Inhibitors of Rho-related coiled-coil-containing protein kinases

Abstract

To provide inhibitors of ROCK1 and / or ROCK2.SOLUTION: The present invention provides methods of inhibiting ROCK1 and / or ROCK2 that are useful for the treatment of disease.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to inhibitors of ROCK1 and / or ROCK2. Methods for inhibiting ROCK1 and / or ROCK2, which are useful for treating diseases, are also provided. [Background technology]

[0002] Rho-related coiled-coil protein kinase (ROCK) is a member of the serine / threonine kinase family. Two isoforms, ROCK1 and ROCK2, have been identified. Both isoforms are activated by the GTP-bound form of Rho GTPases, and upon activation, phosphorylate various downstream substrates. ROCK plays a crucial role in numerous cellular processes, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Therefore, ROCK inhibitors have potential therapeutic applications in a wide range of pathological conditions, including, for example, asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, renal failure, pulmonary hypertension, neurodegeneration, and osteoporosis.

[0003] ROCK is an important intracellular regulator of cytoskeletal dynamics and cell motility. Through phosphorylation, ROCK modulates several downstream targets of RhoA, including, for example, myosin light chain, myosin light chain phosphatase-binding subunit, and LIM kinase 2. These substrates regulate the organization and contractility of actin filaments. In smooth muscle cells, ROCK mediates calcium sensitization and smooth muscle contraction. Inhibition of Rho kinase blocks 5-HT and phenylephrine agonist-induced muscle contraction. When introduced into non-smooth muscle cells, ROCK induces stress fiber formation, which is required for RhoA-mediated cell transformation. ROCK is involved in a variety of cellular processes, including, but not limited to, cell adhesion, cell motility and migration, growth regulation, cell contraction, and cytokinesis. ROCK is also involved in Na / H exchange system activation, stress fiber formation, adusin activation, and physiological processes such as vascular stenosis, bronchial smooth muscle contraction, vascular smooth muscle and endothelial cell proliferation, and platelet aggregation.

[0004] Inhibition of ROCK activity in animal models has demonstrated several benefits of inhibiting ROCK to treat human diseases. These include models of cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, intraocular pressure, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, central nervous system disorders such as neurodegeneration and spinal cord injury, and neoplasia. Inhibition of ROCK activity has been shown to inhibit tumor cell proliferation and metastasis, angiogenesis, arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, asthma, regulation of intraocular pressure, and bone resorption. In patients, inhibiting ROCK activity has benefits in controlling cerebral vasospasm and ischemia after subarachnoid hemorrhage, lowering intraocular pressure, increasing aqueous humor outflow due to relaxation of the trabecular reticular tissue, improving blood flow to the optic nerve, and protecting healthy ganglion cells.

[0005] A considerable amount of in vivo data has been compiled focusing on ROCK activity in the CNS. Abnormal activation of the ROCK pathway has been recorded in many central nervous system disorders. For example, axonal growth and synaptic plasticity depend on the structural regulation of the actin cytoskeleton. The Rho-ROCK cascade plays a central role in synaptic plasticity, in both dendritic morphogenesis and stability, and even in growth cone motility and collapse. In addition, since several axonal growth inhibitory molecules concentrate in RhoA / ROCK in neurons, this makes it an attractive pathway for intervening in CNS disorders.

[0006] The Nogo receptor (NgR) (and other complex members, including LINGO-1) and their ligands are perhaps the best-characterized and potent inhibitors of neurite outgrowth. Some of the earliest downstream events of receptor activation by myelin-related inhibitors are the upregulation of RhoA and ROCK. These events result in increased contractility and have a strong inhibitory effect on axonal growth in mature neurons. Therefore, being able to inhibit this signaling cascade provides a very promising therapeutic strategy in spinal cord and optic nerve injuries. Neurodegenerative conditions such as Huntington's disease and Alzheimer's disease (AD) have also been investigated as responding to inhibition of NgR signaling. Not only are NgR family members associated with APP processing, but the intracellular localization of NgR and Nogo is also altered in the AD brain.

[0007] Alzheimer's disease (AD), the most common cause of dementia in the elderly, is a progressive neurodegenerative disorder associated with the gradual decline of many cognitive functions, including memory impairment (Selkoe, 2001). Synaptic loss is commonly observed in AD pathogenesis and is a prominent feature of synaptic dysfunction in AD (Tanzi and Bertram, 2005). Oligomerized β-amyloid peptides are thought to be involved in the loss of synaptic plasticity and neural network dysfunction. Synaptic plasticity is dependent on the structural regulation of the actin cytoskeleton in dendritic spines. The Rho-ROCK cascade plays a central role in synaptic plasticity, in both dendritic morphogenesis and stability, as well as in the motility and collapse of the growth cone (Govek et al., 2005; Linseman and Loucks, 2008). Several studies have demonstrated that ROCK kinase can induce the production of toxic β-amyloid peptides, and that inhibition of ROCK can inhibit toxic peptide processing. In a feedforward mechanism, β-amyloid increases Rho GTPase activity, which inhibits neurite outgrowth and synapse formation via ROCK activation (Petratos et al., 2008). Therefore, ROCK inhibitors may have the potential to prevent synaptic and neuronal degeneration, and even promote regeneration processes, in AD. A recent study by Herskowitz et al. showed that knockdown of ROCK reduces aβ levels. These effects demonstrate the need for highly ROCK-selective inhibitors to provide effective treatment for Alzheimer's disease (AD). To demonstrate the use of ROCK inhibition for AD by altering BACE-1 distribution and amyloid precursor protein (APP) transport to lysosomes, the model compound SR3677 was tested in a rodent model of AD. SR3677 showed promising effects in reducing sAPPβ after direct intrapial (ip) injection into the hippocampus, due to its inadequate oral PK properties (5%F and half-life of less than 1 hour) and lack of cerebral permeability.

[0008] Huntington's disease (HD) is a destructive, untreated, and primarily hereditary neurodegenerative disorder characterized by psychiatric impairment, motor impairment, and dementia. Misfolding and aggregation of the Htt protein, a product of the huntingtin gene, leads to HD pathology (Shao and Diamond, 2007). A very small number of mechanism-based therapeutic lead substances have been developed to treat HD. While scientific investigation is still ongoing, several pieces of evidence suggest that ROCK inhibition may be an effective treatment for HD. In a mouse model of HD, ROCK inhibition significantly reduced soluble Htt levels, reversed aggregate formation and neurite regression, and was protective against neuronal cell death (Deyts et al., 2009; Li et al., 2009). Similar results were obtained in studies in Drosophila, where ROCK inhibition controlled Htt aggregation (Shao et al., 2008a; Shao et al., 2008b). The ROCK signaling pathway is a promising therapeutic target for hemorrhagic disease (HD).

[0009] ROCK signaling has also been linked to Parkinson's disease and amyotrophic lateral sclerosis (ALD). See, for example, Tonges, L. et al. (2012). “Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson's disease.” Brain. 135(11):3355-70.

[0010] ROCK phosphorylates multiple downstream substrates, including myosin light chains (MLC, at threonine 18 and serine 19) and myosin light chain phosphatase (MYPT1, at threonine 853), driving polymerization from globular G-actin to filamentous F-actin and assembling the actomyosin contraction mechanism. This pathway has been shown to contribute to the pathogenesis of several CNS disorders, such as spinal cord injury, stroke, and Alzheimer's disease (AD). In adult CNS, damaged axons regenerate only inadequately due to the presence of myelin-related axonal growth inhibitors. Myelin-related inhibitors, such as myelin-associated glycoprotein (MAG), Nogo, oligodendrocyte-myelin glycoprotein (OMgp), and repulsive guidance molecules (RGM), limit axonal regeneration in damaged brain and spinal cord. The common mechanism among various myelin-related inhibitors is that they all activate Rho and its downstream effector kinase ROCK, thereby inhibiting neurite outgrowth.

[0011] Blocking the Rho / ROCK pathway with small molecules is a desirable strategy in central nervous system (CNS) disorders. However, the blood-brain barrier (BBB), while playing a crucial role in brain homeostasis, significantly hinders the penetration of many small molecule inhibitors. With growing interest in the development of selective and potent inhibitors to treat CNS diseases, there is an urgent need for ROCK1 and / or ROCK2 inhibitors, particularly those that can cross the blood-brain barrier. [Overview of the project]

[0012] In one embodiment, the present invention relates to a compound of formula I: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 N-C(=O)-(CR 12 R 13 ) c selected from the group consisting of; each R 10 is independently selected from H, lower alkyl, and C3-C6 cycloalkyl; each R 11 is independently selected from H, lower alkyl, and C3-C6 cycloalkyl; each R 12 is independently selected from H and lower alkyl; each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 may together form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is 2 - 4; d is 1 - 4; R 2 is selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which may be unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2These atoms combine to form a monocyclic or bicyclic group, where the monocyclic group has 4 to 7 ring atoms including up to 2 ring heteroatoms, and the bicyclic group has 8 to 10 ring atoms including up to 3 ring heteroatoms; the monocyclic and bicyclic groups are either unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, aryl, and heteroaryl; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl; R 4 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; R 5 The C3-C6 cycloalkyl group is selected from H, lower alkyl groups, and C3-C6 cycloalkyl groups; a is either 0 or 1; b is between 0 and 2; and Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or, by other means, R and R' together form a 5-6 membered heterocyclic ring.

[0013] The present invention includes a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0014] The present invention comprises compositions comprising substantially pure compounds of the present invention and pharmaceutically acceptable salts, stereoisomers, or hydrates thereof, and a pharmaceutically acceptable carrier.

[0015] In one embodiment, the present invention provides a method for inhibiting ROCK in mammals, comprising administering an effective amount of one or more compounds of formula I. The present invention also provides a method for treating a patient suffering from a disease, comprising administering a therapeutically effective amount of a compound of formula I to a patient requiring such treatment. In certain such embodiments, the compound of formula I inhibits ROCK1 and / or ROCK2. In certain such embodiments, the compound of formula I selectively inhibits ROCK1 and / or ROCK2. Non-limited diseases and conditions treated according to the present invention include central nervous system disorders, e.g., neurodegeneration and spinal cord injury; cardiovascular diseases, e.g., hypertension, atherosclerosis, restenosis, cardiac hypertrophy, intraocular pressure, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction; arterial thrombotic disorders, e.g., platelet aggregation and leukocyte aggregation; asthma; regulation of intraocular pressure; and bone resorption. In neoplasia, inhibition of Rho kinase inhibits tumor cell proliferation and metastasis, as well as angiogenesis.

[0016] The present invention provides a method for treating a central nervous system disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Central nervous system disorders include, but are not limited to, neurodegenerative or spinal cord injury, as well as Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or multiple sclerosis.

[0017] The present invention provides a method for treating an autoimmune disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Autoimmune disorders include, but are not limited to, rheumatoid arthritis, (multiple sclerosis), systemic lupus erythematosus (SLE; lupus), psoriasis, Crohn's disease, atopic dermatitis, eczema, or graft-versus-host disease (GVHD).

[0018] The present invention provides a method for treating a cardiovascular disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Cardiovascular disorders include, but are not limited to, hypertension, atherosclerosis, angina pectoris, arterial occlusion, peripheral artery disease, peripheral circulatory disorders, cavernous hemangioma, restenosis, cardiac hypertrophy, intraocular hypertension, cerebral ischemia, cerebral vasospasm, acute respiratory distress syndrome (ARDS), or erectile dysfunction.

[0019] The present invention provides a method for treating inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Inflammation includes, but is not limited to, asthma, cardiovascular inflammation, nephritis, or arteriosclerosis.

[0020] The present invention provides a method for treating arterial thrombotic disorders in a subject, comprising administering to the subject a therapeutically effective dose of a compound of formula I. Non-limiting examples of arterial thrombotic disorders include platelet aggregation or leukocyte aggregation.

[0021] The present invention provides a method for treating fibrotic disorders in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Non-limiting examples of fibrotic disorders include pulmonary fibrosis, including cystic and idiopathic pulmonary fibrosis; radiation-induced lung injury; hepatic fibrosis, including cirrhosis; cardiac fibrosis, including arterial fibrosis; endocardial myocardial fibrosis; old myocardial infarction; arterial stiffness; atherosclerosis; restenosis; joint fibrosis; Crohn's disease; myelofibrosis; Peyronie's disease; nephrogenic systemic fibrosis; progressive nodular fibrosis; retroperitoneal cavity fibrosis; scleroderma / systemic sclerosis; mediastinal fibrosis; keloids and hypertrophic scars; glial scars; or renal fibrosis.

[0022] The present invention provides a method for maintaining epithelial stability, comprising administering a therapeutically effective amount of a compound of formula I to a subject.

[0023] The present invention provides a method for treating glaucoma in a subject or regulating intraocular pressure in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Non-limiting examples of glaucoma include primary open-angle glaucoma, acute closed-angle glaucoma, pigmentary glaucoma, neovascular glaucoma, congenital glaucoma, normal-tension glaucoma, or secondary glaucoma.

[0024] The present invention provides a method for treating a neoplastic disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I. Neoplastic diseases include, but are not limited to, lymphomas, carcinomas, leukemias, sarcomas, or blastomas, such as squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, glioblastoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, or head and neck cancer.

[0025] The present invention also provides a method for treating metabolic syndrome, insulin resistance, hyperinsulinemia, type 2 diabetes, or glucose intolerance in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0026] Furthermore, the present invention provides a method for treating osteoporosis or promoting bone formation in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0027] The present invention provides a method for treating an eye disorder having a vascular component, comprising administering to a subject a therapeutically effective amount of a compound of formula I and an angiogenesis inhibitor. Non-limiting examples of such eye disorders include age-related macular degeneration (AMD), choroidal neovascularization (CNV), diabetic macular edema (DME), iris neovascularization, uveitis, neovascular glaucoma, or retinitis of prematurity (ROP). [Brief explanation of the drawing]

[0028] [Figure 1] This is the result of a typical Z'-Lyte assay. The ROCK inhibitor of the present invention exhibits low nanomolar efficacy (single order of magnitude) against both ROCK isoforms. [Figure 2] The compounds of the present invention inhibit both ROCK isoforms in cells. HCT116 cells in which either ROCK1 or ROCK2 was knocked out (ROCK1KO and ROCK2KO, respectively) were treated with the inhibitor for 90 minutes, and pMypt(T853) levels were visualized by Western blotting. The ROCK inhibitors efficiently blocked ROCK-targeted MYPT1 phosphorylation at 110 nM. [Figure 3] Human oligodendrocytes / neuronal progenitor cells were cultured in vitro for 2 and 14 days with and without a ROCK inhibitor (Compound 2 of Example). Different stages of neuronal differentiation were identified by visualizing nestin and MAP2 proteins with commercially available antibodies. ROCK inhibitors significantly promoted MAP2 expression, a marker of mature neurons, while improving neurite outgrowth, as evidenced by the marked increase in MAP2 signaling in cells differentiated in the presence of the ROCK inhibitor. [Figure 4] Under co-culture conditions of rat oligodendrocytes and rat dorsal root ganglion (DRG) explants, treatment with a ROCK inhibitor (Compound 2 in Example 2) altered the cytoskeleton, generating numerous short, aligned myelin segments, which were identified by nerve filament staining. Simultaneously, the ROCK inhibitor also promoted axonal support in oligodendrocytes, as demonstrated by the alignment of oligodendrocytes (stained MBP) along with the direction of axonal elongation (stained nerve filaments). [Modes for carrying out the invention]

[0029] ROCK inhibitors The compounds according to the present invention include compounds having formula I: [ka] Includes [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR 12 R 13 ) c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; R 2These are selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which may be unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2 These atoms combine to form a monocyclic or bicyclic group, where the monocyclic group has 4 to 7 ring atoms including up to 2 ring heteroatoms, and the bicyclic group has 8 to 10 ring atoms including up to 3 ring heteroatoms; the monocyclic and bicyclic groups are either unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, aryl, and heteroaryl; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl; R 4 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; R 5 The C3-C6 cycloalkyl group is selected from H, lower alkyl groups, and C3-C6 cycloalkyl groups; a is either 0 or 1; b is between 0 and 2; and Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5- to 6-membered heterocyclic ring.

[0030] Compounds according to the invention include compounds having the formula II:

Chemical formula

[0031] In a particular embodiment of the present invention, a compound of formula III: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR 12 R 13 ) c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; R 2These are selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which may be unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2 These atoms together form a monocyclic or bicyclic group, where the monocyclic group has 4 to 7 ring atoms including up to 2 ring heteroatoms, and the bicyclic group has 8 to 10 ring atoms including up to 3 ring heteroatoms; the monocyclic and bicyclic groups are either unsubstituted or optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, aryl, and heteroaryl; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl)- and Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or, by other means, R and R' together form a 5-6 membered heterocyclic ring.

[0032] In a particular embodiment of the present invention, a compound of formula IV: [ka] Provide [In the formula, Ring A is a five- or six-membered aromatic ring containing up to three ring heteroatoms of any choice; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl; R 4 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; b is between 0 and 2; R 6 This is selected from the group consisting of H, halo, lower alkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl, and carboxyl; R 7 This is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, and RCONR'-; Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or, by other means, R and R' together form a 5-6 membered heterocyclic ring; and m is between 1 and 3.

[0033] In a particular embodiment of the present invention, a compound of formula V: [ka] Provide [In the formula, Ring B is a five- or six-membered aromatic ring containing up to three ring heteroatoms of any choice; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl; R 4H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; b is between 0 and 2; R 8 is selected from the group consisting of H, halo, lower alkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl, and carboxyl; R 9 This is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy and carboxyl, RR'N-, RR'NCO-, RCONH-, and RCONR'-; Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or, by other means, R and R' together form a 5-6 membered heterocyclic ring; and m is between 1 and 3.

[0034] In a particular embodiment of the present invention, a compound of formula VI: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR12 R 13 ) c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; R 3 These are H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 Selected from alkyl; R 4 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; b is between 0 and 2; Each R 21 is independently selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; and n is between 0 and 3.

[0035] In a particular embodiment of the present invention, a compound of formula VII: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR 12 R 13 ) c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; R 4H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 Alkyl)-, and RR'N-(C 2~4 Selected from the group consisting of alkyl)-O-; b is between 0 and 2; Each R 21 is independently selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; and n is between 0 and 3.

[0036] In a particular embodiment of the present invention, a compound of formula VIII: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR 12 R 13 ) c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6-cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; Each R 21 is independently selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO2C-, aryl-O-, and heteroaryl-O-; and n is between 0 and 3.

[0037] In a particular embodiment of the present invention, a compound of formula IX: [ka] Provide [In the formula, R 1 This includes lower alkyl groups, substituted lower alkyl groups, C3-C6 cycloalkyl groups, substituted C3-C6 cycloalkyl groups, and R 10 R 11 N(CR 12 R 13 ) c -, R 10 O(CR 12 R 13 ) c -, W(CR 12 R 13 ) d - and R 10 R 11 NC(=O)-(CR 12 R 13 )c -Selected from the group consisting of; Each R 10 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 11 This is independently selected from H, lower alkyl, and C3-C6 cycloalkyl groups; Each R 12 It is independently selected from H and lower alkyl groups; Each R 13 It is independently selected from H and lower alkyl groups; In addition, or by other means, R bonded to the same carbon atom 12 and R 13 They may also come together to form a C3-C6 cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring heteroatoms; c is between 2 and 4; d is 1 to 4; Each R 22 is independently selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, amino, C1-C3 perfluoroalkyl, and C1-C3 perfluoroalkoxy; and n is between 0 and 3.

[0038] In preferred embodiments of formulas I to IX, R 1 is selected to be a lower alkyl group. More preferably, R 1 is a C1-C3 alkyl group, and more preferably R 1 It is either methyl or ethyl.

[0039] The term "alkyl" refers to radicals of saturated aliphatic groups, including linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, linear or branched alkyl groups have 8 or fewer carbon atoms in their main chain (e.g., C1-C8 for linear groups, and C3-C8 branched chains for branched groups), more preferably 6 or fewer. Similarly, preferred cycloalkyl groups have 3-8 carbon atoms in their ring structure, more preferably 3-6 carbon atoms in their ring structure.

[0040] Unless otherwise specified, “lower alkyl” as used herein means an alkyl group having 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, as defined above. In preferred embodiments, substituents shown herein as alkyl are lower alkyl groups. Lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0041] The term "cycloalkyl" refers to a saturated carbocyclic group having 3 to 6 carbon atoms in the ring. Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0042] The term "substituted alkyl" refers to an alkyl group having 1 to 3 substituents, as defined above. The substituents are selected from the group consisting of halo, hydroxyl, lower alkoxy, amino, lower alkylamino, nitro, cyano, perfluorolower alkyl, perfluorolower alkoxy, and carboxyl.

[0043] A "substituted lower alkyl" refers to a lower alkyl group that has 1 to 3 substituents, as defined above. The substituents are selected from the group consisting of halo, hydroxyl, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy, and carboxyl.

[0044] "Substituted cycloalkyls," such as "substituted C3-C6 cycloalkyls," refer to cycloalkyl groups that meet the definition above and have 1 to 3 substituents. The substituents are selected from the group consisting of halo, hydroxyl, lower alkyl, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy, and carboxyl.

[0045] As used herein, the term "halogen" or "halo" refers to -F, -Cl, -Br, or -I, preferably F, Cl, or Br.

[0046] As used herein, the terms "alkoxyl" or "alkoxy" refer to alkyl groups bonded via an oxygen atom as defined above. Typical alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. The term "lower alkoxy" refers to an alkoxy substituent to which a lower alkyl group is bonded via an oxygen atom, where the "lower alkyl" portion is as defined above.

[0047] The terms "amine" and "amino" refer to both unsubstituted and substituted amines, for example, in the general formula: [ka] This refers to the part that can be represented by [In the formula, R and R' are independently selected from H and lower alkyl groups, respectively.]

[0048] As used herein, the term “aryl” includes five- and six-membered monocyclic aromatic groups that may contain 0 to 4 heteroatoms, such as benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. These aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocyclic” or “heteroaryl” groups. The aromatic ring may be substituted at one or more ring positions with substituents as described above. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings (these rings are “fused rings”) in which two or more carbons are shared by two adjacent rings, and at least one of the rings is aromatic.

[0049] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group. Preferably, the alkyl group is a lower alkyl group as described above.

[0050] The term "heterocyclic" refers to a non-aromatic heterocyclic ring that has 4 to 7 ring atoms and contains 1 to 3 ring heteroatoms.

[0051] As used herein, the term “heteroatom” means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur. Most preferred are nitrogen and oxygen.

[0052] As used herein, each expression present more than once in any of the structures, such as alkyl, m, n, R 1 , R 2 Definitions such as these are intended to be independent of their definitions elsewhere within the same structure.

[0053] It will be understood that the terms "substitution" or "substituted with" implicitly include the conditions that such substitution conforms to the permissible valencies of the atom and substituent being substituted, and that the substitution results in a stable compound, one that does not undergo spontaneous transformation, such as rearrangement, cyclization, or removal.

[0054] As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. In a broad range of embodiments, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Examples of substituents include, for example, those listed herein. For a given organic compound, there may be one or more acceptable substituents, and they may be the same or different. For the purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any acceptable substituents of the organic compounds described herein that satisfy the valence of the heteroatom. The present invention is not intended to be limited in any way by the acceptable substituents of the organic compound.

[0055] As used herein, the term “protecting group” refers to a transient substituent that protects a potentially reactive functional group from undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been studied (Greene, TW; Wuts, PGMP Rotective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

[0056] Certain compounds of the present invention may exist in geometric or stereoisomeric forms. The present invention aims to encompass all such compounds, including cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, racemic mixtures thereof, and other mixtures thereof, which fall within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, and even mixtures thereof, are included in the present invention. The present invention also aims to encompass isotopic substitutions of atoms in compounds, for example, deuterium in the case of hydrogen.

[0057] In one embodiment, the present invention provides compounds of formulas I to IX that are inhibitors of ROCK. ROCK exists in two forms, namely ROCK1 (ROCKβ; p160-ROCK) and ROCK2 (ROCKα). In some embodiments, compounds of formulas I to IX selectively inhibit ROCK1. In some embodiments, compounds of formulas I to IX selectively inhibit ROCK2. In some embodiments, compounds of formulas I to IX are non-selective with respect to the inhibition of ROCK1 and ROCK2. In the context of the present invention, selective means that the inhibitor does not inhibit IC1 of other kinases. 50 Compared to that, the IC50 is at least 2 times, at least 5 times, at least 10 times, or at least 25 times lower for a given type of kinase. 50 It means to indicate.

[0058] Methods for determining kinase inhibition are known in the art. For example, the kinase activity and inhibitory capacity of an enzyme of a test compound can be determined by measuring the enzyme-specific phosphorylation of the substrate. Commercially available assays and kits are available and can be used. For example, kinase inhibition can be determined using the IMAP® assay (Molecular Devices). This assay method involves the use of a fluorescently labeled peptide substrate. Phosphorylation of the labeled peptide by the kinase of interest promotes the binding of the peptide to trivalent metal-based nanoparticles via a specific high-affinity interaction between the phospho group and the trivalent metal. Proximity to the nanoparticles results in increased fluorescence polarization. Inhibition of the kinase by a kinase inhibitor prevents the phosphorylation of the substrate, thereby limiting the binding of the fluorescently labeled substrate to the nanoparticles. Such assays can be adapted to a microwell assay format and allow for the IC50 detection of numerous compounds. 50 This makes it possible to decide both simultaneously.

[0059] Methods for treating diseases One aspect of the present invention provides a method for treating a patient suffering from a disease, comprising administering a therapeutically effective amount of the compound of the present invention to the patient in need of such treatment. As used herein, the term “therapeutably effective amount” means an amount of the compound, substance, or composition containing the compound of the present invention that is effective in producing some desired therapeutic effect in at least a subpopulation of animal cells with a reasonable benefit-to-risk ratio applicable to any medical treatment, e.g., reasonable side effects applicable to any medical treatment.

[0060] CNS failure The compounds of formulas I to IX exhibit effective blood-brain barrier (BBB) ​​penetration and distribution to central nervous system tissues. Therefore, the compounds of the present invention are useful in treating central nervous system disorders, as well as certain eye disorders that benefit from the ability to cross the BBB. Such disorders may include, but are not limited to, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, dementia, neurodegeneration, spinal cord injury, cerebral vasospasm, or multiple sclerosis, and may involve neurodegeneration or physical damage to nerve tissue.

[0061] Cardiovascular and other diseases The compounds of the present invention that inhibit ROCK and / or ROCK-mediated phosphorylation are useful for treating patients suffering from cardiovascular and non-cardiovascular diseases involving Rho kinase function, such as hypertension, pulmonary hypertension, atherosclerosis, restenosis, ischemic heart disease, cardiac hypertrophy, intraocular pressure, retinal disorders, ischemic diseases, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, peripheral circulatory disorders, peripheral artery occlusive disease, glaucoma (e.g., regulation of intraocular pressure), pulmonary fibrosis, hepatic fibrosis, renal fibrosis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, and central nervous system disorders, such as neurodegeneration and spinal cord injury. Furthermore, the ROCK inhibitors of the present invention can be used to treat arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, as well as bone resorption.

[0062] In one embodiment of the present invention, the compound is used to treat cerebral cavernous hemangioma (CCM). CCM is a vascular lesion consisting of a dense network of leaky, dilated capillaries and is associated with central nervous system (CNS) disorders, including seizures and strokes. Loss of vascular integrity is thought to involve RhoA activation and ROCK activation, leading to changes in cytoskeletal stability and increased vascular permeability. The compound of the present invention inhibits ROCK activation and regenerates vascular endothelial function.

[0063] Glaucoma In one embodiment of the present invention, compounds of formulas I to IX are used to treat glaucoma. The two most common types of primary open-angle glaucoma and acute closed-angle glaucoma are characterized by high intraocular pressure. Pigmentary glaucoma and congenital glaucoma are also characterized by reduced fluid outflow and high intraocular pressure (IOP). Normal-tension glaucoma is thought to be due to a different mechanism, particularly insufficient blood flow to the optic nerve. Secondary glaucoma can result from injury, infection, inflammation, tumor or cataract, and is also associated with long-term steroid use, systemic hypertension, diabetic retinopathy, and central retinal vein occlusion. Glaucoma with neovascularization may benefit from the administration of angiogenesis inhibitors in addition to ROCK inhibitors.

[0064] inflammation The present invention provides a method for treating inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formulas I to IX. Inflammation includes, but is not limited to, asthma, cardiovascular inflammation, nephritis, atherosclerosis and arteriosclerosis, and sepsis. Other inflammatory conditions that can be treated by the method of the present invention include fibrotic conditions (e.g., idiopathic pulmonary fibrosis, NASH, scleroderma, systemic sclerosis, and cirrhosis).

[0065] Autoimmune disorders The present invention provides a method for treating an autoimmune disorder in a subject, comprising administering a therapeutically effective amount of a compound of formulas I to IX to the subject. Autoimmune disorders include, but are not limited to, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE; lupus), psoriasis, Crohn's disease, atopic dermatitis, eczema, or graft-versus-host disease (GVHD), acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, and autoimmune disorders. Hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinal disorders, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuroneuropathy, Barlow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitisOstomyelitis (CRMO), Churg-Strauss syndrome, pemphigoid scarring / benign mucosal pemphigoid, Crohn's disease, Kogan's syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, herpetiform dermatitis, dermatomyositis, Dovic's disease (neuromyelitis optica), lupus discoid, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, nodules Erythema malformation, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrotic alveolar septitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA) (formerly known as Wegner granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes zoster of pregnancy, hypogammaglobulinemia, idiopathic Thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, lignite conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (M CTD), Mohren's ulcer, Mucher-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Dovic's disease), neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune streptococcal neuropsychiatric disorder), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Personnage-Turner syndromeSyndrome, squamous cell cartilage (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and III polyglandular autoimmune syndromes, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis This includes inflammation, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, autoimmune diseases of sperm and testes, generalized rigidus syndrome, subacute bacterial endocarditis (SBE), Suzac syndrome, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Trosa-Hunt syndrome, transverse myelitis, type 1 diabetes mellitus, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular and bullous skin diseases, and vitiligo.

[0066] According to the present invention, targeting of Th17 (IL-17 secreting) cells by ROCK inhibition provides a method for treating, in humans, autoimmune disorders, such as RA, MS, SLE, psoriasis, and Crohn's disease, and Th17 cell-mediated diseases including GVHD. In one embodiment of the present invention, the ROCK inhibitor is a compound of formula I.

[0067] Treg development and function depend on the activation of specific signal transduction pathways. Both TGF-β and IL-2 activate the expression of Foxp3 and STAT5 transcription factors, which play essential roles in regulating Treg repression. On the other hand, pro-inflammatory cytokines inhibit Foxp3 expression by upregulating STAT3 phosphorylation. According to the present invention, pharmacological inhibition of ROCK2 can modulate Treg function.

[0068] Neoplastic disease The ROCK inhibitors of the present invention inhibit the proliferation and metastasis of tumor cells, as well as angiogenesis, and are useful for treating neoplastic diseases. Neoplastic diseases include any malignant growth or tumor resulting from abnormal or uncontrolled cell division, which can spread to other parts of the body via the lymphatic system or bloodstream. Neoplastic diseases include, but are not limited to, lymphoma (a neoplasm of lymphoid tissue that is usually malignant), carcinoma (any malignant tumor of epithelial tissue), leukemia (a malignant neoplasm of hematopoietic tissue characterized by abnormal proliferation of white blood cells), sarcoma (a usually malignant tumor arising from connective tissue (such as bone or muscle)), and blastoma (a malignant lesion in precursor cells). Non-limiting examples include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, glioa astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancers.

[0069] Weight gain / loss According to the present invention, ROCK inhibitors are used to induce weight loss and / or limit weight gain. ROCK inhibitors promote weight loss in normal subjects and limit weight gain in subjects prone to obesity.

[0070] Insulin resistance In one embodiment of the present invention, a ROCK inhibitor is used to reduce or prevent insulin resistance or to restore insulin sensitivity. Therefore, in one embodiment, the compounds of the present invention are used to promote or restore insulin-dependent glucose uptake. In another embodiment of the present invention, the ROCK inhibitor is used to promote or restore glucose tolerance. In yet another embodiment of the present invention, the ROCK inhibitor is used to treat metabolic syndromes. In yet another embodiment, the ROCK inhibitor is used to reduce or prevent hyperinsulinemia. In one embodiment of the present invention, the ROCK inhibitor is used to treat diabetes (particularly type 2 diabetes). The ROCK inhibitor may also be used to promote or restore insulin-mediated relaxation of vascular smooth muscle cells (VSMCs).

[0071] angiogenesis The present invention provides methods and compounds for treating diseases and disorders involving vascular components. According to the present invention, in certain embodiments, such diseases and disorders are treated by administering an effective amount of a ROCK inhibitor to the subject. According to the present invention, such diseases and disorders can also be treated by administering an effective amount of a rho kinase inhibitor and an effective amount of angiogenesis inhibitor. According to the present invention, eye diseases and disorders having vascular components are treated by this method. In one embodiment, the present invention provides a method for treating age-related macular degeneration (AMD) occurring in "dry" and "exudative" forms. The "exudative" form of AMD results in vision loss due to abnormal vascular growth (neovascularization). Bleeding, leakage, and scarring from these retinal blood vessels ultimately result in irreversible damage to photoreceptors. The dry form results from atrophy of the retinal pigment epithelium, which results in vision loss due to the loss of photoreceptors (rods and cones) in the central part of the eye. In another embodiment, the present invention provides a method for treating choroidal neovascularization (CNV). Choroidal neovascularization is the process by which new blood vessels grow in the choroid via Bruch's membrane and invade the subretinal space, and is a symptom of age-related macular degeneration, myopia, and ocular trauma, among other causes. In another embodiment, the present invention provides a method for treating diabetic macular edema (DME). In another embodiment, the present invention provides a method for treating macular edema secondary to branch retinal vein occlusion (BRVO) or central retinal vein occlusion (CRVO). In other embodiments, diseases to be treated include, but are not limited to, infectious and non-infectious retinal neovascularization, infectious and non-infectious corneal neovascularization, iris neovascularization, uveitis, neovascular glaucoma, and retinitis of prematurity (ROP). The treatment method may be prophylactic, such as to avoid corneal neovascularization after corneal transplantation or to regulate the wound healing process in palisade excision surgery. These diseases and disorders may be characterized by having a vascular component. According to the present invention, such disorders are treated by administering ROCK inhibitors and angiogenesis inhibitors.

[0072] Therefore, in one such embodiment, the disease or disorder is AMD, and a subject requiring treatment for AMD is administered a ROCK inhibitor in an amount effective for treating AMD. In another embodiment, the subject is administered a ROCK inhibitor and an angiogenesis inhibitor in amounts effective for treating AMD. In some embodiments, the angiogenesis inhibitor is a VEGFR2 antagonist. In certain such embodiments, the VEGFR2 antagonist binds to VEGF. In other such embodiments, the VEGFR2 antagonist binds to VEGFR2. Such VEGFR2-binding inhibitors include, but are not limited to, antibodies and their VEGFR2-binding fragments, which are activators that bind to the extracellular domain of VEGFR2, as well as activators that interact with the intracellular domain of VEGFR2 and block the activation of VEGFR2-dependent signaling. VEGFR2 antagonists further include activators that interact with other cellular components to block VEGFR2-dependent signaling. Other embodiments of the present invention similarly treat other ophthalmic diseases and disorders having vascular-derived components as described above.

[0073] According to the present invention, ROCK inhibitors and angiogenesis inhibitors are administered to a subject in an effective amount to treat or prevent pathological conditions characterized by excessive angiogenesis. For example, such conditions involving angiogenesis and / or inflammation include atherosclerosis, rheumatoid arthritis (RA), hemangiomas, angiofibromas, and psoriasis. Other non-specific examples of vascular diseases include retinopathy of prematurity (posterior fibrous proliferation of the lens), corneal graft rejection, corneal neovascularization associated with complications of refractive surgery, corneal neovascularization associated with contact lens complications, corneal neovascularization associated with pterygium and recurrent pterygium, corneal ulcerative diseases, and nonspecific ocular surface diseases, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, Crohn's disease, autoimmune nephritis, primary biliary cirrhosis, acute pancreatitis, allochromic graft rejection, allergic inflammation, contact dermatitis and delayed hypersensitivity reactions, inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis, cognitive impairment induced by neuronal inflammation, Osler-Weber syndrome, restenosis, and fungal, parasitic and viral infections, including cytomegalovirus infection.

[0074] This invention provides pan-ROCK inhibitors (i.e., compounds that inhibit ROCK1 and ROCK2). Some studies have observed that ROCK2 is often overexpressed in hepatocellular carcinoma compared to non-tumor liver, while ROCK1 expression remains unchanged. Other cancers that may benefit from treatment with ROCK2 selective inhibitors include, but are not limited to, colorectal and bladder cancer. In contrast, higher ROCK1 expression levels have been observed in breast tumors. Any cancer can be tested to determine whether ROCK1 and / or ROCK2 overexpression is present and treated accordingly. In certain circumstances, ROCK1 and ROCK2 isoforms may exhibit similarities in modulating certain downstream targets, and neither isoform is considered dominant. In such cases, pan-ROCK inhibitors may be preferred.

[0075] Combination with other active substances The compounds of the present invention can be advantageously administered together with a second active agent to patients who require it. When a ROCK inhibitor is administered together with a second active agent, the ROCK inhibitor and the second active agent can be administered sequentially or incidentally. Sequentially means that one active agent is administered over a period of time, followed by the administration of the other active agent, which may be after the administration of the first active agent. When the active agents are administered sequentially, the level of one active agent does not need to be maintained at a therapeutically effective level when the second active agent is administered, and vice versa. Incidentally means that the first and second active agents are administered according to a schedule in which the active agents are not administered simultaneously, but both active agents are maintained at substantially therapeutically effective levels. Each active agent can be administered in single or multiple doses, and the dose can be administered on any schedule, including, but not limited to, twice daily, daily, weekly, every two weeks, and monthly.

[0076] The present invention also includes adjunctive administration. Adjunctive administration means that a second active substance is administered to a patient in addition to a first active substance that has already been administered to treat a disease or disease symptoms. In some embodiments, adjunctive administration involves administering the second active substance to a patient whose disease or disease symptoms have not been adequately treated by the administration of the first active substance. In other embodiments, adjunctive administration involves administering the second active substance to a patient whose disease has been effectively treated by the administration of the first active substance, but to whom the adjunctive administration is expected to improve the treatment outcome. In some embodiments, the effects of administering the first and second active substances are synergistic. In some embodiments, administering the first and second active substances prevents relapse or prolongs the time to relapse compared to administering either active substance alone. In some embodiments, administering the first and second active substances allows for a reduction in the dosage and / or frequency of administration of the first and second active substances.

[0077] Anti-inflammatory and immunosuppressant agents that can be administered in combination with the compounds of the present invention include steroids, such as glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus), cyclosporine, fingolimod, interferons, such as IFNβ or IFNγ, tumor necrosis factor alpha (TNF-α) binding proteins, such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira), mycophenolic acid, MMF, methotrexate, NSAIDs, statins, and cyanopropyl alcohol. This includes lolimus / temsirolimus / everolimus, abatacept (Orencia), anakinra (Kineret), certolizumab (Cimzia), golimumab (Simponi), ixekizumab (Taltz), natalizumab (Tysabri), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect), daclizumab (Zinbryta), muromonab (Orthoclone OKT3), Jakafi (luxolitinib), Xeljanz (Tofacitnib), and Otezla (apremilast).

[0078] In one embodiment, the RHO kinase inhibitor and antitumor agent of the present invention are administered to a subject in need. In another embodiment, the RHO kinase inhibitor and angiogenesis inhibitor of the present invention are administered to a subject in need. In yet another embodiment, the RHO kinase inhibitor and anti-inflammatory agent of the present invention are administered to a subject in need. In yet another embodiment, the ROCK inhibitor and immunosuppressant of the present invention are administered. The second active substance may be, but is not limited to, a small molecule, an antibody or its antigen-binding fragment, or radiation.

[0079] Antineoplastic agents include, but are not limited to, cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation. In addition to the rho kinase inhibitors of the present invention, compounds and agents that can be administered to treat tumors include: irinotecan, etoposide, camptothecin, 5-fluorouracil, hydroxyurea, tamoxifen, paclitaxel, capcitabine, carboplatin, cisplatin, bleomycin, dactomycin, gemcitabine, doxorubicin, danorubicin, cyclophosphamide, and radiotherapy, which may be extracorporeal (e.g., external beam radiation therapy (EBRT)) or intracorporeal (e.g., brachytherapy (BT)).

[0080] Targeted small molecules and biological molecules include, but are not limited to, inhibitors of components of signaling pathways, such as tyrosine kinase modulators and receptor tyrosine kinase inhibitors, as well as activators that bind to tumor-specific antigens. Examples include inhibitors of epidermal growth factor receptor (EGFR), including gefitinib, erlotinib, and cetuximab; inhibitors of HER2 (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DM1; T-DM1), and pertuzumab); anti-VEGF antibodies and fragments (e.g., bevacizumab); antibodies that inhibit CD20 (e.g., rituximab, ibritumomab); anti-VEGFR antibodies (e.g., ramucirumab (IMC-1121B), IMC-1C11, and CDP791); anti-PDGFR antibodies; and imatinib. Small molecule kinase inhibitors may be specific to one particular tyrosine kinase or may be inhibitors of two or more kinases. For example, the compound N-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydrocyclopenta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazoline-4-amine (also known as XL647, EXEL-7647, and KD-019) is an in vitro inhibitor of several receptor tyrosine kinases (RTKs), including EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3), and ErbB2, and is also an inhibitor of SRC kinases involved in pathways that result in tumor nonresponsiveness to certain TKIs. In one embodiment of the present invention, the treatment of the target to be required includes administration of ROCK inhibitors of formulas I to IX and administration of KD-019.

[0081] Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orally bioavailable ATP site-competitive Src inhibitor. Dasatinib targets Bcr-Abl (approved by the FDA for use in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL)), as well as c-Kit, PDGFR, c-FMS, EphA2, and Src family kinases. Two other oral tyrosine kinase inhibitors of Src and Bcr-Abl are bosutinib (SKI-606) and salakatinib (AZD0530).

[0082] According to the present invention, angiogenesis inhibitors can be administered to a subject in combination with the compounds of the present invention. Angiogenesis inhibitors include any substance that inhibits the growth of new blood vessels. For example, angiogenesis inhibitors include VEGF, PlGF, and VEGF receptor antagonists, including antibodies disclosed herein. VEGF antagonists reduce or block VEGF-related functions in cells. VEGF antagonists may act on VEGF by binding to VEGF and blocking its binding to its receptor, and / or on other cellular components involved in VEGF-mediated signaling. Similarly, VEGFR2 antagonists are substances that reduce or block VEGFR2-mediated signaling by blocking binding to and ligand binding to VEGFR2 or by interacting with VEGFR2 substrates, or by acting on other cellular components to reduce or block VEGFR2-mediated signaling. Therefore, angiogenesis inhibitors include, but are not limited to, anti-VEGFR2 antibodies, as well as VEGF, VEGFR1, VEGFR2, PDGF, PDGFR-β, neuropilin-1 (NRP1), and complement antagonists.

[0083] Angiogenesis inhibitors include, for example, intracellular agents that block signaling mediated by VEGF, PDGF, ligands for VEGF or PDGF receptors, or complement. Intracellular agents that inhibit angiogenesis inhibitors include, but are not limited to, the following: Sunitinib (Sutent; SU11248) is a panspecific small molecule inhibitor of VEGFR1-VEGFR3, PDGFRα and PDGFRβ, stem cell factor receptor (cKIT), Flt-3, and colony-stimulating factor-1 receptor (CSF-1R). Axitinib (AG013736; Inlyta) is another small molecule tyrosine kinase inhibitor that inhibits VEGFR-1-VEGFR-3, PDGFR, and cKIT. Cedilanib (AZD2171) is an inhibitor of VEGFR-1-VEGFR-3, PDGFRβ, and cKIT. Sorafenib (Nexavar) is another small molecule inhibitor of several tyrosine protein kinases, including VEGFR, PDGFR, and Raf kinase. Pazopanib (Votrient; (GW786034)) inhibits VEGFR-1, -2, and -3, cKIT, and PDGFR. Foretinib (GSK1363089; XL880) inhibits VEGFR2 and MET. CP-547632 is a potent inhibitor of VEGFR-2 and basic fibroblast growth factor (FGF) kinase. E-3810 ((6-(7-((1-aminocyclopropyl)methoxy)-6-methoxyquinoline-4-yloxy)-N-methyl-1-naphthamide) inhibits VEGFR-1, -2, and -3 as well as FGFR-1 in the nanomolar range. It inhibits EGFR-2 kinase. Brivanib (BMS-582664) is a VEGFR-2 inhibitor that also inhibits FGF receptor signaling. CT-322 (Adonectin) is a small protein based on the human fibronectin domain that binds to VEGFR2 and inhibits its activation. Vandetanib (Caprelas; Zactima; ZD6474) is an inhibitor of VEGFR2, EGFR, and RET tyrosine kinase. X-82 (Xcovery) is a small molecule indolinone inhibitor of signaling mediated by growth factor receptors VEGFR and PDGFR.

[0084] Pharmaceutical composition In one embodiment, the present invention provides a pharmaceutically acceptable composition comprising one or more compounds of formulas I to IX in a therapeutically effective amount, formulated together with one or more pharmaceutical excipients. As described below, the pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including (1) oral administration (e.g., oral tablets (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for buccal, sublingual, and systemic absorption, boluses, powders, granules, and pastes for application to the tongue), (2) parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or epidural injection) (e.g., sterile solutions or suspensions, or as sustained-release formulations), (3) topical application (e.g., as creams, ointments, or controlled-release patches or sprays applied to the skin), (4) vaginal or rectal administration (e.g., as vaginal suppositories, creams, or foams), (5) sublingual, (6) intraocular, (7) transdermal, or (8) transnasal.

[0085] The term "pharmaceutically acceptable" is used herein to mean a compound, substance, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, within the bounds of proper medical judgment, with a reasonable benefit-to-risk ratio of toxicity, irritation, allergic response, or other problems or complications.

[0086] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid extender, diluent, additive, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, that is involved in transporting or delivering the compound from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and must not be harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose, and derivatives of cellulose such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) additives such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) propylene glycosides. (11) Glycols such as (11) Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol, (12) Esters such as ethyl oleate and ethyl laurate, (13) Agar, (14) Buffers such as magnesium hydroxide and aluminum hydroxide, (15) Alginic acid, (16) Pyrolytic water-free, (17) Isotonic saline, (18) Ringer's solution, (19) Ethyl alcohol, (20) pH buffer solution, (21) Polyesters, polycarbonates and / or polyanhydrides, and (22) Other non-toxic and suitable substances used in pharmaceutical formulations.

[0087] As presented above, certain embodiments of the compound may contain basic functional groups such as amino or alkylamino, and therefore can form pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term “pharmaceutically acceptable salt” in this context refers to relatively non-toxic, inorganic, and organic acid addition salts of the compound of the present invention. These salts can be prepared in situ in the administration vehicle or during the dosage form manufacturing process, or by reacting the purified compound of the present invention in free base form separately with a suitable organic or inorganic acid, and then isolating the salt thus formed during subsequent purification. Typical salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptate, lactobionate, and lauryl sulfonate (see, for example, Berge et al. (1977) "Pharmaceutical Salts, J. Pharm. Sci. 66:1-19").

[0088] pharmaceutically acceptable salts of this compound include conventional non-toxic salts or quaternary ammonium salts of the compound from non-toxic organic or inorganic acids, for example. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isothionic acid.

[0089] In other cases, the compounds of the present invention may contain one or more acidic functional groups and can therefore form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these examples, the term “pharmaceutically acceptable salt” refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ in the administration vehicle or in the dosage form manufacturing process, or by reacting the purified compound in free base form separately with a suitable base such as a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate, ammonia, or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Typical alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Typical organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine (see, e.g., Berge et al., cited above).

[0090] Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0091] Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0092] The formulations of the present invention include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations can be conveniently presented in unit dosage forms and can be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier to produce a single dosage form varies depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier to produce a single dosage form is the amount of the compound that produces the therapeutic effect. Generally, out of 100%, this amount is in the range of about 0.1% to about 99%, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0093] In certain embodiments, the formulation of the present invention comprises cyclodextrin, cellulose, liposomes, micellar-forming agents such as bile acids, and polymer carriers such as polyesters and polyanhydrides; as well as the compound of the present invention. In certain embodiments, the above-described formulation makes the compound of the present invention orally bioavailable.

[0094] The preparation methods for these formulations or compositions include the step of mixing the compound of the present invention with a carrier and optionally one or more minor components. Generally, formulations are prepared by homogeneously and closely mixing the compound of the present invention with a liquid carrier or a fine solid carrier, or both, and then, if necessary, shaping the product.

[0095] Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges, powders, granules, or liquids or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, each containing a predetermined amount of the compound of the present invention as an active ingredient. The compound of the present invention may also be administered as a bolus, lick, or paste.

[0096] In the solid dosage forms of the present invention for oral administration (capsules, tablets, pills, sugars, powders, granules, lozenges, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable additives, including sodium citrate or dicalcium phosphate and / or any of the following pharmaceutically acceptable carriers: (1) fillers and bulking agents such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants such as glycerol; (4) agar, calcium carbonate, potato or tapioca starch, alginic acid, and / or other specific (5) Disintegrants such as certain silicates and sodium carbonate; (6) Dissolution retarders such as paraffin; (7) Absorption enhancers such as quaternary ammonium compounds and surfactants such as poloxamer and sodium lauryl sulfate; (8) Wetting agents such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (9) Absorbents such as kaolin and bentonite clay; (10) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (11) Colorants; and (12) Release control agents such as crospovidone or ethylcellulose. In the case of capsules, tablets and pills, the pharmaceutical composition may further contain buffers. Similar types of solid compositions may be used as fillers in soft and hard gelatin capsules, using lactose or lactose, and even high molecular weight polyethylene glycol as such additives.

[0097] Tablets may be prepared by compressing or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or sodium cross-linked carboxymethylcellulose), a surfactant or dispersant. Wet tablets may be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent using suitable machinery.

[0098] Tablets of the pharmaceutical compositions of the present invention, as well as other solid dosage forms such as sugar-coated tablets, capsules, pills and granules, may optionally be scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical art. These dosage forms may also be formulated to provide for sustained or controlled release of the active ingredient in the dosage form using, for example, various proportions of hydroxypropylmethylcellulose, or using other polymeric matrices, liposomes and / or microparticles, to obtain the desired release profile. They may be formulated for rapid release, for example, by lyophilization. They may be sterilized, for example, by filtration through a bacteria-retentive filter, or by incorporating a sterilizing agent in the form of a sterile solid composition which is soluble in sterile water or some other sterile injectable medium immediately prior to use. These compositions may optionally contain an opacifying agent and may be compositions which release the active ingredient(s) only, or preferentially, in a particular part of the digestive tract, optionally with a delay. Examples of implant compositions which can be used include polymeric substances and waxes. The active ingredient may, if appropriate, be in the form of microcapsules containing one or more of the above additives.

[0099] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may include solubilizing and emulsifying agents such as inert diluents commonly used in the art, such as water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, sorbitan polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof.

[0100] In addition to the diluent, the oral composition may also contain additional additives such as wetting agents, emulsifying agents and suspending agents, sweetening, flavoring, coloring, perfuming and preserving agents.

[0101] The suspending agent may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof, in addition to the active compound.

[0102] The pharmaceutical compositions of the present invention for rectal or vaginal administration may be presented as suppositories, which are prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating additives or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate, which are solid at room temperature but liquid at body temperature and thus dissolve in the rectal or vaginal cavity to release the active compound.

[0103] Preparations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers, which are known in the art to be suitable.

[0104] Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0105] In addition to the active compound of the present invention, ointments, pastes, creams, and gels may contain additives such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0106] Powders and sprays may contain additives such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof, in addition to the compounds of the present invention. Sprays may also contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0107] Transdermal patches offer the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the compounds in a suitable medium. Absorption enhancers can also be used to increase the flow rate of the compounds through the skin. The rate of such flow can be controlled by providing a rate-controlled membrane or by dispersing the compounds in a polymer matrix or gel.

[0108] Ophthalmic preparations, eye ointments, powders, liquids, and the like are also intended to be within the scope of the present invention.

[0109] A pharmaceutical composition of the present invention suitable for parenteral administration comprises one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous aqueous solutions, dispersions, suspensions or emulsions, or one or more compounds of the present invention in combination with sterile powders, which can be reconstituted into sterile injectable solutions or dispersants immediately before use, and which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes to make the formulation isotonic with the blood of the intended recipient, suspending agents or thickeners.

[0110] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants.

[0111] These compositions may also contain additional additives such as preservatives, humectants, emulsifiers, and dispersants. The inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, can ensure the inhibition of microbial activity in the compound. It may also be desirable for the composition to include isotonic agents such as sugars and sodium chloride. In addition, prolonging the absorption of the injectable pharmaceutical form is achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin.

[0112] In some cases, it is desirable to slow down the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This can be achieved by using a liquid suspension of a crystalline or amorphous substance that is poorly soluble in water. The absorption rate of the drug then depends on its dissolution rate, which may further depend on the crystal size and form. Alternatively, the absorption delay of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oily vehicle.

[0113] Injectable depot formulations are prepared by forming a microcapsule substrate of the compound in a biodegradable polymer such as polylactide-polyglycolide. The drug release rate can be controlled depending on the ratio of the drug to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot injection formulations can also be prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

[0114] When the compounds of the present invention are administered to humans and animals as pharmaceuticals, they can be administered as is or in combination with a pharmaceutically acceptable carrier, as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of the active ingredient.

[0115] Route of administration and dosage The formulations of the present invention can be administered orally, parenterally, topically, or rectally. These are, of course, administered in a form appropriate to each route of administration. For example, they can be administered in the form of tablets or capsules, by injection, inhalation, eye drops, ointment, suppositories, etc.; by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository. Oral administration is preferred.

[0116] As used in this invention, the expressions "parenteral administration" and "administered parenterally" refer to administration methods other than intestinal and local administration, which are usually administered by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, percutaneous intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, and intrasternal injections and infusions.

[0117] As used in the present invention, the expressions "systemic administration", "administered systemically", "peripheral administration" and "administered peripherally" mean the administration (e.g., subcutaneous administration) of a compound, drug or other substance that enters the patient's system and thus undergoes metabolism and other similar processes, other than entering directly into the central nervous system.

[0118] These compounds may be administered to humans and other animals for therapeutic purposes by any suitable route of administration, including oral, nasal (e.g., by aerosol), rectal, intravaginal, parenteral, intravesical, and topical administration by powders, ointments or drops (including buccal and sublingual administration).

[0119] Regardless of the selected route of administration, the compounds of the present invention, and / or the pharmaceutical compositions of the present invention, which may be used in the appropriate hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0120] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may vary depending upon the particular patient, composition, and mode of administration in order to obtain an amount of the active ingredient that is not toxic to the patient and effective to achieve the desired therapeutic response.

[0121] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention, or ester, salt or amide thereof, used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being used, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or substances used in combination with the particular compound being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known to the medical and veterinary arts.

[0122] [[ID=​

[0123] Generally, the appropriate daily dose of the compound of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose generally depends on the factors mentioned above. Generally, when used for the indicated analgesic effect, the oral, intravenous, intraventricular, and subcutaneous doses of the compound of the present invention to a patient vary in the range of about 0.0001 to about 100 mg per kg of body weight per day.

[0124] In certain embodiments, a dose of the compound or composition is administered daily, every other day, every two days, every three days, once a week, twice a week, three times a week, or once every two weeks. If desired, the effective daily dose of the active compound may be administered as sub-doses in two, three, four, five, six or more doses at appropriate intervals throughout the day, and optionally, in unit dosage form. In some embodiments, a dose(s) of the compound or composition is administered over two, three, five, seven, fourteen, or twenty-one days. In certain embodiments, a dose of the compound or composition is administered over one month, one and a half months, two months, two and a half months, three months, four months, five months, six months, or longer.

[0125] The above dosing schedule is provided for illustrative purposes only and should not be considered limiting. Those skilled in the art will readily understand that any dosage falls within the scope of the present invention.

[0126] Patients receiving this treatment include primates, particularly humans, as well as other mammals such as horses, cattle, pigs, and sheep; and any necessary animals, including common poultry and pets.

[0127] The compounds used in the methods of the present invention can be administered as is or mixed with a pharmaceutically acceptable carrier, and can also be administered in combination with antimicrobial agents such as penicillin, cephalosporins, aminoglycosides, and glycopeptides. Therefore, combination therapy involves administering active compounds sequentially, simultaneously, and separately in such a manner that the therapeutic effect of the first administered formulation is not completely lost when the subsequent formulation is administered.

[0128] Adding the active compound of the present invention to animal feed is preferably achieved by preparing a suitable feed premix containing an effective amount of the active compound and incorporating the premix into the finished feed.

[0129] Alternatively, intermediate concentrates or feed supplements containing active ingredients can be blended into the feed. Methods for preparing and administering such feed premixes and finished feeds are described in the reference books (e.g., “Applied Animal Nutrition”, WH Freedman and CO., San Francisco, USA, 1969, or “Livestock Feeds and Feeding”, O and B books, Corvallis, Ore., USA, 1977).

[0130] Microemulsification techniques may be used to improve the bioavailability of lipophilic (water-insoluble) pharmaceuticals. Examples include Trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, PC, et al., J Pharm Sci 80(7), 712-714, 1991). Microemulsification, in particular, enhances bioavailability by preferentially inducing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing the breakdown of the compound in the hepatobiliary circulation.

[0131] Controlled release The release characteristics of the formulations of the present invention depend on the encapsulation material, the concentration of the encapsulated drug, and the presence of release modifiers. For example, release can be manipulated in a pH-dependent manner by using pH-sensitive coatings that release only at low pH, such as in the stomach, or only at high pH, ​​such as in the intestines. Enteric coatings can be used to prevent release from occurring until the drug has finished passing through the stomach. Multilayer coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain initial release in the stomach followed by a later release in the intestines. Release can also be manipulated by including salts or pore-forming agents, which can increase drug release by water uptake or diffusion from the capsule. Additives that alter the solubility of the drug can also be used to control the release rate. Active ingredients that enhance matrix breakdown or release from the matrix can also be incorporated. Depending on the compound, these can be added to the drug, added as a separation phase (i.e., as microparticles), or co-dissolved with the polymer phase. The types of decomposition accelerators include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, as well as organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, and surfactants such as Tween® and Pluronic®. Pore-forming agents (i.e., water-soluble compounds such as inorganic salts and sugars) are added as microparticles to add microstructure to the matrix. The range must be between 1 and 30% (w / w polymer).

[0132] Uptake can also be manipulated by altering the residence time of particles in the digestive tract. This can be achieved, for example, by coating the particles with a mucosal adhesive polymer or by selecting such a polymer as an encapsulation material. Examples include chitosan, cellulose, and most polymers having free carboxyl groups, especially polyacrylates (in the context of this invention, polyacrylate refers to polymers containing acrylate groups as well as modified acrylate groups such as cyanoacrylate and methacrylate).

[0133] Those skilled in the art should understand and anticipate that they can create variations within the principles of the invention disclosed herein, and that such modifications are intended to fall within the scope of the invention. The following examples further illustrate the invention, but should not be construed as limiting the scope of the invention in any way. All references cited herein are incorporated herein by reference in their entirety. [Examples]

[0134] Example 1 All solvents and reagents were commercially obtained and used as obtained. 1¹H NMR spectra were recorded using a Bruker instrument (300 MHz or 400 MHz) in the specified deuterated solvent. Chemical shifts are expressed in ppm, and coupling constants are in Hertz. All final compounds were purified using CH3CN / water as the solvent by flash chromatography using 220–400 mesh silica gel or reverse-phase HPLC. Thin-layer chromatography was performed on silica gel 60F-254 (0.25 nm thick) plates. Visualization was achieved with UV light and / or 10% phosphomolybdic acid in ethanol. Reference (low-resolution) mass spectra were obtained using either a Waters LCT or an Applied Biosystems API 3000 mass spectrometer. High-resolution mass spectra (HRMS) were obtained using either a Waters LCT or an Agilent TOF mass spectrometer. All other LC-MS experiments were performed using an Agilent 1100 HPLC coupled to an Agilent single quadrupole mass spectrometer. The purity of the compounds was determined by LC-MS at wavelengths of 230 nM and 254 nM. All final compounds reported herein have a purity of 95% or higher.

[0135] General Procedure A [ka] EDCI coupling: Compound (1 equivalent) of general structural formula 1, EDCI (1 equivalent), HOBt (0.2 equivalents), and DIEA (2 equivalents) were dissolved in DMF and stirred at 25°C for 5 minutes. Then, amine (1 equivalent) was added to the mixture. The mixture was stirred at 25°C for 16 hours. The mixture was poured into water and extracted with ethyl acetate. The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain the compound of general structural formula 2.

[0136] HATU coupling: Compound (1 equivalent) of general structural formula 1, HATU (1.25 equivalents), and DIPEA (1.5 equivalents) were dissolved in DMF and stirred at 23°C for 15 minutes. 1H-indazole-5-amine (1 equivalent) was introduced into the reaction mixture, and the solution was stirred for a further 16 hours at 23°C. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 2.

[0137] Boc deprotection: The compound with general structural formula 2 was dissolved in DCM, and 4N HCl in dioxane was introduced into the solution. The reaction mixture was stirred at 23°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the crude reaction mixture, which was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 3.

[0138] Benzyl group removal: The compound with general structural formula 2 (1 equivalent) and concentrated HCl (1.2 equivalents) were dissolved in MeOH, and 10% anhydrous Pd / C was added. The reaction mixture was stirred at 50°C for 5 hours under an atmosphere of H2 (1 atm). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 3.

[0139] General Procedure B [ka] Two equivalents of an amine HCl salt were dissolved in methanol, and two equivalents of triethylamine were added. The reaction solution was stirred for 15 minutes, after which one equivalent of a ketone or aldehyde of general structural formula 4 was added together with four equivalents of HOAc. Stirring was continued for another 15 minutes, and NaBH3CN was added. The reaction temperature was raised to 60°C, and stirring was continued for 16 hours. The reaction mixture was diluted with water and extracted with ethylacetin. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 5.

[0140] K2CO3 (2 equivalents), LiI (0.05 equivalents), and benzyl bromide were added at room temperature to a solution of the compound with general structural formula 5 (1 equivalent) in MeCN. The reaction temperature was raised to 60°C and stirring was continued for 16 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound with general structural formula 6.

[0141] A solution of the compound with general structural formula 6 (1 equivalent), Pd(PPh3)4 (0.15 equivalents), and K3PO4 (3 equivalents) in THF was purged with nitrogen at room temperature for 5 minutes. The reaction temperature was increased to 120°C, and stirring was continued for 48 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain the desired compound with general structural formula 7.

[0142] One equivalent of the compound with general structural formula 7 was dissolved in a methanol / water mixture, and two equivalents of NaOH were added. The reaction mixture was stirred continuously at 30°C for 16 hours. The reaction solution was acidified to pH=5. The crude residue was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 8.

[0143] One equivalent of the compound with general structural formula 8, 1.25 equivalents of HATU, and 1.5 equivalents of DIPEA were dissolved in DMF and stirred at 23°C for 15 minutes. One equivalent of 1H-indazole-5-amine was introduced into the reaction mixture, and the solution was stirred for a further 16 hours at 23°C. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound with general structural formula 9.

[0144] One equivalent of the compound with general structural formula 9 and 1.2 equivalents of concentrated HCl were dissolved in MeOH, and 10% anhydrous Pd / C was added. The reaction mixture was stirred at 50°C for 5 hours under an H2 atmosphere (1 atm). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 10.

[0145] General Procedure C [ka] One equivalent of the compound with general structural formula 11 was dissolved in acetic acid, and PtO2 (0.1 equivalents) was added. The reaction solution was stirred at room temperature for 16 hours under a hydrogen atmosphere (1 atm). The resulting solution was filtered and concentrated under reduced pressure. The residue was made basic with 2N NaOH to pH=9, and the mixture was extracted with SiO2. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound with general structural formula 12.

[0146] One equivalent of the compound with general structural formula 12 was dissolved in DCM, and Boc2O (1.5 equivalents) and DIPEA (2 equivalents) were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was then purified on silica gel to obtain the desired compound with general structural formula 13.

[0147] One equivalent of the compound with general structural formula 13 was dissolved in a methanol / water mixture, and two equivalents of NaOH were added. The reaction mixture was stirred continuously at 30°C for 16 hours. The reaction solution was acidified to pH=5. The crude residue was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 14.

[0148] One equivalent of the compound with general structural formula 14, 1.25 equivalents of HATU, and 1.5 equivalents of DIPEA were dissolved in DMF and stirred at 23°C for 15 minutes. One equivalent of 1H-indazole-5-amine was introduced into the reaction mixture, and the solution was stirred for a further 16 hours at 23°C. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude substance, which was purified on silica gel to obtain the desired compound with general structural formula 15.

[0149] The compound with general structural formula 15 was dissolved in DCM, and 4N HCl in dioxane was introduced into the solution. The reaction mixture was stirred at 23°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the crude reaction mixture, which was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 16.

[0150] General Procedure D [ka] A solution of the compound with general structural formula 17 (1 equivalent) and an aldehyde or ketone (1 equivalent) in MeOH (3.00 mL) was stirred at 23°C for 16 hours. After 16 hours, NaBH3CN (1.2 equivalents) was added, and the mixture was stirred for a further 3 hours. The reaction mixture was quenched by adding water, and the resulting mixture was extracted with phenylethylamine. The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by preparative HPLC to obtain the compound with general structural formula 18.

[0151] General Procedure E [ka] One equivalent of the compound with general structural formula 19 was dissolved in CCl4, and AIBN (0.1 equivalent) followed by NBS (1.2 equivalents) were added at room temperature. The reaction temperature was raised to 60°C, and stirring was continued for 16 hours. The reaction mixture was filtered off, and the solvent was removed under reduced pressure. The crude substance 20 was used in the next step without further purification.

[0152] The compound with general structural formula 20 (1 equivalent) and an alkylamine (2 equivalents) in THF were stirred at 70°C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the substance was purified by silica gel chromatography to obtain the compound with general structural formula 21.

[0153] Two equivalents of NaOH were introduced into a reaction vessel containing one equivalent of the compound with general structural formula 21, dissolved in a MeOH / H2O mixture. The reaction mixture was stirred at 20°C for 16 hours. The solvent was removed under reduced pressure. The crude residue was dissolved in 10 mL of water and carefully neutralized with 6 N HCl until the pH reached 8. The suspension was filtered, the solid was collected and dried to obtain the compound with general structural formula 22.

[0154] One equivalent of the compound with general structural formula 22, HATU (1.25 equivalents), and DIPEA (2 equivalents) were dissolved in DMF and stirred at 20°C for 15 minutes. One equivalent of 1H-indazole-5-amine was added to the reaction mixture and stirred at 20°C for 15.8 hours. The reaction product was diluted with water and extracted with ethyl acetate. The combined organic layer was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to obtain the compound with general structural formula 23.

[0155] General Procedure F [ka] One equivalent of benzylamine was dissolved in dichloromethane. One equivalent of the corresponding arylboronic acid 24 was added to the reaction solution, followed by the addition of one equivalent of glyoxylic acid under a nitrogen atmosphere. The reaction mixture was heated to 40°C and maintained at that temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to obtain the compound with general structural formula 25.

[0156] One equivalent of the compound with general structural formula 25, HATU (1.25 equivalents), and DIPEA (2 equivalents) were dissolved in DMF and stirred at 20°C for 15 minutes. One equivalent of 1H-indazole-5-amine was added to the reaction mixture and stirred at 20°C for 16 hours. The reaction product was diluted with water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by normal-phase silica gel chromatography to obtain the compound with general structural formula 26.

[0157] One equivalent of the compound with general structural formula 26 and 1.2 equivalents of concentrated HCl were dissolved in MeOH, and 10% anhydrous Pd / C was added. The reaction mixture was stirred at 60°C for 3 hours under an H2 atmosphere (1 atm). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse-phase preparative HPLC to obtain the desired compound with general structural formula 27.

[0158] Example 2 N-(1H-indazole-5-yl)-2-(methylamino)-2-phenylacetamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-phenylacetamide as an off-white solid (80%). 1H NMR (400 MHz, CD3OD) δ 8.34 (s, 1H), 8.27 (s, 1H), 7.68-7.65 (m, 2H), 7.60-7.53 (m, 5H), 5.1 (s, 1H), 2.69 (s, 3H). MS (ES+) m / e 281.1 (M+H).

[0159] Example 3 N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide as a yellow solid (23%). 1 H NMR (400 MHz, MeOD-d3) δ 13.75 (s, 1H), 10.84 (s, 1H), 8.94 (s, 1H), 8.80 (s, 1H), 8.26 (m, 2H), 7.71 (m, 2H), 7.28 (m, 2H), 6.63 (s, 1H), 4.21 (m, 1H), 3.97 (m, 1H), 3.65 (m, 3H). MS (ES+) m / e 293.1 (M+H) + .

[0160] Example 4 N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide as a yellow solid (51%). 1H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 9.96 (s, 1H), 8.21 (s, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 7.53-7.48 (m, 2H), 7.20-7.12 (m, MS (ES+) m / e 293.0 (M+H) + .

[0161] Example 5 N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide as a white solid (18%). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.16 (s, 1H), 8.21(s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.52-7.46 (m, 2H), 7.37-7.35 (m, MS (ES+) m / e 293.1 (M+H) + .

[0162] Example 6 N-(1H-indazole-5-yl)isoindoline-1-carboxamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)isoindoline-1-carboxamide as a white solid (10%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 10.09 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.56-7.44 (m, 3H), 7.34-7.20 (m, 3H), 4.96 (brs, 1H), 4.40-4.26 (m, 2H), 3.79 (brs, 1H). MS (ES+) m / e 279.1 (M+H) + .

[0163] Example 7 N-(2-(dimethylamino)ethyl)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(2-(dimethylamino)ethyl)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (50%). 1 H NMR (400 MHz, DMSO-d6) δ 13.28 (brs, 1H), 8.07 (brs, 1H), 7.78-6.91 (m, 5H), 6.54 (d, J = 7.6 Hz, 1H), 6.48 (s, 1H), 4.01-3.67 (m, 3H), 3.61 (s, 3H), 2.32-2.11 (m, 3H), 2.11-2.10 (m, 9H). MS (ES+) m / e 382.1 (M+H) + .

[0164] Example 8 N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide [ka] The reaction was carried out according to general protocol C. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide as a light brown solid (46%). 1 H NMR (400 MHz, DMSO-d6) δ 13.01 (brs, 1H), 10.59 (s, 1H), 8.23 ​​(s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.50-7.48 (m, 1H), 7.44-7.42 (m, 1H), 7.29-7.22 (m, 1H), 7.20-7.13 (m, 3H), 4.05-4.00 (m, 1H), 3.95-3.85 (m, 1H), 3.79-3.75 (m, 1H), 3.37-3.33 (m, 1H), 3.22-3.19 (m, 1H). MS (ES+) m / e 293.0 (M+H) + .

[0165] Example 9 N-(1H-indazole-5-yl)-3-methylisoindoline-1-carboxamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-3-methylisoindoline-1-carboxamide as a white solid (29%). 1H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 10.04 (s, 1H), 8.15 (s, 1H), 8.02 (s, 1H), 7.53-7.42 (m, 3H), 7.35-7.21 (m, 3H), 4.89 (s, 1H), 4.58-4.57 (m, 1H), 4.01 (s, 1H), 1.47 (d, J = 6.4 Hz, 3H). MS (ES+) m / e 293.0 (M+H) + .

[0166] Example 10 N-(1H-indazole-5-yl)pyrrorizin-2-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)pyrrolidine-2-carboxamide as a white solid (33%). MS (ES+) m / e 231.1 (M+H) + .

[0167] Example 11 N-(1H-indazole-5-yl)pyrroridine-3-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)pyrrolidine-3-carboxamide as a white solid (41%). MS (ES+) m / e 231.1 (M+H) + .

[0168] Example 12 (2S,5R)-N-(1H-indazole-5-yl)-5-phenylpyrrolidine-2-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain (2S,5R)-N-(1H-indazole-5-yl)-5-phenylpyrrolidine-2-carboxamide carboxamide as a white solid (26%). MS (ES+) m / e 307.0 (M+H) + .

[0169] Example 13 N-(1H-indazole-5-yl)piperidine-2-carboxamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)piperidine-2-carboxamide as a white solid (39%). MS (ES+) m / e 245.0 (M+H) + .

[0170] Example 14 N-(1H-indazole-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamide as a white solid (7%). 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.47 (s, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.00-6.93 (m, 4H), 6.89 (d, J = 8.4 Hz, 2H), 4.20 (d, J = 7.2 Hz, 1H), 3.75 (s, 3H), 2.60 (m, 1H), 2.30 (d, J = 4.8 Hz, 3H). MS (ES+) m / e 403.1 (M+H) + .

[0171] Example 15 N-(1H-indazole-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol A. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamide as a white solid (8%). 1 H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.08 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.51-7.48 (m, 4H), 7.27 (t, J = 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.72 (dd, J = 8.4, 2.0 Hz, 1H), 6.58 (t, J = 2.4 Hz, 1H), 6.53 (dd, J = 8.0, 2.4 Hz, 1H), 4.23 (s, 1H), 3.73 (s, 3H), 2.31 (s, 3H). MS (ES+) m / e 403.1 (M+H) + .

[0172] Example 16 2-(4-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (7%). 1 H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.10 (s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.52-7.40 (m, 6H), 4.24 (s, 1H), 2.72 (m, 1H), 2.28 (s, 3H). MS (ES+) m / e 315.0 (M+H) + .

[0173] Example 17 2-(4-chlorophenyl)-2-(ethylamino)-N-(1H-indazole-5-yl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (12%). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.13 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.49-7.39 (m, 4H), 4.39 (s, 1H), 2.51-2.50 (m, 2H), 1.08 (t, J = 6.4 Hz, 3H). MS (ES+) m / e 329.1 (M+H) + .

[0174] Example 18 2-(4-chlorophenyl)-N-(1H-indazole-5-yl)-2-(isopropylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-chlorophenyl)-N-(1H-indazole-5-yl)-2-(isopropylamino)acetamide as a white solid (49%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.18 (s, 1H), 8.21 (s, 1H), 8.09 (d, J = 1.2 Hz, 1H), 8.00 (d, J = 0.8 Hz, 1H), 7.52-7.45 (m, MS (ES+) m / e 343.1 (M+H) + .

[0175] Example 19 2-(4-fluorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-fluorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (11%). 1H NMR (400 MHz, DMSO-d6) δ 13.08 (s, 1H), 10.64 (s, 1H), 9.59-9.31 (m, 2H), 8.07 (d, J = 9.2 Hz, 2H), 7.67-7.64 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.41-7.34 (m, 3H), 5.04 (s, 1H), 2.52 (s, 3H). MS (ES+) m / e 299.0 (M+H) + .

[0176] Example 20 2-(4-fluorophenyl)-N-(1H-indazole-5-yl)-2-(isopropylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-fluorophenyl)-N-(1H-indazole-5-yl)-2-(isopropylamino)acetamide as a white solid (54%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.17 (s, 1H), 8.20 (s, 1H), 8.10 (d, J = 1.2 Hz, 1H), 8.00 (d, J = 0.8 Hz, 1H), 7.52 (dd, J = MS (ES+) m / e 327.1 (M+H) + .

[0177] Example 21 2-(ethylamino)-N-(1H-indazole-5-yl)-2-phenylacetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-N-(1H-indazole-5-yl)-2-phenylacetamide as a white solid (39%). 1 H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 10.17 (s, 1H), 8.23 ​​(s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.52-7.45 (m, 4H), 7.35 (t, J = MS (ES+) m / e 295.0 (M+H) + .

[0178] Example 22 N-(1H-indazole-5-yl)-2-(isopropylamino)-2-phenylacetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(isopropylamino)-2-phenylacetamide as a white solid (63%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.17 (s, 1H), 8.20 (s, 1H), 8.11 (d, J = 1.2 Hz, 1H), 8.00 (d, J = 0.8 Hz, 1H), 7.52-7.41 (m, MS (ES+) m / e 309.1 (M+H) + .

[0179] Example 23 2-(ethylamino)-2-(4-fluorophenyl)-N-(1H-indazole-5-yl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-2-(4-fluorophenyl)-N-(1H-indazole-5-yl)acetamide as a white solid (8%). 1 H NMR (400 MHz, CD3OD) δ 8.45 (s, 1H), 8.09 (d, J = 1.2 Hz, 1H), 8.00 (s, 1H), 7.67-7.61 (m, 2H), 7.50 (d, J = 8.8 Hz, 1H), 7.42 (dd, J = MS (ES+) m / e 313.1 (M+H) + .

[0180] Example 24 N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as a white solid (4%). 1H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 10.01 (s, 1H), 8.12 (s, 1H), 7.99 (s, 1H), 7.46 (s, 2H), 7.39 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 4.15 (s, 1H), 3.72 (s, 3H), 2.28 (s, 3H). MS (ES+) m / e 311.1 (M+H) + .

[0181] Example 25 N-(1H-indazole-5-yl)-2-(isopropylamino)-2-(4-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as a white solid (52%). 1 H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.11 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.48-7.39 (m, 4H), 6.90 (d, J = MS (ES+) m / e 339.1(M+H) + .

[0182] Example 26 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide as a white solid (5%). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.10 (s, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.50-7.41 (m, 4H), 6.91 (d, J = MS (ES+) m / e 325.1 (M+H) + .

[0183] Example 27 2-(cyclopropylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(cyclopropylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide as a white solid (10%). 1 H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 10.07 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 7.99 (d, J = 0.8 Hz, 1H), 7.49-7.41 (m, 2H), 7.39 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.39 (s, 1H), 3.72 (s, 3H), 2.05-2.00 (m, 1H), 0.42-0.33 (m, 4H). MS (ES+) m / e 337.0 (M+H)+ .

[0184] Example 28 N-(1H-indazole-5-yl)-2-(methylamino)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(p-tolyl)acetamide as a white solid (7%). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.04 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.46 (s, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 4.19 (s, 1H), 2.28 (d, J = 7.2 Hz, 6H). MS (ES+) m / e 295.1 (M+H) + .

[0185] Example 29 N-(1H-indazole-5-yl)-2-(isopropylamino)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(isopropylamino)-2-(p-tolyl)acetamide as a white solid (44%). 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.17 (s, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.48-7.37 (m, 4H), 7.16 (d, J = MS (ES+) m / e 323.1 (M+H) + .

[0186] Example 30 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide as a white solid (13%). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.08 (s, 1H), 8.24 (s, 1H), 8.10 (s, 1H), 7.99 (s, 1H), 7.48-7.44 (m, 2H), 7.37 (d, J = MS (ES+) m / e 309.1 (M+H) + .

[0187] Example 31 2-(cyclopropylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(cyclopropylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide as a white solid (23%). 1 H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 10.07 (s, 1H), 8.22 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.48-7.42 (m, 2H), 7.35 (d, J = MS (ES+) m / e 321.1 (M+H) + .

[0188] Example 32 N-(1H-indazole-5-yl)-2-(2'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(2'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide as a yellow solid (16%). 1H NMR (400 MHz, DMSO-d6) δ 13.00 (brs, 1H), 10.13 (s, 2H), 8.28 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.53-7.43 (m, 6H), 7.33-7.30 (m, 1H), 7.27 (dd, J = 7.6, 2.0 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.01 (t, J = 6.4 Hz, 1H), 4.27 (s, 1H), 2.34 (s, 3H). MS (ES+) m / e 387.1 (M+H) + .

[0189] Example 33 2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (10%). 1 H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.14 (s, 1H), 8.31 (s, 2H), 8.14 (s, 1H), 8.00 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.54-7.48 (m, 5H), 7.42-7.38 (m, 1H), 7.33-7.26 (m, 2H), 4.30 (s, 1H), 2.33 (s, 3H). MS (ES+) m / e 375.1 (M+H) + .

[0190] Example 34 2-((2-(dimethylamino)ethyl)amino)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain 2-((2-(dimethylamino)ethyl)amino)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)acetamide as a white solid (27%). 1 H NMR (400 MHz, DMSO-d6) δ 13.01 (brs, 1H), 10.19 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.49-7.43 (m, 2H), 7.27 (t, J = 7.8 Hz, 1H), 7.07-7.04 (m, 2H), 6.86 (dd, J = 8.0, 2.0 Hz, 1H), 4.36 (s, 1H), 3.75 (s, 3H), 2.65-2.57 (m, 2H), 2.55-2.53 (m, 2H), 2.25 (s, 6H). MS (ES+) m / e 368.1 (M+H) + .

[0191] Example 35 N-(1H-indazole-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide as a pink solid (10%). 1H NMR (400 MHz, DMSO-d6) δ 13.03 (brs, 1H), 9.99 (brs, 1H), 8.20 (s, 2H), 8.13 (s, 1H), 8.01 (s, 1H), 7.52-7.46 (m, 2H), 7.40 (dd, J = 7.6, 1.6 Hz, 1H), 7.32-7.27 (m, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H), 4.59 (s, 1H), 3.83 (s, 3H), 2.34 (s, 3H). MS (ES+) m / e 311.0 (M+H) + .

[0192] Example 36 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-(4-methoxyphenyl)acetamide as a light brown solid (35%). 1 H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.14 (m, 2H), 8.06 (s, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.34 (dd, J = MS (ES+) m / e 353.1 (M+H) + .

[0193] Example 37 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-phenylacetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-phenylacetamide as a purple solid (4%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.26 (s, 1H), 8.35 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.49-7.46 (m, 4H), 7.32 (t, J = 7.6 Hz, 2H), 7.26-7.22 (m, 1H), 4.50 (s, 1H), 1.10 (s, 9H). MS (ES+) m / e 323.1 (M+H) + .

[0194] Example 38 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(tert-butylamino)-N-(1H-indazole-5-yl)-2-(p-tolyl)acetamide as a white solid (12%). 1 H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.20 (s, 1H), 8.09 (s, 1H), 8.00 (s, 1H), 7.49-7.43 (m, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 4.44 (s, 1H), 2.26 (s, 3H), 1.09 (s, 9H). MS (ES+) m / e 337.1 (M+H) + .

[0195] Example 39 N-(1H-indazole-5-yl)-2-(methylamino)-2-(pyridine-3-yl)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(pyridine-3-yl)acetamide as a white solid (9%). 1 H NMR (400 MHz, DMSO-d6) δ 13.02 (brs, 1H), 10.21 (s, 1H), 8.69 (d, J = 1.6 Hz, 1H), 8.50 (dd, J = 4.8, 1.6 Hz, 1H), 8.24 (s, 1H), 8.12 MS (ES+) m / e 282.0 (M+H) + .

[0196] Example 40 N-(1H-indazole-5-yl)-2-(methylamino)-2-(pyridine-2-yl)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(pyridine-2-yl)acetamide as an off-white solid (15%). 1H NMR (400 MHz, DMSO-d6) δ 12.98 (brs, 1H), 10.19 (s, 1H), 8.54 (dd, J = 4.8, 0.8 Hz, 1H), 8.21 (s, 1H), 8.14 (d, J = 1.2 Hz, 1H), 8.00 (s, 1H), 7.84-7.79 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.48 (s, 2H), 7.34-7.30 (m, 1H), 4.44 (s, 1H), 2.36 (s, 3H). MS (ES+) m / e 282.0 (M+H) + .

[0197] Example 41 N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as an off-white solid (42%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.11 (s, 1H), 8.23 ​​(s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.48-7.44 (m, 2H), 7.27 (t, J = MS (ES+) m / e 311.0 (M+H) + .

[0198] Example 42 2-(3-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(3-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as an off-white solid (63%). 1 H NMR (400 MHz, DMSO-d6) δ 13.00 (brs, 1H), 10.16 (brs, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.59 (s, 1H), 7.51-7.43 (m, 3H), 7.42-7.31 (m, 2H), 4.31 (s, 1H), 2.30 (s, 3H). MS (ES+) m / e 315.0 (M+H) + .

[0199] Example 43 2-(4-ethoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-ethoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as an off-white solid (59%). 1 H NMR (400 MHz, DMSO-d6) δ 13.02 (brs, 1H), 10.11 (brs, 1H), 8.23 ​​(s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.49-7.43 (m, 2H), 7.40 (d, J = MS (ES+) m / e 324.1 (M+H) +.

[0200] Example 44 N-(1H-indazole-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide as an off-white solid (67%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 10.10 (s, 1H), 8.23 ​​(s, 1H), 8.14-8.13 (m, 1H), 8.01 (s, 1H), 7.47 (s, 2H), 7.38 (d, J = MS (ES+) m / e 339.1 (M+H) + .

[0201] Example 45 N-(1H-indazole-5-yl)-2-(4'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(4'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide as a white solid (34%). 1H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 10.14 (s, 1H), 8.21 (s, 1H), 8.14 (t, J = 1.6 Hz, 1H), 8.01 (s, 1H), 7.62-7.54 (m, 6H), 7.48-7.46 (m, 2H), 7.03-7.00 (m, 2H), 4.31 (s, 1H), 3.79 (s, 3H), 2.34 (s, 3H). MS (ES+) m / e 387.0 (M+H) + .

[0202] Example 46 N-(1H-indazole-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (16%). 1 H NMR (400 MHz, DMSO-d6) δ 13.07 (s, 1H), 10.61 (s, 1H), 9.55 (s, 2H), 8.07 (d, J = 7.6 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.45-7.36 (m, 2H), 7.25-7.21 (m, 2H), 7.07-7.04 (m, 1H), 5.07 (s, 1H), 3.80 (s, 3H), 3.78-3.59 (m, 2H), 3.31 (s, 3H), 3.11 (m, 1H), 2.99 (m, 1H). MS (ES+) m / e 355.0 (M+H) + .

[0203] Example 47 2-(2-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(2-chlorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as an off-white solid (32%). 1 H NMR (400 MHz, DMSO-d6) δ 13.03 (brs, 1H), 10.14 (s, 1H), 8.17-8.14 (m, 2H), 8.02 (s, 1H), 7.61-7.58 (m, 1H), 7.50-7.46 (m, 3H), 7.37-7.30 (m, 2H), 4.67 (s, 1H), 2.35 (s, 3H). MS (ES+) m / e 315.0 (M+H) + .

[0204] Example 48 2-(4-fluoro-3-methoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-fluoro-3-methoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (49%). 1 H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 10.15 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50-7.44 (m, 2H), 7.34 (dd, J = MS (ES+) m / e 329.1 (M+H)+ .

[0205] Example 49 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(m-tolyl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-N-(1H-indazole-5-yl)-2-(m-tolyl)acetamide as a yellow solid (30%). 1 H NMR (400 MHz, DMSO-d6) δ 13.08 (s, 1H), 10.66 (s, 1H), 9.44-9.37 (m, 2H), 8.10-8.06 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.47-7.36 (m, 4H), 7.31 (d, J = 7.6 Hz, 1H), 5.04 (t, J = 6.0 Hz, 1H), 2.98 -2.84 (m, 2H), 2.36 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 309.1 (M+H) + .

[0206] Example 50 N-(1H-indazole-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)-phenyl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)phenyl)acetamide as a white solid (27%). 1H NMR (400 MHz, DMSO-d6) δ 13.00 (brs, 1H), 10.18 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.54-7.44 (m, 5H), 7.30-7.28 (m, 1H), 4.34 (s, 1H), 2.30 (s, 3H). MS (ES+) m / e 365.0 (M+H) + .

[0207] Example 51 2-(ethylamino)-2-(3-fluorophenyl)-N-(1H-indazole-5-yl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(ethylamino)-2-(3-fluorophenyl)-N-(1H-indazole-5-yl)acetamide as a white solid (21%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.16 (s, 1H), 8.18 (s, 1H), 8.11 (d, J = 0.8 Hz, 1H), 8.01 (d, J = 0.8 Hz, 1H), 7.50-7.33 (m, 5H), 7.14-7.09 (m, 1H), 4.43 (s, 1H), 2.60-2.53 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H).MS (ES+) m / e 313.1 (M+H) + .

[0208] Example 52 N-(1H-indazole-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide as a white solid (11%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.11 (s, 1H), 8.20 (s, 2H), 8.13 (s, 1H), 8.01 (s, 1H), 7.49-7.45 (m, 2H), 7.32-7.23 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 4.25 (s, 1H), 2.31 (s, 6H). MS (ES+) m / e 295.1 (M+H) + .

[0209] Example 53 2-(3-fluorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(3-fluorophenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (11%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.14 (s, 1H), 8.21 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50-7.32 (m, 5H), 7.14-7.10 (m, 1H), 4.30 (s, 1H), 2.30 (s, 3H). MS (ES+) m / e 299.1 (M+H) + .

[0210] Example 54 (R)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain (R)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (17%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AD column) (mobile phase: 55% EtOH containing 0.1% NH4OH in CO2, flow rate 70 g / min) to obtain the desired compound with enantiomer purity of 99%. 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.47 (s, 2H), 7.26 (t, J = 8.0 Hz, 1H), 7.09-7.05 (m, 2H), 6.85 (dd, J = 8.0, 2.0 Hz, 1H), 4.21 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H).MS (ES+) m / e 311.1 (M+H) + . [α] 25°C D = +98.86 (c = 0.2 in MeOH).

[0211] Example 55 (S)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain (S)-N-(1H-indazole-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (12%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AD column) (mobile phase: 55% EtOH containing 0.1% NH4OH in CO2, flow rate 70 g / min) to obtain the desired compound with enantiomer purity of 99%. 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.47 (s, 2H), 7.26 (t, J = 8.0 Hz, 1H), 7.09-7.05 (m, 2H), 6.85 (dd, J = 8.0, 2.4 Hz, 1H), 4.20 (s, 1H), 3.75 (s, 3H), 2.30 (s, 3H). MS (ES+) m / e 311.1 (M+H) + . [α] 25°C D = -96.44 (c=0.2 in MeOH).

[0212] Example 56 N-(1H-indazole-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazole-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide as a brown solid (19%). 1 H NMR (400 MHz, DMSO-d6) δ12.99 (s, 1H), 10.15 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.50-7.45 (m, 2H), 7.36 (d, J = 8.4 Hz, 2H), 4.30 (s, 1H), 2.30 (s, 3H). MS (ES+) m / e 365.1 (M+H) + .

[0213] Example 57 2-(3-fluoro-4-methoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(3-fluoro-4-methoxyphenyl)-N-(1H-indazole-5-yl)-2-(methylamino)acetamide as a white solid (29%). 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.07 (s, 1H), 8.18 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.49-7.44 (m, 2H), 7.35 (dd, J = MS (ES+) m / e 329.0 (M+H) + .

[0214] Example 58 2-(3-chlorophenyl)-2-(ethylamino)-N-(1H-indazole-5-yl)acetamide [ka] The reaction was carried out according to general procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(3-chlorophenyl)-2-(ethylamino)-N-(1H-indazole-5-yl)acetamide as a brown solid (19%). 1 H NMR (400 MHz, CD3OD) δ 8.53 (s, 1H), 8.08 (d, J = 1.2 Hz, 1H), 8.02 (s, 1H), 7.62 (s, 1H), 7.53-7.39 (m, 5H), 4.55 (s, 1H), 2.83-2.68 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 329.1 (M+H) + .

[0215] Comparative Example 1 [ka]

[0216] Comparative Example 2 [ka]

[0217] Comparative Example 3 [ka]

[0218] Comparative Example 4 [ka]

[0219] Comparative Example 5 [ka]

[0220] Comparative Example 6 [ka]

[0221] Comparative Example 7 [ka]

[0222] Example 59 Determination of ROCK inhibitory activity of compounds in vitro (Z'lyte assay): Recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) proteins were purchased from ThermoFisher Scientific. The activity of the compounds was measured using the Z'-lyte kinase kit (ThermoFisher Scientific), and IC2 was determined. 50I calculated it.

[0223] Determination of ROCK inhibitory activity of compounds in A7R5 cells: Rat aortic smooth muscle cell line A7R5 cells were maintained and treated in DMEM medium containing 10% fetal bovine serum. Cells were seeded at a density of 5,000 cells / well in 96-well plates for 24 hours, and then treated with the test compound for 90 minutes. Cells were then fixed and treated according to the In-Cell ELISA Colorimetric Detection Kit manual (Thermo Scientific). Using the In-Cell ELISA kit, cellular phosphomyosin light chain (ppMlc, Thr18 / Ser19) levels were determined after treatment with DMSO control or the test compound. The inhibition percentage was calculated by fitting the data obtained from 1 μM (or 10 μM) compound treatment to the formula: [1 - (compound / DMSO)] × 100%. The ppMlc data obtained from nine 2x series dilutions of the compound were fitted to a nonlinear regression curve fitting function in GraphPad Prism software for in-cell IC. 50 The value was calculated.

[0224] NIH3T3 cell Acta2-promoter-driven luciferase assay: We established an NIH3T3 cell line that stably expresses a luciferase reporter driven by the human ACTA2 gene promoter (-1000-1 bp) (NIH3T3-Acta2-luciferase). Cells were plated to confluence and treated with the test compound and TGFβ1 for 24 hours. Subsequently, the cells were lysed, and luciferase activity was measured using the LightSwitch luciferase kit from Active Motif.

[0225] ROCK inhibitors potently inhibited ROCK kinase activity both in vitro and in cells. As shown in Figure 1, the ROCK inhibitors of the present invention potently inhibited both isoforms of ROCK kinase activity, as measured in vitro by the Z'-Lyte kit, at concentrations of less than 20 nanomolars.

[0226] As measured by the A7R5 in-cell Elisa assay, the compounds of the present invention inhibited ROCK in cells. A7R5 cells were treated with 9-point 2-fold series dilutions of the compounds, and ppMlc(T18 / S19) levels were determined to assess the cellular IC5 levels of the compounds. 50 The values ​​were calculated. The results are shown in Table 1 below. [Table 1]

[0227] The ROCK inhibitors of the present invention inhibit both isoforms of ROCK. HCT116 cells in which either ROCK1 or ROCK2 was knocked out were generated using a CRISPR / CAS9 system. ROCK1KO and ROCK2KO cells were treated with the compound from Example 2 for 90 minutes, and pMypt(T853) levels were visualized by Western blotting. The ROCK inhibitors effectively blocked ROCK-targeted MYPT1 phosphorylation at 110 nM. See Figure 2.

[0228] Table 2 below shows the in vitro and intracellular inhibition of ROCK by the compounds of the present invention. The activity of the compounds was measured using the Z'-lyte kinase kit (ThermoFisher Scientific). The inhibition percentage was calculated by normalizing the kinase activity values ​​obtained from 1 μM compound treatments against the DMSO control value. GraphPad Prism software was used, along with kinase activity data collected from nine point series dilutions of compound treatments, to calculate IC50. 50 The following calculations were performed. ROCK inhibition was carried out in A7R5 cells and NIH3T3(Acta2-Luc) cells as described above.

[0229] pMLC / A7R5 IC 50 Therefore, a two-step measurement was employed. Inhibition percentages were calculated for single-dose (1 μM or 10 μM) compound treatments to screen for active compounds, and then only compounds with inhibition rates greater than 50% were used for cellular IC. 50 We measured it. [Table 2-1] [Table 2-2] [Table 2-3]

[0230] The comparative example lacks the alkyl substituent on the 2-amino group that is present in the compound of the present invention (i.e., in the comparative compound, the 2-amino group is NH2). Addition of 2-alkylamino (i.e., R 1 (where is alkyl, etc.) In particular, the lower alkylamine at the 2 position (i.e., R 1 In the case of a lower alkyl group, enhanced cellular ROCK activity is associated. For example, compare Comparative Example 2 (containing a primary 2-amino) with Examples 16, 17, and 18, which have a secondary 2-alkylamino, and in particular, a small alkyl group. All four compounds show high in vitro inhibition of ROCK1 and ROCK2, but Examples 16, 17, and 18 enhance cellular ROCK inhibition, as measured by phosphorylation of ROCK target pMLCs in A7R5 cells. For example, Example 2 has a more than 17-fold lower IC50 than Comparative Example 1. 50 Therefore, it exhibits cellular ROCK inhibition (252 vs 4410 ICs, respectively). 50 Similarly, cellular IC in Examples 24, 25, 26, 27, and 36 50 This is lower than that of Comparative Example 4, which has a primary 2-amino group.

[0231] Example 59 ROCK inhibitors improved the oligodendroglioblastic process in cultured human oligodendroglioblasts / neuronal progenitor cells. Human oligodendroglioblasts / neuronal progenitor cells were cultured in vitro for 2 and 14 days with and without ROCK inhibitors. Different stages of neuronal differentiation were identified by visualizing nestin and MAP2 proteins by staining with commercially available antibodies. As demonstrated by the significant increase in MAP2 signaling in cells differentiated in the presence of the ROCK inhibitor (compound of Example 2), the ROCK inhibitors of the present invention significantly promoted MAP2 expression, a marker of mature neurons, while improving neurite outgrowth. See Figure 3.

[0232] Example 60 ROCK inhibitors improved neurite outgrowth and axon ensheathment in oligodendrocytes (oligoblasts) in a co-culture system with rat dorsal root ganglion (DRG) explants. As shown in Figure 4, under co-culture conditions of rat oligodendrocytes and rat dorsal root ganglion (DRG) explants, treatment with ROCK inhibitors altered the cytoskeleton, generating numerous short, arranged myelin segments, which were identified by neurofilament staining. Simultaneously, ROCK inhibitors systematically promoted axon support in oligodendrocytes, which was visualized by staining for MBP, a marker for mature oligodendrocytes.

[0233] Example 61 ROCK inhibitors crossed the blood-brain barrier. In the mouse pharmacokinetic study, animal tissue was collected 2 hours after administration to determine the compound distribution. As shown in Table 3, the compound in Example 2 exhibited excellent blood-brain barrier (BBB) ​​permeability. [Table 3]

[0234] The brain and plasma concentrations of the selected ROCK inhibitors were also evaluated in mice by HPLC / MS / MS at 15 minutes and 2 hours after intravenous administration of 2.5 mg / kg. The results are shown in Table 4 below. [Table 4]

[0235] List of references: Deyts, C., Galan-Rodriguez, B., Martin, E., Bouveyron, N., Roze, E., Charvin, D., Caboche, J., and Betuing, S. (2009).Dopamine D2 receptor stimulation potentiates PolyQ-Huntingtin-induced mouse striatal neuron dysfunctions via Rho / ROCK-II activation.PLoS One 4,e8287. Govek, EE, Newey, SE, and Van Aelst, L. (2005). The role of the Rho GTPases in neural development. Genes Dev 19,1-49. Li,M.,Huang,Y.,Ma,AA,Lin,E.,and Diamond,MI(2009).Y-27632 improves rotarod performance and reduces huntingtin levels in R6 / 2 mice.Neurobiol Dis 36,413-420. Linseman, DA, and Loucks, FA (2008). Diverse roles of Rho family GTPases in neuronal development, survival, and death. Front Biosci 13, 657-676. Petratos,S.,Li,Q.X.,George,A.J.,Hou,X.,Kerr,M.L.,Unabia,S.E.,Hatzinisiriou,I.,Maksel,D.,Aguilar,M.I.,and Small,D.H.(2008).The beta-amyloid protein of Alzheimer’s disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism.Brain 131,90-108. Selkoe,D.J.(2001).Alzheimer’s disease:genes,proteins,and therapy.Physiol Rev 81,741-766. Shao,J.,and Diamond,M.I.(2007).Polyglutamine diseases:emerging concepts in pathogenesis and therapy.Hum Mol Genet 16 Spec No.2,R115-123. Shao,J.,Welch,W.J.,and Diamond,M.I.(2008a).ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation.FEBS Lett 582,1637-1642. Shao,J.,Welch,W.J.,Diprospero,N.A.,and Diamond,M.I.(2008b).Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation.Mol Cell Biol 28,5196-5208. Tanzi,R.E.,and Bertram,L.(2005).Twenty years of the Alzheimer’s disease amyloid hypothesis:a genetic perspective.Cell 120,545-555.

Claims

1. Compounds having formula I: 【Chemistry 1】 [In the formula, R 1 is a lower alkyl, C 3 ~C 6 Cycloalkyl, and R 10 O(CR 12 R 13 ) c Selected from the group consisting of: Each R 10 It is independently selected from lower alkyl groups; Each R 12 is H; Each R 13 H is; c is 2; R 2 This includes unsubstituted pyridinyl or halo, lower alkyl, lower alkoxy, C1-C1 3 A phenyl compound optionally substituted with one or two substituents independently selected from the group consisting of perfluoroalkoxys and aryl-O- compounds. However, if R1 is a lower alkyl group, R2 is an unsubstituted pyridinyl or phenyl group optionally substituted with one or two substituents independently selected from the group consisting of halo, lower alkyl, lower alkoxy, C1-C3 perfluoroalkoxy, and aryl-O-. When R1 is a C3-C6 cycloalkyl group, R2 is a phenyl compound optionally substituted with one substituent independently selected from the group consisting of lower alkyl and lower alkoxy groups. If R1 is R10O(CR12R13)c-, then R2 is a phenyl molecule optionally substituted with one substituent independently selected from the group consisting of lower alkoxys; R 3 H and RR'N-(C 2~4 Selected from alkyl; R 4 H is; R 5 H is; b is 1; and Each R and R' is independently selected from lower alkyl groups, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, having formula III: 【Chemistry 2】 [In the formula, R 1 These are lower alkyl or cyclopropyl compounds; R 2 These are halo, lower alkyl, lower alkoxy, C1-C 3 phenyl optionally substituted with one or two substituents independently selected from the group consisting of perfluoroalkoxys and aryl-O-; However, if R1 is a lower alkyl group, R2 is a phenyl group that may be substituted with one or two substituents independently selected from the group consisting of halo, lower alkyl, lower alkoxy, C1-C3 perfluoroalkoxy, and aryl-O-. If R1 is a C3-C6 cycloalkyl group, R2 is a phenyl compound optionally substituted with one substituent independently selected from the group consisting of lower alkyl and lower alkoxy compounds, or a pharmaceutically acceptable salt thereof.

3. Compounds having formula VI: 【Transformation 3】 [In the formula, R 1 is a lower alkyl, C 3 ~C 6 Cycloalkyl, and R 10 O(CH 2 ) c Selected from the group consisting of: Each R 10 It is a lower alkyl; c is 2; R 3 H and RR'N-(C 2~4 Selected from alkyl; R 4 H is; b is 1; Each R 21 H, halo, lower alkyl, lower alkoxy, C1-C 3 Independently selected from the group consisting of perfluoroalkoxys and aryl-O-; However, if R1 is a lower alkyl group, R21 is independently selected from the group consisting of H, halo, lower alkyl, lower alkoxy, C1-C3 perfluoroalkoxy, and aryl-O-; If R1 is a C3-C6 cycloalkyl group, R21 is independently selected from the group consisting of lower alkyl and lower alkoxy groups; If R1 is R10O(CH2)c-, then R21 is independently selected from the group consisting of lower alkoxys; Each R and R' is independently selected from H and lower alkyl groups; and n is 0 to 2], or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, having formula VII: 【Chemistry 4】 [wherein the formula, the substituents are as defined in claim 3], or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 3, having formula VIII: 【Transformation 5】 [In the formula, R 1 It is a lower alkyl; R 21 [as defined in claim 3], or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 1, having formula IX: 【Transformation 6】 [In the formula, R 1 These are lower alkyl or cyclopropyl compounds; Each R 22 These are H, halo, lower alkyl, lower alkoxy and C 1 ~C 3 Independently selected from the group consisting of perfluoroalkoxys; However, if R1 is a lower alkyl group, R22 is independently selected from the group consisting of H, halo, lower alkyl, lower alkoxy, and C1-C3 perfluoroalkoxy; If R1 is a C3-C6 cycloalkyl group, R22 is independently selected from the group consisting of H, halo, lower alkyl, lower alkoxy, and C1-C3 perfluoroalkoxy; and n is 0 to 2], or a pharmaceutically acceptable salt thereof.

7. Compounds having the following formula: 【Transformation 7】 [In the formula, R 1 is a lower alkyl, C 3 -C 6 Cycloalkyl, or R 10 O(CH 2 ) c - and; R 10 is a lower alkyl group; R 2 These are halo, lower alkyl, lower alkoxy, C1-C 3 Phenyl or pyridyl optionally substituted with one or two substituents independently selected from perfluoroalkoxy, aryl, and aryl-O-, wherein the aryl and -O-aryl substituents are optionally substituted with lower alkoxys; However, if R1 is a lower alkyl group, R2 is an unsubstituted pyridinyl or a phenyl group optionally substituted with one or two substituents independently selected from the group consisting of halo, lower alkyl, lower alkoxy, C1-C3 perfluoroalkoxy, aryl, and aryl-O-. When R1 is a C3-C6 cycloalkyl group, R2 is a phenyl compound optionally substituted with one substituent independently selected from the group consisting of lower alkyl and lower alkoxy groups. If R1 is R10 O(CH2)c-, then R2 is a phenyl molecule optionally substituted with one substituent independently selected from the group consisting of lower alkoxys; R 3 is H, or RR'N-(C 2-4 Selected from alkyl; Each R and R' is independently selected from lower alkyl groups; and c is 2], or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 7 having the following formula: 【Transformation 8】 [In the formula, R 1 It is a lower alkyl or cyclopropyl; Each R 22 The elements are selected from halo, lower alkyl, and lower alkoxy; However, if R1 is a lower alkyl group, R22 is independently selected from the group consisting of halo, lower alkyl, and lower alkoxy groups; If R1 is a C3-C6 cycloalkyl group, R22 is independently selected from the group consisting of lower alkyl and lower alkoxy groups; n is between 0 and 2], or a pharmaceutically acceptable salt thereof.

9. R 1 The compound according to claim 8, wherein is a lower alkyl group and n is 1 or 2.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, which is formed with one or more pharmaceutically acceptable carriers.

11. The pharmaceutical composition according to claim 10, which is formulated as a solid dosage form for oral administration.

12. A pharmaceutical composition according to claim 10 or 11 for the treatment of fibrous disorders in a subject.

13. The pharmaceutical composition according to claim 12, wherein the fibrous disorder is selected from the group consisting of pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, hepatic fibrosis, cirrhosis, cardiac fibrosis, arterial fibrosis, endocardial myocardial fibrosis, old myocardial infarction, arteriosclerosis, atherosclerosis, restenosis, arthritis fibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive nodular fibrosis, retroperitoneal fibrosis, scleroderma or systemic sclerosis, mediastinal fibrosis, keloid, hypertrophic scar, glial scar, and renal fibrosis.

14. A pharmaceutical composition according to claim 10 or 11 for the treatment of a target central nervous system disorder.

15. The pharmaceutical composition according to claim 14, wherein the central nervous system disorder is selected from the group consisting of Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Batten's disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, neuronal degeneration, spinal cord injury, cerebral vasospasm, and multiple sclerosis.

16. A pharmaceutical composition according to claim 10 or 11 for the treatment of a target glaucoma.

17. A pharmaceutical composition according to claim 10 or 11 for the treatment of inflammation in a subject.

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