EA043363B1 - INHIBITORS OF RHO-ASSOCIATED SUPERCOIL-CONTAINING PROTEIN KINASE - Google Patents

Description

Technical field of the invention

This invention relates to inhibitors of ROCK1 and/or ROCK2. Methods of inhibiting ROCK1 and/or ROCK2 useful for treating a disease are also provided.

BACKGROUND OF THE INVENTION

Rho-associated coiled-coil containing protein kinases (ROCK) are members of the serine/threonine kinase family. Two isoforms have been identified, ROCK 1 and ROCK 2. Both isoforms are activated by GTP-bound forms of Rho GTPase and upon activation phosphorylate a number of downstream substrates. ROCKs play important roles in numerous cellular processes, including smooth muscle cell contraction, proliferation, cell adhesion, and cell migration. Therefore, ROCK inhibitors have potential therapeutic utility in a wide range of pathological conditions, including, for example, asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, renal failure, pulmonary hypertension, neuronal degeneration and osteoporosis.

ROCK is a key intracellular regulator of cytoskeletal dynamics and cell motility. ROCK regulates a number of downstream targets of RhoA through phosphorylation, including for example myosin light chain, myosin light chain phosphatase binding subunit, and LIM kinase 2. These substrates regulate actin filament organization and contractility. In smooth muscle cells, ROCK mediates calcium sensitivity and smooth muscle contraction. Inhibition of Rho kinase blocks 5-HT and adrenaline-induced muscle contraction. When applied to non-smooth muscle cells, ROCK induces stress fiber formation and is required for RhoA-mediated cellular transformation. ROCK is involved in a number of cellular processes including, but not limited to, cell adhesion, cell motility and migration, growth regulation, cell contraction and cytokinesis. ROCK is also involved in the activation of the transport system with Na/H exchange, the formation of stress fibers, the activation of adducin and physiological processes such as vasoconstriction, contraction of bronchial smooth muscle, proliferation of smooth muscle cells and vascular endothelium, platelet aggregation and others.

Inhibition of ROCK activity in animal models has demonstrated a number of beneficial effects of ROCK inhibition for the treatment of human diseases. These include models of cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy, intraocular hypertension, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, central nervous system disorders such as neuronal degeneration and spinal cord injury, and neoplasia. Inhibition of ROCK activity has been shown to inhibit tumor cell growth and metastasis, angiogenesis, arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, asthma, intraocular pressure regulation, and bone resorption. Inhibiting ROCK activity in patients has beneficial effects on vasospasm and cerebral ischemia after subarachnoid hemorrhage, reducing intraocular pressure, increasing ocular aqueous outflow through relaxation of trabecular meshwork tissue, improving blood flow to the optic nerve, and protecting healthy ganglion cells.

A significant body of in vivo data has been generated focusing on ROCK activity in the CNS. Abnormal activation of the ROCK pathway has been reported in many central nervous system disorders. For example, axonal growth and synaptic plasticity depend on the structural regulation of the actin cytoskeleton. The Rho-ROCK cascade plays a key role in synaptic plasticity, dendritic morphogenesis and stability, and growth cone motility and collapse. In addition, many molecules that inhibit axonal growth accumulate on RhoA/ROCK in neurons, making it an attractive route for intervention in CNS disorders.

Nogo receptors (NgRs) (along with other members of the complex, including LINGO-1) and their ligands are perhaps the most well-studied and effective inhibitors of neurite outgrowth. Some of the earliest events following receptor activation by myelin-associated inhibitors are 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 for spinal cord and optic nerve injuries. Neurodegenerative pathological conditions such as Huntington's disease and Alzheimer's disease (AD) have also been investigated as susceptible to inhibition of NgR signaling. Members of the NgR family are not only associated with APP processing, but also the subcellular localization of NgR and Nogo is altered in the AD brain.

Alzheimer's disease (AD), the most common cause of dementia in older adults, is a progressive neurodegenerative disorder that involves a gradual decline in many cognitive functions, including memory impairment ( Selkoe, 2001 ). Synaptic loss is commonly observed in AD pathology and is an indicator of synaptic dysfunction in AD (Tanzi and Bertram, 2005). Oligomerized amyloid-β peptides have been associated with loss of synaptic plasticity and neuronal network dysfunction. Synaptic plasticity depends on the structural regulation of the actin cytoskeleton in dendritic spines. The Rho-ROCK cascade plays a key role in synaptic plasticity, dendritic morphogenesis and stability, and growth cone motility and collapse (Govek et al., 2005; Linseman and Loucks, 2008). Several studies have demonstrated that ROCK kinases can induce the generation of toxic amyloid-β peptide and, in addition, inhibition of ROCK can inhibit toxic peptide processing. In the anticipatory neural signaling mechanism, β-amyloid increases the activity of Rho-GTPase, which, through activation of ROCK, inhibits neurite outgrowth and synapse formation (Petratos et al., 2008). Thus, ROCK inhibitors may have the potential to prevent synaptic and neuronal degradation, as well as stimulate regeneration processes in AD. A recent study by Herskowitz et al. showed that knockdown of ROCK reduced levels of ae. These effects demonstrate that highly selective ROCK inhibitors are needed to provide effective treatment for Alzheimer's disease (AD). The model compound SR3677 was tested in a rodent model of AD in an attempt to demonstrate the utility of ROCK inhibition in AD by altering the distribution of BACE-1 and translocating amyloid precursor protein (APP) to lysosomes. Following direct IP injection into the hippocampus, due to its poor oral PK properties (5% F and half-life < 1 h) and lack of brain penetration, SR3677 had a promising sAPPe-lowering effect.

Huntington's disease (HD) is a debilitating, incurable, predominantly inherited neurodegenerative disease characterized by mental disorder, motor impairment, and dementia.

Misfolding and aggregation of the Htt protein, a product of the huntingtin gene, leads to HD pathology (Shao and Diamond, 2007). Very few mechanism-based therapeutics have been developed for the treatment of HD. Although research is still ongoing, there is ample evidence to suggest that ROCK inhibition may be an effective treatment for HD. In mouse models of HD, ROCK inhibition significantly reduced soluble Htt levels, reversed aggregate formation, neurite retraction, and protected against neuronal cell death (Deyts et al., 2009; Li et al., 2009). Similar results were obtained in studies in Drosophila, in which ROCK inhibition regulated Htt aggregation (Shao et al., 2008a; Shao et al., 2008b). The ROCK signaling pathway is a promising therapeutic target in HD.

ROCK signaling has also been linked to 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.

ROCKs phosphorylate multiple downstream substrates, including myosin light chain (MLC, at threonine 18 and serine 19) and myosin light chain phosphatase (MYPT1, at threonine 853), to drive the polymerization of globular G-actin into filamentous F-actin and assembly of the actomyosin contraction machinery . It has been recognized that this pathway may be involved in the pathogenesis 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 axonal growth inhibitors. Myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo, oligodendrocyte-myelin glycoprotein (OMgp), and repulsive guidance molecule (RGM) limit axonal regeneration in the injured brain and spinal cord. The common mechanism of the various myelin-associated inhibitors is that they all activate Rho and its downstream effector ROCK kinases to inhibit neurite outgrowth.

Blocking the Rho/ROCK pathway with a small molecule is a necessary strategy for central nervous system (CNS) disorders. However, the blood-brain barrier (BBB), although playing a key role in brain homeostasis, also significantly impedes the penetration of most small molecule inhibitors. Given the growing interest in developing selective and effective inhibitors for the treatment of CNS diseases, there is a pressing need for ROCK 1 and/or ROCK 2 inhibitors, particularly those that cross the blood-brain barrier.

The essence of the invention

The present invention relates to a compound having the formula

or a pharmaceutically acceptable salt thereof, wherein

R ¹ represents Cl-C4-alkyl, cyclopropyl or -(C _2-4 alkyl)-OCH ₃ ;

R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C ₁ - 2 043363

C ₄ -alkyl, C ₁ -C ₄ -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR'N-,

RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-;

R ³ is selected from H, C _g C ₄ -alkyl, substituted C _g C ₄ -alkyl and RR'N-(C _2-4 alkyl)-;

R ⁴ is selected from the group consisting of halogen, hydroxy, C 1 -C ₄ -alkyl, C _g C ₄ -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR' N-, RR'NCO-, RCONR'-, RR'N-(C _2-4 alkyl) and RR'N-(C _2-4 alkyl)-O-;

b is from 0 to 2 and each R and R' are independently selected from H, C1- _C4 -alkyl and _C3 - _C6 -cycloalkyl; where aryl represents a 6-membered single-ring aromatic group; heteroaryl is a 5- or 6-membered single-ring aromatic group that may contain from one to four heteroatoms; a heteroatom is nitrogen, oxygen or sulfur; substituted C ₁ -C ₄ -alkyl is a C ₁ -C ₄ -alkyl having one to three substituents selected from the group consisting of halogen, hydroxy, C 1 -C ₄ -alkoxy, amino, nitro, cyano, perfluoro C1 -C ₄ -alkyl, perfluoro C1-C ₄ -alkoxy and carboxyl.

In a preferred embodiment of the present invention, R ¹ represents C1-C4 alkyl; R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, hydroxy, C 1 -C ₄ -alkyl, C 1 -C ₄ -alkoxy, C ₁ -C ₃ - perfluoroalkyl and C1-C ₃ -perfluoroalkoxy; R ³ is selected from H or RR'N-(C _2-4 alkyl)- and b is 0.

In yet another preferred embodiment of the present invention, said compound has formula VI wherein each R ²¹ is independently selected from the group consisting of H, halogen, hydroxy, C1- _C4 alkyl, C1- _C4 alkoxy, nitro, cyano, _C1 -C _3- perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3.

In yet another preferred embodiment of the present invention, said compound has formula VII (VII).

In yet another preferred embodiment of the present invention, said compound has formula VIII where

R ¹ represents C1- _C4 alkyl;

each R ²¹ is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR' N-, RR'NCO-, RCONR'-, RO ₂ C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3.

In yet another preferred embodiment of the present invention, said compound has formula IX where

R ¹ represents C1- _C4 alkyl;

each R ²² is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 -alkoxy, amino, C ₁ -C ₃ -perfluoroalkyl and C 1 -C ₃ -perfluoroalkoxy; and n is from 0 to 3.

In yet another aspect, the present invention provides a compound having the following formula:

or a pharmaceutically acceptable salt thereof; where R ¹ represents C ₁ -C ₄ -alkyl, C ₃ -C ₆ cycloalkyl or R ¹⁰ O(CH2)c-; or alternatively R ¹ may be R ¹⁰ R ¹¹ N(CH2)c- when R 2 is phenyl, which may be unsubstituted or substituted with methoxy;

R ¹⁰ and R ¹¹ are C ₁ -C ₄ -alkyl;

R2 is phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C ₁ C ₄ -alkyl, C 1 -C ₄ -alkoxy, C ₁ -C _3- perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, aryl and aryl-O-, where the aryl and -O-aryl substituent, in turn, may be unsubstituted or substituted with C1-C ₄ -alkoxy;

R ³ is selected from H, C ₁ -C ₄ -alkyl and RR'N-(C _2-4 alkyl)-;

each R and R' are independently selected from H, C1- _C4 -alkyl and _C3 - _C6 -cycloalkyl and c is from 2 to 4; where aryl is a 6-membered single ring aromatic group.

In a preferred embodiment of the present invention, said compound has the following formula:

Where

R ¹ represents C1- _C4 alkyl or cyclopropyl;

each R ²² is selected from halo, C ₁ -C ₄ -alkyl and C ₁ -C ₄ -alkoxy;

n is from 0 to 2.

Preferably, R ¹ is C ₁ -C ₄ alkyl and n is 1 or 2.

In another aspect, the present invention relates to a pharmaceutical composition containing one of the above compounds or a pharmaceutically acceptable salt thereof, formulated with one or more pharmaceutically acceptable carriers. Preferably in the form of a solid dosage form for oral administration.

In another aspect, the present invention relates to the use of the above composition for the preparation of a medicament for the treatment of a fibrotic disorder in a subject. The fibrotic disorder may be selected from the group consisting of pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, liver cirrhosis, myocardial fibrosis, arterial fibrosis, endomyocardial fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma or systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars, glial scarring and renal fibrosis.

In yet another aspect, the present invention relates to the use of the above composition for the preparation of a medicament for the treatment of an autoimmune disorder, a cardiovascular disorder or a central nervous system disorder in a subject. Wherein the autoimmune disorder, cardiovascular disorder or central nervous system disorder is selected from Crohn's disease, previous myocardial infarction, arterial stiffness, atherosclerosis, restenosis, cerebral vasospasm, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), disease Batten, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, neuronal degeneration, spinal cord injury and multiple sclerosis.

In another aspect, the present invention relates to the use of the above composition for the preparation of a medicament for the treatment of glaucoma in a subject.

In another aspect, the present invention relates to the use of the above composition for the preparation of a medicament for treating inflammation in a subject.

Brief description of the figures

Fig. 1: Representative results of the Z'-Lyte assay. The ROCK inhibitors of this invention exhibit only single-digit nanomolar potency against both ROCK isoforms.

Fig. 2: The compounds of this invention inhibit both isoforms of ROCK in cells. Cells

- 4 043363

HCT116 with ROCK1 or ROCK2 knockout (ROCK1KO and ROCK2KO, respectively) were treated with the inhibitor for 90 min, and pMypt (T853) levels were visualized by Western blotting. The ROCK inhibitor effectively blocked phosphorylation of the ROCK target MYPT1 at 110 nM.

Fig. 3: Human oligodendrocyte/neuron progenitor cells were cultured in vitro with or without ROCK inhibitor (compound of Example 2) for 2 and 14 days. Nestin and MAP2 proteins were visualized by staining with commercial antibodies to determine the different stages of neuronal cell differentiation. ROCK inhibitor significantly facilitated the expression of the cellular marker of mature neurons MAP2 while improving neurite outgrowth, as evidenced by a strong increase in MAP2 signal in cells differentiated in the presence of ROCK inhibitor.

Fig. 4: Under conditions of co-culture of rat oligodendrocytes with rat dorsal root ganglion (DRG) explants, treatment with a ROCK inhibitor (compound from Example 2) altered the organization of the cytoskeleton with the formation of a large number of shorter ordered myelin segments, which were identified by neurofilament staining.

At the same time, ROCK inhibitor also facilitated axonal support of oligodendrocytes, demonstrated by the alignment of oligodendrocytes (stained for MBP) along the direction of axonal length (stained for neurofilaments).

Detailed Description of the Invention

ROCK inhibitors.

Compounds of this invention include those having the formula I(I,n

About ΛγΆ

R ² R ³ or pharmaceutically acceptable salts thereof, where:

R ¹ represents C1- _C4 alkyl, cyclopropyl or -( _C2-4 alkyl) _-OCH3 ;

R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C ₁ C ₄ -alkyl, C ₁ -C ₄ -alkoxy, nitro, cyano , C ₁ -C ₃ -perfluoroalkyl, C1-C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-;

R ³ is selected from H, C1-C4 alkyl, substituted C1-C4 alkyl and RR'N-(C2-4alkyl)-; R ⁴ is selected from the group consisting of halogen, hydroxy, C 1 -C ₄ -alkyl, C ₁ -C ₄ -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR 'N-, RR'NCO-, RCONR'-, RR'N-(C _2-4 alkyl)- and RR'N-(C _2-4 alkyl)O-;

b is from 0 to 2; and each R and R' are independently selected from H, C1- _C4 -alkyl and _C3 - _C6 -cycloalkyl; where aryl represents a 6-membered single-ring aromatic group; heteroaryl is a 5- or 6-membered single-ring aromatic group that may contain from one to four heteroatoms; a heteroatom is nitrogen, oxygen or sulfur;

substituted C1- _C4 alkyl is a C1- _C4 alkyl having one to three substituents selected from the group consisting of halogen, hydroxy, _C1 - _C4 alkoxy, amino, nitro, cyano, _perfluoroC1 -C ₄ -alkyl, perfluoro C ₁ -C ₄ -alkoxy and carboxyl. In certain embodiments of these compounds:

R ¹ represents C ₁ -C ₄ -alkyl;

R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C ₁ C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ - C ₃ -perfluoroalkyl, and C ₁ -C ₃ -perfluoroalkoxy;

R ³ is selected from H or RR'N-(C _2-4 alkyl)- and b is 0.

In certain embodiments, the compounds of the present invention have formula VI

where each R ²¹ is independently selected from the group consisting of H, halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl , RR'N-, RR'NCO-, RCONR'-, RO ₂ C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3.

In certain embodiments, the compounds of the present invention have formula VII

- 5 043363

In certain embodiments, the compounds of the present invention have formula VIII

Where

R ¹ represents C ₁ -C ₄ -alkyl;

each R ²¹ is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, Ci-C ₄ alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3.

In certain embodiments, the compounds of the present invention have formula IX

Where

R ¹ represents C ₁ -C ₄ -alkyl;

each R ²² is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 -alkoxy, amino, C ₁ -C ₃ -perfluoroalkyl and C ₁ -C ₃ -perfluoroalkoxy; and n is from 0 to 3.

Additionally, compounds of this invention include those having the formula

or pharmaceutically acceptable salts thereof, wherein

R ¹ is C1-C4 alkyl, C3-C6 cycloalkyl or R ¹⁰ O(CH2) _c - or alternatively R ¹ may be R ¹⁰ R ¹¹ N(CH ₂ ) _c - when R ² is phenyl, which may be unsubstituted or substituted with methoxy;

R ¹⁰ and R ¹¹ are C1- _C4 alkyl;

R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C1C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ -C _3- perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, aryl and aryl-O-, where the aryl and -O-aryl substituent, in turn, may be unsubstituted or substituted with C ₁ -C ₄ -alkoxy;

R ³ is selected from H, C ₁ -C ₄ -alkyl and RR'N-(C ₂ - ₄ alkyl)-;

each R and R' are independently selected from H, C ₁ -C ₄ -alkyl and C ₃ -C ₆ -cycloalkyl and c is from 2 to 4;

where aryl is a 6-membered single ring aromatic group.

In certain embodiments, said compounds have the formula

Where

R ¹ represents C ₁ -C ₄ -alkyl or cyclopropyl;

- 6 043363 each R ²² is selected from halo, ^-^-alkyl and C1-C4-alkoxy;

n is from 0 to 2. Preferably R ¹ is C ₁ -C ₄ alkyl and n is 1 or 2.

The term alkyl refers to a radical of saturated aliphatic groups, including straight alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups. In preferred embodiments, the straight-chain or branched alkyl has 8 or fewer carbon atoms on its backbone (eg, C ₁ -C ₈ in the case of a straight chain, C ₃ -C ₈ in the case of a branched chain), and more preferably 6 or less. Likewise, preferred cycloalkyls have 3-8 carbon atoms in their ring structure and more preferably have 3-6 carbon atoms in their ring structure.

Unless the number of carbon atoms is specifically stated, as used herein, lower alkyl means an alkyl group as defined above, but having from one to four carbon atoms, and more preferably from one to three carbon atoms. In preferred embodiments, the substituent designated herein as alkyl is lower alkyl. Lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl and tert-butyl.

The term cycloalkyl refers to saturated carbocyclic groups having 3-6 carbon atoms in the ring. Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term substituted alkyl refers to an alkyl group as defined above having one to three substituents. The substituents are selected from the group consisting of halogen, hydroxy, lower alkoxy, amino, lower alkylamino, nitro, cyano, lower perfluoroalkyl, lower perfluoroalkoxy and carboxyl.

Substituted lower alkyl refers to a lower alkyl group as defined above having one to three substituents. The substituents are selected from the group consisting of halogen, hydroxy, lower alkoxy, amino, nitro, cyano, lower perfluoroalkyl, lower perfluoroalkoxy and carboxyl.

The term substituted cycloalkyl, such as substituted C3-C6 cycloalkyl, refers to a cycloalkyl group as defined above having from one to three substituents. The substituents are selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, lower perfluoroalkyl, lower perfluoroalkoxy and carboxyl.

As used herein, the term halogen or halo means -F, -Cl, -Br or -I and preferably F, Cl or Br.

As used herein, the terms alkoxy or alkoxy refer to an alkyl group as defined above that is attached via an oxygen atom. Representative alkoxy groups include methoxy, ethoxy, propyloxy, t-butoxy and the like. The term lower alkoxy refers to an alkoxy substituent in which the lower alkyl is linked via an oxygen atom, wherein the lower alkyl moiety is as defined above.

The terms amine and amino refer to both unsubstituted and substituted amines, for example a moiety that can be represented by the general formula

R where R and R' are each independently selected from H and lower alkyl.

As used herein, the term aryl includes 5- and 6-membered single-ring aromatic groups that may contain zero to four heteroatoms, such as benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine , pyridazine and pyrimidine, etc. Those aryl groups that contain heteroatoms in the ring structure may also be called aryl heterocycles or heteroaryl groups. The aromatic ring may be substituted at one or more positions on the ring with substituents such as those described above. The term aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are common to the two connected rings (the rings are fused rings), wherein at least one of the rings is aromatic.

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

The term heterocycle or heterocyclyl refers to non-aromatic heterocycles having 47 ring atoms and containing 1-3 ring heteroatoms.

As used herein, the term heteroatom means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur. The most preferred are nitrogen and oxygen.

In the context of this document, it is assumed that the definition of each expression, for example, alkyl, m, n, R ¹ , R ² , etc., if it appears more than once in any structure, is independent of its definition elsewhere in the same structure.

It should be understood that substitution or substituted includes the indirect clause that such substitution is made in accordance with the permissible valency of the replaced atom and substituent and that the substitution results in a stable compound, for example, which is not subject to spontaneous transformations, for example, due to rearrangement, cyclization, elimination etc.

As used herein, the term substituted is intended to include all valid substituents for organic compounds. In broad terms, acceptable substituents include acyclic and cyclic, branched and straight-chain, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include, for example, those described above herein. Acceptable substituents may be one or more and may be the same or different for the respective organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, may contain hydrogen substituents and/or any permissible organic substituents described herein that satisfy the valency of the heteroatoms. This invention is not intended to be limited in any way by the permissible organic substituents.

As used herein, the expression protecting group refers to temporary substituents that protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include carboxylic acid esters, alcohol silyl esters, and aldehyde and ketone acetals and ketals, respectively. There is a review of the field of protecting group chemistry (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

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

The proposed compounds are ROCK inhibitors. ROCK exists in two forms, ROCK 1 (ROCKe; p160-ROCK) and ROCK 2 (ROCKa). In some embodiments, a compound of formulas I-IX selectively inhibits ROCK1. In some embodiments, a compound of Formulas IIX selectively inhibits ROCK2. In some embodiments, the compound of formulas I-IX is non-selective for inhibition of ROCK1 and ROCK2. In the context of this invention, selectivity means that the inhibitor exhibits a TC ₅₀ that is at least 2-fold, at least 5-fold, at least 10-fold, or at least 25-fold lower for a single kinase compared to the IC ₅₀ for another kinase.

Methods for determining kinase inhibition are known in the art. For example, the kinase activity of an enzyme and the inhibitory ability of a test compound can be determined by measuring enzyme-specific phosphorylation of a substrate. Commercial assays and kits are available and can be used for this purpose. For example, kinase inhibition can be determined using the IMAP® assay (Molecular Devices). This analytical method involves the use of a fluorescently labeled peptide substrate. Phosphorylation of the labeled peptide by the kinase of interest stimulates the binding of the peptide to the trivalent metal nanoparticle through a specific high-affinity interaction between the phosphogroup and the trivalent metal. Proximity to the nanoparticle results in increased fluorescent polarization. Inhibition of the kinase by a kinase inhibitor prevents phosphorylation of the substrate and thus limits the binding of the fluorescently labeled substrate to the nanoparticle. Such an assay can be compatible with a microwell assay format, allowing simultaneous determination of the IC50 of multiple compounds.

Methods of treating diseases

In one aspect of the present invention, the present compounds in a pharmaceutical composition are used to prepare a medicament for treating diseases in a subject, such as a fibrotic disorder, an autoimmune disorder, a cardiovascular or central nervous system disorder, glaucoma and inflammation.

Central nervous system disorders

The compounds of the invention demonstrate effective penetration through the blood-brain barrier (BBB) and distribution into the tissues of the central nervous system. Thus, the compounds of the invention are useful for the treatment of disorders of the central nervous system, as well as disorders, such as certain ocular disorders, in which the ability to cross the BBB is beneficial. Such disorders may include neuronal degeneration or physical damage to nerve tissue, including, but not limited to, Huntington's disease, Parkinson's disease, Alzheimer's disease,

- 8 043363 amyotrophic lateral sclerosis (ABS), Batten disease, dementia, spinal muscular atrophy, motor neuron diseases, spinocerebellar ataxia, acute or chronic pain, dementia, neuronal degeneration, spinal cord injury, cerebral vasospasm or multiple sclerosis.

Cardiovascular and other diseases

The compounds of the invention, which inhibit ROCK and/or ROCK-mediated phosphorylation, are useful for the treatment of patients suffering from cardiovascular and non-cardiovascular diseases in which Rho kinase function plays a role, such as hypertension, pulmonary hypertension, atherosclerosis, restenosis, coronary heart disease, cardiac hypertrophy, intraocular hypertension, retinopathy, ischemic diseases, cerebral ischemia, cerebral vasospasm, penile erectile dysfunction, peripheral circulatory disorder, peripheral arterial occlusive disease, glaucoma (eg, affecting intraocular pressure), pulmonary fibrosis, liver fibrosis , kidney fibrosis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, central nervous system disorders such as neuronal degeneration and spinal cord injury. In addition, the ROCK inhibitors of the invention can be used to treat arterial thrombotic disorders such as platelet aggregation and leukocyte aggregation, as well as bone resorption.

In an embodiment of the invention, the compounds are used for the treatment of brain cavity malformation (CBM). MPGMs are vascular lesions consisting of clusters of leaky, dilated capillaries and are associated with central nervous system (CNS) disorders, including seizures and stroke. Loss of vascular integrity is thought to be associated with RhoA activation and ROCK activation, leading to changes in cytoskeletal stability and increased vascular permeability. The compounds of the invention inhibit ROCK activation and restore vascular endothelial function.

Glaucoma

In an embodiment of the invention, the compound of the invention is used for the treatment of glaucoma. The two most common variants, primary angle-closure glaucoma and acute angle-closure glaucoma, are characterized by high intraocular pressure. Pigmentary glaucoma and congenital glaucoma are also characterized by decreased aqueous outflow and high intraocular pressure (IOP). Normal-tension glaucoma is thought to be due to a different mechanism, specifically poor blood flow to the optic nerve. Secondary glaucoma can result from injury, infection, inflammation, tumor, or cataract and is also associated with long-term steroid use, systemic hypertension, diabetic retinopathy, and central retinal vein occlusion. Glaucomas that have a neovascular component may benefit from administration of an angiogenesis inhibitor in addition to the ROCK inhibitor.

Inflammation

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

Autoimmune disorders

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 (glomerular basement membrane)/anti-TBM (tubular basement membrane) nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dys Tonya , autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (IAD), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroiditis, autoimmune urticaria, axonal and neuronal neuropathy, Balo'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 multiple osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid , Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome (calcinosis, Raynaud's disease, esophagitis, sclerodactyly and telangiectasia), primary mixed type cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optic nerve), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia,

- 9 043363 Fibrosing alveolitis, gigantic cell arteritis (temporal arteritis), gigantic cell myocarditis, glomerulonephritis, goodspaper syndrome, granulomatosis with polyiangiath (GPA) (previously called vegene granulomatosis), Graves syndrome, Hashimoto encephalitis, tireolid Khashimo Hashimo. then hemolytic anemia , Purpura Shenlein-Genoha, gestational herpes, hypogrammaglobulinemia, idiopathic thrombocytopenic purple (ITP), IGA-nefropathy, IgG4-tied sclerosing disease, immunoregulatory lipoproteins, myositis with Taurus, interstitial cystitis, juvenal. Arthritis, juvenile diabetes (type 1 diabetes) , juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, ligneous conjunctivitis, IgA-dependent linear dermatosis (ADLD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed disease connective tissue (CTD), Moray ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devica), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (pediatric autoimmune neuro- mental disorders associated with streptococcal infections), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Personage-Turner syndrome, parsplanitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, the presence of M-protein and skin lesions), polyarteritis nodosa, autoimmune polyglandular syndromes type I, II and III, polymyalgia rheumatica, polymyositis, post-infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis , psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, true red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome , scleritis, scleroderma, Sjogren's syndrome, testicular sperm autoimmunity, muscle stiffness syndrome, subacute bacterial endocarditis (SBE), Susak syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis and vitiligo.

In accordance with the invention, targeting Th17 (IL-17-secreting) cells through inhibition of ROCK provides treatments for Th17 cell-mediated diseases, including, but not limited to, autoimmune diseases such as RA, MS, SLE, psoriasis and Crohn's disease, and GVHD in humans. In an embodiment of the invention, the ROCK inhibitor is a compound of formula I.

The development and function of Tregs depend on the activation of specific signal transduction pathways. TGF-β and IL2 activate the expression of the transcription factors Foxp3 and STAT5, which both play important roles in regulating Treg suppressive function. On the other hand, proinflammatory cytokines inhibit Foxp3 expression through up-regulation of STAT3 phosphorylation. According to the invention, pharmacological inhibition of ROCK2 can regulate Treg function.

Pharmaceutical compositions

In one aspect, the present invention provides pharmaceutically acceptable compositions that contain a therapeutically effective amount of one or more compounds of the present invention formulated together with one or more pharmaceutical excipients. As described below, the pharmaceutical compositions of this invention can be prepared, in particular, in solid or liquid form, including those adapted for the following: (1) oral administration, for example, liquid dosage forms (aqueous or non-aqueous solutions or suspensions), tablets, for example , intended for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection in the form of, for example, a sterile solution or suspension, or a sustained release form; (3) topical application, such as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) intravaginal or intrarectal application, for example, in the form of a pessary, cream or foam; (5) sublingual use; (6) ophthalmic use; (7) transdermal application; or (8) nasal administration.

The expression pharmaceutically acceptable is used herein to refer to those compounds, materials, compositions and/or dosage forms that are medically suitable for use in contact with human and animal tissue and are characterized by toxicity, irritation, allergic reactions or other problems or complications. corresponding to an acceptable benefit/risk ratio.

As used herein, the expression pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or base solution, such as a liquid or solid.

- 10 043363 excipient, diluent, excipient, manufacturing aid (for example, lubricant, magnesium silicate talc, calcium or zinc stearate or stearic acid) or solvent-encapsulating material involved in the transfer or transport of a given compound from one organ or part body to another organ or other part of the body. Each carrier must be acceptable in terms of compatibility with the other ingredients of the dosage form and not cause harm to the patient. Some examples of materials that may 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 carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as coconut oil and suppository waxes; (9) oils such as peanut oil, cottonseed oil, sunflower 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 solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH-buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical forms.

As noted above, some embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this regard, the term pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of this invention. These salts can be prepared in situ during the manufacture of the stock administration solution or dosage form or by separately reacting the purified compound of the invention in its free base form with a suitable organic or inorganic acid and isolating the salt thus obtained during subsequent purification. Representative salts include hydrobromic, hydrochloric, 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 salts, etc. (See, for example, Berge et al. (1977) Pharmaceutical Salts, J. Pharm. Sci. 66:1-19).

Pharmaceutically acceptable salts of the subject compounds include conventional non-toxic salts or quaternary ammonium salts of compounds, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and salts derived from organic acids, such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfamic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, etc.

In other cases, the compounds of this invention may contain one or more acidic functional groups and, therefore, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these cases, the term pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds of this invention. These salts can similarly be prepared in situ during the manufacture of the stock administration solution or dosage form, or by separately reacting the purified compound of the invention in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, ammonium or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (see, for example, Berge et al., above).

Also present in the compositions may be wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators such as acetic acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

- 11 043363

Dosage forms of this invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The dosage forms may be conveniently presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will generally be that amount of the compound that produces a therapeutic effect. In general, of one hundred percent, this amount will range from about 0.1 to about 99% of the active ingredient, preferably from about 5 to about 70%, most preferably from about 10 to about 30%.

In certain embodiments, the dosage form of this invention contains an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents, such as bile acids, and polymeric carriers, such as polyesters and polyanhydrides; and a compound of this invention. In certain embodiments, the above dosage form makes the compound of this invention orally bioavailable.

Methods for preparing dosage forms or compositions include the steps of combining a compound of this invention with a carrier and, optionally, one or more auxiliary ingredients. In general, dosage forms are prepared by uniformly and carefully combining the compound of this invention with liquid carriers or particulate solid carriers, or both, and then, if necessary, molding the product.

The dosage forms of the invention suitable for oral administration may be in the form of capsules, starch capsules, pills, tablets, lozenges, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion. or water in oil, or in the form of an elixir or syrup, or in the form of lozenges (using an inert base such as gelatin or glycerin, or sucrose and gum arabic) and/or in the form of mouth rinses, etc., which are all contain a predetermined amount of a compound of this invention as an active ingredient. The compound of this invention can also be administered as a bolus, electuary or paste.

In the solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, lozenges, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable excipients, including a pharmaceutically acceptable carrier such as citrate sodium or dicalcium phosphate, and/or any of the following: (1) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds and surfactants such as poloxamer and sodium lauryl sulfate; (7) wetting agents such as, for example, 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 glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (10) dyes; and (11) controlled release agents such as crospovidone or ethylcellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard shell gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using a binder (eg, gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (eg, sodium starch glycolate or crosslinked sodium carboxymethylcellulose), surfactant or dispersing agent. Molded tablets can be prepared by molding in a suitable device a mixture of a powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions of this invention, such as dragees, capsules, pills and granules, may optionally be scored or formulated with coatings or coatings, such as enteric coatings and other coatings well known in the art of pharmaceutical drug formulation. forms They can also be prepared to provide a delayed or controlled release of the active

- 12 043363 ingredients, using, for example, hydroxypropyl methylcellulose in different proportions to provide the desired release profile, other polymer matrices, liposomes and/or microcapsules. They can be prepared for rapid release, for example by lyophilizing. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions may also optionally contain opacifiers and may be formulated such that they release only the active ingredient(s) or, preferably, in a specific part of the gastrointestinal tract, optionally in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances and waxes. The active ingredient may also be present in microencapsulated form, optionally with one or more of the excipients described above.

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

In addition to diluents, oral compositions may also contain additional excipients such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, colors, flavoring agents and preservatives.

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

Dosage forms of the pharmaceutical compositions of the invention for rectal or vaginal administration can be presented in the form of a suppository, which can be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers containing, for example, coconut oil, polyethylene glycol, wax for suppositories or salicylate, and which is solid at room temperature but liquid at body temperature and therefore melts in the rectal or vaginal cavity and releases the active compound.

Dosage forms of this invention that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or sprays containing such carriers as are suitable as known in the art.

Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalers. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be necessary.

Ointments, pastes, creams and gels may contain, in addition to the active compound of the invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and oxide zinc, or mixtures thereof.

Powders and sprays may contain, in addition to the compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Transdermal patches have the additional advantage of providing controlled delivery of the compound of this invention into the body. Such dosage forms can be prepared by dissolving or dispersing the compound in an appropriate medium. Suction enhancers can also be used to increase the flow of the compound through the skin. The rate of such flow can be controlled by installing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

Ophthalmic dosage forms, eye ointments, powders, solutions, etc. are also intended to be within the scope of this invention.

The pharmaceutical compositions of this invention, suitable for parenteral administration, contain one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted as sterile 13 043363 solutions or dispersions for injection immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that render the dosage form isotonic with the blood of the intended recipient, or suspending or thickening agents.

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

These compositions may also contain additional excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of exposure of the compounds in question to microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example paraben, chlorobutanol, phenol, sorbic acid, and the like. It may be necessary to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drug, it is necessary to slow down the absorption of the drug after subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material having poor solubility in water. Therefore, the rate of absorption of a drug depends on the rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug is achieved by dissolving or suspending the drug in an oil vehicle.

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

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

Routes of administration and doses

The preparations of this invention can be administered orally, parenterally, topically or rectally. Of course, they are used in forms suitable for each route of administration. For example, they are administered in the form of tablets or capsules, by injection, inhalation, in the form of ophthalmic lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; local administration via lotion or ointment; and rectal administration via suppositories. Oral administration is preferred.

As used herein, the expressions parenteral and parenterally administered mean modes of administration other than enteral and local administration, typically by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal , subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrathoracic injection and infusion.

As used herein, the expressions systemically administered, systemically administered, peripherally administered, and peripherally administered mean the administration of a compound, drug, or other material other than directly into the central nervous system so that it enters the patient's system and thereby becomes metabolized. and similar processes, for example, as in the case of subcutaneous administration.

These compounds can be administered to humans and other animals for therapy by any suitable route of administration, including oral, nasal, e.g., spray, rectal, intravaginal, parenteral, intracisternal, and topical, e.g., powders, ointments, or drops, including buccal and sublingual.

Regardless of the route of administration chosen, the compounds of this invention, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions of this invention are prepared in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without toxicity to the patient.

The dosage level chosen will depend on a number of factors, including the potency of the particular compound of this invention or its ester, salt or amide used, route of administration, time of administration, rate of elimination or metabolism of the particular compound used, rate and extent of absorption, duration of treatment, other drugs drugs, compounds and/or materials used in combination with the particular compound used, age, sex, weight, pathological condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

A physician or veterinarian appropriately skilled in the art can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician or veterinarian may begin with dosages of the compounds of the invention used in a pharmaceutical composition at levels below those required to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of compound that is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above. In general, oral, intravenous, intracerebroventricular and subcutaneous dosages of the compounds of this invention to a patient, when used for the indicated analgesic effect, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.

In certain embodiments, a dose of the compound or composition is administered to the subject every day, every other day, every two days, every three days, once a week, twice a week, thrice a week, or once every two weeks. If necessary, an effective daily dose of the active compound can be administered in two, three, four, five, six or more dosage units administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the dose(s) of the compound or composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, the dose of the compound or composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more.

The above dosing regimens are for illustrative purposes only and should not be considered limiting. One skilled in the art will understand that all dosages are within the scope of this invention.

The patient receiving such treatment is any animal in need thereof, including primates, particularly humans, and other mammals such as horses, cattle, pigs and sheep; as well as poultry and domestic animals in general.

Compounds for use in the methods of this invention can be administered alone or in the form of mixtures with pharmaceutically acceptable carriers, and can also be administered in combination with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Thus, combination therapy includes sequential, simultaneous and separate administration of the active compound in such a way that the therapeutic effect of the first administered component does not completely disappear during the administration of the subsequent one.

The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate pre-feed containing the active compound in an effective amount and incorporating the pre-feed into the complete diet.

Alternatively, an intermediate concentrate or feed additive containing the active ingredient may be mixed into the feed. The manner in which such premixes and complete rations can be prepared and administered is described in books (such as 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).

Microemulsification technology can be used to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. 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). Among other things, microemulsification provides increased bioavailability due to preferential absorption in the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing degradation of compounds in the hepatobiliary circulation.

Controlled release

The release characteristics of the dosage form of this invention depend on the encapsulating material, the concentration of the encapsulated drug and the presence

- 15 043363 release modifying substances. Release can be made pH dependent, for example by using a pH sensitive coating that allows release only at low pH, such as in the stomach, or higher pH, such as in the intestine. An enteric coating may be used to prevent release before passing through the stomach. Several types of coating or mixture of cyanamide encapsulated in different materials can be used to provide initial release in the stomach followed by release in the intestine. Release can also be controlled by the inclusion of salts or pore-forming agents, which can enhance water uptake or drug release by diffusion from the capsule. Can also be used in excipients that modify the solubility of the drug to control the rate of release. Agents that enhance matrix degradation or release from the matrix may also be included. They can be added to the drug product, added as a separate phase (ie, particulate), or co-dissolved in the polymer phase, depending on the compound. Some types of degradation enhancers 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, 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®. Pore-forming agents that impart microstructure to the matrices (i.e., water-soluble compounds such as inorganic salts and sugars) are added in particulate form. Their range should be from one to thirty percent (w/w, polymer).

Absorption can also be adjusted by changing the residence time of the particles in the intestine. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or selecting it as an encapsulating material. Examples include most polymers with free carboxyl groups, such as chitosan, celluloses and, in particular, polyacrylates (as used herein, polyacrylates refer to polymers containing acrylate groups and modified acrylate groups, such as cyanoacrylates and methacrylates).

It is understood and expected that variations in the principles of the invention described herein may be made by one skilled in the art, and such modifications are intended to be included within the scope of the invention. The following examples further illustrate the present invention, but should not be construed as limiting in any way the scope of the present invention. All references cited herein are incorporated by reference in their entirety.

Examples

Example 1.

All solvents and reagents were obtained commercially and used as received. 1H NMR spectra were recorded on a Bruker instrument (300 MHz or 400 MHz) in the indicated deuterated solvents. Chemical shift values are given in ppm and interaction constants are given in hertz. All final compounds were purified by flash chromatography using silica gel 220-400 sieve or reverse phase HPLC with CH ₃ CN/water as solvents. Thin layer chromatography was carried out on silica gel plates 60 F-254 (0.25 mm thick). Visualization was performed using UV light and/or 10% phosphomolybdic acid in ethanol. Nominal (low resolution) mass spectra were recorded on a Waters LCT or Applied Biosystems API 3000 mass spectrometer. High resolution mass spectra (HRMS) were recorded on a Waters LCT or Agilent TOF mass spectrometer. All other LC-MS experiments were performed on an Agilent 1100 HPLC system coupled to an Agilent single quadrupole mass spectrometer. The purity of the compound was determined by LC-MS with wavelengths of 230 and 254 nm. All final compounds reported herein are >95% pure.

General Procedure A:

2 z

EDCI conjugation: general structure compound 1 (1 eq), EDCI (1 eq), HOBt (0.2 eq) and DIEA (2 eq) were dissolved in DMF stirred at 25°C for 5 min . Amine (1 eq.) was then added to the mixture. The mixture was stirred at 25°C for 16 hours. The mixture was poured into water and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave a residue. This residue was purified by column chromatography to obtain a compound with general structure 2.

HATU conjugation: General structure compound 1 (1 eq), HATU (1.25 eq) and DIPEA (1.5 eq) were dissolved in DMF stirred at 23°C for 15 min. 1Hindazol-5-amine (1 equiv.) was added to the reaction mixture and the solution continued to be stirred at 23°C for another 16 hours.

- 16 043363 the mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 2.

Deprotection: compound of general structure 2 was dissolved in DCM and added to a solution of 4 N HCl in dioxane. 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 structure 3.

Removal of benzyl group: compound of general structure 2 (1 eq.) and conc. HCl (1.2 eq.) was dissolved in MeOH and 10% dry Pd/C was added. The reaction mixture was stirred under H ₂ (1 atm) at 50°C for 5 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse phase preparative HPLC to obtain the desired compound of the general structure 3.

General Procedure B:

5 6 7 8

10

Amine salt HCl (2 eq.) was dissolved in methanol and triethylamine (2 eq.) was added. The reaction solution was stirred for 15 min after which ketone or aldehyde of general structure 4 (1 eq.) was added along with HOAc (4 eq.). Stirring was continued for another 15 minutes and NaBH ₃ CN was added. The reaction temperature was raised to 60°C and stirring was continued for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 5.

K2CO3 (2 eq), LiI (0.05 eq) and benzyl bromide were added to a solution of general structure 5 (1 eq) 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, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 6.

A solution of compound of general structure 6 (1 eq.), Pd(PPh ₃ ) ₄ (0.15 eq.)), K3PO4 (3 eq.) in THF was purged with nitrogen for 5 min at room temperature. 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 structure 7.

Compound of general structure 7 (1 eq.) was dissolved in an aqueous mixture of methanol and NaOH (2 eq.) was added. The reaction mixture 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 structure 8.

General compound 8 (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 eq.) was added to the reaction mixture and the solution was continued to stir at 23°C for another 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 , filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 9.

Compound of general structure 9 (1 eq.) and conc. HCl (1.2 eq.) was dissolved in MeOH and 10% dry Pd/C was added. The reaction mixture was stirred under H ₂ (1 atm) at 50°C for 5 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse phase preparative HPLC to obtain the desired compound of the general structure 10.

- 17 043363

General Procedure C:

N^C° ₂ R BocN^^° ^2H BocN^g° ^2H

12 13 1

16

The compound of general structure 11 (1 eq.) was dissolved in acetic acid and PtO ₂ (0.1 eq.) was added. The reaction solution was stirred under a hydrogen atmosphere (1 atm) at room temperature for 16 hours. The resulting solution was filtered and concentrated under reduced pressure. The residue was made alkaline with 2 N NaOH to pH = 9 and the mixture was extracted. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 12.

The compound of general structure 12 (1 eq.) was dissolved in DCM and Boc ₂ O (1.5 eq.), DIPEA (2 eq.) was added. The reaction mixture 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 Na2SO4, filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 13.

The compound of general structure 13 (1 eq.) was dissolved in an aqueous mixture of methanol and NaOH (2 eq.) was added. The reaction mixture 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 structure 14.

General compound 14 (1 eq), HATU (1.25 eq) and DIPEA (1.5 eq) were dissolved in DMF and stirred at 23°C for 15 min. 1H-Indazol-5-amine (1 eq.) was added to the reaction mixture and the solution was continued to stir at 23°C for another 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 , filtered and concentrated under reduced pressure to give the crude material, which was purified on silica gel to give the desired compound of general structure 15.

The compound of general structure 15 was dissolved in DCM and added to the solution 4 N. HCl in dioxane. 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 structure 16.

General procedure D:

18

A solution of compound 17 (1 eq.) and aldehyde or ketone (1 eq.) in MeOH (3.00 ml) was stirred at 23°C for 16 hours. After 16 hours, NaBH ₃ CN (1.2 eq.) was added. ) and stirred the mixture for an additional 3 hours. The reaction mixture was quenched by adding water and the resulting mixture was extracted with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by preparative HPLC to obtain a compound with general structure 18.

General procedure E:

20 21 22 23

The general structure compound 19 (1 eq.) was dissolved in CCl ₄ and AIBN (0.1 eq.) was added followed by NBS (1.2 eq.) at room temperature. The reaction temperature was raised to 60°C and stirring was continued for 16 hours. The reaction mixture was filtered and the solvent was removed under reduced pressure. Crude material 20 was used in the next step without further purification.

The compound of general structure 20 (1 eq.) and alkylamine (2 eq.) in THF were stirred at 70°C for 16 hours. The reaction mixture was concentrated under reduced pressure and the material was purified by

- 18 043363 house of chromatography on silica gel to obtain a compound of general structure 21.

NaOH (2 eq.) was added to a reaction vessel containing a compound of general structure 21 (1 eq.) dissolved in a MeOH/H ₂ O mixture. The reaction mixture was stirred at 20°C for 16 hours. The solvent was removed under reduced pressure. The crude residue was dissolved in water (10 ml) and carefully neutralized with 6 N HCl to pH = 8. The suspension was filtered and the solids were collected and dried to give a compound of general structure 22.

General compound 22 (1 eq), HATU (1.25 eq) and DIPEA (2 eq) were dissolved in DMF and stirred at 20°C for 15 min. 1H-Indazol-5-amine (1 eq.) was added to the reaction mixture and stirred at 20°C for 15.8 hours. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine (15 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to obtain a compound with general structure 23.

General Procedure F:

25 26 27

Benzylamine (1 eq.) was dissolved in dichloromethane. The corresponding arylboronic acid 24 (1 eq.) was added to the reaction mixture, followed by glyoxylic acid (1 eq.) under a nitrogen atmosphere. The reaction mixture was heated to 40°C and the temperature was maintained for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase preparative HPLC to obtain a compound with general structure 25.

General compound 25 (1 eq), HATU (1.25 eq) and DIPEA (2 eq) were dissolved in DMF and stirred at 20°C for 15 min. 1H-Indazol-5-amine (1 eq.) was added to the reaction mixture and stirred at 20°C for 16. 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 , filtered and concentrated under reduced pressure. The crude reaction mixture was purified by normal phase chromatography on silica gel to obtain a compound with general structure 26.

Compound of general structure 26 (1 eq.) and conc. HCl (1.2 eq.) was dissolved in MeOH and 10% dry Pd/C was added. The reaction mixture was stirred under H ₂ (1 atm) at 60°C for 3 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by reverse phase preparative HPLC to obtain the desired compound of the general structure 27.

Example 2. N-(1H-undazol-5-yl)-2-(methylamino)-2-phenylacetamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-phenylacetamide as an off-white solid (80%).

^1H NMR (400 MHz, CD3OD) δ 8.34 (s, 1H), 8.27 (s, 1H), 7.68-7.65 (m, 2H), 7.60-7.53 (m , 5H), 5.1 (s, 1H), 2.69 (s, 3H). MS (ER+) m/e 281.1 (M+H).

Example 3 N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroquinoline-3carboxamide as a yellow solid (23%).

^1H NMR (400 MHz, MeOD-d3) δ 13.75 (s, 1H), 10.84 (s, 1H), 8.94 (s, 1H), 8.80 (s, 1H), 8. 26 (m, 2H), 7.71 (m, 2H), 7.28 (m, 2H), 6.63 (s, 1H), 4.21 (m, 1H), 3.97 (m, 1H ), 3.65 (m, 3H). MS (ER+) m/e 293.1 (M+H) ⁺ .

Example 4. N-(1H-undazol-5-ul)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide

- 19 043363

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-3carboxamide as a yellow solid (51%).

1H NMR (400 MHz, DMSO-d6) δ 12.99 (br s, 1H), 9.96 (s, 1H), 8.21 (s, 1H), 8.18 (s, 1H), 8 .03 (s, 1H), 7.53-7.48 (m, 2H), 7.20-7.12 (m, 3H), 7.10-7.08 (m, 1H), 4.10 -3.93 (m, 3H), 3.75-3.64 (m, 1H), 3.07-3.02 (m, 1H), 2.93-2.91 (m, 1H). MS (ER+) m/e 293.0 (M+H)+.

Example 5 N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-1carboxamide as a white solid (18%).

1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.16 (s, 1H), 8.21 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.52-7.46 (m, 2H), 7.37-7.35 (m, 1H), 7.18-7.13 (m, 3H), 4.64 (d , J=4.0 Hz, 1H), 3.25-3.22 (m, 1H), 2.97-2.81 (m, 2H), 2.75-2.65 (m, 1H). MS (ER+) m/e 293.1 (M+H)+.

Example 6 N-(1H-indazol-5-yl)isoindoline-1-carboxamide

The reaction was carried out following General Protocol B. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)isoindolin-1-carboxamide as a white solid (10%).

1H NMR (400 MHz, DMSO- _d6 ) δ 12.99 (br s, 1H), 10.09 (s, 1H), 8.18 (s, 1H), 8.01 (s, 1H), 7.56-7.44 (m, 3H), 7.34-7.20 (m, 3H), 4.96 (br. s, 1H), 4.40-4.26 (m, 2H), 3.79 (br. s, 1H). MS (ER+) m/e 279.1 (M+H)+.

Example 7 N-(2-(dimethylamino)ethyl)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give K-(2-(dimethylamino)ethyl)-K-(1H-indazol-5-yl)-2-(3methoxyphenyl)-2-(methylamino )acetamide as a white solid (50%).

1H NMR (400 MHz, DMSO-d6) δ 13.28 (br s, 1H), 8.07 (br s, 1H), 7.78-6.91 (m, 5H), 6.54 ( d, J= 7.6 Hz, 1H), 6.48 (s, 1H), 4.01-3.67 (m, 3H), 3.61 (s, 3H), 2.32-2.11 (m, 3H), 2.11-2.10 (m, 9H). MS (ER+) m/e 382.1 (M+H)+.

Example 8 N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide

The reaction was carried out following General Protocol C. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-4carboxamide as a light brown solid (46% ).

1H NMR (400 MHz, DMSO-d6) δ 13.01 (br s, 1H), 10.59 (s, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 8 .01 (s, 1H), 7.50-7.48 (m, 1H), 7.44-7.42 (m, 1H), 7.29-7.22 (m, 1H), 7.20 -7.13 (m, 3H), 4.05-4.00 (m, 1H), 3.95-3.85 (m, 1H), 3.79-3.75 (m, 1H), 3 .37-3.33 (m, 1H), 3.22-3.19 (m, 1H). MS (ER+) m/e 293.0 (M+H)+.

Example 9 N-(1H-indazol-5-yl)-3-methylisoindoline-1-carboxamide

The reaction was carried out following general protocol B. The final residue was purified by reverse phase preparative HPLC to give N(N-indazol-5-yl)-3-methylisoindoline-1-carboxamide as a white solid (29%).

1H NMR (400 MHz, DMSO-Y6) δ 12.99 (br s, 1H), 10.04 (s, 1H), 8.15 (s, 1H), 8.02 (s, 1H), 7 .53-7.42 (m, 3H), 7.35-7.21 (m, 3H), 4.89 (s, 1H), 4.58-4.57 (m, 1H), 4.01 (s, 1H), 1.47 (d, J=6.4 Hz, 3H). MS (ER+) m/e 293.0 (M+H) ⁺ .

Example 10. N-(1H-indazol-5-yl)pyrrolidin-2-carboxamide n

_Nv

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)pyrrolidin-2-carboxamide as a white solid (33%). MS (ER+) m/e 231.1 (M+H) ⁺ .

Example 11 N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)pyrrolidin-3-carboxamide as a white solid (41%). MS (ER+) m/e 231.1 (M+H) ⁺ .

Example 12. (2S,5R)-N-(1H-indazol-5-yl)-5-phenylpyrrolidine-2-carboxamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give (2S,5R)-N-(1H-indazol-5-yl)-5-phenylpyrrolidine-2carboxamide as a white solid (26%). MS (ER+) m/e 307.0 (M+H) ⁺ .

Example 13 N-(1H-indazol-5-yl)piperidine-2-carboxamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)piperidin-2-carboxamide as a white solid (39%). MS (ER+) m/e 245.0 (M+H) ⁺ .

Example 14 N-(1H-indazol-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2(methylamino)acetamide as white solids (7%).

^1H NMR (400 MHz, DMSO-I6) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7, 47 (s, 2H), 7.45 (d, J=8.8 Hz, 2H), 7.00-6.93 (m, 4H), 6.89 (d, J=8.4 Hz, 2H ), 4.20 (d, J=7.2 Hz, 1H), 3.75 (s, 3H), 2.60 (m, 1H), 2.30 (d, J=4.8 Hz, 3H ). MS (ER+) m/e 403.1 (M+H) ⁺ .

Example 15 N-(1H-indazol-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

The reaction was carried out following General Protocol A. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2(methylamino)acetamide as white solids (8%).

1H NMR (400 MHz, DMSO-I6) δ 12.98 (s, 1H), 10.08 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.51 -7.48 (m, 4H), 7.27 (t, J=8.4 Hz, 1H), 7.00 (d, J=8.4 Hz, 2H), 6.72 (dd, J= 8.4, 2.0 Hz, 1H), 6.58 (t, J=2.4 Hz, 1H), 6.53 (dd, J=8.0, 2.4 Hz, 1H), 4, 23 (s, 1H), 3.73 (s, 3H), 2.31 (s, 3H). MS (ER+) m/e 403.1 (M+H) ⁺ .

- 21 043363

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2(methylamino)acetamide as a white solid (7% ).

1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.10 (s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.52 -7.40 (m, 6H), 4.24 (s, 1H), 2.72 (m, 1H), 2.28 (s, 3H). MS (ER+) m/e 315.0 (M+H) ⁺ .

Example 17 2-(4-Chloropheni)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(4-chloropheni)-2-(ethylamino)-N-(1H-indazol-5yl)acetamide as a white solid (12%) .

^1H NMR (400 MHz, DMSO) δ 12.97 (s, 1H), 10.13 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.01 ( s, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.49-7.39 (m, 4H), 4.39 (s, 1H), 2.51-2.50 (m, 2H), 1.08 (t, J=6.4 Hz, 3H). MS (ER+) m/e 329.1 (M+H) ⁺ .

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide as a white solid (49 %).

1H NMR (400 MHz, DMSO) δ 12.99 (s, 1H), 10.18 (s, 1H), 8.21 (s, 1H), 8.09 (d, J= 1.2 Hz, 1H ), 8.00 (d, J=0.8 Hz, 1H), 7.52-7.45 (m, 3H), 7.44-7.39 (m, 3H), 4.51 (s, 1H), 2.70-2.66 (m, 1H), 1.03 (t, J=6.4 Hz, 1H). MS (ER+) m/e 343.1 (M+H) ⁺ .

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2(methylamino)acetamide as a white solid (11% ).

1H NMR (400 MHz, DMSOL) δ 13.08 (s, 1H), 10.64 (s, 1H), 9.59-9.31 (m, 2H), 8.07 (d, J=9, 2 Hz, 2H), 7.67-7.64 (m, 2H), 7.52 (d, J=8.8 Hz, 1H), 7.41-7.34 (m, 3H), 5, 04 (s, 1H), 2.52 (s, 3H). MS (ER+) m/e 299.0 (m+n) ⁺ .

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide as a white solid (54 %).

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.99 (s, 1H), 10.17 (s, 1H), 8.20 (s, 1H), 8.10 (d, J=1.2 Hz, 1H), 8.00 (d, J=0.8 Hz, 1H), 7.52 (dd, J=8.4, 5.6 Hz, 2H), 7.49-7.39 (m , 2H), 7.17 (t, J=8.8 Hz, 2H), 4.51 (s, 1H), 2.72-2.66

- 22 043363 (m, 1H), 1.04 (t, J=6.8 Hz, 6H). MS (ER+) m/e 327.1 (M+H)+.

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(ethylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide as a white solid (39%).

^1H NMR (400 MHz, DMSO- _d6 ) δ 13.00 (s, 1H), 10.17 (s, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 8 .00 (s, 1H), 7.52-7.45 (m, 4H), 7.35 (t, J=7.2 Hz, 2H), 7.30-7.28 (m, 1H), 4.43 (s, 1H), 2.63-2.52 (m, 2H), 1.09 (t, J=7.2 Hz, 3H). MS (ER+) m/e 295.0 (M+H) ⁺ .

Example 22 N-(1H-indazol-5-yl)-2-(isopropylamino)-2-phenylacetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(isopropylamino)-2phenylacetamide as a white solid (63%).

1H NMR (400 MHz, DMSO-66) δ 12.99 (s, 1H), 10.17 (s, 1H), 8.20 (s, 1H), 8.11 (d, J=1.2 Hz , 1H), 8.00 (d, J=0.8 Hz, 1H), 7.52-7.41 (m, 4H), 7.34 (t, J=7.2 Hz, 2H), 7 .30-7.27 (m, 1H), 4.51 (s, 1H), 2.72-2.69 (m, 1H), 1.04 (dd, J=7.6, 6.4 Hz , 6H). MS (ER+) m/e 309.1 (M+H) ⁺ .

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(ethylamino)-2-(4-fluorophenyl)-N-(1H-indazol-5yl)acetamide as a white solid (8%) .

¹ H NMR (400 MHz, CD ₃ OD) δ 8.45 (s, 1H), 8.09 (d, J=1.2 Hz, 1H), 8.00 (s, 1H), 7.67- 7.61 (m, 2H), 7.50 (d, J=8.8 Hz, 1H), 7.42 (dd, J=8.8, 1.6 Hz, 1H), 7.26-7 .19 (m, 2H), 4.82 (s, 1H), 2.96-2.88 (m, 2H), 1.29 (t, J=7.2 Hz, 3H). MS (ER+) m/e 313.1 (M+H) ⁺ .

Example 24 N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following 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 (4% ).

1H NMR (400 MHz, DMSO- _d6 ) δ 12.96 (s, 1H), 10.01 (s, 1H), 8.12 (s, 1H), 7.99 (s, 1H), 7. 46 (s, 2H), 7.39 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 4.15 (s, 1H), 3, 72 (s, 3H), 2.28 (s, 3H). MS (ER+) m/e 311.1 (M+H)+.

Example 25 N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(4-methoxyphenyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(4methoxyphenyl)acetamide as a white solid (52%) .

^1H NMR (400 MHz, DMSO- _d6 ) δ 12.98 (s, 1H), 10.11 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 8 .00 (s, 1H), 7.48-7.39 (m, 4H), 6.90 (d, J=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),

- 23 043363

1.02 (d, J=6.4 Hz, 3H). MS (ER+) m/e 339.1 (M+H) ⁺ .

Example 26 2-(Ethylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

The reaction was carried out following 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 %).

^1H NMR (400 MHz, DMSO- _d6 ) δ 12.97 (s, 1H), 10.10 (s, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 8 .00 (s, 1H), 7.50-7.41 (m, 4H), 6.91 (d, J=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 (ER+) m/e 325.1 (M+H) ⁺ .

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

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(4methoxyphenyl)acetamide as a white solid (10%) .

1H NMR (400 MHz, DMSO- _d6 ) δ 12.95 (s, 1H), 10.07 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 7. 99 (d, J=0.8 Hz, 1H), 7.49-7.41 (m, 2H), 7.39 (d, J=8.8 Hz, 2H), 6.89 (d, J =8.8 Hz, 2H), 4.39 (s, 1H), 3.72 (s, 3H), 2.05-2.00 (m, 1H), 0.42-0.33 (m, 4H). MS (ER+) m/e 337.0 (M+H) ⁺ .

Example 28 N-(1H-indazol-5-yl)-2-(methylamino)-2-(n-tolyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(n-tolyl)acetamide as a white solid (7 %).

1H NMR (400 MHz, DMSO) δ 12.97 (s, 1H), 10.04 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.46 (s , 2H), 7.36 (d, J=8.0 Hz, 2H), 7.14 (d, J=8.0 Hz, 2H), 4.19 (s, 1H), 2.28 (d , J=7.2 Hz, 6H). MS (ER+) m/e 295.1 (M+H) ⁺ .

Example 29 N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(n-tolyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(itolyl)acetamide as a white solid (44%) .

1H NMR (400 MHz, DMSO- _d6 ) δ 12.98 (s, 1H), 10.17 (s, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 8. 00 (s, 1H),

7.48-7.37 (m, 4H), 7.16 (d, J=7.6 Hz, 2H), 4.51 (s, 1H), 2.76-2.70 (m, 1H) , 2.27 (s, 3H), 1.05 (dd, J=8.4, 6.4 Hz, 6H). MS (ER+) m/e 323.1 (M+H) ⁺ .

Example 30 2-(Ethylamino)-N-(1H-indazol-5-yl)-2-(n-tolyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(ethylamino)-N-(1H-indazol-5-yl)-2-(n-tolyl)acetamide as a white solid (13 %).

Ή NMR (400 MHz, DMSO) δ 12.97 (s, 1H), 10.08 (s, 1H), 8.24 (s, 1H), 8.10 (s, 1H), 7.99 (s , 1H),

- 24 043363

7.48-7.44 (m, 2H), 7.37 (d, J=8.0 Hz, 2H), 7.15 (d, J=8.0 Hz, 2H), 4.34 (s , 1H), 2.59-2.51 (m, 2H), 2.27 (s, 3H),

1.07 (t, J=7.2 Hz, 3H). MS (ER+) m/e 309.1 (M+H) ⁺ .

Example 31 2-(Cyclopropylamino)-N-(1H-indazol-5-yl)-2-(n-tolyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(ntolyl)acetamide as a white solid (23%) .

1H NMR (400 MHz, DMSO- _d6 ) δ 12.96 (s, 1H), 10.07 (s, 1H), 8.22 (s, 1H), 8.11 (s, 1H), 7. 99 (s, 1H), 7.48-7.42 (m, 2H), 7.35 (d, J=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 (ER+) m/e 321.1 (M+H) ⁺ .

Example 32 N-(1H-indazol-5-yl)-2-(2'-methoxy-[1,1'-biphenyl]-4-yl)-2-(methylamino)acetamide

The reaction was carried out following 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%).

1H NMR (400 MHz, DMSO-d ₆ ) δ 13.00 (br s, 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 (ER+) m/e 387.1 (M+H) ⁺ .

Example 33 2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was carried out following 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%).

1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.14 (s, 1H), 8.31 (s, 2H), 8.14 (s, 1H), 8.00 (s, 1H), 7.60 (d, J=8.4 Hz, 2H), 7.54-7.48 (m, 5H), 7.42-7.38 (m, 1H), 7. 33-7.26 (m, 2H), 4.30 (s, 1H), 2.33 (s, 3H). MS (ER+) m/e 375.1 (M+H) ⁺ .

Example 34 2-((2-(Dimethylamino)ethyl)amino)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Protocol D. The final residue was purified by reverse phase preparative HPLC to give 2-((2-(dimethylamino)ethyl)amino)-N-(1H-indazol-5-yl)-2(3-methoxyphenyl)acetamide as a white solid (27%).

1H NMR (400 MHz, DMSO-d ₆ ) δ 13.01 (br s, 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 (ER+) m/e 368.1 (M+H)+.

Example 35 N-(1H-indazol-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following general protocol D. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(2-methoxyphenyl)-2-(methyl- 25 043363 amino)acetamide as pink solids (10%).

^1H NMR (400 MHz, DMSO-66) δ 13.03 (br s, 1H), 9.99 (br s, 1H), 8.20 (s, 2H), 8.13 (s, 1H ), 8.01 (s, 1H), 7.52-7.46 (m, 2H), 7.40 (dd, J=7.6, 1.6 Hz, 1H), 7.32-7, 27 (m, 1H), 7.03 (d, J=8.0 Hz, 1H), 6.96 (t, J=7.2 Hz, 1H), 4.59 (s, 1H), 3, 83 (s, 3H), 2.34 (s, 3H). MS (ER+) m/e 311.0 (M+H) ⁺ .

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

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(4methoxyphenyl)acetamide as a light brown solid (35%).

¹ H NMR (400 MHz, DMSO-66) δ 10.69 (s, 1H), 9.14 (m, 2H), 8.06 (s, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.8 Hz, 1H), 7.34 (dd, J=8.8, 2.0 Hz, 1H), 7.07 (d, J=8 .8 Hz, 2H), 5.20-5.18 (m, 1H), 3.78 (s, 3H), 1.32 (s, 9H). MS (ER+) m/e 353.1 (M+H)+.

Example 3 7. 2-(tert-Butylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2phenylacetamide as a purple solid (4%).

1H NMR (400 MHz, DMSO-66) 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 (ER+) m/e 323.1 (M+H) ⁺ .

Example 3 8. 2-(tert-Butylamino)-N-( 1 H-indazol-5 -yl)-2-(n-tolyl)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(ntolyl)acetamide as a white solid (12 %).

1H NMR (400 MHz, DMSO-06) 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 (ER+) m/e 337.1 (M+H) ⁺ .

Example 39 N-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-3-yl)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(niridin-3yl)acetamide as a white solid (9 %).

1H NMR (400 MHz, DMSO-06) δ 13.02 (br s, 1H), 10.21 (s, 1H), 8.69 (d, J=1.6 Hz, 1H), 8.50 (dd, J=4.8, 1.6 Hz, 1H), 8.24 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.90-7, 88 (m, 1H), 7.50-7.45 (m, 2H), 7.41-7.39 (m, 1H), 4.34 (s, 1H), 2.32 (s, 3H) . MS (ER+) m/e 282.0 (M+H) ⁺ .

Example 40 N-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-2-yl)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-2

- 26 043363 yl)acetamide in the form of an off-white solid (15%).

1H NMR (400 MHz, DMSO-d6) δ 12.98 (br s, 1H), 10.19 (s, 1H), 8.54 (dd, J=4.8, 0.8 Hz, 1H) , 8.21 (s, 1H), 8.14 (d, J=1.2 Hz, 1H), 8.00 (s, 1H), 7.84-7.79 (m, 1H), 7, 60 (d, J=8.0 Hz, 1H), 7.48 (s, 2H), 7.34-7.30 (m,

1H), 4.44 (s, 1H), 2.36 (s, 3H). MS (ER+) w/e 282.0 (M+N) ⁺ .

Example 41 N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as an off-white solid (42%).

^1H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.11 (s, 1H), 8.23 (s, 1H), 8.12 (s, 1H), 8. 01 (s, 1H), 7.48-7.44 (m, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.11-7.04 (m, 2H), 6 .88-6.83 (m, 1H), 4.26 (s, 1H), 3.76 (s, 3H), 2.31 (s, 3H). MS (ER+) m/e 311.0 (M+H) ⁺ .

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

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(3-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as an off-white solid (63%).

1H NMR (400 MHz, DMSO-d6) δ 13.00 (br s, 1H), 10.16 (br s, 1H), 8.19 (s, 1H), 8.12 (s, 1H) , 8.01 (s, 1H), 7.59 (s, 1H), 7.51-7.43 (m, 3H), 7.42-7.31 (m, 2H), 4.31 (s , 1H), 2.30 (s, 3H). MS (ER+) w/e 315.0 (M+N) ⁺ .

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

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(4-ethoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as an off-white solid (59%).

1H NMR (400 MHz, DMSO- _d6 ) δ 13.02 (br s, 1H), 10.11 (br s, 1H), 8.23 (s, 1H), 8.12 (s, 1H ), 8.01 (s, 1H), 7.49-7.43 (m, 2H), 7.40 (d, J=8.8 Hz, 2H), 6.90 (d, J=8, 8 Hz, 2H), 4.26 (s, 1H), 4.00 (kv, J=6.8 Hz, 2H), 2.31 (s, 3H), 1.31 (t, J=6, 8 Hz, 3H). MS (ER+) w/e 324.1 (M+N) ⁺ .

Example 44 N-(1H-indazol-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(4-isopropoxyphenyl)-2(methylamino)acetamide as an off-white solid ( 67%).

1H NMR (400 MHz, DMSO-d6) δ 12.99 (br s, 1H), 10.10 (s, 1H), 8.23 (s, 1H), 8.14-8.13 (m, 1H), 8.01 (s, 1H), 7.47 (s, 2H), 7.38 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 4.61-4.54 (m, 1H), 4.23 (s, 1H), 2.31 (s, 3H), 1.24 (d, J=6.0 Hz, 6H). MS (ER+) w/e 339.1 (M+N) ⁺ .

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

- 27 043363

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(4'-methoxy-[1,1'-biphenyl]-4-yl) -2(methylamino)acetamide as a white solid (34%).

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.99 (br s, 1H), 10.14 (s, 1H), 8.21 (s, 1H), 8.14 (t, J=1 .6 Hz, 1H), 8.01 (s, 1H), 7.62-7.54 (m, 6H), 7.48-7.46 (m, 2H), 7.03-7.00 ( m, 2H), 4.31 (s, 1H), 3.79 (s, 3H), 2.34 (s, 3H). MS (ER+) m/e 387.0 (M+H) ⁺ .

Example 46 N-(1H-indazol-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following 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-(3methoxyphenyl)acetamide as dirty white solid (16%).

1H NMR (400 MHz, DMSO-Y6) δ 13.07 (s, 1H), 10.61 (s, 1H), 9.55 (s, 2H), 8.07 (d, J=7.6 Hz , 2H), 7.52 (d, J=8.8 Hz, 1H), 7.45-7.36 (m, 2H), 7.25-7.21 (m, 2H), 7.07- 7.04 (m, 1H), 5.07 (s, 1H), 3.80 (s, 3H), 3.78-3.59 (m, 2H), 3.31 (s, 3H), 3 .11 (m, 1H), 2.99 (m, 1H). MS (ER+) m/e 355.0 (M+H) ⁺ .

Example 47 2-(2-Chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was carried out following general protocol E. The final residue was purified by reverse phase preparative HPLC to give 2-(2-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as an off-white solid (32%).

1H NMR (400 MHz, DMSO-d6) δ 13.03 (br s, 1H), 10.14 (s, 1H), 8.17-8.14 (m, 2H), 8.02 (s, 1H), 7.617.58 (m, 1H), 7.50-7.46 (m, 3H), 7.37-7.30 (m, 2H), 4.67 (s, 1H), 2.35 (s, 3H). MS (ER+) m/e 315.0 (M+H) ⁺ .

Example 48 2-(4-Fluoro-3-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(4-fluoro-3-methoxyphenyl)-I-(1H-indazol-5-yl)-2(methylamino)acetamide as a white solid substances (49%).

1H NMR (400 MHz, DMSO-66) 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 (ER+) m/e 329.1 (M+H) ⁺ .

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

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(ethylamino)-X-(1H-indazol-5-yl)-2-(m-tolyl)acetamide as a yellow solid (30 %).

1H NMR (400 MHz, DMSO-I6) δ 13.08 (s, 1H), 10.66 (s, 1H), 9.44-9.37 (m, 2H), 8.10-8.06 ( m, 2H), 7.52 (d, J=8.8 Hz, 1H), 7.47-7.36 (m, 4H), 7.31 (d, J=7.6 Hz, 1H), 5.04 (t, J=6.0 Hz, 1H), 2.98-2.84 (m, 2H), 2.36 (s, 3H), 1.23 (t, J=7.2 Hz , 3H). MS (ER+) m/e 309.1 (M+H) ⁺ .

Example 50 X-(1H-indazol-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)phenyl)acetamide

- 28 043363

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)phenyl)acetamide as white solids (27%).

1H NMR (400 MHz, DMSO-06) δ 13.00 (br s, 1H), 10.18 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 8 .01 (s, 1H), 7.54-7.44 (m, 5H), 7.30-7.28 (m, 1H), 4.34 (s, 1H), 2.30 (s, 3H ). MS (ER+) m/e 365.0 (M+H) ⁺ .

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

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(ethylamino)-2-(3-fluorophenyl)-N-(1H-indazol-5yl)acetamide as a white solid (21%) .

1H NMR (400 MHz, DMSO-06) δ 12.99 (s, 1H), 10.16 (s, 1H), 8.18 (s, 1H), 8.11 (d, J=0.8 Hz , 1H), 8.01 (d, J=0.8 Hz, 1H), 7.50-7.33 (m, 5H), 7.14-7.09 (m, 1H), 4.43 ( s, 1H), 2.60-2.53 (m, 2H), 1.09 (t, J=7.2 Hz, 3H). MS (ER+) m/e 313.1 (M+H) ⁺ .

Example 52 N-(1H-indazol-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide as a white solid (11 %).

^1H NMR (400 MHz, DMSO-06) δ 12.99 (s, 1H), 10.11 (s, 1H), 8.20 (s, 2H), 8.13 (s, 1H), 8. 01 (s, 1H), 7.49-7.45 (m, 2H), 7.32-7.23 (m, 3H), 7.11 (d, J=7.2 Hz, 1H), 4 .25 (s, 1H), 2.31 (s, 6H). MS (ER+) m/e 295.1 (M+H) ⁺ .

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

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(3-fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as a white solid (11 %).

1H NMR (400 MHz, DMSO-06) δ 12.99 (s, 1H), 10.14 (s, 1H), 8.21 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50-7.32 (m, 5H), 7.14-7.10 (m, 1H), 4.30 (s, 1H), 2.30 (s, 3H). MS (ER+) m/e 299.1 (M+H) ⁺ .

Example 54 (R)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give (R)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (17%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AD column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO ₂ , flow rate 70 g/min to obtain the desired compound at 99% enantiomeric purity.

1H NMR (400 MHz, DMSO-46) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.47 (s, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.09-7.05 (m, 2H), 6.85 (dd, J=8.0, 2.0 Hz, 1H), 4.21 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H). MS (ER+) m/e 311.1 (M+H) ⁺ . [a] ²⁵ ° ^C _D = +98.86 (c = 0.2 in MeOH).

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

- 29 043363

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give (S)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (12%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AD column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO ₂ , flow rate 70 g/min to obtain the desired compound at 99% enantiomeric purity.

^1H NMR (400 MHz, DMSO- _d6 ) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7 .47 (s, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.09-7.05 (m, 2H), 6.85 (dd, J=8.0, 2 .4 Hz, 1H), 4.20 (s, 1H), 3.75 (s, 3H), 2.30 (s, 3H). MS (ER+) m/e 311.1 (M+H) ⁺ . [a] ²⁵ ° ^C d = -96.44 (c = 0.2 in MeOH).

Example 56. N-(1H-indazol-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide

OCF ₃

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give N-(1H-indazol-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide as a brown solid substances (19%).

^1H NMR (400 MHz, DMSO- _d6 ) δ 12.99 (s, 1H), 10.15 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 8 .01 (s, 1H), 7.62 (d, J=8.8 Hz, 2H), 7.50-7.45 (m, 2H), 7.36 (d, J=8.4 Hz, 2H), 4.30 (s, 1H), 2.30 (s, 3H). MS (ER+) m/e 365.1 (M+H) ⁺ .

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

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(3-fluoro-4-methoxyphenyl)-N-(1H-indazol-5-yl)-2(methylamino)acetamide as a white solid substances (29%).

^1H NMR (400 MHz, DMSO- ₀₆ ) δ 12.99 (s, 1H), 10.07 (s, 1H), 8.18 (s, 1H), 8.11 (s, 1H), 8 .01 (s, 1H), 7.49-7.44 (m, 2H), 7.35 (dd, J=12.8, 1.2 Hz, 1H), 7.25 (d, J=8 .4 Hz, 1H), 7.16-7.12 (m, 1H), 4.21 (s, 1H), 3.82 (s, 3H), 2.28 (s, 3H). MS (ER+) m/e 329.0 (M+H) ⁺ .

Example 58 2-(3-Chloropheni)-2-(ethylαamino)-N-(1H-inαzol-5-yl)acetamide

The reaction was carried out following General Procedure F. The final residue was purified by reverse phase preparative HPLC to give 2-(3-chloropheni)-2-(ethylamino)-N-(1H-inαzol-5yl)acetamide as a brown solid (19%) .

¹ H NMR (400 MHz, CD ₃ OD) δ 8.53 (s, 1H), 8.08 (d, J=1.2 Hz, 1H), 8.02 (s, 1H), 7.62 ( s, 1H), 7.53-7.39 (m, 5H), 4.55 (s, 1H), 2.83-2.68 (m, 2H), 1.24 (t, J=7, 2 Hz, 3H). MS (ER+) m/e 329.1 (M+H) ⁺ .

Comparative example 1.

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Comparative example 2.

Comparative example 3.

Comparative example 4.

Comparative example 5.

Comparative example 6.

Comparative example 7.

Example 59.

Determination of ROCK inhibitory activity of compounds in vitro (Z'lyte assay): Recombinant proteins ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) were purchased from ThermoFisher Scientific. The activity of the compounds was determined using the Z'-lyte kinase kit (ThermoFisher Scientific) and the IC50 was calculated.

Determination of ROCK inhibitory activity of compounds in A7R5 cells: A7R5 cells of the rat aortic smooth muscle cell line were maintained in DMEM with 10% fetal bovine serum. Cells were seeded at a density of 5000 cells/well in 96-well plates for 24 hours and then treated with test compounds for 90 minutes. Cells were then fixed and processed according to the Intracellular Colorimetric ELISA kit manual (Thermo Scientific). Cellular levels of phospho-myosin light chains (ppMlc, Thr18/Ser19) were determined after treatment with control DMSO or test compounds using an intracellular ELISA kit. The percentage level of inhibition was calculated by substituting the data obtained for 1 μM (or 10 μM) of the compound into the formula [1-(compound/DMSO)]x100%. ppMlc data obtained from a 9-point 2-fold serial dilution of compounds was plugged into the fitted nonlinear regression curve function of GraphPad Prism software to calculate the IC50 in cells.

Analysis of NIH3T3 cells using an Acta2 promoter-driven luciferase reporter gene: An NIH3T3 cell line stably expressing a luciferase reporter gene driven by the human ACTA2 promoter (-1000-1 bp) was generated (NIH3T3-Acta2-luciferase). Cells were seeded to confluency and treated with test compounds plus TGFe1 for 24 h. Cells were then lysed and luciferase activity was measured using the LightSwitch Luciferase Kit from Active Motif.

ROCK inhibitors effectively inhibit ROCK kinase activity in vitro and in cells. As illustrated in FIG. 1, the ROCK inhibitors of the invention at nanomolar concentrations of less than 20 effectively inhibited the activity of both ROCK kinase isoforms in vitro as measured using the Z'-Lyte kit.

The compounds of the invention inhibited ROCK in cells as measured by intracellular ELISA for A7R5. A7R5 cells were treated with a 9-point 2-fold serial dilution of compounds and ppMlc levels (T18/S19) were determined to calculate cellular IC50 values of compounds. The results are shown in table. 1 below.

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The ROCK inhibitors of this invention inhibit both isoforms of ROCK. The CRISPR/CAS9 system was used to generate HCT116 cells with ROCK1 or ROCK2 knockout. ROCK1KO and ROCK2KO cells were treated with the compound from Example 2 for 90 min and pMypt(T853) levels were visualized by Western blotting. The ROCK inhibitor effectively blocked phosphorylation of the ROCK target MYPT1 at 110 nM (see Fig. 2).

In table 2 below shows data on in vitro and cellular inhibition of ROCK by compounds of the invention. The activity of the compounds was determined using the Z'-lyte kinase kit (ThermoFisher Scientific). The percentage level of inhibition was calculated by normalizing the value of kinase activity obtained by treatment with 1 μM of the compound relative to the value of the DMSO control. IC50 was calculated using GraphPad Prism software and kinase activity data obtained from treatment with 9-point serial dilutions of compounds. ROCK inhibition in A7R5 cells and NIH3T3 cells (Acta2-Luc) was performed as described above.

Two measurement steps were adapted for pMLC/A7R5 IC50. Percentage inhibition levels were calculated from single dose (1 μM or 10 μM) treatments of compounds to screen for active compounds, and cellular IC50 values were then measured only for compounds that had inhibition levels greater than 50%.

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table 2

The ex. No. % ING. ROCK2 (3 µM)* 1C ₅₀ ROCK2 (nM) % ING. ROCK1 (3 µM)* IC50 ROCK1 (nM) % ING. pMLC or 1C ₅₀ (nM) A7R5 NIH3T3, Acta2-luc, 1C ₅₀ (nM) 24 98.8 3.6 99 3,892 224 321 29 98.8 6.3 99 65.7 934 25 98.8 9.7 99 10.6 633 36 98.8 9.9 99 24.7 766 15 10 13 124 219 26 98.8 12 99 24 261 845 thirty 98.8 13 99 16 502 960 16 99 @ 1 µM 15 93.2 3.8 252 915 42 16 8.8 454 882 41 99.5 @ 1 µM 17 3.2 304 539 2 98.7 21 98.8 25 304.0 885 50 93 @0.5 µM 21 93 @0.5 µM 28 98.8 21.2 99 17.78 834

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38 100 27.4 100 61.44 1342 23 95.3 43 94.3 495 1056 27 66 79 2977 48 76 @0.5 µM 71 87 @0.5 µM 43 98 @1 µM 73 4% ing. @ 10 µM 37 98.8 74.3 99 98.1 2601 5 77 38 35810 49 76 @0.5 µM 87 88 @0.5 µM 47 135 8.3 461 659 20 94.6 152 18.4 561 2507 19 96.6 160 21.4 173 1604 39 213 250 4205 17 96.3 248 93.3 < 10 556

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34 438 49% ing. @ 10 µM 40 453 143 37% ing. @ 10 µM 44 455 42% ing. @ 10 µM 7 eleven @ 1 µM 1628 16 @0.5 µM 2% ing. @ 1 µM 4 > 1000 > 1000 18% ing. @ 10 µM 31 > 1000 228 35% ing. @ 10 µM 6 > 1000 421 1% ing. @ 10 µM 32 > 1000 310 5% ing. @ 10 µM 21 95.8 91.5 2386 22 96.7 94.3 1802 18 94.1 89.6 2507 8 76.7 @ 1 µM 2620 33 66.5 @1 µM 48% ing. @ 10 µM

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45 22 @1 µM 4% ing. @ 10 µM 46 67 @1 µM 27% ing. @ 10 µM 14 100 @0.5 µM 32.6 207 678 35 71 @1 µM 16% ing. @ 1 µM 3 0 0 25% ing. @ 10 µM 13 11% ing. @ 1 µM 9 4% ing. @ 1 µM 10 1% ing. @ 1 µM AND 1% ing. @ 1 µM 12 1% ing. @ 1 µM 51 53 1660 5453 52 34 532 1512 53 41 914 1159

-36043363

54 14 200 268 55 thirty 589 773 56 46 546 666 57 95% @0.5 µM 25 339 473 58 61% @0.5 µM 64 85% @0.5 µM 722 1090 Connection 1 85.7 @ 1 µM 10.6 34% ing. @ 10 µM Connection 2 97.5 92.2 4410 Connection 3 97.4 91.5 10920 Connection 4 96.5 @ 1 µM 95.8 6158 Connection 5 96.2 @ 1 µM 33 99 1310 Connection 6 98.8 41.76 99 41.32 6990 Connection 7 100 303 100 166 18390

*% inhibition in the ROCK1/ROCK2 assay was tested at 3 μM unless otherwise stated.

The comparative examples lack the alkyl substituent on the 2-amino group (ie, in the comparative compounds the 2-amino group is NH ₂ ), which is present in the compounds of the invention. The addition of 2-alkylamino (i.e. R ¹ is alkyl, etc.) is associated with increased cellular ROCK activity, particularly for lower alkyl amines at the 2-position (i.e. R ¹ is lower alkyl) . Compare, for example, Comparative Example 2 (with a primary 2-amino) with Examples 16, 17 and 18, which have a secondary 2-alkylamino and, in particular, in which the alkyl group is small. All four compounds exhibit potent inhibition of ROCK1 and ROCK 2 in vitro, but Examples 16, 17 and 18 exhibit increased cellular ROCK inhibition as measured by phosphorylation of the ROCK target pMLC in A7R5 cells. For example, Example 2 provides cellular ROCK inhibition with an IC50 greater than 17 times lower than Comparative Example 1 (IC50 of 252 and 4410, respectively). Likewise, the cellular IC50s for Examples 24, 25, 26, 27 and 36 are lower than for Comparative Example 4, which has a primary 2-amino group.

Example 59.

ROCK inhibitors improve oligodendrocyte process in human oligodendrocyte/neuron progenitor cells in culture.

Human oligodendrocyte/neuron progenitor cells were cultured in vitro with or without ROCK inhibitor for 2 and 14 days. Nestin and MAP2 proteins were visualized by staining with commercial antibodies to determine the different stages of neuronal cell differentiation. The ROCK inhibitors of the present invention significantly facilitated the expression of the mature neuronal cellular marker MAP2 while improving neurite outgrowth, as evidenced by the strong increase in MAP2 signal in cells differentiated in the presence of the ROCK inhibitor (compound of Example 2). See fig. 3.

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Example 60.

ROCK inhibitor improved neurite extension and axonal sheathing in oligodendrocytes in a coculture system with rat dorsal root ganglia (DRG) explants. Under co-culture conditions of rat oligodendrocytes with rat dorsal root ganglion (DRG) explants, treatment with ROCK inhibitor, as illustrated in FIG. 4, changed the organization of the cytoskeleton with the formation of a large number of shorter, ordered myelin segments, which were identified by neurofilament staining. At the same time, ROCK inhibitor also facilitated axonal support of oligodendrocytes in an orderly manner, as visualized by staining for the mature oligodendrocyte marker MBP.

Example 61.

ROCK inhibitors cross the blood-brain barrier.

In pharmacokinetic studies in mice, animal tissue was collected 2 hours after dosing to determine compound distribution. As demonstrated in Table. 3, the compound of Example 2 had excellent BBB penetration properties.

Table 3

ID connections Path Dose (mg active substance/kg) Plasma concentration [ng/ml]* Brain concentration [ng/g] * Brain/plasma ratio EXAMPLE 2 IV 10 211 699 3.31 By 10 405 700 1.73 By thirty 1400 2856 2.04 By 100 9288 19363 2.08

*Plasma and brain tissue were obtained 2 hours after dosing.

Brain and plasma concentrations of selected ROCK inhibitors were also assessed in mice by HPLC/MS/MS 15 min and 2 h after IV administration of 2.5 mg/kg drug. The results are shown in table. 4 below.

Table 4

ID connections Dose level Path Place Average CONC. after 0.25 hours (ng/ml) or (ng/g) Average END, after 2 hours (ng/ml) or (ng/g) Average brain/plasma ratio at 0.25 h Average brain/plasma ratio at 2 hours EXAMPLE 15 2.5 IV Plasma 394 41.4 2.17 6.99 Head brain 853 288 EXAMPLE 41 2.5 IV Plasma 427 2.14 1.33 8.41 Head brain 566 17.7 Head brain 541 76.6

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Claims (17)

Bibliography Deyts, S., Galan-Rodriguez, V., 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, 90108. 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, Rl 15-123, 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. CLAIM 1. A compound having the formula R ² R ³ or a pharmaceutically acceptable salt thereof, where R ¹ represents C ₁ -C ₄ -alkyl, cyclopropyl or -(C ₂ . ₄ alkyl)-OSH ₃ ; R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C1C ₄ -alkyl, C ₁ -C ₄ -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; R ³ is selected from H, C ₁ -C ₄ -alkyl, substituted C ₁ -C ₄ -alkyl and RR'N-(C ₂ . ₄ alkyl)-; R ⁴ is selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RR'N-(C ₂ . ₄ alkyl) and RR'N-(C ₂ . ₄ alkyl)-O-; b is from 0 to 2 and each R and R' are independently selected from H, C ₁ -C ₄ -alkyl and C ₃ -C ₆ -cycloalkyl; where aryl represents a 6-membered single-ring aromatic group; heteroaryl is a 5- or 6-membered single-ring aromatic group that may contain from one to four heteroatoms; a heteroatom is nitrogen, oxygen or sulfur; substituted C ₁ -C ₄ -alkyl is a C ₁ -C ₄ -alkyl having from one to three substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkoxy, amino, nitro , cyano, perfluoro C ₁ -C ₄ -alkyl, perfluoro C ₁ -C ₄ -alkoxy and carboxyl. 2. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, where R ¹ represents C ₁ -C ₄ -alkyl; R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C1C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ -C ₃ -perfluoroalkyl and C ₁ -C ₃ -perfluoroalkoxy; R ³ is selected from H or RR'N-(C _2-4 alkyl)- and b is 0. 3. A compound according to claim 1, having formula VI or a pharmaceutically acceptable salt thereof, wherein each R ²¹ is independently selected from the group consisting of H, halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ - C ₃ -perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3. 4. A compound according to claim 3, having formula VII or a pharmaceutically acceptable salt thereof. 5. A compound according to claim 1, having formula VIII or a pharmaceutically acceptable salt thereof, where R ¹ represents C ₁ -C ₄ -alkyl; each R ²¹ is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ -alkyl, C 1 -C 4 -alkoxy, nitro, cyano, C ₁ -C ₃ -perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, carboxyl, RR' N-, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; and n is from 0 to 3. 6. A compound according to claim 1, having formula IX or a pharmaceutically acceptable salt thereof, wherein R ¹ represents C ₁ -C ₄ -alkyl; each R ²² is independently selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ alkyl, C 1 -C 4 alkoxy, amino, C ₁ -C ₃ perfluoroalkyl and C ₁ -C ₃ perfluoroalkoxy; and n is from 0 to 3. 7. A compound having the formula where R ¹ represents C ₁ -C ₄ -alkyl, C ₃ -C ₆ -cycloalkyl or R ¹⁰ O(CH2)c- or alternatively R ¹ - 40 043363 may mean R ¹⁰ R ⁿ N(CH2) _c - when R2 represents phenyl, which may be unsubstituted or substituted with methoxy; R ¹⁰ and R ¹¹ are C ₁ -C ₄ -alkyl; R ² represents phenyl or pyridyl, each of which may be unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, C1C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ -C _3- perfluoroalkyl, C ₁ -C ₃ -perfluoroalkoxy, aryl and aryl-O-, where the aryl and -O-aryl substituent, in turn, may be unsubstituted or substituted with C ₁ -C ₄ -alkoxy; R ³ is selected from H, C ₁ -C ₄ -alkyl and RR'N-(C _2-4 alkyl)-; each R and R' are independently selected from H, C1- _C4 -alkyl and _C3 - _C6 -cycloalkyl and c is from 2 to 4; or a pharmaceutically acceptable salt thereof; where aryl is a 6-membered single ring aromatic group 8. A compound according to claim 7, having the formula where R ¹ represents C ₁ -C ₄ -alkyl or cyclopropyl; each R ²² is selected from halo, C ₁ -C ₄ -alkyl and C ₁ -C ₄ -alkoxy; n is from 0 to 2; or a pharmaceutically acceptable salt thereof. 9. A compound according to claim 8, where R ¹ means C ₁ -C ₄ -alkyl and n is 1 or 2. 10. A pharmaceutical composition containing a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, formulated with one or more pharmaceutically acceptable carriers. 11. Pharmaceutical composition according to claim 10, formulated in the form of a solid dosage form for oral administration. 12. Use of a composition according to claim 10 or 11 for the preparation of a medicament for the treatment of a fibrotic disorder in a subject. 13. Use according to claim 12, wherein the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, liver cirrhosis, myocardial fibrosis, arterial fibrosis, endomyocardial fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma or systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars, glial scarring and renal fibrosis. 14. Use of a composition according to claim 10 or 11 for the preparation of a medicament for treating an autoimmune disorder, a cardiovascular disorder or a central nervous system disorder in a subject. 15. Use according to claim 14, wherein the autoimmune disorder, cardiovascular disorder or central nervous system disorder is selected from Crohn's disease, previous myocardial infarction, arterial stiffness, atherosclerosis, restenosis, cerebral vasospasm, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ABS), Batten disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, neuronal degeneration, spinal cord injury and multiple sclerosis. 16. Use of a composition according to claim 10 or 11 for the preparation of a medicament for the treatment of glaucoma in a subject. 17. Use of a composition according to claim 10 or 11 for the preparation of a medicament for treating inflammation in a subject. -