Inhibitors of rho associated coiled-coil containing protein kinase

Abstract

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

Description

Technical Field

[0001] The present invention relates to inhibitors of ROCK1 and / or ROCK2. Also provided are methods of inhibiting ROCK1 and / or ROCK2 useful for treating diseases.

Background Art

[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, when activated, phosphorylate various downstream substrates. ROCK plays an important role in a number of cellular processes including smooth muscle cell contraction, cell proliferation, adhesion and migration. Thus, ROCK inhibitors have potential therapeutic applicability in a wide variety of pathological conditions including, for example, asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, renal failure, pulmonary hypertension, neurodegeneration, and osteoporosis.

[0003] ROCK is an important intracellular regulator of cytoskeletal dynamics and cell motility. ROCK regulates some downstream targets of RhoA via phosphorylation, including, for example, myosin light chain, myosin light chain phosphatase binding subunit and LIM kinase 2. These substrates regulate the organization and contractility of actin filaments. In smooth muscle cells, ROCK mediates calcium sensitization and smooth muscle contraction. Inhibition of Rho kinase blocks 5-HT and phenylephrine agonist-induced muscle contraction. When introduced into non-smooth muscle cells, ROCK induces stress fiber formation and is required for RhoA-mediated cell 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 transport system activation, stress fiber formation, adducin activation, and physiological processes such as vascular stenosis, bronchial smooth muscle contraction, vascular smooth muscle and endothelial cell proliferation, platelet aggregation, and the like.

[0004] Inhibition of ROCK activity in animal models has shown several advantages of inhibiting ROCK for treating human diseases. These include cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, central nervous system disorders such as neurodegeneration and spinal cord injury, and models of 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. Inhibiting ROCK activity in patients has advantages for the control of cerebral vasospasm and ischemia after subarachnoid hemorrhage, reduction of intraocular pressure, increased aqueous humor outflow due to relaxation of the trabecular meshwork tissue, improvement of blood flow to the optic nerve, and protection of healthy ganglion cells.

[0005] A significant amount of in vivo data has been generated focusing on the activity of ROCK in the CNS. Aberrant activation of the ROCK pathway has been documented in many disorders of the central nervous system. For example, axonal growth and synaptic plasticity are influenced by the structural regulation of the actin cytoskeleton. The Rho-ROCK cascade plays a central role in synaptic plasticity, in both dendritic spine morphogenesis and stability, and even in the motility and collapse of growth cones. In addition, since multiple axonal growth inhibitory molecules converge on neuronal RhoA / ROCK, this represents an attractive pathway for intervening in CNS disorders.

[0006] Nogo receptors (NgR) (along with 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 the upregulation of RhoA and ROCK. These events lead to increased contractility and have a strong inhibitory effect on axonal growth in mature neurons. Thus, the ability to inhibit this signaling cascade provides a very promising therapeutic strategy in spinal cord and optic nerve injury. Neurodegenerative conditions such as Huntington's disease and Alzheimer's (AD) disease are also being investigated as responding to inhibition of NgR signaling. Not only are NgR family members associated with APP processing, but the intracellular localization of NgR and Nogo also changes in the AD brain.

[0007] Alzheimer's disease (AD), the most common cause of dementia in the elderly, is a progressive neurodegenerative disorder that is associated with a gradual decline in many cognitive functions, including memory impairment (Selkoe, 2001). In the AD pathology, synaptic loss is commonly observed and is a prominent feature of synaptic dysfunction in AD (Tanzi and Bertram, 2005). Oligomerized β-amyloid peptide has been implicated in the loss of synaptic plasticity and neural circuit dysfunction. Synaptic plasticity is governed by the structural regulation of the actin cytoskeleton in dendritic spines. The Rho-ROCK cascade plays a central role in synaptic plasticity, in both dendritic morphogenesis and stability, and furthermore in the motility and collapse of growth cones (Govek et al., 2005; Linseman and Loucks, 2008). Several studies have demonstrated that ROCK kinase can induce the production of toxic β-amyloid peptide and that inhibition of ROCK can inhibit toxic peptide processing. In a feed-forward mechanism, β-amyloid increases Rho GTPase activity, which inhibits neurite outgrowth and synapse formation via ROCK activation (Petratos et al., 2008). Therefore, ROCK inhibitors may have the potential to prevent synaptic and neuronal degeneration in AD and furthermore promote the regeneration process. A recent study by Herskowitz et al. showed that knockdown of ROCK decreased aβ levels. These effects demonstrate that a highly ROCK-selective inhibitor is required to provide an effective treatment for Alzheimer's disease (AD). To demonstrate the use of ROCK inhibition for AD, the model compound SR3677 was tested in a rodent model of AD by changes in BACE-1 distribution and amyloid precursor protein (APP) transport to lysosomes. SR3677 had a promising effect of reducing sAPPβ after direct intraparenchymal (i.p.) injection into the hippocampus due to its poor oral PK properties (5% F and a half-life of less than 1 hour) and lack of brain penetration.

[0008] Huntington's disease (HD) is a destructive, incurable, mainly hereditary neurodegenerative disease characterized by psychiatric disorders, movement disorders, and dementia. The misfolding and aggregation of the Htt protein, the product of the huntingtin gene, result in HD pathology (Shao and Diamond, 2007). To treat HD, a very small number of mechanism-based therapeutic lead compounds have been developed. Although scientific investigations are still ongoing, several lines of evidence suggest that ROCK inhibition could be an effective treatment for HD. In a mouse model of HD, ROCK inhibition significantly reduced soluble Htt levels, reversed aggregate formation, neurite retraction, and was protective against neuronal death (Deyts et al., 2009; Li et al., 2009). Similar results were obtained in studies in Drosophila, where Htt aggregation was controlled by inhibition of ROCK (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 (ALS). See, for example, Tonges, L., et al. (2012). “Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson’s disease.” Brain. 135(11):3355-70.

[0010] ROCK phosphorylates multiple downstream substrates, including myosin light chain (MLC, at threonine 18 and serine 19) and myosin light chain phosphatase (MYPT1, at threonine 853), driving the polymerization of globular G-actin into filamentous F-actin and assembling the actomyosin contractile machinery. It has been recognized that this pathway may contribute to the etiology of several CNS disorders, such as spinal cord injury, stroke, and AD. In the adult CNS, damaged 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), restrict axon regeneration in the damaged brain and spinal cord. A common mechanism among various myelin-associated inhibitors is that they all activate Rho and its downstream effector kinase ROCK to inhibit neurite outgrowth.

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

[0012] In one aspect, the present invention provides a compound of formula I:

Chemical formula

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

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

[0015] In one aspect, the present invention provides a method of inhibiting ROCK in a mammal, comprising administering an effective amount of one or more compounds of Formula I. The present invention provides a method of 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 ROCK1 and / or ROCK2. In certain such embodiments, the compound of Formula I selectively inhibits ROCK1 and / or ROCK2. Non-limiting diseases and conditions treated according to the present invention include central nervous system disorders, such as neurodegeneration and spinal cord injury, cardiovascular diseases, such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, arterial thrombotic disorders, such as platelet aggregation and leukocyte aggregation, asthma, regulation of intraocular pressure, and bone resorption. In neoplasia, inhibition of Rho kinase inhibits tumor cell growth and metastasis, and angiogenesis.

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

[0017] The present invention provides a method for treating an autoimmune disorder in a subject, the method 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, the method 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 for treating inflammation in a subject, the method 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, kidney inflammation or atherosclerosis.

[0020] The present invention provides a method for treating an arterial thrombotic disorder in a subject, the method 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 for treating a fibrotic disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I. Non-limiting examples of fibrotic disorders include pulmonary fibrosis including cystic and idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis including cirrhosis, cardiac fibrosis including arterial fibrosis, endomyocardial fibrosis, old myocardial infarction, arterial stiffness, atherosclerosis, restenosis, joint 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 scar, glial scar, or kidney fibrosis.

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

[0023] The present invention provides a method for treating glaucoma or regulating intraocular pressure in a subject, which comprises 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 for treating a neoplastic disease in a subject, which comprises 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, glioblastoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, 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, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, or head and neck cancer.

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

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

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

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0029] ROCK inhibitor The compounds according to the present invention include compounds having the formula I:

Chemical Formula

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

Chemical formula

[0031] In certain embodiments of the invention, the compound of formula III:

Chemical Formula

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

[0033] In certain embodiments of the invention, a compound of formula V:

Chemical formula

[0034] In certain embodiments of the invention, a compound of formula VI:

Chemical formula

[0035] In certain embodiments of the present invention, a compound of formula VII:

Chemical formula

[0036] In certain embodiments of the present invention, a compound of formula VIII:

Chemical formula

[0037] In certain embodiments of the present invention, a compound of Formula IX:

Chemical formula

[0038] In preferred embodiments for Formulas I to IX, R 1 is selected to be lower alkyl. More preferably, R 1 is C 1 ~C 3 alkyl, and even more preferably, R 1 is methyl or ethyl.

[0039] The term "alkyl" refers to a radical of a saturated aliphatic group including a straight-chain alkyl group, a branched-chain alkyl group, a cycloalkyl (alicyclic) group, an alkyl-substituted cycloalkyl group, and a cycloalkyl-substituted alkyl group. In a preferred embodiment, the straight-chain or branched-chain alkyl has 8 or fewer carbon atoms in its main chain (e.g., C1-C8 for straight-chain and C3-C8 for branched-chain), more preferably 6 or fewer. Similarly, preferred cycloalkyls have 3-8 carbon atoms in their ring structures, more preferably 3-6 carbons in the ring structure.

[0040] Unless otherwise specified as to the number of carbons, "lower alkyl" as used herein is as defined above, but means an alkyl group having 1-4 carbons, more preferably 1-3 carbon atoms. In a preferred embodiment, the substituents shown herein as alkyl are lower alkyl. Lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, and tert-butyl.

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

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

[0043] "Substituted lower alkyl" is as defined above and refers to a lower alkyl group having 1-3 substituents. The substituents are selected from the group consisting of halo, hydroxy, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy and carboxyl.

[0044] "Substituted C3 ~C 6 "Substituted cycloalkyl" such as "cycloalkyl" is as defined above and refers to a cycloalkyl group having 1 to 3 substituents. The substituents are selected from the group consisting of halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy and carboxyl.

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

[0046] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group as defined above bonded 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 lower alkyl is bonded through an oxygen atom, where the "lower alkyl" moiety is as defined above.

[0047] The terms "amine" and "amino" refer to both unsubstituted and substituted amines, for example, of the general formula:

Chemical formula

[0048] As used herein, the term "aryl" includes 5- and 6-membered monocyclic aromatic groups which may contain 0 to 4 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 may also be 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 (the rings are "fused rings") in which two or more carbons are shared between two adjacent rings and at least one of the rings is aromatic.

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

[0050] The term "heterocycle" or "heterocyclyl" refers to a non-aromatic heterocycle having 4 to 7 ring atoms and containing 1 to 3 ring heteroatoms.

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

[0052] As used herein, each expression that occurs more than once in any structure, such as alkyl, m, n, R 1 , R 2 etc. are intended to be independent of their definitions at other locations in the same structure.

[0053] "Substituted" or "substituted with" is understood to include the implicit conditions that such substitution follows the acceptable valences of the atoms being substituted and the substituents, and that the substitution results in a stable compound, i.e., a stable compound that does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, etc.

[0054] As used herein, the term "substituted (substitued with)" is intended to include all acceptable substituents of organic compounds. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Examples of substituents include, for example, those described hereinabove. Acceptable substituents can be one or more and can be the same or different for appropriate organic compounds. For the purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any acceptable substituents of the organic compounds described herein that satisfy the valences of the heteroatoms. It is not intended that the present invention be limited by acceptable substituents of organic compounds.

[0055] As used herein, the expression "protecting group" means a temporary substituent that protects a potentially reactive functional group from unwanted chemical transformation. 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 investigated (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

[0056] Certain compounds of the present invention may exist, in particular, in 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 that fall within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included in the present invention. The present invention also contemplates substitution of isotopes of atoms in the compounds, for example, deuterium for hydrogen.

[0057] In one aspect, the present invention provides compounds of formulas I - IX that are inhibitors of ROCK. ROCK exists in two forms, namely, ROCK1 (ROCKβ; p160 - ROCK) and ROCK2 (ROCKα). In some embodiments, the compounds of formulas I - IX selectively inhibit ROCK1. In some embodiments, the compounds of formulas I - IX selectively inhibit ROCK2. In some embodiments, the compounds of formulas I - IX are non - selective with respect to the inhibition of ROCK1 and ROCK2. In the context of the present invention, selective means that the inhibitor has an IC 50 that is at least 2 - fold, at least 5 - fold, at least 10 - fold, or at least 25 - fold lower for one kinase compared to the IC 50 shown.

[0058] Methods for determining kinase inhibition are known in the art. For example, the kinase activity and inhibitory ability of an enzyme 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 the binding of the peptide to a trivalent metal-based nanoparticle via a specific high-affinity interaction between the phosphate group and the trivalent metal. Proximity to the nanoparticle results in an increase in fluorescence polarization. Inhibition of the kinase by a kinase inhibitor blocks phosphorylation of the substrate, thereby limiting the binding of the fluorescently labeled substrate to the nanoparticle. Such an assay can be compatible with a microwell assay format and allows for the simultaneous determination of the IC 50 of a number of compounds.

[0059] Methods of treating a disease In one aspect of the 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 invention. As used herein, the phrase "therapeutically effective amount" means an amount of a compound, substance, or composition comprising a compound of the invention effective to produce some desired therapeutic effect in at least a subpopulation of cells of an animal, with a reasonable benefit / risk ratio applicable to any medical treatment, e.g., with reasonable side effects applicable to any medical treatment.

[0060] CNS disorder Compounds of Formulas I - IX exhibit effective blood - brain barrier (BBB) penetration and distribution into central nervous system tissues. Thus, the compounds of the present invention are useful for treating central nervous system disorders and also certain eye disorders that benefit from the ability to cross the BBB. Such disorders include, but are not limited to, Huntington's disease, Parkinson's disease, Alzheimer's, 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, and can be involved in neurodegeneration or physical damage to neural tissue.

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

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

[0063] Glaucoma In one embodiment of the present invention, the compounds of Formulas I-IX are used for treating glaucoma. The two most common types of primary open-angle glaucoma and acute angle-closure glaucoma are characterized by high intraocular pressure. Pigmentary glaucoma and congenital glaucoma are also characterized by reduced fluid outflow and high intraocular pressure (IOP). Normal-tension glaucoma is thought to be due to another mechanism, particularly inadequate blood flow to the optic nerve. Secondary glaucoma can result from injury, infection, inflammation, tumors or cataracts, and is also associated with long-term use of steroids, systemic hypertension, diabetic retinopathy, and central retinal vein occlusion. Glaucoma with an angiogenesis component may benefit from administration of an angiogenesis inhibitor in addition to a ROCK inhibitor.

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

[0065] autoimmune disorder The present invention provides a method of treating an autoimmune disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I-IX. 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 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 neuronal neuropathies, Barlow'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 osteomyelitis (Chronic recurrent multifocalosteomyelitis) (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan 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 syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto encephalopathy, Hashimoto thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing diseases, immunomodulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonnage-Turner syndrome (Parsonnage-Turnersyndrome), pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and III polyglandular autoimmune syndrome, polymyalgia rheumatica, polymyositis, post myocardial infarction syndrome, post pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, erythroleukemia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous skin diseases, and vitiligo are included.

[0066] According to the present invention, targeting of Th17 (IL-17 secreting) cells by ROCK inhibition provides a method for treating, in humans, but not limited to, Th17 cell-mediated diseases including autoimmune disorders such as RA, MS, SLE, psoriasis, and Crohn's disease, and GVHD. In one embodiment of the invention, the ROCK inhibitor is a compound of formula I.

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

[0068] Neoplastic diseases The ROCK inhibitor of the present invention inhibits the proliferation and metastasis of tumor cells, as well as angiogenesis, and is useful for treating neoplastic diseases. Neoplastic diseases include any malignant growth or tumor resulting from abnormal or uncontrolled cell division, which can spread to other parts of the body via the lymphatic system or bloodstream. Neoplastic diseases include, but are not limited to, lymphoma (a neoplasm of lymphatic tissue, usually malignant), carcinoma (any malignant tumor derived from epithelial tissue), leukemia (a malignant neoplasm of hematopoietic tissue characterized by abnormal proliferation of white blood cells), sarcoma (a usually malignant tumor arising from connective tissue such as bone or muscle), and blastoma (a malignant lesion in precursor cells). Non-limiting examples include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, glioblastoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, 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, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancers.

[0069] Weight gain / loss According to the present invention, the ROCK inhibitor is used to cause weight loss and / or limit weight gain. The ROCK inhibitor promotes weight loss in normal subjects and limits weight gain in subjects prone to obesity.

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

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

[0072] Thus, in such an embodiment, the disease or disorder is AMD, and an effective amount of a ROCK inhibitor is administered to a subject in need of treatment for AMD. In another embodiment, a ROCK inhibitor and an angiogenesis inhibitor are administered to the subject in an effective amount for treating AMD. In some embodiments, the angiogenesis inhibitor is a VEGFR2 antagonist. In certain such embodiments, the VEGFR2 antagonist binds to VEGF. In other such embodiments, the VEGFR2 antagonist binds to VEGFR2. Such VEGFR2-binding inhibitors include, but are not limited to, agents that bind to the extracellular domain of VEGFR2, including antibodies and their VEGFR2-binding fragments, as well as agents that interact with the intracellular domain of VEGFR2 and block the activation of VEGFR2-dependent signaling. The VEGFR2 antagonist further includes agents that interact with other cellular components to block VEGFR2-dependent signaling. In other embodiments of the present invention, other eye diseases and disorders having vascular-derived components as shown above are similarly treated.

[0073] 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. For example, such conditions associated with angiogenesis and / or inflammation include atherosclerosis, rheumatoid arthritis (RA), hemangioma, angiofibroma, and psoriasis. Other non-limiting examples of vascular-derived diseases are retinopathy of prematurity (retrolental fibroplasia), corneal graft rejection, corneal angiogenesis associated with complications of refractive surgery, corneal angiogenesis associated with contact lens complications, corneal angiogenesis associated with pterygium and recurrent pterygium, corneal ulcer disease, and non-specific ocular surface diseases, 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-type hypersensitivity, inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis, cognitive impairment induced by neuronal inflammation, Osler-Weber syndrome, restenosis, and fungal, parasitic, and viral infections including cytomegalovirus infection.

[0074] The present invention provides a pan-ROCK inhibitor (i.e., a compound that inhibits ROCK1 and ROCK2). A study observed that ROCK2 is often overexpressed in hepatocellular carcinoma compared to non-tumor liver, while ROCK1 expression remains unchanged. Other cancers that may benefit from treatment with a ROCK2-selective inhibitor include, but are not limited to, colon and bladder cancers. In contrast, ROCK1 expression levels have been observed to be higher in breast tumors. Any cancer can be tested to determine whether overexpression of ROCK1 and / or ROCK2 is present and treated accordingly. In certain situations, the ROCK1 and ROCK2 isoforms show similarity in the regulation of certain downstream targets and neither isoform is dominant. In such cases, a pan-ROCK inhibitor may be preferred.

[0075] Combination with other agents The compounds of the present invention can advantageously be administered to a patient 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. By sequentially, it is meant that one agent is administered over a period of time and subsequently the other agent is administered, which means that it may be after the administration of the first agent. When the agents are administered sequentially, the level of one agent need not be maintained at a therapeutically effective level when the second agent is administered, and vice versa. By concomitantly, it is meant that the first and second agents are administered according to a schedule in which the agents are not administered simultaneously, but both agents are maintained at substantially therapeutically effective levels. Each agent can be administered in a single or multiple administrations, and the dosage can be administered according to any schedule including, but not limited to, twice a day, daily, weekly, every two weeks, and monthly.

[0076] The present invention also includes adjunctive administration. Adjunctive administration means that a second agent is administered to a patient in addition to a first agent that is already being administered to treat a disease or disease symptom. In some embodiments, adjunctive administration involves administering the second agent to a patient in whom the administration of the first agent has not sufficiently treated the disease or disease symptom. In other embodiments, adjunctive administration involves administering the second agent to a patient in whom the disease is effectively treated by the administration of the first agent, but adjunctive treatment is predicted to improve the treatment outcome. In some embodiments, the effects of the administration of the first and second agents are synergistic. In some embodiments, the administration of the first and second agents prevents recurrence or prolongs the time to recurrence as compared to the administration of either agent alone. In some embodiments, the administration of the first and second agents allows for a reduction in the dosage and / or frequency of administration of the first and second agents.

[0077] Anti-inflammatory and immunosuppressive agents that can be administered in combination with the compounds of the present invention include steroid drugs, such as glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus), cyclosporine, fingolimod, interferons, such as IFNβ or IFNγ, tumor necrosis factor alpha (TNF-α) binding proteins, such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira), mycophenolic acid, MMF, methotrexate, NSAID, statin, sirolimus / 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 (tofacitinib), and Otezla (apremilast).

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

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

[0080] Targeted small molecules and biological molecules include, but are not limited to, inhibitors of components of signal transduction pathways, such as modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, as well as agents that bind to tumor-specific antigens. Examples include inhibitors of epidermal growth factor receptor (EGFR) such as gefitinib, erlotinib, and cetuximab; inhibitors of HER2 (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DM1; T-DM1) and pertuzumab); anti-VEGF antibodies and fragments (e.g., bevacizumab); antibodies that inhibit CD20 (e.g., rituximab, ibritumomab); anti-VEGFR antibodies (e.g., ramucirumab (IMC-1121B), IMC-1C11, and CDP791); anti-PDGFR antibodies; and imatinib. Small molecule kinase inhibitors may be specific for 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 kinase involved in pathways that result in tumor non-responsiveness to certain TKIs. In one embodiment of the invention, the treatment of a subject in need comprises administration of a ROCK inhibitor of Formula I-IX and administration of KD-019.

[0081] Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orally bioavailable ATP-site competitive Src inhibitor. Dasatinib has been approved by the FDA for use in patients with Bcr-Abl (chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)), and also targets 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).

[0082] According to the present invention, an angiogenesis inhibitor can be administered to a subject in combination with the compound of the present invention. The angiogenesis inhibitor includes any substance that inhibits the growth of new blood vessels. For example, the angiogenesis inhibitor includes antagonists of VEGF, PlGF, and VEGF receptors, including the antibodies disclosed herein. A VEGF antagonist reduces or blocks the functions associated with VEGF in cells. A VEGF antagonist can act on VEGF by binding to VEGF and blocking its binding to the receptor, and / or can act on another cellular component involved in VEGF-mediated signal transduction. Similarly, a VEGFR2 antagonist is an agent that reduces or blocks VEGFR2-mediated signal transduction by blocking the binding to VEGFR2 and ligand binding or the interaction with VEGFR2 substrates, or acts on another cellular component to reduce or block VEGFR2-mediated signal transduction. Thus, the angiogenesis inhibitor includes anti-VEGFR2 antibodies, and includes, but is not limited to, antagonists of VEGF, VEGFR1, VEGFR2, PDGF, PDGFR-β, neuropilin-1 (NRP1), and complement.

[0083] Angiogenesis inhibitors include, for example, VEGF, PDGF, ligands of VEGF or PDGF receptors, or intracellular agents that block signal transduction mediated by the 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 and FGFR-1 and -2 kinases in the nanomolar range. 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 signal transduction via the growth factor receptors VEGFR and PDGFR.

[0084] Pharmaceutical composition In one aspect, the present invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more of the compounds of Formulas I-IX formulated together with one or more pharmaceutical additives. As follows, the pharmaceutical compositions of the present invention are (1) for oral administration (e.g., drinking medicine (aqueous or non-aqueous liquid or suspension), tablets, e.g., for buccal, sublingual, and systemic absorption purposes, bolus agents, powders, granules, pastes for application to the tongue), (2) parenteral administration (e.g., by subcutaneous, intramuscular, intravenous, or epidural injection) (e.g., as a sterile liquid or suspension, or as a sustained release formulation), (3) topical application (e.g., as a cream, ointment, or controlled release patch or spray applied to the skin), (4) intravaginal or rectal (e.g., as a vaginal suppository, cream, or foam), (5) sublingual, (6) intraocular, (7) transdermal, or (8) adapted for nasal, and can be specially formulated for administration as a solid or a liquid, including those adapted for.

[0085] As used herein, the expression "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment, with reasonable benefit / risk ratios, and without undue toxicity, irritation, allergic response, or other problems or complications.

[0086] As used herein, the expression "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or vehicle involved in transporting or conveying the compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid diluent, excipient, additive, manufacturing aid (e.g., lubricant, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not being harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose, (2) starches such as corn starch and potato starch, (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, (4) tragacanth powder, (5) malt, (6) gelatin, (7) talc, (8) additives such as cocoa butter and suppository wax, (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffering agents 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 buffering solutions, (21) polyesters, polycarbonates and / or polyanhydrides, and (22) other non-toxic and compatible substances used in pharmaceutical formulations.

[0087] As presented above, certain embodiments of the present compound may contain basic functional groups such as amino or alkylamino, and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this regard 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 by reacting the free base form of the purified compound of the present invention with a suitable organic or inorganic acid either in the administration vehicle or in the dosage form manufacturing process, and then 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, glucoheptonate, lactobionate, and lauryl sulfonate, etc. (see, for example, Berge et al. (1977) "Pharmaceutical Salts, J. Pharm. Sci. 66:1-19").

[0088] Pharmaceutically acceptable salts of the present compound include, for example, conventional non-toxic salts of the compound from non-toxic organic or inorganic acids or quaternary ammonium salts. 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 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, isothionic acid, etc.

[0089] In other cases, the compounds of the invention may contain one or more acidic functional groups and can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the invention. These salts can likewise be prepared in situ by reacting the free base form of the purified compound with a suitable base such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary or tertiary amine, either in the administration vehicle or in the process of manufacturing the dosage form, or when the purified compound is in the free base form. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, among others. 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).

[0090] Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition.

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

[0092] The formulations of the present invention include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, intravaginal, and / or parenteral administration. These formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of the active ingredient that can be combined with the carrier substance to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of the active ingredient that can be combined with the carrier substance to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, out of 100%, this amount ranges 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%.

[0093] In certain embodiments, the formulations of the present invention include additives selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle formers such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and the compounds of the present invention. In certain embodiments, the above-described formulations make the compounds of the present invention biologically available orally.

[0094] The method for preparing these formulations or compositions includes the step of mixing the compounds of the present invention with a carrier and optionally one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately mixing the compounds of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0095] The 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 water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as troches (using inert bases such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, each containing a predetermined amount of the compounds of the present invention as the active ingredient. The compounds of the present invention may also be administered as boluses, linctuses, or pastes.

[0096] In the solid dosage forms of the present invention for oral administration (such as capsules, tablets, pills, lozenges, powders, granules, troches, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable additives containing sodium citrate or calcium phosphate, and / or a pharmaceutically acceptable carrier such as 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) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) dissolution retardants such as paraffin, (6) absorption promoters such as quaternary ammonium compounds and surfactants such as poloxamer and sodium lauryl sulfate, (7) wetting agents such as cetyl alcohol, glycerol monostearate, and nonionic surfactants, (8) absorbents such as kaolin and bentonite clay, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, (10) coloring agents, and (11) release control agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets, and pills, the pharmaceutical composition may further contain a buffering agent. Such additives may be used as lactose or milk sugar, and furthermore, high molecular weight polyethylene glycol, etc., and solid compositions of the same kind may be used as fillers in soft and hard gelatin capsules.

[0097] The tablets may be prepared by compressing or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a binder (such as gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (such as sodium starch glycolate or croscarmellose sodium), a surfactant or a dispersing agent. Wet tablets may be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent using a suitable machine.

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

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

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

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

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

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

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

[0105] Ointments, pastes, creams and gels may contain, in addition to the active compound of the present invention, additives such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

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

[0107] 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 promoters can also be used to increase the flux of the compound through the skin. The rate of such flow can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0108] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated within the scope of the present invention.

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

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

[0111] These compositions may also contain additional additives such as preservatives, wetting agents, emulsifying agents and dispersing agents. By containing various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc., the activity of microorganisms with the present compound can be surely prevented. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, the prolonged absorption of injectable pharmaceutical forms is brought about by containing agents that delay absorption, such as aluminum monostearate and gelatin.

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

[0113] Injectable depot dosage forms are prepared by forming microcapsule matrices of the compounds in biodegradable polymers such as polylactide - polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injection formulations are also prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

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

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

[0116] As used in the present invention, the expressions "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, which includes, but is not limited to, intravenous, intramuscular, intra - arterial, intrathecal, intracapsular, intra - orbital, intracardiac, intradermal, intraperitoneal, percutaneous tracheal, subcutaneous, subepidermal, intra - articular, subcapsular, subarachnoid, intrathecal and intrasternal injection and infusion.

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

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

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

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

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

[0122] 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 may start with a dosage level lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage as desired effects are obtained.

[0123] Generally, a suitable daily dosage of the compounds of the present invention is the amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective dosages generally depend on the factors described above. Generally, when used for the indicated analgesic effect, the oral, intravenous, intraventricular, and subcutaneous dosages of the compounds of the present invention to a patient vary in the range of about 0.0001 to about 100 mg per kg of body weight per day.

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

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

[0126] The patients to be treated are any animals as necessary, including primates, particularly humans, as well as other mammals such as horses, cows, pigs and sheep; and common poultry and pets.

[0127] The compounds for use in the method of the present invention can be administered as such or in admixture with a pharmaceutically acceptable carrier and can also be administered in combination with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Thus, combination therapy includes administering the active compounds sequentially, simultaneously and separately in such a way that the therapeutic effect of the initially administered formulation has not completely disappeared when the subsequent formulation is administered.

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

[0129] 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 complete feeds are described in reference books (“Applied Animal Nutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969, or “Livestock Feeds and Feeding” O and B books, Corvallis, Ore., U.S.A., 1977, etc.).

[0130] Microemulsion technology may be used to improve the bioavailability of lipophilic (water-insoluble) pharmaceuticals. Examples include Trimetrine (Dordunoo, S.K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, P.C., et al., J Pharm Sci 80(7), 712-714, 1991). In particular, microemulsion preferentially induces absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing the destruction of the compound in the enterohepatic circulation, resulting in enhanced bioavailability.

[0131] Controlled release The release characteristics of the formulations of the present invention depend on the encapsulating material, the concentration of the encapsulated drug, and the presence of a release modifier. For example, pH-dependent manipulation of release can be achieved 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. An enteric coating can be used to prevent release from occurring until after passing through the stomach. A multilayer coating or mixture of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach followed by a subsequent release in the intestine. Release can also be manipulated by including salts or pore-forming agents, which can increase drug release by water uptake or diffusion from the capsule. Additives that change 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. These can be added to the drug, either according to the compound, as a separate phase (i.e., as microparticles), or co-dissolved with the polymer phase. The 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, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, as well as surfactants such as Tween® and Pluronic®. A pore-forming agent (i.e., a water-soluble compound such as an inorganic salt and a sugar) that adds a fine structure to the matrix is added as microparticles. The range must be between 1 and 30% (w / w polymer).

[0132] By changing the residence time of the particles in the digestive tract, uptake can also be manipulated. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting such a polymer as the encapsulating material. Examples include chitosan, cellulose, and most polymers having free carboxyl groups such as polyacrylates (when used in the present invention, polyacrylates refer to polymers containing acrylate groups as well as modified acrylate groups such as cyanoacrylates and methacrylates).

[0133] Those skilled in the art should understand and expect that variant forms can be created within the principles of the invention disclosed herein, and such variant forms are also intended to be included within the scope of the present invention. The following examples further illustrate and explain the present invention, but they should not be construed as limiting the scope of the present invention in any way. All reference documents cited herein are hereby incorporated by reference in their entirety.

Examples

[0134] Example 1 All solvents and reagents were obtained commercially and used as received. 1 1H NMR spectra were recorded on a Bruker instrument (300 MHz or 400 MHz) in the deuterated solvent specified. Chemical shifts are reported in ppm and coupling constants in Hertz. All final compounds were purified by flash chromatography using 220 - 400 mesh silica gel or by reverse phase HPLC with CH as the solvent 3Purified using water. Thin layer chromatography was performed on silica gel 60F-254 (0.25 nm thick) plates. Visualization was achieved with UV light and / or 10% phosphomolybdic acid in ethanol. Reference (low resolution) mass spectra were obtained using either a Waters LCT or an Applied Biosystems API 3000 mass spectrometer. High resolution mass spectra (HRMS) were obtained using either a Waters LCT or an Agilent TOF mass spectrometer. All other LC-MS experiments were performed on an Agilent 1100 HPLC coupled to an Agilent single quadrupole mass spectrometer. Compound purity was determined by LC-MS at wavelengths of 230 nM and 254 nM. All final compounds reported herein have a purity of 95% or greater.

[0135] General procedure A

Chemical formula

[0136] HATU coupling: A compound of general structural formula 1 (1 equiv), HATU (1.25 equiv) and DIPEA (1.5 equiv) were dissolved in DMF and stirred at 23 °C for 15 min. 1H-Indazol-5-amine (1 equiv) was introduced into the reaction mixture and the solution was stirred at 23 °C for an additional 16 h. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4It was dried above, filtered, concentrated under reduced pressure to obtain a crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 2.

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

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

[0139] General Procedure B

Chemical formula

[0140] K 2 CO 3(2 equivalents), LiI (0.05 equivalents), and benzyl bromide were added to a solution of the compound of general structural formula 5 (1 equivalent) in MeCN at room temperature. The reaction temperature was raised to 60 °C, and stirring was continued for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and dried over Na 2 SO 4 then filtered and concentrated under reduced pressure to obtain a crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 6.

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

[0142] The compound of general structural formula 7 (1 equivalent) was dissolved in a methanol / water mixture, and NaOH (2 equivalents) was added. The reaction was continuously stirred at 30 °C for 16 hours. The reaction solution was acidified to pH = 5. The crude residue was purified by reverse-phase preparative HPLC to obtain the desired compound of general structural formula 8.

[0143] The compound of general structural formula 8 (1 equivalent), HATU (1.25 equivalents), and DIPEA (1.5 equivalents) were dissolved in DMF and stirred at 23 °C for 15 minutes. 1H-indazole-5-amine (1 equivalent) was introduced into the reaction mixture, and the solution was continuously stirred 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 and dried over Na 2 SO 4 then filtered and concentrated under reduced pressure to obtain a crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 9.

[0144] The compound of general structural formula 9 (1 equivalent) and concentrated HCl (1.2 equivalents) were dissolved in MeOH, and 10% anhydrous Pd / C was added. The reaction mixture was stirred at 50 °C for 5 hours under a H 2 atmosphere (1 atm). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by reverse-phase preparative HPLC to obtain the desired compound of general structural formula 10.

[0145] General procedure C [Chemical formula] The compound of general structural formula 11 (1 equivalent) was dissolved in acetic acid, and PtO 2 (0.1 equivalent) was added. The reaction solution was stirred at room temperature for 16 hours under a hydrogen atmosphere (1 atm). The resulting solution was filtered off and concentrated under reduced pressure. The residue was made basic to pH = 9 with 2N NaOH, and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to obtain a crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 12.

[0146] The compound of general structural formula 12 (1 equivalent) was dissolved in DCM, and Boc 2 O (1.5 equivalents) and DIPEA (2 equivalents) were added. The reaction was continued to stir at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to obtain a crude substance, which was purified on silica gel to obtain the desired compound of general structural formula 13.

[0147] The compound of general structural formula 13 (1 equivalent) was dissolved in a methanol / water mixture, and NaOH (2 equivalents) was added. The reaction was continued to stir at 30 °C for 16 hours. The reaction solution was acidified to pH = 5. The crude residue was purified by reverse-phase preparative HPLC to obtain the desired compound of general structural formula 14.

[0148] A compound of general structural formula 14 (1 equivalent), HATU (1.25 equivalents), and DIPEA (1.5 equivalents) were dissolved in DMF and stirred at 23 °C for 15 minutes. 1H-indazole-5-amine (1 equivalent) was introduced into the reaction mixture, and the solution was continuously stirred 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 and dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to obtain a crude material, which was purified on silica gel to obtain the desired compound of general structural formula 15.

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

[0150] General procedure D

Chemical formula

[0151] General procedure E

Chemical formula

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

[0153] NaOH (2 equivalents) was introduced into a reaction vessel containing the compound of general structural formula 21 (1 equivalent) dissolved in a MeOH / H 2 O 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 6N HCl until pH = 8. The suspension was filtered, and the solid was collected and dried to obtain the compound of general structural formula 22.

[0154] The compound of general structural formula 22 (1 equivalent), HATU (1.25 equivalents) and DIPEA (2 equivalents) were dissolved in DMF and stirred at 20 °C for 15 minutes. 1H-indazole-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 Na 2 SO 4 dried, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to obtain the compound of general structural formula 23.

[0155] General procedure F

Chemical formula

[0156] The compound of general structural formula 25 (1 equiv), HATU (1.25 equiv), and DIPEA (2 equiv) were dissolved in DMF and stirred at 20 °C for 15 min. 1H-Indazol-5-amine (1 equiv) was introduced into the reaction mixture and stirred at 20 °C for 16 h. The reaction was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by normal-phase silica gel chromatography to afford the compound of general structural formula 26.

[0157] The compound of general structural formula 26 (1 equiv) and concentrated HCl (1.2 equiv) were dissolved in MeOH, and 10% anhydrous Pd / C was added. The reaction mixture was stirred at 60 °C for 3 h under an H 2 atmosphere (1 atm). 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 afford the desired compound of general structural formula 27.

[0158] Example 2 N-(1H-Indazol-5-yl)-2-(methylamino)-2-phenylacetamide

Chemical formula

[0159] Example 3 N-(1H-Indazol-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide

Chemical Structure

[0160] Example 4 N-(1H-Indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide

Chemical Structure

[0161] Example 5 N-(1H-Indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide

Chemical formula

[0162] Example 6 N-(1H-Indazol-5-yl)isoindoline-1-carboxamide

Chemical formula

[0163] Example 7 N-(2-(Dimethylamino)ethyl)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

Chem.

[0164] Example 8 N-(1H-Indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide

Chem.

[0165] Example 9 N-(1H-Indazol-5-yl)-3-methylisoindoline-1-carboxamide

Chem.

[0166] Example 10 N-(1H-Indazol-5-yl)pyrrolidine-2-carboxamide

Chemical formula

[0167] Example 11 N-(1H-Indazol-5-yl)pyrrolidine-3-carboxamide

Chemical formula

[0168] Example 12 (2S,5R)-N-(1H-Indazol-5-yl)-5-phenylpyrrolidine-2-carboxamide

Chemical formula

[0169] Example 13 N-(1H-Indazol-5-yl)piperidine-2-carboxamide

Chemical formula

[0170] Example 14 N-(1H-Indazol-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

Chemical formula

[0171] Example 15 N-(1H-Indazol-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

Chemical Structure

[0172] Example 16 2-(4-Chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chem.

[0173] Example 17 2-(4-Chlorophenyl)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide

Chem.

[0174] Example 18 2-(4-Chlorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide

Chemical Structure

[0175] Example 19 2-(4-Fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chemical Structure

[0176] Example 20 2-(4-Fluorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide

Chem.

[0177] Example 21 2-(Ethylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide

Chem.

[0178] Example 22 N-(1H-Indazol-5-yl)-2-(isopropylamino)-2-phenylacetamide

Chemical Structure

[0179] Example 23 2-(Ethylamino)-2-(4-fluorophenyl)-N-(1H-indazol-5-yl)acetamide

Chemical formula

[0180] Example 24 N-(1H-Indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide

Chemical formula

[0181] Example 25 N-(1H-Indazol-5-yl)-2-(isopropylamino)-2-(4-methoxyphenyl)acetamide [Chemical formula] The reaction was carried out according to General Protocol D. The final residue was purified by reverse-phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as a white solid (52%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.98 (s, 1H), 10.11 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.48-7.39 (m, 4H), 6.90 (d, J = 8.4 Hz, 2H), 4.45 (s, 1H), 3.72 (s, 3H), 2.72-2.68 (m, 1H), 1.05 (d, J = 6.4 Hz, 3H), 1.02 (d, J = 6.4 Hz, 3H). MS (ES+) m / e 339.1(M+H) + .

[0182] Example 26 2-(ethylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide [Chemical formula] The reaction was carried out according to General Protocol D. The final residue was purified by reverse-phase preparative HPLC to give 2-(ethylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide as a white solid (5%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.97 (s, 1H), 10.10 (s, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.50-7.41 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 4.37 (s, 1H), 3.73 (s, 1H), 2.60-2.53 (m, 2H), 2.27 (s, 3H), 1.08 (t, J = 7.2 Hz, 3H). MS (ES+) m / e 325.1 (M+H) + .

[0183] Example 27 2-(Cyclopropylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

Chemical Structure

[0184] Example 28 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(p-tolyl)acetamide

Chemical Structure

[0185] Example 29 N-(1H-Indazol-5-yl)-2-(isopropylamino)-2-(p-tolyl)acetamide

Chemical Structure

[0186] Example 30 2-(Ethylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide

Chemical Structure

[0187] Example 31 2-(Cyclopropylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide [Chemical formula] The reaction was carried out according to General Protocol D. The final residue was purified by reverse-phase preparative HPLC to give 2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide as a white solid (23%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.96 (s, 1H), 10.07 (s, 1H), 8.22 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.48 - 7.42 (m, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 4.40 (s, 1H), 2.26 (s, 3H), 2.07 - 1.99 (m, 1H), 0.42 - 0.29 (m, 4H). MS (ES+) m / e 321.1 (M+H) + .

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

[0189] Example 33 2-(2'-Fluoro-[1,1'-biphenyl]-4-yl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide [Chemical formula] The reaction was carried out according to General Protocol D. The final residue was purified by reverse-phase preparative HPLC to give 2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as a white solid (10%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.98 (s, 1H), 10.14 (s, 1H), 8.31 (s, 2H), 8.14 (s, 1H), 8.00 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.54 - 7.48 (m, 5H), 7.42 - 7.38 (m, 1H), 7.33 - 7.26 (m, 2H), 4.30 (s, 1H), 2.33 (s, 3H). MS (ES+) m / e 375.1 (M+H) + .

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

[0191] Example 35 N-(1H-Indazol-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide [Chemical formula] The reaction was carried out according to General Protocol D. The final residue was purified by reverse-phase preparative HPLC to obtain N-(1H-indazol-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide as a pink solid (10%). 1 1H NMR (400 MHz, DMSO-d 6) δ 13.03 (broad singlet, 1H), 9.99 (broad singlet, 1H), 8.20 (singlet, 2H), 8.13 (singlet, 1H), 8.01 (singlet, 1H), 7.52 - 7.46 (multiplet, 2H), 7.40 (doublet, J = 7.6, 1.6 Hz, 1H), 7.32 - 7.27 (multiplet, 1H), 7.03 (doublet, J = 8.0 Hz, 1H), 6.96 (triplet, J = 7.2 Hz, 1H), 4.59 (singlet, 1H), 3.83 (singlet, 3H), 2.34 (singlet, 3H). MS (ES+) m / e 311.0 (M+H) + .

[0192] Example 36 2-(tert-Butylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

Chemical Structure

[0193] Example 37 2-(tert-Butylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide [Chemistry] The reaction was carried out according to General Protocol E. The final residue was purified by reverse-phase preparative HPLC to give 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide as a purple solid (4%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.99 (s, 1H), 10.26 (s, 1H), 8.35 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.49-7.46 (m, 4H), 7.32 (t, J = 7.6 Hz, 2H), 7.26-7.22 (m, 1H), 4.50 (s, 1H), 1.10 (s, 9H). MS (ES+) m / e 323.1 (M+H) + .

[0194] Example 38 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide [Chemistry] The reaction was carried out according to General Protocol E. The final residue was purified by reverse-phase preparative HPLC to give 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide as a white solid (12%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.98 (s, 1H), 10.20 (s, 1H), 8.09 (s, 1H), 8.00 (s, 1H), 7.49-7.43 (m, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 4.44 (s, 1H), 2.26 (s, 3H), 1.09 (s, 9H). MS (ES+) m / e 337.1 (M+H) + .

[0195] Example 39 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(pyridin-3-yl)acetamide

Chemical Structure

[0196] Example 40 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(pyridin-2-yl)acetamide

Chemical Structure

[0197] Example 41 N-(1H-Indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

Chemical Structure

[0198] Example 42 2-(3-Chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chemical Structure

[0199] Example 43 2-(4-Ethoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chemical Structure

[0200] Example 44 N-(1H-Indazol-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide

Chemical Structure

[0201] Example 45 N-(1H-Indazol-5-yl)-2-(4'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide

Chemical Structure

[0202] Example 46 N-(1H-Indazol-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide [Chemical formula] The reaction was carried out according to General Protocol E. The final residue was purified by reverse-phase preparative HPLC to give N-(1H-indazol-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide as an off-white solid (16%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.07 (singlet, 1H), 10.61 (singlet, 1H), 9.55 (singlets, 2H), 8.07 (doublet, J = 7.6 Hz, 2H), 7.52 (doublet, J = 8.8 Hz, 1H), 7.45 - 7.36 (multiplet, 2H), 7.25 - 7.21 (multiplet, 2H), 7.07 - 7.04 (multiplet, 1H), 5.07 (singlet, 1H), 3.80 (singlet, 3H), 3.78 - 3.59 (multiplet, 2H), 3.31 (singlet, 3H), 3.11 (multiplet, 1H), 2.99 (multiplet, 1H). MS (ES+) m / e 355.0 (M+H) + .

[0203] Example 47 2-(2-Chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide [Chemical formula] The reaction was carried out according to General Protocol E. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(2-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as an off-white solid (32%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.03 (brs, 1H), 10.14 (s, 1H), 8.17 - 8.14 (m, 2H), 8.02 (s, 1H), 7.61 - 7.58 (m, 1H), 7.50 - 7.46 (m, 3H), 7.37 - 7.30 (m, 2H), 4.67 (s, 1H), 2.35 (s, 3H). MS (ES+) m / e 315.0 (M+H) + .

[0204] Example 48 2-(4-Fluoro-3-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide [Chemical formula] The reaction was carried out according to General Procedure F. The final residue was purified by reverse-phase preparative HPLC to obtain 2-(4-fluoro-3-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as a white solid (49%). 1 H NMR (400 MHz, DMSO-d 6) δ 13.00 (s, 1H), 10.15 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50 - 7.44 (m, 2H), 7.34 (dd, J = 8.4, 2.0 Hz, 1H), 7.21 - 7.16 (m, 1H), 7.07 - 7.03 (m, 1H), 4.31 (s, 1H), 3.86 (s, 3H), 2.32 (s, 3H). MS (ES+) m / e 329.1 (M+H) + .

[0205] Example 49 2-(Ethylamino)-N-(1H-indazol-5-yl)-2-(m-tolyl)acetamide

Chem.

[0206] Example 50 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)-phenyl)acetamide

Chem.

[0207] Example 51 2-(Ethylamino)-2-(3-fluorophenyl)-N-(1H-indazol-5-yl)acetamide

Chemical Structure

[0208] Example 52 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide

Chemical Structure

[0209] Example 53 2-(3-Fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chemical Structure

[0210] Example 54 (R)-N-(1H-Indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

Chem.

[0211] Example 55 (S)-N-(1H-Indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

Chem.

[0212] Example 56 N-(1H-Indazol-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide

Chemical Structure

[0213] Example 57 2-(3-Fluoro-4-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

Chemical Structure

[0214] Example 58 2-(3-Chlorophenyl)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide

Chemical Structure

[0215] Comparative Example 1

Chemical Structure

[0216] Comparative Example 2

Chemical Structure

[0217] Comparative Example 3

Chemical Structure

[0218] Comparative Example 4

Chemical Structure

[0219] Comparative Example 5

Chemical Structure

[0220] Comparative Example 6

Chemical Structure

[0221] Comparative Example 7

Chem.

[0222] Example 59 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 the compounds was measured using a Z’-lyte kinase kit (ThermoFisher Scientific), and IC 50 was calculated.

[0223] 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 at a density of 5,000 cells / well in a 96-well plate for 24 hours and then treated with the test compounds for 90 minutes. The cells were then fixed and processed according to the In-Cell ELISA Colorimetric Detection Kit manual (Thermo Scientific). Using the In-Cell ELISA kit, the cellular phospho-myosin light chain (ppMlc, Thr18 / Ser19) levels were determined after treatment with DMSO control or test compounds. The inhibition percentage was calculated by applying the data obtained with 1 μM (or 10 μM) compound treatment to the formula: [1 - (compound / DMSO)] × 100%. The ppMlc data obtained with 9-point two-fold serial dilutions of the compounds were fitted to the non-linear regression curve fitting function of GraphPad Prism software to calculate the in-cell IC 50 value.

[0224] 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 the test compound and TGFβ1 for 24 hours. The cells were then lysed, and luciferase activity was measured using the LightSwitch luciferase kit from Active Motif.

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

[0226] As measured by the A7R5 In-Cell Elisa assay, the compounds of the present invention inhibited ROCK in cells. A7R5 cells were treated with 9-point two-fold serial dilutions of the compound, and the ppMlc (T18 / S19) level was determined to calculate the cellular IC 50 value of the compound. The results are shown in Table 1 below.

Table 1

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

[0228] Table 2 below shows the in vitro and cellular inhibition of ROCK by the compounds of the present invention. The activity of the compounds was measured using a Z’-lyte kinase kit (ThermoFisher Scientific). The inhibition percentage was calculated by normalizing the kinase activity value obtained with 1 μM compound treatment against the DMSO control value. GraphPad Prism software was used with the kinase activity data collected from 9-point serial dilutions of compound treatment to calculate the IC 50 . ROCK inhibition in A7R5 cells and NIH3T3 (Acta2-Luc) cells was performed as described above.

[0229] For pMLC / A7R5 IC 50 , a two-step measurement was employed. The inhibition percentage was calculated with a single-dose (1 μM or 10 μM) of compound treatment to screen for active compounds, and then the cellular IC 50 was measured only for compounds with an inhibition rate exceeding 50%.

Table 2-1

Table 2-2

Table 2-3

[0230] The comparative examples do not have an alkyl substituent on the 2-amino group, which is present in the compounds of the present invention (i.e., in the comparative compounds, the 2-amino group is NH 2 ). The addition of 2-alkylamino (i.e., R 1 is alkyl, etc.) is especially the lower alkylamine at the 2-position (i.e., R 1(wherein R is lower alkyl) is associated with enhanced cellular ROCK activity. For example, Comparative Example 2 (containing a primary 2-amino group) is to be compared with Examples 16, 17, and 18 having a secondary 2-alkylamino, especially when the alkyl group is small. All four compounds show high in vitro inhibition of ROCK1 and ROCK2, but Examples 16, 17, and 18 enhance cellular ROCK inhibition as measured by phosphorylation of the ROCK target pMLC in A7R5 cells. For example, Example 2 has an IC 50 showing cellular ROCK inhibition (252 vs. 4410 IC 50 ), respectively). Similarly, the cellular IC 50 in Examples 24, 25, 26, 27, and 36 is lower than that of Comparative Example 4 having a primary 2-amino group.

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

[0232] Example 60 The ROCK inhibitor improved neurite outgrowth and axon ensheathment in oligodendroglial cells in a co - culture system with rat dorsal root ganglion (DRG) explants. As shown in Figure 4, under co - culture conditions of rat oligodendroglial cells and rat dorsal root ganglion (DRG) explants, treatment with the ROCK inhibitor changed the cytoskeletal system, generating many short - arrayed myelin segments, which were identified by neurofilament staining. At the same time, the ROCK inhibitor also promoted axon support by oligodendroglial cells organizationally, which was visualized by staining for MBP, a mature oligodendroglial cell marker.

[0233] Example 61 The ROCK inhibitor passed through the blood - brain barrier. In a mouse pharmacokinetic study, 2 hours after administration, animal tissues were collected to determine compound distribution. As shown in Table 3, the compound of Example 2 had excellent BBB permeation characteristics.

Table 3

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

Table 4

[0235] List of references: Deyts, C., Galan-Rodriguez, B., Martin, E., Bouveyron, N., Roze, E., Charvin, D., Caboche, J., and Betuing, S. (2009). Dopamine D2 receptor stimulation potentiates PolyQ-Huntingtin-induced mouse striatal neuron dysfunctions via Rho / ROCK-II activation. PLoS One 4, e8287. Govek, 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 I: 【Chemistry 1】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 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 may be unsubstituted or optionally substituted with halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 optionally substituted with 1 to 3 substituents independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2 are taken together to 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 halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 substituted with 1 to 3 substituents independently selected from the group consisting of 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, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 alkyl)-O-; R 5 is H, lower alkyl and C 3 ~C 6 cycloalkyl; a is 0 or 1; b is 0 to 2; and Each R and R′ is selected from H, lower alkyl, and C 3 ~C 6 cycloalkyl, or alternatively, R and R' taken together form a 5-6 membered heterocyclic ring.

2. The compound of claim 1 having formula II: 【Chemistry 2】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 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 may be unsubstituted or optionally substituted with halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 optionally substituted with 1 to 3 substituents independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2 are taken together to 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 halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 substituted with 1 to 3 substituents independently selected from the group consisting of 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, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 alkyl)-O-; b is 0 to 2; and Each R and R′ is selected from H, lower alkyl, and C 3 ~C 6 cycloalkyl, or alternatively, R and R' taken together form a 5-6 membered heterocyclic ring.

3. The compound of claim 1 having formula III: 【Chemistry 3】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 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 may be unsubstituted or optionally substituted with halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 optionally substituted with 1 to 3 substituents independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; Alternatively, R 1 and R 2 are taken together to 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 halo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 substituted with 1 to 3 substituents independently selected from the group consisting of perfluoroalkoxy, carboxyl, aryl and heteroaryl; R 3 is H, lower alkyl, substituted lower alkyl, and RR'N-(C 2~4 alkyl)-; and Each R and R′ is selected from H, lower alkyl, and C 3 ~C 6 cycloalkyl, or alternatively, R and R' taken together form a 5-6 membered heterocyclic ring.

4. The compound of claim 1 having formula IV: 【Chemistry 4】 [In the formula, Ring A is a 5- or 6-membered aromatic ring optionally containing up to 3 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, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 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 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 selected from the group consisting of perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, and RCONR'-; Each R and R′ is selected from H, lower alkyl, and C 3 ~C 6 cycloalkyl, or alternatively, R and R′ taken together form a 5-6 membered heterocyclic ring; and m is 1 to 3.

5. The compound of claim 1 having formula V: 【Chemistry 5】 [In the formula, Ring B is a 5- or 6-membered aromatic ring optionally containing up to 3 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, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 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 H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 selected from the group consisting of perfluoroalkoxy and carboxyl, RR'N-, RR'NCO-, RCONH-, and RCONR'-; each R and R' is independently selected from H, lower alkyl, and C3-C6 cycloalkyl, or alternatively, R and R' taken together form a 5-6 membered heterocyclic ring; and m is 1 to 3.

6. The compound of claim 1 having formula VI: 【Chemistry 6】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 ring heteroatoms; c is 2 to 4; d is 1 to 4; 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, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 alkyl)-O-; b is 0 to 2; Each R 21 is H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; and n is 0 to 3.

7. The compound of claim 1 having formula VII: 【Chemistry 7】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R 4 is H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RR'N-(C 2~4 alkyl)- and RR'N-(C 2~4 alkyl)-O-; b is 0 to 2; Each R 21 is H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; and n is 0 to 3.

8. The compound of claim 1 having formula VIII: 【Chemistry 8】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 ring heteroatoms; c is 2 to 4; d is 1 to 4; Each R 21 is H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C 1 ~C 3 Perfluoroalkyl, C 1 ~C 3 Perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR'-, RO 2 independently selected from the group consisting of C-, aryl-O-, and heteroaryl-O-; and n is 0 to 3.

9. The compound of claim 1 having formula IX: 【Chemistry 9】 [In the formula, R 1 is lower alkyl, substituted lower alkyl, C 3 ~C 6 Cycloalkyl, substituted C 3 ~C 6 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 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 11 is H, lower alkyl, and C 3 ~C 6 cycloalkyl; Each R 12 is independently selected from H and lower alkyl; Each R 13 is independently selected from H and lower alkyl; In addition, or alternatively, R 12 and R 13 Together, C 3 ~C 6 It may form a cycloalkyl group; W is a 3-7 membered heterocyclic ring having 1-3 ring heteroatoms; c is 2 to 4; d is 1 to 4; Each R 22 is H, halo, hydroxy, lower alkyl, lower alkoxy, amino, C 1 ~C 3 Perfluoroalkyl, and C 1 ~C 3 perfluoroalkoxy; and n is 0 to 3.

10. 10. A method of treating a fibrotic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9.

11. 11. The method of claim 10, wherein the fibrotic disorder is selected from the group consisting of pulmonary fibrosis including cystic and idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis including cirrhosis, cardiac fibrosis including arterial fibrosis, endomyocardial fibrosis, old myocardial infarction, arterial stiffness, atherosclerosis, restenosis, arthritic fibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars, glial scars, or renal fibrosis.

12. 10. A method of treating a central nervous system disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9.

13. 13. The method of claim 12, wherein the central nervous system disorder is selected from the group consisting of Huntington's disease, Parkinson's disease, Alzheimer's, 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.

14. 10. A method of treating glaucoma in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9.

15. 10. A method of treating inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9.

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