EA046894B1 - INHIBITORS OF RHO-ASSOCIATED SUPERCOIL PROTEIN KINASE - Google Patents

Description

Technical field of the invention

This invention relates to compounds that are inhibitors of ROCK1 and/or ROCK2. The use of ROCK1 and/or ROCK2 inhibitors for the treatment of the disease has also been proposed.

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, ROCK1 and ROCK2. 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. Rho kinase 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 the organization and contractility of actin filaments. In smooth muscle cells, ROCK mediates calcium sensitivity and smooth muscle contraction. Inhibition of ROCK blocks 5-HT and epinephrine-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 activation of the transport system with Na/H metabolism, formation of stress fibers, 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 Rho kinase 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 β-amyloid peptides have been associated with loss of synaptic plasticity

- 1 046894 sti and dysfunction of the neural network. 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 ROCK knockdown reduced Av levels. 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):335570.

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

In one aspect, this invention provides a compound having Formula I

Where

A represents

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R ¹ is selected from the group consisting of lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, R ¹⁰ R ¹¹ N(CR ¹ 2R ¹³ )c-, R ¹⁰ O(CR ¹² R ¹³ )c -, W(CR ¹² R ¹³ )d- and R ¹⁰ R ¹¹ NC(=O)-(CR ¹² R ¹³ ) _c -;

each R ¹⁰ is independently selected from H, lower alkyl and C3-C6 cycloalkyl; each R ¹¹ is independently selected from H, lower alkyl and C3-C6 cycloalkyl; each R ¹² is independently selected from H and lower alkyl; each R ¹³ is independently selected from H and lower alkyl; in an additional or alternative embodiment, R ¹² and R ¹³ attached to the same carbon atom may together form a C3-C6 cycloalkyl group; W represents a 3-7 membered heterocyclic ring having 1 to 3 ring heteroatoms, or a 5-6 membered heteroaryl ring having 1 to 3 ring heteroatoms; c is equal to 2 to 4; d is from 1 to 4;

R ² is selected from the group consisting of aryl, heteroaryl and heterocyclyl, where heteroaryl and heterocyclyl may be 5- or 6-membered monocyclic or 9- or 10-membered bicyclic rings having 1 to 3 ring heteroatoms, and each of aryl, heteroaryl and heterocyclyl may be optionally substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, RR'N -, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-;

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

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

R ⁵ is selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl;

alternatively, R ³ and R ⁵ together with the nitrogen atoms to which they are attached form a cyclic group having from 5 to 7 ring atoms, including 2-3 ring heteroatoms, and the cyclic group is unsubstituted or substituted with 1-3 substituents , independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, carboxyl, aryl and heteroaryl;

a is equal to 0 or 1;

b is from 0 to 2 and each R and R' are independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl or, alternatively, R and R' together with the nitrogen atom to which they are attached form 5-6 -membered heterocyclic ring;

where lower alkyl represents C _1-4 alkyl;

substituted lower alkyl is a C _1-4 alkyl having 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, RR'NCO- and carboxyl;

lower alkoxy is -O-C _1-4 alkyl;

carboxyl represents -CO ₂ H;

substituted C3-C6 cycloalkyl is a C3-C6 cycloalkyl having one to three substituents selected from the group consisting of halogen, hydroxy, _C1-4 alkyl, C1- _C4 alkoxy, amino, nitro, cyano, perfluoro C1- C4 alkyl, perfluoro C1-C ₄ alkoxy and carboxyl;

aryl is phenyl;

heteroaryl, unless otherwise stated, is a 5- or 6-membered aromatic group containing from one to four heteroatoms; and the heteroatom is nitrogen, oxygen or sulfur;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the present invention provides a compound that has formula II

R ² R ³ (II) or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the present invention there is provided a compound which has the formula V

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Where

R ¹ is selected from the group consisting of lower alkyl and substituted lower alkyl;

each R ²¹ is independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C3 perfluoroalkyl, C ₁ -C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH- , RCONR'-, RO2C-, aryl-O- and heteroaryl-O-;

each R and R' are independently selected from H, lower alkyl and C3-C6 cycloalkyl or, alternatively, R and R' together form a 5-6 membered heterocyclic ring and n is from 0 to 3;

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the present invention there is provided a compound which has formula VI

Where

R ¹ is selected from the group consisting of lower alkyl and substituted lower alkyl;

each R ²² is independently selected from halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR '-, RO2C-, aryla-O- and heteroaryl-O-;

each R and R' are independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl or, alternatively, R and R' together form a 5-6 membered heterocyclic ring;

and n is from 0 to 3;

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the present invention provides a compound that has the formula

Where

R ¹ is selected from the group consisting of lower alkyl and lower alkyl substituted -O-C _1-4 alkyl, perfluoro C ₁ -C ₄ alkoxy, -C(=O)-NH ₂ , -C(=O)-NH (C _1- ^kk3) or -C^O^N^^alkyl)^

R ²² is selected from halogen, lower alkyl, lower alkoxy and -C(=O)-NH(C _1-4 alkyl) and n is 1 or 2;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the above compound is a compound in which: R ¹ represents lower alkyl; or a pharmaceutically acceptable salt thereof.

Preferably, the above compound is a compound in which

R ⁵ represents H;

R ²² is selected from halogen, lower alkyl and lower alkoxy and n is 1;

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment of the present invention there is provided a compound which is

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N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; (R)-N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; (8)-M-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; N-(4-(l H-pyrazol-4-yl)phenyl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-methoxyphenyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(p-tolyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-fluorophenyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-methoxyphenyl)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(m-tolyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-fluorophenyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(ethylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(methylamino)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(p-tolyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(ethylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)-2-(methylamino)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide; N-(4-(l H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2-methylbenzo[c1]thiazol-6-yl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-(dimethylamino)phenyl)-2-(methylamino)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxypyridine in-4-yl)-2-(methylamino)acetamide; Methyl 3 -(2-((4-( 1 H-pyrazol-4-yl)phenyl)amino)-1 -(methylamino)-2-oxoethyl)benzoate;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)-2-(methylamino)acetamide;

-(2-((4-( 1 H-pyrazol-4-yl)phenyl)amino)-1 -(methylamino)-2-oxoethyl)benzoic acid; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methyl-1H-indazol-6-yl)-2-(methylamino)acetamide; Methyl 4(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate;

T4-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methyl-1H-benzo[(1]imidazol-6-yl)-2(methylamino)acetamide;

M-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-methoxypropyl)amino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2-(3-methoxyphenyl)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-((3 -(dimethylamino)propyl)amino)-2-(3 methoxyphenyl)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2-oxoethyl)amino)-2-(3 methoxyphenyl)acetamide; M-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3methoxyphenyl)acetamide;

T4-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3 -(methylamino)propyl)amino)acetamide; M-(4-(1H-pyrazol-4-yl)phenyl)-2-(('1-methoxy-2-methylpropan-2yl)amino)-2-3-methoxyphenyl)acetamide;

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N-(4-(l H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)-2oxoethyl)amino)acetamide;

2-(((1 H-pyrazol-4-yl)methyl)am hho)-N-(4-(1 H-pyrazol-4-yl)phenyl)-2-(3 methoxyphenyl)acetamide;

N-(4-(l H-pyrazol-4-yl)phenyl)-2-(ethyl(2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3 methoxyphenyl)acetamide;

X-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide;

M-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)-Mmethyl acetamide;

M-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-morpholinoethyl)amino)acetamide; N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl) acetamide;

N-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(pyrrolidin-1 yl)ethyl)amino)acetamide;

N-(4-( 1 H-pyrazol-4-yl)phenyl)-2-(( 1 -(methox and methyl)cyclopropyl)amino)-2-(3 methoxyphenyl)acetamide;

18G-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(2,2,2trifluoroethoxy)ethyl)amino)acetamide;

N-(3-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3 methoxyphenyl)acetamide;

N-(3-methox and-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3 methoxyphenyl)acetamide;

(K)-T4-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; (S)-N-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; (8)-18G-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3methoxyphenyl)acetamide;

(K)-M-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3methoxyphenyl)acetamide;

(K)-T\G-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(methylamino)ethyl)amino)acetamide;

(8)-M-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2methyl amino)ethyl)amino)acetamide;

(8)-M-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide;

(K)-M-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide;

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N-(2-fluoro-5-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide;

3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-M-methylbenzamide;

4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid;

4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-M-methylbenzamide; N(2-(3 -(dimethylamino)propoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide;

T\G-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide;

(K)-M-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)2-(methylamino)acetamide;

(8)-M-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)2-(methylamino)acetamide ;

or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above compounds and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention relates to the use of any of the above compounds or the above pharmaceutical compositions for treating a fibrotic disorder in a subject.

In this case, the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, cystic and idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, myocardial fibrosis, arterial fibrosis, endomyocardial fibrosis, previous myocardial infarction, arterial stiffness, atherosclerosis, restenosis, arthrofibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars, glial scarring and renal fibrosis.

In a preferred application, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

In yet another aspect, the present invention relates to the use of any of the above compounds or the above pharmaceutical compositions for treating a central nervous system disorder in a subject.

Wherein the central nervous system disorder is selected from the group consisting of Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Batten disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia, acute or chronic pain, neuronal degeneration, spinal cord damage, cerebral vasospasm and multiple sclerosis.

In another aspect, the present invention relates to the use of any of the above compounds or the above pharmaceutical compositions for the treatment of glaucoma in a subject.

In yet another aspect, the present invention relates to the use of any of the above compounds or pharmaceutical compositions for treating inflammation in a subject.

Brief description of the figures

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

Fig. 1b: ELISA analysis in A7R5 cells. 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. Representative compounds of the invention gave IC50 in cells of up to 200 nM.

Fig. 1c: ROCK inhibitors can be efficiently sequenced using a cell-based ACTA2 promoter-driven luciferase reporter gene assay. NIH3T3 cells stably expressing ACTA2 promoter-driven luciferase were seeded in 96-well plates until confluent and treated with a 9-point serial dilution of the compounds in combination in TGF$$1 for 24 h. Luciferase activity was measured and the IC50 of the compounds was calculated. Representative compounds of the invention exhibited IC50s of up to 200 nM.

Fig. 1d: Western blot of SVEC4-10 cell lysates to visualize the levels of ppMlc (T18/S19) and pMYPT (T853) after treatment with the compound of Example 2 for 120 min. SVEC4-10 cells were treated with the compound and phosphorylated proteins were visualized by Western blotting. Repre

- 7 046894 zentative compounds effectively blocked phosphorylation of the ROCK targets MLC and MYPT1 at 110 nM.

Fig. 2a and b: ROCK inhibitors dose-dependently stimulate neurite outgrowth in human oligodendrocyte/neuron progenitor cells in culture. Human oligodendrocyte/neuronal progenitor cells were cultured in vitro with or without ROCK inhibitor for 3 days.

Fig. 2c and d: ROCK inhibitors increase differentiation and maturation of neuronal cells. Human oligodendrocyte/neuron progenitor cells were cultured for 14 days. ROCK inhibitors significantly facilitated the expression of the mature neuronal cell marker beta-III tubulin and improved neurite outgrowth. Fig. 2s. Representative images; Fig. 2d. Quantification of neurite length.

Fig. 2e, f and g: ROCK inhibitors block the inhibitory effect of chondroitin sulfate proteoglycan (CSPG) on neurite outgrowth. Human oligodendrocyte/neuron progenitor cells were cultured on 0.3 mg/ml CSPG-coated plates for 3 days. Quantification of neurite length was performed using the IncuCyte NeuroTrack software module. Statistical analysis for the 72 hour time point was performed using ANOVA. Fig. 2e: Representative images; Fig. 2f: Temporal dynamics of neurite length; Fig. 2g: Quantification of neurite length 72 h after treatment.

Fig. 3a, b, c, d and f: ROCK inhibitors protected neuronal cells from Av 1-42-induced cell death. Human oligodendrocyte/neuron progenitor cells were preincubated with ROCK inhibitors for 30 min before exposure to 10.5 μM Av 1-42. Annexin V red reagent from IncuCyte was added to the culture medium to detect cell apoptosis. Neurite length and total annexin V positive area were analyzed using IncuCyte S3 software. Fig. 3a: Representative images of human oligodendrocyte progenitor cells/neurons treated with Av 1-42 in combination with or without compounds; Fig. 3b: Time course of total annexin V positive area; Fig. 3c: Temporal dynamics of neurite length; Fig. 3d: Dose dependence of total annexin V positive area; Fig. 3e: Quantification of dose-dependent neurite length.

Fig. 4: ROCK inhibitor reduced TGFe1-induced fibrotic gene expression in the mouse fibroblast cell line NIH3T3. NIH3T3 cells were treated for 24 h with TGFe1 in the presence or absence of ROCK inhibitor, and mRNA expression was quantified by Taqman qPCR. Fig. 4a: aSMA; fig. 4b. CTGF, fig. 4s. CCN1 mRNA.

Fig. 4d: ROCK inhibitor inhibited TGFe1-induced CCN1 secretion. Human lung fibroblasts CCD18Lu were treated with ROCK inhibitor and stimulated with TGFe1 for 40 h before measuring CCN1 levels by ELISA.

Fig. 5a: ROCK inhibitor is active in a mouse model of pulmonary fibrosis. Representative images of lung sections stained with Masson's trichrome to visualize fibrosis in mouse lungs after 21 days of ROCK inhibitor treatment in a model of bleomycin-induced lung injury.

Fig. 5b: Quantification of positive ROCK inhibitor activity in an in vivo model of pulmonary fibrosis. Ashcroft fibrosis scores, percentage a-smooth muscle actin (aSMA) positive area, and total leukocyte counts in bronchoalveolar lavage fluid obtained from the lungs of mice in a model of bleomycin-induced lung fibrosis treated with the compound of Example 2.

Fig. 6a: ROCK inhibitor is therapeutically active in a mouse model of pulmonary fibrosis. Representative images of lung sections stained with Masson's trichrome to visualize fibrosis in mouse lungs following therapeutic treatment with ROCK inhibitor in a model of bleomycin-induced lung injury.

Fig. 6b: Quantification of reduction in fibrosis score in histopathological analysis of images obtained from ROCK inhibitor- and vehicle-treated mice in a model of bleomycin-induced lung injury.

Fig. 7a: ROCK inhibition stabilizes endothelial barrier function in a mouse model of histamine-induced vascular permeability. Representative images of histamine-induced Evans blue dye sweating in the skin of ROCK inhibitor and basal solution-treated mice compared to the control compound (salbutamol). The ROCK inhibitor induces stabilization of the vascular barrier after administration of a single dose of the compound 1 hour before histamine injection.

Fig. 7b: Quantification of reduction in spot size and Evans blue dye amount in the skin of ROCK inhibitor-treated mice following capillary leak induction by histamine injection.

- 8 046894

Detailed Description of the Invention

ROCK inhibitors

ROCK inhibitors are compounds having Formula I

Where

A represents

R ¹ is selected from the group consisting of lower alkyl, substituted lower alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, R ¹⁰ R ¹¹ N(CR ¹² R ¹³ )c-, R ¹⁰ O(CR ¹² R ¹³ )c -, W(CR ¹² R ¹³ )d- and R ¹⁰ R ¹¹ NC(=O)-(CR ¹² R ¹³ ) _c -;

each R ¹⁰ is independently selected from H, lower alkyl and C3-C6 cycloalkyl;

each R ¹¹ is independently selected from H, lower alkyl and C3-C6 cycloalkyl;

each R ¹² is independently selected from H and lower alkyl;

each R ¹³ is independently selected from H and lower alkyl;

in an additional or alternative embodiment, R ¹² and R ¹³ attached to the same carbon atom may together form a C3-C6 cycloalkyl group;

W represents a 3-7 membered heterocyclic ring having 1 to 3 ring heteroatoms, or a 5-6 membered heteroaryl ring having 1 to 3 ring heteroatoms;

c is equal to 2 to 4;

d is from 1 to 4;

R ² is selected from the group consisting of aryl, heteroaryl and heterocyclyl, where heteroaryl and heterocyclyl may be 5- or 6-membered monocyclic or 9- or 10-membered bicyclic rings having 1 to 3 ring heteroatoms, and each of aryl, heteroaryl and heterocyclyl may be optionally substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, RR'N -, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-;

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

R ⁴ is selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C1-C3 perfluoroalkoxy, RR'N-, RR'NCO-, RCONR'-, II' \-(C _2-4 a.p<i.1a)- and II'\-(T _2-4 alki.1a)-(')-;

R ⁵ is selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl;

alternatively, R ³ and R ⁵ together with the nitrogen atoms to which they are attached form a cyclic group having from 5 to 7 ring atoms, including 2-3 ring heteroatoms, and the cyclic group is unsubstituted or substituted with 1-3 substituents , independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C 1 -C 3 perfluoroalkoxy, carboxyl, aryl and heteroaryl;

a is equal to 0 or 1;

b is from 0 to 2 and each R and R' are independently selected from H, lower alkyl and C3-C6 cycloalkyl or, alternatively, R and R' together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring;

where lower alkyl represents C _1-4 alkyl;

substituted lower alkyl is a _C1-4 alkyl having one to three substituents selected from the group consisting of halogen, hydroxy, C1-C4 alkoxy, amino, nitro, cyano, perfluoro C1-C4 alkyl, perfluoro C1-C4 alkoxy , RR'NCO- and carboxyl;

lower alkoxy is -O-C _1-4 alkyl; carboxyl represents -CO ₂ H;

substituted C3-C6 cycloalkyl is a C3-C6 cycloalkyl having one to three substituents selected from the group consisting of halogen, hydroxy, _C1-4 alkyl, C1-C4 alkoxy, amino, nitro, cyano, C1-C4 perfluoro alkyl, perfluoro C1-C ₄ -alkoxy and carboxyl;

aryl is phenyl;

heteroaryl, unless otherwise stated, is a 5- or 6-membered aromatic group containing from one to four heteroatoms; and the heteroatom is nitrogen, oxygen or sulfur; or pharmaceutically acceptable salts thereof.

As used herein, the term alkyl refers to a saturated aliphatic radical

- 9 046894 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, C1-C8 in the case of a straight chain, C3-C8 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, t-butyl, cyclopropyl and cyclobutyl.

The term cycloalkyl refers to saturated carbocyclic groups having 3-8 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 C ₃ -C ₆ cycloalkyl, refers to a cycloalkyl group as defined above having 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:

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, for example, benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, etc. Those aryl groups which 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 such same structure.

It should be understood that the substitution or superseded includes an indirect clause that such substitution

- 10 046894 is carried out in accordance with the permissible valence of the replaced atom and substituent and that the replacement leads to 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. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (O)-isomers. (b)-isomers. their racemic mixtures, as well as other mixtures thereof, are included in 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 compounds of the present invention are ROCK inhibitors. ROCK exists in two forms, ROCK1 (ROCKe; p160-ROCK) and ROCK2 (ROCKa). In some embodiments, the present compounds selectively inhibit ROCK1. In some embodiments, the present compounds selectively inhibit ROCK2. In some embodiments, the present compound is non-selective for inhibition of ROCK1 and ROCK2. In the context of this invention, selectivity means that the inhibitor exhibits an IC50 that is at least 2-fold, at least 5-fold, at least 10-fold, or at least 25-fold lower for one kinase compared to the IC50 for another kinases.

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 ΙΜΑΡβ 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 phospho group 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 IC ₅₀ of multiple compounds.

Methods of treating diseases

In one aspect of the present invention, the use of the compounds is provided for treating a patient suffering from a disease. Treatment involves administering to a patient in need of such treatment a therapeutically effective amount of a compound of this invention. As used herein, the expression "therapeutically effective amount" means an amount of a compound, material, or composition containing a compound of this invention that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in the animal's body at an acceptable benefit/risk ratio, applicable to any medical treatment, for example, if side effects are acceptable, applies to any medical treatment.

Central nervous system disorders

The compounds proposed in the present invention demonstrate effective penetration through the blood-brain barrier (BBB) and distribution throughout the tissues of the central nervous system. Thus, the compounds of the invention are useful for the treatment of disorders of the central nervous

- 11 046894 systems, as well as disorders, such as certain ocular disorders, in which the ability to cross the BBB is useful. 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, amyotrophic lateral sclerosis (ALS), 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.

Glaucoma

In an embodiment of the present invention, the proposed compounds are 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

In an embodiment of the present invention, the compounds of the invention are used to treat inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formulas I-X. Inflammation includes, but is not limited to, asthma, cardiovascular inflammation, renal inflammation, atherosclerosis and arteriosclerosis, and sepsis. Other inflammatory 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).

Pharmaceutical compositions

In one aspect, the present invention provides pharmaceutical compositions that contain a therapeutically effective amount of one or more compounds of the 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 filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium silicate talc, calcium or zinc stearate, or stearic acid). acid) or solvent-encapsulating material involved in the transfer or transport of a given compound from one organ or part of the 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

- 12 046894 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, lucoheptonate , 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 are wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, 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.

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

- 13 046894 no.

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, can optionally be scored or formulated with coatings or coatings, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. dosage forms. They can also be formulated to provide sustained or controlled release of the active ingredient, using, for example, hydroxypropyl methylcellulose in varying 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 the active ingredient(s) only or preferentially 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,

- 14 046894 lubricating agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular 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 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, etc. 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

- 15 046894 This form can be provided by the inclusion of 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 chosen route of administration, 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 dosing the compounds of the invention used in a pharmaceutical composition at levels below those required to obtain the desired therapeutic benefit.

- 16 046894 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 desired, an effective daily dose of the active compound may 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, which allows it to bypass the liver and prevents the 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 of release modifying agents. 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 intestines. 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

- 17 046894 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 (wt/wt of 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 CHsCN/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.

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 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 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 the 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.

- 18 046894

Compound of general structure 4 (1 eq.) was dissolved in CCl ₄ and AIBN (0.1 eq.) was added at room temperature, followed by NBS (1.2 eq.). 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 5 was used in the next step without further purification.

The compound of general structure 5 (1 eq.) and the 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 chromatography on silica gel to obtain the compound of general structure 6.

NaOH (2 eq.) was added to a reaction vessel containing a compound of general structure 6 (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 7.

General compound 7 (1 eq), HATU (1.25 eq) and DIPEA (2 eq) were dissolved in DMF and stirred at 20°C for 15 min. N-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 8.

Example 2. N(4-(III-pyrazol-4-yl)phenyl)-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 N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (77%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.09 (br s, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J=8, 8 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.08-7.04 (m, 2H) , 6.86 (dd, J=8.0, 2.4 Hz, 1H), 4.23 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H). MS (ER+) m/e 337.0 (M+H) ⁺ .

Example 3: (R)-N-(4-(1H-pyrazol-4-yl)phenyl)-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 N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (77%). Enantiomers were separated by SFC (Chiralpak AD-H column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO ₂ , flow rate 70 g/min. ¹ H NMR (400 MHz, DMSO-a ₆ ) δ 10.09 (br s, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J= 8.8 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.08-7.04 (m, 2H), 6.86 (dd, J=8.0, 2.4 Hz, 1H), 4.23 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H). MS (ER+) m/e 337.0 (M+H) ⁺ . [α]ρ = +102.3 (c=0.5, CH3OH, 25°C).

Example 4. ^)-M-(4-(III-pyrazol-4-yl)phenyl)-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 N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (77%). Enantiomers were separated by SFC

- 19 046894 (Chiralpak AD-H column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO2, flow rate 70 g/min. 1H NMR (400 MHz, DMSO-06) δ 10.09 (br s, 1H), 8.20 (s, 1H), 7.99 (s, 2H), 7.62 (d, J=8, 8 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.08-7.04 (m, 2H) , 6.86 (dd, J=8.0, 2.4 Hz, 1H), 4.23 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H). MS (ER+) m/e 337.0 (M+H) ⁺ . [a]D = -97.2 (c = 0.5, CH3OH, 25°C).

Example 5 2-(4-Chlorophenyl)-2-(methylamino)-N(4-(pyridin-4-yl)phenyl)acetamide

L ij ⁰

X

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

Example 6. 2-(4-Methoxyphenyl)-2-(methylamino)-^(4-(pyridin-4-yl)phenyl)acetamide

The reaction was carried out following General Protocol B. The final residue was purified by reverse phase preparative HPLC to give 2-(4-methoxyphenyl)-2-(methylamino)-^(4-(pyridin-4yl)phenyl)acetamide as an off-white solid (16%). ¹ H NMR (400 MHz, DMSO-dg) δ 10.93 (s, 1H), 9.66-9.27 (m, 2H), 8.77 (d, J=6.4 Hz, 2H), 8.02 (d, J=6.4 Hz, 2H), 7.96 (d, J=8.7 Hz, 2H), 7.78 (d,

J=8.7 Hz, 2H), 7.52 (d, J=8.8 Hz, 2H), 7.08 (d, J=8.8 Hz, 2H), 5.03 (s, 1H) , 3.79 (s, 3H), 2.67 (s, 3H). MS (ER+) m/e 348.0 (M+H) ⁺ .

Example 7. 2-(Methylamino)-N(4-(pyridin-4-yl)phenyl)-2-(p-tolyl)acetamide

The reaction was carried out following general protocol B. The final residue was purified by reverse phase preparative HPLC to give 2-(methylamino)-Na(4-(pyridin-4-yl)phenyl)-2-(ptolyl)acetamide as a white solid (28 %). 1H NMR (400 MHz, DMSO-dg) δ 10.25 (br s, 1H), 8.59 (dd, J=4.4, 1.6 Hz, 2H), 8.32 (s, 1H) , 7.81-7.76 (m, 4H), 7.68 (dd, J=4.4, 1.6 Hz, 2H), 7.37 (d, J=8.0 Hz, 2H), 7.16 (d, J=8.0 Hz, 2H), 4.21 (s, 1H), 2.29 (s, 3H), 2.28 (s, 3H). MS (ER+) m/e 332.1 (M+H) ⁺ .

Example 8. N-H-(J11-11irazol-4-yl)phe11yl)-2-(2-\1ethoxyphe11yl)-2-(>1eti.la\1u11o)a11eta\1id

The reaction was carried out following general protocol B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(2-methoxyphenyl)-2(methylamino)acetamide as a white solid substances (32%). 1H NMR (400 MHz, DMSO-dg) δ 9.98 (br s, 1H), 8.21 (s, 1H), 7.99 (s, 2H), 7.64 (d, J=8, 4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.38 (d, J=7.6 Hz, 1H), 7.30 (t, J=8.0 Hz , 1H), 7.03 (d, J=8.4 Hz, 1H), 6.96 (t, J=7.2 Hz, 1H), 4.58 (s, 1H), 3.82 (s , 3H), 2.33 (s, 3H). MS (ER+) m/e 337.1 (m+H) ⁺ .

Example 9. N(4-(III-Pyrazol-4-yl)phenyl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide

- 20 046894

The reaction was carried out following general protocol B. The final residue was purified by reverse-phase preparative HPLC to give N-(4-(111-11irazod-4-id)phe11yl)-2-(4-metho1<syphenyl)-2(methylamino)acetamide in as a white solid (32%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.60 (s, 1H), 9.46-9.30 (m, 2H), 8.01 (s, 2H), 7.59-7.50 ( m, 6H), 7.07 (d, J=8.8 Hz, 2H), 4.95-4.94 (m, 1H), 3.79 (s, 3H), 2.54 (s, 3H ). MS (ER+) m/e 337.1 (M+H) ⁺ .

Example 10 N-(4-(1H-Pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-methoxyphenyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N-(4-(111-11irazod-4-yl)phe11yl)-2-(ethylami11o)-2-(4methoxyphenyl)acetamide as a white solid substances (16%). 1H NMR (400 MHz, CD ₃ OD) δ 8.55 (s, 1H), 7.93 (s, 2H), 7.59-7.51 (m, 6H), 7.04 (d, J= 8.8 Hz, 2H), 4.77 (s, 1H), 3.83 (s, 3H), 2.98-2.83 (m, 2H), 1.30 (t, J=7.2 Hz, 3H). MS (ER+) m/e 351.1 (M+H) ⁺ .

Example 11

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(ptolyl)acetamide as a white solid (8%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.19 (s, 1H), 8.18 (s, 1H), 7.99 (s, 2H), 7.59 (d, J=8.0 Hz , 2H), 7.53 (d, J=8.0 Hz, 2H), 7.38 (d, J=7.6 Hz, 2H), 7.17 (d, J=8.0 Hz, 2H ), 4.44 (s, 1H), 2.62-2.54 (m, 2H), 2.28 (s, 3H), 1.09 (t, J=7.2 Hz, 3H). MS (ER+) m/e 335.1 (M+H) ⁺ .

Example 12 N-(4-(1H-Pyrαzol-4-yl)phenyl)-2-(ethylamino)-2-(4-fluorophenyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4fluorophenyl)acetamide as a white solid substances (20%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.76 (s, 1H), 9.53-9.47 (m, 2H), 8.01 (s, 2H), 7.69-7.60 ( m, 2H), 7.59-7.53 (m, 4H), 7.37 (dd, J=8.8, 8.8 Hz, 2H), 5.14 (s, 11), 2.96 -2.83 (m, 2H), 1.22 (t, J=7.2 Hz, 3H). MS (ER+) m/e 339.0 (M+H) ⁺ .

Example 13. N(4-(III-Pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3methoxyphenyl)acetamide as a pink solid (20%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.09 (s, 1H), 8.26 (s, 1H), 7.99 (s, 2H), 7.61 (d, J=8.4 Hz , 2H), 7.53 (d, J=8.4 Hz, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.08

- 21 046894

7.04 (m, 2H), 6.86-6.84 (m, 1H), 4.33 (s, 1H), 3.76 (s, 3H), 2.56-2.53 (m, 2H), 1.08 (t, J=7.2 Hz, 3H). MS (ER+) m/e 351.1 (M+H) ⁺ .

Example 14. N(4-(III-Pyrazol-4-yl)phenyl)-2-(methylamino)-2-(m-tolyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(mtolyl)acetamide as a white solid. (14%). ^1H NMR (400 MHz, DMSO^ ₆ ) δ 10.05 (s, 1H),

8.20 (s, 1H), 7.99 (s, 2H), 7.63-7.61 (m, 2H), 7.54-7.52 (m, 2H), 7.29-7. 09 (m, 3H), 7.10 (d, J=6.8 Hz, 1H), 4.18 (s, 1H), 2.31 (s, 3H), 2.28 (s, 3H). MS (ER+) m/e 321.1 (M+H) ⁺ .

Example 15. N(4-(III-Pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-fluorophenyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3fluorophenyl)acetamide as a white solid substances (20%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.14 (s, 1H), 8.17 (s, 1H), 8.00 (s, 2H), 7.61-7.59 (m, 2H) , 7.55-7.53 (m, 2H), 7.43-7.31 (m, 3H), 7.14-7.09 (m, 1H), 4.40 (s, 1H), 2 .56-2.53 (m, 2H), 1.07 (t, J=7.2 Hz, 3H). MS (ER+) m/e 339.1 (M+H) ⁺ .

Example 16. N(4-(III-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(ethylamino)acetamide

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

Example 17. N(4-(III-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2(methylamino)acetamide as a white solid substances (8%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.11 (s, 1H), 8.21 (s, 1H), 7.99 (s, 2H), 7.62-7.60 (m, 2H) , 7.54-7.49 (m, 4H), 7.43-7.41 (m, 2H), 4.25 (s, 1H), 2.28 (s, 3H). MS (ER+) m/e 341.0 (M+H) ⁺ .

Example 18. N(4-(III-Pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2

- 22 046894 (methylamino)acetamide in the form of a white solid (23%). 1H NMR (400 MHz, DMSOM) δ 10.14 (s, 1H), 8.18 (s, 1H), 7.99 (s, 2H), 7.63-7.60 (m, 2H), 7 .55-7.53 (m, 2H), 7.43-7.31 (m, 3H), 7.15-7.10 (m, 1H), 4.30 (s, 1h), 2.29 (c, 3H). MS (ER+) m/e 325.1 (M+n) ⁺ .

Example 19. X-(4-(III-Pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give X-(4-(N-pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2(methylamino)acetamide as dirty -white solid (25%). 1H NMR (400 MHz, DMSO-dg) δ 10.11 (s, 1H), 8.20 (s, 2H), 7.99 (s, 2H), 7.62-7.50 (m, 6H) , 7.21-7.17 (m, 2H), 4.27 (s, 1H), 2.29 (s, 3H). MS (ER+) m/e 325.1 (M+H)+.

Example 20. X-(4-(III-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give X-(4-(N-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2(methylamino)acetamide as dirty -white solid (3%). 1H NMR (400 MHz, DMSO^6) δ 10.13 (s, 1H), 8.33 (s, 2H), 7.99 (s, 2H), 7.63-7.61 (m, 2H) , 7.56-7.53 (m, 3H), 7.45-7.34 (m, 3H), 4.26 (s, 1H), 2.28 (s, 3H). MS (ER+) m/e 341.0 (M+H)+.

Example 21. X-(4-(III-Pyrazol-4-yl)phenyl)-2-(methylamino)-2-(p-tolyl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give X-(4-(N-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(ptolyl)acetamide as a yellow oil (12%). 1H NMR (400 MHz, DMSO-de) δ 12.88 (s, 1H), 10.01 (s, 1H), 8.11 (s, 1H), 7.86 (s, 1H), 7.63 -7.61 (m, 2H), 7.54-7.52 (m, 2H), 7.35 (d, J=8.0 Hz, 2H), 7.15 (d, J=8.0 Hz, 2H), 4.16 (s, 1H), 2.28 (s, 6H). MS (ER+) m/e 321.1 (M+H)+.

Example 22. X-(4-(III-Pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(ethylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give X-(4-(N-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2(ethylamino)acetamide as white solids (6%). 1H NMR (400 MHz, DMSO-66) δ 10.13 (s, 1H), 8.23 (s, 1H), 7.99 (s, 2H), 7.61-7.50 (m, 6H) , 7.43-7.41 (m, 2H), 4.38 (s, 1H), 2.57-2.54 (m, 2H), 1.07 (t, J= 7.2 Hz, 3H ). MS (ER+) m/e 355.1 (M+H)+.

Example 23. X-(4-(III-Pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)-2-(methylamino)acetamide

- 23 046894

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)2-(methylamino)acetamide as a white solid (5%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 12.86 (s, 1H), 10.03 (s, 1H), 7.99 (s, 2H), 7.60 (d, J=8.8 Hz , 2H), 7.52 (d, J=8.8 Hz, 2H), 7.32 (d, J=12.8 Hz, 1H), 7.23 (d, J=8.8 Hz, 1H ), 7.12 (t, J=8.8 Hz, 1H), 4.15 (s, 1H), 3.80 (s, 3H), 2.67 (s, 1H), 2.25 (s , 3H). MS (ER+) m/e 353.2 (M+H)+.

Example 24

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide as an off-white solid (18%). 1H NMR (400 MHz, DMSO-06) δ 10.12 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.62-7.60 (m, 2H) , 7.55-7.53 (m, 2H), 7.44-7.05 (m, 5H), 4.28 (s, 1H), 2.29 (s, 3H). MS (ER+) m/e 373.3 (M+H)+.

Example 25. N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2(3-methoxyphenyl) acetamide as a white solid (12%). 1H NMR (400 MHz, DMSO^6) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H) , 7.27 (t, J= 4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H ), 3.75 (s, 3H), 3.44 (t, J=5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J=5.2 Hz, 2H ). MS (ER+) m/e 381.3 (M+H)+.

Example 26. No. (4-(III-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(methylamino)ethyl )amino)acetamide as a white solid (29%). 1H NMR (400 MHz, CD3OD) δ 8.49 (s, 1H), 7.91 (s, 2H), 7.58-7.52 (m, 4H), 7.31 (t, J=8, 0 Hz, 1H), 7.10-7.08 (m, 2H), 6.92-6.89 (m, 1H), 4.43 (s, 1H), 3.81 (s, 3H), 3.14-3.04 (m, 2H), 2.92-2.81 (m, 2H), 2.71 (s, 3H). MS (ER+) m/e 380.3 (M+H)+.

Example 27. N(4-(III-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2-methylbenzo[d]thiazol-6-yl)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse

- 24 046894 phase preparative HPLC to obtain N(4-(III-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2methylbenzo[d]thiazol-6-yl)acetamide in the form of a white solid ( 6%). 1H NMR (400 MHz, DMSO d ₆ ) δ 10.34 (s, 1H), 8.16 (d, J=6.4 Hz, 2H), 8.00-7.94 (m, 3H), 7 .63-7.54 (m, 5H), 4.67 (s, 1H), 2.81 (s, 3H), 2.40 (s, 3H). MS (ER+) m/e 378.3 (M+H) ⁺ .

Example 28 N-(4-(1H-Pyrazol-4-yl)phenyl)-2-(3-(dimethylamino)phenyl)-2-(methylamino)acetamide

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

EXAMPLE 29

The reaction was carried out following general procedure B. The final residue was purified by reverse phase preparative HPLC to give N-N-(Ί11-11irazod-4-id)phe11id)-2-(2-metho1<ci11iridi11-4-ide)2-(methylamino )acetamide as a white solid (2%). 1H NMR (400 MHz, DMSO-dg) δ 10.12 (s, 1H), 8.15 (s, 1H), 8.12 (d, J=5.6 Hz, 1H), 7.98 (s , 2H), 7.61-7.52 (m, 4H), 7.07 (d, J=4.4 Hz, 1H), 6.91 (s, 1H), 4.24 (s, 1H) , 3.83 (s, 3H), 2.28 (s, 3H). MS (ER+) m/e 338.2 (M+H) ⁺ .

Example 30 Methyl 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give methyl 3-(2-((4-(N-pyrazol-4-yl)phenyl)amino)-1(methylamino)-2-oxoethyl) benzoate as a white solid (15%). 1H NMR (400 MHz, DMSO-dg) δ 10.71 (s, 1H), 9.65-9.51 (m, 2H), 8.25 (s, 1H), 8.07 (d, J= 8.0 Hz, 1H), 8.00 (s, 2H), 7.86 (d, J=8.0 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H), 7 .59-7.52 (m, 4H), 5.15 (s, 1H), 3.89 (s, 3H), 2.54 (s, 3H). MS (ER+) m/e 365.3 (M+H) ⁺ .

Example 31. N(4-(III-Pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)2-(methylamino)acetamide in as a white solid (4%). 1H NMR (400 MHz, CD ₃ OD) δ 7.90 (s, 2H), 7.57-7.50 (m, 4H), 7.30 (d, J=8.8 Hz, 2H), 6 .76 (d, J=8.8 Hz, 2H), 4.15 (s, 1H), 2.91 (s, 6H), 2.37 (s, 3H). MS (ER+) m/e 350.3 (M+H) ⁺ .

Example 32. 3-(2-((4-(III-Pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid

- 25 046894

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give 3-(2-((4-(N-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2oxoethyl)benzoic acid as a white solid (15%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.15 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.98 (s, 2H), 7.86 (d, J=7.6 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.61 (d, J=8.8 Hz, 2H), 7.53 (d , J=8.4 Hz, 2H), 7.47 (t, J=7.6 Hz, 1H), 4.33 (s, 1H), 2.29 (s, 3H). MS (ER+) m/e 351.3 (M+H) ⁺ .

Example 33. N(4-(III-Pyrazol-4-yl)phenyl)-2-(3-methyl-III-indazol-6-yl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methyl-N-indazol-6yl)-2-( methylamino)acetamide as a white solid (8%). 1H NMR (400 MHz, CD ₃ OD) δ 7.93 (s, 2H), 7.89 (d, J=8.4 Hz, 1H), 7.78 (s, 1H), 7.58-7 .54 (m, 4H), 7.33 (d, J=8.8 Hz, 1H), 5.12 (s, 1H), 2.69 (s, 3H), 2.57 (s, 3H) . MS (ER+) m/e 361.3 (M+H) ⁺ .

Example 34 Methyl 4-(2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give methyl 4-(2-((4-(N-pyrazol-4-yl)phenyl)amino)-1(methylamino)-2-oxoethyl) benzoate as an off-white solid (11%). 1H NMR (400 MHz, CD ₃ OD) δ 8.17 (dd, J=6.8, 2.0 Hz, 2H), 7.92 (s, 2H), 7.74 (d, J=8, 4 Hz, 2H), 7.58-7.53 (m, 4H), 5.07 (s, 1H), 3.92 (s, 3H), 2.70 (s, 3H). MS (ER+) m/e 365.1 (M+H) ⁺ .

Example 35. N(4-(III-pyrazol-4-yl)phenyl)-2-(2-methyl-III-benzo[d]imidazol-6-yl)-2-(methylamino)acetamide

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

Example 36. N(4-(III-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-methoxypropyl)amino)acetamide

- 26 046894

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3methoxypropyl)amino) acetamide as an off-white solid (21%). 1H NMR (400 MHz,

CD3OD) δ 8.55 (s, 2H), 7.72-7.65 (m, 4H), 7.44 (t, J=7.6 Hz, 1H), 7.26-7.25 (m , 2H), 7.09 (dd, J=8.0, 2.0 Hz, 1H), 5.15 (s, 1H), 3.85 (s, 3H), 3.51 (t, J= 5.6 Hz, 2H), 3.35 (s, 3H), 3.30-3.08 (m, 2H), 2.03-1.98 (m, 2H). MS (ER+) m/e 395.3 (M+H) ⁺ .

Example 37. N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2(3-methoxyphenyl) acetamide as a white solid (8%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 12.89 (s, 1H), 10.09 (s, 1H), 8.12 (s, 1H), 7.87 (s, 1H), 7.60 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.8 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.04-7 .02 (m, 2H), 6.85 (dd, J=8.0, 2.0 Hz, 1H), 4.33 (s, 1H), 3.75 (s, 3H), 3.48- 3.41 (m, 4H), 2.66-2.63 (m, 2H), 1.11 (t, J=7.2 Hz, 3H). MS (ER+) m/e 395.3 (M+H)+.

Example 38. N(4-(III-Pyrazol-4-yl)phenyl)-2-((3-(dimethylamino)propyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((3(dimethylamino)propyl)amino)-2-(3 -methoxyphenyl)acetamide as an off-white solid (4%). 1H NMR (400 MHz, CD ₃ OD) δ 7.93 (s, 2H), 7.60-7.53 (m, 4H), 7.30 (t, J=8.0 Hz, 1H), 7 .09-7.08 (m, 2H), 6.90 (dd, J=8.4, 1.6 Hz, 1H), 4.34 (s, 1H), 3.82 (s, 3H), 2.68-2.61 (m, 2H), 2.48-2.42 (m, 2H), 2.30 (s, 6H), 1.831.74 (m, 2H). MS (ER+) m/e 408.3 (M+H)+.

Example 39. No. (4-(III-Pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2oxoethyl)amino)-2- (3-methoxyphenyl)acetamide as a white solid (7%). 1H NMR (400 MHz, DMSO-06) δ 12.87 (br s, 1H), 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7 .62 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.28 (t, J=8.0 Hz, 1H), 7.06 -7.03 (m, 2H), 6.87 (dd, J=8.0, 2.4 Hz, 1H), 4.38 (s, 1H), 3.75 (s, 3H), 3, 38 (m, 2H), 2.87 (s, 3H), 2.84 (s, 3H). MS (ER+) m/e 408.3 (M+H)+.

Example 40. N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

- 27 046894

The reaction was carried out following general procedure B. The final residue was purified by reverse phase preparative HPLC to give N-(4-(N-pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3- methoxyphenyl)acetamide as a white solid (28%). 1H NMR (400 MHz, CD3OD) δ 8.03 (s, 2H), 7.61-7.55 (m, 4H), 7.44 (t, J=8.0 Hz, 1H), 7.23 -7.20 (m, 2H), 7.10-7.07 (m, 1H), 5.13 (s, 1H), 3.85 (s, 3H), 3.74-3.70 (m , 3H), 3.21-3.13 (m, 2H), 1.22 (dd, J=6.4, 3.2 Hz, 6H). MS (ER+) me 409.3 (M+n) ⁺ .

Example 41. N-(4-(N-Pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N-(4-(N-pyrazol-4-yl)phenyl)-2-((2-amino-2oxoethyl)amino)-2-( 3-methoxyphenyl)acetamide as a white solid (6%). 1H NMR (400

MHz, DMSO^) δ 10.13 (s, 1H), 8.22 (s, 1H), 7.99 (s, 2H), 7.62-7.53 (m, 4H), 7.45 ( s, 1H), 7.28 (t, J=8.0 Hz,

1H), 7.12-7.01 (m, 3H), 6.87 (dd, J=8.0, 2.0 Hz, 1H), 4.34 (s, 1H), 3.76 (s , 3H), 3.04 (s, 2H). MS (ER+) m/e 380.2 (M+H)+.

Example 42. N-(4-(N-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-(methylamino)propyl)amino)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N-(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3(methylamino) propyl)amino)acetamide as a white solid (6%). 1H NMR (400 MHz, DMSOd6) δ 10.81 (s, 1H), 9.70 (s, 2H), 8.65 (s, 2H), 8.00 (s, 2H), 7.56 (s , 4H), 7.43 (t, J=8.0 Hz, 1H), 7.22-7.19 (m, 2H), 7.06 (dd, J=8.0, 1.6 Hz, 1H), 5.11 (s, 1H), 3.79 (s, 3H), 2.96-2.90 (m, 4H), 2.54 (s, 3H), 2.03-1.96 (m, 2H). MS (ER+) m/e 394.3 (M+H)+.

Example 43. N-(4-(N-Pyrazol-4-yl)phenyl)-2-((1-methoxy-2-methylpropan-2-yl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by preparative reverse phase HPLC to give N-(4-(N-pyrazol-4-yl)phenyl)-2-((1-methoxy-2methylpropan-2-yl) amino)-2-(3-methoxyphenyl)acetamide as a white solid (17%). 1H NMR (400 MHz, DMSO-d^δ 10.60 (s, 1H), 9.23-9.19 (m, 2H), 7.99 (s, 2H), 7.58-7.52 ( m, 4H), 7.41 (t, J=8.0 Hz, 1H), 7.21-7.19 (m, 2H), 7.04 (dd, J=8.8, 2.8 Hz , 1H), 5.22 (s, 1H), 3.79 (s, 3H), 3.42-3.35 (m, 2H), 3.17 (s, 3H), 1.33 (s, 3H), 1.31 (s, 3H). MS (ER+) m/e 409.3 (M+H)+.

Example 44. N-(4-(N-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)-2-oxoethyl)amino)acetamide

- 28 046894

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(methylamino)- 2-oxoethyl)amino)acetamide as a yellow solid (3%). 1H NMR (400 MHz,

CD3OD) δ 7.95 (s, 2H), 7.59-7.53 (m, 4H), 7.44 (t, J=8.4 Hz, 1H), 7.18-7.17 (m , 2H), 7.16-7.10 (m, 1H), 5.12 (s, 1H), 3.84 (s, 3H), 3.68 (s, 2H), 2.79 (s, 3H). MS (ER+) m/e 394.3 (M+H) ⁺ .

Example 45

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give 2-(((N-pyrazol-4-yl)methyl)amino)-N-(4-(N-pyrazol-4-yl)phenyl)- 2-(3-methoxyphenyl)acetamide as a white solid (1%). 1H NMR (400 MHz,

DMSO-b6) δ 10.58 (s, 1H), 9.77 (s, 2H), 7.99 (s, 2H), 7.70 (s, 2H), 7.57 (d, J=8 ,8 Hz, 2H), 7.51 (d, J=8.8 Hz,

2H), 7.43 (t, J=8.0 Hz, 1H), 7.20-7.17 (m, 2H), 7.06 (dd, J=8.0, 2.0 Hz, 1H ), 4.91 (s, 1H), 4.01 (s, 2H), 3.79 (s, 3H). MS (ER+) m/e 403.3 (M+H) ⁺ .

Example nyl)acetamide

46.

N(4-(III-Pyrazol-4-yl)phenyl)-2-(ethyl(2-methoxyethyl)amino)-2-(3-methoxyphen-

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

Example 47. N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-( 3-methoxyphenyl)acetamide as an off-white solid (24%). 1H NMR (400 MHz, acetone^) δ 10.85 (s, 1H), 8.01 (s, 2H), 7.71 (d, J=8.8 Hz, 2H), 7.61 (d, J=8.4 Hz, 2H), 7.46 (t, J=8.0 Hz, 1H), 7.41 (s, 1H), 7.33 (d, J=7.6 Hz, 1H) , 7.11 (dd, J=8.0.2 Hz, 1H), 5.80 (d, J=4.4 Hz, 1H), 3.953.92 (m, 2H), 3.87 (s, 3H), 3.72-3.60 (m, 2H), 3.42 (s, 3H), 3.06 (s, 3H). MS (ER+) m/e 395.3 (M+H) ⁺ .

Example 48. No. (2-(2-(Dimethylamino)ethoxy)-5-fluoro-4-(III-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3- methoxyphenyl)acetamide

- 29 046894

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

Example 49. N-(4-(N-Pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)-N-methyl acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N-(4-(N-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2(3-methoxyphenyl )-N-methylacetamide as a yellow solid (15%). 1H NMR (400 MHz, D2O) δ 8.07 (s, 2H), 7.48 (m, 2H), 7.27-7.23 (m, 2H), 6.95 (m, 2H), 6 .68 (d, J=6.8 Hz, 1H), 6.28 (s, 1H), 5.02 (s, 1H), 3.61 (m, 2H), 3.52 (s, 3H) , 3.30 (s, 3H), 3.24 (s, 3H), 3.15-3.11 (m, 1H), 3.02-2.97 (m, 1H). MS (ER+) m/e 395.3 (M+H) ⁺ .

Example 50. N-(4-(N-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-morpholinoethyl)amino)acetamide

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

Example 51. N-(2-(2-(Dimethylamino)ethoxy)-5-fluoro-4-(N-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2 -(3-methoxyphenyl)acetamide

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

Example 52. N-(4-(N-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(pyrrolidin-1-yl)ethyl)amino)acetamide

- 30 046894

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(pyrrolidin-1 -yl)ethyl)amino)acetamide as a white solid (35%). 1H NMR (400 MHz,

DMSO-de) δ 10.64 (s, 1H), 8.00 (s, 2H), 7.57 (m, 4H), 7.41 (t, J=8.0, 1H), 7.19 -7.16 (m, 2H), 7.05-7.03 (m, 1H), 5.01 (s, 1H), 3.79 (s, 3H), 3.44-3.27 (m , 8H), 1.94 (m, 4H). MS (ER+) m/e 420.3 (M+H) ⁺ .

Example 53

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((1-(methoxymethyl)cyclopropyl)amino)-2-( 3-methoxyphenyl)acetamide as an off-white solid (27%). ^1H NMR (400 MHz, DMSO-06) δ 10.65 (br s, 1H), 9.58 (br s, 1H), 8.00 (s, 2H), 7.62-7.52 (m, 4H), 7.42 (t, J=7.6 Hz, 1H), 7.24-7.15 (m, 2H), 7.08-7.02 (m, 1H), 5, 25 (s, 1H), 3.80 (s, 3H), 3.64 (d, J=11.6 Hz, 1H), 3.37 (d, J=11.6 Hz, 1H), 3, 23 (s, 3H), 1.23-2.21 (m, 1H), 1.02-0.91 (m, 1H), 0.89-0.76 (m, 2H). MS (ER+) m/e 407.3 (M+H) ⁺ .

Example 54. N(4-(III-Pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(2,2,2-trifluoroethoxy)ethyl)amino)acetamide

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

Example 55. N(3-Fluoro-4-(III-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

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

Example 56

- 31 046894

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(3-methoxy-4-(N-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)amino)-2-(3 -methoxyphenyl)acetamide as a yellow solid (17%). 1H NMR (400 MHz, DMSO-06) δ 10.79 (s, 1H), 9.62 (s, 2H), 8.00 (s, 2H), 7.58 (d, J=8.4 Hz , 1H), 7.41 (t, J=8.0 Hz, 1H), 7.32 (d, J=1.6 Hz, 1H), 7.26-7.21 (m, 3H), 7 .06-7.02 (m, 1H), 5.13 (s, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.64-3.59 (m, 2H ), 3.30 (s, 3H), 3.09-3.00 (m, 2H). MS (ER+) m/e 411.3 (M+H) ⁺ .

Example 57. N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N(4-(N-pyrazol-4-yl)phenyl)-2-((2(dimethylamino)ethyl)amino)-2-(3 -methoxyphenyl)acetamide as a white solid (41%). 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J=8.4 Hz , 2H), 7.53 (d, J=8.8 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.05-7.02 (m, 2H), 6 .86 (dd, J=8.4, 2.4 Hz, 1H), 4.34 (s, 1H), 3.75 (s, 3H), 2.652.51 (m, 4H), 2.30 ( s, 6H). MS (ER+) m/e 394.3 (M+H) ⁺ .

Example 58. ^)-N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide r-NH ⁰ ίι ίΓ

......Eh

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N)-N4-(N-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)amino)-2-(3-methoxyphenyl) acetamide as a white solid (12%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AS column). Mobile phase: 40% EtOH with 0.1% NH ₄ OH in CO ₂ , flow rate 70 g/min to obtain the desired compound with 99% enantiomeric purity. 1H NMR (400 MHz, DMSO-06) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H) , 7.27 (t, J=4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H ), 3.75 (s, 3H), 3.44 (t, J=5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J=5.2 Hz, 2H ). MS (ER+) m/e 381.3 (M+H) ⁺ . [α] ²⁵ °% = +114 (c = 0.2 in MeOH).

Example 59

The reaction was carried out following General Procedure B. The final residue was purified by reverse phase preparative HPLC to give N)-M-(4-(N-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)amino)-2-(3 -methoxyphenyl)acetamide as a white solid (12%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AS column). Mobile phase: 40% EtOH with 0.1% NH ₄ OH in CO ₂ , flow rate 70 g/min to obtain the desired compound with 99% enantiomeric purity. 1H NMR (400 MHz, DMSO-06) δ 10.18 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.61-7.53 (m, 4H) , 7.27 (t, J=4.6 Hz, 1H), 7.06-7.03 (m, 2H), 6.87-6.84 (m, 1H), 4.40 (s, 1H ), 3.75 (s, 3H), 3.44 (t, J=5.2 Hz, 2H), 3.24 (s, 3H), 2.67 (t, J=5.2 Hz, 2H ). MS (ER+) m/e 381.3 (M+H) ⁺ . ^)-N4-(III-pyrazol-4-yl)phenyl)-2-((2methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide. [a] ²⁵ ° ^C D = -103.5 (c = 0.2 in MeOH).

- 32 046894

Example 60: Phenyl)acetamide

^)-N(4-(III-Pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxy-

The reaction was carried out following General Procedure B. The final residue was purified by reverse-phase preparative HPLC to give N)-N(4-(N-pyrazol-4-yl)phenyl)-2-((2(dimethylamino)ethyl)amino)-2 -(3-methoxyphenyl)acetamide as a white solid (41%).

Enantiomers were separated by SFC (DAICEL CHIRALPAK AS column). Mobile phase: 50% EtOH with 0.1% NH ₄ OH in CO ₂ , flow rate 70 g/min to obtain the desired compound with 99% enantiomeric purity. ^1H NMR (400 MHz, DMSO-06) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J=8.4 Hz, 2H), 7.53 (d, J=8.8 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.05-7.02 (m, 2H), 6.86 (dd, J=8.4, 2.4 Hz, 1H), 4.34 (s, 1H), 3.75 (s, 3H), 2.65-2.51 (m, 4H) , 2.30 (s, 6H). MS (ER+) m/e 394.3 (M+H) ⁺ . [a] ²⁵ ° ^C _D = +95.7 (c = 0.2 in MeOH).

Example 61

The reaction was carried out following General Procedure B. The final residue was purified by reverse-phase preparative HPLC to give N)-N(4-(N-pyrazol-4-yl)phenyl)-2-((2(dimethylamino)ethyl)amino)-2 -(3-methoxyphenyl)acetamide as a white solid (41%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AS column). Mobile phase: 50% EtOH with 0.1% NH4OH in CO2, flow rate 70 g/min to obtain the desired compound at 99% enantiomeric purity. ¹ H NMR (400 MHz, DMSO-06) δ 10.18 (s, 1H), 8.25 (s, 1H), 7.99 (s, 2H), 7.60 (d, J=8.4 Hz, 2H), 7.53 (d, J=8.8 Hz, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.05-7.02 (m, 2H), 6.86 (dd, J=8.4, 2.4 Hz, 1H), 4.34 (s, 1H), 3.75 (s, 3H), 2.65-2.51 (m, 4H) , 2.30 (s, 6H). MS (ER+) m/e 394.3 (M+H) ⁺ . [a] ²⁵ ° ^C _D = -92.8 (c = 0.2 in MeOH).

Example 62

Step 1: Compound tert-butyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)-(1-(3methoxyphenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran -2-yl)-N-pyrazol-4-yl)phenyl)amino)ethyl)carbamate was dissolved in EtOH (0.15 M) and p-toluenesulfonic acid (2 eq.) was added. The solution was stirred for 20 hours at 15°C. The reaction mixture was quenched with NaHCO ₃ and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give tert-butyl (2-((4-(III-pyrazol-4-yl)phenyl)amino)-1(3- methoxyphenyl)-2-oxoethyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamate as a yellow solid in 92% yield. Enantiomers were separated by SFC (DAICEL CHIRALPAK OD column). Mobile phase: 45% EtOH with 0.1% NH4OH in CO ₂ , flow rate 70 g/min to produce tert-butyl^)-(2-((2-((4-(III-pyrazol-4-yl) phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate with 99% enantiomeric purity. ^1H NMR (400 MHz, DMSO- _s16 ) δ δ 12.84 (s, 1H), 10.19 (s, 1H), 8.04 (s, 2H), 7.71-7.49 (m , 4H), 7.37-7.33 (m, 1H), 7.03-6.89 (m, 3H), 6.02-5.33 (m, 1H), 3.76 (s, 3H ), 3.42-2.70 (m, 7H), 1.55-1.25 (m, 18H).

Step 2: Compound tert-butyl ^)-(2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)-2oxoethyl)(2-((tert-butoxycarbonyl) (methyl)amino)ethyl)carbamate was dissolved in dichloromethane and 4 N HCl in dioxane was added. The reaction mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The final residue was purified by reverse-phase preparative HPLC to give ^)-N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(methylamino)ethyl)amino) acetamide as a white solid (12%). ¹ H NMR (400 MHz, CD3OD)

- 33 046894 δ δ 8.51 (s, 1H), 7.91 (s, 2H), 7.58-7.51 (m, 4H), 7.31 (t, J=7.6 Hz, 1H ), 7.09-7.08 (m, 2H), 6.91-6.89 (m, 1H), 4.42 (s, 1H), 3.81 (s, 3H), 3.14- 3.04 (m, 2H), 2.92-2.82 (m, 2H), 2.72 (s, 3H). MS (ER+) m/e 380.4 (M+H) ⁺ . [a] ²⁵ ° ^C D = -86.02 (0.5 in MeOH).

Example 63

A.

Step 1: Compound tert-butyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(1-(3-methoxyphenyl)2-oxo-2-((4-(1-(tetrahydro-2H-pyran -2-yl)-N-pyrazol-4-yl)phenyl)amino)ethyl)carbamate was dissolved in EtOH (0.15 M) and p-toluenesulfonic acid (2 eq.) was added. The solution was stirred for 20 hours at 15°C. The reaction mixture was quenched with NaHCO ₃ and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give tert-butyl (2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(3 -methoxyphenyl)-2oxoethyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamate as a yellow solid in 92% yield. Enantiomers were separated by SFC (DAICEL CHIRALPAK OD column). Mobile phase: 45% EtOH with 0.1% NH ₄ OH in CO ₂ , flow rate 70 g/min to produce tert-butyl (S)-(2-((2-((4(III-pyrazole-4- yl)phenyl)amino)-1-(3-methoxyphenyl)-2-oxoethyl)(tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate with 99% enantiomeric purity. 1H NMR (400 MHz, DMSOL6) δ 5 12.84 (s, 1H), 10.19 (s, 1H), 8.04 (s, 2H), 7.71-7.49 (m, 4H), 7.37-7.33 (m, 1H), 7.03-6.89 (m, 3H), 6.02-5.33 (m, 1H), 3.76 (s, 3H), 3.422, 70 (m, 7H), 1.55-1.25 (m, 18H). [a] ²⁵ ° ^C D = -92.8 (c = 0.2 in MeOH).

Step 2: Compound tert-butyl ^)-(2-((2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(3-methoxyphenyl)2-oxoethyl)(tert-butoxycarbonyl )Amino)ethyl)(methyl)carbamate was dissolved in dichloromethane and 4 N HCl in dioxane was added. The reaction mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The final residue was purified by reverse-phase preparative HPLC to give ^)-N(4-(N-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2(methylamino)ethyl)amino) acetamide as a white solid (12%). 1H NMR (400 MHz, CD ₃ OD) δ 8.51 (s, 1H), 7.91 (s, 2H), 7.58-7.51 (m, 4H), 7.31 (t, J= 7.6 Hz, 1H), 7.09-7.08 (m, 2H), 6.91-6.89 (m, 1H), 4.42 (s, 1H), 3.81 (s, 3H ), 3.14-3.04 (m, 2H), 2.92-2.82 (m, 2H), 2.72 (s, 3H). MS (ER+) m/e 380.4 (M+H) ⁺ . [a] ²⁵ ° ^C D = + 82.43 (0.7 in MeOH).

Example 64. ^)-N(4-(III-Pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide

Step 1: CbzNH ₂ (3 eq.) was dissolved in n-propyl alcohol. 0.4 N NaOH (3 eq.) was added to the solution, followed by the addition of tert-butyl hypochlorite (1.05 eq.) in one part. After 5 minutes, a solution of (DHQ) ₂ PHAL (0.05 eq.) in n-propyl alcohol was added to the reaction mixture in one part. A solution of p-chlorostyrene compound (1 eq.) in n-propyl alcohol was added in one part, followed by the addition of K ₂ OsO ₂ (OH) ₄ (0.03 eq.). The light green solution was stirred at 20°C for an hour. A saturated solution of Na ₂ SO ₃ was added to the reaction mixture and the mixture was stirred at 20°C for 10 minutes. The organic layer was separated and the aqueous phase was extracted with EtOAc. The combined organic layer was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by preparative HPLC (TPA conditions) and lyophilized to give benzyl ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (62%, 94.9% ee) as a white solid. . ^1H NMR (400 MHz, CDCl ₃ ) δ 7.34-7.31 (m, 7H), 7.28-7.22 (m, 2H), 5.64 (s, 1H), 5.13- 5.05 (m, 2H), 4.80 (s, 1H), 3.86-3.78 (m, 2H), 2.24 (m, 1H).

Step 2: To a mixture of compound ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (1 eq.) and imidazole (2 eq.) in DMF, TBS-Cl (1.5 eq.) was added in one part. at 30°C in a nitrogen atmosphere. The mixture was stirred at 30°C for 1 hour. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiO ₂ , petroleum ether/EtOAc = 15/1) to give benzyl ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (95% , 89.3% ee) as a colorless oil. 1H NMR (400 MHz, CDCl ₃ ) δ 7.38-7.25 (m, 9H), 5.59 (s, 1H), 5.16-5.07 (m, 2H), 4.77

- 34 046894 (s, 1H), 3.95 (dd, J=10.0, 4.0 Hz, 1H), 3.75 (m, 1H), 0.89 (s, 9H), -0, 03 (s, 3H), -0.06 (s, 3H).

Step 3: To a solution of compound ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4chlorophenyl)ethyl)carbamate (1 equiv.) in DMF, NaH (2 equiv.) was added portionwise at 0°C in atmosphere nitrogen. The mixture was stirred at 0°C for 10 min, and then a solution of MeI (1.5 eq.) in DMF was added dropwise to the mixture. The mixture was heated to 20°C and stirred for 50 minutes. The mixture was poured into saturated NH ₄ Cl solution and extracted with EtOAc. The combined organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The mixture was purified by chromatography on silica gel (SiO ₂ , petroleum ether/ESAAc = 20/1) to obtain benzyl ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (93 %, 94.4% ee) in the form of a colorless oil. 1H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.19 (m, 9H), 5.37-5.22 (m, 1H), 5.19-5.04 (m, 2H), 4 .05 (m, 2H), 2.80 (s, 3H), 0.88 (s, 9H), 0.07 (s, 6H).

Stage 4:

One part of the compound ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (1 eq.) in MeOH/ _H2O (7:3) was added KHSO4 (4 eq.) at 20°C in N2 atmosphere. Water was added to the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography ( _SiO2 , petroleum ether/EtOAc = 2/1) to give the compound benzyl (S)-(1-(4chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (99%, 94% e.g. i.) in the form of a colorless oil. 1H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.20 (m, 9H), 5.33 (dd, J=8.0, 6.4 Hz, 1H), 5.20 (m, 2H ), 4.17-4.07 (m, 2H), 2.78 (s, 3H).

Step 5: A solution of compound ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (1 eq.) was dissolved in sat. KN ₂ PO ₄ and MeCN at 0°C. NaClO ₂ (2 eq.) and TEMPO (0.2 eq.) were added to the reaction mixture at 0-5°C, followed by dropwise addition of NaClO (2 eq.) at 0-5°C under N2 atmosphere. The mixture turned deep purple and the reaction mixture was stirred at 10-15°C for 2 hours. The reaction mixture was diluted with H ₂ O (100 ml) and extracted with a mixture of DCM (100 ml x 3). The combined organic phase was washed with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO ₂ , petroleum ether/EtOAc = 1/1, 0/1) to give the compound ^)-2-(((benzyloxy)carbonyl)(methyl)amino)-2-(4chlorophenyl)acetic acid acids (95%, 94% ee) as a yellow oil.

Step 6: Into a mixture of compound ^)-2-(((benzyloxy)carbonyl)-(methyl)amino)-2-(4-chlorophenyl)acetic acid (1 eq.) and compound 4-(1-(tetrahydro-2H -pyran-2-yl)-III-pyrazol-4-yl)aniline (1.1 equiv.) in THF was added in one part with DMT-MM (1.05) at 10°C in an N2 atmosphere. The mixture was stirred at 20°C for 3 hours. Water was added and the mixture was extracted with EtOAc. The combined organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether/EAAc = 2:1) to obtain the compound benzyl ((1S)-1 -(4-chlorophenyl)-2-oxo-2-((4-( 1 -(tetrahydro-2H- pyran-2-yl)-1 H-pyrazol-4-yl)phenyl)amino)ethyl)(methyl)carbamate (59%, 86% ee) as a yellow solid. 1H NMR (400 MHz, CDCl ₃ ) δ 7.75 (s, 1H), 7.71 (s, 1H), 7.40-7.31 (m, 4H), 7.28-7.18 (m , 9H), 5.88 (s, 1H), 5.41 (dd, J=8.8, 2.8 Hz, 1H), 5.16-5.09 (m, 2H), 4.05- 4.00 (m, 1H), 3.68-3.41 (m, 1H), 2.80 (s, 3H), 2.09-2.04 (m, 2H), 1.66-1, 53 (m, 4H).

Step 7: Into a solution of the compound ((^)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)III-pyrazol-4-yl )phenyl)amino)ethyl)(methyl)carbamate (1 equiv.) in MeCN was added TMSI (8 equiv.) at 0°C in an N ₂ atmosphere. H ₂ O was added and the mixture was extracted with petroleum ether. The aqueous phase was purified by preparative HPLC (MK conditions) to obtain N)-N-(4-(N-pyrazol-4-yl)phenyl)-2-(4chlorophenyl)-2-(methylamino)acetamide (9%, 87. 5% ee) in the form of a white powder. 1H NMR (400 MHz, DMSO-06) δ 10.08 (s, 1H), 8.20 (s, 1H), 7.98 (s, 2H), 7.61-7.42 (m, 8H) , 4.24 (s, 1H), 2.27 (s, 3H). MS (ER+) m/e 341.0 (M+H) ⁺ .

Example 65

Step 1: CbzNH ₂ (3 eq.) was dissolved in n-propyl alcohol. 0.4 N NaOH (3 eq.) was added to the solution, followed by the addition of tert-butyl hypochlorite (1.05 eq.) in one part. After 5 minutes, a solution of (DHQD) ₂ PHAL (0.05 eq.) in n-propyl alcohol was added to the reaction mixture in one part. A solution of the compound p-chlorostyrene (1 eq.) in n-propyl alcohol was added in one part, followed by the addition of K ₂ OsO ₂ (OH) ₄ (0.03 eq.). The light green solution was stirred at 20°C for an hour. A saturated solution of Na ₂ SO ₃ was added to the reaction mixture and the mixture was stirred at 20°C for 10 minutes. The organic layer was separated and the aqueous phase was extracted with EtOAc. The combined organic layer was washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated

- 35 046894 in a vacuum. The residue was purified by preparative HPLC (TPA conditions) and lyophilized to give benzyl ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (71%, 94.5% ee) as a white solid. . 1H NMR (400 MHz, DMSOD ₆ ) δ 7.76 (d, J=8.4 Hz, 1H), 7.39-7.10 (m, 9H), 5.05-4.97 (m, 2H ), 4.87 (t, J=5.6 Hz, 1H), 4.62-4.56 (m, 1H), 3.58-3.48 (m, 2H).

Step 2: To a mixture of compound ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)carbamate (1 eq.) and imidazole (2 eq.) in DMF, TBS-Cl (1.5 eq.) was added in one part. at 30°C in a nitrogen atmosphere. The mixture was stirred at 30°C for 1 hour. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/ESAAc = 15/1) to obtain benzyl ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)carbamate (71% , 90% ee) in the form of a colorless oil.

Step 3: To a solution of compound ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4chlorophenyl)ethyl)carbamate (1 equiv.) in DMF, NaH (2 equiv.) was added portionwise at 0°C in atmosphere nitrogen. The mixture was stirred at 0°C for 10 min, and then a solution of MeI (1.5 eq.) in DMF was added dropwise to the mixture. The mixture was heated to 20°C and stirred for 50 minutes. The mixture was poured into saturated NH4Cl solution and extracted with EtOAc. The combined organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The mixture was purified by chromatography on silica gel (SiO ₂ , petroleum ether/EYAc = 20/1) to obtain benzyl ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (75 %, 93% ee) in the form of a colorless oil.

Step 4: In a mixture of compound ^)-(2-((tert-butyldimethylsilyl)oxy)-1-(4-chlorophenyl)ethyl)(methyl)carbamate (1 eq.) in MeOH/H ₂ O (7:3) KHSO4 (4 eq.) was added in one part at 20°C in an N2 atmosphere. Water was added to the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/EOAc = 2/1) to obtain the compound benzyl ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (91%, 92% e .i.) in the form of a colorless oil.

Step 5: A solution of compound ^)-(1-(4-chlorophenyl)-2-hydroxyethyl)(methyl)carbamate (1 eq.) was dissolved in sat. KH2PO4 and MeCN at 0°C. NaClO ₂ (2 eq.) and TEMPO (0.2 eq.) were added to the reaction mixture at 0-5°C, followed by dropwise addition of NaClO (2 eq.) at 0-5°C under N2 atmosphere. The mixture turned deep purple and the reaction mixture was stirred at 10-15°C for 2 hours. The reaction mixture was diluted with H ₂ O (100 ml) and extracted with a mixture of DCM (100 ml x 3). The combined organic phase was washed with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/EYAc = 1/1, 0/1) to obtain the compound d-2-(((benzyloxy)carbonyl)(methyl)amino)-2-(4chlorophenyl)acetic acid (94% ee) as a yellow oil.

Step 6: Into a mixture of compound ^)-2-(((benzyloxy)carbonyl)(methyl)amino)-2-(4-chlorophenyl)acetic acid (1 eq.) and compound 4-(1-(tetrahydro-2H- pyran-2-yl)-1H-pyrazol-4-yl)aniline (1.1 equiv.) in THF was added in one part with DMT-MM (1.05) at 10°C under N2 atmosphere. The mixture was stirred at 20°C for 3 hours. Water was added and the mixture was extracted with EtOAc. The combined organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether/EAAc = 2:1) to obtain the compound benzyl ((^)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H- pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)(methyl)carbamate (83%, 87% ee) as a yellow solid. ^1H NMR (400 MHz, CDCl ₃ ) δ 7.75 (s, 1H), 7.71 (s, 1H), 7.40-7.31 (m, 4H), 7.28-7.18 ( m, 9H), 5.88 (s, 1H), 5.41 (dd, J=8.8, 2.8 Hz, 1H), 5.165.09 (m, 2H), 4.05-4.00 (m, 1H), 3.68-3.41(m, 1H), 2.80 (s, 3H), 2.09-2.04 (m, 2H), 1.66-1.53 (m , 4H).

Step 7: Into a solution of the compound ((^)-1-(4-chlorophenyl)-2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)1H-pyrazol-4-yl )phenyl)amino)ethyl)(methyl)carbamate (1 equiv.) in MeCN was added TMSI (8 equiv.) at 0°C in an N ₂ atmosphere. H ₂ O was added and the mixture was extracted with petroleum ether. The aqueous phase was purified by preparative HPLC (MK conditions) to obtain ^)-H-(4-(N-pyrazol-4-yl)phenyl)-2-(4chlorophenyl)-2-(methylamino)acetamide (28%, 87% e.i.) in the form of a white powder. 1H NMR (400 MHz, DMSOd6) δ 10.15 (s, 1H), 8.14 (s, 1H), 7.98 (s, 2H), 7.59 (d, J=8.8 Hz, 4H ), 7.54-7.49 (m, 4H), 7.44-7.42 (m, 2H), 4.32 (s, 1H), 2.30 (s, 3H). MS (ER+) m/e 341.3 (M+H) ⁺ .

Example 66. N(2-Fluoro-5-methoxy-4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

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The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(2-fluoro-5-methoxy-4-(N-pyrazol-4-yl)phenyl)-2-(3methoxyphenyl)-2-( methylamino)acetamide as a yellow oil (1%). 1H NMR (400 MHz, DMSOL ₆ ) δ 12.91 (s, 1H), 9.91 (br s, 1H), 8.07 (s, 2H), 7.74 (d, J=7.2 Hz, 1H), 7.58 (d, J=12.4 Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 7.04-7.01 (m, 2H), 6.86 (dd, J=7.2, 1.6 Hz, 1H), 4.28 (s, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 2.31 (c, 3H). MS (ER+) m/e 385.2 (M+H) ⁺ .

Example 67 3-(2-((4-(1H-Pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-I-methylbenzamide

Step 1: Into a solution of the compound methyl 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1(tetrahydro-2H-pyran-2-yl)-III- pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 eq.) in MeOH and water, NaOH (2 eq.) was added. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (MK conditions) to obtain the compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1(tetrahydro-2H-pyran-2- yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (58%) as an off-white solid. 1H NMR (400 MHz, DMSO-06) δ 10.21 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.957.90 (m, 2H), 7.88 (d, J=8.0 Hz, 1H), 7.63-7.55 (m, 5H), 7.27 (d, J=8.8 Hz, 2H), 6.89 (d, J= 8.8 Hz, 2H), 5.38 (dd, J=10.4, 2.4 Hz, 1H), 4.33 (s, 1H), 3.92 (d, J=11.6 Hz, 1H), 3.73 (s, 3H), 3.66-3.63 (m, 1H), 3.51-3.42 (m, 2H), 2.16-2.09 (m, 4H) , 1.94 (s, 2H), 1.69-1.65 (m, 1H), 1.58-1.54 (m, 2H).

Step 2: Into a solution of the compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro2H-pyran-2-yl)-1H-pyrazol- 4-yl)phenyl)amino)ethyl)benzoic acid (1 eq.), methylamine (1 eq.)) in CH ₃ CN was added T ₃ P (1.5 eq.), DIPEA (1.5 eq.) at 20°C. The mixture was heated to 80°C and stirred for 15 hours. The reaction mixture was diluted with H ₂ O 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 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4- (1(tetrahydro-2H-pyran-2-yl)-N-pyrazol-4-yl)phenyl)amino)ethyl)-M-methylbenzamide as a white solid.

Step 3: Mixture of compound 3-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro-2Hpyran-2-yl)-1H-pyrazol-4 -yl)phenyl)amino)ethyl)-M-methylbenzamide in TPA was stirred at 75°C for 16 hours. The mixture was concentrated in vacuo to leave a residue. The residue was purified by preparative HPLC (TPA conditions) to obtain 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)2-oxoethyl)-N-methylbenzamide as white solid matter (43%). 1H NMR (400 MHz, DMSOL ₆ ) δ 10.69 (s, 1H), 9.61-9.47 (m, 2H), 8.56 (d, J=4.8 Hz, 1H), 8, 12 (s, 1H), 8.01 (s, 2H), 7.93 (d, J=7.6 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7, 63-7.54 (m, 5H), 5.09 (s, 1H), 2.81 (s, 3H), 2.53 (s, 3H). MS (ER+) m/e 364.4 (M+H) ⁺ .

Example 68 4-(2-((4-(1H-Pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid

Step 1: Mixture of the compound methyl 4-(1-((4-methoxybenzyl)-(methyl)amino)-2-oxo-2-((4-(1(tetrahydro-2H-pyran-2-yl)-III- pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 eq.) in TPA was stirred at 70°C for 112 hours. The reaction mixture was diluted with MeOH and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TPA conditions) to obtain the compound methyl 4-(2-((4-(N-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate (19% ) as an off-white solid. 1H NMR (400 MHz, CD3OD) δ 8.17 (dd, J=6.8, 2.0 Hz, 2H), 7.92 (s, 2H), 7.74 (d, J=8.4 Hz , 2H), 7.58-7.53 (m, 4H), 5.07 (s, 1H), 3.92 (s, 3H), 2.70 (s, 3H).

Step 2: Into a solution of the compound 4-(2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2

- 37 046894 oxoethyl)benzoate (1 eq.) NaOH (3 eq.) was added to MeOH and H2O. The mixture was stirred at 30°C for 16 hours. The mixture was acidified to pH = 5 with 1 N HCl. The resulting mixture was purified by preparative HPLC (MK conditions) to obtain 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1(methylamino)-2-oxoethyl)benzoic acid (18 %) as a white solid. 1H NMR (400 MHz, DMSO-06) δ 10.13 (s, 1H), 8.27 (s, 1H), 7.98 (s, 2H), 7.90 (d, J=8.4 Hz , 2H), 7.65-7.50 (m, 6H), 4.30 (s, 1H), 2.28 (s, 3H). MS (ER+) m/e 351.1 (M+H) ⁺ .

Example 69 4-(2-((4-(1H-Pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-M-methylbenzamide

Step 1: Into a solution of the compound methyl 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1(tetrahydro-2H-pyran-2-yl)-1H- pyrazol-4-yl)phenyl)amino)ethyl)benzoate (1 eq.) in MeOH and H ₂ O was added with NaOH (2 eq.). The mixture was stirred at 35°C for 36 hours. The reaction mixture was diluted with H ₂ O. The resulting solution was purified by preparative HPLC (MK conditions) to obtain the compound 4-(1-((4-methoxybenzyl)(methyl)amino)- 2-oxo-2-((4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)benzoic acid (70%, 98% purity) in as a white solid. 1H NMR (400 MHz, DMSO-06) δ 12.92 (s, 1H), 10.19 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.88 (s , 1H), 7.69 (d, J=8.4 Hz, 2H), 7.65-7.60 (m, 2H), 7.59-7.52 (m, 2H), 7.28 ( d, J=8.8 Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 5.38 (dd, J=10.0, 2.4 Hz, 1H), 4, 33 (s, 1H), 3.94-3.92 (m, 1H), 3.74 (s, 3H), 3.68-3.58 (m, 1H), 3.54-3.40 ( m, 2H), 2.18-2.03 (m, 4H), 2.00-1.87 (m, 2H), 1.75-1.61 (m, 1H), 1.59-1, 48 (m, 2H).

Step 2: Into a solution of the compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro2H-pyran-2-yl)-1H-pyrazol- 4-yl)phenyl)amino)ethyl)benzoic acid (1 eq.) in MeCN was added with T ₃ P (1.5 eq.), MeNH ₂ (2.5 eq.) and DIPEA (4 eq.). The mixture was stirred at 30°C for 36 hours. The reaction mixture was cooled to room temperature and quenched by adding H ₂ O at 15°C. The resulting mixture was diluted with EtOAc and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1- (tetrahydro-2H-pyran-2yl)-1H-pyrazol-4-yl)phenyl)amino)ethyl)-M-methylbenzamide (98%, 87% purity) as a white solid.

Step 3: Into a solution of the compound 4-(1-((4-methoxybenzyl)(methyl)amino)-2-oxo-2-((4-(1-(tetrahydro2H-pyran-2-yl)-1H-pyrazol- 4-yl)phenyl)amino)ethyl)-M-methylbenzamide (1 eq.) in THF and MeOH was added with dry Pd/C (0.1 eq.) and HCl (2 eq.). The mixture was stirred at 50°C for 2 hours in an atmosphere of H ₂ (1 atm.). The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (MK conditions) to obtain 4-(2-((4-(III-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-Mmethylbenzamide (98% purity ) as a white solid. 1H NMR (400 MHz, DMSO^ ₆ ) δ 10.13 (s, 1H), 8.44-8.36 (m, 1H), 8.16 (s, 1H), 7.98 (s, 2H) , 7.80 (d, J=8.4 Hz, 2H), 7.63-7.58 (m, 2H), 7.57-7.51 (m, 4H), 4.30 (s, 1H ), 2.76 (d, J=4.4 Hz, 3H), 2.29 (s, 3H). MS (ER+) m/e 364.3 (M+H) ⁺ .

Example 70. No. (2-(3-(Dimethylamino)propoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(2-(3-(dimethylamino)propoxy)-5-fluoro-4-(N-pyrazol-4yl)phenyl)-2-(3 -methoxyphenyl)-2-(methylamino)acetamide as a white solid (13%). 1H NMR (400 MHz, CD3OD) δ 8.04 (d, J=12.8 Hz, 1H), 8.00 (s, 2H), 7.30-7.23 (m, 2H), 7.03 -7.02 (m, 2H), 6.88 (dd, J=8.4, 0.8 Hz, 1H), 4.20 (s, 1H), 4.16 (t, J=6.0 Hz, 2H), 3.79 (s, 3H), 2.62-2.58 (m, 2H), 2.45 (s, 3H), 2.30 (s, 6H), 2.07-2 .00 (m, 2H). MS (ER+) m/e 456.4 (M+H) ⁺ .

Example 71

- 38 046894

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(N-pyrazol-4yl)phenyl)-2-(3 -methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). 1H NMR (400 MHz, CD3OD) δ 8.52 (s, 2H), 8.04-8.0 (m, 3H), 7.42 (t, J=8.0 Hz, 1H), 7.28 (d, J=6.8 Hz, 1H), 7.247.19 (m, 2H), 7.05 (dd, J=8.8, 2.4 Hz, 1H), 5.43 (s, 1H) , 4.44-4.41 (m, 1H), 4.27-4.25 (m, 1H), 3.84 (s, 3H), 3.413.36 (m, 2H), 2.81 (s , 6H), 2.65 (s, 3H). MS (ER+) m/e 442.4 (M+H) ⁺ .

Example 72

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(N-pyrazol-4yl)phenyl)-2-(3 -methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). 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 ^)-N2-(2-(dimethylamino)ethoxy)-5-fluoro4-(III-pyrazol-4-yl )phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide with 99% enantiomeric purity. 1H NMR (400 MHz, DMSO-06) δ 11.05 (s, 1H), 10.59 (s, 1H), 9.98-9.96 (m, 1H), 9.72-9.69 ( m, 1H), 8.08 (m, 2H), 7.94 (d, J=12.4 Hz, 1H), 7.47 (s, 1H), 7.43-7.34 (m, 3H ), 7.04-7.01 (m, 1H), 6.05 (t, J=6.4 Hz, 1H), 4.47-4.34 (m, 2H), 3.79 (s, 3H), 3.58-3.49 (m, 2H), 2.83 (dd, J=7.2, 5.2 Hz, 6H), 2.48 (s, 3H). MS (ER+) m/e 442.3 (M+H) ⁺ .

Example 73 )acetamide

The reaction was carried out following General Procedure A. The final residue was purified by reverse phase preparative HPLC to give N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(N-pyrazol-4yl)phenyl)-2-(3 -methoxyphenyl)-2-(methylamino)acetamide as a white solid (37%). Enantiomers were separated by SFC (DAICEL CHIRALPAK AD column). Mobile phase: 55% EtOH with 0.1% NH4OH in CO2, flow rate 70 g/min to give ^)-M-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4(III-pyrazol- 4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide with 99% enantiomeric purity. 1H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 10.59 (s, 1H), 9.97-9.94 (m, 1H), 9.71-9.68 (m, 1H), 8.08 (m, 2H), 7.94 (d, J=12.4 Hz, 1H), 7.47 (s, 1H), 7.43-7.36 (m, 3H), 7.04-7.02 (m, 1H), 6.05 (t, J=6.4 Hz, 1H), 4.484.34 (m, 2H), 3.79 (s, 3H), 3.58 -3.47 (m, 2H), 2.83 (dd, J=7.2, 5.2 Hz, 6H), 2.48 (s, 3H). MS (ER+) m/e 442.3 (M+H) ⁺ .

Example 74 1-(4-(1H-Pyrazol-4-yl)phenyl)-4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1,4-diazepan2-one

Step 1: In a mixture of the compound benzyl 2-(3-methoxyphenyl)-3-oxo-1,4-diazepane-1-carboxylate (1 eq.) and the compound 4-(4-iodophenyl)-1-(tetrahydro-2H- pyran-2-yl)-III-pyrazole (1 equiv.) in dioxane was added

- 39 046894

Cs ₂ CO ₃ (4 equiv.) and Xantphos-Pd-G3 (0.05 equiv.) at 20°C in an N2 atmosphere. The mixture was stirred at 100°C for 16 hours. The mixture was extracted with EtOAc (100 ml x 4). The combined organic phase was washed with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/EOAc = 2/1, 1/1) to obtain the compound benzyl 2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro- 2H-pyran-2-yl)-Spirazol-4-yl)phenyl)-1,4-diazepan-1-carboxylate (25%) as a yellow oil.

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

Step 3: Into the mixture of the compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-III-pyrazol-4yl)phenyl)-1,4-diazepan-2 -one (1 equiv.) and aldehyde (2 equiv.) in MeOH, TEA (3) was added in one part at 25°C in an N ₂ atmosphere. The mixture was stirred at 25°C for 30 minutes. NaBH(OAc) ₃ (4 eq.) was added to the reaction mixture and the reaction mixture was stirred at 25°C for 4 hours. The mixture was poured into water and extracted with EtAOc. The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/ESAAc = 2/1, 1/1) to obtain the compound 4-(cyclopropylmethyl)-3-(3-methoxyphenyl)-1-(4-(1(tetrahydro- 2H-pyran-2-yl)-III-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (32%) as a colorless oil.

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

Example 75 1-(4-(1H-Pyrazol-4-yl)phenyl)-4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1,4diazepan-2-one

Step 1: Into the mixture of the compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-III-pyrazol-4yl)phenyl)-1,4-diazepan-2 -one (1 equiv.) and aldehyde (2 equiv.) in MeOH, TEA (3) was added in one part at 25°C in an N ₂ atmosphere. The mixture was stirred at 25°C for 30 minutes. NaBH(OAc) ₃ (4 eq.) was added to the reaction mixture and the reaction mixture was stirred at 25°C for 4 hours. The mixture was poured into water and extracted with EtAOc. The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/ESAAc = 2/1, 1/1) to obtain the compound 4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1-(4( 1-(tetrahydro-2H-pyran-2-yl)-III-pyrazol-4-yl)phenyl)-1,4-diazepan-2-one (13%) as a colorless oil.

Step 2: Into the mixture of the compound 4-(2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2Hpyran-2-yl)-III-pyrazol-4-yl )phenyl)-1,4-diazepan-2-one (1 equiv.) in DCM was added to TPA at 0°C under N ₂ atmosphere. The mixture was stirred at 0°C for 10 minutes and the reaction mixture was heated to 25°C. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure at 45°C, and the residue was purified by preparative HPLC to give 1-(4-(1Hpyrazol-4-yl)phenyl)-4- (2-(dimethylamino)ethyl)-3-(3-methoxyphenyl)-1,4-diazepan-2-one (40%) as a yellow solid. ^1H NMR (400 MHz, CD3OD) δ 8.00 (s, 2H), 7.68 (d, J=8.8 Hz, 2H), 7.42 (t, J=8.0 Hz, 1H) , 7.30 (d, J=8.4 Hz, 2H), 7.19-7.15 (m, 2H), 6.98 (dd, J=8.0, 2.4 Hz, 1H), 5.06 (s, 1H), 3.93-3.86 (m, 4H), 3.77-3.73 (m, 1H), 3.48-3.35 (m, 4H), 3. 22-3.14 (m, 2H), 2.94 (s, 3H), 2.87 (s, 3H), 2.19-2.17 (m, 1H), 1.77 (m, 1H) . MS (ER+) m/e 434.3 (M+H)+.

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Example 76 1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-methoxyphenyl)-4-(2-(methylamino)ethyl)-1,4-diazepan-2-one

Step 1: Into the mixture of the compound 3-(3-methoxyphenyl)-1-(4-(1-(tetrahydro-2H-pyran-2-yl)-III-pyrazol-4yl)phenyl)-1,4-diazepan-2 -one (1 equiv.) and aldehyde (2 equiv.) in MeOH, TEA (3) was added in one part at 25°C in an N ₂ atmosphere. The mixture was stirred at 25°C for 30 minutes. NaBH(OAc) ₃ (4 eq.) was added to the reaction mixture and the reaction mixture was stirred at 25°C for 4 hours. The mixture was poured into water and extracted with EtAOc. The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (SiO ₂ , petroleum ether/ESAAc = 2/1, 1/1) to obtain the compound tert-butyl (2-(2-(3-methoxyphenyl)-3-oxo-4-(4- (1(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-4-yl)phenyl)-1,4-diazepan-1 -yl)ethyl)(methyl)carbamate (77%) as dirty white oil.

Step 2: Into the mixture of the compound tert-butyl (2-(2-(3-methoxyphenyl)-3-oxo-4-(4-(1-(tetrahydro-2Hpyran-2-yl)-III-pyrazol-4-yl )phenyl)-1,4-diazepan-1-yl)ethyl)(methyl)carbamate (1 equiv.) in DCM was added with TFA at 0°C under N2 atmosphere. The mixture was stirred at 0°C for 10 minutes and the reaction mixture was heated to 25°C. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure at 45°C, and the residue was purified by preparative HPLC to give 1-(4-(1H-pyrazol-4-yl)phenyl) -3-(3-methoxyphenyl)-4-(2-(methylamino)ethyl)-1,4-diazepan-2-one (66%) as a white solid. ^1H NMR (400 MHz, CD3OD) δ 8.00 (s, 2H), 7.67 (d, J=8.4 Hz, 2H), 7.40 (t, J=8.0 Hz, 1H) , 7.28 (d, J=8.8 Hz, 2H), 7.16-7.13 (m, 2H), 7.67 (dd, J=8.4, 2.4 Hz, 1H), 5.03 (s, 1H), 3.90-3.87 (m, 1H), 3.84 (s, 3H), 3.73-3.66 (m, 1H), 3.39-3. 34 (m, 1H), 3.23-3.08 (m, 5H), 2.73 (s, 3H), 2.17-2.14 (m, 1H), 1.71-1.68 ( m, 1H). MS (ER+) m/e 420.3 (M+H) ⁺ .

Example 77.

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.

Example 78.

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).

Example 79.

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 plated until confluent 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.

Example 80.

Thawing and proliferation of human oligodendrocyte/neuron progenitor cells (OPCs). Cryopreserved human oligodendrocyte/neuron progenitor cells (Millipore, cat. no. CS204496) were recovered and cultured in Matrigel (BD, cat. no. 356324) T-25 flasks in expansion medium containing OPO/neuron basal medium, media supplement N21, recombinant human bFGF, PDGF-AA and NT-3. When the cells reached 80% confluency, 3 ml of Accutase (Millipore SCR005) was applied to passage the cells at a ratio of 1:4. Subcultured cells were maintained in expansion medium to proliferate.

Example 81.

Spontaneous differentiation of human oligodendrocyte/neuron progenitor cells in 24-well and 96-well plates. The plate was coated with 10 μg/ml polyJ-ornithine (Sigma, cat. no. P4957) overnight at room temperature. The polyJ-ornithine solution was removed and the plate was washed 1X

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PBS, 10 μg/ml laminin (Millipore, cat. no. CC095) was added and incubated at 37°C for at least 2 hours. Cells were seeded onto coated plates in expansion medium and left overnight to attach. The next day, the expansion medium was removed and replaced with freshly prepared spontaneous differentiation medium containing only KPO/neuron basal medium and N21 medium supplement. In the case of a 14-day culture, the differentiation medium was changed every 3-4 days. Cells were visualized and neurite length analyzed using an IncuCyte S3 live cell assay system every 4 hours. After 14 days of differentiation, cells were fixed with 4% paraformaldehyde and stained with DAPI and neuron-specific β-III tubulin antibody (R&D systems, NL1195R).

Example 82.

Chondroitin sulfate proteoglycan (CSPG) assay of human oligodendrocyte/neuron progenitor cells in a 96-well plate. After overnight incubation with 10 μg/ml poly-Lornithine, 96-well plates were coated with a well-mixed solution of 10 μg/ml laminin in combination with 0.3 μg/ml CSPG (Millipore CC117) for at least 2 h at room temperature. . Cells were seeded onto coated plates at a density of 6000 cells per well in expansion medium. The next day, the propagation medium was replaced with fresh spontaneous differentiation medium containing the test compounds. Cells were imaged and neurite length analyzed using an IncuCyte S3 Live Cell Analysis System every 4 h.

Example 83.

Αβ 1-42-induced cell death assay for human oligodendrocyte/neuron progenitor cells in a 96-well plate. Cells were seeded onto poly-L-ornithine and laminin-coated plates at a density of 8000 cells per well and maintained in expansion medium for up to 3 days, then the medium was replaced with fresh spontaneous differentiation medium containing test compounds and Annexin V red IncuCyte reagent (dilution 1:200) (Essen BioScience, cat. no. 4641). After a 30-minute pre-incubation period at 37°C, 10.5 μM Αβ 1-42 (Bachem Product, No. H-8146) was added directly to the pretreated solution. Neurite length and total annexin V positive area were analyzed using an IncuCyte S3 Live Cell Assay System every 2 h.

Example 84.

Bleomycin-induced pulmonary fibrosis in mice (therapeutic administration): Male C57B/L6 mice were kept acclimatized for at least 7 days. After an acclimatization period, animals were randomly assigned to treatment groups. On day 0, mice were anesthetized with 5% isoflurane by inhalation for 10–15 min and then suspended by their anterior teeth from a wire attached to a fixed animal operating plate. 50 μl of saline or bleomycin was administered by intratracheal infusion. Mice in the treatment groups received an intratracheal infusion of 2.0 U/kg (mg/kg) bleomycin hydrochloride (BLM) in sterile saline. On day 7 after bleomycin administration, half of the animals in the basal solution treatment group were sacrificed to establish the baseline level of fibrosis at the start of treatment. Mice in the normal control group received saline po once a day, from day 7 to day 20. Mice in the treatment group received the compound orally once a day, from day 7 to day 20. On day 20, 2-3 hours after the last dose, all mice were sacrificed. The lungs were gently washed through a tracheal cannula with 0.8 ml PBS containing 1% BSA and 0.6 mM EDTA. After washing, lung tissues were removed from each animal. Lung tissues were divided into 3 parts: the larger proportion of the right part was fixed in 10% neutral formalin for paraffin embedding and histopathology. The remaining right-hand portions were snap-frozen and protein lysates were prepared for target binding assays.

Example 85.

Bleomycin-induced pulmonary fibrosis in mice (prophylactic administration): Male C57B/L6 mice were ear-tagged and weighed prior to the study. Animals from the treatment group were treated with the compound on day -1 before bleomycin administration and on all subsequent days after bleomycin administration. On day 0, animals from the disease groups received a dose of 1.5 U/kg bleomycin by the oropharyngeal route. The control group without bleomycin received sterile saline. The animals were closely monitored daily until the end of the study. On day 21 after bleomycin administration, all animals were sacrificed and blood, lungs, and bronchoalveolar lavage fluid (BAL) were obtained. Blood samples were used to obtain plasma; The total number of leukocytes in the BAL fluid was counted; and the lungs were fixed in 10% neutral buffered formalin for histopathological analysis.

Example 86

Mouse model of histamine-induced vascular permeability: 7-8 week old female Balb/c mice were acclimatized for at least 3 days and randomly assigned to groups. Animals received an intravenous injection of 1% Evans blue at a dose of 100 μl/mouse. In 10

- 42 046894 min animals from the disease groups received an intradermal injection into the back of 1 μg of histamine in 20 μl of PBS and were killed 20 minutes after administration. Animals from the control group were injected intradermally into the back of animals with 20 μl of PBS. Dosing of the base solution and test compounds was carried out through a gastric tube, respectively. The positive control salbutamol was administered 15 min before Evans blue. Animals were sacrificed 20 min after histamine stimulation. The skin was then everted and the diameter of the lesions was measured. The area of lesions was calculated and expressed in square millimeters. After measuring the diameter, Evans blue dye was extracted from the skin tissue and the optical density was measured. Data were calculated relative to peak data on an Evans blue standard curve.

Example 87.

ROCK inhibitors effectively inhibit ROCK kinase activity in vitro and in cells. 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.

Table 1

Compound IC50 ppMlc (nM) Example 2 86 Example 8 517

In table 2 below provides in vitro and cellular ROCK inhibition data for the compounds of the invention. The activity of the compounds was determined using the Z'-lyte kinase kit (ThermoFisher Scientific). The percentage inhibition level was calculated by normalizing the kinase activity value 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.

table 2

The ex. No. % ING. ROCK2 (0.5 µM) IC50 ROCK 2 (nM) % ING. ROCK1 (0.5 µM) IC50 ROCK1 (nM) IC50 pMLC (nM), A7R5 NIH3T3, Acta2luc, IC50 (nM) 8 3 12 517 4 3 108 BY 2 4 17 120 352 3 12 222 157 9 15.4 355 10 21 255 1277 eleven 4 69 106 12 thirty 458 735 13 6.3 425 1568 14 6.8 353 496 15 24 655 1049 16 3.5 292 376 17 98% @0.5 µM 6.3 99% @ 0.5 µM 60 108

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18 89% @0.5 µM 10 92% @ 0.5 µM 504 464 19 90% @0.5 µM 4.8 96% @ 0.5 µM 355 190 20 94% @0.5 µM 1.7 98% @ 0.5 µM 263 157 21 3.1 47 66 22 8.9 169 155 23 91% @0.5 µM 21 435 252 24 97% @0.5 µM 22 88 334 1711 25 94 692 663 26 5.3 152 251 57 10 74 175 59 239 759 4173;5553 >5000 58 12 57 484 74 60 98 258 695 1413 61 2.9 7.7 35 48 27 65 1676 699 28 82 947 335 62 1.8 3 58 108 63 22 81 548 633 29 14 647 146 thirty 18 247 >5000 31 55 667 1229 32 31 >5000 >5000 33 29 85 913 1476 64 24 379 227 65 1.2 65 51 66 18 56 332 396 34 310 >5000 >5000 67 1.9 269 296 68 451 >5000 >5000 69 400 >5000 >5000

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35 486 >5000 >5000 70 7.7 134 71 3 57 301 36 16 1,577 1546 37 37 977 2314 38 8.7 338 1622 39 16 1,668 >5000 40 45 632 826 41 12 1248 >5000 42 6.5 359 43 47 1491 44 42 2,021 2229 45 53 1 358 1463 46 107 1,877 1200 47 thirty 321 465 48 67 314 1013 49 > 1000 74 > 1000 50 2.4 97 92 51 8.1 54 101 52 17 106 675 54 68 1,516 2666 53 31 759 1260 55 17 506 601 56 5.3 115 78 75 > 1000 72 46 382 984 73 1.6 15 122 76 950

The compounds of this invention contain an alkyl substituent (optionally substituted) at the 2-amino group (i.e. R ¹ is alkyl, etc.), which is associated with increased cellular ROCK activity compared to compounds containing a primary amino group at this position and in particular for lower alkyl amines at the 2-position (ie R ¹ is lower alkyl).

Example 88.

ROCK inhibitors cross the blood-brain barrier.

Brain and plasma concentrations of selected ROCK inhibitors were 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. 3 below.

Table 3.

Compound Dose level Path Place Average conc. after 0.25 hours (ng/ml) or (ng/g) Average conc. after 2 hours (ng/ml) or (ng/g) Average brain/plasma ratio at 0.25 h Average brain/plasma ratio at 2 hours EXAMPLE 2 2.5 IV Plasma 268 25.7 2.01 2.99 Head brain 541 76.6

The ROCK inhibitors of the invention effectively inhibited ROCK kinase activity in vitro and in cells.

As illustrated in FIG. 1a, compounds of the invention at a concentration of less than 20 nanomo

- 45 046894 lei effectively inhibited the activity of both ROCK isoforms in vitro as measured using the Z'-Lyte kit (ThermoFisher Scientific). The percentage inhibition rate was calculated by normalizing the kinase activity value obtained from compound treatment to that of the DMSO control and then plugging it into the equation [DMSO - (compound/DMSO)] x 100%. IC ₅₀ was calculated using the fitted nonlinear regression curve function of GraphPad Prism software and kinase activity data obtained from treatment with a 9-point 2-fold serial dilution of the compounds.

Intracellular A7R5 ELISA was performed to determine the ROCK inhibition of the test compounds. A7R5 cells were treated with a 9-point 2-fold serial dilution of compounds and ppMlc (T18/S19) levels were determined to calculate cellular IC ₅₀ values of compounds. The compounds of the invention exhibited activity from IC ₅₀ to 200 nM. See fig. 1b. 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 using the fitted nonlinear regression curve function of GraphPad Prism software.

ROCK inhibitors can be efficiently screened using a cell-based assay of the ACTA2 promoter-driven luciferase reporter gene. NIH3T3 cells stably expressing the ACTA2 promoter-driven luciferase gene were used to measure the functional IC ₅₀ value of compounds on ROCK in cells. Cells were seeded in 96-well plates until confluent and treated with a 9-point serial dilution of the compounds in combination in TGFe1 for 24 hours. Luciferase activity was measured and the IC ₅₀ of the compounds was calculated using the fitted nonlinear regression curve function of GraphPad Prism software. The compounds of the invention gave IC ₅₀ in cells of up to 200 nM, see FIG. 1s.

The ROCK activity of the compounds was tested in additional cell types. Svec 4-10 murine endothelial cells were treated with the compound of Example 2 for 120 min and the levels of ppMlc(T18/S19) and pMypt(T853) were visualized by Western blotting. The compounds of Example 2 at 110 nM effectively blocked phosphorylation of the ROCK targets MLC and MYPT1, see FIG. 1d.

Example 89: ROCK Inhibitors Stimulate Neurite Outgrowth and Neuronal Differentiation

Neuronal development requires highly coordinated regulation of the cytoskeleton. Activation of the RhoA/ROCK pathway leads to cytoskeletal changes, such as increased actin backflow and decreased actin depolymerization, and ultimately leads to growth cone collapse, which in turn inhibits neurite growth and propagation. Here, we show that ROCK inhibitors facilitate neuronal sprouting and neurite outgrowth in a dose-dependent manner. In fig. 2a, b demonstrate that the compounds of Example 2 and Example 17 significantly increase neurite length at different concentrations in cultured human oligodendrocyte/neuron progenitor cells. The compounds of Example 2 and Example 17 also facilitate neuronal differentiation and maturation, see FIG. 2c, d.

ROCK inhibitors block the inhibitory effect of chondroitin sulfate proteoglycan (CSPG) on neurite outgrowth. Neurons in the central nervous system of adult mammals are not capable of spontaneous regeneration after traumatic injury or neurodegeneration. This lack of regeneration was due to both the innate regenerative capacity of adult neurons and the inhibitory tissue environment. The main obstacles in the favorable environment are chondroitin sulfate proteoglycans (CSPGs), secreted by reactive astrocytes, and myelin-associated inhibitors produced by oligodendrocytes. These growth inhibitory glial cell molecules are overexpressed after neuronal injury, accumulate downstream of the RhoA/ROCK signaling pathway, and block neuroregeneration. To stimulate regeneration, inactivation of growth-inhibiting factors from the tissue environment is necessary. Here we show that by blocking the Rho/ROCK pathway, through which glial growth inhibitory factor signaling occurs, the inhibitory effect of CSPG on neurite elongation can be reversed. In fig. Figure 2 shows representative images on day 3. As illustrated in FIG. 2f and 2g, both 1 μM and 3 μM of the compounds of Example 2 and Example 17 significantly counteracted the 0.3 mg/ml CSPG-mediated limitation of neurite outgrowth in human oligodendrocyte/neuron progenitor cells. Statistical analysis for the 72 hour time point was performed using ANOVA.

Example 90.

ROCK inhibitors protect neuronal cells from Av 1-42-induced cell death.

In addition to stimulating neuroregeneration, ROCK inhibitors have been shown to have neuroprotective effects and prolong neuronal survival. Here, we examined the neuroprotective effect of ROCK inhibitors against Av 1-42. Human oligodendrocyte/neuron progenitor cells were preincubated with ROCK inhibitors for 30 min before exposure to 10.5 μM Ae1-42. Annexation reagent V red from Incu was added to the culture medium

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Cyte to detect cell apoptosis. Images were acquired using IncuCyte S3, and the total annexin V positive area was analyzed using IncuCyte S3 software. Representative images are illustrated in FIGS. 3a. Neurite length was quantified using the IncuCyte NeuroTrack software module, see FIG. 3b and 3c. In fig. 3e and 3f illustrate statistical analysis of annexin V area and neurite length for the 24 hour time point, which shows that 3 μM of the compounds of Example 2 and Example 17 significantly protected against Av 1-42-induced cell death, and 3 μM of the compound of Example 17 stimulated neurite growth in the presence of Av 1-42.

Example 91.

The ROCK inhibitors of the invention reduced the expression of profibrotic genes.

Expression of α-smooth muscle actin (aSMA or Acta2) by fibroblasts is a key marker indicating the transdifferentiation of fibroblasts into more profibrotic myofibroblasts, which are characterized by increased expression of fibrogenic factors. Such profibrotic factors, including TGFe signaling, lead to the expression of aSMA and are involved in the differentiation, proliferation and prolongation of myofibroblast survival. As illustrated in FIG. 4a, TGFe1-induced aSMA expression was significantly reduced by ROCK inhibitor. NIH3T3 cells were treated for 24 h with TGFe1 in a combination of ROCK inhibitors, and mRNA expression was quantified by Taqman qPCR.

In addition to expressing aSMA, myofibroblasts isolated from the lungs of patients with IPF showed signs of aging. They are resistant to apoptosis and have a very active metabolism, constantly producing large amounts of ECM proteins to promote the development of fibrosis. CCN1 (or CYR61) and CTGF belong to these classes of proteins. Treatment of human lung fibroblasts with ROCK inhibitor upon induction of TGFe strongly inhibited the expression of CTGF and CCN1, see Fig. 4b and c. The amount of secreted CCN1 proteins demonstrated by ELISA analysis of cell culture medium, as illustrated in FIG. 4d was also reduced by ROCK inhibition. CCD18Lu fibroblast cells were treated with ROCK inhibitor in combination with TGFe for 40 hours. CCN1 content was measured using a human CCN1 ELISA kit from R&D Systems. These results suggest that ROCK inhibitors may attenuate prosurvival and aging signaling in myofibroblasts under fibrotic conditions.

Example 92.

The ROCK inhibitor was effective in reducing some fibrotic parameters in a mouse model of bleomycin-induced pulmonary fibrosis.

Given the potent antifibrotic activity of the ROCK inhibitors of this invention, the ability of such inhibitors to reduce fibrosis in vivo was assessed in several mouse models of fibrotic diseases, including bleomycin-induced pulmonary fibrosis. After intratracheal infusion of bleomycin, mice were orally administered ROCK inhibitor at a dose of 3, 10, or 30 mg/kg. Control animals received only the basic solution according to the same administration schedule once a day. After 20 days of treatment, the animals were sacrificed and the lungs were removed, weighed and fixed for histopathological evaluation. In fig. Figure 5a shows representative images of Masson's trichrome-stained lung sections at 10X magnification from each treatment group. While treatment groups still exhibited fibrous mass formation (arrowhead), alveolar septal thickening, and nodule-like formations (arrow), all were less severe compared to bleomycin-induced controls. (B = bronchiole). Overall, in this model, ROCK inhibitor improved lung architecture and collagen deposition in a dose-dependent manner. However, mice receiving doses >10 mg/kg showed clear and statistically significant improvement.

Improvement in overall lung fibrosis, percentage aSmooth muscle actin (aSMA) positive area and total leukocyte count in bronchoalveolar lavage fluid were quantified by applying the Ashcroft grading scale to histopathological images, IGS quantification and flow cytometry, respectively. Administration of >10 mg/kg ROCK inhibitor resulted in a significant reduction in fibrosis and less inflammatory leukocyte infiltration compared to basal-treated controls, as indicated by lower group average fibrosis scores, aSMA-positive area, and total leukocyte counts in bronchoalveolar lavage fluid. . See fig. 5b.

Example 93.

ROCK inhibition is effective in reducing fibrosis scores when administered therapeutically in a mouse model of bleomycin-induced pulmonary fibrosis.

The activity of ROCK inhibitors in reducing pulmonary fibrosis at therapeutic dosing was assessed in a bleomycin-induced model following intratracheal infusion of bleomycin to induce lung tissue damage and subsequent pulmonary fibrosis. 7 days after the infusion of bleomycin, which ensured the development of fibrosis before the start of treatment, mice were treated with the compound

- 47 046894 nenium or control base solution. All mice were sacrificed on day 20 and lungs were obtained. A large portion of the right lung was fixed in 10% neutral formaldehyde, sectioned, and stained with Masson's trichrome. Clear antifibrotic effects were observed in the 10 and 30 mg/kg ROCK inhibitor treatment groups, as evidenced by lower collagen content and decreased Masson trichrome staining scores. See fig. 6a.

Improvement in overall pulmonary fibrosis was quantified for each animal in the control and treatment groups. See fig. 6b. Masson's trichrome-stained lung sections from individual mice are shown, with group averages and standard errors of the mean (SEM). The scores of normal control mice were 0. The amount of collagen in the lungs of mice treated with the compound of Example 2 was significantly less.

Example 94 Inhibition of ROCK stabilizes endothelial barrier function in a mouse model of histamine-induced vascular permeability.

In addition to damage to the organ epithelium, fibrotic diseases also cause damage to endothelial cells, leading to endothelial barrier destabilization and capillary leakage, which are hallmarks of fibrotic tissues. The ROCK signaling pathway is upregulated in damaged endothelial cells and leads to additional barrier destabilization and vascular leakage. The involvement of ROCK in the induction of vascular sweating led us to hypothesize that administration of a ROCK inhibitor may stabilize this vascular barrier function in vivo. We examined the efficacy of the blocking activity of a ROCK inhibitor following a single dose in a mouse model of histamine-induced dermal vascular sweating. In this model, intradermal injection of histamine rapidly induced sweating of Evans blue dye at the site of histamine injection. Administration of the ROCK inhibitor of Example 2 1 hour before histamine injection blocked histamine-induced vascular permeability in a dose-dependent manner, suggesting that in addition to antifibrotic activity, the ROCK inhibitors of this invention can also be used in diseases associated with induced vascular permeability. In fig. Figure 7a shows representative images of histamine-induced Evans blue dye sweating.

The degree of effectiveness of the compound was quantified by calculating the total area of the spot on the skin and the amount of Evans blue dye that penetrated the skin. Animals were sacrificed 20 min after histamine stimulation, and the diameter of the lesions was measured. The area of lesions was calculated and expressed in square millimeters. After measuring the diameter, Evans blue dye was extracted from the skin tissue and the optical density was measured. Data were calculated relative to peak data on an Evans blue standard curve. ** p < 0.01 compared with the basal solution group using one-way ANOVA followed by Dunnett's post hoc test. See fig. 7b.

Claims (16)

1. A compound having formula I R ¹ R ² R ³ (I) where A represents R ¹ is selected from the group consisting of lower alkyl, substituted lower alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₃ -C ₆ cycloalkyl, R ¹⁰ R ¹¹ N(CR ¹² R ¹³ )c-, R ¹⁰ O(CR ¹² R ¹³ )c-, W(CR ¹² R ¹³ )d- and R ¹⁰ R ¹¹ NC(=O)-(CR ¹² R ¹³ )c-; each R ¹⁰ is independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl; each R ¹¹ is independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl; each R ¹² is independently selected from H and lower alkyl; each R ¹³ is independently selected from H and lower alkyl; in an additional or alternative embodiment, R ¹² and R ¹³ attached to the same carbon atom may together form a C3-C6 cycloalkyl group; W represents a 3-7 membered heterocyclic ring having 1 to 3 ring heteroatoms, or a 5-6 membered heteroaryl ring having 1 to 3 ring heteroatoms; c is equal to 2 to 4; d is from 1 to 4; - 48 046894 R ² is selected from the group consisting of aryl, heteroaryl and heterocyclyl, where heteroaryl and heterocyclyl may be 5- or 6-membered monocyclic or 9- or 10-membered bicyclic rings having 1 to 3 ring heteroatoms, and each of aryl, heteroaryl and heterocyclyl may be optionally substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, RR'N -, RR'NCO-, RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; R ³ is selected from H, lower alkyl, substituted lower alkyl and RR'N-(C _2-4 alkyl)-; R ⁴ is selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, RR'N-, RR'NCO-, RCONR'-, RR'N-(C _2-4 alkyl)- and RR'N-(C _2-4 alkyl)-O-; R ⁵ is selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl; alternatively, R ³ and R ⁵ together with the nitrogen atoms to which they are attached form a cyclic group having from 5 to 7 ring atoms, including 2-3 ring heteroatoms, and the cyclic group is unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C1- _C3 perfluoroalkyl, C1- _C3 perfluoroalkoxy, carboxyl, aryl and heteroaryl; a is equal to 0 or 1; b is from 0 to 2 and each R and R' are independently selected from H, lower alkyl and C3-C6 cycloalkyl or, alternatively, R and R' together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic personal ring; where lower alkyl represents C _1-4 alkyl; substituted lower alkyl is _C1-4 alkyl having one to three substituents selected from the group consisting of halogen, hydroxy, C1- _C4 alkoxy, amino, nitro, cyano, perfluoro C1- _C4 alkyl, perfluoro C1- With ₄ alkoxy, RR'NCO- and carboxyl; lower alkoxy is -O-C _1-4 alkyl; carboxyl represents -CO2H; substituted C ₃ -C ₆ cycloalkyl is a C ₃ -C ₆ cycloalkyl having one to three substituents selected from the group consisting of halogen, hydroxy, C _1-4 alkyl, C 1-C ₄ alkoxy, amino, nitro, cyano , perfluoro C1-C ₄ alkyl, perfluoro C1-C ₄ alkoxy and carboxyl; aryl is phenyl; heteroaryl, unless otherwise stated, is a 5- or 6-membered aromatic group containing from one to four heteroatoms; and the heteroatom is nitrogen, oxygen or sulfur; or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, having formula II n l £ / R ² R ³ (II) or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1, having formula V Where R ¹ is selected from the group consisting of lower alkyl and substituted lower alkyl; each R ²¹ is independently selected from the group consisting of halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C1- _C3 perfluoroalkyl, C1- _C3 perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH- , RCONR'-, RO2C-, aryl-O- and heteroaryl-O-; each R and R' are independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl or, alternatively, R and R' together form a 5-6 membered heterocyclic ring and n is from 0 to 3; or a pharmaceutically acceptable salt thereof. 4. A compound according to claim 1, having formula VI - 49 046894 Where R ¹ is selected from the group consisting of lower alkyl and substituted lower alkyl; each R ²² is independently selected from halogen, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C ₁ -C ₃ perfluoroalkyl, C ₁ -C ₃ perfluoroalkoxy, carboxyl, RR'N-, RR'NCO-, RCONH-, RCONR '-, RO2C-, aryla-O- and heteroaryl-O-; each R and R' are independently selected from H, lower alkyl and C ₃ -C ₆ cycloalkyl or, alternatively, R and R' together form a 5-6 membered heterocyclic ring and n is from 0 to 3; or a pharmaceutically acceptable salt thereof. 5. A compound according to claim 1, having the formula n R ⁵ O G if 'Ϋ C3~ ^(R22)n where R ¹ is selected from the group consisting of lower alkyl and lower alkyl substituted -O^^alkyl, perfluoro C ₁ -C ₄ alkoxy, -C(=O)-NH ₂ , -C^ODC^C^alkyl) or - S^OD^S^kilD; R ²² is selected from halogen, lower alkyl, lower alkoxy and -C^O^Al^kil) and n is 1 or 2; or a pharmaceutically acceptable salt thereof. 6. A compound according to claim 5, where R ¹ represents lower alkyl; or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 6, where R ⁵ represents H; R ²² is selected from halogen, lower alkyl and lower alkoxy and n is 1; or a pharmaceutically acceptable salt thereof. 8. The compound according to claim 1, where the compound is N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; ^)-N(4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; ^)-N(4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-methoxyphenyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(p-tolyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(4-fluorophenyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(m-tolyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(ethylamino)-2-(3-fluorophenyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(ethylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-fluorophenyl)-2-(methylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(4-fluorophenyl)-2-(methylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-chlorophenyl)-2-(methylamino)acetamide; N(4-(N-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(p-tolyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(ethylamino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-fluoro-4-methoxyphenyl)-2-(methylamino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-(difluoromethoxy)phenyl)-2-(methylamino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(methylamino)-2-(2-methylbenzo^]thiazol-6-yl)acetamide; N(4-(III-pyrazol-4-yl)phenyl)-2-(3-(dimethylamino)phenyl)-2-(methylamino)acetamide; - 50 046894 No. (4-(1H-pyrazol-4-yl)phenyl)-2-(2-methoxypyridin-4-yl)-2-(methylamino)acetamide; methyl 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate; N(4-(1H-pyrazol-4-yl)phenyl)-2-(4-(dimethylamino)phenyl)-2-(methylamino)acetamide; 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid; H-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methyl-1H-indazol-6-yl)-2-(methylamino)acetamide; methyl 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoate; H-(4-(1H-pyrazol-4-yl)phenyl)-2-(2-methyl-1H-benzo[imidazol-6-yl)-2-(methylamino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-methoxypropyl)amino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-ethoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((3-(dimethylamino)propyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-isopropoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-amino-2-oxoethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((3-(methylamino)propyl)amino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((1-methoxy-2-methylpropan-2-yl)amino)-2-3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)-2-oxoethyl)amino)acetamide; 2-(((1H-pyrazol-4-yl)methyl)amino)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(ethyl(2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3-methoxyphenyl)acetamide; N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3methoxy(enyl)acetamide ; N(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)-H-methylacetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-morpholinoethyl)amino)acetamide; No.(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)(methyl)amino)-2-(3 - methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(pyrrolidin-1-yl)ethyl)amino)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-((1-(methoxymethyl)cyclopropyl)amino)-2-(3-methoxyphenyl)acetamide; N(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(2,2,2-trifluoroethoxy)ethyl)amino)acetamide; N(3-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; N(3-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-((2-(dimethylamino)ethyl)amino)-2-(3-methoxyphenyl)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-(methylamino)ethyl)amino)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-((2-methylamino)ethyl)amino)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide; ^)-H-(4-(1H-pyrazol-4-yl)phenyl)-2-(4-chlorophenyl)-2-(methylamino)acetamide; N(2-fluoro-5-methoxy-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide; 3-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-H-methylbenzamide; 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)benzoic acid; 4-(2-((4-(1H-pyrazol-4-yl)phenyl)amino)-1-(methylamino)-2-oxoethyl)-H-methylbenzamide; N(2-(3-(dimethylamino)propoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide; N(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide; ^)-H-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide; ^)-H-(2-(2-(dimethylamino)ethoxy)-5-fluoro-4-(III-pyrazol-4-yl)phenyl)-2-(3-methoxyphenyl)-2(methylamino)acetamide; or a pharmaceutically acceptable salt thereof. 9. A pharmaceutical composition containing a therapeutically effective amount of a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 10. Use of a compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for the treatment of a fibrotic disorder in a subject. 11. Use according to claim 10, wherein the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, cystic and idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, myocardial fibrosis, arterial fibrosis, endomyocardial fibrosis - 51 046894 fibrosis, previous myocardial infarction, arterial stiffness, atherosclerosis, restenosis, arthrofibrosis, Crohn's disease, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, mediastinal fibrosis, keloid and hypertrophic scars , scarring of glial tissue and renal fibrosis. 12. Use according to claim 11, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis. 13. Use of a compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for treating a central nervous system disorder in a subject 14. Use according to claim 13, wherein the central nervous system disorder is selected from the group consisting of Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Batten disease, dementia, spinal muscular atrophy, motor neuron disease, spinocerebellar ataxia , acute or chronic pain, neuronal degeneration, spinal cord injury, cerebral vasospasm and multiple sclerosis. 15. Use of a compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for the treatment of glaucoma in a subject. 16. Use of a compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for the treatment of inflammation in a subject.