Inhibitors of rho-associated coiled-coil containing protein kinases

Abstract

The present invention relates to inhibitors of ROCK1 and / or ROCK2.SOLUTION: Also provided are methods of inhibiting ROCK1 and / or ROCK2 useful for treating diseases.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present 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-associated coiled-coil-containing 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 GTPase, and upon activation, they phosphorylate various downstream substrates. ROCK plays an important 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 variety of pathological conditions, including asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, renal failure, pulmonary hypertension, neurodegeneration, and osteoporosis.

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

[0004] Inhibition of ROCK activity in animal models has shown several benefits of inhibiting Rho kinase to treat human diseases. These include models of cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, high 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. Inhibition of ROCK activity in patients has the benefits of controlling cerebral vasospasm and ischemia after subarachnoid hemorrhage, reducing intraocular pressure, relaxing the trabecular meshwork tissue to increase aqueous humor outflow, improving blood flow to the optic nerve, and protecting healthy ganglion cells.

[0005] A substantial amount of in vivo data has been generated that highlights the activity of ROCK in the CNS. Abnormal activation of the ROCK pathway has been documented in many disorders of the central nervous system. For example, axonal growth and synaptic plasticity depend on structural regulation of the actin cytoskeleton. The Rho-ROCK cascade plays a central role in synaptic plasticity, both dendritic morphogenesis and stability, as well as growth cone motility and collapse. In addition, multiple axonal growth inhibitory molecules converge on RhoA / ROCK in neurons, making this an attractive route for intervention in CNS disorders.

[0006] Nogo receptors (NgRs) (as well as other complex members, including LINGO-1) and their ligands are perhaps the best-characterized and most potent inhibitors of neurite outgrowth. Some of the earliest events downstream of receptor activation by myelin-associated inhibitors are upregulation of RhoA and ROCK. These events result in increased contractility and have potent inhibitory effects on axon growth in mature neurons. Therefore, being able to inhibit this signaling cascade offers a highly promising therapeutic strategy for spinal cord and optic nerve injury. Neurodegenerative conditions, such as Huntington's disease and Alzheimer's disease (AD), are also being investigated as responding to inhibition of NgR signaling. Not only are NgR family members involved in APP processing, but the subcellular localization of NgR and Nogo is also altered in AD brains.

[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). Synapse loss is commonly observed in AD pathology and is a hallmark of synaptic dysfunction in AD (Tanzi and Bertram, 2005). Oligomerized β-amyloid peptides have been implicated in the loss of synaptic plasticity and neuronal network dysfunction. Synaptic plasticity depends on structural regulation of the actin cytoskeleton in dendritic spines. The Rho-ROCK cascade plays a central role in synaptic plasticity, both in the morphogenesis and stability of dendrites, and in growth cone motility and collapse (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 ROCK inhibition can inhibit toxic peptide processing. In a feedforward mechanism, β-amyloid increases Rho GTPase activity, which inhibits neurite outgrowth and synaptogenesis via ROCK activation (Petratos et al., 2008). Therefore, ROCK inhibitors may have the potential to prevent synaptic and neuronal degeneration in AD and even promote regeneration processes. A recent study by Herskowitz et al. showed that ROCK knockdown reduces aβ levels. These effects demonstrate the need for highly selective ROCK inhibitors to provide effective treatment for Alzheimer's disease (AD). To validate the use of ROCK inhibition for AD by altering BACE-1 distribution and amyloid precursor protein (APP) trafficking to lysosomes, a model compound, SR3677, was tested in a rodent model of AD. SR3677 had promising effects in reducing sAPPβ after direct intrathecal (ip) injection into the hippocampus due to its poor oral PK properties (5% F and half-life of less than 1 hour) and lack of brain penetration.

[0008] Huntington's disease (HD) is a devastating, incurable, primarily inherited neurodegenerative disorder characterized by psychiatric disorders, movement disorders, and dementia. Misfolding and aggregation of the Htt protein, the product of the huntingtin gene, leads to HD pathology (Shao and Diamond, 2007). A very small number of mechanism-based therapeutic leads have been developed to treat HD. While scientific investigations are still ongoing, several lines of evidence suggest that ROCK inhibition could be an effective treatment for HD. In mouse models of HD, ROCK inhibition significantly reduced soluble Htt levels, reversed aggregate formation and neurite retraction, and protected against neuronal cell death (Deyts et al., 2009; Li et al., 2009). Similar results were obtained in Drosophila studies, where ROCK inhibition suppressed Htt aggregation (Shao et al., 2008a; Shao et al., 2008b). The ROCK signaling pathway is a promising therapeutic target for HD.

[0009] ROCK signaling has also been implicated in Parkinson's disease and amyotrophic lateral sclerosis (ALD). See, e.g., 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 chain (MLC, at threonine 18 and serine 19) and myosin light chain phosphatase (MYPT1, at threonine 853), to drive the polymerization of globular G-actin into filamentous F-actin and assemble the actomyosin contractile machinery. It has been recognized that this pathway may contribute to the pathogenesis of several CNS disorders, such as spinal cord injury, stroke, and AD. In the adult CNS, injured axons regenerate poorly due to the presence of myelin-associated axon growth inhibitors. Myelin-associated inhibitors, such as myelin-associated glycoprotein (MAG), Nogo, oligodendrocyte-myelin glycoprotein (OMgp), and repulsive guidance molecule (RGM), limit axon regeneration in the injured brain and spinal cord. A common mechanism shared by various myelin-associated inhibitors is that they all activate Rho and its downstream effector kinase ROCK, thereby inhibiting neurite outgrowth.

[0011] Blockade of the Rho / ROCK pathway with small molecules is a desirable strategy for central nervous system (CNS) disorders. However, the blood-brain barrier (BBB), while playing a critical role in brain homeostasis, significantly impedes the penetration of many small molecule inhibitors. Because of growing interest in developing selective and potent inhibitors for treating CNS diseases, there is an urgent need for inhibitors of ROCK1 and / or ROCK2, especially those that cross the blood-brain barrier. Summary of the Invention

[0012] In one aspect, the present invention provides a compound of formula I: [ka] provide During the ceremony, A is, [ka] is selected from the group consisting of R 1is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; R 2 is selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which is 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 together form a monocyclic or bicyclic group, wherein 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, and the monocyclic and bicyclic groups are 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 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4 is 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 is selected from the group consisting of alkyl)-O-; R 5 is selected from H, lower alkyl, and C3-C6 cycloalkyl; Alternatively, R 3 and R 5 are taken together to form a cyclic group having 5 to 7 ring atoms including 2 to 3 ring heteroatoms, wherein the cyclic group is 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; a is 0 or 1, b is 0 to 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 5-6 membered heterocyclic ring.

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

[0014] The present invention includes compositions comprising a substantially pure compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, and a pharmaceutically acceptable carrier.

[0015] In one aspect, the present invention provides a method for inhibiting ROCK in a mammal, comprising administering an effective amount of one or more compounds of Formula I. The present invention 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 in need of such treatment. In certain such embodiments, the compound of Formula I inhibits ROCK2. In certain such embodiments, the compound of Formula I selectively inhibits ROCK2. Non-limiting 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, ocular hypertension, 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 ROCK inhibits tumor cell proliferation and metastasis and angiogenesis.

[0016] The present invention provides a method of 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, neurodegeneration 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 of 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 arterial disease, peripheral circulatory disorders, cerebral cavernous hemangioma, restenosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, acute respiratory distress syndrome (ARDS), or erectile dysfunction.

[0019] The present invention provides a method of 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, renal inflammation, or arteriosclerosis.

[0020] The present invention provides a method of treating an arterial thrombotic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I. Non-limiting examples of arterial thrombotic disorders are platelet aggregation or leukocyte aggregation.

[0021] The present invention provides a method of treating a fibrotic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I. Non-limiting examples of fibrotic disorders are pulmonary fibrosis, including cystic and idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, including cirrhosis, cardiac fibrosis, including arterial fibrosis, endomyocardial fibrosis, previous myocardial infarction, arterial stiffness, atherosclerosis, restenosis, arthritic fibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal cavity fibrosis, scleroderma / systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars, glial scars, or renal fibrosis.

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

[0023] The present invention provides a method of 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 angle-closure glaucoma, pigmentary glaucoma, neovascular glaucoma, congenital glaucoma, normal-tension glaucoma, or secondary glaucoma.

[0024] The present invention provides a method of 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, lymphoma, carcinoma, leukemia, sarcoma, or blastoma, such as squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, 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, gastric cancer, melanoma, or head and neck cancer.

[0025] The present invention also provides a method of 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] Additionally, the present invention provides a method of 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 of treating an ocular disorder having an angiogenic 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 ocular 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 drawings]

[0028] [Figure 1A] Representative Z'-Lyte assay results: ROCK inhibitors of the present invention exhibit low single-digit nanomolar potency against both isoforms of ROCK. [Figure 1B] A7R5 in-cell ELISA assay. A7R5 cells were treated with 9-point 2-fold serial dilutions of compounds, and the ppMlc (T18 / S19) level was determined to calculate the cellular IC50 value of the compound. Representative compounds of the present invention showed an IC50 of less than 200 nM in cells. [Figure 1C] ROCK inhibitors were effectively ranked using an ACTA2 promoter-driven luciferase reporter cell assay. NIH3T3 cells stably expressing ACTA2 promoter-driven luciferase were plated to confluence in a 96-well plate and treated with a 9-point serial dilution of the compound in combination with TGFβ1 for 24 hours. Luciferase activity was measured, and the IC50 of the compound was calculated. Representative compounds of the present invention showed an IC50 of less than 200 nM. [Figure 1D] Figure 1 shows Western blots of SVEC4-10 cell lysates to visualize ppMlc (T18 / S19) and pMYPT (T853) levels after 120 minutes of treatment with the compound of Example 2. SVEC4-10 cells were treated with the compounds, and phosphorylated proteins were visualized by Western blotting. Representative compounds efficiently blocked ROCK target MLC and MYPT1 phosphorylation at 110 nM. [Figure 2A]ROCK inhibitors dose-dependently promote neurite outgrowth in cultured human oligodendrocyte / neuronal progenitor cells. Human oligodendrocyte / neuronal progenitor cells were cultured in vitro with or without ROCK inhibitors for 3 days. [Figure 2B] ROCK inhibitors dose-dependently promote neurite outgrowth in cultured human oligodendrocyte / neuronal progenitor cells. Human oligodendrocyte / neuronal progenitor cells were cultured in vitro with or without ROCK inhibitors for 3 days. [Figure 2C] ROCK inhibitors enhance neuronal differentiation and maturation. Human oligodendrocyte / neuron progenitor cells were cultured for 14 days. ROCK inhibitors significantly promoted the expression of beta III tubulin, a marker of mature neurons, and improved neurite outgrowth. Representative images. [Figure 2D] ROCK inhibitors enhance neuronal differentiation and maturation. Human oligodendrocyte / neuron progenitor cells were cultured for 14 days. ROCK inhibitors significantly promoted the expression of beta III tubulin, a marker of mature neurons, and improved neurite outgrowth. Quantification of neurite length. [Figure 2E] ROCK inhibitors block the inhibitory effects of chondroitin sulfate proteoglycans (CSPGs) on neurite outgrowth. Human oligodendrocyte / neuronal progenitor cells were cultured on plates coated with 0.3 mg / ml CSPG for 3 days. Neurite length was quantified using the IncuCyte NeuroTrack Software Module. Statistical analysis at 72 hours was performed by ANOVA. Representative images. [Figure 2F]ROCK inhibitors block the inhibitory effects of chondroitin sulfate proteoglycans (CSPGs) on neurite outgrowth. Human oligodendrocyte / neuronal progenitor cells were cultured on plates coated with 0.3 mg / ml CSPG for 3 days. Neurite length was quantified using the IncuCyte NeuroTrack Software Module. Statistical analysis for the 72-hour time point was performed by ANOVA. Time course of neurite length. [Figure 2G] ROCK inhibitors block the inhibitory effects of chondroitin sulfate proteoglycans (CSPGs) on neurite outgrowth. Human oligodendrocyte / neuronal progenitor cells were cultured on plates coated with 0.3 mg / ml CSPG for 3 days. Neurite length was quantified using the IncuCyte NeuroTrack Software Module. Statistical analysis for the 72-hour time point was performed by ANOVA. Quantification of neurite length at 72 hours of treatment. [Figure 3A] ROCK inhibitors protect neurons from Aβ1-42-induced cell death. Human oligodendrocytes / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. IncuCyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Neurite length and total Annexin V-positive area were analyzed using IncuCyte S3 software. Representative images of human oligodendrocytes / neuronal progenitor cells treated with Aβ1-42 with or without compound combination. [Figure 3B] ROCK inhibitors protect neurons from Aβ1-42-induced cell death. Human oligodendrocyte / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. IncuCyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Neurite length and total Annexin V-positive area were analyzed using IncuCyte S3 software. Time course of total Annexin V-positive area. [Figure 3C] ROCK inhibitors protect neurons from Aβ1-42-induced cell death. Human oligodendrocyte / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. IncuCyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Neurite length and total Annexin V-positive area were analyzed using IncuCyte S3 software. Time course of neurite length. [Figure 3D] ROCK inhibitors protect neurons from Aβ1-42-induced cell death. Human oligodendrocyte / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. IncuCyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Neurite length and total Annexin V-positive area were analyzed using IncuCyte S3 software. Dose response of total Annexin V-positive area. [Figure 3E] ROCK inhibitors protect neurons from Aβ1-42-induced cell death. Human oligodendrocyte / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. IncuCyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Neurite length and total Annexin V-positive area were analyzed using IncuCyte S3 software. Dose-response quantification of neurite length. [Figure 4A] ROCK inhibitors reduced TGFβ1-induced profibrotic gene expression in the NIH3T3 mouse fibroblast cell line. NIH3T3 cells were treated with TGFβ1 for 24 hours in the presence or absence of ROCK inhibitors, and mRNA expression of αSMA was quantified by Taqman qPCR. [Figure 4B]ROCK inhibitors reduced TGFβ1-induced profibrotic gene expression in the NIH3T3 mouse fibroblast cell line. NIH3T3 cells were treated with TGFβ1 for 24 hours in the presence or absence of ROCK inhibitors, and mRNA expression was quantified by Taqman qPCR. CTGF mRNA expression was also quantified by Taqman qPCR. [Figure 4C] ROCK inhibitors reduced TGFβ1-induced profibrotic gene expression in the NIH3T3 mouse fibroblast cell line. NIH3T3 cells were treated with TGFβ1 for 24 hours in the presence or absence of ROCK inhibitors, and mRNA expression was quantified by Taqman qPCR. CCN1 mRNA. [Figure 4D] ROCK inhibitors inhibited TGFβ1-induced CCN1 secretion. CCD18Lu human lung fibroblasts were treated with ROCK inhibitors and stimulated with TGFβ1 for 40 hours, after which secreted CCN1 levels were measured by ELISA. [Figure 5A] ROCK inhibitors are active in a mouse model of pulmonary fibrosis. Representative images of lung sections stained with Masson's trichrome to visualize fibrosis in the lungs of mice after treatment with ROCK inhibitors for 21 days in a bleomycin-induced lung injury model. [Figure 5B-1] Quantification of the positive activity of ROCK inhibitors in an in vivo model of pulmonary fibrosis. Ashcroft fibrotic indices obtained from the lungs of mice in a bleomycin pulmonary fibrosis model treated with the compound of Example 2. Quantification of the positive activity of ROCK inhibitors in an in vivo model of pulmonary fibrosis. α-smooth muscle actin (αSMA) positive area percentage and total leukocyte count in bronchoalveolar lavage fluid obtained from the lungs of mice in a bleomycin pulmonary fibrosis model treated with the compound of Example 2. [Figure 5B-2]Quantification of the positive activity of ROCK inhibitors in an in vivo model of pulmonary fibrosis. Ashcroft fibrotic indices obtained from the lungs of mice in a bleomycin pulmonary fibrosis model treated with the compound of Example 2. Quantification of the positive activity of ROCK inhibitors in an in vivo model of pulmonary fibrosis. α-smooth muscle actin (αSMA) positive area percentage and total leukocyte count in bronchoalveolar lavage fluid obtained from the lungs of mice in a bleomycin pulmonary fibrosis model treated with the compound of Example 2. [Figure 6A] ROCK inhibitors are therapeutically active in a mouse model of pulmonary fibrosis. Representative images of lung sections stained with Masson's trichrome to visualize lung fibrosis in mice following therapeutic treatment with a ROCK inhibitor in a bleomycin-induced lung injury model. [Figure 6B] Quantification of the reduction in fibrotic index in histopathological analysis of images from ROCK inhibitor-treated and vehicle-treated mice in a bleomycin-induced lung injury model. [Figure 7A] ROCK inhibition stabilizes endothelial barrier function in a histamine-induced vascular permeability model in mice. Representative images of histamine-induced Evans blue dye extravasation in the skin of ROCK inhibitor-treated and vehicle-treated mice compared to a control compound (salbutamol). ROCK inhibitors induce vascular barrier stabilization after a single dose of the compound administered 1 hour before histamine injection. [Figure 7B] Quantification of the reduction in spot area size and Evans blue dye amount in the skin of ROCK inhibitor-treated mice after induction of capillary leakage by histamine injection. DETAILED DESCRIPTION OF THE INVENTION

[0029] ROCK inhibitors Compounds according to the present invention include compounds having formula I: [ka] Contains, During the ceremony, A is, [ka] is selected from the group consisting of R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; R 2is selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which is 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 together form a monocyclic or bicyclic group, wherein 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, and the monocyclic and bicyclic groups are 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 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4 is 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 is selected from the group consisting of alkyl)-O-; R 5 is selected from H, lower alkyl, and C3-C6 cycloalkyl; Alternatively, R 3 and R 5are taken together to form a cyclic group having 5 to 7 ring atoms including 2 to 3 ring heteroatoms, wherein the cyclic group is 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; a is 0 or 1, b is 0 to 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 5-6 membered heterocyclic ring.

[0030] In certain embodiments of the present invention, compounds of formula II: [ka] provide During the ceremony, R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; R 2 is selected from the group consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each of which is 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 together form a monocyclic or bicyclic group, wherein 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, and the monocyclic and bicyclic groups are 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 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 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 5-6 membered heterocyclic ring.

[0031] In certain embodiments of the present invention, compounds of formula III: [ka] provide During the ceremony, Ring A is a 5- or 6-membered aromatic ring optionally containing 0-2 ring heteroatoms; R 3 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4 is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 2; R 6 is selected from the group consisting of H, halo, lower alkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl, and carboxyl; R 7is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'NCO-, RCONH-, and RCONR'-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and m is 1 or 2.

[0032] In certain embodiments of the present invention, compounds of formula IV: [ka] provide During the ceremony, Ring B is a 5- or 6-membered aromatic ring optionally containing 0-2 ring heteroatoms; R 3 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4 is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 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 is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy and carboxyl, RR'NCO-, RCONH-, and RCONR'-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and m is 1 to 3.

[0033] In certain embodiments of the present invention, a compound of formula V: [ka] provide During the ceremony, R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; R 4 is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 2; Each R 21 are 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'-, ROC-, aryl-O-, and heteroaryl-O-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and n is 0 to 3.

[0034] In certain embodiments of the present invention, compounds of formula VI: [ka] provide During the ceremony, R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 R13 ) c - selected from the group consisting of Each R 10 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; Each R 22 are independently selected from 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-; Each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring.

[0035] In certain embodiments of the present invention, a compound of formula VII: [ka] provide During the ceremony, Ring A is a 5- or 6-membered aromatic ring optionally containing 0-2 ring heteroatoms; R 3 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4alkyl)-, R 4 is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 2; R 6 is selected from the group consisting of H, halo, lower alkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl, and carboxyl; R 7 is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, carboxyl, RR'NCO-, RCONH-, and RCONR'-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and m is 1 or 2.

[0036] In certain embodiments of the present invention, a compound of formula VIII: [ka] provide During the ceremony, Ring B is a 5- or 6-membered aromatic ring optionally containing 0-2 ring heteroatoms; R 3 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-, R 4is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 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 is selected from the group consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy and carboxyl, RR'NCO-, RCONH-, and RCONR'-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and m is 1 to 3.

[0037] In certain embodiments of the present invention, compounds of formula IX: [ka] provide During the ceremony, R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; R 4 is 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 is selected from the group consisting of alkyl)-O-; b is 0 to 2; Each R 21 are 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'-, ROC-, aryl-O-, and heteroaryl-O-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' together form a 5-6 membered heterocyclic ring; and n is 0 to 3.

[0038] In certain embodiments of the present invention, compounds of formula X: [ka] provide During the ceremony, R 1 is lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, 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 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 11 are independently selected from H, lower alkyl, and C-C cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; Additionally or alternatively, R 12 and R 13 may be taken 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 2 to 4; d is 1 to 4; Each R 22are independently selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, C1-C3 perfluoroalkyl, and C1-C3 perfluoroalkoxy; and n is 0 to 3.

[0039] In a preferred embodiment for Formulas I-X, R 1 is selected to be lower alkyl or substituted lower alkyl. In certain embodiments, R 1 is C1-C3 alkyl, and even more preferably, R 1 is methyl or ethyl.

[0040] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, straight-chain or branched-chain alkyls have 8 or fewer carbon atoms in their backbone (e.g., C1-C8 for straight chain, C3-C8 branched chain for branched chain), and more preferably 6 or fewer. Similarly, preferred cycloalkyls have 3-8 carbon atoms in their ring structure, and more preferably 3-6 carbons in the ring structure.

[0041] Unless the number of carbons is otherwise specified, "lower alkyl," as used herein, means an alkyl group, as defined above, having 1 to 4 carbons, more preferably 1 to 3 carbon atoms. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl. Lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, and cyclobutyl.

[0042] The term "cycloalkyl" refers to a saturated carbocyclic group having from 3 to 8 carbons in the ring. Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0043] The term "substituted alkyl" refers to an alkyl group as defined above and having from 1 to 3 substituents selected from the group consisting of halo, hydroxy, lower alkoxy, amino, lower alkylamino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy, and carboxyl.

[0044] "Substituted lower alkyl" refers to a lower alkyl group as defined above and having 1 to 3 substituents selected from the group consisting of halo, hydroxy, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy, and carboxyl.

[0045] "Substituted cycloalkyl," such as "substituted C3-C6 cycloalkyl," refers to a cycloalkyl group as defined above and having 1 to 3 substituents selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy, and carboxyl.

[0046] As used herein, the term "halogen" or "halo" refers to -F, -Cl, -Br or -I, preferably F, Cl or Br.

[0047] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, attached through an oxygen atom. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. The term "lower alkoxy" refers to an alkoxy substituent in which a lower alkyl is attached through an oxygen atom, where the "lower alkyl" portion is as defined above.

[0048] The terms "amine" and "amino" refer to both unsubstituted and substituted amines, for example, amines of the general formula: [ka] and wherein R and R' are each independently selected from H and lower alkyl.

[0049] As used herein, the term "aryl" includes 5- and 6-membered monocyclic aromatic groups that may contain zero to four heteroatoms, such as benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Those aryl groups having heteroatoms in the ring structure are sometimes referred to as "aryl heterocycles" 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 in which two or more carbons are shared between two adjacent rings (the rings are "fused rings"), and in which at least one of the rings is aromatic.

[0050] The term "aralkyl" as used herein refers to an alkyl group substituted with an aryl group. Preferably, the alkyl group is a lower alkyl as defined above.

[0051] The term "heterocycle" or "heterocyclyl" refers to a non-aromatic heterocycle having from 4 to 7 ring atoms and containing from 1 to 3 ring heteroatoms.

[0052] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur. Most preferred are nitrogen and oxygen.

[0053] As used herein, each expression that occurs more than once in any structure, e.g., alkyl, m, n, R 1 , R 2 etc. definition is intended to be independent of its definition elsewhere in the same structure.

[0054] It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with the allowed valences of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., one that does not undergo spontaneous transformation by rearrangement, cyclization, elimination, etc.

[0055] As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. It is not intended that this invention be limited in any way by the permissible substituents of organic compounds.

[0056] As used herein, the term "protecting group" refers to temporary substituents that protect potentially reactive functional groups from undesired 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 reviewed (Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

[0057] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the present invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are included in the present invention. The present invention also contemplates the substitution of atomic isotopes in the compounds, for example, deuterium for hydrogen, etc.

[0058] In one aspect, the present invention provides compounds of Formulas I-X that are inhibitors of ROCK. ROCK exists in two forms: ROCK1 (ROCKβ; p160-ROCK) and ROCK2 (ROCKα). In some embodiments, compounds of Formulas I-X selectively inhibit ROCK1. In some embodiments, compounds of Formulas I-X selectively inhibit ROCK2. In some embodiments, compounds of Formulas I-X are non-selective with respect to the inhibition of ROCK1 and ROCK2. In the context of this invention, selective means that the inhibitor has a lower IC50 than other kinases. 50 an IC50 that is at least 2-fold, at least 5-fold, at least 10-fold, or at least 25-fold lower for a given kinase compared to 50 This means that

[0059] Methods for determining kinase inhibition are known in the art. For example, the kinase activity and inhibitory ability of a test compound can be determined by measuring the enzyme-specific phosphorylation of a 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 binding of the peptide to trivalent metal-based nanoparticles through a specific, high-affinity interaction between the phosphogroup and the trivalent metal. Proximity to the nanoparticles results in an increase in fluorescence polarization. Inhibition of the kinase by a kinase inhibitor prevents phosphorylation of the substrate, thereby limiting binding of the fluorescently labeled substrate to the nanoparticles. Such assays can be adapted to a microwell assay format, allowing for the determination of IC values ​​for a large number of compounds. 50 This allows simultaneous determination of

[0060] Methods of Treating Disease In one aspect of the present invention, there is provided a method of treating a patient suffering from a disease, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of the present invention. As used herein, the phrase "therapeutically effective amount" means an amount of a compound, substance, or composition comprising a compound of the present invention that is effective to produce some desired therapeutic effect in at least a subpopulation of cells of an animal, at a reasonable benefit / risk ratio applicable to any medical treatment, e.g., with reasonable side effects applicable to any medical treatment.

[0061] CNS disorders The compounds of Formulas I-X exhibit effective blood-brain barrier (BBB) ​​penetration and distribution to tissues of the central nervous system. Accordingly, the compounds of the present invention are useful for treating disorders of the central nervous system, including certain ophthalmic disorders, that benefit from the ability to cross the BBB. Such disorders may involve neurodegeneration or physical damage to neural tissue, including, but not limited to, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Batten disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, dementia, neurodegeneration, spinal cord injury, cerebral vasospasm, or multiple sclerosis.

[0062] cardiovascular 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 in which ROCK function is involved, such as hypertension, pulmonary hypertension, atherosclerosis, restenosis, ischemic heart disease, cardiac hypertrophy, ocular hypertension, retinopathy, ischemic disease, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, peripheral circulatory disorders, peripheral arterial occlusive disease, glaucoma (e.g., regulation of intraocular pressure), pulmonary fibrosis, liver 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.

[0063] In one embodiment of the present invention, the compounds are used to treat cerebral cavernous hemangiomas (CCMs). CCMs are vascular lesions consisting of a dense network of leaky, dilated capillaries and are associated with central nervous system (CNS) disorders, including seizures and stroke. Loss of vascular integrity is thought to involve activation of RhoA and ROCK, leading to altered cytoskeletal stability and increased vascular permeability. The compounds of the present invention inhibit ROCK activation and restore vascular endothelial function.

[0064] glaucoma In one embodiment of the present invention, compounds of Formulae I-X are used to treat glaucoma. The two most common types, primary open-angle glaucoma and acute angle-closure glaucoma, are characterized by elevated intraocular pressure. Pigmentary and congenital glaucoma are also characterized by reduced fluid outflow and elevated intraocular pressure (IOP). Normal-tension glaucoma is thought to be due to other mechanisms, 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 an angiogenic component may benefit from the administration of an angiogenesis inhibitor in addition to a ROCK inhibitor.

[0065] 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-X. Inflammation includes, but is not limited to, asthma, cardiovascular inflammation, renal inflammation, atherosclerosis and arteriosclerosis, and sepsis. Other inflammatory conditions that can be treated by the methods of the present invention include fibrotic conditions (including, for example, idiopathic pulmonary fibrosis, NASH, scleroderma, systemic sclerosis, and cirrhosis).

[0066] Autoimmune disorders The present invention provides methods for treating an autoimmune disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formulas I-X. 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, autoimmune vasculopathy, and vasculopathy. Hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and nerve neuropathy, Barrow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitisostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, nodules Erythema urticaria, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (formerly called Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic IgA thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (M) CTD), Mooren's ulcer, Mucher-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Dwig's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal disease), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndromesyndrome), pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, types I, II, and III polyglandular autoimmune syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progestational 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, recurrent multiple cartilage ulcers, These include: inflammatory bowel disease, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular-bullous skin diseases, and vitiligo.

[0067] According to the present invention, targeting Th17 (IL-17-secreting) cells by ROCK inhibition provides a method for treating Th17 cell-mediated diseases in humans, including, but not limited to, autoimmune disorders such as RA, MS, SLE, psoriasis, and Crohn's disease, and GVHD. In one embodiment of the present invention, the ROCK inhibitor is a compound of Formulas I-X.

[0068] Neoplastic disease The ROCK inhibitors of the present invention inhibit tumor cell proliferation and metastasis, 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, lymphomas (usually malignant neoplasms of lymphoid tissue), carcinomas (any malignant tumor derived from epithelial tissue), leukemias (malignant neoplasms of hematopoietic tissue characterized by abnormal proliferation of white blood cells), sarcomas (usually malignant tumors arising from connective tissues (such as bone or muscle)), and blastomas (malignant lesions in precursor cells). Non-limiting examples include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, 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 cancer.

[0069] Insulin resistance In one embodiment of the present invention, a ROCK inhibitor is used to reduce or prevent insulin resistance or restore insulin sensitivity. Thus, in one embodiment, a compound of the present invention is used to promote or restore insulin-dependent glucose uptake. In another embodiment of the present invention, a ROCK inhibitor of the present invention is used to promote or restore glucose tolerance. In another embodiment of the present invention, a ROCK inhibitor of the present invention is used to treat metabolic syndrome. In another embodiment, a ROCK inhibitor of the present invention is used to reduce or prevent hyperinsulinemia. In one embodiment of the present invention, a ROCK inhibitor is used to treat diabetes (particularly type 2 diabetes). A ROCK inhibitor of the present invention can also be used to promote or restore insulin-mediated relaxation of vascular smooth muscle cells (VSMCs).

[0070] angiogenesis The present invention provides methods and compounds for treating diseases and disorders with an angiogenic component. According to the present invention, in certain embodiments, such diseases and disorders are treated by administering an effective amount of a ROCK inhibitor to a 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 an angiogenesis inhibitor. According to the present invention, ocular diseases and disorders with an angiogenic component are treated in this manner. In one embodiment, the present invention provides a method for treating age-related macular degeneration (AMD), which occurs in "dry" and "wet" forms. The "wet" form of AMD results in vision loss due to abnormal blood vessel growth (neovascularization). Bleeding, leakage, and scarring from these retinal vessels ultimately lead to irreversible damage to the photoreceptors. The dry form results from atrophy of the retinal pigment epithelium, which leads to 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 through Bruch's membrane and invade the subretinal space. It 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, the diseases 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). Treatment methods may be prophylactic, such as to avoid corneal neovascularization after corneal transplantation or to modulate the wound healing process in trabeculectomy surgery. These diseases and disorders may be characterized as having an angiogenic component. According to the present invention, such disorders are treated by administering a ROCK inhibitor and an angiogenesis inhibitor.

[0071] Thus, in one such embodiment, the disease or disorder is AMD, and a subject in need of AMD treatment is administered a ROCK inhibitor in an amount effective to treat AMD. In another embodiment, a ROCK inhibitor and an angiogenesis inhibitor are administered to the subject in amounts effective to treat 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, agents that bind to the extracellular domain of VEGFR2, including antibodies and VEGFR2-binding fragments thereof, as well as agents that interact with the intracellular domain of VEGFR2 and block activation of VEGFR2-dependent signaling. VEGFR2 antagonists further include agents that interact with other cellular components to block VEGFR2-dependent signaling. In other embodiments of the present invention, other ocular diseases and disorders with an angiogenic component, as identified above, are similarly treated.

[0072] According to the present invention, a ROCK inhibitor and an angiogenesis inhibitor are administered to a subject in an amount effective to treat or prevent a pathological condition characterized by excessive angiogenesis, such as atherosclerosis, rheumatoid arthritis (RA), hemangiomas, angiofibromas, and psoriasis. Other non-limiting examples of angiogenic diseases are retinopathy of prematurity (retrolental fibroplasia), corneal graft rejection, corneal neovascularization associated with refractive surgery complications, corneal neovascularization associated with contact lens complications, corneal neovascularization associated with pterygium and recurrent pterygium, corneal ulcer disease, and nonspecific ocular surface disease, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, Crohn's disease, autoimmune nephritis, primary biliary cirrhosis, acute pancreatitis, allograft 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, restinosis, and fungal, parasitic, and viral infections, including cytomegalovirus infection.

[0073] The present invention provides pan-ROCK inhibitors (i.e., compounds that inhibit ROCK1 and ROCK2). One study observed that ROCK2 is often overexpressed in hepatocellular carcinoma compared to non-tumorous liver, while ROCK1 expression remains unchanged. Other cancers that may benefit from treatment with a ROCK2-selective inhibitor include, but are not limited to, colon and bladder cancer. In contrast, ROCK1 expression levels have been observed to be higher in breast tumors. Any cancer can be tested to determine whether ROCK1 and / or ROCK2 are overexpressed and treated accordingly. In certain situations, ROCK1 and ROCK2 isoforms may exhibit similarities in regulating certain downstream targets, with neither isoform predominating. In such cases, a pan-ROCK inhibitor may be preferred.

[0074] Combination with other agents The compounds of the present invention can be advantageously administered to patients in need thereof together with a second agent. When a ROCK inhibitor is administered together with a second agent, the ROCK inhibitor and the second agent can be administered sequentially or concomitantly. Sequentially means that one agent is administered over a period of time, followed by the administration of another agent, which may occur after the administration of the first agent. When agents are administered sequentially, the level of one agent may not be maintained at a therapeutically effective level when the second agent is administered, and vice versa. Concomitantly means that the first and second agents are not administered simultaneously, but are administered according to a schedule in which both agents are maintained at substantially therapeutically effective levels. Each agent can be administered in a single or multiple doses, and the doses can be administered according to any schedule, including, but not limited to, twice daily, daily, weekly, every two weeks, and monthly.

[0075] The present invention also encompasses adjunctive administration. Adjunctive administration refers to the administration of a second agent to a patient in addition to a first agent already being administered to treat a disease or disease symptom. In some embodiments, adjunctive administration involves administering a second agent to a patient whose disease or disease symptom has not been adequately treated by administration of a first agent. In other embodiments, adjunctive administration involves administering a second agent to a patient whose disease has been effectively treated by administration of a first agent, but whose adjunctive treatment is expected to improve the treatment outcome. In some embodiments, the effect of administering a first and second agent is synergistic. In some embodiments, administration of a first and second agent prevents relapse or prolongs the time to relapse compared to administration of either agent alone. In some embodiments, administration of a first and second agent allows for a reduction in dosage and / or frequency of administration of the first and second agents.

[0076] Anti-inflammatory and immunosuppressive agents that may be administered in combination with the compounds of the invention include steroids, e.g., glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus), cyclosporine, fingolimod, interferons, e.g., IFNβ or IFNγ, tumor necrosis factor alpha (TNF-α) binding proteins, e.g., infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira), mycophenolic acid, MMF, methotrexate, NSAIDs, statins, cyclosporine ... These include rolimus / 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 (ruxolitinib), Xeljanz (Tofacitnib), and Otezla (apremilast).

[0077] In one embodiment of the present invention, a ROCK inhibitor of the present invention and an anti-tumor drug are administered to a subject in need thereof. In another embodiment, a ROCK inhibitor of the present invention and an angiogenesis inhibitor are administered to a subject in need thereof. In another embodiment, a ROCK inhibitor of the present invention and an anti-inflammatory drug are administered to a subject in need thereof. In yet another embodiment, a ROCK inhibitor of the present invention and an immunosuppressant drug are administered. The second agent may be, but is not limited to, a small molecule, an antibody or antigen-binding fragment thereof, or radiation.

[0078] Anti-neoplastic agents include, but are not limited to, cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation. In addition to the ROCK 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 radiation therapy, which can be external (e.g., external beam radiation therapy (EBRT)) or internal (e.g., brachytherapy (BT)).

[0079] Targeting small molecules and biological molecules include, but are not limited to, inhibitors of signal transduction pathway components, such as tyrosine kinase modulators and receptor tyrosine kinase inhibitors, and agents that bind to tumor-specific antigens. Examples include epidermal growth factor receptor (EGFR) inhibitors, including gefitinib, erlotinib, and cetuximab, HER2 inhibitors (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DM1; T-DM1), and pertuzumab), anti-VEGF antibodies and fragments (e.g., bevacizumab), CD20-inhibiting antibodies (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)quinazolin-4-amine (also known as XL647, EXEL-7647, and KD-019) is an in vitro inhibitor of multiple receptor tyrosine kinases (RTKs), including EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3), and ErbB2, and is also an inhibitor of SRC kinases, which are involved in pathways that lead to tumor non-responsiveness to certain TKIs.

[0080] 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 saracatinib (AZD0530).

[0081] According to the present invention, an angiogenesis inhibitor can be administered to a subject in combination with a compound of the present invention. Angiogenesis inhibitors include any substance that inhibits the growth of new blood vessels. For example, angiogenesis inhibitors include antagonists of VEGF, PlGF, and VEGF receptors, including the antibodies disclosed herein. VEGF antagonists reduce or block VEGF-related functions in cells. VEGF antagonists can act on VEGF by binding to VEGF and blocking its binding to its receptor, and / or can act on another cellular component involved in VEGF-mediated signaling. Similarly, a VEGFR2 antagonist is an agent that reduces or blocks VEGFR2-mediated signaling by binding to VEGFR2 and blocking ligand binding or interacting with a VEGFR2 substrate, or by acting on another cellular component to reduce or block VEGFR2-mediated signaling. Thus, angiogenesis inhibitors include anti-VEGFR2 antibodies, as well as, but are not limited to, antagonists of VEGF, VEGFR1, VEGFR2, PDGF, PDGFR-β, neuropilin-1 (NRP1), and complement.

[0082] Angiogenesis inhibitors include, for example, intracellular agents that block signal transduction mediated by VEGF, PDGF, ligands of 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 pan-specific 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. Cediranib (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-methoxyquinolin-4-yloxy)-N-methyl-1-naphthamide) inhibits VEGFR-1, -2, and -3 as well as FGFR-1 and -3 in the nanomolar range. It inhibits VEGFR-2 kinase. Brivanib (BMS-582664) is a VEGFR-2 inhibitor that also inhibits FGF receptor signaling. CT-322 (Adnectin) 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 kinases. X-82 (Xcovery) is a small molecule indolinone inhibitor of signaling through the growth factor receptors VEGFR and PDGFR.

[0083] Pharmaceutical Composition In one aspect, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more compounds of Formulas I-X formulated with one or more pharmaceutical excipients. As described below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration (e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, or pastes for application to the tongue), (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection (e.g., as a sterile solution or suspension, or sustained-release formulation), (3) topical application (e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin), (4) vaginal or rectal (e.g., as a vaginal suppository, cream, or foam), (5) sublingual, (6) ocular, (7) transdermal, or (8) nasal.

[0084] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals with no toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0085] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, that is involved in carrying or transporting the compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious 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 cellulose derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (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 glycol; (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) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0086] As noted above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and therefore may form pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this regard, the term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ in the administration vehicle or dosage form manufacturing process, or by separately reacting the purified compounds of the present invention in their free base form with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative 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 salts (see, e.g., Berge et al. (1977) "Pharmaceutical Salts, J. Pharm. Sci. 66:1-19").

[0087] Pharmaceutically acceptable salts of the present compounds include, for example, conventional non-toxic salts or quaternary ammonium salts of the compounds from non-toxic organic or inorganic acids. 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, nitric acid, and the like; 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, isothioic acid, and the like.

[0088] In other cases, compounds of the present invention may contain one or more acidic functional groups and, therefore, can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" refers to the 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 dosage form manufacturing process, or by separately reacting the purified compound in its free base form with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, e.g., Berge et al., supra).

[0089] Wetting agents, emulsifying agents and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0090] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, 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 chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0091] The formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0092] In certain embodiments, a formulation of the invention comprises an additive selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and a compound of the invention. In certain embodiments, the formulations described above render the compound of the invention orally bioavailable.

[0093] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier(s), and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0094] Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges, powders, granules, or solutions 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 (with an inert base such as gelatin and glycerin, or sucrose and gum acacia), and / or as mouthwashes, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compounds of the present invention may also be administered as a bolus, electuary, or paste.

[0095] In solid dosage forms of the present invention for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, lozenges, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable excipients, including pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers and extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerol; (4) agar, calcium carbonate, potato or tapioca starch, alginic acid, certain sorbitols, and / or sorbitols; (5) disintegrating agents such as certain silicates and sodium carbonate; (6) solution retarders such as paraffin; (7) absorption enhancers such as quaternary ammonium compounds and surfactants such as poloxamers 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) coloring agents; and (12) release-controlling agents such as crospovidone or ethylcellulose. In the case of capsules, tablets, and pills, pharmaceutical compositions may further comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules, using such additives as lactose or milk sugar, as well as high molecular weight polyethylene glycols.

[0096] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets may also be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0097] Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, 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 arts. These dosage forms may also be formulated to achieve sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose to achieve the desired release profile, or using other polymer matrices, liposomes, and / or microparticles. They may also be formulated for rapid release, e.g., lyophilization. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may optionally contain opacifying agents and may be composed to release the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally with a delayed release. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned additives.

[0098] 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 contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as 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, tetrahydrofuryl alcohol, sorbitan polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.

[0099] Besides diluents, the oral compositions may also include additional additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0100] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0101] Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active compound.

[0102] Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0103] Dosage forms for topical or transdermal administration of a compound of this 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 with any preservatives, buffers, or propellants which may be required.

[0104] The ointments, pastes, creams and gels may contain, in addition to the active compounds of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0105] Powders and sprays can contain, in addition to the compounds of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0106] Transdermal patches have 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 compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0107] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the present invention.

[0108] Pharmaceutical compositions of the invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents or thickening agents.

[0109] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0110] These compositions may also contain additional additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity on the compound can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0111] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous substances that are poorly soluble in water.The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0112] Injectable depot forms are prepared by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0113] When the compound of the present invention is administered to humans and animals as a pharmaceutical, it 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.

[0114] Route of administration and dosage The formulations of the present invention can be administered orally, parenterally, topically, or rectally. They are, of course, administered in a form appropriate for each administration route. For example, they can be administered in tablet or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc.; by injection, infusion, or inhalation; by topical administration in lotion or ointment; and by rectal administration in suppositories. Oral administration is preferred.

[0115] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, percutaneous intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion.

[0116] The terms "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally," as used herein, refer to the administration of a compound, drug, or other substance other than directly into the central nervous system (e.g., subcutaneous administration) so that it enters the patient's system and is therefore subject to metabolic and other similar processes.

[0117] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally (e.g., by aerosol), rectally, vaginally, parenterally, intracisternally, and topically (including buccal and sublingually) by powders, ointments, or drops.

[0118] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0119] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective for a particular patient, composition, and mode of administration to achieve the desired therapeutic response without being toxic to the patient.

[0120] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the invention, or ester, salt, or amide thereof, being used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound being used, the duration of treatment, other drugs, compounds, and / or substances being 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 similar factors well known to those skilled in the art of medicine.

[0121] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start dosages of the compounds of the present invention used in the pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0122] In general, a suitable daily dose of a compound of the present invention is that amount of the compound that is the lowest effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, when used for the indicated analgesic effect, oral, intravenous, intracerebroventricular, and subcutaneous doses of a compound of the present invention to a patient will range from about 0.0001 to about 100 mg per kg of body weight per day.

[0123] In certain embodiments, a dose of a compound or composition is administered to a subject daily, every other day, every third day, every third day, 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 two, three, four, five, six, or more subdoses administered at appropriate intervals throughout the day, and optionally in unit dosage form. In some embodiments, a dose (or multiple) of a compound or composition is administered over a 2-, 3-, 5-, 7-, 14-, or 21-day period. In certain embodiments, a dose of a compound or composition is administered over a 1-, 1.5-, 2-, 2.5-, 3-, 4-, 5-, 6-, or more-month period.

[0124] The above dosing schedules are provided for illustrative purposes only and should not be considered limiting. One of ordinary skill in the art will readily appreciate that any dose is within the scope of the present invention.

[0125] Patients for this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as horses, cattle, pigs and sheep; and common poultry and pets.

[0126] The compounds for use in the methods of the present invention can be administered neat or mixed with a pharmaceutically acceptable carrier, and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Combination therapy thus includes sequential, simultaneous, and separate administration of active compounds in such a manner that the therapeutic effect of the first administered formulation is not completely diminished when the subsequent formulation is administered.

[0127] Addition of the active compounds of the present invention to animal feed is preferably accomplished by preparing a suitable feed premix containing an effective amount of the active compound and incorporating the premix into the finished feed.

[0128] Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. Methods for preparing and administering such feed premixes and finished feeds are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, USA, 1969, or "Livestock Feeds and Feeding", O and B books, Corvallis, Ore., USA, 1977).

[0129] Microemulsification technology can 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 directing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing breakdown of the compound in the hepatobiliary circulation.

[0130] 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 using a pH-sensitive coating that releases only at low pH, such as in the stomach, or only at high pH, ​​such as in the intestine. Enteric coatings can be used to prevent release from occurring until after the stomach has been completely filled. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by including salts or pore-forming agents, which can increase drug release via water uptake or diffusion from the capsule. Additives that modify the solubility of the drug can also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix can also be incorporated. Depending on the compound, these can be added to the drug, added as a separate phase (i.e., as microparticles), or co-dissolved with the polymer phase. Types of degradation 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; 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 should be between 1 and 30% (w / w polymer).

[0131] Uptake can also be manipulated by altering the residence time of the particle in the gastrointestinal tract. This can be achieved, for example, by coating the particle with a mucoadhesive polymer or by selecting that polymer as the encapsulating material. Examples include chitosan, cellulose, and most polymers with free carboxyl groups, such as polyacrylates, in particular (as used herein, polyacrylate refers to polymers containing acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).

[0132] It is to be understood and expected that one skilled in the art may make variations within the principles of the invention disclosed herein, and that such variations are intended to be included 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 by reference in their entirety. [Example]

[0133] Example 1 All solvents and reagents were obtained commercially and used as received. 1H NMR spectra were recorded on a Bruker instrument (300 MHz or 400 MHz) in deuterated solvents as specified. Chemical shifts are given in ppm, and coupling constants are in Hertz. All final compounds were purified by flash chromatography using 220-400 mesh silica gel or by reverse-phase HPLC using CH3CN / water as the solvent. 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 on either a Waters LCT or an Applied Biosystems API 3000 mass spectrometer. High-resolution mass spectra (HRMS) were obtained on either a Waters LCT or an Agilent TOF mass spectrometer. All other LC-MS experiments were performed on an Agilent 1100 HPLC coupled to an Agilent single quadrupole mass spectrometer. Compound purity was determined by LC-MS at 230 nM and 254 nM wavelengths. All final compounds reported herein have a purity of 95% or greater.

[0134] General Procedure A [ka] EDCI coupling: The compound of general structural formula 1 (1 equivalent), EDCI (1 equivalent), HOBt (0.2 equivalents), and DIEA (2 equivalents) were dissolved in DMF and stirred at 25° C. for 5 minutes. Then, an 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 EtOAc. The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the compound of general structural formula 2.

[0135] HATU coupling: The compound of general structure 1 (1 equivalent), HATU (1.25 equivalents), and DIPEA (1.5 equivalents) were dissolved in DMF and stirred at 23° C. for 15 minutes. 1H-indazol-5-amine (1 equivalent) was introduced into the reaction mixture, and the solution was continued to stir at 23° C. for an additional 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 2.

[0136] Boc deprotection: Compounds of general structure 2 were 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 give the crude reaction mixture, which was purified by reverse-phase preparative HPLC to give the desired compounds of general structure 3.

[0137] Benzyl group removal: Compounds of general structure 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 under an atmosphere of H (1 atm) at 50° C. for 5 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by reverse-phase preparative HPLC to give the desired compounds of general structure 3.

[0138] General Procedure B [ka] The compound of general structure 4 (1 equivalent) was dissolved in CCl4, and AIBN (0.1 equivalent) was added followed by NBS (1.2 equivalents) at room temperature. The reaction temperature was raised to 60 °C, and stirring was continued for 16 hours. The reaction mixture was filtered, and the solvent was removed under reduced pressure. The crude material 5 was used in the next step without further purification.

[0139] A compound of general structure 5 (1 equivalent) and an alkylamine (2 equivalents) in THF were stirred for 16 hours at 70° C. The reaction mixture was concentrated under reduced pressure and the material was purified by silica gel chromatography to give a compound of general structure 6.

[0140] NaOH (2 equivalents) was introduced into a reaction vessel containing a compound of general structure 6 (1 equivalent) dissolved in a MeOH / HO mixture. The reaction was stirred at 20° C. for 16 hours. The solvent was removed under reduced pressure. The crude residue was dissolved in water (10 mL) and carefully neutralized with 6 N HCl until pH=8. The suspension was filtered, and the solid was collected and dried to give a compound of general structure 7.

[0141] The compound of general structure 7 (1 equivalent), HATU (1.25 equivalents), and DIPEA (2 equivalents) were dissolved in DMF and stirred at 20° C. for 15 minutes. 1H-Indazol-5-amine (1 equivalent) was introduced into the reaction mixture and stirred at 20° C. for 15.8 hours. The reaction was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine (15 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to give the compound of general structure 8.

[0142] Example 2 N-(4-(1H-pyrazol-4-yl)phenyl)-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 give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (77%). 1H NMR (400 MHz, DMSO-d6) δ 10.09 (brs, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.27 (t, J = MS (ES+) m / e 337.0 (M+H) + .

[0143] Example 3 (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-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 give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (77%). The enantiomers were separated by SFC (Chiralpak AD-H column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO2, flow rate 70 g / min. 1 H NMR (400 MHz, DMSO-d6) δ 10.09 (brs, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.27 (t, J = MS (ES+) m / e 337.0 (M+H) + .[α] D = +102.3 (c = 0.5, CH3OH, 25℃).

[0144] Example 4 (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-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 give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (77%). The enantiomers were separated by SFC (Chiralpak AD-H column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO2, flow rate 70 g / min. 1 H NMR (400 MHz, DMSO-d6) δ 10.09 (brs, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.27 (t, J = MS (ES+) m / e 337.0 (M+H) + [α]D = -97.2 (c = 0.5, CH3OH, 25℃).

[0145] Example 5 2-(4-chlorophenyl)-2-(methylamino)-N-(4-(pyridin-4-yl)phenyl)acetamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to give 2-(4-chlorophenyl)-2-(methylamino)-N-(4-(pyridin-4-yl)phenyl)acetamide as a yellow solid (24%). 1 H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.62 (brs, 2H), 8.78 (d, J = 6.4 Hz, 2H), 8.02 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.62 (s, 4H), 5.16 (s, 1H), 2.55 (s, 3H). MS (ES+) m / e 352.0 (M+H) + .

[0146] Example 6 2-(4-Methoxyphenyl)-2-(methylamino)-N-(4-(pyridin-4-yl)phenyl)acetamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to give 2-(4-methoxyphenyl)-2-(methylamino)-N-(4-(pyridin-4-yl)phenyl)acetamide as an off-white solid (16%). 1 H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 9.66-9.27 (m, 2H), 8.77 (d, J = 6.4 Hz, 2H), 8.02 (d, J = 6.4 Hz, 2H), 7.96 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 5.03 (s, 1H), 3.79 (s, 3H), 2.67 (s, 3H). MS (ES+) m / e 348.0 (M+H) + .

[0147] Example 7 2-(Methylamino)-N-(4-(pyridin-4-yl)phenyl)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to give 2-(methylamino)-N-(4-(pyridin-4-yl)phenyl)-2-(p-tolyl)acetamide as a white solid (28%). 1 H NMR (400 MHz, DMSO-d6) δ 10.25 (brs, 1H), 8.59 (dd, J = 4.4, 1.6 Hz, 2H), 8.32 (s, 1H), 7.81-7.76 (m, 4H), 7.68 (dd, J = 4.4, 1.6 Hz, MS (ES+) m / e 332.1 (M+H) + .

[0148] Example 8 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide as a white solid (32%). 1H NMR (400 MHz, DMSO-d6) δ 9.98 (brs, 1H), 8.21 (s, 1H), 7.99 (s, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 7.6 MS (ES+) m / e 337.1 (M+H) + .

[0149] Example 9 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general protocol B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as a white solid (32%). 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.46-9.30 (m, 2H), 8.01 (s, 2H), 7.59-7.50 (m, 6H), 7.07 (d, J = 8.8 Hz, 2H), 4.95-4.94 (m, 1H), 3.79 (s, 3H), 2.54 (s, 3H). MS (ES+) m / e 337.1 (M+H) + .

[0150] Example 10 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-methoxyphenyl)acetamide as a white solid (16%). 1 H NMR (400 MHz, CD3OD) δ 8.55 (s, 1H), 7.93 (s, 2H), 7.59-7.51 (m, 6H), 7.04 (d, J = 8.8 Hz, 2H), 4.77 (s, 1H), 3.83 (s, 3H), 2.98-2.83 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 351.1 (M+H) + .

[0151] Example 11 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(p-tolyl)acetamide as a white solid (8%). 1 H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.18 (s, 1H), 7.99 (s, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 7.6 MS (ES+) m / e 335.1 (M+H) + .

[0152] Example 12 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-fluorophenyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(p-tolyl)acetamide as a white solid (20%). 1 H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 9.53-9.47 (m, 2H), 8.01 (s, 2H), 7.69-7.60 (m, 2H), 7.59-7.53 (m, 4H), 7.37 (dd, J = 8.8, MS (ES+) m / e 339.0 (M+H) + .

[0153] Example 13 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-methoxyphenyl)acetamide as a pink solid (20%). 1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.26 (s, 1H), 7.99 (s, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.26 (t, J = 8.0 MS (ES+) m / e 351.1 (M+H) + .

[0154] Example 14 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(m-tolyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(m-tolyl)acetamide as a white solid (14%). 1 H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.63-7.61 (m, 2H), 7.54-7.52 (m, 2H), 7.29-7.09 (m, 3H), 7.10 (d, J = 6.8 Hz, 1H), 4.18 (s, 1H), 2.31 (s, 3H), 2.28 (s, 3H). MS (ES+) m / e 321.1 (M+H) + .

[0155] Example 15 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-fluorophenyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-fluorophenyl)acetamide as a white solid (20%). 1 H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.17 (s, 1H), 8.00 (s, 2H), 7.61-7.59 (m, 2H), 7.55-7.53 (m, 2H), 7.43-7.31 (m, 3H), 7.14-7.09 (m, 1H), 4.40 (s, 1H), 2.56-2.53 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 339.1 (M+H) + .

[0156] Example 16 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(ethylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(ethylamino)acetamide as a white solid (6%). 1 H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.19 (s, 1H), 8.00 (s, 2H), 7.62-7.53 (m, 5H), 7.45-7.34 (m, 3H), 4.39 (s, 1H), 2.53 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 355.1 (M+H) + .

[0157] Example 17 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide as a white solid (8%). 1 H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.21 (s, 1H), 7.99 (s, 2H), 7.62-7.60 (m, 2H), 7.54-7.49 (m, 4H), 7.43-7.41 (m, 2H), 4.25 (s, 1H), 2.28 (s, 3H). MS (ES+) m / e 341.0 (M+H) + .

[0158] Example 18 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2-(methylamino)acetamide as a white solid (23%). 1 H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.18 (s, 1H), 7.99 (s, 2H), 7.63-7.60 (m, 2H), 7.55-7.53 (m, 2H), 7.43-7.31 (m, 3H), 7.15-7.10 (m, 1H), 4.30 (s, 1H), 2.29 (s, 3H). MS (ES+) m / e 325.1 (M+H) + .

[0159] Example 19 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2-(methylamino)acetamide as an off-white solid (25%). 1 H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.20 (s, 2H), 7.99 (s, 2H), 7.62-7.50 (m, 6H), 7.21-7.17 (m, 2H), 4.27 (s, 1H), 2.29 (s, 3H). MS (ES+) m / e 325.1 (M+H) + .

[0160] Example 20 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(methylamino)acetamide as an off-white solid (3%). 1 H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.33 (s, 2H), 7.99 (s, 2H), 7.63-7.61 (m, 2H), 7.56-7.53 (m, 3H), 7.45-7.34 (m, 3H), 4.26 (s, 1H), 2.28 (s, 3H). MS (ES+) m / e 341.0 (M+H) + .

[0161] Example 21 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(p-tolyl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(p-tolyl)acetamide as a yellow oil (12%). 1 H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 10.01 (s, 1H), 8.11 (s, 1H), 7.86 (s, 1H), 7.63-7.61 (m, 2H), 7.54-7.52 (m, 2H), 7.35 (d, MS (ES+) m / e 321.1 (M+H) + .

[0162] Example 22 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(ethylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(ethylamino)acetamide as a white solid (6%). 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.23 ​​(s, 1H), 7.99 (s, 2H), 7.61-7.50 (m, 6H), 7.43-7.41 (m, 2H), 4.38 (s, 1H), 2.57-2.54 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 355.1 (M+H) + .

[0163] Example 23 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)-2-(methylamino)acetamide as a white solid (5%). 1 H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 10.03 (s, 1H), 7.99 (s, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.32 (d, J = MS (ES+) m / e 353.2 (M+H) + .

[0164] Example 24 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide as an off-white solid (18%). 1 H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.62-7.60 (m, 2H), 7.55-7.53 (m, 2H), 7.44-7.05 (m, 5H), 4.28 (s, 1H), 2.29 (s, 3H). MS (ES+) m / e 373.3 (M+H) + .

[0165] Example 25 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide as a white solid (12%). 1 H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H), 7.27 (t, J = 4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H), 3.75 (s, 3H), 3.44 (t, J = 5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J = 5.2 Hz, 2H). MS (ES+) m / e 381.3 (M+H) + .

[0166] Example 26 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide as a white solid (29%). 1 H NMR (400 MHz, CD3OD) δ 8.49 (s, 1H), 7.91 (s, 2H), 7.58-7.52 (m, 4H), 7.31 (t, J = 8.0 Hz, 1H), 7.10-7.08 (m, 2H), 6.92-6.89 (m, MS (ES+) m / e 380.3 (M+H) + .

[0167] Example 27 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2-methylbenzo[d]thiazol-6-yl)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2-methylbenzo[d]thiazol-6-yl)acetamide as a white solid (6%). 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.16 (d, J = 6.4 Hz, 2H), 8.00-7.94 (m, 3H), 7.63-7.54 (m, 5H), 4.67 (s, 1H), 2.81 (s, 3H), 2.40 (s, 3H). MS (ES+) m / e 378.3 (M+H) + .

[0168] Example 28 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(dimethylamino)phenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(dimethylamino)phenyl)-2-(methylamino)acetamide as a yellow solid (17%). 1 H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.19 (s, 1H), 7.98 (s, 2H), 7.62-7.51 (m, 4H), 7.15 (t, J = 8.0 Hz, 1H), 6.87 (s, 1H), 6.76 (d, J = 7.2 Hz, 1H), 6.66-6.63 (m, 1H), 4.25 (s, 1H), 2.89 (s, 6H), 2.32 (s, 3H). MS (ES+) m / e 350.3 (M+H) + .

[0169] Example 29 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxypyridin-4-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxypyridin-4-yl)-2-(methylamino)acetamide as a white solid (2%). 1 H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.15 (s, 1H), 8.12 (d, J = 5.6 Hz, 1H), 7.98 (s, 2H), 7.61-7.52 (m, 4H), 7.07 (d, J = 4.4 Hz, 1H), 6.91 (s, 1H), 4.24 (s, 1H), 3.83 (s, 3H), 2.28 (s, 3H). MS (ES+) m / e 338.2 (M+H) + .

[0170] Example 30 Methyl 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give methyl 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate as a white solid (15%). 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.65-9.51 (m, 2H), 8.25 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.00 (s, 2H), 7.86 (d, J = 8.0 Hz, MS (ES+) m / e 365.3 (M+H) + .

[0171] Example 31 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give methyl N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)-2-(methylamino)acetamide as a white solid (4%). 1 H NMR (400 MHz, CD3OD) δ 7.90 (s, 2H), 7.57-7.50 (m, 4H), 7.30 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 4.15 (s, 1H), 2.91 (s, 6H), 2.37 (s, 3H). MS (ES+) m / e 350.3 (M+H) + .

[0172] Example 32 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid as a white solid (15%). 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.23 ​​(s, 1H), 8.07 (s, 1H), 7.98 (s, 2H), 7.86 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 4.33 (s, 1H), 2.29 (s, 3H). MS (ES+) m / e 351.3 (M+H) + .

[0173] Example 33 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methyl-1H-indazol-6-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methyl-1H-indazol-6-yl)-2-(methylamino)acetamide as a white solid (8%). 1 H NMR (400 MHz, CD3OD) δ 7.93 (s, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.58-7.54 (m, 4H), 7.33 (d, J = 8.8 Hz, 1H), 5.12 (s, 1H), 2.69 (s, 3H), 2.57 (s, 3H). MS (ES+) m / e 361.3 (M+H) + .

[0174] Example 34 Methyl 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give methyl 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate as an off-white solid (11%). 1 H NMR (400 MHz, CD3OD) δ 8.17 (dd, J = 6.8, 2.0 Hz, 2H), 7.92 (s, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.58-7.53 (m, 4H), 5.07 (s, 1H), 3.92 (s, 3H), 2.70 (s, 3H). MS (ES+) m / e 365.1 (M+H) + .

[0175] Example 35 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methyl-1H-benzo[d]imidazol-6-yl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methyl-1H-benzo[d]imidazol-6-yl)-2-(methylamino)acetamide as a yellow oil (4%). 1 H NMR (400 MHz, CD3OD) δ 8.02 (d, J = 1.2 Hz, 1H), 7.92 (s, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.75 (dd, J = 8.0, 1.6 Hz, 1H), 7.59-7.52 (m, 4H), 5.21 (s, 1H), 2.84 (s, 3H), 2.71 (s, 3H). MS (ES+) m / e 361.2 (M+H) + .

[0176] Example 36 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-methoxypropyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-methoxypropyl)amino)acetamide as an off-white solid (21%). 1 H NMR (400 MHz, CD3OD) δ 8.55 (s, 2H), 7.72-7.65 (m, 4H), 7.44 (t, J = 7.6 Hz, 1H), 7.26-7.25 (m, 2H), 7.09 (dd, J = 8.0, 2.0 Hz, 1H), 5.15 (s, 1H), 3.85 (s, 3H), 3.51 (t, J = 5.6 Hz, 2H), 3.35 (s, 3H), 3.30-3.08 (m, 2H), 2.03-1.98 (m, 2H). MS (ES+) m / e 395.3 (M+H) + .

[0177] Example 37 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (8%). 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 10.09 (s, 1H), 8.12 (s, 1H), 7.87 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.04-7.02 (m, 2H), 6.85 (dd, J = 8.0, 2.0 Hz, 1H), 4.33 (s, 1H), 3.75 (s, 3H), 3.48-3.41 (m, 4H), 2.66-2.63 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 395.3 (M+H) + .

[0178] Example 38 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((3-(dimethylamino)propyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((3-(dimethylamino)propyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (4%). 1 H NMR (400 MHz, CD3OD) δ 7.93 (s, 2H), 7.60-7.53 (m, 4H), 7.30 (t, J = 8.0 Hz, 1H), 7.09-7.08 (m, 2H), 6.90 (dd, J = 8.4, 1.6 Hz, 1H), 4.34 (s, 1H), 3.82 (s, 3H), 2.68-2.61 (m, 2H), 2.48-2.42 (m, 2H), 2.30 (s, 6H), 1.83-1.74 (m, 2H). MS (ES+) m / e 408.3 (M+H) + .

[0179] Example 39 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (7%). 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (brs, 1H), 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.28 (t, J = 8.0 Hz, 1H), 7.06-7.03 (m, 2H), 6.87 (dd, J = 8.0, 2.4 Hz, 1H), 4.38 (s, 1H), 3.75 (s, 3H), 3.38 (m, 2H), 2.87 (s, 3H), 2.84 (s, 3H). MS (ES+) m / e 408.3 (M+H) + .

[0180] Example 40 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (28%). 1H NMR (400 MHz, CD3OD) δ 8.03 (s, 2H), 7.61-7.55 (m, 4H), 7.44 (t, J = 8.0 Hz, 1H), 7.23-7.20 (m, 2H), 7.10-7.07 (m, 1H), 5.13 (s, MS (ES+) m / e 409.3 (M+H) + .

[0181] Example 41 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (6%). 1 H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.22 (s, 1H), 7.99 (s, 2H), 7.62-7.53 (m, 4H), 7.45 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.12-7.01 (m, 3H), 6.87 (dd, J = 8.0, 2.0 Hz, 1H), 4.34 (s, 1H), 3.76 (s, 3H), 3.04 (s, 2H). MS (ES+) m / e 380.2 (M+H) + .

[0182] Example 42 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-(methylamino)-propyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (6%). 1 H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 9.70 (s, 2H), 8.65 (s, 2H), 8.00 (s, 2H), 7.56 (s, 4H), 7.43 (t, J = 8.0 Hz, 1H), 7.22-7.19 (m, 2H), 7.06 (dd, J = 8.0, 1.6 Hz, 1H), 5.11 (s, 1H), 3.79 (s, 3H), 2.96-2.90 (m, 4H), 2.54 (s, 3H), 2.03-1.96 (m, 2H). MS (ES+) m / e 394.3 (M+H) + .

[0183] Example 43 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((1-methoxy-2-methylpropan-2-yl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((1-methoxy-2-methylpropan-2-yl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (17%). 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.23-9.19 (m, 2H), 7.99 (s, 2H), 7.58-7.52 (m, 4H), 7.41 (t, J = 8.0 Hz, 1H), 7.21-7.19 (m, MS (ES+) m / e 409.3 (M+H) + .

[0184] Example 44 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)-2-oxoethyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)-2-oxoethyl)amino)acetamide as a yellow solid (3%). 1 H NMR (400 MHz, CD3OD) δ 7.95 (s, 2H), 7.59-7.53 (m, 4H), 7.44 (t, J = 8.4 Hz, 1H), 7.18-7.17 (m, 2H), 7.16-7.10 (m, 1H), 5.12 (s, 1H), 3.84 (s, 3H), 3.68 (s, 2H), 2.79 (s, 3H). MS (ES+) m / e 394.3 (M+H) + .

[0185] Example 45 2-(((1H-pyrazol-4-yl)methyl)amino)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give 2-(((1H-pyrazol-4-yl)methyl)amino)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)acetamide as a white solid (1%). 1 H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.77 (s, 2H), 7.99 (s, 2H), 7.70 (s, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.20-7.17 (m, 2H), 7.06 (dd, J = 8.0, 2.0 Hz, 1H), 4.91 (s, 1H), 4.01 (s, 2H), 3.79 (s, 3H). MS (ES+) m / e 403.3 (M+H) + .

[0186] Example 46 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethyl(2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(ethyl(2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (19%). 1H NMR (400 MHz, acetone-d6) δ 11.21 (s, 1H), 8.01 (s, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.47-7.43 (m, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.11-7.09 (m, 1H), 6.03-6.01 (m, 1H), 3.93-3.80 (m, 5H), 3.74-3.53 (m, 3H), 3.42 (m, 4H), 1.44 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 409.3 (M+H) + .

[0187] Example 47 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (24%). 1 H NMR (400 MHz, acetone-d6) δ 10.85 (s, 1H), 8.01 (s, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.46 (t, J = 8.0 Hz, 1H), 7.41 (s, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.11 (dd, J = 8.0, 2 Hz, 1H), 5.80 (d, J = 4.4 Hz, 1H), 3.95-3.92 (m, 2H), 3.87 (s, 3H), 3.72-3.60 (m, 2H), 3.42 (s, 3H), 3.06 (s, 3H). MS (ES+) m / e 395.3 (M+H)+ .

[0188] Example 48 N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (13%). 1 H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.92 (s, 1H), 9.66 (s, 2H), 8.07 (s, 2H), 7.87 (d, J = 12.4 Hz, 1H), 7.46-7.39 (m, 2H), 7.23-7.19 (m, 2H), 7.07 (dd, J = 8.0, 6.0 Hz, 1H), 5.36 (s, 1H), 4.44-4.35 (m, 2H), 3.80 (s, 3H), 3.66-3.64 (m, 2H), 3.63-3.49 (m, 2H), 3.31 (s, 3H), 3.07-3.00 (m, 2H), 2.76 (s, 6H). MS (ES+) m / e 486.3 (M+H) + .

[0189] Example 49 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)-N-methylacetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)-N-methylacetamide as a yellow solid (15%). 1 H NMR (400 MHz, D2O) δ 8.07 (s, 2H), 7.48 (m, 2H), 7.27-7.23 (m, 2H), 6.95 (m, 2H), 6.68 (d, J = 6.8 Hz, 1H), 6.28 (s, 1H), 5.02 (s, MS (ES+) m / e 395.3 (M+H) + .

[0190] Example 50 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-morpholinoethyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-morpholinoethyl)amino)acetamide as a white solid (38%). 1 H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.00 (s, 2H), 7.59-7.57 (m, 4H), 7.42 (t, J = 7.6 Hz, 1H), 7.21-7.17 (m, 2H), 7.07-7.04 (m, 1H), 5.09 (s, 1H), 3.79 (m, 7H), 3.31-3.12 (m, 8H). MS (ES+) m / e 436.3 (M+H) + .

[0191] Example 51 N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide as a yellow solid (31%). 1 H NMR (400 MHz, D2O) δ 8.04 (s, 2H), 7.56-7.48 (m, 2H), 7.19-7.14 (m, 4H), 5.27 (s, 1H), 4.23-4.21 (m, 1H), 4.11-4.10 (m, 1H), 3.80 (m, 5H), 3.50 (m, 2H), 3.33 (m, 4H), 3.19-3.16 (m, 1H), 2.82-2.62 (m, 9H). MS (ES+) m / e 500.3 (M+H) + .

[0192] Example 52 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(pyrrolidin-1-yl)ethyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(pyrrolidin-1-yl)ethyl)amino)acetamide as a white solid (35%). 1H NMR (400 MHz, DMOS-d6) δ 10.64 (s, 1H), 8.00 (s, 2H), 7.57 (m, 4H), 7.41 (t, J = 8.0, 1H), 7.19-7.16 (m, 2H), 7.05- 7.03 (m, 1H), 5.01 (s, 1H), 3.79 (s, 3H), 3.44-3.27 (m, 8H), 1.94 (m, 4H). MS (ES+) m / e 420.3 (M+H) + .

[0193] Example 53 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((1-(methoxymethyl)cyclopropyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((1-(methoxymethyl)cyclopropyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (27%). 1 H NMR (400 MHz, DMOS-d6) δ 10.65 (brs, 1H), 9.58 (brs, 1H), 8.00 (s, 2H), 7.62-7.52 (m, 4H), 7.42 (t, J = 7.6 Hz, 1H), 7.24-7.15 (m, 2H), 7.08-7.02 (m, 1H), 5.25 (s, 1H), 3.80 (s, 3H), 3.64 (d, J = 11.6 Hz, 1H), 3.37 (d, J = 11.6 Hz, 1H), 3.23 (s, 3H), 1.23-2.21 (m, 1H), 1.02-0.91 (m, 1H), 0.89-0.76 (m, 2H). MS (ES+) m / e 407.3 (M+H) + .

[0194] Example 54 N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(2,2,2-trifluoroethoxy)ethyl)amino)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(2,2,2-trifluoroethoxy)ethyl)amino)acetamide as an off-white solid (6%). 1 H NMR (400 MHz, DMOS-d6) δ 12.86 (s, 1H), 10.06 (s, 1H), 8.11 (s, 1H), 7.86 (s, 1H), 7.60-7.52 (m, 4H), 7.26 (t, J = 7.9 Hz, 1H), 7.05-7.02 (m, 2H), 6.85 (dd, J = 8.2, 1.8 Hz, 1H), 4.34 (d, J = 7.6 Hz, 1H), 4.10-4.03 (m, 2H), 3.75 (s, 3H), 3.69 (t, J = 5.6 Hz, 2H), 2.68 (s, 3H). MS (ES+) m / e 449.2 (M+H) + .

[0195] Example 55 N-(3-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(3-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a yellow solid (21%). 1H NMR (400 MHz, DMOS-d6) δ 10.97 (s, 1H), 9.64 (s, 2H), 8.00 (d, J = 1.6 Hz, 2H), 7.70 (t, J = 8.0 Hz, 1H), 7.62 (dd, J = 13.2, 2.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 7.24-7.19 (m, 2H), 7.06-7.03 (m, 1H), 5.14 (s, 1H), 3.79 (s, 3H), 3.64-3.57 (m, 2H), 3.29 (s, 3H), 3.09-3.00 (m, 2H). MS (ES+) m / e 399.2 (M+H) + .

[0196] Example 56 N-(3-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(3-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a yellow solid (17%). 1H NMR (400 MHz, DMOS-d6) δ 10.79 (s, 1H), 9.62 (s, 2H), 8.00 (s, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 1.6 Hz, 1H), 7.26-7.21 (m, 3H), 7.06-7.02 (m, 1H), 5.13 (s, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.64-3.59 (m, 2H), 3.30 (s, 3H), 3.09-3.00 (m, 2H). MS (ES+) m / e 411.3 (M+H) + .

[0197] Example 57 N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (41%). 1 H NMR (400 MHz, DMOS-d6) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 MS (ES+) m / e 394.3 (M+H) + .

[0198] Example 58 (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (12%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AS column) (mobile phase: 40% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give the desired compound in 99% enantiomeric purity. 1 H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H), 7.27 (t, J = 4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H), 3.75 (s, 3H), 3.44 (t, J = 5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J = 5.2 Hz, 2H). MS (ES+) m / e 381.3 (M+H) + .[α] 25°C D =+114 (c=0.2 in MeOH).

[0199] Example 59 (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (12%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AS column) (mobile phase: 40% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give the desired compound in 99% enantiomeric purity. 1 H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H), 7.27 (t, J = 4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H), 3.75 (s, 3H), 3.44 (t, J = 5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J = 5.2 Hz, 2H). MS (ES+) m / e 381.3 (M+H) + (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide. [α] 25°C D =-103.5 (c=0.2 in MeOH).

[0200] Example 60 (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give (S)—N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (41%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AS column) (mobile phase: 50% EtOH containing 0.1% NH4OH in CO2, flow rate 70 g / min) to give the desired compound in 99% enantiomeric purity. 1 H NMR (400 MHz, DMOS-d6) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 MS (ES+) m / e 394.3 (M+H) + .[α] 25°C D =+95.7 (c=0.2 in MeOH).

[0201] Example 61 (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide [ka] The reaction was carried out according to general procedure B. The final residue was purified by reverse-phase preparative HPLC to give (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide as a white solid (41%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AS column) (mobile phase: 50% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give the desired compound in 99% enantiomeric purity. 1 H NMR (400 MHz, DMOS-d6) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 MS (ES+) m / e 394.3 (M+H) + .[α] 25°C D =-92.8 (c=0.2 in MeOH).

[0202] Example 62 (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide [ka] Step 1: Compound tert-butyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)-(1-(3-methoxyphenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)carbamate was dissolved in EtOH (0.15 M) and para-toluenesulfonic acid (2 equivalents) was added. The solution was stirred for 20 hours at 15°C. The reaction mixture was quenched with NaHCO3 and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford tert-butyl (2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamate as a yellow solid in 92% yield. The enantiomers were separated by SFC (DAICEL CHIRALPAK OD column) (mobile phase: 45% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give tert-butyl (R)-(2-((2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(tert-butoxycarbonyl)amino)ethyl)-(methyl)carbamate with an enantiomeric purity of 99%. 1 H NMR (400 MHz, DMOS-d6) δ δ 12.84 (s, 1H), 10.19 (s, 1H), 8.04 (s, 2H), 7.71-7.49 (m, 4H), 7.37-7.33 (m, 1H), 7.03-6.89 (m, 3H), 6.02-5.33 (m, 1H), 3.76 (s, 3H), 3.42-2.70 (m, 7H), 1.55-1.25 (m, 18H).

[0203] Step 2: Compound tert-butyl (R)-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamate was dissolved in dichloromethane and 4N HCl in dioxane was added. The reaction mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The final residue was purified by reverse-phase preparative HPLC to give (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide as a white solid (12%). 1 H NMR (400 MHz, CD3OD) δ δ 8.51 (s, 1H), 7.91 (s, 2H), 7.58-7.51 (m, 4H), 7.31 (t, J = 7.6 Hz, 1H), 7.09-7.08 (m, 2H), 6.91-6.89 (m, MS (ES+) m / e 380.4 (M+H) + .[α] 25°C D = -86.02 (0.5 in MeOH).

[0204] Example 63 (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide [ka] Step 1: Compound tert-butyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(1-(3-methoxyphenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)carbamate was dissolved in EtOH (0.15 M) and para-toluenesulfonic acid (2 equivalents) was added. The solution was stirred for 20 hours at 15°C. The reaction mixture was quenched with NaHCO3 and extracted with EtOAc. The combined organic fractions were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give tert-butyl (2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamate as a yellow solid in 92% yield. The enantiomers were separated by SFC (DAICEL CHIRALPAK OD column) (mobile phase: 45% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give tert-butyl (S)-(2-((2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate in 99% enantiomeric purity. 1 H NMR (400 MHz, DMOS-d6) δ δ 12.84 (s, 1H), 10.19 (s, 1H), 8.04 (s, 2H), 7.71-7.49 (m, 4H), 7.37-7.33 (m, 1H), 7.03-6.89 (m, 3H), 6.02-5.33 (m, 1H), 3.76 (s, 3H), 3.42-2.70 (m, 7H), 1.55-1.25 (m, 18H).[α] 25°C D =-92.8 (c=0.2 in MeOH)

[0205] Step 2: Compound tert-butyl (S)-(2-((2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate was dissolved in dichloromethane and 4N HCl in dioxane was added. The reaction mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The final residue was purified by reverse-phase preparative HPLC to give (S)—N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide as a white solid (12%). 1 H NMR (400 MHz, CD3OD) δ 8.51 (s, 1H), 7.91 (s, 2H), 7.58-7.51 (m, 4H), 7.31 (t, J = 7.6 Hz, 1H), 7.09-7.08 (m, 2H), 6.91-6.89 (m, MS (ES+) m / e 380.4 (M+H) + .[α] 25°C D = +82.43 (0.7 in MeOH).

[0206] Example 64 (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide [ka] Step 1: CbzNH2 (3 equivalents) was dissolved in n-propyl alcohol. 0.4 N NaOH (3 equivalents) was added to the solution, followed by the addition of tert-butyl hypochlorite (1.05 equivalents) in one portion. After 5 minutes, a solution of (DHQ)2PHAL (0.05 equivalents) in n-propyl alcohol was added in one portion to the reaction mixture. A solution of compound p-chlorostyrene (1 equivalent) in n-propyl alcohol was added in one portion, followed by the addition of KOsO2(OH)4 (0.03 equivalents). The light green solution was stirred at 20 °C for 1 hour. A saturated solution of Na2SO3 was added to the reaction mixture, and the mixture was stirred at 20 °C for 10 minutes. The organic layer was separated, and the aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (TFA condition) and lyophilized to obtain the compound benzyl (S)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (62%, 94.9% ee) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.34-7.31 (m, 7H), 7.28-7.22 (m, 2H), 5.64 (s, 1H), 5.13-5.05 (m, 2H), 4.80 (s, 1H), 3.86-3.78 (m, 2H), 2.24 (m, 1H).

[0207] Step 2: To a mixture of compound (S)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (1 equivalent) and imidazole (2 equivalents) in DMF was added TBSCl (1.5 equivalents) in one portion at 30° C. under nitrogen. The mixture was stirred at 30° C. for 1 hour. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiO2, petroleum ether / EtOAc=15 / 1) to give benzyl (S)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (95%, 89.3% ee) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.38-7.25 (m, 9H), 5.59 (s, 1H), 5.16-5.07 (m, 2H), 4.77 (s, 1H), 3.95 (dd, J = 10.0, 4.0 Hz, 1H), 3.75 (m, 1H), 0.89 (s, 9H), -0.03 (s, 3H), -0.06 (s, 3H).

[0208] Step 3: To a solution of compound (S)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (1 equivalent) in DMF was added NaH (2 equivalents) portionwise at 0° C. under nitrogen. The mixture was stirred at 0° C. for 10 minutes, and then a solution of MeI (1.5 equivalents) in DMF was added dropwise to the mixture. The mixture was warmed to 20° C. and stirred for 50 minutes. The mixture was poured into a saturated solution of NH4Cl and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The mixture was purified by silica gel chromatography (SiO2, petroleum ether / EtOAc=20 / 1) to give benzyl (S)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (93%, 94.4% ee) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.36-7.19 (m, 9H), 5.37-5.22 (m, 1H), 5.19-5.04 (m, 2H), 4.05 (m, 2H), 2.80 (s, 3H), 0.88 (s, 9H), 0.07 (s, 6H).

[0209] Step 4: To a mixture of compound (S)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (1 equivalent) in MeOH / HO (7:3) was added KHSO (4 equivalents) in one portion at 20 °C under N. Water was added to the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1) to give compound benzyl (S)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (99%, 94% ee) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.36-7.20 (m, 9H), 5.33 (dd, J = 8.0, 6.4 Hz, 1H), 5.20 (m, 2H), 4.17-4.07 (m, 2H), 2.78 (s, 3H).

[0210] Step 5: A solution of compound (S)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (1 equivalent) was dissolved in saturated KH2PO4 and MeCN at 0 °C. NaClO2 (2 equivalents) and TEMPO (0.2 equivalents) were added to the reaction mixture at 0-5 °C, followed by dropwise addition of NaClO (2 equivalents) at 0-5 °C under N2. The mixture turned deep purple, and the reaction mixture was stirred at 10-15 °C for 2 h. The reaction mixture was diluted with HO (100 mL), and the mixture was extracted with DCM (100 mL × 3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO2, petroleum ether / EtOAc = 1 / 1, 0 / 1) to give compound (S)-2-(((benzyloxy)carbonyl)(methyl)amino)-2-(4-chlorophenyl)acetic acid (95% 94% ee) as a yellow oil.

[0211] Step 6: To a mixture of the compound (S)-2-(((benzyloxy)carbonyl)-(methyl)amino)-2-(4-chlorophenyl)acetic acid (1 equivalent) and the compound 4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)aniline (1.1 equivalents) in THF, DMT-MM (1.05) was added in one portion at 10° C. under N2. The mixture was stirred at 20° C. for 3 hours. Water was added and the mixture was extracted with EtOAc. The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether / EtOAc=2:1) ​​to obtain the compound benzyl ((1S)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-(methyl)carbamate (59%, 86% ee) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.71 (s, 1H), 7.40-7.31 (m, 4H), 7.28-7.18 (m, 9H), 5.88 (s, 1H), 5.41 (dd, J = 8.8, 2.8 Hz, 1H), 5.16-5.09 (m, 2H), 4.05-4.00 (m, 1H), 3.68-3.41(m, 1H), 2.80 (s, 3H), 2.09-2.04 (m, 2H), 1.66-1.53 ​​(m, 4H).

[0212] Step 7: To a solution of the compound ((1S)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)(methyl)carbamate (1 equivalent) in MeCN was added TMSI (8 equivalents) at 0 °C under N. HO was added and the mixture was extracted with petroleum ether. The aqueous phase was purified by preparative HPLC (FA conditions) to give (S)—N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide (9%, 87.5% ee) as a white powder. 1 H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.20 (s, 1H), 7.98 (s, 2H), 7.61-7.42 (m, 8H), 4.24 (s, 1H), 2.27 (s, 3H). MS (ES+) m / e 341.0 (M+H) + .

[0213] Example 65 (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide [ka] Step 1: CbzNH2 (3 equivalents) was dissolved in n-propyl alcohol. 0.4 N NaOH (3 equivalents) was added to the solution, followed by the addition of tert-butyl hypochlorite (1.05 equivalents) in one portion. After 5 minutes, a solution of (DHQD)2PHAL (0.05 equivalents) in n-propyl alcohol was added in one portion to the reaction mixture. A solution of compound p-chlorostyrene (1 equivalent) in n-propyl alcohol was added in one portion, followed by the addition of K2OsO2(OH)4 (0.03 equivalents). The light green solution was stirred at 20 °C for 1 hour. A saturated solution of Na2SO3 was added to the reaction mixture, and the mixture was stirred at 20 °C for 10 minutes. The organic layer was separated, and the aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (TFA condition) and lyophilized to obtain compound benzyl (R)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (71%, 94.5% ee) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J = 8.4 Hz, 1H), 7.39-7.10 (m, 9H), 5.05-4.97 (m, 2H), 4.87 (t, J = 5.6 Hz, 1H), 4.62-4.56 (m, 1H), 3.58-3.48 (m, 2H).

[0214] Step 2: To a mixture of compound (R)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (1 equivalent) and imidazole (2 equivalents) in DMF was added TBSCl (1.5 equivalents) in one portion at 30° C. under nitrogen. The mixture was stirred at 30° C. for 1 hour. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiO , petroleum ether / EtOAc=15 / 1) to give benzyl (R)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (71%, 90% ee) as a colorless oil.

[0215] Step 3: To a solution of compound (R)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (1 equivalent) in DMF was added NaH (2 equivalents) portionwise at 0° C. under nitrogen. The mixture was stirred at 0° C. for 10 minutes, and then a solution of MeI (1.5 equivalents) in DMF was added dropwise to the mixture. The mixture was warmed to 20° C. and stirred for 50 minutes. The mixture was poured into a saturated solution of NH4Cl and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The mixture was purified by silica gel chromatography (SiO2, petroleum ether / EtOAc=20 / 1) to give benzyl (R)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (75%, 93% ee) as a colorless oil.

[0216] Step 4: To a mixture of compound (R)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (1 equivalent) in MeOH / HO (7:3) was added KHSO (4 equivalents) in one portion at 20 °C under N. Water was added to the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1) to give compound benzyl (R)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (91%, 92% ee) as a colorless oil.

[0217] Step 5: A solution of compound (R)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (1 equivalent) was dissolved in saturated KH2PO4 and MeCN at 0 °C. NaClO2 (2 equivalents) and TEMPO (0.2 equivalents) were added to the reaction mixture at 0-5 °C, followed by dropwise addition of NaClO (2 equivalents) at 0-5 °C under N2. The mixture turned deep purple, and the reaction mixture was stirred at 10-15 °C for 2 h. The reaction mixture was diluted with HO (100 mL), and the mixture was extracted with DCM (100 mL × 3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO2, petroleum ether / EtOAc = 1 / 1, 0 / 1) to give the compound (R)-2-(((benzyloxy)carbonyl)-(methyl)amino)-2-(4-chlorophenyl)acetic acid (94% ee) as a yellow oil.

[0218] Step 6: To a mixture of the compound (R)-2-(((benzyloxy)carbonyl)(methyl)amino)-2-(4-chlorophenyl)acetic acid (1 equivalent) and the compound 4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)aniline (1.1 equivalents) in THF, DMT-MM (1.05) was added in one portion at 10° C. under N2. The mixture was stirred at 20° C. for 3 hours. Water was added and the mixture was extracted with EtOAc. The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether / EtOAc=2:1) ​​to give the compound benzyl ((1R)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)(methyl)carbamate (83%, 87% ee) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.71 (s, 1H), 7.40-7.31 (m, 4H), 7.28-7.18 (m, 9H), 5.88 (s, 1H), 5.41 (dd, J = 8.8, 2.8 Hz, 1H), 5.16-5.09 (m, 2H), 4.05-4.00 (m, 1H), 3.68-3.41(m, 1H), 2.80 (s, 3H), 2.09-2.04 (m, 2H), 1.66-1.53 ​​(m, 4H).

[0219] Step 7: To a solution of the compound ((1R)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)(methyl)carbamate (1 equivalent) in MeCN was added TMSI (8 equivalents) at 0 °C under N. HO was added and the mixture was extracted with petroleum ether. The aqueous phase was purified by preparative HPLC (FA conditions) to give (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide (28%, 87% ee) as a white powder. 1 H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.14 (s, 1H), 7.98 (s, 2H), 7.59 (d, J = 8.8 Hz, 4H), 7.54-7.49 (m, 4H), 7.44-7.42 (m, 2H), 4.32 (s, 1H), 2.30 (s, 3H). MS (ES+) m / e 341.3 (M+H) + .

[0220] Example 66 N-(2-fluoro-5-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(2-fluoro-5-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a yellow oil (1%). 1 H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 9.91 (brs, 1H), 8.07 (s, 2H), 7.74 (d, J = 7.2 Hz, 1H), 7.58 (d, J = 12.4 Hz, 1H), 7.27 (t, J = MS (ES+) m / e 385.2 (M+H) + .

[0221] Example 67 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-N-methylbenzamide [ka] Step 1: To a solution of compound methyl 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 equivalent) in MeOH and water, NaOH (2 equivalents) was added. The mixture was concentrated under vacuum. The residue was purified by preparative HPLC (FA conditions) to give compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (58%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.95-7.90 (m, 2H), 7.88 (d, J = 8.0Hz, 1H), 7.63-7.55 (m, 5H), 7.27 (d, J = 8.8Hz, 2H), 6.89 (d, J = 8.8Hz, 2H), 5.38 (dd, J = 10.4, 2.4 Hz, 1H), 4.33 (s, 1H), 3.92 (d, J = 11.6Hz, 1H), 3.73 (s, 3H), 3.66-3.63 (m, 1H), 3.51-3.42 (m, 2H), 2.16-2.09 (m, 4H), 1.94 (s, 2H), 1.69-1.65 (m, 1H), 1.58-1.54 (m, 2H).

[0222] Step 2: To a solution of compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (1 equivalent), methylamine (1 equivalent)) in CH3CN was added T3P (1.5 equivalents), DIPEA (1.5 equivalents) at 20°C. The mixture was heated to 80°C and stirred for 15 hours. The reaction mixture was diluted with HO and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-N-methylbenzamide as a white solid.

[0223] Step 3: A mixture of compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-N-methylbenzamide in TFA was stirred at 75° C. for 16 hours. The mixture was concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (TFA condition) to give 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-N-methylbenzamide as a white solid (43%). 1 H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.61-9.47 (m, 2H), 8.56 (d, J = 4.8 Hz, 1H), 8.12 (s, 1H), 8.01 (s, 2H), 7.93 (d, J = 7.6 Hz, MS (ES+) m / e 364.4 (M+H) + .

[0224] Example 68 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid [ka] Step 1: A mixture of compound methyl 4-(1-((4-methoxybenzyl)-(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 equivalent) in TFA was stirred at 70° C. for 112 hours. The reaction mixture was diluted with MeOH and concentrated under reduced pressure to provide a residue. The residue was purified by preparative HPLC (TFA condition) to provide compound methyl 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate (19%) as an off-white solid. 1 H NMR (400 MHz, CD3OD) δ 8.17 (dd, J = 6.8, 2.0 Hz, 2H), 7.92 (s, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.58-7.53 (m, 4H), 5.07 (s, 1H), 3.92 (s, 3H), 2.70 (s, 3H).

[0225] Step 2: To a solution of compound methyl 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate (1 equivalent) in MeOH and HO was added NaOH (3 equivalents). The mixture was stirred at 30° C. for 16 hours. The mixture was acidified with 1 N HCl to pH=5. The resulting mixture was purified by preparative HPLC (FA conditions) to afford 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid (, 18%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.27 (s, 1H), 7.98 (s, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.65-7.50 (m, 6H), 4.30 (s, 1H), 2.28 (s, 3H). MS (ES+) m / e 351.1 (M+H) + .

[0226] Example 69 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-N-methylbenzamide [ka] Step 1: To a solution of compound methyl 4-(1-((4-methoxybenzyl)-(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 equivalent) in MeOH and HO was added NaOH (2 equivalents). The mixture was stirred at 35° C. for 36 hours. The reaction mixture was diluted with HO. The resulting solution was purified by preparative HPLC (FA conditions) to afford compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (70%, purity 98%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 10.19 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.88 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.65-7.60 (m, 2H), 7.59-7.52 (m, 2H), 7.28 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 5.38 (dd, J = 10.0, 2.4 Hz, 1H), 4.33 (s, 1H), 3.94-3.92 (m, 1H), 3.74 (s, 3H), 3.68-3.58 (m, 1H), 3.54-3.40 (m, 2H), 2.18-2.03 (m, 4H), 2.00-1.87 (m, 2H), 1.75-1.61 (m, 1H), 1.59-1.48 (m, 2H).

[0227] Step 2: To a solution of compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (1 equivalent) in MeCN, T3P (1.5 equivalents), MeNH2 (2.5 equivalents) and DIPEA (4 equivalents) were added. The mixture was stirred at 30°C for 36 hours. The reaction mixture was cooled to room temperature and quenched at 15°C by adding HO. The resulting mixture was diluted with EtOAc and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-N-methylbenzamide (98%, purity 87%) as a white solid.

[0228] Step 3: To a solution of compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-N-methylbenzamide (1 equivalent) in THF and MeOH, anhydrous Pd / C (0.1 equivalent) and HCl (2 equivalents) were added. The mixture was stirred at 50°C for 2 hours under an atmosphere of H (1 atmosphere). The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (FA conditions) to give 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-N-methylbenzamide (purity 98%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.44-8.36 (m, 1H), 8.16 (s, 1H), 7.98 (s, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.63-7.58 (m, 2H), 7.57-7.51 (m, 4H), 4.30 (s, 1H), 2.76 (d, J = 4.4 Hz, 3H), 2.29 (s, 3H). MS (ES+) m / e 364.3(M+H) + .

[0229] Example 70 N-(2-(3-(dimethylamino)propoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(3-(dimethylamino)propoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (13%). 1H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 12.8 Hz, 1H), 8.00 (s, 2H), 7.30-7.23 (m, 2H), 7.03- 7.02 (m, 2H), 6.88 (dd, J = 8.4, 0.8Hz, 1H), 4.20 (s, 1H), 4.16 (t, J = 6.0 Hz, 2H), 3.79 (s, 3H), 2.62-2.58 (m, 2H), 2.45 (s, 3H), 2.30 (s, 6H), 2.07-2.00 (m, 2H). MS (ES+) m / e 456.4 (M+H) + .

[0230] Example 71 N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). 1 H NMR (400 MHz, CD3OD) δ 8.52 (s, 2H), 8.04-8.0 (m, 3H), 7.42 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 6.8 Hz, 1H), 7.24-7.19 (m, 2H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 5.43 (s, 1H), 4.44-4.41 (m, 1H), 4.27-4.25 (m, 1H), 3.84 (s, 3H), 3.41-3.36 (m, 2H), 2.81 (s, 6H), 2.65 (s, 3H). MS (ES+) m / e 442.4 (M+H) + .

[0231] Example 72 (R)-N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AD column) (mobile phase: 55% EtOH 5% containing 0.1% NHOH in CO, flow rate 70 g / min) to give (R)-N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide in 99% enantiomeric purity. 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.59 (s, 1H), 9.98-9.96 (m, 1H), 9.72-9.69 (m, 1H), 8.08 (m, 2H), 7.94 (d, J = 12.4 Hz, 1H), 7.47 (s, 1H), 7.43-7.34 (m, 3H), 7.04-7.01 (m, 1H), 6.05 (t, J = 6.4 Hz, 1H), 4.47-4.34 (m, 2H), 3.79 (s, 3H), 3.58-3.49 (m, 2H), 2.83 (dd, J = 7.2, 5.2 Hz, 6H), 2.48 (s, 3H). MS (ES+) m / e 442.3 (M+H) + .

[0232] Example 73 (S)-N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide [ka] The reaction was carried out according to general procedure A. The final residue was purified by reverse-phase preparative HPLC to give N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). The enantiomers were separated by SFC (DAICEL CHIRALPAK AD column) (mobile phase: 55% EtOH containing 0.1% NHOH in CO, flow rate 70 g / min) to give (S)—N-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide in 99% enantiomeric purity. 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.59 (s, 1H), 9.97-9.94 (m, 1H), 9.71-9.68 (m, 1H), 8.08 (m, 2H), 7.94 (d, J = 12.4 Hz, 1H), 7.47 (s, 1H), 7.43-7.36 (m, 3H), 7.04-7.02 (m, 1H), 6.05 (t, J = 6.4 Hz, 1H), 4.48-4.34 (m, 2H), 3.79 (s, 3H), 3.58-3.47 (m, 2H), 2.83 (dd, J = 7.2, 5.2 Hz, 6H), 2.48 (s, 3H). MS (ES+) m / e 442.3 (M+H) + .

[0233] Example 74 1-(4-(1H-pyrazol-4-yl)phenyl)-4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1,4-diazepan-2-one [ka] Step 1: To a mixture of compound benzyl 2-(3-methoxyphenyl)-3-oxo-1,4-diazepane-1-carboxylate (1 equivalent) and compound 4-(4-iodophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (1 equivalent) in dioxane, CsCO (4 equivalents) and Xantphos-Pd-G (0.05 equivalents) were added at 20° C. under N2. The mixture was stirred at 100° C. for 16 hours. The mixture was extracted with EtOAc (100 mL × 4). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1, 1 / 1) to give the compound benzyl 2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepane-1-carboxylate (25%) as a yellow oil.

[0234] Step 2: To a solution of compound benzyl 2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepane-1-carboxylate (1 equivalent) in MeOH, dry Pd / C (0.1 equivalent) was added under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred under H (15 psi) at 20 °C for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated to give a residue. The residue was purified by preparative HPLC (FA conditions) to give compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (67%) as a white solid.

[0235] Step 3: To a mixture of compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (1 equivalent) and aldehyde (2 equivalents) in MeOH, TEA (3) was added in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 30 minutes. NaBH(OAc)3 (4 equivalents) was added to the reaction, and the reaction mixture was stirred at 25° C. for 4 hours. The mixture was poured into water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1, 1 / 1) to give the compound 4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (32%) as a colorless oil.

[0236] Step 4: To a mixture of compound 4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (1 equivalent) in DCM was added TFA at 0° C. under N2. The mixture was stirred at 0° C. for 10 minutes and the reaction mixture was warmed to 25° C. The reaction mixture was stirred at 25° C. for 0.5 hours. The reaction mixture was concentrated under reduced pressure at 45° C. and the residue was purified by preparative HPLC to give 1-(4-(1H-pyrazol-4-yl)phenyl)-4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1,4-diazepan-2-one (7%) as an off-white solid. 1H NMR (400 MHz, CD3OD) δ 7.99 (s, 2H), 7.69-7.65 (m, 2H), 7.45 (t, J = 8.0 Hz, 1H), 7.34-7.30 (m, 2H), 7.26-7.24 (m, 2H), 7.09 (dd, J = 8.8, 1.6 Hz, 1H), 5.73 (s, 1H), 4.40 (m, 1H), 3.99-3.83 (m, 6H), 3.38-3.32 (m, 1H), 3.31-3.20 (m, 1H), 2.38 (m, 2H), 1.15-1.10 (m, 1H), 0.78-0.73 (m, 2H), 0.38-0.34 (m, 2H). MS (ES+) m / e 417.3 (M+H) + .

[0237] Example 75 1-(4-(1H-pyrazol-4-yl)phenyl)-4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1,4-diazepan-2-one [ka] Step 1: To a mixture of compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (1 equivalent) and aldehyde (2 equivalents) in MeOH, TEA (3) was added in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 30 minutes. NaBH(OAc)3 (4 equivalents) was added to the reaction, and the reaction mixture was stirred at 25° C. for 4 hours. The mixture was poured into water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1, 1 / 1) to give the compound 4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (13%) as a colorless oil.

[0238] Step 2: To a mixture of compound 4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (1 equivalent) in DCM was added TFA at 0° C. under N2. The mixture was stirred at 0° C. for 10 minutes, and the reaction mixture was warmed to 25° C. The reaction mixture was stirred at 25° C. for 0.5 hours. The reaction mixture was concentrated under reduced pressure at 45° C., and the residue was purified by preparative HPLC to give 1-(4-(1H-pyrazol-4-yl)phenyl)-4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1,4-diazepan-2-one (40%) as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 8.00 (s, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.42 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.19-7.15 (m, 2H), 6.98 (dd, J = 8.0, 2.4 Hz, 1H), 5.06 (s, 1H), 3.93-3.86 (m, 4H), 3.77-3.73 (m, 1H), 3.48-3.35 (m, 4H), 3.22-3.14 (m, 2H), 2.94 (s, 3H), 2.87 (s, 3H), 2.19-2.17 (m, 1H), 1.77 (m, 1H). MS (ES+) m / e 434.3 (M+H) + .

[0239] Example 76 1-(4-(1H-pyrazol-4-yl)phenyl)-3-(3-methoxyphenyl)-4-(2-(methylamino)ethyl)-1,4-diazepan-2-one [ka] Step 1: To a mixture of compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (1 equivalent) and aldehyde (2 equivalents) in MeOH, TEA (3) was added in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 30 minutes. NaBH(OAc)3 (4 equivalents) was added to the reaction, and the reaction mixture was stirred at 25° C. for 4 hours. The mixture was poured into water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO, petroleum ether / EtOAc = 2 / 1, 1 / 1) to give the compound tert-butyl (2-(2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-1-yl)ethyl)(methyl)carbamate (77%) as an off-white oil.

[0240] Step 2: To a mixture of compound tert-butyl (2-(2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)-1,4-diazepan-1-yl)ethyl)(methyl)carbamate (1 equivalent) in DCM was added TFA at 0° C. under N2. The mixture was stirred at 0° C. for 10 minutes, and the reaction mixture was warmed to 25° C. The reaction mixture was stirred at 25° C. for 0.5 hours. The reaction mixture was concentrated under reduced pressure at 45° C., and the residue was purified by preparative HPLC to give 11-(4-(1H-pyrazol-4-yl)phenyl)-3-(3-methoxyphenyl)-4-(2-(methylamino)ethyl)-1,4-diazepan-2-one (66%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 8.00 (s, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.16-7.13 (m, 2H), 7.67 (dd, J = 8.4, 2.4 Hz, 1H), 5.03 (s, 1H), 3.90-3.87 (m, 1H), 3.84 (s, 3H), 3.73-3.66 (m, 1H), 3.39-3.34 (m, 1H), 3.23-3.08 (m, 5H), 2.73 (s, 3H), 2.17-2.14 (m, 1H), 1.71-1.68 (m, 1H). MS (ES+) m / e 420.3 (M+H) + .

[0241] Example 77 Determination of the 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 compounds was measured by Z'-lyte kinase kit (ThermoFisher Scientific) and calculated as IC 50 was calculated.

[0242] Example 78 Determination of the 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. The cells were seeded in a 96-well plate at a density of 5,000 cells / well for 24 hours, and then treated with test compounds for 90 minutes. The cells were then fixed and processed according to the In-Cell ELISA Colorimetric Detection Kit manual (Thermo Scientific).

[0243] Example 79 NIH3T3 cell Acta2-promoter-driven luciferase assay: An NIH3T3 cell line stably expressing a luciferase reporter driven by the human ACTA2 gene promoter (-1000-1bp) was established (NIH3T3-Acta2-luciferase). Cells were plated to confluence and treated with test compounds and TGFβ1 for 24 hours. Cells were then lysed, and luciferase activity was measured using the LightSwitch Luciferase Kit from Active Motif.

[0244] Example 80 Thawing and expansion of human oligodendrocyte / neuron progenitor cells (OPCs). Cryopreserved human oligodendrocyte / neuron progenitor cells (Millipore Cat. No. CS204496) were harvested and cultured in Maltrigel (BD Cat. No. 356324)-coated T-25 flasks in growth medium containing OPC / neuron basal medium, N21 medium supplement, recombinant human bFGF, PDGF-AA, and NT-3. When cells reached 80% confluence, 3 ml of Accutase (Millipore SCR005) was added at a 1:4 ratio to the passaged cells. Subcultured cells were maintained and expanded in growth medium.

[0245] Example 81 Spontaneous differentiation of human oligodendrocyte / neuron progenitor cells in 24-well and 96-well plates. Plates were coated overnight at room temperature with 10 μg / ml poly-L-ornithine (Sigma Cat. No. P4957). The poly-L-ornithine solution was removed, the plates were rinsed with 1x PBS, and 10 μg / ml laminin (Millipore Cat. No. CC095) was added and incubated at 37°C for at least 2 hours. Cells were seeded onto the coated plates in culture medium and allowed to adhere overnight. The next day, the culture medium was removed and replaced with freshly prepared spontaneous differentiation medium containing only OPC / Neuron Basal Medium and N21 Medium Supplement. The differentiation medium was changed every 3–4 days for 14 days of culture. Cells were imaged, and neurite length was analyzed every 4 hours using an IncuCyte S3 Live Cell Analysis System. After 14 days of differentiation, cells were fixed with 4% paraformaldehyde and stained with DAPI and neuron-specific β-III tubulin antibody (R&D Systems, NL1195R).

[0246] Example 82 Chondroitin sulfate proteoglycan (CSPG) assay in human oligodendrocyte / neuron progenitor cells in 96-well plates. After overnight incubation with 10 μg / ml poly-L-ornithine, 96-well plates were coated with a well-mix solution of 10 μg / ml laminin combined with 0.3 μg / ml CSPG (Millipore CC117) for at least 2 hours at room temperature. Cells were seeded onto the coated plates at a density of 6,000 cells per well in growth medium. The next day, the growth medium was replaced with fresh spontaneous differentiation medium containing test compounds. Cells were imaged, and neurite length was analyzed every 4 hours using an IncuCyte S3 Live Cell Analysis System.

[0247] Example 83 Aβ1-42-induced cell death assay in human oligodendrocyte / neuron progenitor cells in 96-well plates. Cells were seeded on poly-L-ornithine and laminin-coated plates at a density of 8,000 cells per well and maintained in growth medium until day 3. The medium was then replaced with fresh spontaneous differentiation medium containing test compound and IncuCyte Annexin V Red reagent (1:200 dilution) (Essen BioScience Cat. No. 4641). After a 30-minute preincubation period at 37°C, 10.5 μM Aβ1-42 (Bachem Product No. H-8146) was added directly to the pretreatment solution. Neurite length and total Annexin V-positive area were analyzed every 2 hours using an IncuCyte S3 Live Cell Analysis System.

[0248] Example 84 Bleomycin-induced pulmonary fibrosis in mice (therapeutic administration): Male C57B / L6 mice were house-fed and acclimated for at least 7 days. After the acclimation period, animals were randomly assigned to treatment groups. On day 0, mice were anesthetized with 5% isoflurane via inhalation for 10–15 minutes, and then their front teeth were attached to a wire attached to a stationary animal operating table. 50 μL of saline or bleomycin solution was administered intratracheally. Mice in the treatment group were intratracheally instilled with 2.0 U / kg (mg / kg) bleomycin hydrochloride (BLM) in sterile saline. Seven days after bleomycin administration, half of the animals in the vehicle group were sacrificed to establish a baseline fibrosis level at the start of treatment. Mice in the normal control group were orally administered saline once daily from days 7 to 20. Mice in the treatment group were orally administered the compound once daily from days 7 to 20. On day 20, 2-3 hours after the last dose, all mice were sacrificed. The lungs were gently irrigated with 0.8 mL of PBS containing 1% BSA and 0.6 mM EDTA via a tracheal cannula. After irrigation, lung tissue was collected from each animal. The lung tissue was divided into three sections. The major lobe of the right section was fixed in 10% neutral formalin for paraffin embedding and histopathology. The remaining lobe of the right section was snap-frozen, and protein lysates were prepared for target engagement analysis.

[0249] Example 85 Bleomycin-induced pulmonary fibrosis in mice (prophylactic administration): Male C57B / L6 mice were ear-tagged and weighed prior to the start of the study. Animals in the treatment group were treated with compound on day -1 before bleomycin administration and on all subsequent days after bleomycin administration. On day 0, animals in the disease group received a 1.5 U / kg dose of bleomycin via the oropharyngeal route. The non-bleomycin control group received sterile saline. Animals were closely monitored daily until the end of the study. On day 21 after bleomycin administration, all animals were euthanized, and blood, lungs, and bronchoalveolar lavage (BAL) fluid were collected. Blood samples were used for plasma preparation. Total leukocytes were counted in the BAL fluid, and lungs were fixed in 10% neutral buffered formalin for histopathological analysis.

[0250] Example 86 Histamine-induced vascular permeability model in mice: 7-8 week-old female Balb / c mice were acclimated for at least 3 days and randomly assigned to each group. Animals were intravenously injected with 1% Evans blue at a dose of 100 μl per mouse. 10 minutes later, animals in the disease group received 1 μg of histamine in 20 μl of PBS intradermally on the back and were sacrificed 20 minutes after administration. 20 μl of PBS was intradermally injected on the back of animals in the sham control group. Vehicle and test substances were administered by oral gavage accordingly. A positive control, salbutamol, was administered 15 minutes before Evans blue administration. 20 minutes after histamine exposure, animals were euthanized. The skin was then everted, and the diameter of the lesion was measured. The area of ​​the lesion was calculated and expressed in square millimeters. After measuring the diameter, Evans blue dye was extracted from the skin tissue, and the optical density was measured. Data are calculated against a spiked Evans Blue standard curve.

[0251] Example 87 ROCK inhibitors potently inhibited ROCK kinase activity in vitro and in cells. Compounds of the present invention inhibited ROCK in cells as measured by an A7R5 in-cell Elisa assay. A7R5 cells were treated with 9-point 2-fold serial dilutions of compounds, and ppMlc (T18 / S19) levels were determined to determine the cellular IC of the compounds.50 The values ​​were calculated and the results are shown in Table 1 below. [Table 1]

[0252] Table 2 below shows in vitro and intracellular ROCK inhibition with compounds of the present invention. Compound activity was measured using a Z'-lyte kinase kit (ThermoFisher Scientific). Percent inhibition was calculated by normalizing kinase activity values ​​obtained with 1 μM compound treatment to the DMSO control value. GraphPad Prism software was used with kinase activity data collected from 9-point serial dilutions of compound treatment to calculate IC 50 ROCK inhibition in A7R5 cells and NIH3T3 (Acta2-Luc) cells was performed as described above. [Table 2-1] [Table 2-2]

[0253] The compounds of the present invention have an alkyl substituent (optionally substituted) on the 2-amino group (i.e., R 1 is alkyl, etc.), which is particularly advantageous for compounds with lower alkyl amines at position 2 (i.e., R 1 is a lower alkyl) is associated with enhanced intracellular ROCK activity.

[0254] Example 88 The ROCK inhibitor crossed the blood-brain barrier. Brain and plasma concentrations of selected ROCK inhibitors were assessed in mice by HPLC / MS / MS at 15 minutes and 2 hours after intravenous drug administration of 2.5 mg / kg. The results are shown in Table 3 below. [Table 3]

[0255] The ROCK inhibitor of the present invention potently inhibited ROCK kinase activity in vitro and in cells.

[0256] As shown in Figure 1A, compounds of the present invention potently inhibited the activity of both ROCK isoforms in vitro at concentrations below 20 nanomolar, as measured by the Z'-Lyte kit (ThermoFisher Scientific). Percent inhibition was calculated by normalizing kinase activity values ​​obtained with compound treatment to DMSO control values ​​and then fitting them to the equation: [DMSO - (compound / DMSO)] x 100%. The nonlinear regression curve fit function of GraphPad Prism software was used with kinase activity data measured at 9-point, 2-fold serial dilutions of compound treatment to determine IC 50 was calculated.

[0257] A7R5 in-cell ELISA assay was performed to determine the ROCK inhibition of test articles. A7R5 cells were treated with 9-point 2-fold serial dilutions of compounds, and the ppMlc (T18 / S19) levels were determined to determine the cellular IC of the compounds. 50 The compounds of the present invention had an IC value of less than 200 nM. 50 See Figure 1B. A7R5 cells were treated with 9-point 2-fold serial dilutions of compounds, and the ppMlc (T18 / S19) levels were determined to calculate the cellular IC of the compounds using the nonlinear regression curve fit function in GraphPad Prism software. 50 values ​​were calculated.

[0258] ROCK inhibitors can be effectively ranked by ACTA2-promoter-driven luciferase reporter cell assay. NIH3T3 cells stably expressing ACTA2-promoter-driven luciferase were used to determine the functional IC of ROCK compounds in cells. 50Cells were plated to confluence in 96-well plates and treated with 9-point serial dilutions of compounds in combination with TGFβ1 for 24 hours. Luciferase activity was measured and the IC values ​​of compounds were calculated using the nonlinear regression curve fit function in GraphPad Prism software. 50 The compounds of the present invention exhibited an IC of less than 200 nM in cells. 50 See Figure 1C.

[0259] Compound ROCK activity was tested in additional cell types. Svec4-10 mouse endothelial cells were treated with the compound of Example 2 for 120 minutes, and ppMlc (T18 / S19) and pMypt (T853) levels were visualized by Western blotting. At 110 nM, Example 2 effectively blocked phosphorylation of the ROCK targets MLC and MYPT1. See Figure ID.

[0260] Example 89 ROCK inhibitors promote neurite outgrowth and neuronal differentiation Neuronal development requires highly coordinated regulation of the cytoskeleton. Activation of the RhoA / ROCK pathway leads to cytoskeletal changes, such as increased actin filament mobility and decreased actin depolymerization, ultimately resulting in growth cone retraction, which then inhibits neurite growth and elongation. Here, we demonstrate that ROCK inhibitors promote neuronal sprouting and neurite outgrowth in a dose-dependent manner. Figures 2A and 2B demonstrate that the compounds of Examples 2 and 17 significantly increase neurite length in cultures of human oligodendrocytes / neuronal progenitor cells at various concentrations. Examples 2 and 17 also promote neuronal differentiation and maturation. See Figures 2C and 2D.

[0261] ROCK inhibitors block the inhibitory effects of chondroitin sulfate proteoglycans (CSPGs) on neurite outgrowth. Mature mammalian CNS neurons do not spontaneously regenerate after traumatic injury or neurodegeneration. This lack of regeneration has been attributed to the low intrinsic regenerative capacity of both mature neurons and the inhibitory tissue environment. The main obstacles in the appropriate environment are chondroitin sulfate proteoglycans (CSPGs) secreted by reactive astrocytes and myelin-associated inhibitory factors produced by oligodendrocytes. These glial cell-derived growth inhibitory molecules are upregulated after neuronal injury and converge downstream of the RhoA / ROCK signaling pathway, blocking neuronal regeneration. Promotion of regeneration requires inactivation of growth inhibitors from the tissue environment. Herein, we demonstrate that blocking the Rho / ROCK pathway reversed the glial growth inhibitor signaling mediated by CSPGs on neurite outgrowth. Figure 2E shows a representative photograph at day 3. As shown in Figures 2F and 2G, both 1 μM and 3 μM of Example 2 and Example 17 significantly antagonized the neurite outgrowth arrest mediated by 0.3 mg / ml of CSPG in human oligodendrocyte / neuron progenitor cells. Statistical analysis at 72 hours was performed by ANOVA.

[0262] Example 90 ROCK inhibitors protect neurons from Aβ1-42-induced cell death. In addition to promoting neural regeneration, ROCK inhibitors have been reported to exert neuroprotective effects and prolong neuronal survival. Here, we test the neuroprotective effects of ROCK inhibitors against Aβ1-42. Human oligodendrocyte / neuronal progenitor cells were preincubated with ROCK inhibitor for 30 minutes and then exposed to 10.5 μM Aβ1-42. Incucyte Annexin V Red reagent was added to the culture medium to label cell apoptosis. Photographs were taken using an Incucyte S3, and the total Annexin V-positive area was analyzed using Incucyte S3-based software. A representative photograph is shown in Figure 3A. Neurite length was quantified using the Incucyte NeuroTrack Software Module. See Figures 3B and 3C. Figures 3E and 3F show statistical analysis of Annexin V area and neurite length at 24 hours, demonstrating that 3 μM of Examples 2 and 17 significantly protected against Aβ1-42-induced cell death, and that 3 μM of Example 17 promoted neurite outgrowth in the presence of Aβ1-42.

[0263] Example 91 The ROCK inhibitor of the present invention suppressed pro-fibrotic gene expression Expression of α-smooth muscle actin (αSMA, or Acta2) by fibroblasts is an important marker of transdifferentiation from fibroblasts to profibrotic myofibroblasts, as exemplified by increased expression of fibrogenic factors. Such profibrotic factors, including TGFβ signaling, result in αSMA expression and contribute to sustained myofibroblast differentiation, proliferation, and survival. As shown in Figure 4A, TGFβ1-induced αSMA expression was significantly reduced by ROCK inhibitor. NIH3T3 cells were treated with TGFβ1 in combination with ROCK inhibitor for 24 hours, and mRNA expression was quantified by Taqman qPCR.

[0264] In addition to expressing αSMA, myofibroblasts isolated from the lungs of IPF patients exhibit senescent characteristics. They are resistant to apoptosis and highly metabolically active, continuously producing large amounts of ECM proteins to drive fibrosis development. CCN1 (or CYR61) and CTGF belong to this class of proteins. Treatment of human lung fibroblasts with a ROCK inhibitor under TGFβ induction significantly inhibited the expression of CTGF and CCN1 (see Figures 4B and C). Secreted CCN1 protein was also reduced by ROCK inhibition, as demonstrated by cell culture medium ELISA assays as shown in Figure 4D. CCD18Lu fibroblasts were treated with a ROCK inhibitor in combination with TGFβ for 40 hours. CCN1 content was measured using a human CCN1 ELISA kit from R&D Systems. These results suggest that a ROCK inhibitor could suppress pro-survival and pro-senescent signals in myofibroblasts under fibrotic conditions.

[0265] Example 92 ROCK inhibitors are effective in reducing multiple fibrotic indices in a bleomycin-induced pulmonary fibrosis model in mice Based on the potent anti-fibrotic cell activity of the ROCK inhibitors of the present invention, the ability of such inhibitors to reduce fibrosis in vivo was evaluated in multiple mouse models of fibrotic diseases, including bleomycin-induced pulmonary fibrosis. Following intratracheal instillation of bleomycin, mice were orally administered ROCK inhibitors at doses of 3 mg / kg, 10 mg / kg, or 30 mg / kg. Control animals received vehicle alone on the same once-daily dosing schedule. After 20 days of treatment, animals were sacrificed, and lungs were removed, weighed, and fixed for histopathological evaluation. Figure 5A shows representative images of Masson's trichrome-stained lung sections from each treatment group, taken under a 10x objective. In the treatment groups, fibrotic tissue formation (arrowheads), enlarged alveolar septa, and nodule-like formations (arrows) were still present, all of which were less severe than in bleomycin-induced controls. (B = bronchioles). In this model, all ROCK inhibitors improved lung structure and collagen deposition in a dose-dependent manner, with mice receiving doses of 10 mg / kg or higher showing clear, statistically significant improvements.

[0266] Improvements in total lung fibrosis, α-smooth muscle actin (αSMA)-positive area percentage, and total white blood cell count in bronchoalveolar lavage fluid were quantified by applying the Ashcoft scoring criteria to histopathology images, IHC quantification, and flow cytometry, respectively. Administration of 10 mg / kg or more of a ROCK inhibitor resulted in significantly less fibrosis and lower inflammatory leukocyte infiltration compared with vehicle-treated controls, as indicated by lower group mean fibrosis index, αSMA-positive area, and total white blood cell count in bronchoalveolar lavage fluid. See Figure 5B.

[0267] Example 93 When administered therapeutically, ROCK inhibition is effective in reducing fibrosis scores in a mouse model of bleomycin-induced pulmonary fibrosis. The activity of ROCK inhibitors in attenuating lung fibrosis in a therapeutic setting was evaluated in a bleomycin-induced model after intratracheal bleomycin instillation, which induces lung tissue damage and subsequent pulmonary fibrosis. Mice were treated with compound or vehicle control 7 days after bleomycin instillation, allowing fibrosis to establish before treatment began. All mice were sacrificed on day 20, and lungs were harvested. The right lung lobe was fixed in 10% neutral formalin, sectioned, and stained with Masson's trichrome staining. A clear anti-fibrotic effect was observed in the 10 mg / kg and 30 mg / kg ROCK inhibitor-treated groups, as evidenced by lower collagen content and reduced Masson's trichrome staining scores. See Figure 6A.

[0268] The improvement in total lung fibrosis was quantified for each animal in the control and treatment groups. See Figure 6B. Masson's trichrome-stained lung sections from individual mice are shown, along with the group mean and standard error of the mean (SEM). Normal control mice received a score of 0. Mice treated with the compound of Example 2 had significantly lower lung collagen content.

[0269] Example 94 ROCK inhibition stabilizes endothelial barrier function in a histamine-induced vascular permeability model in mice In addition to damage to organ epithelial cells, damage also occurs to endothelial cells in fibrotic diseases, leading to endothelial barrier destabilization and capillary leakage, both of which are characteristic of fibrotic tissue. The ROCK signaling pathway is upregulated in damaged endothelial cells, leading to further barrier destabilization and vascular leakage. The involvement of ROCK in inducing vascular leakage led us to hypothesize that administration of a ROCK inhibitor could stabilize such vascular barrier function in vivo. We tested the efficacy of a blocking ROCK inhibitor after single-dose administration in a mouse model of histamine-induced cutaneous vascular leakage. In this model, intradermal injection of histamine rapidly induced Evans blue dye extravasation at the site of histamine administration. Administration of the ROCK inhibitor of Example 2 1 hour before histamine injection dose-dependently blocked histamine-induced vascular permeability, suggesting that in addition to its antifibrotic activity, the ROCK inhibitors of the present invention may also be useful in diseases associated with induced vascular permeability. Figure 7A shows representative images of histamine-induced Evans blue dye extravasation.

[0270] The degree of compound efficacy was quantified by calculating both the total skin spot area and the amount of Evans blue dye that penetrated the skin.Twenty minutes after histamine exposure, the animals were euthanized and the lesion diameter was measured.The area of ​​the lesion was calculated and expressed in square millimeters.After measuring the diameter, Evans blue dye was extracted from the skin tissue and the optical density was measured.Data was again calculated against a spiked Evans blue standard curve. ** p<0.01 (vs. vehicle group; using one-way ANOVA followed by Dunnett's post-hoc test). See Figure 7B.

[0271] Bibliography: 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,E.E.,Newey,S.E.,and Van Aelst,L.(2005).The role of the Rho GTPases in neuronal development.Genes Dev 19,1-49. Li,M.,Huang,Y.,Ma,A.A.,Lin,E.,and Diamond,M.I.(2009).Y-27632 improves rotarod performance and reduces huntingtin levels in R6 / 2 mice.Neurobiol Dis 36,413-420. Linseman,D.A.,and Loucks,F.A.(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 VI: 【Chemistry 1】 [In the formula, R1 is selected from the group consisting of R10 R11 N-C(=O)-(CR12 R13) c-; Each R10 is independently selected from H and methyl; Each R11 is independently selected from H and methyl; Each R 12 is H; Each R 13 is H; c is 1; Each R ２２ Each is independently selected from lower alkoxy or lower alkyl; n is between 1 and 2. or a salt of which is permitted as a medicine.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 22 is a lower alkoxy group and n is 1.

3. The compound is as follows: 【Chemistry 2】 A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the above.

4. A pharmaceutical composition comprising a therapeutically effective amount of the compound described in any one of claims 1 to 3, and a pharmaceutically acceptable carrier or additive.

5. The pharmaceutical composition according to claim 4 for treating fibrosis in a subject.

6. The pharmaceutical composition according to claim 5, wherein the fibrosis is pulmonary fibrosis, cystic or idiopathic pulmonary fibrosis, radiation-induced lung injury, hepatic fibrosis, cirrhosis, cardiac fibrosis, arterial fibrosis, endocardial fibrosis, former myocardial infarction, arteriosclerosis, atherosclerosis, restenosis, articular fibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive widespread fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, mediastinal fibrosis, keloid or hypertrophic scar, glial scar, or renal fibrosis.

7. The pharmaceutical composition according to claim 5, wherein the fibrosis is idiopathic pulmonary fibrosis.

8. The pharmaceutical composition according to claim 4 for treating central nervous system disorders in a subject.

9. The pharmaceutical composition according to claim 8, wherein the central nervous system disorder is 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, neurodegeneration, spinal cord injury, cerebral vasospasm, or multiple sclerosis.

10. The pharmaceutical composition according to claim 4 for treating inflammation or glaucoma in a subject.

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