis, osteopenia, hormone deficiency, hormone excess, Paget's disease, osteoarthritis, renal osteodystrophy, osteogenesis imperfecta, and hypophosphatase syndrome. More specifically, the term refers to osteoporosis.

[0200] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from diseases involving impaired bone turnover, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term "disease involving impaired bone turnover" refers to osteoporosis, osteopenia, hormone deficiency, hormone excess, Paget's disease, osteoarthritis, renal osteodystrophy, osteogenesis imperfecta, and hypophosphatase syndrome. More specifically, the term refers to osteoporosis.

[0201] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of diseases associated with excessive IL6 secretion. Specifically, the term diseases associated with excessive IL6 secretion refers to Kaseman disease, multiple myeloma, psoriasis, Kaposi's sarcoma, and / or mesangial proliferative glomerulonephritis.

[0202] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases associated with excessive IL6 secretion. Specifically, the term diseases associated with excessive IL6 secretion refers to Kaseman disease, multiple myeloma, psoriasis, Kaposi's sarcoma, and / or mesangial proliferative glomerulonephritis.

[0203] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from diseases associated with excessive IL6 secretion, said methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term diseases associated with excessive IL6 secretion refers to Kaseman disease, multiple myeloma, psoriasis, Kaposi's sarcoma, and / or mesangial proliferative glomerulonephritis.

[0204] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23. Specifically, the term "diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23" refers to systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, and / or Crohn's disease. More specifically, the term refers to Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, and / or Crohn's disease.

[0205] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23. Specifically, the term "diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23" refers to systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis and / or Crohn's disease. More specifically, the term refers to Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis and / or Crohn's disease.

[0206] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23, said methods comprising administering an effective amount of the compounds of the present invention described herein or one or more pharmaceutical compositions for the treatment or prevention of said conditions. Specifically, the term "diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17, and / or IL-23" refers to systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, and / or Crohn's disease. More specifically, the term refers to Sjögren's syndrome, psoriasis, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Down syndrome, ulcerative colitis, and / or Crohn's disease.

[0207] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of respiratory diseases. Specifically, the term "respiratory disease" refers to asthma, adult respiratory distress syndrome, isocarbon dioxide hyperventilation, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, emphysema, pulmonary hypertension, interstitial pulmonary fibrosis, cystic fibrosis, or hypoxia. More specifically, the term refers to pulmonary hypertension or interstitial pulmonary fibrosis.

[0208] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of respiratory diseases. Specifically, the term "respiratory disease" refers to asthma, adult respiratory distress syndrome, isocarbon dioxide hyperventilation, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, emphysema, pulmonary hypertension, interstitial pulmonary fibrosis, cystic fibrosis, or hypoxia. More specifically, the term refers to pulmonary hypertension or interstitial pulmonary fibrosis.

[0209] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from respiratory diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term respiratory disease refers to asthma, adult respiratory distress syndrome, isocarbon dioxide hyperventilation, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, emphysema, pulmonary hypertension, interstitial pulmonary fibrosis, cystic fibrosis, or hypoxia. More specifically, the term refers to pulmonary hypertension or interstitial pulmonary fibrosis.

[0210] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of endocrine disorders. Specifically, the term endocrine disorder refers to hypothyroidism, congenital adrenal hyperplasia, parathyroid disease, diabetes, adrenal gland disorders, Cushing's syndrome and Addison's disease, ovarian dysfunction, and polycystic ovary syndrome.

[0211] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of endocrine disorders. Specifically, the term endocrine disorder refers to hypothyroidism, congenital adrenal hyperplasia, parathyroid disorders, diabetes, adrenal disorders, Cushing's syndrome and Addison's disease, ovarian dysfunction, and polycystic ovary syndrome.

[0212] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from endocrine disorders, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term endocrine disorder refers to hypothyroidism, congenital adrenal hyperplasia, parathyroid disease, diabetes, adrenal disorders, Cushing's syndrome and Addison's disease, ovarian dysfunction, and polycystic ovary syndrome.

[0213] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of metabolic diseases. Specifically, the term metabolic disease refers to cystic fibrosis, phenylketonuria (PKU), diabetes, hyperlipidemia, gout, type II diabetic nephropathy, and rickets. More specifically, the term refers to obesity and / or type II diabetes.

[0214] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of metabolic diseases. Specifically, the term metabolic disease refers to cystic fibrosis, phenylketonuria (PKU), diabetes, hyperlipidemia, gout, type II diabetic nephropathy, and rickets. More specifically, the term refers to obesity and / or type II diabetes.

[0215] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from metabolic diseases, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term metabolic disease refers to cystic fibrosis, phenylketonuria (PKU), diabetes, hyperlipidemia, gout, type II diabetic nephropathy, and rickets. More specifically, the term refers to obesity and / or type II diabetes.

[0216] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of cardiovascular diseases. Specifically, the term cardiovascular disease refers to arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac arrhythmia; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis; stroke; peripheral obstructive arterial disease of limbs, organs, or tissues; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxic shock, surgical shock, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, inflammation, or dysfunction. More specifically, cardiovascular disease refers to atherosclerosis or giant cell arteritis.

[0217] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases. Specifically, the term cardiovascular disease refers to arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac arrhythmia; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis; stroke; peripheral obstructive arterial disease of limbs, organs, or tissues; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxic shock, surgical shock, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, inflammation, or dysfunction. More specifically, cardiovascular disease refers to atherosclerosis or giant cell arteritis.

[0218] In other aspects of treatment, the present invention provides methods for preventing and / or treating cardiovascular diseases in mammals, the methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term cardiovascular disease refers to arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac rhythm disorders; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis; stroke; peripheral obstructive arterial disease of limbs, organs, or tissues; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxic shock, surgical shock, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, inflammation, or dysfunction. More specifically, cardiovascular disease refers to atherosclerosis or giant cell arteritis.

[0219] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of skin diseases. Specifically, the term skin disease refers to atopic dermatitis, bullous diseases, collagen diseases, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, vitiligo, pruritus, scleroderma, wound healing, scarring, hypertrophic scarring, keloids, Kawasaki disease, rosacea, Sjögren's-Lasson syndrome, or urticaria. More specifically, the term refers to psoriasis, psoriatic lesions, scleroderma, and vitiligo.

[0220] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of skin diseases. Specifically, the term skin disease refers to atopic dermatitis, bullous diseases, collagen diseases, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, vitiligo, pruritus, scleroderma, wound healing, scarring, hypertrophic scarring, keloids, Kawasaki disease, rosacea, Sjögren's-Lasson syndrome, or urticaria. More specifically, the term refers to psoriasis, psoriatic lesions, scleroderma, and vitiligo.

[0221] In other aspects of treatment, the present invention provides methods for preventing and / or treating mammals suffering from skin diseases, said methods comprising administering an effective amount of the compounds or one or more pharmaceutical compositions of the present invention described herein for the treatment or prevention of said conditions. Specifically, the term skin disease refers to atopic dermatitis, bullous diseases, collagen diseases, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, vitiligo, pruritus, scleroderma, wound healing, scar formation, hypertrophic scar formation, keloids, Kawasaki disease, rosacea, Sjögren's-Lasson syndrome, or urticaria. More specifically, the term refers to psoriasis, psoriatic lesions, scleroderma, and vitiligo.

[0222] In one embodiment, the present invention provides compounds of the present invention or pharmaceutical compositions comprising compounds of the present invention for the prevention and / or treatment of diseases associated with abnormal angiogenesis. Specifically, the term "diseases associated with abnormal angiogenesis" refers to atherosclerosis, hypertension, tumor growth, inflammation, rheumatoid arthritis, wet macular degeneration, choroidal neovascularization, retinal neovascularization, and diabetic retinopathy. More specifically, the term refers to atherosclerosis, hypertension, or diabetic retinopathy.

[0223] In another embodiment, the present invention provides the use of the compounds of the present invention or pharmaceutical compositions comprising the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal angiogenesis. Specifically, the term diseases associated with abnormal angiogenesis refers to atherosclerosis, hypertension, tumor growth, inflammation, rheumatoid arthritis, wet macular degeneration, choroidal neovascularization, retinal neovascularization, and diabetic retinopathy. More specifically, the term refers to atherosclerosis, hypertension, or diabetic retinopathy.

[0224] In another aspect of treatment, the present invention provides methods for preventing and / or treating mammals suffering from diseases associated with abnormal angiogenesis, said methods comprising administering an effective amount of the compounds of the present invention described herein or one or more pharmaceutical compositions for treating or preventing said conditions. Specifically, the term diseases associated with abnormal angiogenesis refers to atherosclerosis, hypertension, tumor growth, inflammation, rheumatoid arthritis, wet macular degeneration, choroidal neovascularization, retinal neovascularization, and diabetic retinopathy. More specifically, the term refers to atherosclerosis, hypertension, or diabetic retinopathy.

[0225] Injection dose levels range from about 0.1 mg / kg / h to at least 10 mg / kg / h, sustained for about 1 to about 120 hours, particularly 24 to 96 hours. Preloading boluses of about 0.1 mg / kg to more than 10 mg / kg may also be administered to achieve adequate steady-state levels. For human patients weighing 40–80 kg, the maximum total dose should not exceed about 1 g / day.

[0226] For the prevention and / or treatment of long-term conditions, such as degenerative conditions, treatment regimens typically last for months or years; therefore, oral administration is preferred for patient convenience and tolerability. For oral administration, the typical regimen is 1–4 times daily (1–4), particularly 1–3 times daily (1–3), typically 1–2 times daily (1–2), and most typically once daily (1). Alternatively, for long-acting medications, oral administration every other week, once weekly, and once daily are representative regimens. In particular, dosing regimens can be every 1–14 days, more particularly 1–10 days, even more particularly 1–7 days, and most particularly 1–3 days.

[0227] Using these dosing methods, each dose provides about 1 to about 1000 mg of the compound of the invention, with specific doses each providing about 10 to about 500 mg, particularly about 30 to about 250 mg.

[0228] Transdermal doses are typically chosen to provide similar or lower blood levels as achieved with injectable doses.

[0229] When used to prevent the onset of the condition, the compounds of the present invention are typically administered at the above-described dosage levels to patients at risk of developing the condition, as advised and under the supervision of a physician. Patients at risk of developing a particular condition typically include those with a family history of the condition, or those who have been identified through genetic testing or screening as particularly susceptible to developing the condition.

[0230] The compounds of the present invention can be administered as single active agents or in combination with other therapeutic agents, including other compounds of the present invention that exhibit the same or similar therapeutic activity and are determined to be safe and effective for such combination administration. In one specific embodiment, co-administration of two (or more) agents allows for the use of significantly lower doses of each agent, thereby reducing observed side effects.

[0231] In one embodiment, the compound of the present invention or a pharmaceutical composition comprising the compound of the present invention is administered as a medicine. In a specific embodiment, the pharmaceutical composition further comprises another active ingredient.

[0232] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for treating and / or preventing diseases involving inflammation, the specific agent including (but not limited to) immunomodulators such as azathioprine, corticosteroids (e.g., prednisolone or dexamethasone), cyclophosphamide, cyclosporine A, tacrolimus, mycophenolate mofetil, moromuzumab-CD3 (OKT3, e.g., orthocolone). ® ), ATG, aspirin, acetaminophen, ibuprofen, naproxen, and piroxicam.

[0233] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for the treatment and / or prevention of arthritis (e.g., rheumatoid arthritis), including but not limited to analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, synthetic DMARDs (e.g., but not limited to methotrexate, leflunomide, sulfasalazine, auronoxine, sodium auronoxate, penicillamine, chloroquine, hydroxychloroquine, azathioprine, JAK inhibitors (e.g., tofacitinib, baricitinib, utpatinib, ruxotinib, or fegnotinib), fantatinib, and cyclosporine), and biological DMARDs (e.g., but not limited to infliximab, etanercept, adalimumab, rituximab, and abatacept).

[0234] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for the treatment and / or prevention of proliferative disorders, including but not limited to: methotrexate, leucovorin, doxorubicin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, and anti-HER2 monoclonal antibodies (e.g., Herceptin). ® ), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g., Iressa) ® Tarceva ®Erbitux ® VEGF inhibitors (such as Avastin) ® ), proteasome inhibitors (e.g., Velcade) ® ), Gleevec ® And HSP90 inhibitors (e.g., 17-AAG). Furthermore, the compounds of Formula I of the present invention can be administered in combination with other therapies, including but not limited to radiation therapy or surgery. In one specific embodiment, the proliferative condition is selected from cancer, myeloproliferative disorders, or leukemia.

[0235] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for the treatment and / or prevention of autoimmune diseases, including but not limited to: glucocorticoids, cell growth inhibitors (e.g., purine analogs), alkylating agents (e.g., nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds of the present invention, etc.), antimetabolites (e.g., methotrexate, azathioprine, and mercaptopurine), cytotoxic antibiotics (e.g., styrosinase, anthracyclines, mitomycin C, bleomycin, and sclerosmycin), antibodies (e.g., anti-CD20, anti-CD25, or anti-CD3 (OTK3) monoclonal antibodies, Atgam). ® and Thymoglobuline ® ), cyclosporine, tacrolimus, rapamycin (sirolimus), interferon (e.g., IFN-β), TNF-binding protein (e.g., infliximab, etanercept, or adalimumab), mycophenolate mofetil, fingolimod, and myriocin.

[0236] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for treating and / or preventing transplant rejection, including but not limited to: calcineurin inhibitors (e.g., cyclosporine or tacrolimus (FK506)), mTOR inhibitors (e.g., sirolimus, everolimus), antiproliferative agents (e.g., azathioprine, mycophenolate mofetil), corticosteroids (e.g., prednisolone, hydrocortisone), antibodies (e.g., monoclonal anti-IL-2Rα receptor antibodies, baliximab, dalizumab), and polyclonal anti-T cell antibodies (e.g., anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

[0237] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for the treatment and / or prevention of asthma and / or rhinitis and / or COPD, including but not limited to: β2-adrenergic receptor agonists (e.g., salbutamol, levosalbutamol, terbutaline, and bitoterol), adrenaline (inhaled or tablet form), anticholinergics (e.g., ipratropium bromide), glucocorticoids (oral or inhaled), and long-acting β2-agonists (e.g., salmeterol, formoterol, bambuterol, and sustained-release oral...). Oral salbutamol), combinations of inhaled steroids and long-acting bronchodilators (e.g., fluticasone / salmeterol, budesonide / formoterol), leukotriene antagonists and synthesis inhibitors (e.g., montelukast, zafirlukast, and zileutone), mediator release inhibitors (e.g., cromoglycate and ketotifen), biological modulators of IgE response (e.g., omalizumab), antihistamines (e.g., cetirizine, cinnarizine, fexofenadine), and vasoconstrictors (e.g., oxymetazoline, xylometazoline, naphazoline, and tramazoline).

[0238] In addition, the compounds of the present invention can be administered in combination with emergency treatments for asthma and / or COPD, such treatments including administration of oxygen or a helium-oxygen mixture, nebulized salbutamol or terbutaline (optionally in combination with an anticholinergic such as ipratropium), systemic steroids (oral or intravenous, such as prednisone, prednisolone, methylprednisolone, dexamethasone, or hydrocortisone), intravenous salbutamol, nonspecific β-agonists (injection or inhalation, such as epinephrine, isoproterenol, isoproterenol, or metaproterenol), anticholinergics (intravenous or nebulized, such as glycopyrronium bromide, atropine, or ipratropium), methylxanthines (theophylline, aminophylline, benzoyltheophylline), inhaled anesthetics with bronchodilatory effects (such as isoflurane, halothane, enflurane), ketamine, and intravenous magnesium sulfate.

[0239] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for the treatment and / or prevention of inflammatory bowel disease (IBD), including but not limited to: glucocorticoids (e.g., prednisone, budesonide), synthetic disease-modifying immunomodulators (e.g., methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurine, and cyclosporine), and biological disease-modifying immunomodulators (infliximab, adalimumab, rituximab, and abatacept).

[0240] In one embodiment, the compound of the present invention is administered in combination with another therapeutic agent for the treatment and / or prevention of SLE, including but not limited to: human monoclonal antibody (Benlysta). ®Disease-modifying antirheumatic drugs (DMARDs) include antimalarial drugs (such as hydroxychloroquine), immunosuppressants (such as methotrexate and azathioprine), cyclophosphamide and mycophenolic acid, immunosuppressants and analgesics such as nonsteroidal anti-inflammatory drugs, opioids (such as dextropropoxyphene and codeine-acetaminophen combination preparations (co-codamo)), opioids (such as hydrocodone, oxycodone, MSContin or methadone), and fentanyl transdermal patches (Duragesic).

[0241] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for treating and / or preventing psoriasis, including but not limited to: topical medications such as bath liquids, moisturizers, ointments and creams containing coal tar, anthraquinol, and corticosteroids such as desoxymethasone. ® Fluocinolone acetonide, vitamin D3 analogs (e.g., calcipotriol), argan oil, and retinoids (etretinate, acitretin, tazarotene); systemic medications such as methotrexate, cyclosporine, retinoids, thioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, fumarate; or biologics such as Amevive. ® Enbrel ® Humira ® Remicade ® Raptiva ® And ustekinumab (IL-12 and IL-23 blockers). Furthermore, the compounds of the present invention can be administered in combination with other therapies, including but not limited to phototherapy or photochemotherapy (e.g., psoralen and ultraviolet A phototherapy (PUVA)).

[0242] In one embodiment, the compounds of the present invention are co-administered with another therapeutic agent for treating and / or preventing allergic reactions, including but not limited to: antihistamines (e.g., cetirizine, diphenhydramine, fexofenadine, levocetirizine), glucocorticoids (e.g., prednisone, betamethasone, beclomethasone, dexamethasone), adrenaline, theophylline or leukotriene antagonists (e.g., montelukast or zafirlukast), anticholinergics and decongestants.

[0243] Co-administration includes any manner in which two or more therapeutic agents are delivered to a patient as part of the same treatment regimen, as will be apparent to those skilled in the art. While two or more pharmaceutical agents may be administered simultaneously in a single formulation, i.e., as a single pharmaceutical composition, this is not necessary. The agents may be administered in different formulations and at different times.

[0244] Chemical synthesis methods

[0245] Universal type

[0246] The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. It should be understood that, given typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), other process conditions may be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through conventional optimization procedures.

[0247] Furthermore, it will be apparent to those skilled in the art that conventional protecting groups may be required to prevent certain functional groups from undergoing undesirable reactions. The selection of appropriate protecting groups for specific functional groups and suitable conditions for protection and deprotection are well known in the art (Wuts & Greene 2006).

[0248] The following methods provide details regarding the preparation of the compounds of the present invention as defined above, and comparative examples. The compounds of the present invention can be prepared by those skilled in the art of organic synthesis from known or commercially available starting materials and reagents.

[0249] Unless otherwise specified, all reagents are commercial grade and used as is without further purification. Commercially available anhydrous solvents are used for reactions carried out under an inert atmosphere. Unless otherwise specified, reagent-grade solvents are used in all other cases. React on silica gel 60 (35-70 μm) or with Biotage. ® SNAP KP-NH, Biotage ® SNAP Ultra or Interchim ® PuriFlash ® Column chromatography was performed using a Si HC rapid chromatography cartridge. Thin-layer chromatography was performed using pre-coated silica F-254 plates (0.25 mm thick). Biotage ® ISOLUTE ® A phase separator (e.g., Cat #120-1907-E) is used for aqueous phase separation. Recordings are performed on a Bruker DPX 400 NMR spectrometer (400 MHz), a Bruker Avance 300 NMR spectrometer (300 MHz), or a Bruker Avance III HD NMR spectrometer (400 MHz). 1 H NMR spectrum. 1Chemical shifts (δ) in the 1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) as an internal standard or a suitable residual solvent peak (e.g., CHCl3 (δ 7.27)). Multiplicity is given as singlets (s), doublets (d), triplets (t), quartets (q), quintets (quin), multiplets (m), and broad peaks (br). Electrospray MS spectra were obtained on a WatersAcquity Class H or Class I UPLC system connected to a UV PDA detector and a Waters SQD, SQD2, or QDa mass spectrometer. The columns used were: Waters Acquity UPLC BEHC18 1.7 µm, 2.1 mm ID × 30 / 50 mm L; Waters Acquity UPLC CSH C18 1.7 μm, 2.1 mm ID × 100 mm L; Waters Acquity UPLC CSH PhenylHexyl 1.7 μm, 2.1 mm ID × 100 mm L; Waters Acquity UPLC HSS PFP 1.8 μm, 2.1 mm ID × 100 mm L. The method used an ACN / water or MeOH / water gradient, with 0.1% formic acid or 0.05% NH3 in either mobile phase. Preparative HPLC was performed on a Waters automated purification system equipped with UV and MS detection, using a Waters XBridge BEH C18 OBD 30mm ID×100mm L column and an ACN / water gradient with 0.1% formic acid in both mobile phases or 0.1% NH4OH in both mobile phases. Biotage was used for this purpose. ® The initiator uses microwave heating.

[0250] Table I. List of abbreviations used in the experimental section:

[0251] Synthesis and preparation of the compounds of the present invention

[0252] Example 1. Preparation of the compound of the present invention and comparative compounds

[0253] 1.1 Intermediate 1

[0254] 1.1.1. Step A: 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyridine

[0255] At 25°C, Pd(OAc)₂ (4.22 g, 18.78 mmol, 0.025 equivalent), KOAc (221.15 g, 2.25 mol, 3 equivalent), and XPhos (17.90 g, 37.56 mmol, 0.05 equivalent) were added to a suspension of 6-bromopyrazolo[1,5-a]pyridine (CAS# 1264193-11-4; 148 g, 751.15 mmol, 1 equivalent) and bis(pinacol)diborane (209.82 g, 826.26 mmol, 1.1 equivalent) in 2-MeTHF (1.5 L). The suspension was degassed and purged three times with N₂ and stirred at 90°C under N₂ for 12 h. The reaction mixture was cooled to 25°C, diluted with DCM (1.5 L), and filtered through a diatomaceous earth thin-layer filter. The mother liquor was extracted with DCM (2 L) and H2O (2 L), dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography (eluting with EtOAc / petroleum ether 12 / 88) to give 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyridine.

[0256] LCMS: MW (calcd): 401.2; m / z MW (obsd): 162.9 (M+H of corresponding boric acid)

[0257] 1 H NMR (400MHz, CDCl3): δ8.79 (d, 1H), 7.90 (d, 1H), 7.41 (dd, 1H), 7.28 (dd, 1H), 6.40 (d, 1H), 1.28 (s, 12H)

[0258] 1.1.2. Step B: 3-bromopyrazolo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyridine

[0259] NBS (126.14 g, 708.74 mmol, 1 equivalent) was added to a solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyridine (173 g, 708.74 mmol, 1 equivalent) in ACN (1.6 L), and the mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with EtOAc (2 L) at 25 °C, washed with H2O (1 L) and brine (1 L), and the organic phase was dried over anhydrous MgSO4, filtered, and concentrated under vacuum to give 3-bromopyrazolo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyridine.

[0260] 1 H NMR: (400MHz, CDCl3): δ8.74(s, 1H), 7.89(s, 1H), 7.39(s, 2H), 1.28(s, 12H)

[0261] 1.1.3. Step C: Intermediate 1

[0262] Sodium hydroxide (2M in water, 659.46 mL, 3 equivalents) and H₂O₂ (168.63 g, 1.49 mol, 142.90 mL, 30% w / w in H₂O, 3.38 equivalents) were added dropwise to a solution of 3-bromopyrazolo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborheocyton-2-yl)pyrazolo[1,5-a]pyridine (142 g, 439.64 mmol, 1 equivalent) in THF (1.25 L). The reaction mixture was stirred at 0–5 °C for 2 h. The mixture was quenched with 10% Na₂SO₃ (2 L) and stirred at 25 °C for 2 h, then examined by potassium iodide starch paper. The mixture was acidified to pH 5 with 2N HCl, filtered, and concentrated under reduced pressure to give the product. The crude product was purified by silica gel column chromatography (eluting with DCM / THF 9 / 1 to 7 / 3) and further purified by SFC (column: Daicel CHIRALPAK IG (250 mm L × 50 mm ID, 10 µm); CO2 back pressure: 100 bar; cosolvent: 0.1% NH4OH in EtOH; flow rate: 220 mL / min; mode: isocratic 15%; temperature: 40°C) to give intermediate 1.

[0263] 1H NMR: (400MHz, DMSO-d6): δ9.85 (s, 1H), 8.15 (dd, 1H), 7.95 (s, 1H), 7.46 (d, 1H), 7.10 (dd, 1H)

[0264] 1.2 Intermediate 2

[0265] DCM (4 L, 6.7 V) and 4-methylbenzenesulfonyl chloride (873 g, 4571 mmol, 0.9 equivalents) were charged into a 15 L single-jacketed process reactor equipped with mechanical stirring (200 rpm) and baffles. Then, 4-dimethylaminopyridine (73 g, 598 mmol, 0.2 equivalents) was added. The container and funnel were rinsed with DCM (1.5 L, 2.5 V) and added to the reaction mixture. The reaction mixture was cooled to 0 °C, and (R)-(+) ethyl lactate (CAS# 7699-00-5; 600 g, 5079 mmol, 1.00 equivalents) was added in a single batch. The container and funnel were rinsed with DCM (0.5 L, 0.8 V) and added to the reaction mixture. Triethylamine (1.42 L, 10200 mmol, 2.01 equivalents) was added dropwise while maintaining the reaction temperature below 5 °C. The reaction mixture was stirred again at 0°C (250 rpm) for 1.5 h. HCl 2N (3.7 L, 6 V) was slowly added under cooling, maintaining the reaction temperature below 5°C until the pH reached 2-3. The layers were separated; the organic layer was washed with water (2 L, 3 V) and a 10% sodium chloride aqueous solution (2 L, 3 V), then concentrated under reduced pressure to give 1.228 kg of crude product. The crude product was purified by rapid chromatography using heptane / EtOAc100 / 0 to 70 / 30 as the elution system to provide intermediate 2.

[0266] 1 H NMR (400MHz, DMSO -d6) δ7.85-7.77(m, 2H), 7.53-7.45(m, 2H), 4.99(q, 1H), 4.05(qd, 2H), 2.43(s, 3H), 1.38(d, 3H), 1.12(t, 3H)

[0267] 1.3 Intermediate 3

[0268] In a baffled 5L reactor under N2 atmosphere, triethylamine (250.32 mL, 182.24 g, 1800.87 mmol, 2 equivalents) was added to a solution of (R)-(+)-lactic acid methyl ester (CAS# 17392-83-5; 93.74 g, 86 mL, 900.44 mmol, 1 equivalent), toluenesulfonyl chloride (163.076 g, 855.42 mmol, 0.95 equivalents), and DMAP (20 g, 163.705 mmol, 0.18 equivalents) in DCM (1000 mL) cooled at -5°C (jacket temperature). The addition continued for 1 h, with the reaction temperature maintained at 7°C. The reaction mixture was then stirred at 0–5°C for 2.2 h. 2M HCl (500 mL, 6V) was added until pH < 4, while maintaining the reaction mixture temperature below 10°C. The organic phase was washed with water (500 mL, 5V) and 10% NaCl aqueous solution (300 mL, 3V). The organic phase was concentrated and purified by chromatography on silica gel (eluting with heptane / EtOAc 100 / 0 to 70 / 30) to give intermediate 3.

[0269] 1.4 Intermediate 4

[0270] 1.4.1. Step A: N-[(1R,2S)-2-fluorocyclopropyl]-2-hydroxy-6-methoxy-benzamide

[0271] Add 1,1'-carbonyldiimidazole (CAS# 530-62-1; 49 g, 297 mmol, 1 equivalent) in portions to a DCM solution of 2-hydroxy-6-methoxybenzoic acid (CAS# 3147-64-6; 50 g, 297 mmol, 1 equivalent) in 300 mL. Then, (1R,2S)-2-fluorocyclopropyl 4-methylbenzenesulfonate (CAS# 143062-84-4; 80 g, 312 mmol, 1.05 equivalent) and triethylamine (50 mL, 359 mmol, 1.21 equivalent) to the mixture. Stir the resulting solution at room temperature for 2.5 hours. Add water and adjust the pH to 2 using 12N HCl solution (60 mL). Separate the organic layers and extract the aqueous layer twice with DCM. Wash the combined organic layers three times with saturated NaHCO3 solution, dry to Na2SO4, filter, and concentrate under reduced pressure. The residue was purified by rapid chromatography on silica gel (eluting with DCM / EtOAc 100 / 0 to 90 / 10) to give N-[(1R,2S)-2-fluorocyclopropyl]-2-hydroxy-6-methoxy-benzamide.

[0272] 1.4.2. Step B: 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide

[0273] At 5°C, potassium hydroxide (73 g, 1298 mmol, 10 equivalents) was added over 1 hour to a three-necked round-bottom flask equipped with a temperature probe and funnel containing a solution of N-[(1R,2S)-2-fluorocyclopropyl]-2-hydroxy-6-methoxy-benzamide (29 g, 130 mmol, 1 equivalent) in an ACN / water mixture (146 mL / 146 mL). Diethylphosphonate bromide difluoromethyl ester (CAS# 65094-22-6; 46 mL, 260 mmol, 2 equivalents) was added dropwise over 40 minutes while maintaining the reaction temperature below 12°C. The reaction mixture was warmed to room temperature and stirred for 40 minutes. The reaction mixture was then extracted twice with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was resuspended in MTBE (80 mL). The suspension was filtered, and the solid was washed with MTBE (20 mL) and dried to give 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide.

[0274] 1.4.3. Step C: 2-(difluoromethoxy)-4-(2,8-dioxa-5-aza-1-boronabicyclo[3.3.0]octane-1-yl)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide

[0275] Add 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentane (CAS#73183-34-3; 19.2 g, 76 mmol, 1.0 equivalence) to a solution of 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide (20.8 g, 76 mmol, 1.0 equivalence) in THF (85 mL, 1040 mmol, 13.8 equivalence) (CAS#73183-34-3; 19.2 g, 76 mmol, 1.0 equivalence), 4,4'-di-tert-butyl-2,2'-bipyridine (CAS#72914-19-3; 0.83 g, 3.0 mmol, 0.04 equivalence), and [Ir(OCH3)(COD)]2 (CAS#72914-19-3; 0.83 g, 3.0 mmol, 0.04 equivalence) to a solution of 2-(difluoromethoxy)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide (CAS#72914-19-3; 0.83 g, 3.0 mmol, 0.04 equivalence) in THF (CAS#72914-19-3). 12148-71-9; 0.51 g, 0.76 mmol, 0.01 equivalent). The reaction mixture was heated to reflux for 1 hour and then cooled to room temperature. Diethanolamine (CAS# 111-42-2; 7.3 mL, 76 mmol, 1.0 equivalent) was added, and the reaction mixture was stirred at room temperature for 30 min. The suspension was filtered. The filter cake was washed with THF (80 mL) and dried to give 2-(difluoromethoxy)-4-(2,8-dioxa-5-aza-1-boronabicyclo[3.3.0]octane-1-yl)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide.

[0276] 1.4.4. Step D: Intermediate 4

[0277] A suspension of 2-(difluoromethoxy)-4-(2,8-dioxa-5-aza-1-boronabicyclo[3.3.0]octane-1-yl)-N-[(1R,2S)-2-fluorocyclopropyl]-6-methoxy-benzamide (29.2 g, 76 mmol, 1.0 equivalent) in deionized water in hydrochloric acid (1 mol / L) (CAS# 7647-01-0; 115 mL, 115 mmol, 1.5 equivalent) was stirred at room temperature for 40 min. The suspension was filtered. The filter cake was washed with water (200 mL, pH 5), and the powder was dried overnight in an oven at 45°C to give intermediate 4.

[0278] 1.5 Intermediate 5

[0279] THF (8.5 L, 17 V) and intermediate 1 (500 g, 2347 mmol, 1.0 equivalent) were charged into a 15 L single-jacketed process reactor equipped with mechanical stirring (200 rpm) and baffles. The reaction mixture was stirred at room temperature for 10 minutes until the substrate was completely dissolved, and then cooled to 11°C. t-BuOK (85 g, 742 mmol, 0.32 equivalent) was added. When the reaction temperature reached 15°C, the jacket temperature was increased from 5°C to 10°C, and the stirring was increased to 250 rpm. When the reaction temperature reached 12°C, another tBuOK (170 g, 1484 mmol, 0.64 equivalent) was added. A thick suspension was rapidly formed after the addition of the base. The jacket temperature was set to 22°C, and the stirring was increased to 300 rpm. After reaching 20°C, the reaction mixture was stirred for another hour. The reaction mixture was filtered over a sintering funnel. The solid was washed with THF (3V), ground with MTBE (3V), filtered, and collected. The resulting solid was dried under vacuum (3 days at 35°C, then 1 day at room temperature) to give intermediate 5.

[0280] 1 H NMR (400MHz, DMSO-d6) δ7.39 (s, 1H), 7.21 (d, 1H), 6.90 (d, 1H), 6.59 (dd, 1H)

[0281] 1.6 Cpd 1 and Cpd 8

[0282] 1.6.1. Step A: Pyrazolo[1,5-a]pyrimidine-6-ol potassium

[0283] In a round-bottom flask, a suspension of pyrazolo[1,5-a]pyrimidin-6-ol (CAS# 1580489-59-3; 25.0 g, 185 mmol, 1.00 equivalent) in THF (500 mL) was heated at 60°C for 1 h. The suspension was cooled to 0°C, and potassium tert-butoxide (19.7 g, 176 mmol, 0.95 equivalent) was added in two portions (9.0 g initially, followed by 10.7 g after 10 min). The reaction mixture was stirred at room temperature for 1.5 h. The suspension was filtered, and the solid was washed with THF. The solid was milled directly in MTBE on a sintered funnel. The solid was collected and dried under vacuum at 35°C for 3 h.

[0284] 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, 1H), 7.43 (d, 1H), 7.30 (dd, 1H), 6.09 (dd, 1H).

[0285] 1.6.2. Step B: Methyl (2S)-2-pyrazolo[1,5-a]pyrimidin-6-yloxypropionate

[0286] In a round-bottom flask, at 0°C, an intermediate 3 (6.6 g, 25 mmol, 1.1 equivalent) in DMF (8.0 mL) was added to a solution of potassium pyrazolo[1,5-a]pyrimidin-6-ol (4.0 g, 23 mmol, 1.0 equivalent) in DMF (32 mL). The reaction mixture was stirred at 0°C for 1 h. The reaction mixture was quenched by slow addition of 10% aqueous K₂CO₃. Water and EtOAc were then added, and the layers were separated. The aqueous layer was extracted with EtOAc (four times), the organic layers were combined, washed twice with 10% LiCl aqueous solution, washed with 10% NaCl aqueous solution, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was subjected to rapid chromatography using Biotage. ® Purification was performed using a Sfär HC 100g column, followed by elution with heptane / EtOAc 100 / 0 to 0 / 100 to obtain the title product.

[0287] LCMS: MW (calcd): 221.0; m / z MW (obsd): 221.8 (M+H)

[0288] 1.6.3. Step C: Methyl (2S)-2-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)oxypropionate

[0289] In a round-bottom flask, NBS (2.1 g, 1.0 mL, 12 mmol, 1.1 equivalent) was added to a solution of (2S)-2-pyrazolo[1,5-a]pyrimidin-6-yloxypropionate (2.4 g, 11 mmol, 1.0 equivalent) in 48 mL of ACN at 0°C. The reaction mixture was stirred at room temperature for 45 minutes. The mixture was diluted in DCM and the organic layer was washed with 5% NaHCO3 aqueous solution and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was subjected to rapid chromatography using Biotage. ® Purified by Sfär HC100g column, eluted with heptane / EtOAc 100 / 0 to 75 / 25 to give the title product.

[0290] LCMS: MW (calcd): 299.0; m / z MW (obsd): 299.7 / 301.7 (M+H)

[0291] 1 H NMR (400 MHz, CD3OD) δ 8.62 (d, 1H), 8.51 (d, 1H), 8.06 (s, 1H), 5.02 (q, 1H), 3.78 (s, 3H), 1.66 (d, 3H)

[0292] 1.6.4. Alternative step C: Methyl (2S)-2-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)oxypropionate

[0293] Acetonitrile (3500 mL), 3-bromopyrazolo[1,5-a]pyrimidin-6-ol (350 g, 1635 mmol, 1.0 equivalence), methyl (2R)-2-[(4-methylbenzenesulfonyl)oxy]propionate (CAS 1314893-97-4, 412 g, 1595 mmol, 1.0 equivalence, 94.8% ee), and potassium carbonate (315 g, 2279 mmol, 1.4 equivalence) were charged into a 15 L single-jacketed reactor. The resulting mixture was heated to 55 °C over 30 minutes, stirred at 55 °C for 17 h, and then cooled to 20 °C. The suspension was filtered, and the filter cake was washed with EtOAc (4000 mL). The filtrate was washed with a 10% NaCl solution (1200 mL). The organic layer was recovered and concentrated to 1-1.1 kg, then MTBE (1000 mL) was added and stirred at 0-5°C for 30 min. The suspension was filtered and washed with MTBE (1000 mL), and the resulting filter cake was dried under vacuum at 40°C to obtain the title product.

[0294] 1.6.5. Step D: (3S)-3-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol

[0295] In a round-bottom flask, at 0°C, methyl magnesium bromide solution (3.0 mol / L in 2-MeTHF; 10 mL, 31 mmol, 3.0 equivalence) was added dropwise to a suspension of (2S)-2-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)oxypropionate (3100 mg, 10 mmol, 1.0 equivalence) in 2-MeTHF (31 mL). The reaction mixture was stirred at 0°C for 15 min. The mixture was then quenched with a solution of hydrochloric acid (1 mol / L) in water (36 mL, 36 mmol, 3.5 equivalence) and extracted twice with 2-MeTHF. The yellow organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was subjected to rapid chromatography using Biotage. ® Purification was performed using a Sfär HC 100g column, followed by elution with heptane / EtOAc 100 / 0 to 0 / 100 to give the title product. The enantiomeric excess was measured to be 84%.

[0296] LCMS: MW (calcd): 299.0; m / z MW (obsd): 299.8 / 301.8 (M+H)

[0297] 1.6.6. Step E: Cpd1 and Cpd8

[0298] In a round-bottom flask, intermediate 4 (1382 mg, 4.33 mmol, 1.30 equivalent), sodium carbonate (1059 mg, 10.0 mmol, 3.00 equivalent), RuPhos (CAS# 787618-22-8; 31.7 mg, 0.067 mmol, 0.02 equivalent), and Pd(OAC)2 (7.5 mg, 0.033 mmol, 0.010 equivalent) were added to a solution of (3S)-3-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol (1000 mg, 3.33 mmol, 1.00 equivalent) in a mixture of 1,4-dioxane (8 mL) and water (2 mL). The reaction mixture was stirred at 90°C for 1 h. Add RuPhos (31.7 mg, 0.067 mmol, 0.02 equivalents) and Pd(OA) C )2 (7.48 mg, 0.033 mmol, 0.01 equivalent) and the reaction mixture was stirred at 90°C for 1 h. The reaction mixture was then transferred to a Clarcel container. ® Filter onto diatomaceous earth and wash with EtOAc. Wash the organic layer with water, dry with Na2SO4, filter, and concentrate under reduced pressure. Pass the crude product through rapid chromatography using Biotage. ®Purification was performed using a Sfär HC 100g column, eluted with heptane / EtOAc 100 / 0 to 0 / 100. Cpd 1 and Cpd 8 were separated by chiral SFC under the following conditions (Cpd 8 retention time: 3.70 min, Cpd 1 retention time: 5.65 min): Chiralpak IA, 150 × 4.6 mm, 5 μm column; CO2 back pressure: 1500 psi; cosolvent: EtOH; flow rate: 3 mL / min; mode: isocratic 30%; temperature: 40°C.

[0299] 1.7. Cpd2

[0300] 1.7.1. Step A: 7-Benzyloxyimidazo[1,2-b]pyridazine

[0301] In a round-bottom flask, 3-amino-5-(benzyloxy)pyridazine (CAS# 2155875-87-7; 1 g, 4.72 mmol, 1.00 equivalent), sodium bicarbonate (0.793 g, 9.44 mmol, 2.00 equivalent), and 2-propanol (20 mL) were introduced. Then, a solution of chloroacetaldehyde in water (55% by mass) (6.74 g, 5.45 mL, 47.21 mmol, 10.00 equivalent) was added. The resulting mixture was heated to 90°C. The reaction mixture was quenched by adding water and then extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was subjected to rapid chromatography on silica gel using Biotage. ® Purified using a SfärHC 25g column, diluted with DCM, and eluted with heptane / EtOAc 98 / 2 to 50 / 50. The fractions were pooled and concentrated under reduced pressure to give the title product.

[0302] LCMS: MW (calcd): 225,0; m / z MW (obsd): 226.3 (M+H)

[0303] 1.7.2. Step B: Imidazolo[1,2-b]pyridazine-7-ol

[0304] In a round-bottom flask under a nitrogen atmosphere, 7-benzyloxyimidazo[1,2-b]pyridazine (0.89 g, 3.95 mmol, 1.0 equivalent) and MeOH (15 mL) were introduced. Then, 20 wt% palladium hydroxide (0.555 g, 0.79 mmol, 0.20 equivalent) was added on carbon. The reaction mixture was degassed, backfilled with H2, and stirred at room temperature for 1.5 hours. The mixture was then transferred to a Clarcel container. ® Filter the solution onto diatomaceous earth. Wash the residue with MeOH and concentrate the filtrate under reduced pressure to obtain the title product.

[0305] 1.7.3. Step C: (2S)-2-imidazo[1,2-b]pyridazine-7-yloxypropionate ethyl ester

[0306] Under N2, intermediate 2 (0.881 g, 3.23 mmol, 0.95 equivalent) and potassium carbonate (0.941 g, 6.81 mmol, 2.00 equivalent) were added to a solution of imidazo[1,2-b]pyridazine-7-ol (0.46 g, 3.41 mmol, 1.00 equivalent) in DMF (6.8 mL). The mixture was stirred at 70°C for 2.5 h. The mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was subjected to rapid chromatography on silica gel using Biotage. ® Purified using a Sfär HC 25g column, diluted with DCM, and eluted with heptane / EtOAc 98 / 2 to 0 / 100. The fractions were pooled and concentrated under reduced pressure to give the title product.

[0307] LCMS: MW (calcd): 235.1; m / z MW (obsd): 236.1 (M+H)

[0308] 1.7.4. Step D: (3S)-3-imidazo[1,2-b]pyridazin-7-yloxy-2-methyl-but-2-ol

[0309] To an ice-cold solution of ethyl (2S)-2-imidazo[1,2-b]pyridazin-7-yloxypropionate (560 mg, 2.38 mmol, 1.00 equivalence) in THF (4.8 mL), a solution of magnesium methyl bromide (3.0 mol / L) in diethyl ether (1.7 mL, 5.24 mmol, 2.20 equivalence) was added. The reaction mixture was stirred at 0°C for 45 min, then allowed to warm to room temperature. After 1 hour, the reaction mixture was quenched by adding water, followed by extraction with EtOAc and a few drops of MeOH. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was subjected to rapid chromatography on silica gel using Biotage. ® Purified using a Sfär KP-NH11g column, diluted with DCM, and eluted with DCM / MeOH at 100 / 0 to 90 / 10. The fractions were pooled and concentrated under reduced pressure to obtain the title product.

[0310] LCMS: MW (calcd): 221.2; m / z MW (obsd): 222.2 (M+H)

[0311] 1.7.5. Step E: (3S)-3-(3-iodoimidazole[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol

[0312] NIS (0.419 g, 1.83 mmol, 1.05 equivalent) was added to a solution of (3S)-3-imidazo[1,2-b]pyridazin-7-yloxy-2-methyl-but-2-ol (0.385 g, 1.74 mmol, 1.00 equivalent) in DMF (7 mL). The mixture was stirred at room temperature for 40 min, then quenched with water and extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the title product.

[0313] LCMS: MW (calcd): 347.0; m / z MW (obsd): 348.0 (M+H)

[0314] 1.7.6. Step F: Cpd 2

[0315] Intermediate 4 (0.610 g, 1.914 mmol, 1.10 equivalent), cesium carbonate (1.70 g, 5.22 mmol, 3.00 equivalent), and Pd(dppf)Cl2·DCM (CAS# 95464-05-4; 0.15 g, 0.174 mmol, 0.10 equivalent) were added to a solution of (3S)-3-(3-ioimidazolo[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol (0.604 g, 1.74 mmol, 1.00 equivalent) in 1,4-dioxane (5.8 mL) and water (0.6 mL). The reaction mixture was heated at 100 °C for 1 h. The reaction mixture was concentrated under reduced pressure. The crude residue was subjected to rapid chromatography on silica gel using Biotage. ® Purification was performed using a Sfär KP-NH 28g column, diluted with DCM, and eluted with DCM / MeOH at 100 / 0 to 95 / 5. The fractions were pooled and concentrated under reduced pressure to obtain the title product.

[0316] 1.8. Cpd 3

[0317] 1.8.1. Step A: Ethyl 2-imidazo[1,2-b]pyridazine-7-yloxypropionate

[0318] Imidazolo[1,2-b]pyridazine-7-ol (CAS# 2410998-35-3; 0.12 g, 0.888 mmol, 1.00 equivalent) and ethyl 2-hydroxypropionate (CAS# 97-64-3; 0.243 g, 1.95 mmol, 2.20 equivalent) were placed in anhydrous toluene (2.14 mL) under a nitrogen atmosphere. Cyanomethylenetributylphosphine (CAS# 157141-27-0; 0.486 g, 0.549 mL, 1.95 mmol, 2.20 equivalent) was added using a syringe. The reaction mixture was heated at 80°C for 6 h. The mixture was concentrated under reduced pressure. The crude product was separated by column chromatography using 10 g Biotage. ® Purified on a Sfär HC column, eluted with DCM / MeOH 100 / 0 to 90 / 10 to obtain the title product.

[0319] LCMS: MW (calcd): 235.1; m / z MW (obsd): 236.1 (M+H)

[0320] 1.8.2. Step B: 3-Imidazolo[1,2-b]pyridazin-7-yloxy-2-methyl-but-2-ol

[0321] A solution of methyl magnesium bromide in Et2O (0.62 mL, 1.8704 mmol, 2.20 equivalence) in 1.7 mL of THF at 0°C was added to a solution of ethyl 2-imidazo[1,2-b]pyridazine-7-yloxypropionate (200 mg, 0.850 mmol, 1.00 equivalence). The reaction mixture was stirred at 0°C and allowed to warm to room temperature overnight. The reaction mixture was quenched with water and then extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was subjected to column chromatography in 5 g Biotage. ® Purified on a Sfär HC column, eluted with DCM / MeOH 100 / 0 to 90 / 10 to obtain the title product.

[0322] LCMS: MW (calcd): 221.1; m / z MW (obsd): 222.0 (M+H)

[0323] 1.8.3. Step C: 3-(3-iodoimidazole[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol

[0324] NIS (0.0536 g, 0.234 mmol, 1.10 equivalent) was added to a solution of 3-imidazo[1,2-b]pyridazin-7-yloxy-2-methyl-but-2-ol (0.047 g, 0.212 mmol, 1.00 equivalent) in DMF (0.425 mL). The mixture was stirred at room temperature for 40 min. The mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the title product.

[0325] LCMS: MW (calcd): 347.0; m / z MW (obsd): 348.4 (M+H)

[0326] 1.8.4. Step D: Cpd 3

[0327] Intermediate 4 (0.06708 g, 0.210 mmol, 1.00 equivalent), cesium carbonate (0.13703 g, 0.420 mmol, 2.00 equivalent), and Pd(dppf)Cl2·DCM (0.0181 g, 0.0210 mmol, 0.10 equivalent) were added to a solution of 3-(3-ioimidazolo[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol (0.073 g, 0.210 mmol, 1.00 equivalent), water (0.07 mL), and water (0.07 mL). The reaction was heated to 95°C for 8 h. The mixture was concentrated under reduced pressure and purified by preparative LCMS to give the title product.

[0328] Preparative LCMS conditions: Waters XBridge Prep C18 OBD 30×100mm 5μm column with XBridge Prep C18 30×10mm 5μm pre-column; Time: 8 min; Solvents: A = water + 0.1% formic acid, B = ACN + 0.1% formic acid; Flow rate: 50 mL / min; Gradient:

[0329] 1.9. Cpd 4

[0330] 1.9.1. Step A: Ethyl 2-pyrazolo[1,5-a]pyrimidin-6-yloxypropionate

[0331] Pyrazolo[1,5-a]pyrimidin-6-ol (CAS# 1580489-59-3; 0.1 g, 0.703 mmol, 1.00 equivalent) and ethyl 2-hydroxypropionate (CAS# 97-64-3; 0.192 g, 1.547 mmol, 2.20 equivalent) were placed in anhydrous toluene (1.70 mL) under N2 atmosphere in a microwave-safe vial. Cyanomethylenetributylphosphine (CAS# 157141-27-0; 0.385 g, 0.434 mL, 1.547 mmol, 2.20 equivalent) was added using a syringe. The reaction mixture was heated at 80°C for 6 h. The mixture was concentrated under reduced pressure. The crude product was separated by column chromatography using 10 g Biotage. ® Purified on a Sfär HC column, eluted with DCM / MeOH 100 / 0 to 90 / 10 to obtain the title product.

[0332] LCMS: MW (calcd): 235.1; m / z MW (obsd): 236.1 (M+H)

[0333] 1.9.2. Step B: 2-Methyl-3-pyrazolo[1,5-a]pyrimidin-6-yloxy-but-2-ol

[0334] A solution of magnesium methyl bromide in Et₂O (0.16 mL, 0.467 mmol, 2.50 equivalence) was added to a solution of ethyl 2-pyrazolo[1,5-a]pyrimidin-6-yloxypropionate (44 mg, 0.187 mmol, 1.00 equivalence) in THF (0.374 mL) at 0°C. The reaction mixture was stirred at 0°C and allowed to warm to room temperature overnight. The reaction mixture was quenched by adding water and then extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. In 5 g Biotage ® The crude residue was purified by rapid chromatography on a Sfär HC column, eluted with DCM / MeOH 100 / 0 to 90 / 10 to obtain the title product.

[0335] LCMS: MW (calcd): 221.1; m / z MW (obsd): 222.0 (M+H)

[0336] 1.9.3. Step C: 3-(3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol

[0337] NIS (0.0342 g, 0.149 mmol, 1.10 equivalent) was added to a solution of 2-methyl-3-pyrazolo[1,5-a]pyrimidin-6-yloxy-but-2-ol (0.03 g, 0.135 mmol, 1.00 equivalent) in DMF (0.271 mL). The mixture was stirred at room temperature for 30 minutes. The mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the title product.

[0338] LCMS: MW (calcd): 347.0; m / z MW (obsd): 348.0 (M+H)

[0339] 1.9.4. Step D: Cpd4

[0340] Intermediate 4 (0.043 g, 0.135 mmol, 1.00 equivalent), cesium carbonate (0.088 g, 0.27 mmol, 2.00 equivalent), and Pd(dppf)Cl2·DCM (0.0116 g, 0.0135 mmol, 0.10 equivalent) were added to a solution of 3-(3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol (0.047 g, 0.135 mmol, 1.00 equivalent), water (0.045 mL), and water (0.045 mL). The reaction mixture was heated to 95°C for 8 h. The reaction mixture was concentrated under reduced pressure and purified by preparative LCMS to give the title product.

[0341] Preparative LCMS conditions: Waters XBridge Prep C18 OBD 30 × 100 mm 5 μm column with XBridge Prep C18 30 × 10 mm 5 μm pre-column; Time: 8 min; Solvents: A = water + 0.1% formic acid, B = CAN + 0.1% formic acid; Flow rate: 50 mL / min; Gradient:

[0342] 1.10. Cpd 5

[0343] 1.10.1. Step A: Dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]imidazo[1,2-b]pyridazin-7-yl]oxy-1,1-dimethyl-propyl]phosphate

[0344] A solution of 1H-tetrazole (0.45 mol / L) in ACN (7.2 mL) was slowly added to a solution of Cpd 2 (800 mg, 1.62 mmol, 1.00 equivalence) in DMF (4 mL) under N2 conditions, followed by dropwise addition of dibenzyl diisopropylphosphonamide (931.3 mg, 0.91 mL, 2.43 mmol, 1.50 equivalence). The resulting mixture was stirred overnight at room temperature. The reaction mixture was cooled to 0°C, and then 3-chloroperoxybenzoic acid (595.6 mg, 2.59 mmol, 1.60 equivalence) was added. The resulting mixture was stirred at 0°C for 15 min, and then warmed to room temperature for 30 min. The reaction mixture was quenched by adding water and then extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO3 solution, 10% Na2S2O3 solution, water, and then brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was subjected to rapid chromatography on silica gel using Biotage. ® Purification was performed using a 25g column, diluted with DCM, and eluted with DCM / MeOH at a ratio of 100 / 0 to 90 / 10. The fractions were pooled and concentrated under reduced pressure to obtain the title product.

[0345] LCMS: MW (calcd): 754.2; m / z MW (obsd): 755.4 (M+H)

[0346] 1.10.2. Step B: Cpd 5

[0347] In a round-bottom flask, Pd / C 10% (0.074 g, 0.035 mmol, 0.04 equivalent) was added to a solution of dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]imidazo[1,2-b]pyridazin-7-yl]oxy-1,1-dimethyl-propyl]phosphate (0.653 g, 0.865 mmol, 1.00 equivalent) in a mixture of EtOH (10.5 mL) and water (2.6 mL). The reaction mixture was placed under vacuum and backfilled three times with H2. The reaction mixture was stirred at room temperature under H2 atmosphere for 2.5 h. The reaction was purged with N2 and another Pd / C 10% (30 mg, 0.014 mmol, 0.016 equivalent) was added. The reactants were placed under vacuum again, backfilled three times with H2, and stirred at room temperature for 2 days. The reaction vessel was purged with nitrogen, heated to 55°C, and filtered through a 0.2 μm PTFE membrane. The filter cake was washed with hot (60°C) EtOH. The filtrate was concentrated under reduced pressure. (Biotage) ®Purified by rapid chromatography on a Sfär C18 Duo 30g column, eluting with H2O + 0.1% HCOOH / ACN + 0.1% HCOOH 100 / 0 to 0 / 100 to obtain the title product.

[0348] 1.11. Cpd 6

[0349] 1.11.1. Step A: Dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]pyrazolo[1,5-a]pyrimidin-6-yl]oxy-1,1-dimethyl-propyl] phosphate

[0350] In a round-bottom flask, place a suspension of Cpd 1 (1270 mg, 2.57 mmol, 1.00 equivalence) and dibenzyldiisopropylphosphonamide (1480 mg, 1.44 mL, 3.85 mmol, 1.50 equivalence) in DCM (12.7 mL). Cool the suspension in an ice bath and add solid 4,5-dicyanimidazole (551 mg, 4.62 mmol, 1.80 equivalence) in portions. Warm the reaction mixture to room temperature. After 1 hour, add dropwise an aqueous solution of hydrogen peroxide (30% by mass) (582 mg, 0.525 mL, 5.14 mmol, 2.00 equivalence) at 0°C and stir the reaction mixture at 0°C for 2.5 hours. Quench the reaction mixture with a 10% Na₂SO₃ aqueous solution, extract with DCM, wash with a saturated NaHCO₃ aqueous solution, and then wash with 10% NaCl. Filter the organic layer on a phase separator and concentrate under reduced pressure. The crude product was processed using Biotage. ® Rapid chromatography purification using a Sfär C18 HD 120g column, eluting with H2O + 0.1% HCOOH / ACN + 0.1% HCOOH 100 / 0 to 0 / 100 to provide the title product.

[0351] LCMS: MW (calcd): 754.2; m / z MW (obsd): 755.1 (M+H)

[0352] 1H NMR (400 MHz, CD3OD) δ 8.67 (d, 1H), 8.53 (s, 1H), 8.49 (d, 1H), 7.72 (t, 1H), 7.60 (s, 1H), 7.26 (dd, 10H), 6.83 (t, 1H), 4.96 (d, 2H), 4.94(d, 2H), 4.81 – 4.78 (m, 0.5H), 4.63 (ddd, 0.5H), 4.58 (q, 1H), 3.94 (s, 3H), 2.93 – 2.87 (m, 1H), 1.63 (s, 3H), 1.59 (s, 3H), 1.35 (d, 3H), 1.23 – 1.14(m, 1H), 1.05 (ddd, 0.5H), 0.99 (ddd, 0.5H)

[0353] 1.11.2. Step B: Cpd 6

[0354] In a round-bottom flask, 10% Pd / C (0.202 g, 0.095 mmol, 0.04 equivalent) was added to a solution of dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]pyrazolo[1,5-a]pyrimidin-6-yl]oxy-1,1-dimethyl-propyl]phosphate (1.79 g, 2.37 mmol, 1.00 equivalent) in a mixture of EtOH (28.6 mL) and water (7.2 mL). The reaction mixture was placed under vacuum and backfilled three times with H2. The reaction mixture was stirred at room temperature under H2 atmosphere for 4.5 h. The reaction vessel was purged with N2, the reaction mixture was heated to 55°C and filtered through a PTFE membrane (0.2 μm). The filter cake was washed with hot (60°C) EtOH. The filtrate was concentrated under reduced pressure. The solids were ground with ACN, filtered, and dried under reduced pressure overnight at 45°C to provide Cpd 6.

[0355] 1.12. Alternative step B - Cpd 6

[0356] In a three-necked flask, 117 g (155 mmol, 1.0 equivalent) of dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]pyrazolo[1,5-a]pyrimidin-6-yl]oxy-1,1-dimethylpropyl] phosphate, ethanol (1450 mL), and water (360 mL) were added. The reaction mixture was placed under N2 and 10% wet Pd / C (11.11 g, 5.22 mmol, 0.03 equivalent) was added.

[0357] The reaction mixture was placed under vacuum, backfilled twice with H2, and then bubbled through the reaction mixture for 1 minute. The reaction mixture was stirred at room temperature under H2 at 1 atm for 23 h.

[0358] The reaction vessel was purged with nitrogen. 1M NaOH (169 mL, 169 mmol, 1.1 equivalents) and water (360 mL) were added to the reaction mixture, which was then heated to 60°C. The solution was filtered through a diatomaceous earth mat several cm high. The resulting filter cake was rinsed with a 250 mL mixture of EtOH / H₂O 3 / 2.

[0359] The filtrate was combined with another filtrate of the same size and heated to 60°C. Then 1M HCl (339 mL, 339 mmol, 2.2 equivalences) was added and precipitation began. The suspension was cooled to room temperature over 30 minutes and filtered. The resulting solid was dried to give Cpd 6.

[0360] Cpd 6 (630 g, 1097 mmol) and ethyl acetate (6300 mL) were charged into a single-jacketed reactor. The yellow suspension was stirred at 50°C for 1 h. The suspension was then cooled to room temperature and maintained for 16 h. The solid was filtered and dried under vacuum (40°C, 24 h) to give Cpd 6 as a crystalline monohydrate (Model A).

[0361] Water content (by Coulomb-Fischer method (Rivera-Quintero et al. 2024)): 3.9 wt%, equal to 1.3 molar equivalents.

[0362] Diffraction patterns were obtained on a Panalytical Aeris reflection diffractometer equipped with a Cu (λ = 1.54 Å) X-ray generator operating at 15 mA and 40 kV. The sample was held on a low-background silicon substrate and analyzed from 5–40° 2θ in 0.022° 2θ steps and 48.84 s steps. The sample was rotated during the measurement to reduce preferred orientation effects.

[0363] XRPD (° 2θ): Main peaks: 9.1, 10.0, 13.2, 13.6, and 14.9. Other peaks: 15.7, 15.9, 16.6, 17.2, 19.0, 19.3, 20.4, 20.8, 24.2, 24.8, and 26.5.

[0364] 1.12.1. Preparation of Mode C

[0365] Weigh 1 g of Cpd 6, Pattern A, into a round-bottom flask. Add anhydrous ethanol (30 mL) and heat the mixture to 50°C, 750 rpm, for 72 hours. Filter the mixture and separate it under vacuum, washing with anhydrous EtOH (5 mL). Dry the separated solid under vacuum for 1 hour. Cpd 6 is obtained as a crystalline hemihydrate (Patent C).

[0366] Water content (by Coulomb-Fischer method (Rivera-Quintero et al. 2024)): 2.0 wt%, equal to 0.65 molar equivalent.

[0367] XRPD (° 2θ): Main peaks: 9.1, 9.6, 15.1, 15.4 and 19.7. Other peaks: 10.6, 12.4, 14.1, 18.1, 18.5, 18.8, 20.0 and 20.3.

[0368] 1.13. Cpd 7

[0369] 1.13.1. Step A: 6-Chloro-4-[(4-methoxyphenyl)methoxy]-3-methyl-pyridazine

[0370] Under an argon atmosphere at 0°C, a solution of sodium hydride (60% by mass) in mineral oil (310 mg, 7.8 mmol, 1.3 equivalents) was added to a DMF (15 mL) solution of 4-methoxybenzyl alcohol (CAS# 105-13-5; 1.1 mL, 8.7 mmol, 1.5 equivalents). The resulting mixture was stirred at 0°C for 15 min, and a solution of 4,6-dichloro-3-methylpyridazine (CAS# 68240-43-7; 1.0 g, 6.0 mmol, 1.0 equivalents) dissolved in DMF (4 mL) was added dropwise. The resulting mixture was heated to room temperature and stirred for 2 hours. Saturated NH4Cl and water were added at 0°C. A precipitate was observed. The resulting mixture was filtered, and the solid was washed with water and dissolved in DCM. The resulting filtrate was filtered on a phase separator and concentrated under reduced pressure. The resulting solid was then subjected to Biotage.® Purified by chromatography on a SfärHC50g column, eluted with n-heptane / EtOAc 100 / 0 to 60 / 40 to give the title product.

[0371] LCMS: MW (calcd): 264.1; m / z MW (obsd): 575.3 (M+H)

[0372] 1.13.2. Step B: N-[5-[(4-methoxyphenyl)methoxy]-6-methyl-pyridazin-3-yl]-1,1-diphenyl-methylimine

[0373] Under an argon atmosphere, cesium carbonate (1.25 g, 3.84 mmol, 2.0 equivalent) and XantPhos Pd G3 (CAS# 1445085-97-1; 190 mg, 0.19 mmol, 0.10 equivalent) were added to a mixture of 6-chloro-4-[(4-methoxyphenyl)methoxy]-3-methylpyridazine (500 mg, 1.9 mmol, 1.0 equivalent) and benzophenone imine (CAS# 1013-88-3) (0.38 mL, 2.3 mmol, 1.2 equivalent) in 1,4-dioxane (9.4 mL). The resulting mixture was heated at 90°C overnight, and then... ® The product was filtered through a pad and concentrated under reduced pressure to obtain the title product.

[0374] LCMS: MW (calcd): 409.2; m / z MW (obsd): 410.3 (M+H)

[0375] 1.13.3. Step C: 7-[(4-methoxyphenyl)methoxy]-6-methyl-imidazo[1,2-b]pyridazine

[0376] Hydrochloric acid (2 mol / L) in water (10 mL, 20 mmol, 11 equivalents) was added dropwise to a solution of N-[5-[(4-methoxyphenyl)methoxy]-6-methyl-pyridazin-3-yl]-1,1-diphenyl-methylimine (1.9 mmol, 1.9 mmol, 1.0 equivalents) dissolved in THF (10 mL). The resulting solution was stirred at room temperature for 1 hour. The solution was quenched with a saturated aqueous solution of NaHCO3 and extracted twice with EtOAc. The resulting organic layer was filtered on a phase separator and concentrated under reduced pressure. Sodium bicarbonate (0.5 g, 6 mmol, 3 equivalents) and an aqueous solution of chloroacetaldehyde (45% by mass) (0.54 mL, 3.8 mmol, 2.0 equivalents) were added to the resulting oily substance dissolved in EtOH (10 mL). The resulting mixture was refluxed for 2 hours. The resulting mixture was then concentrated under reduced pressure, and the residue was diluted between water and EtOAc. The organic layer was filtered on a phase separator and concentrated under reduced pressure. The resulting residue was then processed in Biotage. ® Purified by rapid chromatography on a Sfär HC 25g column, eluted with n-heptane / EtOAc 100 / 0 to 20 / 80 to give the title product.

[0377] LCMS: MW (calcd): 269.2; m / z MW (obsd): 270.1 (M+H)

[0378] 1.13.4. Step D: 6-Methylimidazo[1,2-b]pyridazine-7-ol

[0379] Under an argon atmosphere, palladium hydroxide on carbon (CAS# 12135-22-7; 15 mg, 0.11 mmol, 0.19 equivalents) was added to a solution of 7-[(4-methoxyphenyl)methoxy]-6-methyl-imidazo[1,2-b]pyridazine (150 mg, 0.56 mmol, 1.0 equivalent) in MeOH (3.0 mL). The reaction mixture was placed under vacuum and backfilled with H2, then stirred at room temperature for 2 hours. The resulting mixture was then subjected to argon atmosphere and purified by Celite. ® The product was filtered, washed with EtOAc, and concentrated under reduced pressure to obtain the title product.

[0380] LCMS: MW (calcd): 149.1; m / z MW (obsd): 149.9 (M+H)

[0381] 1.13.5. Step E: Methyl (2S)-2-(6-methylimidazo[1,2-b]pyridazin-7-yl)oxypropionate

[0382] Preparation of potassium 6-methylimidazo[1,2-b]pyridazine-7-ol: t-BuOK (280 mg, 2.4 mmol, 0.68 equivalent) was added in a single batch to a solution of 6-methylimidazo[1,2-b]pyridazine-7-ol (530 mg, 3.6 mmol, 1.0 equivalent) in THF (18 mL). The resulting mixture was stirred at room temperature for 1 hour. The mixture was filtered, and the resulting solid was washed with THF and then with Et2O to obtain potassium 6-methylimidazo[1,2-b]pyridazine-7-ol.

[0383] Under an argon atmosphere at 0°C, intermediate 3 (1.43 g, 5.54 mmol, 1.0 equivalent) dissolved in DMF (2 mL) was added dropwise to a solution of potassium 6-methylimidazo[1,2-b]pyridazine-7-ol (990 mg, 5.3 mmol, 1.0 equivalent) in DMF (26 mL). The resulting solution was stirred at 0°C for 1 h. The resulting mixture was quenched with aqueous NH4Cl solution at 0°C and extracted several times with EtOAc. The combined organic layers were filtered on a phase separator and concentrated under reduced pressure. The residue was then processed in Biotage. ® Purified by rapid chromatography on a Sfär HC 25g column, eluted with DCM / MeOH 100 / 0 to 96 / 04 to obtain the title product.

[0384] LCMS: MW (calcd): 249.1; m / z MW (obsd): 250.2 (M+H)

[0385] 1.13.6. Step F: (3S)-2-methyl-3-(6-methylimidazo[1,2-b]pyridazin-7-yl)oxy-but-2-ol

[0386] Under an argon atmosphere at 0°C, a solution of methyl magnesium bromide in Et2O (2.5 mL, 7.5 mmol, 3.0 equivalence) in 12 mL of THF was added to a solution of methyl (2S)-2-(6-methylimidazo[1,2-b]pyridazin-7-yl)oxypropionate (580 mg, 2.47 mmol, 1.0 equivalence). The resulting mixture was stirred at 0°C for 1 h. The resulting solution was quenched with a saturated aqueous NH4Cl solution, alkalized with NaHCO3, and finally extracted three times with EtOAc. The combined organic layers were filtered on a phase separator and concentrated under reduced pressure. The resulting oil was then subjected to Biotage. ®The product was purified by rapid chromatography on a SNAP HC 25g column, eluted with CH2Cl2 / MeOH at 100 / 0 to 95 / 05 to obtain the title product.

[0387] LCMS: MW (calcd): 235.1; m / z MW (obsd): 236.2 (M+H)

[0388] 1.13.7. Step G: (3S)-3-(3-iodo-6-methyl-imidazo[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol

[0389] NIS (1.0 g, 4.4 mmol, 1.3 equivalent) was added in a single batch to a solution of (3S)-2-methyl-3-(6-methylimidazo[1,2-b]pyridazin-7-yl)oxy-but-2-ol (800 mg, 3.4 mmol, 1.0 equivalent) in DMF (15 mL). The resulting solution was stirred at room temperature for 1 hour. The resulting mixture was quenched with Na2S2O3 at 0°C, alkalized with NaHCO3, and extracted three times with EtOAc. The combined organic layers were filtered on a phase separator and concentrated under reduced pressure. The resulting residue was subjected to Biotage. ® The product was purified by rapid chromatography on a SNAP HC 25g column, eluting with CH2Cl2 / MeOH at 100 / 0 to 96 / 04 to obtain the title product.

[0390] LCMS: MW (calcd): 361.0; m / z MW (obsd): 362.0 (M+H)

[0391] 1.13.8. Step H: Cpd 7

[0392] Under an argon atmosphere, Pd(dppf)Cl2 (CAS# 72287-26-4; 220 mg, 0.28 mmol, 0.10 equivalent) and potassium carbonate dissolved in water (4.5 mL) were added to a solution of (3S)-3-(3-iodo-6-methyl-imidazo[1,2-b]pyridazin-7-yl)oxy-2-methyl-but-2-ol (1.03 g, 2.85 mmol, 1.00 equivalent) and intermediate 4 (1.09 g, 3.42 mmol, 1.20 equivalent) in 1,4-dioxane (20 mL). The resulting mixture was heated at 80°C for 2 h. The mixture was then diluted with EtOAc and water. The organic layer was filtered on a phase separator and concentrated under reduced pressure. The residue was then subjected to Biotage. ® Purified by rapid chromatography on a Sfär HC 50g column, eluting with n-heptane / (EtOAc / EtOH, 3 / 1) 100 / 0 to 0 / 100 to give the title product.

[0393] 1.14. Cpd 9

[0394] 1.14.1. Step A: Dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]-6-methyl-imidazo[1,2-b]pyridazin-7-yl]oxy-1,1-dimethyl-propyl] phosphate

[0395] At 0°C, 4,5-dicyanimidazolium (280 mg, 2.3 mmol, 1.0 equivalent) was added in a single addition to a suspension of Cpd 7 (670 mg, 1.3 mmol, 1.0 equivalent) and dibenzyl diisopropylphosphonamide (0.74 mL, 2.0 mmol, 1.5 equivalent) in DCM (7.0 mL). The reaction mixture was stirred at room temperature for 1 h. Hydrogen peroxide (30% by mass) (298.8 mg, 0.269 mL, 2.64 mmol, 2.0 equivalent) in water was added dropwise at 0°C, and the reaction mixture was stirred at 0°C for 1 h. The reaction mixture was quenched with 10% Na₂SO₃ aqueous solution, extracted with DCM, washed with 10% NaHCO₃ aqueous solution, and then washed with 10% NaCl aqueous solution. The organic layer was filtered through a hydrophobic filter and concentrated under reduced pressure. The resulting oil was processed using Biotage. ®The title product was purified by rapid chromatography using a Sfär C18 HD 120g column, eluted with H2O + 0.1% HCOOH / ACN + 0.1% HCOOH 100 / 0 to 0 / 100.

[0396] LCMS: MW (calcd): 768.2; m / z MW (obsd): 769.3 (M+H)

[0397] 1.14.2. Step B: Cpd 9

[0398] Under an argon atmosphere, 10% Pd / C (100 mg, 0.047 mmol, 0.038 equivalent) was added in a single batch to a solution of dibenzyl[(2S)-2-[3-[3-(difluoromethoxy)-4-[[(1R,2S)-2-fluorocyclopropyl]carbamoyl]-5-methoxy-phenyl]-6-methyl-imidazo[1,2-b]pyridazin-7-yl]oxy-1,1-dimethyl-propyl]phosphate (950 mg, 1.2 mmol, 1.0 equivalent) in EtOH (20 mL) and water (3 mL). The reaction mixture was placed under vacuum and backfilled three times with H2. The resulting black mixture was stirred overnight at room temperature, then heated at 60°C and filtered through a PTFE membrane (0.2 μm). The filter cake was washed with hot EtOH. The obtained filtrate was concentrated, the solid residue was ground with Et2O and dried under high vacuum overnight to obtain the title product.

[0399] 1.15. Cpd 10 and Cpd 11

[0400] 1.15.1. Step A: 3,7-Dichloro-5-methylpyrazolo[1,5-a]pyrimidine

[0401] 7-Chloro-5-methylpyrazolo[1,5-a]pyrimidine (CAS#16082-27-2; 2 g, 11.93 mmol, 1 equivalent) and anhydrous DMF (18.4 mL) were added to a 100 mL round-bottom flask under N2 atmosphere. NCS (1.75 g, 13.13 mmol, 1.1 equivalent) was added to the solution. The mixture was stirred at 60°C for 2 h. 10 V (180 mL) of water was added to the mixture, and the mixture was stirred at room temperature for 10 min. A precipitate formed. The suspension was filtered under vacuum to obtain a solid, which was absorbed in acetone. The suspension was filtered again. The solid was washed and rinsed with acetone. The filtrate was concentrated under reduced pressure to give the title product.

[0402] LCMS: MW (calcd): 201.0; m / z MW (obsd): 202.1-204.0 (M+H)

[0403] 1.15.2. Step B: 3-Chloro-5-methyl-pyrazolo[1,5-a]pyrimidine-7-amine

[0404] 3,7-Dichloro-5-methylpyrazolo[1,5-a]pyrimidine (1.49 g, 7.37 mmol, 1 equivalent) and ammonium hydroxide (15 mL) were charged into a pressure tube at room temperature. The mixture was stirred at room temperature and heated to 120°C for 2 h. The mixture was cooled to room temperature, and 10 V of water (150 mL) was added. The mixture was stirred at room temperature for 15 min. A precipitate formed. The suspension was filtered under vacuum to obtain the title product.

[0405] LCMS: MW (calcd): 182.0; m / z MW (obsd): 183.1-185.0 (M+H)

[0406] 1.15.3. Step C: 3-chloro-6-iodo-5-methyl-pyrazolo[1,5-a]pyrimidine-7-amine

[0407] At room temperature and under a nitrogen atmosphere, 0.76 g (4.13 mmol, 1 equivalent) of 3-chloro-5-methylpyrazolo[1,5-a]pyrimidine-7-amine and 7 mL of anhydrous DMF were added to a 50 mL round-bottom flask. NIS (1.12 g, 4.96 mmol, 1.2 equivalent) was added to the solution. The mixture was cooled to room temperature, and 70 mL of water (10 V) was added. The reaction mixture was stirred at room temperature for 15 minutes to form a precipitate. The suspension was filtered under vacuum to give the title product.

[0408] LCMS: MW (calcd): 307.9; m / z MW (obsd): 308.9-310.9 (M+H)

[0409] 1.15.4. Step D: 3-Chloro-6-iodo-5-methylpyrazolo[1,5-a]pyrimidine

[0410] 3-Chloro-6-iodo-5-methylpyrazolo[1,5-a]pyrimidin-7-amine (1.16 g, 3.76 mmol, 1 equivalent) and anhydrous THF (10 mL) were added to a 50 mL round-bottom flask under a nitrogen atmosphere. Salicylic acid (0.104 g, 0.75 mmol, 0.2 equivalent) was added to the solution, followed by dropwise addition of tert-butyl nitrite (3.88 g, 4.509 mL, 37.6 mmol, 10 equivalent). The mixture was heated to 70°C and stirred for 2 hours. The mixture was cooled to room temperature and water (70 mL) was added. The mixture was extracted with EtOAc. The organic layer was washed with 50 mL of Na₂S₂O₃ and 50 mL of brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to obtain a solid, which was dissolved in ACN to give the title product.

[0411] LCMS: MW (calcd): 292.9; m / z MW (obsd): 294.0-295.9 (M+H)

[0412] 1.15.5. Step E: 3-chloro-5-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyrimidine

[0413] At room temperature, under a N2 atmosphere, 3-chloro-6-iodo-5-methylpyrazolo[1,5-a]pyrimidine (686 mg, 2.34 mmol, 1 equivalent), bis(pinacol)diboron (CAS# 73183-34-3; 712 mg, 2.80 mmol, 1.2 equivalent), and anhydrous dioxane (10 mL) were added to a screw-cap sample vial, and the mixture was bubbled with N2 for 10 minutes. Pd(dppf)Cl2·DCM (CAS# 95464-05-4; 191 mg, 0.23 mmol, 0.1 equivalent) and potassium neopentate (CAS# 19455-23-3; 983 mg, 7.01 mmol, 3 equivalent) were added to the mixture. The mixture was stirred at 100°C for 50 minutes, then cooled to room temperature and passed through a pre-filled Celite slurry. ® Filter the solid using a pad filter. Wash the solid with EtOAc. Concentrate the filtrate under reduced pressure to obtain the title product, which can be used directly in the next step.

[0414] LCMS: MW (calcd): 293.1; m / z MW (obsd): 294.0-296.1 (M+H)

[0415] 1.15.6. Step F: 3-chloro-5-methyl-pyrazolo[1,5-a]pyrimidin-6-ol

[0416] Under a nitrogen atmosphere, at 0°C, 3-chloro-5-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-2-yl)pyrazolo[1,5-a]pyrimidine (1.93 g, 2.35 mmol, 1 equivalent) and acetone (10 mL) were added to a 100 mL round-bottom flask. A solution of potassium persulfate (0.46 g, 3.05 mmol, 1.3 equivalent) in water (20 mL) was added to the mixture. After 5 minutes, a 10% (w / w) aqueous solution of Na₂S₂O₃ (20 mL) was added to the mixture. The mixture was extracted with EtOAc (20 mL). The organic layer was washed with a 10% (w / w) Na₂S₂O₃ solution and brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by rapid chromatography and eluted with heptane / EtOAc 100 / 0 to 0 / 100 to obtain the title product.

[0417] LCMS: MW (calcd): 183.0; m / z MW (obsd): 184.0-185.9 (M+H)

[0418] 1.15.7. Step G: 3-(3-chloro-5-methyl-pyrazolo[1,5-a]pyrimidin-6-yl)oxybutane-2-one

[0419] 3-Chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-ol (235 mg, 1.28 mmol, 1 equivalent) and anhydrous DMF (3 mL) were charged into a 50 mL bottom flask at 0°C under a N2 atmosphere. K2CO3 (265.34 mg, 1.92 mmol, 1.5 equivalent) was added to the mixture, and the mixture was stirred at room temperature for 10 min. 3-Chloro-2-butanone (205 mg, 0.19 mL, 1.92 mmol, 1.5 equivalent) was added dropwise to the mixture. The mixture was stirred at room temperature for 1.2 h. 15 mL of a 10% K2CO3 aqueous solution and 15 mL of water were added to the mixture. The mixture was extracted with EtOAc. The organic layer was washed with brine and concentrated under reduced pressure. The crude product was purified by rapid chromatography, eluting with heptane / EtOAc 100 / 0 to 50 / 50 to give the title product.

[0420] LCMS: MW (calcd): 253.1; m / z MW (obsd): 254.1-256.1 (M+H)

[0421] 1.15.8. Step H: (3S)-3-(3-chloro-5-methyl-pyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol and (3R)-3-(3-chloro-5-methyl-pyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol

[0422] Under a nitrogen atmosphere at -20°C, a solution of MeMgBr (3M in Et2O) (0.34 mL, 1.034 mmol, 1.2 equivalent) was added dropwise to a solution of 3-(3-chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)oxybutane-2-one (235 mg, 0.86 mmol, 1 equivalent) and anhydrous MeTHF (2.79 mL) in a 100 mL round-bottom flask. The mixture was stirred at -20°C. The reaction mixture was then hydrolyzed at 0°C with a saturated aqueous ammonium chloride solution and stirred at room temperature for 5 minutes. The mixture was extracted with EtOAc. The organic layer was washed twice with a saturated aqueous ammonium chloride solution and brine, dried over anhydrous MgSO4, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography, eluting with DCM / (EtOAc / EtOH 3 / 1) 100 / 0 to 80 / 20 to give a mixture of enantiomers. (The text then abruptly shifts to a seemingly unrelated topic: "Through Waters ACQUITY UPC2...") ® The mixture of enantiomers was separated using SFC on the system. Column: Chiralpak IA, 150 × 21 mm, 5 μm, with guard column 50 × 21 mm, 5 μm; CO2 back pressure: 100 bar; cosolvent: EtOH, cosolvent percentage: 20%; flow rate: 60 mL / min; isocratic mode; temperature: 40°C. First eluted compound: (3R) )-3-(3-chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol, second eluting compound: (3S )-3-(3-chloro-5-methyl-pyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol.

[0423] 1.15.9. Step I: Cpd 10

[0424] In the sealed tube, (3S) 3-(3-chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol (60 mg, 0.22 mmol, 1 equivalent), intermediate 4 (85.16 mg, 0.27 mmol, 1.2 equivalent), K2CO3 (92.2 mg, 0.67 mmol, 3 equivalent), XPhos Pd G3 (CAS# 1445085-55-1; 18.8 mg, 0.022 mmol, 0.1 equivalent), dioxane (1.28 mL), and water (0.32 mL) were degassed with N2 and stirred at 100°C for 2 h. EtOAc and water were added. The organic layer was washed with brine, dried over MgSO4, filtered, evaporated, and purified by rapid chromatography, eluting with heptane / EtOAc 100 / 0 to 0 / 100 to give the title product.

[0425] 1.15.10. Step J: Cpd 11

[0426] Intermediate 4 (87.43 mg, 0.27 mmol, 1.2 equivalents), (3R) was added to a screw cap at room temperature. 3-(3-chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)oxy-2-methyl-but-2-ol (61.6 mg, 0.23 mmol, 1 equivalent), K2CO3 (94.69 mg, 0.69 mmol, 3 equivalent), anhydrous dioxane (4 mL), and water (1 mL) were added to the mixture at room temperature. XPHOS Pd G3 (CAS# 1445085-55-1; 19.3 mg, 0.023 mmol, 0.1 equivalent) was added. The mixture was heated to 110°C and stirred at 110°C for 3.5 h. The mixture was then passed through a Celite filter. ® The mixture was filtered through a pad, the solid was washed with EtOAc, and the filtrate was concentrated under reduced pressure. Water was added to the crude product. The mixture was extracted with DCM using a phase separator. The organic layer was concentrated under reduced pressure. The crude product was purified by rapid chromatography, eluting with heptane / (EtOAc / EtOH 3 / 1) 100 / 0 to 50 / 50. The product was further purified by rapid chromatography, eluting with DCM / ACN 100 / 0 to 50 / 50, to give the desired product.

[0427] 1.16. Cpd 12&13

[0428] 1.16.1. Step A: In a sealed tube, under air, a mixture of 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine (1 equivalent, 1.5 g, 8.74 mmol), Me₂NH in THF (3.6 equivalent, 15.74 mL, 31.48 mmol), and THF (30 mL) was stirred at 70°C for 1 h. DCM was added, and the mixture was concentrated under reduced pressure. The crude residue was purified by rapid chromatography on silica gel: elution with a heptane / EtOAc gradient of 100:0 to 50:50 to give 6-fluoro-N,N-dimethylpyrazolo[1,5-a]pyrimidine-5-amine (1.42 g, 90% yield).

[0429] LCMS: MW (calcd): 180.2; m / z MW (obsd): 181.1 (M+H)

[0430] 1.16.2. Step B: In a sealed circular tube, under air, a mixture of 6-fluoro-N,N-dimethylpyrazolo[1,5-a]pyrimidin-5-amine (1 equivalent, 1,400 mg, 7.77 mmol) and 10 M sodium hydroxide aqueous solution (19.31 equivalent, 15 mL, 150 mmol) in methanol (30 mL) was stirred at 100°C for 3 hours. The mixture was filtered, concentrated under reduced pressure, dissolved in MeOH, and diatomaceous earth was added. The mixture was evaporated and purified by rapid chromatography on silica gel: first with DCM / MEOH 100:0 to 90:10, then with a DCM / MEOH 50:50 gradient to give 6-fluoropyrazolo[1,5-a]pyrimidin-5-ol (530 mg, 38% yield).

[0431] LCMS: MW (calcd): 178.2; m / z MW (obsd): 179.1 (M+H)

[0432] 1.16.3. Step C: In a sealed tube under air, a mixture of 6-fluopyrazolo[1,5-a]pyrimidin-5-ol (1 equivalent, 530 mg, 2.97 mmol), 3-chloro-2-butanone (1.5 equivalent, 475.4 mg, 0.45 mL, 4.46 mmol), K₂CO₃ (1.5 equivalent, 616.6 mg, 4.46 mmol), and DMF (6 mL) was stirred at room temperature for 1 h. EtOAc (20 mL) was added, and the organic layer was washed with water (3 x 15 mL) and brine (2 x 15 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue was purified on silica gel by rapid chromatography: elution with a gradient of heptane / EtOAc 100:0 to 0:100 to give 3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (416 mg, 56% yield).

[0433] LCMS: MW (calcd): 248.3; m / z MW (obsd): 249.2 (M+H)

[0434] 1.16.4. Step D: In a round-bottom flask, under N2 and at -20°C, 3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (1 equivalent, 410 mg, 1.65 mmol) in MeTHF (26.78 mL) was added. The reaction mixture was degassed three times by vacuum / N2 circulation, maintained under N2 atmosphere, and MeMgBr 3M (2 equivalents, 1.10 mL, 3.30 mmol) in Et2O was added at -20°C. The reaction mixture was stirred at -20°C for 1 h. EtOAc (30 mL) and an aqueous solution of NH4Cl (30 mL) were added. The organic layer was washed with brine (20 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified on silica gel by rapid chromatography: elution with a gradient of heptane / EtOAc 100:0 to 0:100 to give a mixture of title enantiomers, which was then purified on Waters ACQUITY UPC2. ® The system was separated using Chiral SFC. Column: LuxCellulose-4, 150 x 21.2 mm, 5 μm; CO2 back pressure: 100 bar; cosolvent: EtOH, cosolvent percentage: 15%; flow rate: 60 mL / min; isocratic mode; temperature: 40°C. (Lux Cellulose-4, 150 x 21.2 mm, 5 μm, yielded the first eluted compound (R) )-3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (109 mg, ee 100%) arbitrarily specified configuration R and second eluting compound (S) )-3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (115 mg, ee 99.9%) in any specified configuration S.

[0435] LCMS: MW (calcd): 264.3; m / z MW (obsd): 265.2 (M+H), for the two enantiomers

[0436] 1.16.5. Step E: In a sealed tube, under air, (R) 3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 109 mg, 0.41 mmol) was added to DMF (1.5 mL). Then, NIS (1.1 equivalent, 102.0 mg, 0.45 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. EtOAc (10 mL) was added, and the organic layer was washed with water (3 x 5 mL) and brine (2 x 5 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give (R )-3-((5-(dimethylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (131.5 mg, 82% yield).

[0437] LCMS: MW (calcd): 390.2; m / z MW (obsd): 391.1 (M+H)

[0438] 1.16.6. Step F: In the sealed tube, under N2, (R) A mixture of 3-((5-(dimethylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 130 mg, 0.33 mmol), intermediate 4 (1.2 equivalent, 127.54 mg, 0.4 mmol), Cs₂CO₃ (1.2 equivalent, 130.2 mg, 0.4 mmol), and PdCl₂dppf.DCM (0.1 equivalent, 27.21 mg, 0.033 mmol) was dissolved in dioxane (10.5 mL) and water (2.5 mL). The mixture was degassed with N₂ for 10 min. Then, the reaction mixture was degassed three times under a vacuum / N₂ cycle while maintaining an N₂ atmosphere. The reaction mixture was stirred at 90°C for 1 h. The reaction mixture was concentrated under reduced pressure and the crude residue was purified by preparative LCMS to give 2-(difluoromethoxy)-4-(5-(dimethylamino)-6-(((R) )-3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide, Cpd 12 (67 mg, 37% yield).

[0439] LCMS: MW (calcd): 537.5; m / z MW (obsd): 538.3 (M+H)

[0440] ee%=100

[0441] 1.16.7. Step G: In a sealed tube, under air, (S) 3-((5-(dimethylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 115 mg, 0.44 mmol) was added to DMF (1.5 mL). Then, NIS (1.1 equivalent, 107.67 mg, 0.48 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. EtOAc (10 mL) was added, and the organic layer was washed with water (3 x 5 mL) and brine (2 x 5 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give (S )-3-((5-(dimethylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (139.4 mg, 82% yield).

[0442] LCMS: MW (calcd): 390.2; m / z MW (obsd): 391.1 (M+H)

[0443] 1.16.8. Step H

[0444] In the sealed tube, under N2, (S) A mixture of 3-((5-(dimethylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 138 mg, 0.35 mmol), intermediate 4 (1.2 equivalent, 135.4 mg, 0.42 mmol), Cs₂CO₃ (1.2 equivalent, 138.3 mg, 0.42 mmol), and PdCl₂dppf.DCM (0.1 equivalent, 28.9 mg, 0.035 mmol) was dissolved in dioxane (10.5 mL) and water (2.5 mL). The mixture was degassed with N₂ for 10 min. Then, the reaction mixture was degassed three times under vacuum / N₂ circulation while maintaining an N₂ atmosphere. The reaction mixture was stirred at 90°C for 1 h. The reaction mixture was concentrated under reduced pressure and the crude residue was purified by preparative LCMS to give 2-(difluoromethoxy)-4-(5-(dimethylamino)-6-(((S )-3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide, Cpd 13 (67 mg, 35% yield).

[0445] LCMS: MW (calcd): 537.5; m / z MW (obsd): 538.5 (M +H)

[0446] ee%=100%

[0447] Cpd 14 and 15

[0448] 1.16.9. Step A: 3-Aminopyrazole (1 equivalent, 10 g, 120.35 mmol) and AcOH (60 mL) were charged into a 250 mL round-bottom flask under air and stirred for 5 min at room temperature. Ethyl propionyl acetate (1 equivalent, 17.35 g, 17.18 mL, 120.35 mmol) was added to the resulting solution at room temperature. The reaction mixture was heated under reflux for 3 h. The reaction was stopped and cooled to room temperature. The resulting suspension was filtered. The resulting white solid was washed twice with MeOH (2 x 1 mL) and dried under vacuum to give 5-ethylpyrazolo[1,5-a]pyrimidin-7-ol (13.51 g, 68% yield).

[0449] LCMS: MW (calcd): 163.2; m / z MW (obsd): 164.2 (M+H)

[0450] 1.16.10. Step B: 5-Ethylpyrazolo[1,5-a]pyrimidine-7-ol (1 equivalent, 500 mg, 3.06 mmol) was added to a 25 mL round-bottom flask and placed under a nitrogen atmosphere. Anhydrous dioxane (2.8 mL) was then added at room temperature, followed by diethylaniline (2 equivalents, 914.5 mg, 0.98 mL, 6.13 mmol) and POCl3 (5 equivalents, 2.35 g, 1.43 mL, 15.32 mmol). The mixture was heated at 105°C for 2 h. The reaction was stopped and cooled to room temperature. The reaction mixture was concentrated under reduced pressure (carefully quenching the evaporated POCl3). The evaporated residue was dissolved in DCM (12 mL) and alkalized with saturated NaHCO3 (pH = 7-8, to V = 20 mL). The two phases were separated, and the aqueous phase was further extracted with DCM. The combined organic phases were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was then purified on silica gel by rapid chromatography: elution with heptane / AcOEt 100 to 80 / 20 to give 7-chloro-5-ethylpyrazolo[1,5-a]pyrimidine (493.5 mg, 89% yield).

[0451] LCMS: MW (calcd): 181.6; m / z MW (obsd): 183.9 (M+H)

[0452] 1.16.11. Step C: Under nitrogen atmosphere, an anhydrous DMF solution (16.91 mL) of 7-chloro-5-ethylpyrazolo[1,5-a]pyrimidine (1 equivalent, 2 g, 11.012 mmol) was added to a 100 mL round-bottom flask, followed by NCS (1.1 equivalent, 1.62 g, 12.11 mmol). The reaction mixture was heated at 60°C for 2 h. The reaction was stopped and cooled to room temperature. 10 V of water (160 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 10 min. The resulting mixture was extracted with EtOAc (40 mL), the two phases were separated, and the aqueous phase was further extracted with EtOAc (twice with 40 mL). The combined organic phases were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was then purified by rapid chromatography on silica gel. Elution with heptane / AcOEt 100 to 80 / 20 yielded 3,7-dichloro-5-ethylpyrazolo[1,5-a]pyrimidine (2.09 g, 88% yield).

[0453] LCMS: MW (calcd): 216.07; m / z MW (obsd): 216.0 – 218.0 (M+H)

[0454] 1.16.12. Step D

[0455] 3,7-Dichloro-5-ethylpyrazolo[1,5-a]pyrimidine (1 equivalent, 2 g, 9.26 mmol) and NH4OH (19.83 mL) were charged into a 10 mL round-bottom flask under air at room temperature. The reaction mixture was heated at 100°C for 2 h. 10 mL of NH4OH was added, and the reaction mixture was heated at 100°C for another 2 h. Heating was stopped, and the reaction mixture was stirred at room temperature for 1.5 days. The reaction was stopped. The resulting suspension was filtered. The yellow solid was washed with water (twice with 4 mL) and dried under vacuum to give 3-chloro-5-ethylpyrazolo[1,5-a]pyrimidine-7-amine (1.8 g, 99% yield).

[0456] LCMS: MW (calcd): 196.6; m / z MW (obsd): 197.1 (M+H)

[0457] 1.16.13. Step E: Under nitrogen atmosphere, an anhydrous DMF solution (18 mL) of 3-chloro-5-ethylpyrazolo[1,5-a]pyrimidine-7-amine (1 equivalent, 1.8 g, 9.15 mmol) was added to a 250 mL round-bottom flask, followed by the addition of NCS (1.3 equivalent, 1.59 g, 11.9 mmol). The reaction mixture was heated at 50°C for 2.5 h. The reaction was stopped and cooled to room temperature. 10 V of water (180 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 10 min. The resulting suspension was filtered. The white solid was washed with water (0.2 mL) and dried under vacuum to give 3,6-dichloro-5-ethylpyrazolo[1,5-a]pyrimidine-7-amine (1.87 g, 88% yield).

[0458] LCMS: MW (calcd): 231.1; m / z MW (obsd): 231.0 – 233.0 (M+H)

[0459] 1.16.14. Step F: Under nitrogen atmosphere, a solution of 3,6-dichloro-5-ethylpyrazolo[1,5-a]pyrimidin-7-amine (1 equivalent, 1.85 g, 8.01 mmol) in anhydrous THF (20.5 mL) was added dropwise to the solution, followed by the addition of tert-butyl nitrite (10 equivalents, 8.26 g, 9.6 mL, 80.06 mmol), and then salicylic acid (0.2 equivalents, 0.22 g, 1.61 mmol). The reaction mixture was heated at 70°C for 1 h. The reaction was stopped and cooled to room temperature. Water (205 mL) was added to the reaction mixture, and the resulting mixture was extracted with EtOAc (150 mL). The two phases were separated, and the aqueous phase was further extracted with EtOAc (twice with 50 mL). The combined organic phases were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was then purified on silica gel by rapid chromatography: elution with heptane / AcOEt 100 to 80 / 20 to give 3,6-dichloro-5-ethylpyrazolo[1,5-a]pyrimidine (957.5 mg, 55% yield).

[0460] LCMS: MW (calcd): 216.1; m / z MW (obsd): 216.0 – 218.0 (M+H)

[0461] 1.16.15. Step G

[0462] Under nitrogen atmosphere, a solution of 3,6-dichloro-5-ethylpyrazolo[1,5-a]pyrimidine (1 equivalent, 950 mg, 4.4 mmol) in 21.0 mL of methanol was added to a 100 mL round-bottom flask, and NaOH was added. 2N (15 equivalents, 33.0 mL, 66.0 mmol) was added to the solution. The reaction mixture was heated at 65°C for 1.15 h. The reaction mixture was then heated at 65°C for another 2 h, stopped, and cooled to room temperature. It was then quenched with HCl 2N (15 equivalents, 33.0 mL, 66.0 mmol) at room temperature and stirred for 5 min at room temperature. The resulting suspension was filtered. The white solid was washed with water (twice with 10 mL) and dried under vacuum to give 3-chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-ol (675.8 mg, 78% yield).

[0463] LCMS: MW (calcd): 197.6; m / z MW (obsd): 198.0 – 200.0 (M+H)

[0464] 1.16.16. Step H: 3-Chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-ol (1 equivalent, 670 mg, 3.4 mmol) was added to a 10 mL round-bottom flask and placed under a nitrogen atmosphere. Anhydrous DMF (13.4 mL) was then added at 0°C (ice / water bath), followed by K₂CO₃ (1.5 equivalent, 702.8 mg, 5.09 mmol). The reaction mixture was stirred at room temperature for 10 min, followed by dropwise addition of 3-chloro-2-butanone (1.5 equivalent, 541.9 mg, 0.52 mL, 5.09 mmol). The reaction mixture was stirred at room temperature for 3 h and then stopped. 25 mL of 10% K₂CO₃ solution and 25 mL of water were added to the mixture. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by rapid chromatography on silica gel: elution with heptane / AcOEt 100 to 50 / 50 to give 3-((3-chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (819.9 mg, 90% yield).

[0465] LCMS: MW (calcd): 267.7; m / z MW (obsd): 267.8 – 270.1 (M+H); 266.1 –268.1 (MH)

[0466] 1.16.17. Step I: Under nitrogen atmosphere at -20°C, a solution of MeMgBr (3M Et2O) (1.2 equivalent, 1.05 mL, 3.14 mmol) was added dropwise to a solution of 3-((3-chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (1 equivalent, 700 mg, 2.61 mmol) and anhydrous MeTHF (10.4 mL) in a 100 mL round-bottom flask. The mixture was stirred at -20°C for 10 min. The reaction was stopped. The reaction mixture was hydrolyzed at 0°C with a saturated ammonium chloride solution and stirred at room temperature for 5 min. The mixture was extracted with EtOAc. The organic layer was washed twice with a saturated ammonium chloride solution and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was then purified by rapid chromatography on silica gel. Elution with heptane / AcOEt 100 to 40 / 60 yielded 3-((3-chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (461.9 mg, 62% yield).

[0467] LCMS: MW (calcd): 283.8; m / z MW (obsd): 284.2 – 286-1 (M+H)

[0468] 1.16.18. Step J: Intermediate 4 (1.2 equivalents, 0.27 g, 0.85 mmol), 3-((3-chloro-5-ethylpyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 0.2 g, 0.71 mmol), XPhos Pd G3 (0.05 equivalents, 0.03 g, 0.035 mmol), and Cs₂CO₃ (1.2 equivalents, 0.28 g, 0.85 mmol) were added to a vial and degassed with argon. Anhydrous degassed water (0.75 mL) and dioxane (3 mL) were added at room temperature, and the reaction mixture was degassed four times under vacuum / argon circulation while maintaining an argon atmosphere. The reaction mixture was then refluxed under an argon atmosphere and heated for 2 h, and the reaction was stopped. The reaction mixture was filtered through a diatomaceous earth pad and the solids were washed with EtOAc. The filtrate was washed twice with water and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Then, it was processed through Waters ACQUITY UPC2. ® The crude product was purified by chiral separation using a single-cell electrophoresis system. Column: Lux Cellulose-1, 150 x 21.2 mm, 5 μm; CO2 back pressure: 100 bar; cosolvent: EtOH, cosolvent percentage: 15%; flow rate: 60 mL / min; isocratic mode; temperature: 40°C. The first elution of the diastereomer (arbitrarily designated R as shown) yielded Cpd 14 (103.8 mg) in 30% yield; the second elution of the diastereomer (arbitrarily designated S) yielded Cpd 15 (53.7 mg) in 16% yield.

[0469] 1.16.19. Cpd 14

[0470] LCMS: MW (calcd): 522.5; m / z MW (obsd): 523.3 (M+H)

[0471] ee%=100%

[0472] 1.16.20. Cpd 15

[0473] LCMS: MW (calcd): 522.5; m / z MW (obsd): 523.4 (M+H)

[0474] ee%=93.6%

[0475] 1.17. Cpd 16 and Cpd 17

[0476] 1.17.1. Step A

[0477] 3-Aminopyrazole (1 equivalent, 10 g, 120.4 mmol) and AcOH (60 mL) were added to a 2,500 mL round-bottom flask under air and stirred for 5 minutes at room temperature. Ethyl 4,4-difluoro-3-oxobutyrate (1 equivalent, 20.0 g, 120.4 mmol) was added to the resulting solution at room temperature. The reaction mixture was heated under reflux. Rapid formation of a white precipitate was observed. 60 mL of NH4OH was added, and the reaction mixture was heated at 100°C for 2 hours, then stirred overnight at room temperature. The resulting suspension was filtered. The yellow solid was washed with water (twice with 25 mL) and dried under vacuum to give 5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-7-ol (11.12 g, 99% yield).

[0478] LCMS: MW (calcd): 185.1; m / z MW (obsd): 186.2 (M+H)

[0479] 1.17.2. Step B

[0480] 5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-7-ol (1 equivalent, 2 g, 10.80 mmol) was added to a 50 mL round-bottom flask and placed under a nitrogen atmosphere. Anhydrous dioxane (10 mL) was then added at room temperature, followed by diethylaniline (2 equivalents, 3.22 g, 3.46 mL, 21.6 mmol) and POCl3 (5 equivalents, 8.28 g, 5.0 mL, 54.0 mmol). The reaction mixture was heated at 105°C for 2 h, then stopped and cooled to room temperature. The reaction mixture was concentrated under reduced pressure (carefully quenching the evaporated POCl3). The evaporated residue was absorbed in DCM (60 mL) and alkalized with saturated NaHCO3 (to pH 7-8). The two phases were separated, and the aqueous phase was further extracted with DCM. The combined organic phases were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was then purified by rapid chromatography on silica gel. Elution with heptane / AcOEt 100-80 / 20 yielded 7-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (1.67 g, 76% yield).

[0481] LCMS: MW (calcd): 203.6; m / z MW (obsd): 203.9 – 205.9 (M+H)

[0482] 1.17.3. Step C

[0483] 7-Chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (1 equivalent, 12.73 g, 62.53 mmol) and anhydrous DMF (400 mL) were charged into a 500 mL bottom flask under nitrogen atmosphere at room temperature. NCS (1.3 equivalent, 10.85 g, 81.3 mmol) was added to the mixture. The mixture was heated to 60°C and stirred at 60°C for 2 h. Water was added to the mixture, and the mixture was stirred at room temperature for 20 min. A precipitate formed and was filtered to give 3,7-dichloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (13.54 g, 91% yield).

[0484] LCMS: MW (calcd): 238.0; m / z MW (obsd): 238.1 – 239.1 (M+H)

[0485] 1.17.4. Step D

[0486] 3,7-Dichloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (1 equivalent, 15.3 g, 64.2 mmol) and ammonium hydroxide (500 mL) were charged into a 1,000 mL round-bottom flask under nitrogen atmosphere at room temperature. The mixture was stirred and heated to 100°C for 1.75 h at room temperature. The mixture was cooled to room temperature, and water (300 mL) was added. The mixture was stirred at room temperature for 15 min, resulting in the formation of a yellow precipitate. The suspension was filtered under vacuum to give 3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine-7-amine (14.56 g, 100% yield).

[0487] LCMS: MW (calcd): 218.6; m / z MW (obsd): 219.0 – 221.0 (M+H)

[0488] 1.17.5. Step E

[0489] 5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine-7-amine (1 equivalent, 14.56 g, 66.6 mmol) and anhydrous DMF (150 mL) were charged into a 250 mL round-bottom flask under nitrogen atmosphere at room temperature. NBS (1.5 equivalent, 17.8 g, 99.90 mmol) was added to the mixture. The mixture was stirred at 50°C for 1 h and cooled to RT, and then water (300 mL) was added to the mixture. The mixture was stirred at room temperature for 15 min. A gray precipitate formed and was filtered to give 6-bromo-3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine-7-amine (18.03 g, 91% yield).

[0490] LCMS: MW (calcd): 297.5; m / z MW (obsd): 297.0-299.0 (M+H)

[0491] 1.17.6. Step F

[0492] At room temperature under nitrogen atmosphere, 6-bromo-3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-7-amine (1 equivalent, 1.1 g, 3.7 mmol) and anhydrous THF (11 mL) were added dropwise to a 100 mL round-bottom flask. Tert-butyl nitrite (10 equivalent, 3.81 g, 4.43 mL, 36.98 mmol) and DMSO (0.1 equivalent, 0.029 g, 0.026 mL, 0.37 mmol) were added dropwise. The mixture was heated to 70°C and stirred at 70°C for 2 h. The mixture was cooled to room temperature and water was added. The mixture was extracted with EtOAc. The organic layer was washed twice with water and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by rapid chromatography, eluting with a gradient of heptane 100%, heptane / EtOAc 100 to 90 / 10 and DCM 100% to give 6-bromo-3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (0.62 g, 59% yield).

[0493] LCMS: MW (calcd): 282.5; m / z MW (obsd): 282.0-284.0 (M+H)

[0494] 1.17.7. Step G

[0495] At room temperature under nitrogen atmosphere, add bis(pinacol)diboron (1.2 equivalents, 1011.91 mg, 3.98 mmol), 6-bromo-3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidine (1 equivalent, 938 mg, 3.32 mmol), and anhydrous dioxane (10 mL) to a 10–20 mL screw-cap vial. Pd(dppf)Cl2·DCM (0.1 equivalents, 271.2 mg, 0.33 mmol) and potassium neopentate (3 equivalents, 1.4 g, 9.96 mmol) to the mixture. Stir the mixture at 100°C for 15 min. Stop the reaction. Cool the mixture to room temperature and filter it on a pre-filled diatomaceous earth mat. Wash the solid with EtOAc. The filtrate was concentrated under reduced pressure to obtain 3-chloro-5-(difluoromethyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyrazolo[1,5-a]pyrimidine (3.13 g), which was used directly in step H.

[0496] 1.17.8. Step H

[0497] Under nitrogen atmosphere and at 0°C (ice / water bath), 3-chloro-5-(difluoromethyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-2-yl)pyrazolo[1,5-a]pyrimidine (1 equivalent, 3.13 g, 3.31 mmol) and acetone (15 mL) were added to a 100 mL round-bottom flask. A solution of Oxone (1.3 equivalent, 0.66 g, 4.31 mmol) in water (30 mL) was added to the mixture at 0°C, and the reaction was monitored directly by LCMS. The reaction was stopped, and a 10% (w / w) Na₂S₂O₃ solution (20 mL) was added to the mixture at 0°C. The mixture was extracted with EtOAc. The organic layer was washed with a 10% (w / w) Na₂S₂O₃ solution and brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude residue was purified on silica gel by rapid chromatography: elution with DCM 100%, DCM / (EtOAc / EtOH 3 / 1) 100 to 90 / 10 gradients to give 3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-ol (495 mg, 68% yield).

[0498] LCMS: MW (calcd): 219.6; m / z MW (obsd): 220.0-222.0 (M+H).

[0499] 1.17.9. Step I

[0500] Under nitrogen atmosphere and at 0°C, 3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-ol (1 equivalent, 495 mg, 2.25 mmol) and anhydrous DMF (5 mL) were added to a 50 mL round-bottom flask. K₂CO₃ (1.5 equivalent, 467.33 mg, 3.38 mmol) was added to the mixture, and the mixture was stirred at room temperature for 10 min. 3-chloro-2-butanone (1.5 equivalent, 360.29 mg, 0.34 mL, 3.38 mmol) was added dropwise to the mixture. The mixture was stirred at room temperature for 3.5 h. Water was added to the mixture. The mixture was extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous MgSO4, filtered, concentrated under reduced pressure, and purified on silica gel by rapid chromatography: eluted with 100% heptane and a gradient of heptane / EtOAc 100 to 50 / 50 to give 3-((3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (376 mg, 58% yield).

[0501] LCMS: MW (calcd): 289.7; m / z MW (obsd): 290.1-292.1 (M+H).

[0502] 1.17.10. Step J

[0503] Under nitrogen atmosphere at -20°C, a solution of MeMgBr (3M Et2O) (1.2 equivalent, 0.52 mL, 1.56 mmol) was added dropwise to a solution of 3-((3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (1 equivalent, 376 mg, 1.3 mmol) and anhydrous MeTHF (5.18 mL) in a 100 mL round-bottom flask. The mixture was stirred at -20°C and monitored by LCMS immediately after the addition of MeMgBr. The reaction was stopped and hydrolyzed with saturated ammonium chloride solution at 0°C, stirred at room temperature for 5 min. The mixture was extracted with EtOAc. The organic layer was washed twice with saturated ammonium chloride solution and brine, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure, and purified by rapid chromatography on silica gel: gradient elution: DCM 100%, DCM / (EtOAc / EtOH 3 / 1) 100 to 80 / 20, to give 3-((3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (176 mg, 44% yield).

[0504] LCMS: MW (calcd): 305.7; m / z MW (obsd): 306.1 – 308.1 (M+H).

[0505] 1.17.11. Step K

[0506] Under nitrogen atmosphere and at room temperature, intermediate 4 (1.2 equivalents, 95.2 mg, 0.3 mmol), 3-((3-chloro-5-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1.16 equivalents, 88 mg, 0.29 mmol), K₂CO₃ (3 equivalents, 103.1 mg, 0.75 mmol), anhydrous dioxane (4 mL), and water (1 mL) were added to the mixture. XPHOS Pd G₃ (0.1 equivalents, 21.0 mg, 0.025 mmol, CAS# 1445085-55-1) was added to the mixture. The mixture was heated to 110°C and stirred at 110°C for 1 h. The mixture was filtered on a diatomaceous earth mat, and the solid was washed with EtOAc. The filtrate was concentrated under reduced pressure to give a black oil. The mixture was extracted with EtOAc. The organic layer was washed twice with water and brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and then purified directly on silica gel by rapid chromatography: gradient elution: heptane 100%, heptane / EtOAc 100 to 50 / 50 and DCM 100%, DCM / ((EtOAc / EtOH 3 / 1)) 100 to 90 / 10, to give 2-(difluoromethoxy)-4-(5-(difluoromethyl)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide (86.8 mg, 55% yield).

[0507] LCMS: MW (calcd): 544.5; m / z MW (obsd): 545.2 (M+H).

[0508] 1.17.12. Step L: Chiral Separation

[0509] At Waters ACQUITY UPC2 ® Two diastereomers were separated in the system using chiral SFC. Column: LuxCellulose-1, 150 x 21.2 mm, 5 μm; CO2 back pressure: 100 bar; cosolvent: EtOH, cosolvent percentage: 15%; flow rate: 60 mL / min; isocratic mode; temperature: 40°C. The resulting product was arbitrarily designated as 2-(difluoromethoxy)-4-(5-(difluoromethyl)-6-(((R) The first eluting diastereomer of 3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd16 (34.1 mg) and arbitrarily designated as 2-(difluoromethoxy)-4-(5-(difluoromethyl)-6-(((S The second eluted diastereomer of 3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd17 (23.4 mg).

[0510] Cpd 16: %ee: 100

[0511] Cpd 17: %ee: 97.0

[0512] 1.18. Cpd 18 and Cpd 19

[0513] 1.18.1. Step A

[0514] In a sealed tube under air, a mixture of 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine (1 equivalent, 500 mg, 2.91 mmol), dibenzylamine (2 equivalents, 1,150 mg, 1.13 mL, 5.83 mmol), and DIPEA (2 equivalents, 753.4 mg, 1.0 mL, 5.83 mmol) in isopropanol (5 mL) was stirred at 100°C for 24 h. The mixture was evaporated. Water and DCM were added. The organic layer was washed with brine, dried over MgSO4, filtered, evaporated, and then purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 50:50 to give N,N-dibenzyl-6-fluoropyrazolo[1,5-a]pyrimidine-5-amine (970 mg, 88% yield).

[0515] LCMS: MW (calcd): 332.4; m / z MW (obsd): 333.1 (M+H).

[0516] 1.18.2. Step B

[0517] In a sealed vial under air, a mixture of N,N-dibenzyl-6-fluoropyrazolo[1,5-a]pyrimidin-5-amine (1 equivalent, 920 mg, 2.44 mmol) and 10 M sodium hydroxide aqueous solution (19.47 equivalent, 4.74 mL, 47.43 mmol) in methanol (9.49 mL) was stirred at 100°C for 6 hours. The mixture was evaporated and purified by rapid chromatography on silica gel: elution with a gradient of DCM / MeOH in 20 mL from 100:0 to 90:10 gave a yellow solid, which was ground with MeOH and filtered to give 5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-ol (215 mg, 27% yield).

[0518] LCMS: MW (calcd): 330.4; m / z MW (obsd): 331.1 (M+H).

[0519] 1.18.3. Step C

[0520] In a round-bottom flask, under air, a mixture of 5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-ol (1 equivalent, 600 mg, 1.82 mmol), 3-chloro-2-butanone (1.5 equivalent, 290.2 mg, 0.28 mL, 2.72 mmol), K₂CO₃ (1.5 equivalent, 376.5 mg, 2.72 mmol), and DMF (3.63 mL) was stirred at room temperature for 2 h. EtOAc and water were added. The organic layer was washed with brine, dried (MgSO₄), filtered, evaporated, and purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 0:100 to give 3-((5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)butanone (571 mg, 79% yield).

[0521] LCMS: MW (calcd): 400.5; m / z MW (obsd): 401.3 (M+H).

[0522] 1.18.4. Step D

[0523] In a round-bottom flask, under N2 and at -20°C, 3M MeMgBr in Et2O (2 equivalents, 0.95 mL, 2.85 mmol) was added to 3-((5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (1 equivalent, 571 mg, 1.43 mmol) in MeTHF (17.2 mL). The mixture was stirred at -20°C for 2 h. EtOAc and an aqueous solution of NH4Cl were added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 0:100 to give 3-((5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (418 mg, 70% yield).

[0524] LCMS: MW (calcd): 416.5; m / z MW (obsd): 417.3 (M+H).

[0525] 1.18.5. Step E

[0526] In a sealed tube, a mixture of 3-((5-(dibenzylamino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 418 mg, 1.01 mmol) and NIS (1.1 equivalent, 248.4 mg, 1.11 mmol) in DMF (4 mL) was stirred at room temperature for 1 h. EtOAc and water were added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 0:100 to give 3-((5-(dibenzylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (528 mg, 97% yield).

[0527] LCMS: MW (calcd): 542.4; m / z MW (obsd): 543.2 (M+H).

[0528] 1.18.6. Step F

[0529] In a sealed tube, a mixture of 3-((5-(dibenzylamino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 528 mg, 0.97 mmol), intermediate 4 (1.2 equivalent, 372.7 mg, 1.17 mmol), Cs₂CO₃ (1.2 equivalent, 380.6 mg, 1.17 mmol), PdCl₂dppf.DCM (0.1 equivalent, 79.5 mg, 0.097 mmol), dioxane (7.5 mL), and water (1.5 mL) was degassed with N₂ and stirred at 90°C for 2 hours. EtOAc and water were then added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified on silica gel by rapid chromatography: eluted with a gradient of heptane / EtOAc 100:0 to 0:100 to give 4-(5-(dibenzylamino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide (342 mg, 51% yield).

[0530] LCMS: MW (calcd): 689.7; m / z MW (obsd): 690.7 (M+H).

[0531] 1.18.7. Step G

[0532] In a round-bottom flask at 0°C, trifluoromethanesulfonic acid (20 equivalents, 217.6 mg, 0.13 mL, 1.45 mmol) was added to a solution of 4-(5-(dibenzylamino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide (1 equivalent, 50 mg, 0.072 mmol) in DCM (0.9 mL). The mixture was stirred at 0°C for 10 min. It was then kept at room temperature for 18 h. DCM and a saturated aqueous solution of NaHCO3 were added until pH 7, and the mixture was stirred at room temperature. The organic layer was separated using an isolate phase separator and evaporated. The crude residue was purified by preparative LCMS to obtain 4-(5-(benzylamino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd18 (10 mg, 23% yield).

[0533] LCMS: MW (calcd): 599.6; m / z MW (obsd): 600.4 (M+H).

[0534] Step H

[0535] Pd(OH)₂ was packed into an H-Cube Nano. A solution of 4-(5-(dibenzylamino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide (1 equivalent, 50 mg, 0.072 mmol) in isopropanol (2 mL) was run at 1 mL / min, 1 bar of hydrogen, and 70°C. The resulting mixture was used for a second run at 1 mL / min, 1 bar of hydrogen, and 70°C. The resulting mixture was used for a third run at 1 mL / min, 80 bar of hydrogen, and 70°C. The resulting mixture was used for a fourth run at 1 mL / min, 80 bar of hydrogen, and 100°C. The mixture was evaporated and purified by preparative LCMS (PrepB 50 mL 35-50 8 min) to give (5-amino-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd 19 (1 mg, 3% yield).

[0536] LCMS: MW (calcd): 509.5; m / z MW (obsd): 510.2 (M+H).

[0537] 1.19. Cpd 20, Cpd 21 and Cpd 22

[0538] 1.19.1. Step A

[0539] In a sealed tube under air, a mixture of 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine (1 equivalent, 500 mg, 2.91 mmol), N-methylbenzylamine (1.2 equivalent, 423.82 mg, 0.45 mL, 3.5 mmol), and THF (10 mL) was stirred at 70°C for 18 h. N-methylbenzylamine (0.5 equivalent, 176.59 mg, 0.19 mL, 1.46 mmol) was added, and the mixture was stirred at 70°C for 18 h. The mixture was evaporated and purified by rapid chromatography on silica gel: elution with a heptane / EtOAc gradient from 100:0 to 50:50 gave N-benzyl-6-fluoro-N-methylpyrazolo[1,5-a]pyrimidine-5-amine (658 mg, 88%).

[0540] LCMS: MW (calcd): 256.3; m / z MW (obsd): 257.2 (M+H).

[0541] 1.19.2. Step B

[0542] In a sealed round vial, under air, a mixture of N-benzyl-6-fluoro-N-methylpyrazolo[1,5-a]pyrimidin-5-amine (1 equivalent, 658 mg, 2.57 mmol) and 10 M NaOH aqueous solution (19.47 equivalent, 5 mL, 50 mmol) in MeOH (10 mL) was stirred at 100°C for 3 hours. The mixture was evaporated and purified by rapid chromatography, eluting with a DCM / MeOH gradient of 100:0 to 90:10, to give 5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-ol (325 mg, 50%).

[0543] LCMS: MW (calcd): 254.3; m / z MW (obsd): 255.4 (M+H).

[0544] 1.19.3. Step C

[0545] In a round-bottom flask, a mixture of 5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-ol (1 equivalent, 325 mg, 1.28 mmol), 3-chloro-2-butanone (1.5 equivalent, 204.26 mg, 0.19 mL, 1.92 mmol), K₂CO₃ (1.5 equivalent, 264.95 mg, 1.92 mmol), and DMF (2.56 mL) was stirred at room temperature for 1 hour. EtOAc and water were added. The organic layer was washed with brine, dried (MgSO₄), filtered, evaporated, and purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 0:100 to give 3-((5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)butanone (300 mg, 72% yield).

[0546] LCMS: MW (calcd): 324.4; m / z MW (obsd): 325.2 (M+H).

[0547] 1.19.4. Step D

[0548] In a round-bottom flask, under N2 and at -20°C, 3M MeMgBr (2 equivalents, 0.62 mL, 1.85 mmol) in Et2O was added to 3-((5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)but-2-one (1 equivalent, 300 mg, 0.92 mmol) in MeTHF (15 mL). The mixture was stirred at -20°C for 1 h. 3M MeMgBr (2 equivalents, 0.62 mL, 1.85 mmol) in Et2O was added at -20°C, and the mixture was stirred and slowly heated to room temperature for 18 h. An aqueous solution of EtOAc and NH4Cl was added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified on silica gel by rapid chromatography: eluted with a gradient of heptane / EtOAc 100:0 to 0:100 to give 3-((5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (147 mg, 47% yield).

[0549] LCMS: MW (calcd): 340.4; m / z MW (obsd): 341.2 (M+H).

[0550] 1.19.5. Step E

[0551] In a round-bottom flask, a mixture of 3-((5-(benzyl(methyl)amino)pyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 135 mg, 0.4 mmol) and NIS (1.1 equivalent, 98.14 mg, 0.44 mmol) in DMF (1.35 mL) was stirred at room temperature for 18 h. EtOAc and water were added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified by rapid chromatography on silica gel: eluted with a heptane / EtOAc gradient of 100:0 to 0:100 to give 3-((5-(benzyl(methyl)amino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (152 mg, 82%).

[0552] LCMS: MW (calcd): 466.3; m / z MW (obsd): 467.1 (M+H).

[0553] 1.19.6. Step F

[0554] In a sealed tube, a mixture of 3-((5-(benzyl(methyl)amino)-3-iodopyrazolo[1,5-a]pyrimidin-6-yl)oxy)-2-methylbut-2-ol (1 equivalent, 14 mg, 0.03 mmol), intermediate 4 (1.2 equivalent, 11.5 mg, 0.036 mmol), Cs₂CO₃ (1.2 equivalent, 11.74 mg, 0.036 mmol), PdCl₂dppf.DCM (0.1 equivalent, 2.45 mg, 0.003 mmol), dioxane (0.5 mL), and water (0.1 mL) was degassed with N₂ and stirred at 90°C for 1.5 hours. EtOAc and water were then added. The organic layer was washed with brine, dried (MgSO4), filtered, evaporated, and purified on silica gel by rapid chromatography: eluted with heptane / EtOAc 100:0 to 0:100, followed by a 100% EtOAc gradient to give 4-(5-(benzyl(methyl)amino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd 20 (7 mg, 38% yield).

[0555] LCMS: MW (calcd): 613.6; m / z MW (obsd): 614.4 (M+H).

[0556] 1.19.7. Step G

[0557] In a round-bottom flask, under air and at 0°C, trifluoromethanesulfonic acid (20 equivalents, 464.7 mg, 0.27 mL, 3.1 mmol) was added to a solution of 4-(5-(benzyl(methyl)amino)-6-((3-hydroxy-3-methylbut-2-yl)oxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-(difluoromethoxy)-N-((1R,2S)-2-fluorocyclopropyl)-6-methoxybenzamide Cpd 20 (1 equivalent, 95 mg, 0.15 mmol) in DCM (1.9 mL). The mixture was stirred at 0°C for 2 h, then stirred at room temperature. DCM and a saturated aqueous solution of NaHCO3 were added (until pH 7). The organic layer was separated, dried (MgSO4), filtered, evaporated, and purified on silica gel by rapid chromatography: eluted with heptane / EtOAc 100:0 to 0:100, followed by a 100% EtOAc gradient, to give the title racemic mixture (50 mg, 62% yield) as a white solid, which was then purified by chiral SFC at Waters ACQUITY UPC2. ® Purification was performed on the system. Column: Chiralpak IA, 150 x 21 mm, 5 μm, with guard column 50 x 21 mm, 5 μm; CO2 back pressure: 100 bar; cosolvent: isopropanol, cosolvent percentage: 30%; flow rate: 60 mL / min; isocratic mode; temperature: 40°C. The system yielded Cpd 21, with the first eluent arbitrarily designated as the (R) configuration (14.8 mg, 18% yield), and Cpd 22, with the second eluent arbitrarily designated as the (S) configuration (14.2 mg, 18%).

[0558] Cpd 21:%ee: 100

[0559] LCMS: MW (calcd): 523.5; m / z MW (obsd): 524.5 (M+H).

[0560] Cpd 22: %ee: 96.0

[0561] LCMS: MW (calcd): 523.5; m / z MW (obsd): 524.5 (M+H).

[0562] 1.20. Cpd B

[0563] 1.20.1. Step A: Ethyl (2S)-2-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxypropionate

[0564] DMF (3.5 L, 8 V) and intermediate 5 (430 g, 1,712 mmol, 1 equivalent) were charged into a 15 L single-jacketed process reactor equipped with mechanical stirring (200 rpm) and baffles. The resulting solution was stirred at room temperature for 10 min and then cooled to 0 °C. Intermediate 2 (490 g, 1763 mmol, 1.03 equivalent) was slowly added to a solution of DMF (860 mL, 2 V) over 1.3 h while maintaining the process temperature between 0 °C and 2 °C. During the addition, the stirring was increased to 275 rpm. After the addition, the reaction mixture was stirred at 0 °C for another 3 h. The reaction mixture was quenched by slowly adding (approximately 45 min) a 10% (1.075 L, 2.5 V) aqueous solution of K₂CO₃ while maintaining the process temperature below 5 °C. Water (2.2 L, 5 V) and EtOAc (4.4 L, 10 V) were added in triplicate, alternating between the two liquids. The jacket temperature was set to 22°C, and the two-phase mixture was stirred at 275 rpm for 5 minutes. The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 1.1 L, 2 × 2.5 V). The organic layers were combined and washed with 10% K2CO3 (860 mL, 2 V), 10% LiCl (1.7 L, 4 V) aqueous solution, water (1.3 L, 3 V), and 10% NaCl (860 mL, 2 V) aqueous solution. The mixture was then concentrated under vacuum to give ethyl (2S)-2-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxypropionate.

[0565] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.45 (dd, 1H), 8.05 (s, 1H), 7.53 (dd,1H), 7.22 (dd, 1H), 5.12 (q, 1H), 4.16 (qd, 2H), 1.54 (d, 3H), 1.18 (t, 3H).

[0566] 1.20.2. Step B: (3S)-3-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxy-2-methyl-but-2-ol

[0567] Ethyl (2S)-2-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxypropionate (432 g, 1170 mmol, 1 equivalent) and 2-MeTHF (4.3 L, 10 V) were charged into a 15 L single-jacketed process reactor equipped with mechanical stirring (200 rpm) and baffles. The resulting solution was cooled to 0°C. A MeMgBr solution (3.4 M, 1 L, 3400 mmol, 2.9 equivalent) was slowly added over 1.25 hours while maintaining the process temperature between 0 and 2°C. After the addition, the reaction mixture was stirred at this temperature for another 30 minutes. The jacket temperature was set at -15°C, and the reaction mixture was quenched by slowly adding 1M HCl (5.5 L, 5500 mmol, 1V) aqueous solution over 55 min while maintaining the process temperature below 10°C. After adding 3.5 L of HCl, the jacket temperature was warmed to 30°C over 30 min. The stirring was set to 250 rpm, and the two-phase mixture was stirred for 30 min. The jacket temperature was maintained at 30°C for 20 min, and then cooled to 23°C. The layers were separated, and the aqueous layer was extracted with 2-MeTHF (4V). The organic layers were combined, washed with 10% NaHCO3 (2V) aqueous solution and brine (3V), filtered through cellulose, and concentrated under vacuum to give (3S)-3-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxy-2-methyl-but-2-ol.

[0568] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.49 (dd, 1H), 8.02 (s, 1H), 7.53 – 7.42(m, 1H), 7.18 (dd, 1H), 4.54 (s, 1H), 4.19 (q, 1H), 1.22 (d, 3H), 1.18 (s,3H), 1.16 (s, 3H).

[0569] 1.20.3. Step C: Cpd B

[0570] At 20°C, a solution of (3S)-3-(3-bromopyrazolo[1,5-a]pyridin-6-yl)oxy-2-methyl-but-2-ol (562 g, 1550 mmol, 1.0 equivalent) in 1,4-dioxane (4.5 L, 8 V) was charged into a 15 L single-jacketed process reactor equipped with a condenser, nitrogen inlet, gas bubbler, mechanical stirrer (150 rpm), and baffles, followed by intermediate 4 (494 g, 1550 mmol, 1.0 equivalent). Water (1.25 L, 2 V) and sodium carbonate (498 g, 4651 mmol, 3.0 equivalent) were then added. The reaction mixture was then degassed by nitrogen injection for 15 min, and the remaining process was carried out under a nitrogen flow. XPhos (59.7 g, 124 mmol, 8 mol%) and Pd2(dba)3 (28.4 g, 31 mmol, 2 mol%) were then added. Stirring was increased to 200 rpm, and the jacket temperature was set to 75°C. The process temperature was gradually increased over 2 hours to a maximum of 77.4°C, after which it was decreased to 72°C. The reaction mixture was stirred at this temperature for another 1.25 h, then cooled to 30–35°C. After cooling, the reaction mixture was filtered over cellulose and washed with 2-MeTHF (2 × 5 V). The organic layer was transferred to a 15 L reactor and washed with water (3 V). The layers were separated, and the aqueous layer was extracted with 2-MeTHF (2 × 1 V). The organic layers were combined, washed with a 10% (3 V) aqueous solution of NaCl, and partially concentrated under vacuum to obtain a suspension. This suspension was transferred to a 15 L reactor and re-slurryed in MTBE (8 V). The solid was filtered, washed with MTBE, and dried in a vacuum oven (at 45°C for 48 h) to obtain Cpd B.

[0571] Table II. Intermediates used in the compounds of this invention.

[0572] SM = Starting Material, Mtd = Method, MS Mes 'd = Measured Mass, NA = Unmeasured

[0573] Table III. Exemplary Compounds of the Invention

[0574] Sm = Starting material, Mtd = Method, MSMes'd = Measured mass

[0575] Table IV. Comparison of Compounds

[0576] Sm = raw material, Mtd = method, MSMes'd = measured quality

[0577] Table IV. NMR data of exemplary and comparative compounds of the present invention.

[0578]

[0579] Biological Examples

[0580] Example 2. In vitro assay

[0581] 2.1. Biochemical determination

[0582] 2.1.1. ADP –Glo TM SIK kinase assay

[0583] 2.1.1.1 Overview

[0584] ADP-Glo TM The kinase assay is a luminescent technique that measures ADP formed by a kinase reaction. In this particular study, the kinase reaction consisted of phosphorylation of the AMARA peptide substrate (SignalChem, Cat# A11-58 or Eurogentec, SEQ ID 1) by SIK1 (Carna Biosciences, Cat# 02-131), SIK2 (Invitrogen, Cat# PV4792 or Cat# PR8353A), or SIK3 FL (SEQ ID 2). In the second step, the kinase reaction was terminated, and all remaining ATP was depleted. In the final step, ADP was converted to ATP, and this newly synthesized ATP was measured using a luciferase / luciferin reaction. The resulting light was measured using an Envision plate reader, where the obtained luminescent signal was positively correlated with kinase activity.

[0585] 2.1.1.2. No pre-incubation protocol

[0586] Starting with the highest concentration of 2 mM, the test compound was prepared as a serially diluted solution with a 10-point dose response by diluting it 1 / 5 in 100% DMSO, diluting it 1 / 20 in water, and transferring 1 μL to the assay plate (PerkinElmer Inc., Cat# 6007290).

[0587] 1% DMSO and 10 μM astrone final concentrations were used as negative and positive controls.

[0588] Enzyme-substrate mixtures are prepared by adding the enzyme and substrate to the assay buffer.

[0589] Add 2 μl of the enzyme-substrate mixture to the assay plate.

[0590] The reaction was initiated by adding 2 μL of ATP (diluted in assay buffer) to the assay plate. The plate was centrifuged at 1000 rpm for a few seconds, gently shaken for 2 minutes, and then incubated at room temperature for 120 minutes.

[0591] Stop the reaction and deplete any unused ATP by adding 5 μL of ADP-Glo ​​reagent (Promega, Cat #V912B). Centrifuge the plate at 1000 rpm for a few seconds and incubate at room temperature for 40 minutes (to deplete ATP).

[0592] ADP was converted to ATP and introduced into luciferase and luciferin to detect ATP by adding 10 μL of kinase assay reagent (Promega, Cat #V913B+V914B) to the reaction. The plate was centrifuged at 1000 rpm for a few seconds and incubated at room temperature for 30 minutes (ADP detection).

[0593] Emissions were measured on an Envision reader (PerkinElmer Inc.).

[0594] 2.1.1.3. Preparation of SIK3 FL Gst-TEV-HsSIK3 protein

[0595] 2.1.1.3.1. Protein Expression

[0596] Construct design and rod preparation: The coding sequence of amino acids 59 to 1321 of human SIK3 protein (UniProt Q9Y2K2-5) was cloned into pFastBac1 (Cat #10360014, ThermoFisher), which has N-ter (Gst) and C-ter (6His) affinity tags + N-ter (Tobacco Etching Virus, TEV) and C-ter (Thr) protease sites, to obtain the pFastBac-Gst-TEV-HsSIK3(59-1321)-Thr-6His construct. The expression cassette of pFastBac-Gst-TEV-HsSIK3(59-1321)-Thr-6His recombination with parental rods in DH10Bac E. coli competent cells (Invitrogen) and EmbacY_DH10Bac E. coli competent cells (Geneva Biotech) forms expression rods.

[0597] Transfection and Virus Stock Preparation: Sf9 insect cells were transfected with CellFectin II reagent (Life Technologies) via DH10Bac or EmbacY_DH10Bac DNA to obtain the P0 virus stock. The P0 virus stock was used to infect Sf9 cells to prepare the P1 virus stock solution, and this latest virus stock solution was used to obtain the P2 virus stock.

[0598] Recombinant protein production: Sf9 insect cells were infected with P2 virus stock solution. After 4 to 6 days, the cells were harvested and the precipitate was stored at -20°C.

[0599] 2.1.1.3.2 Protein purification

[0600] Cell lysis and sample clarification: The precipitated cells were resuspended on ice (5 vol / g cell pellet) in equilibration buffer (50 mM Tris pH 8, 250 mM NaCl; 2 mM DTT, 1 mM EDTA, 10% glycerol, 0.05% Brij-35) + a mixture of EDTA-free protease inhibitors (Roche). After sonication, Benzonase Nuclease (Merck Millipore) was added to remove DNA viscosity. The sample was clarified by ultracentrifugation and filtration through a 22 µm (polyethersulfone) low-binding filter (Corning).

[0601] GST Trap affinity chromatography: Samples were loaded onto a GST-Trap 4B column (GE) pre-equilibrated with 50 mM Tris pH 8, 250 mM NaCl; 2 mM DTT, 1 mM EDTA, 10% glycerol, and 0.05% Brij-35, and the sample was recycled through the column for 24 hours to improve binding efficiency. The column was washed with 10 CV equilibration buffer. POI was successfully eluted by stepwise elution with 2 CV of 10 mM reduced glutathione. Samples were dialyzed relative to (50 mM Tris pH 8, 250 mM NaCl; 1 mM DTT, 1 mM EDTA, 10% glycerol, 0.05% Brij-35) and stored at -80°C, aliquoted, and rapidly frozen in liquid nitrogen.

[0602] His Trap affinity chromatography: Load the sample onto a pre-equilibrated HisTrap HP (GE) column. Wash the column with 10 CV equilibration buffer and elute stepwise with 300 mM imidazole (6 CV). Combine the elution fractions, centrifuge, and perform size exclusion chromatography to remove potential insoluble aggregates.

[0603] Preparative size exclusion chromatography: The sample was injected into a pre-equilibrated (50 mM Tris pH 8 + 250 mM NaCl + 1 mM MTT + 1 mM EDTA + 10% glycerol + 0.05% Brij-35) Hiload 16 / 600 Superdex 75 preparative chromatography (GE) system. A single peak was detected at a retention volume of approximately 48 mL. The elution fractions were combined, the sample was aliquoted, rapidly frozen in liquid nitrogen, and then stored at -80°C.

[0604] Table VI. Human SIK kinase ADP-Glo TM Measurement conditions

[0605] 2.1.1.4. Data Analysis and Results

[0606] Raw data were generated after reading from TopCount, and dose-response curves were plotted to calculate the percentage inhibition (PIN) and mean IC50 of each SIK homologue, as reported in the table below.

[0607] Table VII. ADP-Globe of Exemplary and Comparative Compounds of the Invention TM SIK kinase assay IC 50 .

[0608]

[0609] 2.1.1.5. Conclusion

[0610] As shown in Table VII, the illustrative compounds of the present invention exhibit enhanced inhibition of SIK3 and / or improved selectivity of SIK3 relative to SIK1 and SIK2, compared to the comparative prior art compound Cpd A.

[0611] 2.2. Cell assay

[0612] 2.2.1. MdM Analysis: LPS-Triggered TNFα / IL-10 (ELISA)

[0613] 2.2.1.1. Overview

[0614] In LPS-triggered monocyte-derived macrophages (MdM) and dendritic cells (MdDCs), inhibition of SIK suppresses TNFα and increases IL-10 release (Clark et al., 2012; Sundberg et al., 2014; Ozanne et al., 2015). This assay evaluated the inhibition of LPS-induced TNFα and LPS-triggered IL-10 secretion in monocyte-derived macrophages by the exemplary compounds of the present invention.

[0615] 2.2.1.2. Research Plan

[0616] PBMCs were isolated from human blood samples (erythrocyte sedimentation rate, brown layer). The brown layer was aseptically transferred to a 50 mL Falcon tube and diluted 1 / 2 in PBS. 20 mL of Lymphoprep™ was filled into the Falcon tube, and 25 mL of the brown layer was carefully added to the top. The tube was centrifuged at 400 × g for 35 min in a temperature-controlled centrifuge at 25°C without brake. PBMCs were aspirated from the white interfacial layer between the sample and Lymphoprep™. The PBMCs were washed 5 times in PBS. The cells were resuspended in RPMI 1640 complete medium supplemented with 10% FBS and 1% P / S, and cell density was determined using a hematology analyzer (Sysmex XS-500i).

[0617] Centrifuge PBMCs at 300 × g for 10 minutes, and then centrifuge at 1.0 × 10⁻⁶ g for 10 minutes. 7 Cells were resuspended at a density of 1 cell / 80 µL Miltenyi buffer (PBS, pH 7.4, 1% FBS, 2 mM EDTA).

[0618] 2.2.1.2.1 Positive marker for CD14+ monocytes.

[0619] From this point in the plan, all steps are performed on ice. Every 1.0 × 10 7 Add 20 µL LCD14+ beads to each cell, mix the tubes, and incubate at 4°C for 15 minutes. Adjust the cell suspension to a total volume of 100 mL using Miltenyi buffer, mix gently, and then centrifuge at 300 × g for 10 min. Discard the supernatant and resuspend the cell pellet in 12 mL of Miltenyi buffer.

[0620] 2.2.1.2.2 Magnetic Cell Sorting

[0621] Four LS columns were placed in a MACS separator (magnet) from Miltenyi Biotec and pre-wetted by rinsing with 3 mL of MACS buffer / column. 3 mL of cell suspension was added to the column (maximum 1.0 × 10⁻⁶). 8 The cells / column were labeled, and the column was then washed three times with 3 mL Miltenyi buffer.

[0622] Remove the column from the magnet and add 5 mL of Miltenyi buffer to the column, flushing away the CD14+ fraction by pushing the plunger into the column. Collect the flushed fraction in 50 mL of fresh Falcon and adjust the volume to 30 mL using Miltenyi buffer. Centrifuge the cells at 300 × g for 10 min. Resuspend the resulting cell pellet in 10 mL of RPMI with / o FBS and determine the cell density using a hematology analyzer (Sysmex XS-500i). Seed 100,000 cells per well of a 96-well plate for differentiation into MdM cells in RPMI 1640 medium supplemented with 10% FBS, 1% P / S, and 100 ng / mL rhM-CSF. On day 5, refresh the medium to 100 µL of RPMI 1640 medium supplemented with 10% FBS, 1% P / S, and 100 ng / mL rhM-CSF.

[0623] On day 10, MdM is triggered and the compound is added.

[0624] Compound dilution plates were prepared by 3-fold dilution of the 10 mM stock solution in 100% DMSO. Intermediate dilution plates (10x final concentration) were prepared by diluting the compound dilution plates 50-fold in RPMI medium.

[0625] Carefully remove the culture medium from the cell plate using a multichannel pipette and replace it with 80 µL of fresh medium. Add 10 µL of the compound at a final concentration of 10x to the cells and incubate at 37°C for 1 hour, then add the trigger. No trigger condition / trigger condition with an equivalent final DMSO concentration of 0.2% DMSO. Add 10 µL of 10x LPS (final concentration 200 ng / mL) solution to all wells except the "no trigger well" (which contains 10 µL of medium). Collect the supernatant 2 hours (IL-10 assay) and 20 hours (TNFα assay) after LPS triggering.

[0626] 2.2.1.2.3 TNFα ELISA

[0627] Lumitrac 600 Greiner 384-well plates were coated with 40 µL of capture antibody (BD Pharmingen, Cat#551220) to a final concentration of 1 µg / mL in 1x PBS and stored overnight at 4°C.

[0628] The plate was then washed once with PBST (PBS + 0.05% Tween 20), once with PBS, and then 100 µL of blocking buffer (1% bovine serum albumin (BSA) - 5% sucrose) was added. The plate was sealed and incubated at room temperature for at least 4 hours. After washing the plate once with PBST and once with PBS, 40 µL of standard or sample was added (TNFα standard curves were prepared using serial dilutions starting at 1 / 2 of 16000 pg / mL; diluted in dilution buffer (PBS + 1% BSA)). The plate was washed twice with PBST and once with PBS, then 35 µL of detection antibody (final concentration diluted in dilution buffer to 0.25 µg / mL) was added, and the plate was incubated at room temperature for at least 2 hours. The plate was washed twice with PBST and once with PBS, then 35 µL of Strep-HRP conjugate (final concentration diluted in dilution buffer to 0.5 µg / mL) was added. The plate was incubated in the dark at room temperature for at least 45 minutes, but no more than 1 hour. The plate was washed twice with PBST and once with PBS. Then, 50 µL of luminol substrate (prepared according to the manufacturer's instructions) was added to each well and incubated at room temperature in the dark for 5 minutes. Chemiluminescence was measured on an Envision 2104.

[0629] 2.2.1.2.1 IL-10 ELISA

[0630] Immulon 2HB 96-well plates (Thermo Electron Co., Cat# 3455) were coated with 40 µL of capture antibody (final concentration of 2 µg / mL in Tris buffer (50 mM Tris; 150 mM NaCl; pH 9 (adjusted with HCl)) and stored overnight at 4°C. The next day, the plates were washed three times with PBST, followed by the addition of 200 µL of blocking buffer (1% BSA + 5% sucrose in PBS-T). After incubation at 37°C for 30 minutes, the plates were washed three times with PBST, and 100 µL of standard or sample was added (IL-10 standard curve samples were prepared using 1 / 2 serial dilutions starting from 1000 pg / mL; diluted in dilution buffer: PBS + 1% BSA). After incubating at 37°C for 1 hour, wash the plate three times with PBST, then add 100 µL of the detection antibody (BD Pharmingen, Cat# 554499) (final concentration 0.25 µg / mL diluted in Tris buffer) and incubate the plate at room temperature for at least 2 hours. Wash the plate three times with PBST, then add 100 µL of the Strep-HRP conjugate (final concentration 0.5 µg / mL diluted in dilution buffer). Incubate the plate in the dark at 37°C for 30 min. Wash the plate three times with PBST. Prepare the substrate solution with a total volume of 20 mL: 18 mL H2O; 2 mL citrate-acetate buffer; 200 µL TMB mixture (101 mg tetramethylbenzidine (TMB), 10 mL DMSO stored at 4°C); and 2.5 µL 30% H2O2. Add 100 µL of the substrate solution to each well and incubate until a bright blue color develops. The reaction was terminated by adding 50 µL of 1 M H2SO4, and the absorbance was then measured at 450 nm on a SpectraMax i3 Molecular Devices.

[0631] 2.2.1.3. Data Analysis and Results

[0632] 2.2.1.3.1 Calculation of TNFα inhibition

[0633] To measure LPS-induced inhibition of TNFα, the percentage inhibition (PIN) value for all tested concentrations was calculated compared to a control. An unstimulated sample (without trigger / loader (0.2% DMSO)) was used as a negative control (100% inhibition). Stimulated samples (with trigger / loader) were used as a positive control (0% inhibition).

[0634]

[0635] Where RLU = Relative Chemiluminescence Units (minus background), and the subscripts p and n refer to the average values ​​of the positive and negative controls, respectively.

[0636] PIN values ​​plotted as concentration-response, EC 50 The values ​​were obtained using GraphPad Prism software, fitted with a 4-parameter nonlinear regression (S-shaped) curve. Because a clear bottom plateau was not obtained, the bottom of the curve was constrained to be equal to 0.

[0637] 2.2.1.3.2 IL-10 Induction Calculation

[0638] IL-10 is induced upon SIK inhibition. To quantify these inductions, the fold change (FC) compared to "LPS only" was calculated for each tested concentration, and the maximum FC (IL-10 FCmax) was calculated:

[0639] Where ABS = absorbance measured at 450 nm.

[0640] The report shows the median maximum FC (median IL-10 FCmax) of the tested compound across two or more assays.

[0641] 2.2.1.3.3 Results and Outcomes

[0642] Data obtained when processing exemplary compounds of the present invention are described in the table below.

[0643] Table VIII. MdM TNFα inhibition and IL-10 induction of exemplary and comparative compounds of the present invention.

[0644]

[0645] 2.2.2. Monocyte assay: LPS-triggered TNFα / IL-10 (ELISA)

[0646] 2.2.2.1. Overview

[0647] In LPS-triggered monocyte-derived macrophages (MdM) and dendritic cells (MdDCs), inhibition of SIK suppresses TNFα and increases IL-10 release (Clark et al., 2012; Sundberg et al., 2014; Ozanne et al., 2015). This assay evaluated the inhibition of LPS-induced TNFα and LPS-triggered IL-10 secretion in monocytes by the exemplary compounds of the present invention.

[0648] 2.2.2.2. Scheme

[0649] PBMCs were isolated from human blood samples (erythrocyte sedimentation rate, brown layer). The brown layer was aseptically transferred to a 50 mL Falcon tube and diluted 1 / 2 in PBS. 20 mL of Lymphoprep™ was filled into the Falcon tube, and 25 mL of the brown layer was carefully added to the top. The tube was centrifuged at 400 × g for 35 min in a temperature-controlled centrifuge at 25°C without brake. PBMCs were aspirated from the white interfacial layer between the sample and Lymphoprep™. The PBMCs were washed 5 times in PBS. The cells were resuspended in RPMI 1640 complete medium supplemented with 10% FBS and 1% P / S, and cell density was determined using a hematology analyzer (Sysmex XS-500i).

[0650] Centrifuge PBMC at 300 × g for 10 minutes, and then centrifuge at 1.0 × 10⁻⁶ g. 7 Cells were resuspended at a density of 1 cell / 80 µL Miltenyi buffer (PBS, pH 7.4, 1% FBS, 2 mM EDTA).

[0651] 2.2.2.2.1 Positive marker for CD14+ monocytes.

[0652] From this point in the plan, all steps are performed on ice. Every 1.0 × 10 7 Add 20 µL LCD14+ beads to each cell, mix the tubes, and incubate at 4°C for 15 minutes. Adjust the cell suspension to a total volume of 100 mL using Miltenyi buffer, mix gently, and then centrifuge at 300 × g for 10 min. Discard the supernatant and resuspend the cell pellet in 12 mL of Miltenyi buffer.

[0653] 2.2.2.2.2 Magnetic Cell Sorting

[0654] Four LS columns were placed in a MACS separator (magnet) from Miltenyi Biotec and pre-wetted by rinsing with 3 mL of MACS buffer / column. 3 mL of cell suspension was added to the column (maximum 1.0 × 10⁻⁶). 8 The cells / column were labeled, and the column was then washed three times with 3 mL Miltenyi buffer.

[0655] Remove the column from the magnet and add 5 mL of Miltenyi buffer to the column. Wash away the CD14+ fraction by pushing the plunger into the column. Collect the washed fraction in 50 mL of fresh Falcon and adjust the volume to 30 mL with Miltenyi buffer. Centrifuge the cells at 300 × g for 10 min. Resuspend the resulting cell pellet in 10 mL of RPMI w / o FBS and determine the cell density using a hematology analyzer (Sysmex XS-500i). Seed 100,000 cells at 80 µL / well in RPMI 1640 medium supplemented with 10% FBS and 1% P / S in 96-well plates.

[0656] Compound dilution plates were prepared by 3-fold dilution of the 10 mM stock solution in 100% DMSO. Intermediate dilution plates (10x final concentration) were prepared by diluting the compound dilution plates 50-fold in RPMI medium.

[0657] Add 10 µL of the compound at a final concentration of 10x to the cells and incubate at 37°C for 1 hour before adding the trigger. Use an equal final DMSO concentration with no trigger or trigger conditions incorporating 0.2% DMSO. Add 10 µL of a 10x LPS solution (final concentration 200 ng / mL) to all wells except the "no trigger well" (which contains 10 µL of culture medium). Collect the supernatant 4 hours after LPS triggering.

[0658] 2.2.2.2.3 TNFα ELISA

[0659] Lumitrac 600 Greiner 384-well plates were coated with 40 µL of capture antibody (BD Pharmingen, Cat#551220) to a final concentration of 1 µg / mL in 1x PBS and stored overnight at 4°C.

[0660] The plate was then washed once with PBST (PBS + 0.05% Tween 20), once with PBS, and then 100 µL of blocking buffer (1% bovine serum albumin (BSA) - 5% sucrose) was added. The plate was sealed and incubated at room temperature for at least 4 hours. After washing the plate once with PBST and once with PBS, 100 µL of blocking buffer (1% BSA - 5% sucrose) was added, the plate was sealed and incubated at room temperature for at least 4 hours. The plate was washed twice with PBST and once with PBS, then 35 µL of the detection antibody (diluted in dilution buffer to a final concentration of 0.25 µg / mL) was added, and the plate was incubated at room temperature for at least 2 hours. The plate was washed twice with PBST and once with PBS, then 35 µL of Strep-HRP conjugate (diluted in dilution buffer to a final concentration of 0.5 µg / mL) was added. The plate was incubated in the dark at room temperature for at least 45 minutes but no more than 1 hour. The plate was washed twice with PBST and once with PBS. Subsequently, 50 µL of luminol substrate (prepared according to the manufacturer's instructions) was added to each well and incubated at room temperature in the dark for 5 minutes. Chemiluminescence was measured on an Envision 2104.

[0661] 2.2.2.2.4 IL-10 ELISA

[0662] Lumitrac 600 Greiner 384-well plates were coated with 40 µL of capture antibody (final concentration 1 µg / mL in 1x PBS) and stored overnight at 4°C. The next day, the plates were washed three times with PBST (PBS + 0.05% Tween 20), once with PBS, and then 100 µL of blocking buffer (1% BSA - 5% sucrose) was added. The plates were sealed and incubated at room temperature for at least 4 hours. After washing the plates once with PBST and once with PBS, 40 µL of standards or samples were added (IL-10 standard curves were prepared using 1 / 2 serial dilutions starting from 2000 pg / mL; dilutions were performed in dilution buffer (PBS + 1% BSA)). The plates were washed twice with PBST and once with PBS, then 35 µL of detection antibody (final concentration 0.143 µg / mL diluted in dilution buffer) was added, and the plates were incubated at room temperature for at least 2 hours. Wash the plate twice with PBST and once with PBS, then add 35 µL of the Strep-HRP conjugate (0.5 µg / mL final concentration diluted in dilution buffer). Incubate the plate in the dark at room temperature for at least 45 minutes but no more than 1 hour. Wash the plate twice with PBST and once with PBS. Afterward, add 50 µL of luminol substrate (prepared according to the manufacturer's instructions) to each well and incubate at room temperature in the dark for 5 minutes. Measure the chemiluminescence on an Envision 2104.

[0663] 2.2.2.3. Data Analysis and Results

[0664] 2.2.2.3.1 Calculation of TNFα Inhibition

[0665] To measure LPS-induced inhibition of TNFα, the percentage inhibition (PIN) value for all tested concentrations was calculated compared to a control. An unstimulated sample (without trigger / loader (0.2% DMSO)) was used as a negative control (100% inhibition). Stimulated samples (with trigger / loader) were used as a positive control (0% inhibition).

[0666]

[0667] Where RLU = Relative Chemiluminescence Units (minus background), and the subscripts p and n refer to the average values ​​of the positive and negative controls, respectively.

[0668] PIN values ​​plotted as concentration-response, EC 50 The values ​​were obtained using GraphPad Prism software, fitted with a 4-parameter nonlinear regression (S-shaped) curve. When a clear bottom plateau was not obtained, the bottom of the curve was constrained to be equal to 0.

[0669] 2.2.2.3.2 IL-10 Induction Calculation

[0670] LPS-induced IL-10 increases after SIK inhibition. To quantify these increases, the fold change (FC) compared to "LPS only" was calculated for the concentration in each test, and the maximum FC (IL-10 FCmax) was calculated:

[0671] The median maximum FC (median IL-10 FCmax) of the tested compounds was reported in more than two trials.

[0672] 2.2.2.3.3 Results and Outcomes

[0673] Data obtained when processing exemplary compounds of the present invention are described in the table below.

[0674] Table IX. Mononuclear cell TNFα inhibition and IL-10 induction by exemplary and comparative compounds of the present invention.

[0675]

[0676] Example 3. Pharmacokinetics, ADME and Toxicity Assays

[0677] 3.1. Assay for transactivation of human pregnane X receptor (PXR)

[0678] 3.1.1. Overview

[0679] PXR is a member of the nuclear receptor superfamily and acts as a transcriptional regulator of the cytochrome P450 gene CYP3A4 by forming a heterodimer with the 9-cis-retinoic acid receptor RXR and binding to the reactive element of the CYP3A4 promoter. Therefore, PXR transactivation assay data can be used to classify the DDI potential of compounds in CYP3A induction (Chu et al., 2009). This assay utilizes the Human PXR Reporter Molecular Assay Kit (Indigo Biosciences, Cat# IB07002) in engineered human cells constitutively expressing a functionally heterozygous form of human PXR, in which the N-terminal sequence encoding the PXR DNA-binding domain (DBD) has been replaced by a sequence from the yeast GAL4-DBD. Upon ligand activation, PXR binds to the GAL4 DNA-binding sequence, which is linked to the luciferase reporter gene. Changes in PXR activity are quantified by luminescence detection on a plate reader.

[0680] 3.1.2. Experimental Procedure

[0681] The test compound was incubated with human PXR reporter cells (from the Indigo PXR kit) for 24 hours. At the end of the incubation period, cell viability and transcriptional activation were measured.

[0682] PXR reporter cells were seeded in 384-well plates and recovered at 37°C in 5% CO2 for 4–6 h, followed by drug administration. Incremental concentrations of the test compound (final DMSO concentration 0.4%; final test compound concentrations were 0.15, 0.48, 1.5, 4.9, 15.6, and 50 µM; each concentration was repeated twice) were administered to the cells as a positive control or load control, and incubated at 37°C for 22–24 h. Rifampin was used as a positive control (final control compound concentrations were 0.032, 0.1, 0.32, 1.0, 3.2, and 10 µM).

[0683] At the end of the incubation period, the culture medium was discarded, and cell viability was determined fluorescently using the Live Cell Multiplex Assay Kit (IndigoBiosciences, Cat# LCM01). Luciferase activity was determined using the luciferase assay kit provided with the Human PXR Reporter Molecular Assay Kit.

[0684] 3.1.3. Data Analysis and Results

[0685] Transcriptional activation was monitored by luminescence. Data were expressed as fold increases in activation relative to the load control, followed by normalization of luciferase activity against cell viability. The use of more than five doses of the test compound and a positive control allowed for the derivation of EC from nonlinear regression analysis of the logarithmic dose-response curve. 50 (i.e., half the concentration that produces the maximum effect) and E max (That is, the maximum activation factor relative to the carrier) value. The maximum activation factor is also calculated as the percentage of activation (%Act) of 10 µM rifampin to allow for interday comparison and classification of test compounds using the following equation:

[0686] The following %Act categories are generally accepted in this study: >40% for high CYP3A4 induction potential; 15% to 40% for medium potential; and <15% for low potential (Chu et al., 2009; Sinz et al., 2006).

[0687] Table X. Human pregnane X receptor activation of exemplary and comparative compounds of the present invention.

[0688]

[0689] 3.1.4. Conclusion

[0690] As shown in Table X, the exemplary compounds of the present invention exhibit surprisingly low transactivation potential of human pregnane X receptors compared to the comparative compound Cpd B.

[0691] Example 4. In vivo analysis

[0692] 4.1. Inflammatory bowel disease: DSS model (mice)

[0693] The mouse model of chronic DSS-induced inflammatory bowel disease (IBD) is a well-validated disease model for inflammatory bowel disease (Sina et al., 2009; Wirtz et al., 2007).

[0694] To induce chronic colitis, female BALB / c mice were fed drinking water containing 4% sodium dextran sulfate (DSS) for 4 days, followed by 3 days of regular drinking water. This cycle was repeated until sacrifice on day 12 or 18. The animals were divided into several groups: a. Normal drinking water (intact water); carrier only, n=10), b. Diseased (DSS; drug carrier only, n=10), c. Sulfasalazine was used as a reference (DSS; 20 mg / kg / day, po, n=10) and d. Test compound (DSS; e.g., 1, 3, 10, 30 mg / kg / day, po, n=10).

[0695] Clinical parameters were measured every other day. The Disease Activity Index (DAI) was a composite measure of individual scores combining weight loss, fecal consistency, and the presence of blood in the feces. Mice were euthanized according to the protocol described by Sina et al. (Sina et al., 2009). At euthanasia, the intact colon was removed and rinsed with sterile PBS. Segments of the distal colon were dissected for histological analysis, gene expression, and protein level measurements.

[0696] 4.2. CIA Model

[0697] 4.2.1. Materials

[0698] Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were purchased from Difco. Bovine type II collagen (CII), lipopolysaccharide (LPS), and Enbrel were also present. ®(Etanercept) was obtained from Chondrex (L'Isle d'Abeau, France), Sigma (P4252, L'Isle d'Abeau, France), and Wyeth (25 mg injectable syringe, France). All other reagents used were reagent-grade, and all solvents were analytical-grade.

[0699] 4.2.2. Animals

[0700] DBA1 / J mice (male, 7-8 weeks old) were obtained from Charles River Laboratories (Écully, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22°C, and food and water were provided freely.

[0701] 4.2.3. Collagen-induced arthritis (CIA)

[0702] One day prior to the experiment, a CII solution (2 mg / mL) was prepared using 0.05 M AcOH and stored at 4°C. Just before immunization, equal volumes of adjuvant (IFA) and CII were mixed in a homogenizer in a pre-chilled glass vial in an ice-water bath. If an emulsion did not form, additional adjuvant and prolonged homogenization may be required. On day 1, 0.2 mL of the emulsion was injected intradermally at the base of the tail of each mouse, followed by a second booster intradermal injection on day 9 (2 mg / mL CII solution in 0.1 mL CFA saline). This immunization method was modified from published methods (I.-Ming Jou et al., 2005; Sims et al., 2004).

[0703] 4.2.4. Research Design

[0704] The therapeutic efficacy of the compound was tested in a mouse model of CIA. Mice were randomly assigned to equal groups of 10 mice each. All mice were immunized on day 1 and boosted on day 21. The negative control group was treated with the carrier (MC 0.5%), and the positive control group was treated with Enbrel. ® Treatment included (10 mg / kg, 3 times / week, subcutaneous). On day 32, animals were randomized between groups based on clinical scores and received therapeutic treatment in their respective groups until day 47. Body weight and clinical scores were recorded at least twice weekly.

[0705] 4.2.5. Clinical assessment of arthritis

[0706] Arthritis was scored according to the methods of Khachigian (2006), Lin et al. (2007), and Nishida et al. (2004) (Khachigian 2006; H.-S. Lin et al. 2007; Nishida, Komiyama, S.-I. Miyazawa et al. 2004). Swelling in each of the four paws was graded using the arthritis score as follows: 0 - asymptomatic; 1 - mild, but with marked redness and swelling in one type of joint (e.g., ankle or wrist), or marked redness and swelling limited to a single toe, regardless of the number of toes affected; 2 - moderate redness and swelling in two or more types of joints; 3 - severe redness and swelling of the entire paw, including the toes; 4 - the most severely inflamed limb involving multiple joints (maximum cumulative clinical arthritis score of 16 per animal) (Nishida et al., 2004).

[0707] 4.2.5.1. Weight change (%) after an arthritis flare-up

[0708] Clinically, weight loss is associated with arthritis (JM Argilés & López-Soriano 1998; Rall & Roubenoff 2004; Shelton et al. 2005; Walsmith et al. 2004). Therefore, changes in body weight after an arthritis flare-up can be used as a nonspecific endpoint to evaluate the effect of treatment in mouse models. The percentage change in body weight after an arthritis flare-up is calculated as follows: Mice:

[0709] 4.2.5.2. Radiology

[0710] X-ray photographs of the hind paws of each animal were taken. Random, blinded identification numbers were assigned to each photograph, and the severity of bone erosion was graded by two independent raters using the radiological Larsen scoring system as follows: 0 - Normal, with intact bone contour and normal joint space; 1 - Slight abnormality showing minor bone erosion in any one or two of the lateral metatarsals; 2 - Clear early abnormality showing bone erosion in any 3-5 of the lateral metatarsals; 3 - Moderately destructive abnormality showing clear bone erosion in all lateral metatarsals and any one or two of the medial metatarsals; 4 - Severely destructive abnormality, with clear bone erosion in all metatarsals and complete erosion of at least one medial metatarsal joint, leaving some partially preserved bone joint contour; 5 - Abnormal abnormality, with no bony contour. This rating system is a modification of Salvemini et al., 2001; Bush et al., 2002; Sims et al., 2004; Jou et al., 2005 (Bush et al. 2002; I.-Ming Jou et al. 2005; D. Salvemini et al. 2001; Sims et al. 2004).

[0711] 4.2.5.3. Steady-state PK

[0712] On day 7, blood samples were collected from the retroorbital sinus at the following time points using lithium heparin as an anticoagulant: before administration, 1, 3, and 6 hours. Whole blood samples were centrifuged, and the resulting plasma samples were stored at -20°C for analysis. Plasma concentrations of each test compound were determined by LC-MS / MS, with the mass spectrometer operated in positive ion electrospray ionization mode.

[0713] 4.3. A mouse model of psoriasis-like epidermal hyperplasia induced by topical application of imiquimod (a TLR7 / 8 agonist).

[0714] 4.3.1. Materials

[0715] Aldara ® The 5% imiquimod cream is from MEDA.

[0716] Purified antibody against mouse IL 12 / IL 23 p40 (C17.8) (Cat# 16 7123 85) was obtained from eBioscience (Frankfurt, Germany).

[0717] 4.3.2. Animals

[0718] Balb / cJ mice (female, 18-20 g body weight) were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22±2℃, and food and water were provided freely.

[0719] 4.3.3. Research Design

[0720] The design of this study is adapted from Van der Fits L. et al. (van der Fits et al. 2009).

[0721] On the first day, the mice were shaved around both ears under mild anesthesia.

[0722] 30 mg of commercially available imiquimod cream (Aldara) ® A 5% cream was applied to the inner and outer surfaces of each ear for four consecutive days, equivalent to a daily dose of 1.5 mg of the active compound. Control animals received the same amount of petroleum jelly.

[0723] From day 1 to day 5, mice were orally administered the test compound in 0.5% methylcellulose twice daily, followed by administration of imiquimod (on day 5, mice were administered only once and euthanized 2 hours later).

[0724] In the positive control group, animals received two intraperitoneal injections of anti-mouse IL 12 / IL 23 p40 antibody, 10 mg / kg, on day 1 and three days prior to day 1.

[0725] 4.3.4. Disease Assessment

[0726] The thickness of both ears was measured daily using a thimometer (Mitutoyo, Absolute Digimatic, 547 321). Body weight was assessed at the start of the experiment and at sacrifice. On day 5, 2 hours after the last administration, mice were sacrificed. The auricle, excluding the cartilage, was removed. The auricle was weighed and then immersed in a solution containing 1 mL of RNAlater. ® The solution was placed in vials to assess gene expression.

[0727] Results are expressed as mean ± SEM and were analyzed using one-way ANOVA, followed by Dunnett's post-hoc test for statistical analysis relative to the imiquimod carrier group.

[0728] 4.3.5. Gene Expression Analysis

[0729] From RNAlater ® Remove the ear from the liquid and use Precellys ® After the 1.4 mm ceramic beads in the device are broken, they are placed in Trizol. ® Then use NucleoSpin. ®Total RNA was purified using an RNA kit. cDNA was prepared, and quantitative PCR was performed using gene-specific primers from Qiagen on a ViiA7 real-time PCR system (Applied Biosystems) using SYBR Green technology. The expression level of each gene was calculated relative to the expression level of the cyclic protein A housekeeping gene. Data are presented as mean ± SEM values. The statistical test used was ANOVA, with Dunnett's post-hoc test relative to the imiquimod carrier group.

[0730] 4.4. Mouse model of psoriasis-like epidermal hyperplasia induced by intradermal injection of IL-23

[0731] 4.4.1. General Overview

[0732] Overexpression of IL-23 in mice (highly expressed in psoriatic plaques, synovium, and endoplasmic reticulum) leads to enthesitis in both the axial and peripheral regions, entheseal new bone formation, and is a hallmark of psoriatic arthritis (Sherlock et al., 2012).

[0733] 4.4.2. Materials

[0734] Recombinant IL-23 in mice (cat#1887-ML / CF), provided by R&D Systems.

[0735] 4.4.3. Animals

[0736] Balb / c mice (female, 18-20 g body weight) were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22°C, and food and water were provided freely.

[0737] 4.4.4. Research Design

[0738] The design of this study was adapted from that of Rizzo HL. et al. (Rizzo et al. 2011). On day 1 (D1), mice were shaved around both ears. For four consecutive days (D1 to D4), under anesthesia, mice received daily intradermal doses of recombinant mouse IL-23 (1 µg / 20 µL in PBS / 0.1% BSA) in the right ear and 20 µL in PBS / 0.1% BSA in the left ear.

[0739] From day 1 to day 5, mice were administered the test compound or carrier one hour before IL-23 injection.

[0740] 4.4.5. Disease Assessment

[0741] The thickness of the right ear was measured daily using a thimometer (Mitutoyo, Absolute Digimatic, Cat# 547 321). Body weight was assessed at start and at sacrifice. Mice were sacrificed on day 5, 2 hours after the last administration. The auricle of the right ear, excluding the cartilage, was excised. The auricle was placed in a container containing 1 mL of RNAlater. ® In the small vial of solution.

[0742] On day 4, blood samples were collected from the posterior orbital sinus before administration (T0) and at 1 hour, 3 hours, and 6 hours after administration for PK analysis.

[0743] Ten mice were in each group. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by Dunnett's post-hoc test for statistical analysis relative to the IL 23 load group.

[0744] 4.4.6. Gene Expression Analysis

[0745] From RNAlater ® Take half an ear from the liquid and use Precellys ® After the 1.4 mm ceramic beads in the device are broken, they are placed in Trizol. ® Then use NucleoSpin. ® Total RNA was purified using an RNA kit. cDNA was prepared and quantitative PCR was performed using gene-specific primers from Qiagen on a ViiA7 real-time PCR system (Applied Biosystems) using SYBR Green technology. The expression level of each gene was calculated relative to the expression level of the cyclic protein A housekeeping gene. Data are presented as mean ± SEM values. Statistical tests used were ANOVA analysis of variance, with Dunnett's post-hoc test for the IL-23 load group.

[0746] 4.5. Imiquimod-induced systemic lupus erythematosus mouse model

[0747] 4.5.1. Materials

[0748] Aldara ® The 5% imiquimod cream is from MEDA.

[0749] The mouse anti-double-stranded DNA antibody ELISA kit was obtained from Alpha Diagnostic International (Cat#5120). The mouse urine albumin ELISA kit was obtained from Abcam (Cat# ab108792). The urine creatinine assay kit was obtained from Abnova (catalog number KA 4344).

[0750] 4.5.2. Animals

[0751] Balb / cJ mice (female, 18-20 g body weight) were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22±2℃, and food and water were provided freely.

[0752] 4.5.3. Research Design

[0753] The study design is adapted from Yokogawa M. et al. (Yokogawa et al. 2014).

[0754] On day 1 (D1), the mice were shaved around their right ears.

[0755] Mice received a topical application of 1.25 mg imiquimod in the right ear auricle three times a week for 12 weeks (D1 to D86). The control group received the same amount of petroleum jelly.

[0756] From D1 to D86, mice were administered the test compound (orally, once daily, in 0.5% methylcellulose) or mixed with a carrier (10 mL / kg).

[0757] 4.5.4. Disease Assessment

[0758] The thickness of the ear is measured weekly using an automatic caliper (Mitutoyo, Absolute Digimatic, 547-321).

[0759] Weight was assessed at the start of treatment, weekly, until euthanasia. Spleen weight was also measured during necropsy. Mice were euthanized 2 hours after the last administration.

[0760] Mice were placed in metabolic cages at different time points (e.g., on days D28, D56, and D84) for urine analysis and assessment of proteinuria (albumin to creatinine ratio).

[0761] Serum was collected at different time points (e.g., D28, D56, and D86) to assess anti-double-stranded DNA IgG levels.

[0762] On day 13, blood samples were also collected from the posterior orbital sinus for PK analysis before administration (T0) and at 1, 3 and 6 hours after administration.

[0763] Each group contained 8–19 mice. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by Dunnett's post-hoc test for statistical analysis relative to the imiquimod load group.

[0764] 4.5.5. Quantification of plasma compound levels

[0765] The plasma concentrations of each test compound were determined by LC-MS / MS, with the mass spectrometer operating in either positive or negative electrospray mode.

[0766] 4.5.5.1. Histopathology

[0767] In each glomerulus, four distinct readings—mesangial proliferation, endocapillary proliferation, mesangial matrix dilatation, and segmental sclerosis—were graded on a scale of 0–2 and then summed. Approximately 50 glomeruli were scored for each kidney, and the average was taken to obtain a glomerular injury score (Yokogawa et al., 2014). Data are presented as mean ± SEM and statistically analyzed using the Kruskal-Wallis test, followed by Dunn's post-hoc test relative to the imiquimod carrier group.

[0768] 4.5.5.2. Cell Quantification

[0769] For each cell type, immunohistochemical analysis of the entire tissue section was performed using image analysis (CaloPix software, TRIBVN Healthcare) at a magnification of ×20. Data are presented as mean ± SEM and analyzed using one-way ANOVA, followed by Dunnett's post-hoc test for statistical analysis relative to the imiquimod carrier group.

[0770] 4.5.5.3. Gene Expression Analysis

[0771] At the time of execution, the second part of the left kidney was placed in a tube containing 1.4 mm ceramic beads and treated with Bertin Instruments Precellys. ® The homogenizer was lysed in 1% DTT RLT lysis buffer (Qiagen, Cat# 79216). Then, RNeasy was used. ®Total RNA was purified using the QIAcube® HT Kit (Qiagen, Cat# 74171). cDNA was prepared, and quantitative PCR was performed using gene-specific primers from Qiagen in a ViiA 7 real-time PCR system (Applied Biosystems) with SYBR Green technology. The expression levels of each gene of interest (GOI = CD3, CD68, CD20, OAS1, Mx1, IFIT1, CXCL11, and Usp18) were calculated relative to the expression levels of cyclophilin, GAPDH, and β-actin housekeeping genes.

[0772] At the time of execution, one-third of the spleen was placed in a tube containing 1.4 mm ceramic beads and treated with Bertin Instruments Precellys. ® Homogenizer in Trizol ® The RNA was broken down in the middle. Total RNA was extracted using the phenol / chloroform method and then processed using RNeasy. ® 96 QIAcube ® The HT Kit (Qiagen, Cat# 74171) was used for purification with the QIAcube. cDNA was prepared and quantitative PCR was performed using gene-specific primers from Qiagen in a ViiA 7 real-time PCR system (Applied Biosystems) using SYBR Green technology. The expression levels of each gene of interest were calculated relative to the expression levels of cyclophilin, GAPDH, and β-actin housekeeping genes.

[0773] 4.6. Systemic lupus erythematosus model in NZB / WF1 / J mice

[0774] The aim of this study was to evaluate the activity of the test compounds of this invention in the treatment of systemic lupus erythematosus (SLE). The NZB / W F1 model, composed of an F1 hybrid between New Zealand black and New Zealand white mice (NZB / W), is the first mouse model described for lupus nephritis. These models can develop lymphadenopathy, splenomegaly, and elevated serum antinuclear autoantibodies (ANA). In particular, they develop renal damage remarkably similar to the pathology described in human lupus (Tejon et al., 2020; Zampeli et al., 2017).

[0775] 4.6.1. Materials

[0776] The test compound was stored as a dry substance in the dark and prepared into a suspension weekly using a carrier solution (5% aqueous methylcellulose solution) with magnetic stirring. The resulting suspension was kept in the dark with magnetic stirring.

[0777] Dexamethasone (DEX; Vet One, Cat# 501012) is prepared in 1% carboxymethyl cellulose and is intended for oral administration at 10 mL / kg.

[0778] 4.6.2. Animals

[0779] NZBW / F1 / J mice (female, 20 weeks old) and NZW mice (female, 8 weeks old) were obtained from Jackson Laboratories (USA). The mice were 28 weeks old at the time of the first treatment.

[0780] 4.6.3. Research Design

[0781] At 27 weeks of age (day 0 of the study), mice with the developmental disease were randomly assigned to groups according to their body weight.

[0782] When the animals were 28 weeks old, treatment began after randomization and continued until they were euthanized at 39 weeks of age.

[0783] Observe the animals daily for significant clinical signs, morbidity, and mortality.

[0784] The activity of the test compounds of this invention was evaluated based on weight, proteinuria level, tissue weight at autopsy (kidney, spleen, and lymph nodes); anti-dsDNA Ab, Igs, cytokine / chemokine and gene expression levels; and histopathology and immunohistochemistry.

[0785] The study was conducted in the following groups (15 mice per group): BID is administered at approximately 10-12 hour intervals – QD is administered at approximately 24 hour intervals.

[0786] The daily dose of the test compound to be administered is calculated in mg / kg based on the animal's latest body weight.

[0787] 4.6.4. End Point

[0788] Using the colorimetric Albustix ® Test strips (Siemens, Cat# 2872A) were used to record proteinuria scores for all animals weekly, starting with fresh urine samples, from week 28 to week 39.

[0789] Within 1 to 2 minutes after sampling, the colors are matched with the code scale to obtain the resulting score, and the following endpoints are given: 0 = None 1 = 1 to 30 mg / dL 2 = 31 to 99 mg / dL 3 = 100 to 299 mg / dL 4 = 300 to 1999 mg / dL 5 = > 2000 mg / dL For all animals from week 28 to week 39, their weight was recorded weekly.

[0790] Blood was collected from all animals under anesthesia at weeks 27, 33, and 38 for dsDNA Ab and Ig.

[0791] Blood samples were collected from the animal group treated with the test compound at the following time points during week 29 for PK analysis: before administration, and 0.25 h, 1 h, 3 h, and 6 h after administration.

[0792] At the time of sacrifice, the spleen, kidneys, and lymph nodes were weighed, and levels of anti-dsDNA Ab, Igs, cytokines / chemokines, and gene expression were maintained; as well as for histopathological and immunohistochemical analysis.

[0793] 4.6.5. Statistical Analysis

[0794] Based on raw data from individual animals, the mean for each group was determined, and the percentage change relative to the disease control was calculated. The treatment group was compared to the disease control group using Dunnett's post-hoc analysis of the measurement (parametric) data or Kruskal-Wallis test using Dunn's post-hoc analysis of the scoring (non-parametric) data.

[0795] Data are reported as 1) all animals, including those that died midway through the study, and 2) only animals that survived to the end of the study (surviving animals). Statistical analysis was performed using Prism 6.0d software (GraphPad).

[0796] Set the significance level for all tests to p < 0.05 and round the p-values ​​to three decimal places. Calculate the inhibition percentage using the following formula:

[0797] 4.7. A mouse model of psoriatic arthritis induced by IL-23 overexpression

[0798] 4.7.1. Materials

[0799] The mouse IL-23 enhanced free expression vector (EEV) was obtained from System Biosciences (Cat#EEV651A-1). The mouse IL-23 Quantikine ELISA kit was obtained from R&D Systems (Cat# M2300). ® 680 and OsteoSense ® 750EX was obtained from PerkinElmer (Cat# NEV10003 and NEV10053EX). RNAlater ® Retrieved from Ambion (Cat# AM7021). Imalgene ® 1000 (Merial) and Rompun ® 2% (Bayer) obtained from Centravet (Cat# IMA004-6827812 and ROM001-6835444).

[0800] 4.7.2. Animals

[0801] B10.RIII mice (male, 8 weeks old) were obtained from Charles River (Écully, France). The mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22±2℃, and food and water were provided on an ad libitum basis.

[0802] 4.7.3. Research Design

[0803] The design of this study is adapted from Sherlock JP. et al. (Sherlock et al. 2012).

[0804] On day 1 (D1), IL-23 EEV in Ringer or Ringer was hydrodynamically injected into the tail vein of mice.

[0805] Starting from day 5, the clinical symptoms of the mice were scored twice a week until the end of the experiment.

[0806] On day 5, blood was collected by puncture of the submandibular vein to assess serum IL-23 concentration.

[0807] On day 9, mice from all groups received ProSense. ® 680 probe (0.8 nmol / 10 g, intraperitoneal). Mice were anesthetized on day 10. Granulocyte infiltration was then measured using in vivo molecular imaging (Bruker In-Vivo Xtreme imaging system).

[0808] In D11, according to ProSense® 680 molecular imaging and scoring were randomized.

[0809] Starting on day 12, mice are given test compounds or carriers.

[0810] On day 19, blood samples were collected at T0, T1h, T3h, and T6h after the last dose. Plasma was separated and maintained at -20°C until bioanalysis was performed.

[0811] On day 36, mice in all groups were sacrificed 2 hours after the last administration of the compound.

[0812] Collect total blood in a serum tube and mix by gently inverting the tube 8-10 times. After clotting, centrifuge the blood sample at 1800 × g for 10 minutes. After centrifugation, store the serum at -80°C.

[0813] 4.7.4. Disease Assessment

[0814] Weight was assessed at the start of the study, then twice a week, and at the time of sacrifice.

[0815] Twice a week, score the clinical signs of inflammation: 0 - normal claw; 1 - if one toe is swollen; 2 - if two or more toes are swollen; 3 - if the entire claw is swollen. Sum the scores for all limbs to produce an overall score.

[0816] On day 32, mice from all groups received ProSense. ® 680 probe (0.8 nmol / 10 g, intraperitoneal) and OsteoSense ® 750EX probe (0.8 nmol / 10 g, intraperitoneal). On day 33, mice were anesthetized, and granulocyte infiltration and bone remodeling were measured using in vivo molecular imaging (Bruker In-Vivo Xtreme imaging system).

[0817] Ten mice were in each group. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's post-hoc test for scoring and imaging analysis in the diseased carrier group, and body weight analysis in the sham carrier group.

[0818] 4.7.5. Results

[0819] When this regimen was implemented, Cpd 6 was administered twice daily at 10 and 30 mg / kg, showing a statistically significant reduction in clinical scores compared to the carrier group with the disease.

[0820] 4.8. Mouse collagen-antibody-induced arthritis model (CAIA)

[0821] 4.8.1. Materials

[0822] The ArthritoMab™ antibody mixture for inducing arthritis and lipopolysaccharide (LPS) from E. coli serotype O55:B5 were purchased from MD Biosciences (Oakdale, MN, USA, Cat# CIA-MAB-50); PBS 1X (GIBCO, Cat#140190-086) was obtained from ThermoFisher Scientific and Enbrel. ® (Etanercept) was purchased from Chondrex (L'Isle d'Abeau, France, Cat# 3771910).

[0823] 4.8.2. Animals

[0824] Five- to seven-week-old female BALB / c mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France). The mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22°C, and food and water were provided freely.

[0825] 4.8.3. Research Design

[0826] The therapeutic effects of the compounds of this invention were tested in a mouse CAIA model (MD Biosciences, Inc. 2008; Nandakumar et al. 2003). On day 1 (D1), mice were randomly assigned to equal groups of 10 mice each. All mice, except the untreated group, were immunized with an ArthritoMab™ mixture (100 mg / kg, IV, 200 µL / mouse) and treatment with either the compound or the carrier was initiated. The weight and clinical score of each mouse were assessed daily, except at the weekend, until the end of the study. On day 4, all mice, except the untreated group, were challenged with LPS (2.5 mg / kg, intraperitoneally). On day 11, all mice were euthanized and blood samples were collected in serum tubes. After centrifugation, the serum was collected and frozen at -80°C for analysis (e.g., cytokine levels, gene expression, compound levels). For histological readings, the right and left hind paws were collected separately in vials (minimum 25 mL) filled with 4% buffered formaldehyde and kept at room temperature for a minimum of 24 hours and a maximum of 4 days.

[0827] 4.8.4. Clinical assessment of arthritis

[0828] Arthritis was scored according to the methods of Khachigian 2006; Lin et al. 2007 and Nishida et al. 2004 (Khachigian 2006; H.-S. Lin et al. 2007; Nishida, Komiyama, S.-I. Miyazawa et al. 2004). The swelling of each of the four paws was graded using the arthritis score as follows:

[0829] The final clinical score was the cumulative score of all four paws (the maximum cumulative clinical arthritis score per animal was 16) (Nishida et al., 2004). Curves of cumulative clinical scores for each group were plotted, and the area under the curve was calculated. Results are expressed as mean ± SEM and analyzed using one-way ANOVA, followed by Dunnett's post-hoc test for statistical analysis relative to the load group.

[0830] 4.9. A mouse model of atopic dermatitis induced by topical application of MC903.

[0831] 4.9.1. General Overview

[0832] Topical application of the vitamin D3 hypocalcemic analog MC903 (calcipotriol) to mice induced the development of chronic eczematous dermatitis, with histopathological changes similar to those of human atopic dermatitis (Li et al., 2006).

[0833] 4.9.2. Materials

[0834] Methylcellulose 0.5% (Cat# AX021233) was obtained from VWR. MC903 (calcipotriol, Cat# 2700 / 50) was obtained from Tocris Bioscience (Bristol, UK). ProSense ® 680 (Cat# NEV10003) was obtained from PerkinElmer (Massachusetts, USA). RNA later ® (Cat# AM7021) was obtained from Ambion (California, USA).

[0835] 4.9.3. Animals

[0836] Balb / cN mice (female, 18–20 g body weight) or CD1 / Swiss mice (female, 24–26 g body weight) were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22 ± 2 °C, and food and water were provided freely.

[0837] 4.9.4. Research Design

[0838] The design of this study was adapted from that of Li M. et al. (Li et al. 2006). On day 1 (D1), mice were anesthetized and the fur around both ears was shaved. Starting from D1, 20 µL of EtOH or 2 nmol of MC903 (in 20 µL of EtOH) was applied topically to each ear of the mice until D9, D11, or D15 (except at the weekend).

[0839] Starting on day 5, mice were administered the test compound (orally, twice daily, 0.5% in methylcellulose) or dexamethasone (5 mg / kg, orally, once daily, 0.5% in methylcellulose), or mixed with a carrier, until day 12.

[0840] 4.9.5. Quantification of plasma compound levels

[0841] The plasma concentrations of each test compound were determined by LC-MS / MS, with the mass spectrometer operated in either positive or negative electrospray mode.

[0842] 4.9.6. Determination of Pharmacokinetic Parameters

[0843] Using Phoenix ® WinNonlin ® (Pharsight ® , USA) calculate pharmacokinetic parameters.

[0844] 4.9.7. Disease Assessment

[0845] The thickness of each ear was measured using a thimometer (Mitutoyo, Absolute Digimatic, Cat# 547 321) before the first application of MC903 (baseline) (three times a week) and at the time of euthanasia.

[0846] Before the first administration of EtOH (baseline) (three times a week) and at the time of sacrifice, assess weight.

[0847] On day 10, mice anesthetized with isoflurane from all groups received ProSense. ® 680 probe (0.8 nmol / 10g, IV retroorbital sinus). Mice were anesthetized on day 2 (D11). Granulocyte infiltration was then measured using in vivo molecular imaging (Bruker In-VivoXtreme imaging system, excitation wavelength: 630 nm, emission wavelength: 700 nm, acquisition time: 5 seconds).

[0848] On day 12, 2 hours after the last administration, the mice were sacrificed, and the total blood was collected in EDTA-coated tubes. The plasma was frozen for further measurements (including circulating compounds).

[0849] Collect the auricle of the ear. Cut one ear longitudinally in half. Fix one half in 3.7% formaldehyde buffer for histology; immerse the other half in RNAlater. ® China and Israel assess gene expression.

[0850] Ten mice were in each group. Results are expressed as mean ± SEM and statistical analysis was performed using one-way ANOVA, followed by Dunnett's post-hoc test for ear thickness relative to the MC903 carrier group (mice treated with MC903 administered with the carrier alone) and / or body weight relative to the EtOH carrier group (mice treated with EtOH administered with the carrier alone).

[0851] Eight mice were in each group. Results are expressed as mean ± SEM and statistical analysis was performed using one-way ANOVA followed by Dunnett post-hoc tests for ear thickness and weight relative to the MC903 load group (mice treated with MC903 alone) and / or body weight relative to the EtOH load group (mice treated with EtOH alone).

[0852] 4.9.8. Histological examination

[0853] After euthanasia, halves of the ears were collected and fixed in 3.7% formaldehyde, then embedded in paraffin. 4 µm thick sections were immunostained with anti-CD3 antibody. The immunostained cell area of ​​the entire section from each mouse was measured by image analysis (CaloPix software, TRIBVN Healthcare, France). Data are presented as mean ± SEM, analyzed using one-way ANOVA, and subsequently statistically analyzed relative to the MC903 load group using Dunnett's post-hoc test.

[0854] 4.9.9. Gene Expression Analysis

[0855] Ear from RNAlater ® Removed from the liquid, at Bertin Instruments Precellys ® After being crushed with 1.4mm ceramic beads in a homogenizer, it was placed in Trizol. ® Then, total RNA was extracted using the phenol / chloroform protocol and processed using RNeasy. ® 96QIAcube ®The HT Kit (Qiagen, Cat# 74171) was purified using the QIAcube. cDNA was prepared and quantitative PCR was performed using gene-specific primers from Qiagen in a ViiA 7 real-time PCR system (Applied Biosystems) using SYBR Green technology. The expression levels of each gene of interest (GOI = IL4, IL5, IL13, TSLP, IL33, ST2, IL25, IL31, IFNγ, IL6, IL10, LCN2, S100A8, and S100A9) were calculated relative to the housekeeping gene expression levels (HPRT, GAPDH, and β-actin).

[0856] All qPCR data are expressed as mean ± SEM of normalized relative amounts (NRQ) calculated as follows: 1- Calculate the geometric mean of the NRQ for each group of animals.

[0857] 2- Calculate the NRQ scale compared to the MC903 loader group.

[0858] The statistical tests used were Dunnett's post-hoc test and ANOVA analysis of variance relative to the EtOH and / or MC903 carrier groups.

[0859] 4.10. LPS-induced endotoxin shock in mice

[0860] 4.10.1. General Overview

[0861] Injection of lipopolysaccharide (LPS) induces rapid release of soluble tumor necrosis factor (TNFα) into the periphery. This model is used to evaluate prospective inhibitors of TNFα release in vivo.

[0862] 4.10.2. Materials

[0863] The lipopolysaccharide (LPS) from Escherich serotype O111:B4 was obtained from Sigma Aldrich (catalog number L2630).

[0864] 4.10.3. Animals

[0865] BALB / cAnNCrl mice (female, 18–20 g body weight) were obtained from Charles River (Calco, Italy). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22 ± 2 °C, and food and water were provided freely.

[0866] 4.10.4. Research Design

[0867] The test compound was administered to mice once via oral (po) or subcutaneous (sc) route in an appropriate carrier.

[0868] At the compound's Tmax, 100 µg of LPS (in H2O) was injected intraperitoneally into mice. A control group was included, which involved administration of the load without LPS stimulation.

[0869] Mice were sacrificed 90 minutes after LPS stimulation, and blood was collected in heparinized tubes. Plasma was obtained by centrifugation at +4°C for 15 minutes (2000 × g) prior to cytokine analysis and then frozen at -80°C.

[0870] 4.10.5. Disease Assessment

[0871] TNFα and IL-10 in plasma were quantified using AlphaLISA assay kits obtained from PerkinElmer, Cat# AL505C and AL502C, respectively.

[0872] Statistical analysis was performed using Prism 5.03 software (GraphPad).

[0873] The active compound was defined as one that, compared with the load cell group, showed a statistically significant reduction in TNFα, whether or not statistically significant IL-10 induction was present.

[0874] 4.10.6. Results

[0875] 4.11. Multidrug Resistance-1a-ablated (MDRa1) model (mice)

[0876] 4.11.1. General Overview

[0877] Abcb1a (MDRa1) deficient mice develop spontaneous colitis, which can be transmitted through infection with Helicobacter bilirubin (MDRa1). Helicobacter bilis This model is used to evaluate the ability of compounds to treat or prevent colitis (Maxwell et al., 2015).

[0878] 4.11.2. Materials

[0879] Sterile PBS (Gibco, Cat# 20012027) was obtained from Thermo Fisher Scientific (Massachusetts, USA); Brucella agar (Cat# 211086) was obtained from Becton Dickinson (New Jersey, USA); Brucella broth (Cat# B3051-500g) was obtained from Sigma Aldrich (Missouri, USA). Fibrin-free sheep blood (Cat# SR0051) and Campygen (Cat# CN0025) were obtained from Thermo Fisher Scientific (Massachusetts, USA). Helicobacter bilirubin ATCC 51360 was obtained from LGC Standards (Molsheim, France), and Combur testE (Cat# 11896857) was obtained from Roche Diagnostics (Basel, Switzerland).

[0880] 4.11.3. Animals

[0881] Female MDR1a (FVB.129P2-Abcb1atm1Bor N7) mice aged 7-9 weeks were obtained from Taconic (Rensselaer, NY, USA), and female FVB mice aged 7-9 weeks were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were kept in a 12-hour light / dark cycle. The temperature was maintained at 22°C, and food and water were provided freely.

[0882] 4.11.4. Preparation of Helicobacter bile inoculum

[0883] Thaw frozen Helicobacter bile spp., place in Brucella broth, and incubate on Brucella agar slants containing 5% defibrinated sheep blood at 37°C under microaerophilic conditions for 4-5 days. On day 1, just before administration, dilute a portion of the Helicobacter bile spp. culture in PBS to obtain a 10⁻⁶ solution. 7 CFU / mouse, and place the second part into fresh Brucella broth and incubate for 7 days as previously described. On D8, just before administration, dilute the Helicobacter bilirubin culture in PBS to obtain 10. 7 CFU / mouse.

[0884] 4.11.5. Research Design

[0885] After a 10-day acclimatization period, the Disease Activity Index (DAI) of each MDR1a mouse was measured to form a homogeneous group based on DAI scores. Then, all mice (n=10) except the sham-operated group were administered an inoculum of *Helicobacter bilirubin* (10 μg / mL) orally (10 μg / mL). 7 CFU / mouse) and treatment was initiated according to the protocol for 6 weeks. A second administration of Helicobacter bilirubin was given 7 days after treatment initiation. DAI was measured twice weekly throughout treatment. Six weeks after treatment initiation, mice were euthanized, blood samples were collected, and intact colons were collected and washed with sterile PBS. The collected colons were measured and weighed to determine the colon weight / length ratio, and samples were subject...

Claims

1. A compound of formula I, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt: I in One of X1 and X2 is N, and the other is C; R 1 It is H or -P(=O)(OH)2; and R 2 yes - H， - Optionally replace C with one or more independently selected halogens. 1-4 alkyl, - -NR 3a R 3b ;and R 3a and R 3b C, each independently selected from H, optionally replacing one or more independently selected halogens. 1-4 Alkyl or phenyl.

2. The compound according to claim 1, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein R 2 It is C 1-4 alkyl.

3. The compound according to any one of claims 1-2, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein R 2 It is -CH3 or -CH2CH3.

4. The compound according to claim 1, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein R 2 It's H.

5. The compound, a pharmaceutically acceptable salt thereof, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of claims 1-5, wherein X1 is N and X2 is C.

6. The compound, a pharmaceutically acceptable salt thereof, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of claims 1-5, wherein X1 is C and X2 is N.

7. The compound of claim 1, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein the compound is a compound of formula IVa, IVb, IVc, or IVd: 。 8. The compound of claim 1, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein the compound is a compound of formula Va, Vb, Vc, or Vd: 。 9. The compound according to any one of claims 1-8, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein R 1 It's H.

10. The compound according to any one of claims 1-8, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, wherein R 1 It is -P(=O)(OH)2.

11. The compound of formula Va according to claim 8, wherein R 1 It is -P(=O)(OH)2, wherein the compound is a crystalline monohydrate, characterized in that, The X-ray powder diffraction pattern using Cu Kα radiation contains peaks at 9.1, 10.0, 13.2, 13.6, and 14.9 ± 0.2° 2θ.

12. The compound of formula Va according to claim 8, wherein R 1 It is -P(=O)(OH)2, wherein the compound is a crystalline hemihydrate, characterized in that, The X-ray powder diffraction pattern using Cu Kα radiation contains peaks at 9.1, 9.6, 15.1, 15.4, and 19.7 ± 0.2° 2θ.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of any one of claims 1-12, a pharmaceutically acceptable salt thereof, a solvate, or a solvate of a pharmaceutically acceptable salt thereof.

14. The compound according to any one of claims 1-12, its pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 13, for use in medicine.

15. The compound of any one of claims 1-12, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 13, for the prevention and / or treatment of: inflammatory diseases, autoinflammatory diseases, autoimmune diseases, proliferative diseases, fibrotic diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, diseases involving impaired bone turnover, diseases associated with excessive IL-6 secretion, diseases associated with excessive secretion of TNFα, interferon, IL-12, IL-17 and / or IL-23, respiratory diseases, endocrine diseases, metabolic diseases, cardiovascular diseases, skin diseases and / or diseases associated with abnormal angiogenesis.

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