Azaindole ROCK inhibitors

Abstract

The present invention provides compounds that inhibit rho-associated protein kinase (ROCK) for therapeutic uses as further described herein.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 436,408, filed December 30, 2022, the entirety of which is incorporated herein by reference for all purposes.

[0002] The present invention provides compounds that inhibit rho-associated protein kinase (ROCK) for therapeutic uses as further described herein. [Background technology]

[0003] ROCK (Rho-associated protein kinase) is a member of the AGC (cAMP-dependent protein kinase (PKA) / protein kinase G (PKG) / protein kinase C (PKC)) family of serine-threonine kinases and is activated by the GTP-bound form of RhoA. Two isoforms of mammalian Rho kinase, ROCK1 and ROCK2, have been reported. They are proteins of approximately 160 kDa, consisting of 1354 and 1388 amino acids, respectively. They contain an N-terminal kinase domain followed by a coiled-coil region containing a Rho-binding domain (RBD), a pleckstrin homology domain (PH), and a C-terminal cysteine-rich region. The RBD binds only to GTP-bound, active RhoA and RhoC. The two ROCK isoforms share approximately 60% amino acid identity overall and approximately 90% identity within the N-terminal kinase domain. The carboxyl-terminal region contains two cysteine-rich zinc finger-like motif domains and a split pleckstrin homology domain, which plays a role in stabilizing the membrane binding of ROCK (see Non-Patent Documents 1, 2, and 3).

[0004] ROCK is enzymatically inactive in its native form. This is caused by autoinhibition of the ROCK kinase domain by the carboxyl-terminal region of ROCK. The best-characterized upstream activators of ROCK are the Rho-GTPase proteins RhoA and RhoC. In their activated GTP-bound state, they interact with the Rho-binding domain of ROCK, causing a conformational change that prevents the autoinhibitory function of the carboxyl-terminal region (Non-Patent Document 2).

[0005] There are numerous downstream targets phosphorylated by ROCK. When ROCK is activated, several central regulatory proteins are phosphorylated, resulting in diverse cellular responses, such as autophagy, cell survival and apoptosis, vesicle dynamics, cytoskeletal regulation, cell growth and reproduction, and cell shape and motility. In response to Rho activators, which stimulate Rho-guanine nucleotide exchange factors (GEFs) and result in the formation of active GTP-bound Rho, ROCKs mediate a wide range of cellular responses involving the actin cytoskeleton. For example, they regulate the assembly of the actin cytoskeleton and cell contractility by phosphorylating various proteins, such as myosin light chain (MLC) phosphatase, LIM kinase, adducin, and ERM (Ezrin / Radixin / Moesin) proteins. ROCK2 acts as a signaling pathway by which MLCK regulates Ca2+ expression. 2+ It is sensitive to Ca in smooth muscle cells. 2+ ROCKs are important regulators of cell growth, migration, metabolism, and apoptosis by controlling the assembly of the actin cytoskeleton and cell contraction. ROCK1 expression is more ubiquitous (ROCK1 messenger RNA and protein are highly expressed in the lung, liver, spleen, kidney, and testis), while ROCK2 is most highly expressed in cardiac and brain tissues. ROCKs regulate cell polarity and migration primarily through actomyosin contraction and focal adhesion enhancement. Elevated ROCK activity has been observed in tumor metastasis, and overexpression of constitutively activated ROCK promotes tumor invasion (Non-Patent Document 4, Non-Patent Document 5).

[0006] Pharmacological inhibitors of ROCK, such as Y-27632, fasudil (HA1077), and hydroxyfasudil, target their ATP-dependent kinase domains and can inhibit both ROCK1 and ROCK2. ROCK inhibitors have been investigated for the treatment of various pathological conditions, including asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, renal failure, neurodegeneration, and osteoporosis. To date, two ROCK inhibitors have been approved for clinical use: fasudil for the treatment of cerebral vasospasm and ripasudil for the treatment of glaucoma.

[0007] Fasudil (an isoquinoline derivative) has been shown to effectively inhibit other kinases, such as PKA, PKG, PKC, and MLCK, in addition to ROCK (Non-Patent Document 6). A randomized, placebo-controlled, double-blind phase IIa clinical trial of the ROCK inhibitor fasudil in amyotrophic lateral sclerosis (ALS) began on February 20, 2019, and is currently ongoing (Clinical Trials Identifier: NCT03792490; Eudra-CT Number: 2017-003676-31).

[0008] Several synthetic ROCK inhibitors identified by researchers at Scripps Research Institute based on indole, 5-azaindole, and 7-azaindole heterocyclic systems have been described in the literature (Non-Patent Document 7, Non-Patent Document 8). The synthesis and inhibitory activity against ROCK of several benzothiazole derivatives have also been previously disclosed (Non-Patent Document 9). Further ROCK inhibitors are described in Patent Document 1 filed by researchers at Scripps Research Institute. The ROCK inhibitory activity of these indole, azaindole, and benzothiazole compounds varies significantly depending on the substituents attached to the heterocycle.

[0009] Patent applications describing ROCK inhibitors and their uses include U.S. Patent No. 6,223,669, ... [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2011 / 050245 [Patent Document 2] International Publication No. 2023 / 209692 [Patent Document 3] International Publication No. 2023 / 139379 [Patent Document 4] International Publication No. 2022 / 020381 [Patent Document 5] International Publication No. 2022 / 150676 [Patent Document 6] International Publication No. 2022 / 042712 [Patent Document 7] International Publication No. 2022 / 012409 [Patent Document 8] International Publication No. 2021 / 214200 [Patent Document 9] International Publication No. 2021 / 095945 [Patent Document 10] International Publication No. 2020 / 177292 [Patent Document 11] International Publication No. 2020 / 094111 [Patent Document 12] International Publication No. 2019 / 000683 [Patent Document 13] International Publication No. 2019 / 000682 [Patent Document 14] International Publication No. 2018 / 130178

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Patent document 16

Patent document 17

Patent document 18

Patent document 19

Patent document 20

Patent document 21

Non-licensed literature

[0011] [Non-licensed document 1] S. Hartmann, AJ Ridley, and S. Lutz. "The Function of Rho-Associated kinases ROCK1 and ROCK2 in the Pathogenesis of Cardiovascular Disease", Frontiers in Pharmacology, November 2015, Vol. 6, Art. 276 [Non-licensed document 2] JC Koch, L. Tatenhorst, A.-E. Roser, K.-A. Saal, L. Toenges, P. Lingor. "ROCK inhibition in models of neurodegeneration and its potential for clinical translation", Pharmacology & Therapeutics 189 (2018) 1-21 [Non-licensed document 3] Y. Feng, PV LoGrasso, O. Defert, and R. Li. "Rho kinase (ROCK) inhibitors and their therapeutic potential", J. Med. Chem. Rev. 2016, 59, 2269–2300

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[0012] Despite these efforts, there remains a need for new ROCK inhibitor compounds for treating disorders mediated by ROCK1 and / or ROCK2 in subjects, eg, humans, in need of such treatment. [Means for solving the problem]

[0013] Azaindole compounds of Formula I, Formula II, and Formula III, as well as pharmaceutically acceptable salts thereof, are provided that inhibit rho-associated protein kinase (ROCK). These compounds exhibit advantageous ADME properties (absorption, distribution, metabolism, and / or excretion) and exhibit greater potency than close analogs in enzyme inhibition assays (see Examples 24 and 25). Surprisingly, these compounds have also been found to have activity against protein kinase X (PRKX; see Tables 2A and 2B). Both ROCK and PRKX activity promote neurodegeneration in amyotrophic lateral sclerosis (ALS). Therefore, by inhibiting both enzymes, certain compounds of the present invention are expected to exhibit advantageous therapeutic efficacy against this disease in humans.

[0014] In certain embodiments, the compound of the present invention is Compound 1: [ka] or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, the compound of the present invention is Compound 2: [ka] or a pharmaceutically acceptable salt thereof.

[0016] Compounds 1 and 2 are highly potent inhibitors of ROCK1, ROCK2, and PRKX (see Tables 2A and 2B). These two compounds have superior efflux ratios compared to eight comparative compounds tested (see Tables 3A and 3B). These properties are important in the treatment of disorders mediated by ROCK1 or ROCK2, such as ALS.

[0017] An effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof and / or a pharmaceutically acceptable composition thereof provided herein can be used to treat a disorder mediated by ROCK1 and / or ROCK2. In some embodiments, methods are provided for treating a subject having a disorder mediated by ROCK1 and / or ROCK2, comprising administering to the subject, typically a human, an effective amount of one or more compounds described herein or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable composition. In certain embodiments, the disorder is mediated by ROCK1. In additional embodiments, the disorder is mediated by ROCK2. In other embodiments, the disorder is mediated by PRKX. In certain embodiments, the disorder is mediated by ROCK1 and PRKX.

[0018] In certain embodiments, a compound of Formula I, Formula II, or Formula III: [ka] (In the formula, R 1 and R 2 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; In certain embodiments, R 1 and R 2 is hydrogen, R 3 is selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl; In certain embodiments, R 3 is H or CH3, R 4 and R 5 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; R 6 is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, halogen, C1-C4 alkyl-OR 7 , and OR 7 is selected from R 7 is selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl) or a pharmaceutically acceptable salt thereof.

[0019] All combinations of variables, substituents, embodiments, and compounds resulting from combinations thereof are considered to be specifically and individually disclosed, as such descriptions are for space purposes only and are not intended to describe only a genus or even a subgenus of compounds.

[0020] In certain embodiments, compounds of the present invention are used to treat disorders mediated by ROCK1 or ROCK2 in the central nervous system (CNS). In other embodiments, compounds of the present invention are used to treat peripheral disorders mediated by ROCK1 or ROCK2.

[0021] In certain embodiments, the compound of formula I has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0022] In other embodiments, the compound of formula I has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0023] In certain embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0024] Non-limiting examples of compounds of Formula I include: [ka] or a pharmaceutically acceptable salt thereof.

[0025] Additional non-limiting examples of compounds of Formula I include: [ka] or a pharmaceutically acceptable salt thereof.

[0026] In other embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0027] Additional examples of compounds of Formula I include: [ka] or a pharmaceutically acceptable salt thereof.

[0028] In certain embodiments, the compound of formula II is [ka] or a pharmaceutically acceptable salt thereof.

[0029] In other embodiments, the compound of formula II is [ka] or a pharmaceutically acceptable salt thereof.

[0030] Non-limiting examples of compounds of Formula II include: [ka] or a pharmaceutically acceptable salt thereof.

[0031] Additional non-limiting examples of compounds of Formula II include: [ka] or a pharmaceutically acceptable salt thereof.

[0032] Additional examples of compounds of formula II include: [ka] or a pharmaceutically acceptable salt thereof.

[0033] In certain embodiments, the compound of formula III is [ka] or a pharmaceutically acceptable salt thereof.

[0034] Non-limiting examples of compounds of formula III include: [ka] or a pharmaceutically acceptable salt thereof.

[0035] In certain embodiments, the compounds of the present invention have sufficient ADME properties to penetrate the blood-brain barrier. Adequate levels of blood penetration are important for the treatment of neurodegenerative disorders. For example, amyotrophic lateral sclerosis primarily affects nerve cells in the brain, and blood penetration is required for the treatment of this disease. In other embodiments, the compounds of the present invention are used to treat stroke, spinal cord injury, Alzheimer's disease, or Parkinson's disease.

[0036] In certain embodiments, methods of treatment are provided that include administering an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable composition, to a subject, e.g., a human, in need thereof. For example, in certain embodiments, a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, is administered to a human to be treated for a neurodegenerative disorder, e.g., amyotrophic lateral sclerosis (ALS) or Parkinson's disease (PD).

[0037] In certain embodiments, compounds of the present invention are used to treat ALS. For example, in certain embodiments, compounds of the present invention are used to treat bulbar, respiratory, flail arm, classical, pyramidal, or flail leg ALS. In certain embodiments, compounds of the present invention are used to treat Parkinson's disease. For example, in certain embodiments, compounds of the present invention are used to treat motor-cognitive, cognitive-predominant, or motor-predominant Parkinson's disease.

[0038] In other embodiments, the compounds of the invention are used to treat a disorder selected from cerebral vasospasm, pulmonary hypertension, acute lung injury exfoliation syndrome, ocular hypertension, or glaucoma (e.g., exfoliation glaucoma). Alternatively, the compounds of the invention can be used to treat edema (e.g., pulmonary edema), inflammatory bowel disease, or inflammation.

[0039] In certain embodiments, compounds of the present invention offer one or more advantages over their close analogs. For example, selected compounds of Formula I, II, or III may demonstrate a) advantageous potency against ROCK1, ROCK2, and / or PRKX, b) advantageous permeability, c) lower efflux ratio, d) lower hERG activity, e) advantageous penetration of the blood-brain barrier, and / or f) advantageous bioavailability compared to their close analogs. As a result of these advantageous properties, in certain embodiments, compounds of the present invention can be taken orally to treat disorders mediated by ROCK1 and / or ROCK2, including neurodegenerative disorders such as amyotrophic lateral sclerosis or Parkinson's disease.

[0040] Selected compounds of the present invention also have advantageous properties compared to fasudil. For example, Compounds 1, 2, 3, 4, and 5, when tested in head-to-head studies with fasudil, possessed superior ROCK1 and ROCK2 inhibitory activity and exhibited lower efflux ratios (see Examples 24 and 25). The concentration of Compound 1 required to inhibit half of ROCK2 activity was less than 50-fold lower than that required for fasudil (Example 24). Similarly, the concentrations of Compounds 2, 3, 4, and 5 required to inhibit half of ROCK2 activity were more than an order of magnitude lower than that required for fasudil (Example 24).

[0041] In certain embodiments, a selected compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, has an isotopic substitution of at least one desired atom at an amount above the natural abundance of the isotope, i.e., enriched. In certain embodiments, a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, comprises a deuterium atom or multiple deuterium atoms. For example, in certain embodiments, a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, has one or more deuterium substitutions at a metabolic site. In other embodiments, a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, has one or more deuterium substitutions adjacent to a metabolic site.

[0042] Other features and advantages of the present application will be apparent from the following detailed description.

[0043] To this end, the present invention includes at least the following features: (a) a compound of Formula I, Formula II, or Formula III described herein, or a pharmaceutically acceptable salt or isotopic derivative thereof (including deuterated derivatives); (b) a method of treating a disorder mediated by ROCK1 and / or ROCK2, e.g., a neurodegenerative disorder (including, e.g., ALS or Parkinson's disease), comprising administering to a subject in need thereof an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof; (c) a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disorder mediated by ROCK1 and / or ROCK2, such as a neurodegenerative disorder (including, for example, ALS or Parkinson's disease); (d) use of an effective amount of a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, in the treatment of a subject, typically a human, in need of treatment, having a disorder mediated by ROCK1 and / or ROCK2, such as a neurodegenerative disorder (including, for example, ALS or Parkinson's disease); (e) use of a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disorder mediated by ROCK1 and / or ROCK2, such as a neurodegenerative disorder (including, for example, ALS or Parkinson's disease); (f) a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, Formula II, or Formula III or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; (g) Any of (a) to (f) above, wherein the compound is of formula I; (h) Any of the above (a) to (f), wherein the compound is Compound 1; (i) any of the above (a) to (f), wherein the compound is Compound 2; (j) Any of the above (a) to (f), wherein the compound is Compound 10; (k) Any of the above (a) to (f), wherein the compound is compound 11; (l) Any of the above (a) to (f), wherein the compound is Compound 12; (m) Any of the above (a) to (f), wherein the compound is compound 13; (n) Any of the above (a) to (f), wherein the compound is compound 14; (o) Any of (a) to (f) above, wherein the compound is of formula II (p) Any of the above (a) to (f), wherein the compound is Compound 3; (q) Any of the above (a) to (f), wherein the compound is Compound 4; (r) any one of the above (a) to (f), wherein the compound is Compound 5; (s) Any of the above (a) to (f), wherein the compound is Compound 7; (t) Any of the above (a) to (f), wherein the compound is Compound 8; (u) Any of the above (a) to (f), wherein the compound is compound 9; (v) Any of (a) to (f) above, wherein the compound is of formula III (w) Any of the above (a) to (f), wherein the compound is Compound 6. DETAILED DESCRIPTION OF THE INVENTION

[0044] I. Definition Compounds are described using their proper names. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The recitation of ranges of values, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein by reference as if it were individually recited herein. The endpoints of all ranges are included within the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of example or exemplary language (e.g., "such as") is intended merely to better describe the invention and does not purport to limit the scope of the invention unless otherwise asserted.

[0046] The present invention includes compounds of Formula I, Formula II, or Formula III, or pharmaceutically acceptable salts thereof, having isotopic substitution of at least one desired atom at greater than the natural abundance of the isotope, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but different number of neutrons.

[0047] Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine, and iodine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 17 O. 18 O. 18 F,36 Cl and 125 In one non-limiting embodiment, the isotopically labeled compounds are used in metabolic studies (e.g., 14 C), reaction kinetic studies (e.g. 2 H or 3 H), drug or substrate tissue distribution assays or radiation treatment of patients, including detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT). 18 F-labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of the invention and prodrugs thereof can generally be prepared by following the procedures disclosed in the schemes or in the examples and preparations below, substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0048] Isotopic substitution, e.g., deuterium substitution, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is replaced with deuterium. In certain embodiments, the isotope is enriched at any position of interest by 90%, 95%, or 99% or more. In one non-limiting embodiment, deuterium is enriched at the desired position by 90%, 95%, or 99%.

[0049] In one non-limiting embodiment, a deuterium atom can be substituted for a hydrogen atom in a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, optionally at a metabolically labile position or adjacent to a metabolically labile position. In one non-limiting embodiment, a deuterium atom is substituted for a hydrogen atom in one or more groups selected from any of the R or variables described herein. For example, an alkyl residue can be deuterated if any of the groups is, or contains, methyl, ethyl, or methoxy (such as, in non-limiting embodiments, CDH2, CD2H, CD3, CH2CD3, CD2CD3, CHDCH2D, CH2CD3, CHDCHD2, OCDH2, OCD2H, or OCD3).

[0050] In certain embodiments, the compounds of the present invention may form solvates with solvents (including, for example, water). Thus, in one non-limiting embodiment, the present invention includes compounds in solvated form. The term "solvate" refers to a molecular complex of a compound of the present invention (including its salts) with one or more solvent molecules. Non-limiting examples of solvents include water, ethanol, isopropanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term "hydrate" refers to a molecular complex comprising a compound of the present invention and water. Pharmaceutically acceptable solvates according to the present invention include those in which the solvent may be isotopically substituted, such as DO, d6-acetone, and d6-DMSO (dimethyl sulfoxide). The solvate may be in liquid or solid form.

[0051] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=O)NH is attached through the carbon of the carbonyl (C=O) group.

[0052] An "alkyl" is a straight-chain, branched-chain, or cyclic saturated aliphatic hydrocarbon group. In certain embodiments, alkyl is C1-C2, C1-C3, or C1-C4; when alkyl is cyclic, it may be, for example, a C3-C4 moiety. As used herein, specified ranges refer to alkyl groups with each member of the range described as a separate species. For example, as used herein, the term C1-C4 alkyl refers to straight-chain or branched-chain alkyl groups having 1, 2, 3, or 4 carbon atoms, each of which is intended to be described as a separate species, and thus each subset is considered separately disclosed. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. The term "alkyl" also encompasses cycloalkyl or carbocyclic groups. For example, when a term containing "alk" is used, "cycloalkyl" or "carbocyclic" can be included as part of the definition unless clearly excluded by context. For example, without limitation, terms such as alkyl, alkoxy, haloalkyl, etc., can all be taken to include cyclic forms of alkyl unless the context clearly excludes otherwise.

[0053] In certain embodiments, "alkyl" is C1-C4 alkyl, C1-C3 alkyl, or C1-C2 alkyl.

[0054] In certain embodiments, an "alkyl" contains one carbon.

[0055] In certain embodiments, an "alkyl" contains two carbons.

[0056] In certain embodiments, the "alkyl" contains 3 carbons.

[0057] In certain embodiments, the "alkyl" contains 4 carbons.

[0058] Non-limiting examples of "alkyl" include methyl, ethyl, propyl, and butyl.

[0059] Additional non-limiting examples of "alkyl" include isopropyl and isobutyl.

[0060] Additional non-limiting examples of "alkyl" include sec-butyl and tert-butyl.

[0061] In certain embodiments, "cycloalkyl" is a C3-C4 cycloalkyl.

[0062] In certain embodiments, the "cycloalkyl" has 3 carbons.

[0063] In certain embodiments, the "cycloalkyl" has 4 carbons.

[0064] Non-limiting examples of "cycloalkyl" include cyclopropyl and cyclobutyl.

[0065] "Halo" and "halogen" refer independently to fluorine, chlorine, bromine, or iodine.

[0066] "Haloalkyl" refers to a linear, branched, or cyclic alkyl group substituted with one or more of the above-mentioned halo atoms, up to the maximum allowable number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Perhaloalkyl" refers to an alkyl group in which all hydrogen atoms have been replaced with halogen atoms. Examples include, but are not limited to, trifluoromethyl and pentafluoroethyl.

[0067] In certain embodiments, "haloalkyl" is C1-C4 haloalkyl, C1-C3 haloalkyl, or C1-C2 haloalkyl.

[0068] In certain embodiments, the "haloalkyl" has 1 carbon.

[0069] In certain embodiments, a "haloalkyl" has one carbon and one halogen.

[0070] In certain embodiments, a "haloalkyl" has 1 carbon and 2 halogens.

[0071] In certain embodiments, a "haloalkyl" has 1 carbon and 3 halogens.

[0072] In certain embodiments, the "haloalkyl" has two carbons.

[0073] In certain embodiments, the "haloalkyl" has 3 carbons.

[0074] In certain embodiments, the "haloalkyl" has 4 carbons.

[0075] In certain embodiments, a "haloalkyl" is a perhaloalkyl.

[0076] Non-limiting examples of "haloalkyl" include: [ka] Examples include:

[0077] Additional non-limiting examples of "haloalkyl" include: [ka] Examples include:

[0078] Additional non-limiting examples of "haloalkyl" include: [ka] Examples include:

[0079] Additional non-limiting examples of "haloalkyl" include: [ka] Examples include:

[0080] "Dosage form" means a unit dose of an active agent. Examples of dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, implants, particles, spheres, creams, ointments, suppositories, inhalable forms, transdermal forms, buccal dosage forms, sublingual dosage forms, topical dosage forms, gels, mucosal dosage forms, etc. "Dosage form" can also include implants, such as optical implants.

[0081] "Effective amount," as used herein, means an amount that produces a therapeutic effect.

[0082] "Parenteral" administration of pharmaceutical compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0083] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject (i.e., palliative treatment), or reducing the cause or effects of the disease or disorder (i.e., disease-modifying treatment).

[0084] As used herein, a "pharmaceutical composition" is a composition containing at least one active agent and at least one other substance, such as a carrier. "Pharmaceutical combinations" are combinations of at least two active agents, which can be combined in a single dosage form or given together in separate dosage forms, where the active agents are indicated for use in combination to treat any of the disorders described herein.

[0085] As used herein, "pharmaceutically acceptable salts" refer to forms of the disclosed compounds in which the parent compound has been modified by making non-toxic inorganic and organic acid or base addition salts thereof. Salts of the present compounds can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid, or by reacting the free acid form of the compounds with a stoichiometric amount of an appropriate base (such as hydroxide, carbonate, bicarbonate, or the like of Na, Ca, Mg, or K). Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical when practical.

[0086] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids, and quaternary ammonium salts. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts derived from acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) n These include salts prepared from organic acids such as —COOH, where n is 0 to 4, or using a different acid that produces the same counterion. Additional lists of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0087] The term "carrier" used in pharmaceutical compositions / combinations of the invention refers to a diluent, excipient, or vehicle provided with an active compound.

[0088] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a carrier or excipient useful in preparing a pharmaceutical composition / combination that is generally safe, suitably non-toxic, and not biologically or otherwise unsuitable for administration to a subject, usually a human.

[0089] A "patient" or "subject" is a human or domestic animal in need of treatment for any of the disorders specifically described herein. Non-limiting examples of domestic animals include dogs, cats, horses, and farm animals. As further described herein, the word patient or subject typically refers to a human patient or subject, and is assumed to refer to a human unless the context indicates otherwise. In alternative embodiments, the patient or subject is a domestic animal in need of and responsive to such therapy. "Livestock" refers to animals typically raised for agricultural purposes, including, for example, cattle, sheep, goats, pigs, and poultry.

[0090] A "therapeutically effective amount" of a pharmaceutical composition / combination of the present invention means an amount that is effective to bring about a therapeutic effect, such as amelioration of symptoms or relief or reduction of the disease itself, when administered to a subject.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not admitted to be prior art to this application. In the case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0092] II. Compounds of Formula I, Formula II and Formula III In certain embodiments, the present invention provides a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof. [ka] wherein all variables are as defined herein.

[0093] In certain embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0094] In certain embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0095] In certain embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0096] In other embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0097] In other embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0098] In certain embodiments, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0099] Non-limiting examples of compounds of Formula I include: [ka] or a pharmaceutically acceptable salt thereof.

[0100] In an alternative embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0101] Alternative examples of compounds of formula I include: [ka] or a pharmaceutically acceptable salt thereof.

[0102] In certain embodiments, the compound of formula II is [ka] or a pharmaceutically acceptable salt thereof.

[0103] In certain embodiments, the compound of formula II is [ka] or a pharmaceutically acceptable salt thereof.

[0104] Non-limiting examples of compounds of Formula II include: [ka] or a pharmaceutically acceptable salt thereof.

[0105] In an alternative embodiment, the compound of formula II is [ka] or a pharmaceutically acceptable salt thereof.

[0106] Additional non-limiting examples of compounds of the present invention include: [ka] or a pharmaceutically acceptable salt thereof.

[0107] R 1 Embodiments of the present invention In certain embodiments, R 1 is hydrogen.

[0108] In certain embodiments, R 1 is CH3.

[0109] In certain embodiments, R 1 is C2H5.

[0110] In certain embodiments, R 2 is CH2CH2CH3.

[0111] In certain embodiments, R 1 is iso-C3H7.

[0112] In certain embodiments, R 1 is F.

[0113] In certain embodiments, R 1 is Cl.

[0114] In certain embodiments, R 1 is Br.

[0115] In certain embodiments, R 1 is CF3.

[0116] In certain embodiments, R 1 is CH2F.

[0117] In certain embodiments, R 1 is CH2CF3.

[0118] R 2 Embodiments of the present invention In certain embodiments, R 2 is hydrogen.

[0119] In certain embodiments, R 2 is CH3.

[0120] In certain embodiments, R 2 is C2H5.

[0121] In certain embodiments, R 2 is CH2CH2CH3.

[0122] In certain embodiments, R 2 is iso-C3H7.

[0123] In certain embodiments, R 2 is F.

[0124] In certain embodiments, R 2 is Cl.

[0125] In certain embodiments, R 2 is Br.

[0126] In certain embodiments, R 2 is CF3.

[0127] In certain embodiments, R 2 is CH2F.

[0128] In certain embodiments, R 2 is CH2CF3.

[0129] R 3 Embodiments of the present invention In certain embodiments, R 3 is hydrogen.

[0130] In certain embodiments, R 3 is CH3.

[0131] In certain embodiments, R 3 is C2H5.

[0132] In certain embodiments, R 3 is CH2CH2CH3.

[0133] In certain embodiments, R 3 is iso-C3H7.

[0134] In certain embodiments, R 3 is CF3.

[0135] In certain embodiments, R 3 is CH2F.

[0136] In certain embodiments, R 3 is CH2CF3.

[0137] R 4 Embodiments of the present invention In certain embodiments, R 4 is hydrogen.

[0138] In certain embodiments, R 4 is CH3.

[0139] In certain embodiments, R 4 is C2H5.

[0140] In certain embodiments, R 4 is CH2CH2CH3.

[0141] In certain embodiments, R 4 is iso-C3H7.

[0142] In certain embodiments, R 4 is F.

[0143] In certain embodiments, R4 is Cl.

[0144] In certain embodiments, R 4 is Br.

[0145] In certain embodiments, R 4 is CF3.

[0146] In certain embodiments, R 4 is CH2F.

[0147] In certain embodiments, R 4 is CH2CF3.

[0148] R 5 Embodiments of the present invention In certain embodiments, R 5 is hydrogen.

[0149] In certain embodiments, R 5 is CH3.

[0150] In certain embodiments, R 5 is C2H5.

[0151] In certain embodiments, R 5 is CH2CH2CH3.

[0152] In certain embodiments, R 5 is iso-C3H7.

[0153] In certain embodiments, R 5 is F.

[0154] In certain embodiments, R 5 is Cl.

[0155] In certain embodiments, R 5 is Br.

[0156] In certain embodiments, R 5 is CF3.

[0157] In certain embodiments, R 5 is CH2F.

[0158] In certain embodiments, R 5 is CH2CF3.

[0159] R 6 Embodiments of the present invention In certain embodiments, R 6 is hydrogen.

[0160] In certain embodiments, R 6 is CH3.

[0161] In certain embodiments, R 6 is C2H5.

[0162] In certain embodiments, R 6 is CH2CH2CH3.

[0163] In certain embodiments, R 6 is iso-C3H7.

[0164] In certain embodiments, R 6 is F.

[0165] In certain embodiments, R 6 is Cl.

[0166] In certain embodiments, R 6 is Br.

[0167] In certain embodiments, R 6 is CF3.

[0168] In certain embodiments, R 6 is CH2F.

[0169] In certain embodiments, R 6 is CH2CF3.

[0170] In certain embodiments, R 6 is OH.

[0171] In certain embodiments, R 6 is OCH3.

[0172] In certain embodiments, R 6 is OC2H5.

[0173] In certain embodiments, R 6 is OCH2CH2CH3.

[0174] In certain embodiments, R 6 is OC3H7-iso.

[0175] In certain embodiments, R 6 is OCF3.

[0176] In certain embodiments, R 6 is OCH2CF3.

[0177] R 7 Embodiments of the present invention In certain embodiments, R 7 is hydrogen.

[0178] In certain embodiments, R 7 is CH3.

[0179] In certain embodiments, R 7 is C2H5.

[0180] In certain embodiments, R 7 is CH2CH2CH3.

[0181] In certain embodiments, R7 is iso-C3H7.

[0182] In certain embodiments, R 7 is CF3.

[0183] In certain embodiments, R 7 is CH2F.

[0184] In certain embodiments, R 7 is CH2CF3.

[0185] In certain embodiments, [ka] The structure: [ka] It is of the type.

[0186] In certain embodiments, [ka] The structure: [ka] It is of the type.

[0187] In certain embodiments, [ka] The structure: [ka] It is of the type.

[0188] The structures of the compounds of the present invention are typically selected to be sufficiently stable to maintain a shelf life of at least 2, 3, 4, or 5 months under ambient conditions. To achieve this, each of the variables described herein is selected so that the resulting compound achieves the desired shelf life of at least 2, 3, 4, or 5 months under ambient conditions. Those skilled in the art are well aware of the stability of chemical moieties and can avoid those that are unstable or overly reactive under the appropriate conditions.

[0189] Additional Embodiments 1. Formula: [ka] (In the formula, R 1 and R 2 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; R 3 is selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl; R 4 and R 5 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; R 6 is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, halogen, C1-C4 alkyl-OR 7 , and OR 7 is selected from R 7 is selected from hydrogen, C1-C4 alkyl, and C1-C4 haloalkyl or a pharmaceutically acceptable salt thereof.

[0190] 2. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0191] 3. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0192] 4. R 5 The compound of any one of embodiments 1-3, wherein is hydrogen.

[0193] 5. R 5 The compound of any one of embodiments 1-3, wherein is halogen.

[0194] 6. R 5 The compound of any one of embodiments 1-3, wherein is fluoro.

[0195] 7. R 5 The compound of any one of embodiments 1-3, wherein is chloro.

[0196] 8. R 5 The compound of any one of embodiments 1-3, wherein is methyl.

[0197] 9. R 5 The compound of any one of embodiments 1-3, wherein is ethyl.

[0198] 10. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0199] 11. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0200] 12. R 6The compound of embodiment 10 or embodiment 11, wherein is hydrogen.

[0201] 13. R 6 The compound of embodiment 10 or embodiment 11, wherein is OMe.

[0202] 14. R 6 The compound of embodiment 10 or embodiment 11, wherein is halogen.

[0203] 15. R 6 The compound of embodiment 10 or embodiment 11, wherein is fluoro.

[0204] 16. R 6 The compound of embodiment 10 or embodiment 11, wherein is chloro.

[0205] 17. R 6 The compound of embodiment 10 or embodiment 11, wherein is methyl.

[0206] 18. R 6 The compound of embodiment 10 or embodiment 11, wherein is ethyl.

[0207] 19. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0208] 20. The compound has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0209] 21. R 7 The compound of embodiment 19 or embodiment 20, wherein is methyl.

[0210] 22. R7 The compound of embodiment 19 or embodiment 20, wherein is hydrogen.

[0211] 23. R 1 The compound of any one of embodiments 1-22, wherein is hydrogen.

[0212] 24. R 1 The compound of any one of embodiments 1-22, wherein is halogen.

[0213] 25. R 1 The compound of any one of embodiments 1-22, wherein is methyl.

[0214] 26. R 2 The compound of any one of embodiments 1-25, wherein is hydrogen.

[0215] 27. R 2 The compound of any one of embodiments 1-25, wherein is halogen.

[0216] 28. R 2 The compound of any one of embodiments 1-25, wherein is methyl.

[0217] 29. R 2 The compound of any one of embodiments 1-25, wherein is C1-C2 haloalkyl.

[0218] 30. R 3 The compound of any one of embodiments 1-29, wherein is hydrogen.

[0219] 31. R 3 The compound of any one of embodiments 1-29, wherein is methyl.

[0220] 32. R 4 The compound of any one of embodiments 1-31, wherein is hydrogen.

[0221] 33. R 4The compound of any one of embodiments 1-31, wherein is halogen.

[0222] 34. R 4 The compound of any one of embodiments 1-31, wherein is fluoro.

[0223] 35. R 4 The compound of any one of embodiments 1-31, wherein is chloro.

[0224] 36. R 4 The compound of any one of embodiments 1-31, wherein is C1-C2 haloalkyl.

[0225] 37. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0226] 38. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0227] 39. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0228] 40. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0229] 41. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0230] 42. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0231] 43. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0232] 44. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0233] 45. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0234] 46. ​​Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0235] 47. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0236] 48. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0237] 49. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0238] 50. Structure: [ka] or a pharmaceutically acceptable salt thereof.

[0239] 51. Formula: [ka] (In the formula, R 1 and R 2 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; R 4 and R 5 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen. or a pharmaceutically acceptable salt thereof.

[0240] 52. R 2 The compound of embodiment 51, wherein is hydrogen.

[0241] 53. R 2 is F.

[0242] 54. R 2 The compound of embodiment 51, wherein is Cl.

[0243] 55. R 2 The compound of embodiment 51, wherein is methyl.

[0244] 56. R 2 The compound of embodiment 51, wherein is C1-C2 haloalkyl.

[0245] 57. Formula: [ka] (In the formula, R 1 is selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen; R 4 and R 5 are independently selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, and halogen. or a pharmaceutically acceptable salt thereof.

[0246] 58. R 1 The compound of any one of embodiments 51-57, wherein is hydrogen.

[0247] 59. R 1 The compound of any one of embodiments 51-57, wherein is F.

[0248] 60. R 1 The compound of any one of embodiments 51-57, wherein is Cl.

[0249] 61. R 1 The compound of any one of embodiments 51-57, wherein is methyl.

[0250] 62. R 1 The compound of any one of embodiments 51-57, wherein is C1-C2 haloalkyl.

[0251] 63. R 4 The compound of any one of embodiments 51-62, wherein is hydrogen.

[0252] 64. R 4 The compound of any one of embodiments 51-62, wherein is halogen.

[0253] 65. R 4 The compound of any one of embodiments 51-62, wherein is fluoro.

[0254] 66. R 4The compound of any one of embodiments 51-62, wherein is chloro.

[0255] 67. R 4 The compound of any one of embodiments 51-62, wherein is C1-C2 haloalkyl.

[0256] 68. R 5 The compound of any one of embodiments 51-67, wherein is hydrogen.

[0257] 69. R 5 The compound of any one of embodiments 51-67, wherein is halogen.

[0258] 70. R 5 The compound of any one of embodiments 51-67, wherein is fluoro.

[0259] 71. R 5 The compound of any one of embodiments 51-67, wherein is chloro.

[0260] 72. R 5 The compound of any one of embodiments 51-67, wherein is C1-C2 haloalkyl.

[0261] 73. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 72 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0262] 74. A pharmaceutical composition described in embodiment 73, suitable for oral administration.

[0263] 75. A pharmaceutical composition described in embodiment 73, suitable for parenteral administration.

[0264] 76. A pharmaceutical composition described in embodiment 73, suitable for intravenous administration.

[0265] 77. A method for treating a disorder mediated by ROCK1 or ROCK2, comprising administering to a subject in need thereof an effective amount of a compound of any one of embodiments 1-72, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition.

[0266] 78. The method of embodiment 77, wherein the subject is a human.

[0267] 79. The method of embodiment 78, wherein the disorder is a neurodegenerative disorder.

[0268] 80. The method of embodiment 79, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis.

[0269] 81. The method of embodiment 78, wherein the neurodegenerative disorder is Parkinson's disease.

[0270] 82. Use of a compound according to any one of embodiments 1-72 or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition, in the manufacture of a medicament for the treatment of a disorder mediated by ROCK1 or ROCK2.

[0271] 83. Use of a compound according to any one of embodiments 1-72 or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition, in the treatment of a disorder mediated by ROCK1 or ROCK2.

[0272] 84. The use according to embodiment 82 or 83, wherein the disorder is a neurodegenerative disorder.

[0273] 85. The use described in embodiment 84, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis.

[0274] 86. The use described in embodiment 82 or 83, wherein the neurodegenerative disorder is Parkinson's disease.

[0275] III. Treatment method Rho-associated coiled-coil kinase (ROCK) isoform 1 and isoform 2 are downstream targets of GTP-bound and activated Rho GTPase proteins that phosphorylate numerous substrates involved in myosin-actin cytoskeletal structure, actin filament dynamics, neurofilaments, and actin-binding proteins. ROCK1 is expressed in various human tissues, including the heart, pancreas, lung, liver, skeletal muscle, and kidney, but is virtually unexpressed in the brain (Fujisawa, K. et al., Identification of the rho-binding domain of p160ROCK, a rho-associated coiled-coil-containing protein kinase. J. Biol. Chem. 271:23022-8(1996)). ROCK2 is preferentially expressed in the brain and skeletal muscle (Nakagawa, O. et al., ROCK-I and ROCK-II, two isoforms of rho-associated coiled-coil forming protein serine / threonine kinase in mice. FEBS Lett. 395:189-93(1996)).Increased ROCK2 activity has been shown to cause defects in dendritic spine structure and function in several model systems (Swanger, SA et al. ROCK1 and ROCK2 inhibition alters dendritic spine morphology in hippocampal neurons. Cell Logist. 5:e1133266(2015); Sellers, KJ et al. Amyloid beta synaptotoxicity is Wnt-PCP dependent and blocked by fasudil. Alzheimers Dement. 14:306-17(2018); Henderson et al. Pharmacologic inhibition of LIMK1 provides dendritic spine resilience against beta-amyloid. Sci Signal. 12:eaaw9318(2019)). In fact, ROCK2 has been implicated in many neurodegenerative and nervous system disorders, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), spinal cord injury, stroke, and neuroinflammation (Weber, AJ et al. Perspectives on ROCK2 as a Therapeutic Target for Alzheimer's Disease. Front in Cell Neurosci. 15:636017(2021)).

[0276] ROCK2 has been shown to regulate several complex neuronal processes associated with neurodegenerative disorders. For example, downregulation of ROCK2 via AAV shRNA rescues dopaminergic neurons in the substantia nigra (SN) and preserves motor behavior in a 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse model (Saal, K. et al. Neurobiol Dis. 73:150-62(2015)). Inhibition of ROCK can be used to regulate these neuronal processes. For example, mouse studies have shown that inhibition of ROCK reduces the expression of human mutant alpha-synuclein (aSyn) in mice. A53T In a mouse model expressing Fasudil, reduction of midbrain α-synuclein symptoms and improvement of motor and cognitive function were observed (Tatenhorst et al. Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease. Acta Neuropathol Commun. 4:39(2016)).

[0277] ROCK proteins are present in many types of neurons in the CNS. Excessive ROCK activity in the CNS can lead to oxidative stress, uncontrolled inflammation, immune abnormalities, impaired energy metabolism, neuronal cell loss, reactive gliosis, and / or impaired synaptic transmission, thereby promoting the development of neurodegenerative diseases. Overexpression of ROCK proteins has been detected in the lesions of Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), suggesting that ROCK proteins may be involved in the pathology of these diseases and may be key initiators of pathogenesis. Inhibition of ROCK proteins has been shown to induce several biological events, including neurite outgrowth, axon regeneration, and activation of the pro-survival protein kinase B (AKT) (Q. Wang et al. "Advantages of Rho-associated kinases and their inhibitor Fasudil for the treatment of neurodegenerative diseases", Neural Regen. Res. 2022, 17(12):2623-2631).

[0278] Because ROCKs play a crucial role in neuronal processes, their overactivity or elevated levels are associated with various neurological defects. For example, elevated ROCK2 protein levels have been observed in the advanced stages of AD (Herskowitz et al. 2013). Also, SOD1 G93A Increased ROCK activity has been observed in mutant ALS model mice (Gunther, R. et al. Rho Kinase Inhibition with Fasudil in the SOD1 G93AMouse Model of Amyotrophic Lateral Sclerosis - Symptomatic Treatment Potential After Disease Onset. Front Pharmacol. 8:17(2017)). Therefore, an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof can be administered to treat a neurological disease. Non-limiting examples of neurological diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and neurological deficits resulting from spinal cord injury.

[0279] Based on the above-described roles of ROCK1 and ROCK2 in central nervous system disorders, methods and uses are provided herein for treating subjects, such as humans, suffering from such disorders. In certain embodiments, a method is provided for treating a subject with a disorder mediated by ROCK1 and / or ROCK2, comprising administering to the subject an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof. Non-limiting examples of disorders mediated by ROCK1 and / or ROCK2 are provided below.

[0280] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat a ROCK1-mediated disorder.

[0281] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat a ROCK1-mediated disorder. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat a ROCK1-mediated disorder. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat a ROCK1-mediated disorder.

[0282] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat a ROCK2-mediated disorder.

[0283] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat a ROCK2-mediated disorder. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat a ROCK2-mediated disorder. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat a ROCK2-mediated disorder.

[0284] In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used to treat a disorder mediated by both ROCK1 and ROCK2.

[0285] In other aspects, Compound 2, or a pharmaceutically acceptable salt thereof, is used to treat a disorder mediated by both ROCK1 and ROCK2. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat a disorder mediated by both ROCK1 and ROCK2. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat a disorder mediated by ROCK1 and ROCK2.

[0286] Specific examples of disorders that can be treated with the compounds described herein, or pharmaceutically acceptable salts thereof, are set forth below.

[0287] Amyotrophic lateral sclerosis (ALS) Amyotrophic lateral sclerosis (ALS) is a relatively rare neurodegenerative disease that affects an estimated 1 million people (Hardiman, O. et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers. 3(17071):1-19(2017)), with varying levels of risk depending on the region. ALS is characterized by degeneration of upper and lower motor neurons, which contributes to both motor and non-motor symptoms. Several subtypes of ALS have been identified, including bulbar, respiratory, flail arm, classic, pyramidal, and flail leg ALS.

[0288] In certain embodiments, compounds of the invention are used to treat ALS, for example, in certain embodiments, compounds of the invention are used to treat bulbar, respiratory, flail arm, classical, pyramidal, or flail leg ALS.

[0289] The present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having ALS or a secondary condition associated with ALS.

[0290] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat ALS.

[0291] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat ALS. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat ALS. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat ALS.

[0292] Parkinson's disease (PD) Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, with an estimated global prevalence of 2–3 per 100,000 people (Parkinson's Foundation. Statistics. 2022). PD is characterized by dysregulation of several mechanisms and pathways that contribute to neuronal loss in the substantia nigra (SN), including α-synuclein proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport, and neuroinflammation (Poewe, W. et al. Parkinson disease. Nat Rev Dis Primers. 3(17013):1-21(2017)). Intracellular α-synuclein aggregates, along with SN neuronal loss and striatal dopamine deficiency, are characteristic findings of PD. Subjects are diagnosed with PD based on the presence of bradykinesia and other motor deficits, as well as non-motor symptoms. Several subtypes of PD have been identified, including motor-cognitive, cognitive-predominant, and motor-predominant PD. Currently, there is no cure for PD, and treatment focuses on slowing the progression of PD and / or alleviating symptoms.

[0293] In certain embodiments, compounds of the invention are used to treat PD, for example, in certain embodiments, compounds of the invention are used to treat motor-cognitive, cognitive-predominant, or motor-predominant PD.

[0294] The present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having PD or a secondary condition associated with PD.

[0295] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat PD.

[0296] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat PD. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat PD. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat PD.

[0297] Alzheimer's disease (AD) Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 40 million people in 2016 (GBD 2016 Dementia Collaborators. Global, regional, and national burden of Alzheimer's disease and other dementias, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 18(1):88-106(2019)), and is expected to increase to 150 million people worldwide by 2050 (Estimation of the global prevalence of dementia in 2019 and forecasted prevalence in 2050: an analysis for the Global Burden of Disease Study 2019. Lancet Public Health. 7(2):e105-e125(2022)). AD is a neurodegenerative disorder characterized by extracellular plaques containing amyloid beta and intracellular neurofibrillary tangles containing tau, leading to neuronal loss (Knopman, DS et al. Alzheimer disease. Nat Rev Dis Primers. 7(33):1-21(2021)). Subjects with AD exhibit cognitive impairment and dementia. Symptoms of cognitive impairment include declines in short-term memory, expressive language skills, visuospatial processing, and executive function. Risk factors for AD include rare, dominantly inherited mutations in APP (encoding amyloid precursor protein), PSEN1 (encoding preserin 1), and PSEN2 (encoding preserin 2), which cause an autosomal dominant form of AD. More common is the development of sporadic, late-onset AD, which is influenced by more common, but incompletely penetrant, genetic polymorphisms in genes such as APOE.

[0298] In certain embodiments, the compounds of the present invention are used to treat AD. Treatment of AD can be beneficial at several stages of disease progression. For example, in certain embodiments, the compounds of the present invention are used to treat subjects who have elevated amyloid beta and / or tau biomarkers without cognitive impairment, who have subjective cognitive decline without declines in cognitive test scores, who have mild cognitive impairment, who have clinically confirmed AD, or who have autosomal dominant AD.

[0299] The present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having AD or a secondary condition associated with AD.

[0300] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat AD.

[0301] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat AD. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat AD. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat AD.

[0302] Spinal cord injury Spinal cord injury can cause complex and diverse neurological effects, including axonal degeneration and mild to severe cognitive decline.

[0303] In certain embodiments, the compounds described herein are used to treat spinal cord injury. For example, the compounds of the present invention can be used to promote axonal regeneration and functional recovery in subjects who have suffered a spinal cord injury.

[0304] The present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having a spinal cord injury or a secondary condition associated with spinal cord injury.

[0305] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat spinal cord injury.

[0306] In another aspect, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat spinal cord injury. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat spinal cord injury. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat spinal cord injury.

[0307] stroke A stroke is a neurological disorder caused by the blockage or rupture of one or more blood vessels supplying blood to the brain. When blood in the brain is deprived, damage occurs to brain tissue in the area previously supplied by the blood vessels. There are two general categories of stroke: ischemic stroke and hemorrhagic stroke. Ischemic stroke occurs when brain tissue becomes ischemic due to a reduced blood supply caused by a blockage. Hemorrhagic stroke, on the other hand, occurs when bleeding occurs when a blood vessel ruptures. Ischemic stroke is the most common form of stroke. During an ischemic stroke, blood flow is blocked, preventing cells from receiving enough oxygen and nutrients.

[0308] In certain embodiments, the compounds described herein are used to treat stroke, non-limiting examples of which include ischemic stroke, acute ischemic stroke, thrombosis, embolism, transient ischemic attack, leukoplakia, and infarction.

[0309] The present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having a stroke or a secondary condition associated with stroke.

[0310] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat stroke.

[0311] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat stroke. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat stroke. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat stroke.

[0312] Other neurodegenerative disorders In addition to playing a key role in regulating several complex neuronal processes, ROCK has also been identified as a regulator of reactive oxygen species (ROS). This biological function allows ROCK inhibition to modulate additional neurodegenerative disorders (Kang H. et al. Chemical Screening Identifies ROCK as a Target for Recovering Mitochondiral Function in Hutchinson-Gilford Progeria Syndrome. Aging Cell 16:541-50(2017) and Sheng W. et al. Reactive Oxygen Species from Human Astrocytes Induce Functional Impairment and Oxidative Damage. Neurochem. Res. 38:2148-59(2013)).

[0313] Non-limiting examples of neurodegenerative diseases include ataxia, Huntington's disease, motor neuron disease, multiple system atrophy, Creutzfeldt-Jakob disease, dementia, non-dementia cognitive impairment, and progressive supranuclear palsy. Non-limiting examples of dementia include senile dementia, vascular dementia, post-traumatic dementia, dementia due to brain tumor, and dementia due to chronic subdural hematoma.

[0314] In other embodiments, the compounds herein are used to prevent stroke or to provide neuroprotection, eg, acute neuroprotection.

[0315] In certain embodiments, the compounds of the present invention are used to reduce inflammation. For example, the compounds of the present invention can be administered to a subject in need thereof to reduce inflammation in the brain.

[0316] Thus, the present invention includes the use of an effective amount of a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having a neurodegenerative disorder, such as one of those listed above, or a secondary condition associated with a neurodegenerative disorder.

[0317] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat a neurodegenerative disorder.

[0318] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat a neurodegenerative disorder. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat a neurodegenerative disorder. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat a neurodegenerative disorder.

[0319] pain disorders Pain is a subjective sensation reflecting tissue damage and can take various forms. Pain is classified as somatic pain and psychogenic pain, with somatic pain further classified as nociceptive pain and neuropathic pain. Nociceptive pain is caused by external stimuli or visceral lesions. Nociceptive pain is primarily acute pain that disappears as the underlying disease heals and serves as a biological signal generated by the injury. Neuropathic pain is chronic pain caused by dysfunction of the peripheral or central nervous system and includes pain caused by various causes, including pain due to diabetes, nerve compression, and spinal cord injury. Psychogenic pain is chronic pain that is caused by mental rather than physical disorders and cannot be explained by organic disorders, including chronic headaches and abdominal pain of unknown cause. Chronic pain is an important treatment target because it can cause significant distress to patients. Non-limiting examples of chronic pain include chronic pain associated with arthritis, diabetes, cancer, etc., which require pain treatment in addition to treatment of the underlying disease.

[0320] ROCK inhibitors have been shown to exert analgesic effects on pain. In certain embodiments, the compounds described herein are used to treat pain disorders. In certain aspects, the compounds of the present invention can be used to treat somatic pain. For example, the compounds of the present invention can be used to treat nociceptive pain or neuropathic pain. In certain embodiments, the compounds of the present invention are used to treat nociceptive pain. Non-limiting examples of nociceptive pain include acute pain caused by an underlying disease. In certain embodiments, the compounds of the present invention are used to treat neuropathic pain. Non-limiting examples of neuropathic pain include chronic pain caused by diabetes, nerve compression, or spinal cord injury. In certain aspects, the compounds of the present invention or pharmaceutically acceptable salts thereof are used to treat pain caused by arthritis, such as osteoarthritis or rheumatoid arthritis.

[0321] Thus, the present invention includes the use of an effective amount of a compound of Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having a pain disorder, such as those listed above, or a secondary condition associated with a pain disorder.

[0322] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat a pain disorder.

[0323] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat pain disorders. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat pain disorders. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat pain disorders.

[0324] cardiovascular disease Studies have demonstrated that the Rho / ROCK pathway is increased in hypertensive patients. For example, in the early stages of atherosclerosis, increased ROCK-dependent smooth muscle contraction is observed in the aorta. During the formation of atherosclerotic lesions, ROCK activity is elevated in certain regions and cell types, including endothelial cells, periadventitial adipocytes, and macrophage foam cells, supporting the role of ROCK in macrophage infiltration and foam cell formation via ERM phosphorylation. ROCK also plays a role in cardiac ischemia / reperfusion injury, which re-enters an area after blood flow has been restricted or blocked. Several in vivo models, including mice, rats, and pigs, have demonstrated a detrimental role for RhoA / ROCK signaling in ischemia / reperfusion injury (M. Surma et al., "Rho kinase as a therapeutic target in cardiovascular disease", Future Cardiol. 2011 September; 7(5): 657-671. Doi:10.2217 / fca.11.51).

[0325] In certain embodiments, the compounds described herein are used to treat cardiovascular diseases, non-limiting examples of which include coronary artery disease, stroke, peripheral artery disease, and aortic disease.

[0326] Thus, the present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having a cardiovascular disease, such as those listed above, or a secondary condition associated with a cardiovascular disease.

[0327] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat cardiovascular disease.

[0328] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat cardiovascular disease. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat cardiovascular disease. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat cardiovascular disease.

[0329] eye disorders The ability of ROCK proteins to mediate ocular smooth muscle contraction makes ROCK a therapeutic target for ocular disorders. ROCK mediates calcium sensitization and smooth muscle contraction. Ca 2+The sensitization effect has been attributed to ROCK-mediated phosphorylation of MYPT-1, a regulatory subunit of myosin light chain phosphatase (MLCP). MYPT-1 is used to inhibit the activity of MLCP in vivo. By inhibiting MLCP, ROCK activates the phosphorylation of MYPT-1, leading to increased myosin light chain phosphorylation and smooth muscle contraction (WO 2005 / 003101 and WO 2005 / 034866). Glaucoma is an eye disease that can lead to irreversible vision loss. It is characterized by progressive optic neuropathy caused in part by the deleterious effects of elevated intraocular pressure. In healthy individuals, intraocular pressure ranges from 12 mmHg to 20 mmHg, with an average of approximately 16 mmHg. However, in individuals with primary open-angle glaucoma, intraocular pressure typically rises to 22 mmHg to over 30 mmHg. In angle-closure or acute glaucoma, intraocular pressure can reach as high as 70 mmHg, leading to blindness within just a few days. In people with abnormally pressure-sensitive eyes, even statistically normal intraocular pressure can lead to vision loss, a condition known as normal-tension glaucoma (see, e.g., PL Kaufman and TW Mittag, "Medical Therapy of Glaucoma," Ch. 9, Sec. II (pp. 9.7-9.30) in PL Kaufman and TW Mittag (eds.): Glaucoma (Vol. 7 of SM Podos and M. Yanoff (eds): Textbook of Ophthalmology Series). London, Mosby-Year Book Europe Ltd. (1994); AC Guyton, Textbook of Medical Physiology (WB Saunders Co., Sixth Ed.), pp. 386-89 (1981)). Open-angle glaucoma accounts for the majority of all primary glaucoma and is characterized by an abnormally high resistance to the drainage of fluid (aqueous humor) from the eye. In glaucomatous eyes, the rate of aqueous humor production remains constant, while increased resistance to outflow causes elevated intraocular pressure.

[0330] In certain embodiments, the compounds of the present invention or pharmaceutically acceptable salts thereof are used to treat ocular disorders, non-limiting examples of which include glaucoma (e.g., open-angle glaucoma, angle-closure glaucoma, acute glaucoma, and normal-tension glaucoma) or intraocular pressure.

[0331] Thus, the present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having an ocular disorder, such as those listed above, or a secondary condition associated with an ocular disorder.

[0332] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat an ocular disorder.

[0333] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat ocular disorders. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat ocular disorders. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat ocular disorders.

[0334] ROCK has recently emerged as a novel therapeutic target for neurodegenerative disorders (Non-Patent Document 2). ROCK1 is preferentially expressed in peripheral tissues, whereas ROCK2 is highly expressed in the central nervous system (CNS). Axon growth inhibitory molecules (e.g., Nogo, MAG, OMgp, ephrins, and semaphorins) bind to specific extracellular receptors and transmit signals through ROCK, resulting in axon degeneration, growth cone collapse, and impaired axon regeneration. In non-neuronal structures, ROCK regulation of actin cytoskeleton plasticity also affects vasoconstriction and vascular remodeling. ROCK levels increase with age, and tissues from ALS patients show elevated levels of ROCK2 as well as its downstream targets, LIMK1 and cofilin. Increased ROCK activity leads to increased levels of phosphorylated adducin, activation of phosphatase and tensin homolog (PTEN), and decreased Akt activity. PTEN activation by ROCK has adverse effects on cell growth, proliferation, and metabolism. ROCK inhibition suppresses neuronal apoptosis and axonal degeneration, while promoting axonal regeneration and modulating microglial activation (Non-Patent Document 6).

[0335] Protein kinase X (PRKX) PRKX (human protein kinase X) is a cAMP-dependent serine / threonine kinase encoded on the X chromosome. cAMP-dependent protein kinases (cAPKs) play a key role in many signal transduction processes and mediate most of the known effects of cAMP in eukaryotic cells. These multisubstrate enzymes regulate the activity of proteins involved in signal transduction, energy metabolism, cell proliferation, or differentiation by phosphorylating Ser or Thr residues, thereby altering the biological properties of the target proteins. Human protein kinase PRKX is related to the catalytic subunit of cAMP-dependent protein kinase but is distinct from the isoforms Cα, Cβ, and Cγ. PRKX shares 53.2% identity with the human Cα subunit of cAPK (PKA-Cα) in the catalytic core region. This degree of homology is much lower than the similarity between the two human isoforms Cα and Cβ (90.5% identity). PRKX mRNA is present in various tissues, with highest expression in fetal and adult brain, kidney, and lung (B. Zimmermann et al., "PRKX Is a Novel Catalytic Subunit of the cAMP-Dependent Protein Kinase Regulated by the Regulatory Subunit Type I," Journal of Biological Chemistry, Vol. 274, No. 9, Issue of February 26, pp. 5370-5378, 1999). In contrast to the ubiquitously expressed Cα subunit, PRKX is primarily active during embryonic organ development and cell differentiation in the hematopoietic lineage. It has been found to be essential for the maturation of macrophages and granulocytes.PRKX has been shown to be involved in kidney development and regulates epithelial cell migration, ureteric bud branching, and glomerular formation (M. Diskar et al., "Regulation of cAMP-dependent protein kinases: the human protein kinase X (PrKX) reveals the role of the catalytic subunit αH-αI loop," Journal of Biological Chemistry, Vol. 285, No. 46, pp. 35910-35918, November 12, 2010).

[0336] The role of PRKX in disease pathology remains unclear. However, recent studies have revealed that dysregulation of PRKX expression may be a molecular cause of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (P. Pontecorvi et al., "Altered Expression of Candidate Genes in Mayer-Rokitansky-Küster-Hauser Syndrome May Influence Vaginal Keratinocyte Biology: A Focus on Protein Kinase X," Biology, 2021, 10, 450.). PRKX has also been implicated in the pathology of amyotrophic lateral sclerosis (ALS) (Oliverira G. et al., "Early Gene Expression Changes in Skeletal Muscle from SOD1(G93A) Amyotrophic Lateral Sclerosis Animal Model").

[0337] In certain embodiments, a method for treating a subject with a PRKX-mediated disorder is provided, comprising administering to the subject an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof. Non-limiting examples of PRKX-mediated disorders are provided herein.

[0338] Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome PRKX has been suggested to be involved in the development of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (P. Pontecorvi et al., "Altered Expression of Candidate Genes in Mayer-Rokitansky-Küster-Hauser Syndrome May Influence Vaginal Keratinocyte Biology: A Focus on Protein Kinase X", Biology, 2021, 10, 450.). Therefore, in certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof is used to treat MRKH syndrome.

[0339] Thus, the present invention includes the use of an effective amount of a compound of Formula I, Formula II, or Formula III, or a pharmaceutically acceptable salt thereof, to treat a subject, such as a human, having Mayer-Rokitansky-Küsterhauser (MRKH) syndrome or a secondary condition associated with Mayer-Rokitansky-Küsterhauser (MRKH) syndrome.

[0340] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used to treat MRKH.

[0341] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used to treat MRKH. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof, can be used to treat pain disorders. In other embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used to treat MRKH.

[0342] IV. Pharmaceutical Compositions The compounds of Formula I, Formula II, or Formula III described herein, or pharmaceutically acceptable salts thereof, can be administered as the neat chemical but are more typically administered as pharmaceutical compositions containing an effective amount to a subject, typically a human, in need of such treatment for the disorders described herein. Accordingly, the present disclosure provides pharmaceutical compositions comprising an effective amount of a compound or a pharmaceutically acceptable salt thereof for use as described herein, together with at least one pharmaceutically acceptable excipient. The pharmaceutical composition may contain only the compound as the active agent, or in alternative embodiments, the compound and at least one additional therapeutic agent.

[0343] In general, the compositions of the present disclosure are administered in a therapeutically effective amount by any of the accepted administration methods. The appropriate dosage range depends on numerous factors, including the severity of the disease being treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication for which administration is intended, and the preferences and experience of the physician involved. Those skilled in the art of treating such diseases can ascertain a therapeutically effective amount of the disclosed compositions for a given disease without undue experimentation, relying on their own knowledge and the disclosure of this application.

[0344] In certain embodiments, the pharmaceutical composition is a dosage form containing about 1 mg to about 1000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, or about 200 mg to about 600 mg of an active compound, and optionally about 1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, or about 200 mg to about 600 mg of an additional therapeutic agent per unit dosage form. Examples are dosage forms containing at least about 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 750 mg of the active compound or salt thereof.

[0345] A pharmaceutically or therapeutically effective amount of the composition is delivered to the subject. The exact effective amount will vary from subject to subject, depending on the species, age, size, and health of the subject, the nature and extent of the condition being treated, the recommendations of the treating physician, and the therapeutic agent or combination of therapeutic agents selected for administration. The effective amount for a given situation can be determined by routine experimentation. The subject can be administered as many doses as necessary to reduce and / or alleviate the signs, symptoms, or causes of the disorder in question, or to effect any other desired change in a biological system. If desired, formulations can be prepared with enteric coatings suitable for sustained- or controlled-release administration of the active ingredient.

[0346] In some embodiments, the compound or pharmaceutically acceptable salt thereof disclosed or used as described herein is administered once daily (QD), twice daily (BID), or three times daily (TID). In some embodiments, a compound disclosed or used as described herein is administered once daily (QD), twice daily (BID), or three times daily (TID) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 35 days, at least 45 days, at least 60 days, at least 75 days, at least 90 days, at least 120 days, at least 150 days, at least 180 days or more (including indefinitely).

[0347] In certain embodiments, the compounds of the invention are administered once daily, twice daily, three times daily, or four times daily.

[0348] In certain embodiments, the compound of the present invention is orally administered once a day. In certain embodiments, the compound of the present invention is orally administered twice a day. In certain embodiments, the compound of the present invention is orally administered three times a day. In certain embodiments, the compound of the present invention is orally administered four times a day.

[0349] In certain embodiments, the compound of the present invention is administered intravenously once a day. In certain embodiments, the compound of the present invention is administered intravenously twice a day. In certain embodiments, the compound of the present invention is administered intravenously three times a day. In certain embodiments, the compound of the present invention is administered intravenously four times a day.

[0350] In some embodiments, the compounds of the invention are administered with a treatment holiday between treatment cycles, for example, the compounds may have a treatment holiday of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days per treatment cycle.

[0351] The pharmaceutical composition may contain a molar ratio of the active compound to the additional active agent. As non-limiting illustrative examples, the pharmaceutical composition may contain a molar ratio of about 0.5:1, about 1:1, about 2:1, about 3:1, or about 1.5:1 to about 4:1 of the anti-inflammatory or immunosuppressant agent.

[0352] These compositions can contain any amount of the active compound that achieves the desired result, for example, 0.1% to 99% by weight (wt%) of the compound, usually at least about 5% by weight of the compound, with some embodiments containing about 25% to about 50% by weight or about 5% to about 75% by weight of the compound.

[0353] In certain embodiments, the compound is administered as a pharmaceutically acceptable salt.Non-limiting examples of pharmaceutically acceptable salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of suitable salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate.

[0354] Thus, the selected compounds of the present invention, or pharmaceutically acceptable salts thereof, can generally be administered as pharmaceutical compositions suitable for systemic, parenteral, or local administration. Non-limiting examples include oral (including buccal and sublingual), rectal, nasal, topical, transdermal, pulmonary, parenteral injection (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous), inhalation or spray, intra-aortic, intracranial, subdermal, intraperitoneal, subcutaneous, or other administration means. Typical administration methods are oral or intravenous, using a convenient daily dosing regimen that can be adjusted according to the level of affliction.

[0355] Depending on the intended method of administration, the pharmaceutical composition may be in the form of a solid, semi-solid or liquid dosage form, such as a tablet, suppository, pill, capsule, powder, liquid, syrup, suspension, cream, ointment, lotion, paste, gel, spray, aerosol, foam or oil, injectable or infusible solution, transdermal patch, subcutaneous patch, inhalation formulation, medical device, suppository, buccal or sublingual formulation, parenteral formulation, or eye drops, preferably in a unit dosage form suitable for single administration of a precise dosage amount.

[0356] Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing an appropriate amount of the active ingredient, e.g., an amount effective to achieve a desired purpose. Oral pharmaceutical compositions contain an effective amount of a selected drug in combination with a pharmaceutically acceptable carrier or excipient, and may further include other medicinal agents, adjuvants, diluents, buffers, etc.

[0357] Pharmaceutically acceptable excipients must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the subject being treated. Pharmaceutically acceptable excipients may be inert or may have medicinal properties of their own. The amount of excipient used in combination with the compound is sufficient to provide a quantity of substance useful for administration per unit dose of the compound.

[0358] Classes of excipients include, but are not limited to, adjuvants, binders, buffers, colors, diluents, disintegrants, emulsifiers, flavors, gels, glidants, lubricants, preservatives, stabilizers, surfactants, solubilizers, tableting agents, wetting agents or solidifying agents.

[0359] Some excipients may be included in more than one class; for example, vegetable oils may be used as lubricants in some formulations and as diluents in others.

[0360] Exemplary pharmaceutically acceptable excipients include sugars, starches, cellulose, powdered tragacanth, malt, gelatin, talc, petrolatum, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and vegetable oils. Any active agent that does not substantially interfere with the activity of the compound of the present invention may be included in the pharmaceutical composition.

[0361] Some excipients include, but are not limited to, liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. The compound can be provided in the form of solid, liquid, spray-dried product, microparticles, nanoparticles, controlled release systems, etc., as desired, for example, depending on the purpose of therapy. Suitable excipients for non-liquid formulations are also known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients and salts can be found in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0362] Additionally, auxiliary substances such as wetting or emulsifying agents, physiological buffer substances, surfactants, etc. may be present in the pharmaceutical composition. The physiological buffer may be any solution that is pharmacologically acceptable and provides the formulation with a desired pH, i.e., a pH within the physiologically acceptable range. Examples of buffer solutions include saline, phosphate-buffered saline, Tris-buffered saline, Hank's buffered saline, etc.

[0363] For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving or dispersing an active compound described herein and any pharmaceutical adjuvants in an excipient, such as water, saline, aqueous dextrose, glycerol, ethanol, or the like, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, supra.

[0364] In yet another embodiment, there is provided the use of penetration-enhancing excipients including polymers such as polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin), polyanions (N-carboxymethylchitosan, polyacrylic acid), and thiolated polymers (carboxymethylcellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0365] Oral tablets and capsules may contain one or more commonly used carriers, such as lactose and corn starch. Lubricants, such as magnesium stearate, are also commonly added. Typically, the compositions of the present disclosure are combined with oral, non-toxic, pharmaceutically acceptable inert excipients, such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, and sorbitol. Furthermore, if desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn syrup, natural and synthetic gums such as gum arabic and gum tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. The disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0366] When a liquid suspension is used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert excipient, such as ethanol, glycerol, water, etc., as well as emulsifying and suspending agents. Flavoring agents, coloring agents, and / or sweetening agents may be added as needed. Other optional ingredients incorporated into the oral formulations herein include, but are not limited to, preservatives, suspending agents, thickening agents, etc.

[0367] For intraocular delivery, the compounds can be administered, for example, by intravitreal, intrastromal, intracameral, subtenon, subretinal, retrobulbar, peribulbar, suprachoroidal, conjunctival, subconjunctival, episcleral, periocular, transscleral, retrobulbar, posterior juxtascleral, periconeal, or lacrimal injection, or in an immediate or controlled release manner via a mucus, mucin, or mucosal barrier, or by an intraocular device, as desired.

[0368] Parenteral preparations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or emulsions. Typically, sterile injectable suspensions are formulated according to techniques known in the art using suitable excipients, dispersing or wetting agents, and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in parenterally acceptable diluents or solvents that are non-toxic to an acceptable extent. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils, fatty acid esters, or polyols are conventionally used as solvents or suspending media. In addition, parenteral administration can involve the use of sustained-release or sustained-release systems to maintain a constant level of dosage.

[0369] Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and includes aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Administration via certain parenteral routes may involve introducing a formulation of the present disclosure into the subject's body through a needle or catheter propelled by a sterile syringe or some other mechanical device, such as a continuous infusion system. The formulations provided by the present disclosure can be administered using a syringe, infuser, pump, or any other device recognized in the art for parenteral administration.

[0370] Preparations according to the present disclosure for parenteral administration include aqueous or non-aqueous sterile solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They can be sterilized, for example, by filtration through a bacteria-retaining filter, incorporating a sterilizing agent into the composition, irradiating the composition, or heating the composition. They can also be prepared using sterile water or some other sterile injectable medium immediately before use.

[0371] Sterile injectable solutions are prepared by incorporating the required amount of one or more compounds of the present disclosure into a suitable solvent, optionally containing various other ingredients as listed above, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. For sterile powders for preparing sterile injectable solutions, typical preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of the active ingredient in 10% by volume of propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0372] Alternatively, the pharmaceutical compositions of the present disclosure can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the active ingredient with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0373] The pharmaceutical compositions of the present disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents.

[0374] Formulations for oral administration include tablets, lozenges, gels, and the like. Alternatively, oral administration can be carried out using transmucosal delivery systems known to those skilled in the art. The compounds of the present disclosure can also be delivered through the skin or mucosal tissue using conventional transdermal drug delivery systems, i.e., transdermal "patches," in which the active ingredient is typically contained within a laminated structure that serves as a drug delivery device attached to a body surface. In such structures, the drug composition is typically contained in a layer, or "reservoir," beneath an upper backing layer. The laminated device may contain a single reservoir or multiple reservoirs. In certain embodiments, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to attach the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, and the like.

[0375] V. Combination Therapy The compounds of Formula I, Formula II, or Formula III, or pharmaceutically acceptable salts thereof, can be used in effective amounts, either alone or in combination with a second therapeutic agent, to treat a subject, such as a human, having a disorder mediated by ROCK1 and / or ROCK2.

[0376] The term "second therapeutic agent" is used to describe an agent other than a selected compound according to the present invention that can be used in combination or alternation with a compound of the present invention to achieve a desired outcome of therapy. In certain embodiments, a compound of the present invention and a second therapeutic agent are administered such that they are active in vivo during an overlapping period, e.g., their Cmax, Tmax, AUC, or other pharmacokinetic parameters overlap. In another embodiment, a compound of the present invention and a second therapeutic agent are administered to a subject in need thereof, and they do not have overlapping pharmacokinetic parameters, but one has a therapeutic impact on the therapeutic effectiveness of the other.

[0377] Non-limiting examples of second therapeutic agents include riluzole, edaravone, sodium phenylbutyrate, taurursodiol, levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, and istradefylline.

[0378] In certain embodiments, a compound of Formula I, Formula II, or Formula III of the present invention, or a pharmaceutically acceptable salt thereof, is used in combination or alternation with tiruzole, edaravone, or taursodiol to treat ALS.

[0379] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used in combination with tiruzole, edaravone, or taursodiol to treat ALS.

[0380] In another aspect, Compound 2 or a pharmaceutically acceptable salt thereof is used in combination with tiruzole, edaravone, or taursodiol to treat ALS. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6 or a pharmaceutically acceptable salt thereof can be used in combination with tiruzole, edaravone, or taursodiol to treat ALS. In certain embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14 or a pharmaceutically acceptable salt thereof can be used in combination with tiruzole, edaravone, or taursodiol to treat ALS.

[0381] In certain embodiments, compounds of the invention or pharmaceutically acceptable salts thereof are used in combination or alternation with levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, and istradefylline to treat PD.

[0382] In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used in combination with levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, or istradefylline to treat PD.

[0383] In another embodiment, Compound 2 or a pharmaceutically acceptable salt thereof is used in combination with levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, or istradefylline to treat PD. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6 or a pharmaceutically acceptable salt thereof can be used in combination with levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, or istradefylline to treat PD. In certain embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14, or a pharmaceutically acceptable salt thereof, can be used in combination with levodopa, selegiline, rasagiline, safinamide, pramipexole, rotigotine, apomorphine, tolcapone, entacapone, trihexyphenidyl, benztropine, orphenadrine, procyclidine, biperiden, amantadine, or istradefylline to treat PD.

[0384] In certain embodiments, a compound of the invention or a pharmaceutically acceptable salt thereof is used in combination with another ROCK inhibitor, non-limiting examples of which include fasudil, netarsudil, and ripasudil.

[0385] In certain embodiments, compounds of the invention or pharmaceutically acceptable salts thereof are used in combination or alternation with fasudil to treat ALS or PD.

[0386] In certain embodiments, Compound 1, or a pharmaceutically acceptable salt thereof, is used in combination with fasudil to treat PD or ALS.

[0387] In other aspects, Compound 2 or a pharmaceutically acceptable salt thereof is used in combination with fasudil to treat PD or ALS. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6 or a pharmaceutically acceptable salt thereof can be used in combination with fasudil to treat PD or ALS. In certain embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14 or a pharmaceutically acceptable salt thereof is used in combination with fasudil to treat PD or ALS.

[0388] In certain embodiments, compounds of the invention or pharmaceutically acceptable salts thereof are used in combination or alternation with netarsudil or ripasudil to treat ocular disorders.

[0389] In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used in combination with netarsudil or ripasudil to treat ocular disorders.

[0390] In another embodiment, Compound 2 or a pharmaceutically acceptable salt thereof is used in combination with netarsudil or ripasudil to treat ocular disorders. Alternatively, Compound 3, Compound 4, Compound 5, or Compound 6 or a pharmaceutically acceptable salt thereof can be used in combination with netarsudil or ripasudil to treat ocular disorders. In certain embodiments, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, or Compound 14 or a pharmaceutically acceptable salt thereof can be used in combination with netarsudil or ripasudil to treat ocular disorders.

[0391] VIII. General synthesis The compounds described herein can be prepared by methods known to those skilled in the art. In one non-limiting example, the disclosed compounds can be made using the following scheme: The abbreviations used in the synthetic procedures have the following definitions:

[0392] TIFF2026502949000066.tif97170

[0393] Example 1: Synthesis of N-(2-fluorobenzyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 1) [ka]

[0394] 6-Chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-3) [ka] To a solution of 6-chloro-3-iodopyridin-2-amine (1-1) (9.00 g, 35.4 mmol), 2-oxopropanoic acid (1-2) (9.34 g, 106 mmol), and DABCO (11.9 g, 106 mmol) in N,N-dimethylformamide (255 mL) was added palladium(II) acetate (0.397 g, 1.77 mmol). The reaction mixture was degassed three times and stirred at 110 °C under a nitrogen atmosphere for 3 h until the reaction was complete and monitored by LCMS. The resulting mixture was concentrated in vacuo. The residue was diluted with ethyl acetate (500 mL) and extracted with aqueous sodium hydroxide (2 M, 500 mL × 3). The combined aqueous layers were concentrated to approximately 500 mL and acidified to pH 3 with aqueous HCl (1 M). The mixture was filtered and the filter cake was collected, washed with water and dried to give 1-3 (5.49 g, 74% yield) as a yellow solid. ESI m / z: 196.9 (M+H) + , retention time 1.23 min, 93.9% at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.41 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.06 (s, 1H) ppm.

[0395] Ethyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (1-4) [ka] To a solution of 1-3 (5.70 g, 35.4 mmol) in ethanol (170 mL) was added 98% sulfuric acid (14.5 g, 145 mmol) at 0 °C, and the reaction mixture was stirred at 80 °C for 18 h and monitored by LCMS. The resulting mixture was neutralized to pH 7-8 with saturated aqueous sodium bicarbonate and then concentrated to remove ethanol. The remaining aqueous mixture was diluted with water (300 mL) and extracted with ethyl acetate (500 mL × 3). The combined organic solution was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v = 2) to give 1-4 (4.29 g, 66% yield) as a yellow solid. ESI m / z: 224.9 (M+H). + , retention time 1.82 min, 98.6% at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.75 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H) ppm.

[0396] Ethyl 6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (1-5) [ka] To a suspension of 1-4 (1.03 g, 4.60 mmol) and potassium carbonate (2.54 g, 18.4 mmol) in N,N-dimethylacetamide (DMA, 21.0 mL) was added iodomethane (1.96 g, 13.8 mmol). The reaction mixture was stirred at room temperature for 72 h and monitored by LCMS. The resulting mixture was poured into water (63 mL). The precipitate was collected by filtration, washed with water (5 mL × 2), and dried. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v = 2) to give 1-5 (766 mg, 70% yield) as a yellow solid. ESI m / z: 238.9 (M+H). + , retention time 2.02 min, 97.5% at 214 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 8.20 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H), 7.28 (d, J = 8.0 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 4.01 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H) ppm.

[0397] Ethyl 1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (1-7) [ka] To a solution of 1-5 (766 mg, 3.22 mmol) in 1,4-dioxane (28.0 mL) and water (7.0 mL) was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 1-6 (1.25 g, 6.44 mol), potassium phosphate (2.05 g, 9.66 mmol), and bis(tri-tert-butylphosphine)palladium (0.164 g, 0.322 mmol) under nitrogen gas. The reaction mixture was stirred at 100 °C under an argon atmosphere for 2 hours, and the reaction progress was monitored by LCMS. The resulting mixture was diluted with water (20 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane / methanol, v / v=20) to give 1-7 (653 mg, 75.1% yield) as a pale yellow solid. ESI m / z: 271.0 (M+H) + , retention time 1.69 min, 98.3% at 214 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 13.03 (s, 1H), 8.29 (s, 2H), 8.08 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.22 (s, 1H), 4.34 (q, J = 7.0 Hz, 2H), 4.08 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H) ppm.

[0398] 1-Methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) [ka] To a solution of compound 1-7 (400 mg, 1.48 mmol) in water (2.6 mL), tetrahydrofuran (THF, 7.8 mL), and methanol (2.6 mL) was added lithium hydroxide monohydrate (0.311 g, 7.41 mmol). The reaction mixture was stirred at 20° C. for 3 hours, and the reaction progress was monitored by LCMS. The reaction mixture was concentrated in vacuo and diluted with water (2 mL). The aqueous mixture was acidified to pH 1 with concentrated aqueous hydrochloric acid and then filtered. The filter cake was dried in vacuo to give 1-8 (357 mg, 99.4% yield). ESI m / z: 243.1 (M+H). + , retention time 1.07 min, purity >99.9% at 254 nm. 1 H NMR (400 MHz, DMSO d6 ) δ 8.30 (s, 2H), 8.07 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.18 (s, 1H), 4.08 (s, 3H) ppm.

[0399] N-(2-fluorobenzyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 1) [ka] To a solution of 1-8 (5.9 g, 24.4 mmol) in N,N-dimethylformamide (236 mL) was added (2-fluorophenyl)methanamine 1-9 (3.66 g, 29.3 mmol, CAS: 89-99-6), EDCI (7.01 g, 36.6 mmol), HOBt (4.94 g, 36.6 mmol), and DIPEA (15.7 g, 122 mmol). The reaction was monitored by LCMS. After stirring at 25 °C for 18 h, the reaction mixture was quenched with water (600 mL) and then extracted with ethyl acetate (600 mL × 3). The combined organic phase was washed with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by preparative HPLC (5% to 95% acetonitrile in aqueous ammonium bicarbonate (10 mM)) to give the desired product, Compound 1 (1.96 g, 23% yield) as a white solid. ESI m / z: 349.9 (M+H). + , retention time 1.59 min, purity >99.9% at 254 nm. 1 H NMR (400 MHz, DMSO d6 ) δ 13.06 (s, 1H), 9.10 (t, J = 5.5 Hz, 1H), 8.26 (s, 2H), 8.05 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.36-7.29 (m, 1H), 7.24-7.13 (m, 3H), 4.54 (d, J = 5.5 Hz, 2H), 4.06 (s, 3H) ppm. 19 F NMR (376 MHz, DMSO d6 ) δ -118.97 ppm.

[0400] Example 2: Synthesis of N-(2-fluorobenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 2) [ka]

[0401] 6-Chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-3) [ka] To a solution of 6-chloro-3-iodopyridin-2-amine (2-1) (9.00 g, 35.4 mmol), 2-oxopropanoic acid (2-2) (9.34 g, 106 mmol), and DABCO (11.9 g, 106 mmol) in N,N-dimethylformamide (255 mL) was added palladium(II) acetate (0.397 g, 1.77 mmol). The reaction mixture was degassed three times and stirred at 110 °C under a N atmosphere for 3 h. LCMS showed the reaction was complete. The mixture was then concentrated in vacuo to remove the solvent. Ethyl acetate (500 mL) was added to the residue, and the mixture was extracted with 2 M aqueous sodium hydroxide solution (500 mL × 3). The aqueous layer was concentrated in vacuo to 500 mL. The aqueous solution was then acidified with HCl to pH = 3. The mixture was then filtered. The filter cake was washed and dried to give 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (5.49 g, 74.1%) as a yellow solid. LCMS: ESI [M+H] + 196.9 was confirmed with a retention time of 1.23 min and a purity of 93.9% at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.41 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.06 (s, 1H) ppm.

[0402] Ethyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (2-4) [ka] To a solution of 2-3 (5.70 g, 35.4 mmol) in ethanol (170 mL) was added 98% sulfuric acid (14.5 g, 145 mmol). The reaction mixture was stirred at 80 °C for 18 hours. LCMS showed the reaction was complete. The mixture was then alkalized with sodium bicarbonate and concentrated under vacuum to remove ethanol. Water (300 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (500 mL × 3). The organic phase was washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated under vacuum. The residue was purified on a silica column (petroleum ether:ethyl acetate = 2:1) to give ethyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (2-4) (4.29 g, 65.9%) as a yellow solid. LCMS: ESI: 224.9 [M+H] + , retention time 1.82 min, purity 98.6% at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.75 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H) ppm.

[0403] Ethyl 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (2-5) [ka] To a solution of 2-4 (3.50 g, 15.6 mmol), 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (6.5 g, 31.3 mol), and potassium phosphate (8.29 g, 39.1 mmol) in 1,4-dioxane (70.0 mL) and water (17.5 mL) was added bis(tri-tert-butylphosphine)palladium (0.799 g, 1.56 mmol). The reaction mixture was stirred at 100 °C for 18 hours under an argon atmosphere. LCMS confirmed the formation of the desired product. Water (100 mL) was then added to the reaction mixture at 25 °C. The reaction mixture was extracted with ethyl acetate (200 mL × 3). The combined organic phase was washed with saturated sodium chloride solution (200 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated. The residue was purified on a silica column (dichloromethane:methanol=20:1) to give ethyl 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (2-5) (1.14 g, 27.0%). LCMS: ESI [M+H] + 271.0, retention time 1.65 min, purity 96.0% at 214 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.73 (s, 1H), 12.26 (s, 1H), 8.04 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 4.33 (q, J = 7.0 Hz, 2H), 2.61 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H) ppm.

[0404] 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-6) [ka] To a solution of 2-5 (1.14 g, 4.22 mmol) in water (3 mL), tetrahydrofuran (9 mL), and methanol (3 mL) was added lithium hydroxide monohydrate (0.886 g, 21.1 mmol). The reaction mixture was stirred at 20 °C for 2 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated in vacuo to remove tetrahydrofuran and methanol. After water (3 mL) was added, 36.5% hydrochloric acid solution was added to adjust the pH to 1, followed by filtration. The filter cake was dried in vacuo to give 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-6) (0.956 g, 93.7%). LCMS: ESI [M+H] + 243.1, retention time 1.12 min, 100% purity at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.15 (s, 1H), 8.15 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 2.63 (s, 3H) ppm.

[0405] N-(2-fluorobenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 2) [ka] To a solution of 2-6 (70.0 mg, 0.289 mmol) in N,N-dimethylformamide (3.00 mL) was added (2-fluorophenyl)methanamine (43.4 mg, 0.347 mmol), N-(3-(dimethylamino)propyl)propanamide dihydrochloride (83.2 mg, 0.434 mmol), 1-hydroxybenzotriazole (58.6 mg, 0.434 mmol), and N,N-diisopropylethylamine (187 mg, 1.45 mmol). The reaction mixture was stirred at 25 °C for 18 h. Water (6 mL) was then added to the reaction mixture. The reaction mixture was then extracted with ethyl acetate (30 mL × 3), and the combined organic phase was washed with saturated sodium chloride solution (10 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated. The residue was purified by preparative HPLC to give N-(2-fluorobenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 2) as a white solid (29.9 mg, 29.6%). LCMS: ESI [M+H] + 350.2, retention time 1.64 min, 100% purity at 254 nm. 1 H NMR (500 MHz, DMSO d6 ) δ 12.68 (s, 1H), 11.88 (s, 1H), 8.94 (t, J = 5.5 Hz, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.45-7.41 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.37-7.30 (m, 1H), 7.24-7.17 (m, 2H), 7.13 (d, J = 2.0 Hz, 1H), 4.56 (d, J = 5.5 Hz, 2H), 2.60 (s, 3H) ppm.

[0406] Example 3: Synthesis of N-(1-(3-methoxyphenyl)cyclopropyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 3) [ka]

[0407] 1-(3-Methoxyphenyl)cyclopropan-1-amine (3-1) [ka] 3-Methoxybenzonitrile (2.2 g, 16.5 mmol) and Ti(O i To a solution of Pr)4 (5.17 g, 18.2 mmol) was added ethylmagnesium bromide (2 M in THF, 18.2 mL) at -78 °C. The yellow solution was stirred at this temperature for 10 minutes and then slowly warmed to room temperature for 1 hour. To this solution, boron trifluoride diethyl etherate (1 M in THF, 33 mL) was added, and the reaction mixture was stirred at room temperature for 1 hour and monitored by LCMS. The reaction was quenched with aqueous HCl (1 N, 60 mL) and diluted with ethyl acetate (100 mL). The resulting mixture was then basified with aqueous sodium hydroxide (10%, 200 mL) and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative HPLC to give compound 3-1 (900 mg, 33% yield) as a colorless oil. ESI m / z: 164.1 [M+H] + , retention time 1.525 min, 96% at 254 nm. 1 HNMR (500 MHz, DMSO d6 ) δ 7.18-7.15 (m, 1H), 6.92-6.91 (m, 1H), 6.81-6.79 (m, 1H), 6.70-6.68 (m, 1H), 3.74 (s, 3H), 0.95-0.91 (m, 2H), 0.90-0.87 (m, 2H)ppm.

[0408] N-(1-(3-methoxyphenyl)cyclopropyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 3) [ka] To a solution of 1-8 (400 mg, 1.65 mmol) in N,N-dimethylformamide (100 mL) were added 1-(3-methoxyphenyl)cyclopropan-1-amine 3-1 (323 mg, 1.98 mmol), HATU (940 mg, 2.5 mmol), and DIPEA (639 mg, 4.95 mmol). The reaction mixture was stirred at room temperature for 15 minutes. The reaction was then quenched with water (300 mL). The resulting mixture was extracted with ethyl acetate (200 mL × 3), and the combined organic solution was washed with saturated sodium chloride solution (300 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was directly purified by reverse-phase flash chromatography (0% → 100% methanol in aqueous ammonium bicarbonate (0.05%)) to give compound 3 (180 mg, 28% yield) as a pale yellow solid. ESI m / z: 388 [M+H] - Retention time: 1.78 min, >99.9% at 214 nm and 254 nm. 1 H NMR (400 MHz, DMSO d6 ) δ 13.1 (s, 1H), 9.25 (s, 1H), 8.34-8.22 (m, 2H), 8.06 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.22-7.19 (m, 2H), 6.82-6.75 (m, 3H), 4.03 (s, 3H), 3.72 (s, 3H), 1.30 (s, 4H) ppm.

[0409] Example 4: Synthesis of N-(1-(2-fluorophenyl)cyclopropyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 4) [ka]

[0410] Methyl 6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxylate [ka] To a stirred mixture of 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (4-1) (14 g, 71.214 mmol, 1.0 equiv.) and KCO (49.21 g, 356.070 mmol, 5.0 equiv.) in N,N-dimethylacetamide (150 mL), CHCl (21.23 g, 149.549 mmol, 2.1 equiv.) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with water at room temperature. The aqueous layer was extracted with ethyl acetate (2 × 500 mL). The organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether and ethyl acetate (3:1) to give methyl 6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (4-2) (10.5 g, 65.6%) as a yellow solid. MS(ESI) m / z: 225 [M+H] + .

[0411] Methyl 1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate [ka] A mixture of methyl 6-chloro-1-methylpyrrolo[2,3-b]pyridine-2-carboxylate (4-2) (10 g, 44.516 mmol, 1.0 equiv.), 1H-pyrazol-4-yl-boronic acid (7.47 g, 66.774 mmol, 1.5 equiv.), Pd(PPh3)4 (5.14 g, 4.452 mmol, 0.1 equiv.), and K2CO3 (24.61 g, 178.064 mmol, 4.0 equiv.) in 1,4-dioxane (90 mL) and HO (30 mL) was stirred at 100 °C overnight under a nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether and ethyl acetate (1:1) to give methyl 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylate (4-3) (4.4 g, 38.6%) as a yellow solid. MS (ESI) m / z: 257 [M+H] + .

[0412] 1-Methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid [ka] A mixture of methyl 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylate (4-3) (4.3 g, 16.78 mmol, 1.0 equiv.) and LiOH (2.01 g, 83.9 mmol, 5.0 equiv.) in tetrahydrofuran (30 mL), methanol (10 mL), and HO (10 mL) was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with ethyl acetate and methanol (5:1) to give 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (4-4) (3.5 g, 86.1%) as a white solid. MS (ESI) m / z: 243 [M+H]+ .

[0413] N-[1-(2-fluorophenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (compound 4) [ka] A mixture of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (4-4) (3.4 g, 14.036 mmol, 1.0 equiv.), DIPEA (9.07 g, 70.180 mmol, 5.0 equiv.), and 1-(2-fluorophenyl)cyclopropan-1-amine (3.18 g, 21.054 mmol, 1.5 equiv.) in N,N-dimethylformamide (25 mL) was stirred at 0° C. for 10 minutes. HATU (8.01 g, 21.054 mmol, 1.5 equiv.) was added in portions to the above mixture at 0° C. The resulting mixture was stirred at room temperature for an additional 2 hours. The reaction was monitored by LCMS. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (2×200 mL). The combined organic layers were dried over anhydrous Na2SO4 and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (0.1% FA), 10% to 50% gradient (40 min); detector, UV 254 nm. This afforded N-[1-(2-fluorophenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (compound 4) (2.8914 g, 52.7%) as a dark yellow solid. MS (ESI) m / z: 376.30 [M+H] + . 1H-NMR (CD3OD) δ: 8.26 (s, 2H), 8.00 (d, J = 8.4 Hz, 1H), 7.66 (td, J = 8.0, 1.6 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.32-7.21 (m, 1H), 7.17-6.96 (m, 3H), 4.02 (s, 3H), 1.31 (s, 4H) ppm.

[0414] Example 5: Synthesis of N-(1-(3-methoxyphenyl)cyclopropyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 5) [ka]

[0415] 1-(3-Methoxyphenyl)cyclopropan-1-amine (5-2) [ka] To a solution of 3-methoxybenzonitrile (5-1) (2.2 g, 16.54 mol) and Ti(Oi-Pr)4 (5.17 g, 18.2 mmol) in 50 mL of tetrahydrofuran was added ethylmagnesium bromide (2 M in THF, 18.2 mL) at -78 °C. The resulting yellow solution was stirred for 10 min. After warming the reaction mixture to room temperature (1 h), BF3·Et2O (1 M in THF, 33 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. To the resulting mixture were added 1 N HCl (60 mL) and ethyl acetate (100 mL), followed by NaOH (10% aqueous solution, 200 mL). The reaction mixture was extracted with ethyl acetate, concentrated, and purified by preparative HPLC to give 1-(3-methoxyphenyl)cyclopropan-1-amine (4-2) (900 mg, 33%) as a colorless oil. LCMS: LC retention time 1.525 minutes. MS(ESI)m / z:164.1[M+H] + , Purity: 98% at 214nm; 96% at 254nm. 1 H NMR (500 MHz, DMSOd6 ): δ 7.18-7.15 (m, 1H), 6.92-6.91 (m, 1H), 6.81-6.79 (m, 1H), 6.70-6.68 (m, 1H), 3.74 (s, 3H), 0.95-0.91 (m, 2H), 0.90-0.87 (m, 2H)ppm.

[0416] N-(1-(3-methoxyphenyl)cyclopropyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 5) [ka] A mixture of 1-(3-methoxyphenyl)cyclopropan-1-amine (5-2) (47 mg, 0.29 mmol), 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-6) (70 mg, 0.29 mmol), EDCI (84 mg, 0.44 mmol), HOBt (54 mg, 0.44 mmol), and DIPEA (187 mg, 1.5 mmol) in N,N-dimethylformamide (2 mL) was stirred at room temperature for 12 hours. LCMS indicated the reaction was complete, and the mixture was purified by preparative HPLC to give the desired product (compound 5) as a white solid (26 mg, 23%). LCMS: LC retention time 1.643 min. MS (ESI) m / z: 388 [M+H] + . Purity: 98% (214nm). 1 H NMR (500 MHz, DMSO d6 ) δ 12.68 (s, 1H), 11.80 (s, 1H), 9.11 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H), 6.74 (m, 2H), 3.71 (s, 3H), 2.59 (s, 3H), 1.27(s, 4H) ppm.

[0417] Example 6: Synthesis of N-(2-chloro-6-(1-hydroxycyclopropyl)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 6) [ka]

[0418] 1-(3-chloro-2-(hydroxymethyl)phenyl)cyclopropan-1-ol (6-2) [ka] To a solution of titanium tetraisopropanolate (1.69 g, 5.93 mmol) in tetrahydrofuran (20 mL) was added MeMgBr (8.90 mL, 1.0 mol / L), and the solution was stirred for 10 min and then cooled to 0 °C. 4-Chloroisobenzofuran-1(3H)-one (6-1) (1.00 g, 5.93 mmol) was then added, and EtMgBr (3.36 mL, 3.0 mol / L) was added dropwise to the reaction mixture. The solution was stirred at room temperature under nitrogen for 1 h. The organic phase was quenched with 10% H2SO4 (7.00 mL), partitioned between water (30.0 mL) and ethyl acetate (30.0 mL x 3), washed with saturated sodium chloride solution (30.0 mL), dried over anhydrous sodium sulfate, and filtered. After filtration, the filtrate was concentrated under reduced pressure and the crude residue was purified by flash column chromatography to give 1-(3-chloro-2-(hydroxymethyl)phenyl)cyclopropan-1-ol (6-2) (600 mg, 2.87 mmol, yield: 48.7%) as a light oil. LCMS: LC retention time 1.49 min. MS(ESI) m / z: 181.0 [M+H-17] + , 100% at UV 254nm. 1H NMR (500 MHz, DMSO-d6) δ, 7.40-7.38 (m, 1H), 7.35-7.33 (m, 1H), 7.29-7.26 (m, 1H), 5.97 (s, 1H), 4.94-4.93 (m, 1H), 4.90-4.89 (d, J = 5 Hz, 2H), 1.01-0.98 (m, 2H), 0.93-0.91 (m, 2H) ppm.

[0419] 2-(2-chloro-6-(1-hydroxycyclopropyl)benzyl)isoindoline-1,3-dione (6-3) [ka] To a stirred solution of 6-2 (500 mg, 2.52 mmol) in tetrahydrofuran (10.0 mL) was added isoindoline-1,3-dione (370 mg, 5.25 mmol) and triphenylphosphine (1.98 g, 7.55 mmol). The solution was cooled to 0 °C, and diisopropyl azodicarboxylate (0.763 g, 0.00378 mol) was added dropwise under nitrogen. The solution was stirred at room temperature for 6 h. The reaction mixture was partitioned between water (10.0 mL) and dichloromethane (10.0 mL × 3). The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, and filtered. After filtration, the solvent was concentrated under reduced pressure and the crude residue was purified by flash column chromatography to give 2-(2-chloro-6-(1-hydroxycyclopropyl)benzyl)isoindoline-1,3-dione (6-3) (700 mg, 76.4%) as a pale oil. LCMS: LC retention time 1.80 min. MS (ESI) m / z: 327.9 [M+H] + ,Purity: 86.9% at UV 254nm. 1 H NMR (500 MHz, DMSO) δ, 7.83 (s, 4H), 7.39-7.37 (m, 1H), 7.35-7.33 (m, 1H), 7.30-7.27 (t, J = 15.5 Hz, 1H), 5.85 (s, 1H), 5.21 (s, 2H), 1.08-1.06 (m, 2H), 0.99-0.91 (m, 2H) ppm.

[0420] 1-(2-(aminomethyl)-3-chlorophenyl)cyclopropan-1-ol (6-4) [ka] To a mixture of 6-3 (700 mg, 2.14 mmol) in ethanol (10.0 mL) was added hydrazine hydrate (160 mg, 3.20 mmol). The reaction mixture was stirred at 78 °C for 4 h. The reaction mixture was partitioned between water (10.0 mL) and dichloromethane (10.0 mL × 3), washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, and filtered. After filtration, the solvent was concentrated under reduced pressure, and the crude residue was purified by reverse-phase column chromatography to give 1-(2-(aminomethyl)-3-chlorophenyl)cyclopropan-1-ol (6-4) (350 mg, 78.8%) as a pink solid. LCMS: LC retention time 1.46 min. MS (ESI) m / z: 198.0 [M+H] + , Purity: 100% at UV 254nm.

[0421] N-(2-chloro-6-(1-hydroxycyclopropyl)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 6) [ka] To a stirred solution of 2-6 (70.0 mg, 0.289 mmol) in N,N-dimethylformamide (4.00 mL) was added 6-4 (5.71 mg, 0.289 mmol), EDCI (83.1 mg, 0.433 mmol), HOBt (58.6 mg, 0.433 mmol), and DIPEA (112 mg, 0.867 mmol). The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was partitioned between water (10.0 mL) and dichloromethane (10.0 mL × 3). The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, and filtered. After filtration, the solvent was concentrated under reduced pressure and the crude residue was purified by preparative HPLC to give N-(2-chloro-6-(1-hydroxycyclopropyl)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (compound 6) (29.0 mg, yield: 23.8%) as a white solid. LCMS: LC retention time 1.93 min. MS (ESI) m / z: 422.1 [M+H] + ,Purity: 98% at UV 254nm. 1 H NMR (500 MHz, DMSO) δ 11.86 (s, 1H), 8.40-8.39 (m, 1H), 7.99-7.98 (m, 2H), 7.46-7.33 (m, 4H), 7.07-7.06 (d, J = 2 Hz, 1H), 6.21 (s, 1H), 4.93-4.92 (d, J = 4 Hz, 1H), 2.58 (s, 3H), 1.03-1.01 (m, 2H), 0.98-0.96 (m, 2H).

[0422] Example 7: Synthesis of N-(1-(3-(1-hydroxycyclopropyl)phenyl)cyclopropyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 7) [ka]

[0423] tert-Butyl N-[1-(3-bromophenyl)cyclopropyl]carbamate (7-2) [ka] A solution of 1-(3-bromophenyl)cyclopropan-1-amine (5.0 g, 23.58 mmol, 1.0 equiv.), di-tert-butyl dicarbonate, and NaHCO3 (0.99 g, 11.79 mmol, 0.5 equiv.) in HO (10 mL) and methanol (40 mL) was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (5:1) to give tert-butyl N-[1-(3-bromophenyl)cyclopropyl]carbamate (7-2) (7.1 g, 96.46%) as a pale yellow solid. MS (ESI) m / z: 312.2 [M+H] + .

[0424] Ethyl 3-{1-[(tert-butoxycarbonyl)amino]cyclopropyl}benzoate (7-3) [ka] A solution of tert-butyl N-[1-(3-bromophenyl)cyclopropyl]carbamate (7-2) (10.0 g, 32.03 mmol, 1.0 equiv.) and Pd(dppf)Cl2 (2.34 g, 3.20 mmol, 0.1 equiv.) in ethanol (20 mL) was stirred at 100 °C under a carbon monoxide atmosphere for 4 h. The desired product could be detected by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (6:1) to give ethyl 3-{1-[(tert-butoxycarbonyl)amino]cyclopropyl}benzoate (7-3) (8.7 g, 88.95%) as a colorless oil. MS (ESI) m / z: 306.2 [M+H] + .

[0425] tert-Butyl N-{1-[3-(1-hydroxycyclopropyl)phenyl]cyclopropyl}carbamate (7-4) [ka] To a stirred solution of ethyl 3-{1-[(tert-butoxycarbonyl)amino]cyclopropyl}benzoate (7-3) (1.0 g, 3.28 mmol, 1.0 equiv.) and Ti(Oi-Pr) (1.40 g, 4.91 mmol, 1.5 equiv.) in tetrahydrofuran (50 mL), ethylmagnesium bromide (2.62 g, 19.65 mmol, 6.0 equiv.) was added portionwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. Upon completion, the reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 × 15 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (9:1) to give tert-butyl N-{1-[3-(1-hydroxycyclopropyl)phenyl]cyclopropyl}carbamate (7-4) (467 mg, 49.28%) as a yellow oil. MS (ESI) m / z: 290.3 [M+H] + .

[0426] 1-(3-(1-aminocyclopropyl)phenyl)cyclopropan-1-ol (7-5) [ka] A solution of tert-butyl N-{1-[3-(1-hydroxycyclopropyl)phenyl]cyclopropyl}carbamate (7-4) (476 mg, 1.65 mmol, 1.0 equiv.) and TFA (2.44 mL, 32.90 mmol, 20 equiv.) in dichloromethane (4 mL) was stirred at 0 °C for 30 min. The reaction was monitored by LCMS. The resulting mixture was neutralized with NaHCO powder at 0 °C, followed by filtration and concentration under reduced pressure. The crude product (7-5) was used directly in the next step without further purification. MS (ESI) m / z: 190.2 [M+H] + .

[0427] N-(1-(3-(1-hydroxycyclopropyl)phenyl)cyclopropyl)-1-methyl-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 7) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (200 mg, 0.826 mmol, 1.0 equiv.), 1-[3-(1-aminocyclopropyl)phenyl]cyclopropan-1-ol (7-5) (234 mg, 1.24 mmol, 1.5 equiv.), EtN (230 μL, 1.65 mmol, 2.0 equiv.), and HATU (471 mg, 1.24 mmol, 1.5 equiv.) in N,N-dimethylformamide (6 mL) was stirred at room temperature for 2 h. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with water and neutralized to pH 7 with HCl (1 M aqueous solution) at 0 °C. The resulting mixture was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with water (2 x 30 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: XBridge Prep OBD C18 Column, 30 mm × 150 mm, 5 μm; Mobile Phase A: Water (10 mmol / L NH4HCO3), Mobile Phase B: MeOH-preparative; Flow Rate: 60 mL / min; Gradient: 40% B → 56% B (10 min), 56% B; Wavelength: 220 nm / 254 nm; RT1 (min): 10.48; Run Number: 0) to give N-{1-[3-(1-hydroxycyclopropyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 7) (24 mg, 97.7% purity, 9.73% yield) as a white solid. MS (ESI) m / z: 414.00 [M+H] + . 1H NMR (400 MHz, DMSO-d6) d: 13.05 (s, 1H), 9.23 (s, 1H), 8.38 (s, 1H), 8.13 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.23-7.16 (m, 3H), 7.05 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 7.6 Hz, 1H), 5.82 (s, 1H), 4.01 (s, 3H), 1.33-1.22 (m, 4H), 1.09-1.04 (m, 2H), 0.92-0.86 (m, 2H) ppm.

[0428] Example 8: Synthesis of N-{1-[2-(fluoromethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 8) [ka]

[0429] N-[1-(2-bromophenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-1) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (1.0 g, 4.13 mmol, 1.0 equiv.) in N,N-dimethylformamide (10 mL) was treated with 1-(2-bromophenyl)cyclopropan-1-amine (0.96 g, 4.54 mmol, 1.1 equiv.), HOBt (558 mg, 4.13 mmol, 1.0 equiv.), EDCI (791 mg, 4.13 mmol, 1.0 equiv.), and DIPEA (2.16 mL, 12.38 mmol, 3.0 equiv.) at room temperature for 2 hours. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with water (10 mL) and then extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated sodium chloride solution (20 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (0.1% FA), 10% to 50% gradient (10 min); detector, UV 254 nm. The resulting mixture was concentrated under reduced pressure. This afforded N-[1-(2-bromophenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-1) (1.14 g, 63.29%) as a pale yellow powder. MS (ESI) m / z: 436.20 [M+H] + .

[0430] N-[1-(2-ethenylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-2) [ka] A solution of N-[1-(2-bromophenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-1) (1.59 g, 3.66 mmol, 1.0 equiv.), potassium ethenyltrifluoroborate (976 mg, 7.29 mmol, 2.0 equiv.), CsCO (3.57 g, 10.94 mmol, 3.0 equiv.), and Pd(dppf)Cl·CHCl (594 mg, 0.73 mmol, 0.2 equiv.) in tetrahydrofuran (36 mL) and HO (3.6 mL) was stirred at 100 °C under a nitrogen atmosphere for 6 h. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with water (100 mL) and then extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed with saturated sodium chloride solution (100 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (1:1) to give N-[1-(2-ethenylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-2) (480 mg, 34.35%) as a yellow solid. MS (ESI) m / z: 384.45 [M+H] + .

[0431] N-[1-(2-formylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-3) [ka] A solution of N-[1-(2-ethenylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-2) (450 mg, 1.17 mmol, 1.0 equiv.), OsO (30.5 mg, 0.12 mmol, 0.1 equiv.), and NaIO (753 mg, 3.52 mmol, 3.0 equiv.) in tetrahydrofuran (2 mL) and HO (2 mL) was stirred at 0 °C for 40 min. The resulting mixture was then stirred at room temperature for an additional 2 h. The reaction was monitored by LCMS. The reaction was quenched by the addition of saturated NaSO (aq.) (3 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (2 × 20 mL). The combined organic layers were washed with saturated sodium chloride solution (2 × 20 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (1:1) to give N-[1-(2-formylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-3) (160 mg, 35.37%) as an orange solid. MS (ESI) m / z: 386.40 [M+H] + .

[0432] N-{1-[2-(hydroxymethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-4) [ka] A solution of N-[1-(2-formylphenyl)cyclopropyl]-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-3) (160 mg, 0.42 mmol, 1.0 equiv.) and NaBH (63 mg, 1.66 mmol, 4.0 equiv.) in methanol (2 mL) was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. Upon completion, the reaction was quenched by the addition of water (1 mL) at room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (10:1) to give N-{1-[2-(hydroxymethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-4) (116 mg, 72.12%) as a pale yellow solid. MS(ESI) m / z: 388.15 [M+H] + .

[0433] N-{1-[2-(fluoromethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (compound 8) [ka] A solution of N-{1-[2-(hydroxymethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (8-4) (110 mg, 0.28 mmol, 1.0 equiv.) and DAST (187 μL, 1.42 mmol, 5.0 equiv.) in dichloromethane (5 mL) was stirred at −78° C. for 30 minutes under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for an additional hour. The reaction was monitored by LCMS. Upon completion, the reaction was quenched by the addition of water (3 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with saturated sodium chloride solution (2×20 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: Column: Sunfire prep C18 column, 30 mm × 150 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: 30% B → 50% B (10 min), 50% B; Wavelength: 254 nm / 220 nm; RT1 (min): 10.78; Run number: 0) to give N-{1-[2-(fluoromethyl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (compound 8) (1.7 mg, 98.6% purity, 1.52% yield) as a white solid. MS (ESI) m / z: 390.20 [M+H] + . 1 H NMR (400 MHz, MeOH-d4) d: 8.44 (s, 1H), 8.19 (s, 2H), 7.94 (d, J = 8.0 Hz, 1H), 7.82-7.78 (m, 1H), 7.46-7.41 (m, 2H), 7.38-7.29 (m, 2H), 6.91 (s, 1H), 5.84 (d, J = 47.6 Hz, 2H), 3.99 (s, 3H), 1.38-1.32 (m, 2H), 1.29-1.23 (m, 2H) ppm.

[0434] Example 9: Synthesis of N-{1-[3-(2-fluoropropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 9) [ka]

[0435] tert-Butyl N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}carbamate (9-1) [ka] A solution of ethyl 3-{1-[(tert-butoxycarbonyl)amino]cyclopropyl}benzoate (7-3) (2 g, 6.549 mmol, 1.0 equiv.) and MeMgBr (1.51 mL, 13.098 mmol, 2.0 equiv.) in tetrahydrofuran (10 mL) was stirred at 0 °C for 4 h under a nitrogen atmosphere. The desired product could be detected by LCMS. After completion, the reaction was quenched by the addition of 20 mL of saturated NH4Cl (aq.) at 0 °C. The resulting mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated sodium chloride solution (20 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (10 mmol / L NH4HCO3), 10% to 60% gradient (30 min); detector, UV 220 nm. This afforded tert-butyl N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}carbamate (9-1) (1.6 g, 83.84%) as a white solid. MS (ESI) m / z: 292.2 [M+H] + .

[0436] 2-[3-(1-aminocyclopropyl)phenyl]propan-2-ol (9-2) [ka] A solution of tert-butyl N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}carbamate (9-1) (1.5 g, 5.15 mmol, 1.0 equiv.) and ZnBr (11.60 g, 51.50 mmol, 10 equiv.) in 1,2-dichloroethane (20 mL) was stirred at room temperature for 1.5 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1:1) to give 2-[3-(1-aminocyclopropyl)phenyl]propan-2-ol (9-2) (330 mg, 33.52%) as a pale gray solid. MS (ESI) m / z: 192.1 [M+H] + .

[0437] N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (9-3) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (300 mg, 1.24 mmol, 1.0 equiv) and 2-[3-(1-aminocyclopropyl)phenyl]propan-2-ol (9-2) (355 mg, 1.86 mmol, 1.5 equiv) in N,N-dimethylformamide (0.5 mL) was stirred at 0 °C for 3 min. To the above mixture, HATU (942 mg, 2.48 mmol, 2.0 equiv) was added in small portions at 0 °C. The resulting mixture was stirred at room temperature for an additional 1 h. The reaction was monitored by LCMS. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (10 mmol / L NH4HCO3), 20% to 70% gradient (30 min); detector, UV 220 nm. This afforded N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (9-3) (46 mg, 8.94%) as a pale yellow solid. MS (ESI) m / z: 416.2 [M+H] + .

[0438] N-{1-[3-(2-fluoropropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 9) [ka] A solution of N-{1-[3-(2-hydroxypropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (9-3) (46 mg, 0.11 mmol, 1.0 equiv.) and DAST (29 μL, 0.22 mmol, 2.0 equiv.) in dichloromethane (0.5 mL) was stirred at −78° C. for 2 minutes under a nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was quenched by the addition of saturated NaHCO (aq.) (1.0 mL) at 0° C. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: (Column: YMC-Actus Triart C18 ExRS, 30 mm × 150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH4HCO3), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: 36% B → 55% B (9 min), 55% B; Wavelength: 254 nm / 220 nm; RT1 (min): 9.53; Run number: 0) to give N-{1-[3-(2-fluoropropan-2-yl)phenyl]cyclopropyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 9) (23.2 mg, 46.73%) as a white solid. MS (ESI) m / z: 418.25 [M+H] + . 1 H NMR (400 MHz, MeOH-d4) d : 8.28-8.16 (m, 2H), 7.99 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.39 (s, 1H), 7.30-7.26 (m, 1H), 7.24-7.20 (m, 2H), 7.07 (s, 1H), 4.07 (s, 3H), 1.66 (s, 3H), 1.61 (s, 3H), 1.40-1.32 (m, 4H) ppm.

[0439] Example 10: Synthesis of N-{[2-(fluoromethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 10) [ka]

[0440] N-{[2-(hydroxymethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (10-1) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (150 mg, 0.619 mmol, 1.0 equiv.), [2-(aminomethyl)phenyl]methanol (127 mg, 0.928 mmol, 1.5 equiv.) in N,N-dimethylformamide (4 mL) was treated with DIPEA (400 mg, 3.095 mmol, 5.0 equiv.) under a nitrogen atmosphere at 0° C. for 10 minutes, followed by the addition of HATU (471 mg, 1.238 mmol, 2.0 equiv.) in portions at 0° C. The resulting mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with water (15 mL) at 0° C. The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with saturated sodium chloride solution (20 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (0.1% TFA), 3% to 50% gradient (30 min); detector, UV 254 nm, 220 nm. This afforded N-{[2-(hydroxymethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (10-1) (110.0 mg, 49.1%) as a white solid. MS (ESI) m / z: 362.1 [M+H] + .

[0441] N-{[2-(fluoromethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 10) [ka] To a stirred solution of N-{[2-(hydroxymethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (10-1) (210 mg, 0.581 mmol, 1.0 equiv.) in dichloromethane (14 mL), DAST (280.9 mg, 1.743 mmol, 3.0 equiv.) was added dropwise at −78° C. under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction was monitored by LCMS. Upon completion, the reaction was quenched with NaHCO (saturated aqueous solution) at room temperature. The precipitated solid was collected by filtration and washed with HO (2×30 mL). The crude product was purified by preparative HPLC using the following conditions: Column: XBridge Shield RP18 OBD Column, 30 mm × 150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH₄HCO₃), Mobile phase B: acetonitrile; Flow rate: 50 mL / min; Gradient: 4% B → 15% B (8 min), 15% B; Wavelength: 254 nm / 220 nm; RT₁ (min): 7.17; Run number: 0) to give N-{[2-(fluoromethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 10) (40.7 mg, 18.5%) as a white solid. MS (ESI) m / z: 364.00 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ: 13.04 (s, 1H), 9.07 (t, J = 6.0 Hz, 1H), 8.37 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.47-7.37 (m, 3H), 7.36-7.28 (m, 1H), 7.16 (s, 1H), 5.64 (d, J = 47.6 Hz, 2H), 4.57 (d, J = 5.6 Hz, 2H), 4.05 (s, 3H) ppm.

[0442] Example 11: Synthesis of N-[(2-fluorophenyl)methyl]-6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 11) [ka]

[0443] Methyl 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (11-2) [ka] A solution of methyl 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (11-1) (1.2 g, 5.697 mmol, 1.0 equiv), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-2H-pyrazole (2.24 g, 8.546 mmol, 1.5 equiv), KCO (1.57 g, 11.394 mmol, 2.0 equiv), XPhos Pd G3 (482.2 mg, 0.570 mmol, 0.1 equiv), and XPhos (271.6 mg, 0.570 mmol, 0.1 equiv) in dioxane (75 mL) and HO (15 mL) was stirred at 100 °C under a nitrogen atmosphere for 4 h. The reaction was monitored by LCMS. The resulting mixture was diluted with water (150 mL) and extracted with ethyl acetate (3 × 150 mL). The combined organic layers were washed with saturated sodium chloride solution (2 × 100 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (5:1) to give methyl 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (11-2) (1.1 g, 62.2%) as a yellow solid. MS (ESI) m / z: 311.1 [M+H] + .

[0444] 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (11-3) [ka] A solution of methyl 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (11-2) (1.2 g, 3.548 mmol, 1.0 equiv) and LiOH·HO (811.5 mg, 19.340 mmol, 5.0 equiv) in tetrahydrofuran (27 mL), methanol (9 mL), and HO (9 mL) was stirred at room temperature for 3 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (30 mL) and acidified to pH 5 with HCl (aq). The precipitated solid was collected by filtration and washed with diethyl ether (3 × 50 mL). This gave 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (11-3) (720.0 mg, crude) as a white solid. MS (ESI) m / z: 297.2 [M+H] + .

[0445] N-[(2-fluorophenyl)methyl]-6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 11) [ka] A solution of 6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (11-3) (300 mg, 1.013 mmol, 1.0 equiv.), HOBt (205.2 mg, 1.519 mmol, 1.5 equiv.), DIEA (529.2 μL, 3.039 mmol, 3.0 equiv.), and 1-(2-fluorophenyl)methanamine (190.1 mg, 1.519 mmol, 1.5 equiv.) in N,N-dimethylformamide (10 mL) was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The resulting mixture was diluted with water (60 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with saturated sodium chloride solution (2 × 100 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product (300 mg) was purified by preparative HPLC using the following conditions: (Column: Sunfire prep C18 column, 30 mm × 150 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: 30% B → 48% B (10 min); Wavelength: 254 nm / 220 nm; RT1 (min): 11.73) to give N-[(2-fluorophenyl)methyl]-6-[3-(trifluoromethyl)-2H-pyrazol-4-yl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 11) (58.0 mg, 14.0%) as a white solid. MS (ESI) m / z: 403.95 [M+H] + . 1 H NMR (300 MHz, DMSO-d6) δ 13.77 (s, 1H), 12.03(s, 1H), 8.99 (t, J = 5.8 Hz, 1H), 8.49-8.34 (m, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.51-7.28 (m, 3H), 7.28-7.10 (m, 3H), 4.57 (d, J = 5.7 Hz, 2H) ppm. 19 F NMR (282 MHz, DMSO-d6) d -58.32 (s), -118.83 (s) ppm.

[0446] Example 12: Synthesis of N-{[2-(difluoromethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 12) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (325.0 mg, 1.342 mmol, 1.0 equiv.), 1-[2-(difluoromethyl)phenyl]methanamine (253.03 mg, 1.610 mmol, 1.2 equiv.), EDCI (385.79 mg, 2.013 mmol, 1.5 equiv.), HOBt (271.94 mg, 2.013 mmol, 1.5 equiv.), and DIPEA (701.10 μL, 4.026 mmol, 3.0 equiv.) in N,N-dimethylformamide (10 mL) was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The resulting mixture was diluted with water (70 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with saturated sodium chloride solution (2 × 100 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product (300.0 mg) was purified by preparative HPLC using the following conditions: Column: Sunfire prep C18 column, 30 mm × 150 mm, 5 μm; Mobile phase A: water (0.05% TFA), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: Isocratic 28% B → 48% B (10 min); Wavelength: 254 nm / 220 nm; RT1 (min): 11.4) to give N-{[2-(difluoromethyl)phenyl]methyl}-1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 12) (59.7 mg, 11.4%) as a white solid. MS(ESI)m / z:382.05[M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.13 (t, J = 6.0 Hz, 1H), 8.27 (s, 2H), 8.06 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.58-7.48 (m, 3H), 7.47-7.40 (m, 1H), 7.37 (t, J = 54.8 Hz, 1H), 7.18 (s, 1H), 4.65 (d, J = 5.9 Hz, 2H), 4.06 (s, 3H) ppm. 19 F NMR (376 MHz, DMSO- d6) δ -74.88 (s), -111.20 (s) ppm.

[0447] Example 13: Synthesis of 1-methyl-6-(1H-pyrazol-4-yl)-N-{[3-(trifluoromethyl)phenyl]methyl}pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 13) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (400 mg, 1.651 mmol, 1.0 equiv.), EDCI (475 mg, 2.477 mmol, 1.5 equiv.), and HOBt (335 mg, 2.477 mmol, 1.5 equiv.) in N,N-dimethylformamide (16 mL) was treated with DIPEA (320 mg, 2.477 mmol, 1.5 equiv.) at room temperature under a nitrogen atmosphere for 3 minutes, followed by the dropwise addition of 1-[3-(trifluoromethyl)phenyl]methanamine (434 mg, 2.477 mmol, 1.5 equiv.) at room temperature. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS. The resulting mixture was diluted with water (40 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 × 20 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product (250 mg) was purified by preparative HPLC using the following conditions: Column: XBridge Prep OBD C18 Column 30 mm × 150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH4HCO3 + 0.1% NH3 HO), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: 32% B → 50% B (10 min); Wavelength: 254 nm / 220 nm; RT1 (min): 1) to give 1-methyl-6-(1H-pyrazol-4-yl)-N-{[3-(trifluoromethyl)phenyl]methyl}pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 13) (63.4 mg, 9.49%) as a white solid. MS(ESI)m / z:400.20[M+H] + . 1 H NMR (300 MHz, DMSO-d6) δ 13.06 (s, 1H), 9.20 (t, J = 6.1 Hz, 1H), 8.39 (s, 1H), 8.14 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.73-7.56 (m, 4H), 7.53 (d, J = 8.2 Hz, 1H), 7.16 (s, 1H), 4.58 (d, J = 6.0 Hz, 2H), 4.06 (s, 3H) ppm. 19F NMR (282 MHZ, DMSO-d6) δ -61.03 (s) ppm.

[0448] Example 14: Synthesis of 1-methyl-6-(1H-pyrazol-4-yl)-N-{[2-(trifluoromethyl)phenyl]methyl}pyrrolo[2,3-b]pyridine-2-carboxamide (Compound 14) [ka] A solution of 1-methyl-6-(1H-pyrazol-4-yl)pyrrolo[2,3-b]pyridine-2-carboxylic acid (1-8) (414 mg, 1.71 mmol, 1.0 equiv.), HATU (651 mg, 1.71 mmol, 1.0 equiv.), and DIPEA (897 μL, 5.14 mmol, 3.0 equiv.) in N,N-dimethylformamide (5 mL) was stirred at room temperature for 10 minutes, followed by the addition of 1-[2-(trifluoromethyl)phenyl]methanamine (300 mg, 1.71 mmol, 1.0 equiv.) at room temperature. The resulting mixture was stirred at room temperature for an additional 2 hours. The reaction was monitored by LCMS. The resulting mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (2 × 50 mL), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: XBridge Prep OBD C18 Column 30 mm × 150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH4HCO3 + 0.1% NH3·HO), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient: 23% B → 36% B (10 min); Wavelength: 254 nm / 220 nm; RT1 (min): 3) to give 1-methyl-6-(1H-pyrazol-4-yl)-N-{[2-(trifluoromethyl)phenyl]methyl}pyrrolo[2,3-b]pyridine-2-carboxamide (59.2 mg, 8.60%) (compound 14) as a white solid. MS(ESI) m / z: 400.20 [M+H] + . 1H NMR (400 MHz, methanol-d4) δ: 8.33-8.11 (m, 2H), 8.00 (d, J = 10.8 Hz, 1H), 7.80-7.70 (m, 1H), 7.69-7.59 (m, 2H), 7.52-7.42 (m, 2H), 7.09 (s, 1H), 4.80 (s, 2H), 4.12 (s, 3H) ppm. 19 F NMR (376 MHz, methanol-d4) δ -61.57 (s) ppm.

[0449] Example 15: Synthesis of N-(2,6-dimethoxybenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 1, see generally U.S. Patent No. 5,949,993) [ka] To a stirred solution of 2-6 (70.0 mg, 0.000298 mol) in N,N-dimethylformamide (4 mL) was added (2,6-dimethoxyphenyl)methanamine (48.3 mg, 0.289 mmol), N-(3-(dimethylamino)propyl)propanamide dihydrochloride (83.1 mg, 0.433 mmol), 1-hydroxybenzotriazole (58.6 mg, 0.433 mmol), and N,N-diisopropylethylamine (112 mg, 0.867 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then partitioned between water (10.0 mL) and dichloromethane (10.0 mL × 3), and the organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by preparative HPLC to give N-(2,6-dimethoxybenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 1) (30.0 mg, 26%) as a white solid. LCMS: LC retention time 1.55 min. MS (ESI) m / z: 391.9 [M+H] + , Purity: 98.85% at 214nm. 1H NMR (500 MHz, DMSO) δ 12.63 (s, 1H), 11.08 (s, 1H), 8.02-8.00 (m, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 9 Hz, 1H), 7.32-7.28 (m, 1H), 7.06 (s, 1H), 6.72 (d, J = 8 Hz, 2H), 4.48 (s, 2H), 3.83 (s, 6H), 2.88(s, 3H) ppm.

[0450] Example 16: Synthesis of N-(2-fluoro-6-methoxybenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 2, see generally U.S. Patent No. 5,949,993) [ka] To a solution of 2-6 (70.0 mg, 0.289 mmol) in N,N-dimethylformamide (3.00 mL) was added (2-fluoro-6-methoxyphenyl)methanamine (53.8 mg, 0.347 mmol), N-(3-(dimethylamino)propyl)propanamide dihydrochloride (83.2 mg, 0.434 mmol), 1-hydroxybenzotriazole (58.6 mg, 0.434 mmol), and N,N-diisopropylethylamine (187 mg, 1.45 mmol). The reaction mixture was stirred at 25 °C for 18 hours. Water (6 mL) was then added to the reaction mixture. The reaction mixture was further extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with saturated sodium chloride solution (10 mL), dried over sodium sulfate, and filtered. The filtrate was concentrated. The residue was purified by preparative HPLC to give N-(2-fluoro-6-methoxybenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 2) (28.8 mg, 26.4%) as a white solid. LCMS: ESI (M+H) + 379.9, retention time 1.56 min, purity 98.7% at 254 nm. 1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 11.81 (s, 1H), 8.39 (t, J =5.0 Hz, 1H), 8.16-7.89 (m, 2H), 7.45-7.23 (m, 2H), 7.08 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.84 (t, J = 8.5 Hz, 1H), 4.51 (d, J = 5.0 Hz, 2H), 3.86 (s, 3H), 2.59 (s, 3H).

[0451] Example 17: Synthesis of N-(2-chloro-6-(oxetan-3-yloxy)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Agent 3, see generally U.S. Patent No. 5,949,993) [ka]

[0452] 2-chloro-6-(oxetan-3-yloxy)benzonitrile (17-2) [ka] To a solution of oxetan-3-ol (1.05 g, 0.0142 mol) in N,N-dimethylformamide (10 mL) was added NaH (0.62 g, 0.0258 mol). The reaction mixture was stirred at room temperature for 1 hour. Then, 2-chloro-6-fluorobenzonitrile (17-1) (2 g, 0.0129 mol) was added to the reaction. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then extracted with dichloromethane (50 mL × 3), washed with water (50 mL), and the organic layer was concentrated and purified by flash column chromatography (ethyl acetate in petroleum ether 0% → 10%) to give 2-chloro-6-(oxetan-3-yloxy)benzonitrile (17-2) (1.2 g, 44%). LCMS: LC retention time 1.58 min. MS (ESI) m / z: 209.9 [M+H] + .

[0453] (2-chloro-6-(oxetan-3-yloxy)phenyl)methanamine (17-3) [ka] A reaction mixture of 2-chloro-6-(oxetan-3-yloxy)benzonitrile (17-2) (1.2 g, 5.7 mmol), NH3·H2O (2 mL), and Raney Ni (0.03 g, 0.57 mmol) in tetrahydrofuran (50 mL) was stirred under H2 at room temperature for 16 h. The reaction mixture was filtered, concentrated in vacuo, and then purified by flash column chromatography (0% to 60% ethyl acetate in petroleum ether) to give (2-chloro-6-(oxetan-3-yloxy)phenyl)methanamine (17-3) (0.28 g, 23%). LCMS: LC retention time 1.33 min. MS (ESI) m / z: 213.9 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.19-7.16 (m, 1H), 7.05 (d, J = 8 Hz, 1H), 6.59 (d, J = 8 Hz, 1H), 5.33-5.29 (m, 1H), 4.94-4.91(m, 2H), 4.59-4.57 (m, 2H), 3.83 (s, 2H) ppm.

[0454] N-(2-chloro-6-(oxetan-3-yloxy)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Control 3) [ka] To a solution of (2-chloro-6-(oxetan-3-yloxy)phenyl)methanamine (17-3) (0.07 g, 0.33 mmol) in N,N-dimethylformamide (2 mL) was added 2-6 (0.07 g, 0.29 mmol), 1-hydroxybenzotriazole (0.058 g, 0.435 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (67 mg, 0.435 mmol), and N,N-diisopropylethylamine (0.012 g, 0.87 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction was extracted with dichloromethane (20 mL × 3) and washed with water (20 mL). The organic layers were combined, concentrated, and then purified by preparative HPLC to give N-(2-chloro-6-(oxetan-3-yloxy)benzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 3) (0.0258 g, 18%) as a white solid. LCMS: LC retention time 1.54 min. MS(ESI) m / z: 437.8 [M+H] + , Purity: 100% (254nm). 1 H NMR (400 MHz, DMSO) δ 11.83 (s, 1H), 8.35 (s, 1H), 7.99 (s, 1H), 7.97 (d, J=6Hz, 1H), 7.39(d, J = 8 Hz, 1H), 7.30-7.28 (m, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.09 (s, 1H), 6.68 (d, J = 8 Hz, 1H), 5.36-5.34 (m, 1H), 4.90-4.88(m 2H), 4.69 (s, 2H), 4.60-4.57(m, 2H), 2.58 (s, 3H).

[0455] Example 18: Synthesis of 6-(5-methyl-1H-pyrazol-4-yl)-N-(2-(oxetan-3-yloxy)benzyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Agent 4, see generally U.S. Patent No. 5,949,993) [ka]

[0456] (2-(oxetan-3-yloxy)phenyl)methanamine [ka] A reaction mixture of 2-chloro-6-(oxetan-3-yloxy)benzonitrile (18-1) (1.2 g, 5.7 mmol), NH3·H2O (2 mL), and Raney Ni (0.03 g, 0.57 mmol) in tetrahydrofuran (50 mL) was stirred under H2 at room temperature for 16 h. The reaction mixture was filtered, concentrated in vacuo, and then purified by flash chromatography (0% to 60% ethyl acetate in petroleum ether) to give (2-(oxetan-3-yloxy)phenyl)methanamine (18-2) (0.18 g, 15%). LCMS: LC retention time 0.55 min. MS (ESI) m / z: 180.1 [M+H] + , Purity: 100% (254nm). 1 H NMR (500 MHz, DMSO-d6) δ 7.37(d, J = 6.5 Hz, 1H), 7.14 (m, 1H), 6.94 (m, 1H), 6.54 (d, J =8 Hz, 1H), 5.28-5.26 (m, 1H), 4.94-4.91(m, 2H), 4.57-4.54(m, 2H), 3.72(s, 2H).

[0457] 6-(5-methyl-1H-pyrazol-4-yl)-N-(2-(oxetan-3-yloxy)benzyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Control 4) [ka] To a solution of 2-(oxetan-3-yloxy)phenyl)methanamine (18-2) (0.06 g, 0.33 mmol) in N,N-dimethylformamide (2 mL) was added 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-6) (0.07 g, 0.29 mmol), 1-hydroxybenzotriazole (0.058 g, 0.435 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.067 g, 0.435 mmol), and N,N-diisopropylethylamine (0.012 g, 0.87 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction was extracted with dichloromethane (20 mL x 3), washed with water (20 mL), concentrated, and purified by preparative HPLC to give 6-(5-methyl-1H-pyrazol-4-yl)-N-(2-(oxetan-3-yloxy)benzyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 4) (38.7 mg, 31.5%) as a white solid. LCMS: LC retention time 1.52 min. MS(ESI) m / z: 403.9 [M+H] + , Purity: 100% (254nm). 1 H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 8.78 (s, 1H), 8.03 (d, J = 8 Hz, 1H), 7.40 (d, J = 8 Hz, 1H), 7.29 (d, J = 1.2Hz, 1H), 7.14 (d, J = 1.2Hz, 1H), 6.96 (s, 1H), 6.63 (d, J = 7.6 Hz, 1H), 5.35-5.34 (m, 1H), 4.95 (m, 2H), 4.62-4.59 (m, 2H), 4.55 (d, J = 6 Hz, 2H), 2.60 (s, 3H).

[0458] Example 19: Synthesis of N-(4-cyano-5-ethoxy-2-fluorobenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Agent 5, see generally U.S. Patent No. 5,949,233) [ka]

[0459] 4-Bromo-2-ethoxy-5-fluorobenzonitrile (19-2) [ka] A mixture of 4-bromo-2,5-difluorobenzonitrile (19-1) (4.36 g, 20 mmol), K2CO3 (8.3 g, 60 mmol), and EtOH (9.4 g, 200 mmol) in N,N-dimethylformamide (70 mL) was stirred at 80 °C for 16 h. LCMS showed the reaction was complete, and the mixture was filtered and concentrated. The mixture was extracted with ethyl acetate (100 mL x 3) and washed with water (120 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified to give 19-2 as a yellow solid (3.9 g, 80%). LCMS: LC retention time 1.861 min. MS (ESI) m / z: 244 [M+H] + . Purity: 80% (214nm).

[0460] Methyl 4-cyano-5-ethoxy-2-fluorobenzoate (19-3) [ka] A mixture of 4-bromo-2-ethoxy-5-fluorobenzonitrile (19-2) (1 g, 4.1 mmol), Pd(OAc) (50 mg, catalytic), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (100 mg, catalytic), and EtN (4.1 g, 41 mmol) in methanol (20 mL) was stirred at 80 °C under carbon monoxide gas for 48 h. LCMS showed the reaction was complete, after which the mixture was concentrated, diluted with HO (60 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with water (60 mL) and subsequently dried over NaSO and filtered. The filtrate was concentrated, and the residue was purified by SGC to give 19-3 as a yellow solid (0.73 g, 80%). LCMS: LC retention time 1.761 min. MS(ESI)m / z:224[M+H]+ . Purity: 100% (214nm).

[0461] 2-Ethoxy-5-fluoro-4-(hydroxymethyl)benzonitrile (19-4) [ka] To a mixture of methyl 4-cyano-5-ethoxy-2-fluorobenzoate (19-3) (730 mg, 3.3 mmol) in methanol (20 mL) was added NaBH4 (371 mg, 9.9 mmol) at 0 °C. After the addition, the mixture was stirred at room temperature for 3 h. LCMS showed the reaction was complete, so the mixture was concentrated and the residue was purified by SGC to give 19-4 as a yellow solid (0.60 g, 94%). LCMS: LC retention time 1.58 min. MS (ESI) m / z: 196 [M+H] + . Purity: 95% (214nm).

[0462] 2-Ethoxy-5-fluoro-4-(hydroxymethyl)benzonitrile (19-5) [ka] To a mixture of 2-ethoxy-5-fluoro-4-(hydroxymethyl)benzonitrile (19-4) (600 mg, 3.1 mmol), isoindoline-1,3-dione (456 mg, 3.1 mmol), and PPh3 (975 mg, 3.72 mmol) in tetrahydrofuran (20 mL) was added DIAD (750 mg, 3.72 mmol) under an argon atmosphere at 0 °C. After the addition, the mixture was stirred at room temperature for 12 h. LCMS showed the reaction was complete, and the mixture was diluted with ethyl acetate (80 mL) and washed with water (50 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified by SGC to give 19-5 as a yellow solid (0.8 g, 80%). LCMS: LC retention time 1.842 min. MS (ESI) m / z: 325 [M+H] + . Purity: 75% (214nm).

[0463] 4-(aminomethyl)-2-ethoxy-5-fluorobenzonitrile (19-6) [ka] A mixture of 4-((1,3-dioxoisoindolin-2-yl)methyl)-2-ethoxy-5-fluorobenzonitrile (19-5) (0.8 g, 2.46 mmol), N2H4·H2O (200 mg, 5 mmol) in ethanol (12 mL) was stirred at 70 °C for 3 h. LCMS showed the reaction was complete, the mixture was concentrated, and the residue was purified by preparative HPLC to give 19-6 as a white solid (300 mg, 63%). LCMS: LC retention time 1.04 min. MS (ESI) m / z: 195 [M+H] + . Purity: 95% (214nm).

[0464] N-(4-cyano-5-ethoxy-2-fluorobenzyl)-6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Control 5) [ka] A mixture of 4-(aminomethyl)-2-ethoxy-5-fluorobenzonitrile (19-6) (56 mg, 0.29 mmol), 6-(5-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (2-6) (70 mg, 0.29 mmol), EDCI (84 mg, 0.44 mmol), HOBt (54 mg, 0.44 mmol), and DIPEA (187 mg, 1.5 mmol) in N,N-dimethylformamide (2 mL) was stirred at room temperature for 12 hours. LCMS showed the reaction was complete, and the mixture was extracted with dichloromethane (20 mL x 3), washed with water (20 mL), concentrated, and purified by preparative HPLC to give reference drug 5 (33 mg, 27%) as a white solid. LCMS: LC retention time 1.523 min. MS(ESI)m / z:419[M+H] + . Purity: 100% (214nm). 1H NMR (500 MHz, DMSO-d6) δ 12.70 (s, 1H), 11.90 (s, 1H), 8.99 (t, J = 6.0 Hz, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 4.58 (d, J = 5.5 Hz, 2H), 4.14 (q, J = 7.0 Hz, 2H), 2.59 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H) ppm.

[0465] Example 20: Synthesis of N-(3-methoxybenzyl)-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 6, see Patent Document 1) [ka]

[0466] 6-chloro-N-(3-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (20-1) [ka] A solution of 6-chloro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (1 g, 5.087 mmol, 1 equiv.) in N,N-dimethylformamide (9 mL) was treated with HATU (2.90 g, 7.630 mmol, 1.5 equiv.) at room temperature for 1.5 hours, followed by the addition of 1-(3-methoxyphenyl)methanamine (1.05 g, 7.630 mmol, 1.5 equiv.) and triethylamine (1.03 g, 10.174 mmol, 2 equiv.) at 0° C. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (0% to 10%) to give the crude product. This was purified by trituration with HO (10 mL) to give 6-chloro-N-[(3-methoxyphenyl)methyl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (20-1) (420 mg, 26.15%) as a yellow solid. LC-MS (ESI, m / z): 316 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 9.12 (t, J = 6.0 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 7.22 (d, J = 2.1 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 6.94-6.89 (m, 2H), 6.83 (dd, J = 9.0, 2.4 Hz, 1H), 4.49 (d, J = 6.0 Hz, 2H), 3.74 (s, 3H).

[0467] N-(3-Methoxybenzyl)-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Control 6) [ka] To a stirred mixture of 6-chloro-N-[(3-methoxyphenyl)methyl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (20-1) (400 mg, 1.267 mmol, 1 equiv.) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (737.42 mg, 3.801 mmol, 3 equiv.) in N,N-dimethylformamide / HO (4 mL / 0.8 mL), Pd(PPh) (146.39 mg, 0.127 mmol, 0.1 equiv.) and KCO (350.15 mg, 2.534 mmol, 2 equiv.) were added at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane / methanol (0% to 10%) to give the crude product, which was purified by trituration with methanol (10 mL) to give N-[(3-methoxyphenyl)methyl]-6-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (Reference Drug 6) (186.2 mg, 42.14%) as a white solid, contaminated with approximately 2% methanol residue. LC-MS (ESI, m / z): 348 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 11.94 (s, 1H), 8.92 (t, J = 6.0 Hz, 1H), 8.30 (s, 1H), 8.06 (s, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 7.12 (d, J = 2.1 Hz, 1H), 6.95-6.89 (m, 2H), 6.85-6.79 (m, 1H), 4.48 (d, J = 5.9 Hz, 2H), 3.74 (s, 3H).

[0468] Example 21: Synthesis of N-(3,4-difluorobenzyl)-6-(1H-pyrazol-4-yl)-1H-indole-3-carboxamide (Reference Drug 7, see Patent Document 1) [ka]

[0469] 6-Bromo-N-(3,4-difluorobenzyl)-1H-indole-3-carboxamide (21-1) [ka] A solution of 6-bromo-1H-indole-3-carboxylic acid (1 g, 4.166 mmol, 1 equiv.) in N,N-dimethylformamide (9 mL) was treated with HATU (2.38 g, 6.249 mmol, 1.5 equiv.) at room temperature for 30 minutes, followed by the addition of 1-(3,4-difluorophenyl)methanamine (0.89 g, 6.249 mmol, 1.5 equiv.) and triethylamine (1.26 g, 12.498 mmol, 3 equiv.) at 0° C. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (0% to 10%) to give the crude product. This was further purified by trituration with HO (10 mL) to give 6-bromo-N-[(3,4-difluorophenyl)methyl]-1H-indole-3-carboxamide (400 mg, 26.29%) as a yellow solid. LC-MS (ESI, m / z): 365, 367 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.56 (t, J = 6.0 Hz, 1H), 8.11-8.06 (m, 2H), 7.64 (s, 1H), 7.50-7.31 (m, 2H), 7.24 (dd, J = 8.6, 1.8 Hz, 1H), 7.22-7.15 (m, 1H), 4.45 (d, J = 6.0 Hz, 2H).

[0470] N-(3,4-difluorobenzyl)-6-(1H-pyrazol-4-yl)-1H-indole-3-carboxamide (control drug 7) [ka] To a stirred mixture of 6-bromo-N-[(3,4-difluorophenyl)methyl]-1H-indole-3-carboxamide (370 mg, 1.013 mmol, 1 equiv.) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (589.81 mg, 3.039 mmol, 3 equiv.) in N,N-dimethylformamide / HO (4 mL / 0.8 mL), Pd(PPh) (117.08 mg, 0.101 mmol, 0.1 equiv.) and KCO (280.06 mg, 2.026 mmol, 2 equiv.) were added at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 16 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (0%→10%) to give the crude product, which was further purified by trituration with methanol (10 mL) to give N-[(3,4-difluorophenyl)methyl]-6-(1H-pyrazol-4-yl)-1H-indole-3-carboxamide (236.4 mg, 65.42%) as a white solid. LC-MS (ESI, m / z): 353 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 11.52 (d, J = 3.0 Hz, 1H), 8.48 (t, J = 6.1 Hz, 1H), 8.23-7.85 (m, 4H), 7.60 (d, J = 1.5 Hz, 1H), 7.46-7.33 (m, 3H), 7.26-7.13 (m, 1H), 4.46 (d, J = 6.0 Hz, 2H).

[0471] Example 22: Synthesis of 6-(1H-pyrazol-4-yl)-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (Reference Drug 8, see Patent Document 1) [ka]

[0472] 6-chloro-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (22-1) [ka] A solution of 6-chloro-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (1 g, 5.087 mmol, 1 equiv.) in N,N-dimethylformamide (10 mL) was treated with HATU (2.90 g, 7.630 mmol, 1.5 equiv.) at room temperature for 30 minutes, followed by the addition of 4-pyridinemethanamine (0.83 g, 7.630 mmol, 1.5 equiv.) and triethylamine (1.03 g, 10.174 mmol, 2 equiv.) at 0° C. The resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (0% to 10%) to give the crude product. This was purified by trituration with water (15 mL) to give 6-chloro-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (22-1) (430 mg, 29.48%) as a yellow solid. LC-MS (ESI, m / z): 287 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 8.75 (t, J = 6.1 Hz, 1H), 8.54-8.49 (m, 2H), 8.46 (d, J = 8.2 Hz, 1H), 8.25 (s, 1H), 7.33 (d, J = 5.0 Hz, 2H), 7.26 (d, J = 8.2 Hz, 1H), 4.51 (d, J = 5.9 Hz, 2H).

[0473] 6-(1H-pyrazol-4-yl)-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (control drug 8) [ka] To a stirred mixture of 6-chloro-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (400 mg, 1.395 mmol, 1 equiv.) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (812.11 mg, 4.185 mmol, 3 equiv.) in N,N-dimethylformamide / HO (4 mL / 0.8 mL), KCO (385.62 mg, 2.790 mmol, 2 equiv.) and Pd(PPh) (161.22 mg, 0.140 mmol, 0.1 equiv.) were added at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane / methanol (10%→30%) to give a crude product. This was further purified by trituration with acetonitrile (10 mL) to give 6-(1H-pyrazol-4-yl)-N-(pyridin-4-ylmethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (220.0 mg, 49.54%) as a white solid. LC-MS (ESI, m / z): 319 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 12.06 (d, J = 2.9 Hz, 1H), 8.65 (t, J = 6.0 Hz, 1H), 8.56-8.47 (m, 2H), 8.47-8.00 (m, 4H), 7.54 (d, J = 8.3 Hz, 1H), 7.40-7.27 (m, 2H), 4.51 (d, J = 5.9 Hz, 2H).

[0474] Example 23: Exemplary compounds of the present invention

[0475] TIFF2026502949000148.tif233170TIFF2026502949000149.tif198170

[0476] TIFF2026502949000150.tif252170

[0477] Example 24: ROCK1, ROCK2, PRKX, and PKA inhibition assays Compound powders were dissolved in dimethyl sulfoxide to make 10 mM stocks. Compounds were serially diluted 3-fold starting from 1 μM to give 10 doses of IC 50 The control compound, staurosporine, was tested in triplicate in the IC2000 mode at 10 doses in 4-fold serial dilutions starting from 20 μM. 50 Tested in mode. 33 Reactions were performed at 10 μM ATP for each test enzyme using the HotSpot kit with P-ATP. The activity ratios relative to the DMSO control at each concentration were then curve-fitted using GraphPad Prism to determine the IC. 50 The IC was determined. Curve fitting was performed when the enzyme activity at the highest compound concentration was less than 65%. 50 Values ​​below 50.8 pM or above 1 μM are estimated based on the best available curve fit. The data obtained for the test compounds are shown in Tables 2A and 2B below. The data for Compound 1 in Table 2B represent IC values ​​obtained from multiple enzyme inhibition experiments. 50 The data are geometric means. Each experiment was performed in the same laboratory using this procedure.

[0478] TIFF2026502949000151.tif100170

[0479] TIFF2026502949000152.tif116170

[0480] Example 25: MDCK-MDR1 efflux assay Experimental Procedure 1: MDCK-MDR1 cells grown in tissue culture flasks were trypsinized, suspended in medium, and the suspension was dispensed into wells of a Millipore 96-well plate. Cells were allowed to grow and differentiate for 5 days with feeding every 2 days.

[0481] For apical-to-basolateral (A→B) permeability, test substances were added to the apical (A) side, and the amount of permeation was measured at the basolateral (B) side. For basolateral-to-apical (B→A) permeability, test substances were added to the B side, and the amount of permeation was measured at the A side. The buffer on the A side contained 100 μM Lucifer Yellow dye in transport buffer (10 mM HEPES, 1.98 g / L glucose in 1x Hank's balanced salt solution) (pH 7.4), and the buffer on the B side was transport buffer (pH 7.4). MDCK-MDR1 cells were incubated with the test substances in these buffers for 2 h. At the end of the assay, donor and receiver solution samples were collected, quenched with 100% methanol containing an internal standard, and centrifuged at 5000 rpm at 4 °C for 10 min. After centrifugation, the supernatants from the donor and receiver samples were analyzed by LC-MS / MS.

[0482] Data Analysis: Data were expressed as permeability (Papp): P app =(dQ / dt) / (C0A) where dQ / dt is the permeation rate, C0 is the initial concentration of the test substance, and A is the monolayer area.

[0483] In the bidirectional permeability test, the discharge ratio (R e ) was calculated: R e =[P app (B→A)] / [P app (A → B)] R e >2 indicates a potential substrate for the P-gp efflux transporter.

[0484] Detailed data are shown in Table 3A.

[0485] TIFF2026502949000153.tif84170

[0486] Experimental Procedure 2: 1. Preparation of MDCKII-MDR1 Cells Cell culture medium (50 μL and 25 mL) was added to each well of the Transwell insert and reservoir, respectively. The HTS Transwell plate was then incubated at 37°C and 5% CO for 1 hour, after which cells were seeded. MDCKII-MDR1 cells were then cultured in culture medium at a density of 1.56 × 10 6 The cells were diluted to 1000 cells / mL, and 50 μL of the cell suspension was dispensed into filter wells of a 96-well HTS Transwell plate. Cells were cultured for 4 to 8 days in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity. The cell culture medium was changed every other day, starting within 24 hours after initial plating.

[0487] 2. Preparation of Stock Solutions Stock solutions (10 mM) of test compounds were prepared in DMSO. Stock solutions of positive controls were prepared in DMSO at a concentration of 10 mM. Metoprolol, digoxin, and fasudil were used as control compounds in this assay.

[0488] 3. Assessment of Cell Monolayer Integrity The medium was removed from the reservoir and each Transwell insert and replaced with fresh prewarmed culture medium. The transepithelial electrical resistance (TEER) across the monolayer was then measured using a Millicell Epithelial Volt-Ohm Measurement System (Millipore, USA). After measurement, the plate was returned to the incubator.

[0489] The TEER value was calculated according to the following formula: TEER measurement value (Ω) × membrane area (cm 2 ) = TEER value (Ω cm 2 ) TEER value is 42 Ω·cm 2The MDCKII-MDR1 monolayer was found to be sufficiently suitable.

[0490] 4. Assay Procedure The MDCKII-MDR1 plate was removed from the incubator, washed twice with preheated HBSS (10 mM HEPES, pH 7.4), and then incubated at 37°C for 30 minutes. Stock solutions of control and test compounds were diluted with DMSO to 200 μM solutions and further diluted with HBSS (10 mM HEPES, pH 7.4) to make 1 μM standard solutions. The final concentration of DMSO in the incubation system was 0.5%.

[0491] The rate of drug transport from the apical to the basolateral direction was determined. 125 μL of standard solution was added to the Transwell insert (apical compartment), and 50 μL of sample was immediately transferred from the apical compartment to 200 μL of acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 100 nM tolbutamide) in a new 96-well plate, which served as the initial donor sample (AB). The sample was vortexed at 1000 rpm for 10 minutes. The wells of the receiver plate (basolateral compartment) were filled with 235 μL of transport buffer.

[0492] The rate of drug transport from the basolateral to the apical direction was determined. 285 μL of standard solution was added to the receiver plate well (basolateral compartment), and 50 μL of sample was immediately transferred from the basolateral compartment to 200 μL of acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 100 nM tolbutamide) in a new 96-well plate, which served as the initial donor sample (BA). The sample was vortexed at 1000 rpm for 10 minutes. The Transwell insert (apical compartment) was filled with 75 μL of transport buffer. Apical-to-basolateral and basolateral-to-apical transport were performed simultaneously.

[0493] The plate was then incubated at 37°C for 2 hours. After the incubation, 50 μL of sample was transferred from the donor side (apical compartment of Ap→B1 flow and basolateral compartment of B1→Ap flow) and the receiver side (basolateral compartment of Ap→B1 flow and apical compartment of B1→Ap flow) to a new 96-well plate. Four volumes of acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 100 nM tolbutamide) were then added. The sample was vortexed for 10 minutes and centrifuged at 3220 g for 40 minutes. A 100 μL aliquot of the supernatant was mixed with an appropriate amount of ultrapure water and subjected to LC-MS / MS analysis.

[0494] To determine Lucifer Yellow leakage after a 2-hour transport period, a Lucifer Yellow stock solution was prepared in DMSO and diluted with HBSS (10 mM HEPES, pH 7.4) to a final concentration of 100 μM. Next, 100 μL of Lucifer Yellow solution was added to each Transwell insert (apical compartment), followed by filling the wells of the receiver plate (basolateral compartment) with 300 μL of HBSS (10 mM HEPES, pH 7.4). The plate was incubated at 37°C for 30 minutes. Afterwards, 80 μL samples were taken directly from the apical and basolateral wells (using the basolateral access hole) and transferred to wells of a new 96-well plate. Lucifer Yellow fluorescence was used to monitor monolayer integrity, and the signal was measured in a fluorescence plate reader at excitation 480 nm and emission 530 nm.

[0495] Detailed data are shown in Table 3B.

[0496] TIFF2026502949000154.tif79170

[0497] Example 26: hERG K d Compounds were dissolved in DMSO to a stock concentration of 10 mM and serially diluted 3-fold from 100 μM to obtain 10 doses of IC 50The control compound E-4031 was tested in triplicate in the IC2000 / 2001 mode. 50 The assay was tested in a competitive binding mode, based on the binding of a fluorescently labeled tracer to a membrane preparation containing 1× Predictor™ hERG membrane and 1 nM Predictor™ hERG Tracer Red in a buffer solution containing 25 mM HEPES (pH 7.5), 15 mM KCl, 1 mM MgCl, 0.05% PF-127, and 1% DMSO.

[0498] Compounds in DMSO were added to the membrane mixture using sonication, and the tracer was added and mixed gently in the dark. Fluorescence was measured after 4 hours of incubation at room temperature. Measurement parameters were as follows: Ex = 531 nm FP and Em = 595 nm P and S.

[0499] Curve fitting was performed using GraphPad Prism software where the activity at the highest compound concentration was less than 65%. The background was established by the average FP signal in the presence of 10 μM E-4031. The data are presented in Table 4 below.

[0500] TIFF2026502949000155.tif24170

[0501] Example 27: Mouse, human and rat liver microsomes Master solutions were prepared according to Table 5 using microsomes of the appropriate species.

[0502] TIFF2026502949000156.tif21170

[0503] Two separate experiments were performed as follows: a) Cofactor (NADPH): 25 μL of 10 mM NADPH was added to the incubations. The final concentrations of microsomes and NADPH were 0.5 mg / mL and 1 mM, respectively. b) No cofactor (NADPH): 25 μL of 100 mM phosphate buffer was added to the incubations. The final concentration of microsomes was 0.5 mg / mL. The mixture was preheated to 37°C for 10 minutes.

[0504] The reaction was initiated by adding 2.5 μL of 100 μM control or test compound solution. Verapamil was used as a positive control in this study. The final concentration of the test or control compound was 1 μM. The incubation solution was incubated in a water bath at 37°C.

[0505] Four 30 μL aliquots were taken from the reaction solution at 0.5, 5, 15, 30, and 60 minutes. The reaction was stopped by adding five volumes of cold acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutamide). The samples were centrifuged at 3220 g for 40 minutes. A 100 μL aliquot of the supernatant was mixed with 100 μL of ultrapure H2O and then used for LC-MS / MS analysis. All calculations were performed using Microsoft Excel. Peak areas were determined from extracted ion chromatograms. The slope value k was determined by linear regression of the natural logarithm of the curve of the parent drug remaining fraction versus incubation time. The in vitro half-life (in vitro t 1 / 2 ) was determined from the slope value: in vitro t 1 / 2 =-(0.693 / k) in vitro t 1 / 2 (min) was used as the in vitro intrinsic clearance (in vitro C Lint The conversion to μL / min / mg protein was performed using the following formula (average of duplicate measurements): In vitro C Lint =0.693 / (t 1 / 2 ) × ((Volume of incubation solution (μL)) / (Amount of protein (mg))

[0506] The data obtained is shown in Table 6 below.

[0507] TIFF2026502949000157.tif84170

[0508] Example 28: Dynamic Solubility Stock solutions of test compounds and the control compound progesterone were prepared at 10 mM in DMSO. 15 μL of each sample stock solution (10 mM) was dispensed sequentially into the appropriate 96-well rack. 485 μL of PBS (pH 7.4) was added to each vial in the uncapped solubility sample plate. Assays were performed in duplicate. Each vial was fitted with a stir bar and sealed using a molded PTFE / silicone plug. The solubility sample plate was then transferred to an Eppendorf Thermomixer Comfort plate shaker and shaken at 1100 rpm at 25°C for 2 hours. After the 2-hour period, the plugs were removed, the stir bars were removed using a large magnet, and the samples were transferred from the solubility sample plate to a filter plate. All samples were filtered using a vacuum manifold. A 5 μL aliquot of DMSO was removed from the filtrate, followed by the addition of 490 μL of a 1:1 mixture of HO and acetonitrile containing the internal standard. Ultrapure water was used to dilute the dilution solution depending on the peak shape, and the dilution factor was changed depending on the solubility value and the signal response of LC-MS.

[0509] From the 10 mM DMSO STD plate, 6 μL was transferred to the remaining empty plate, and then 194 μL of DMSO was added to that plate to achieve a 300 μM STD concentration. From the 300 μM DMSO STD plate, 5 μL of DMSO STD and 5 μL of PBS (pH 7.4) were transferred to the remaining empty plate, followed by the addition of 490 μL of a 1:1 mixture of H2O and acetonitrile containing the internal standard to that plate to achieve a final STD concentration of 3 μM. The dilution solution was diluted with a fixed proportion of ultrapure water depending on the peak shape. The concentration of the standard sample was varied depending on the LC-MS signal response. The plate was loaded into a well-plate autosampler. The samples were evaluated by LC-MS / MS analysis.

[0510] All calculations were performed using Microsoft Excel. The filtrates were analyzed and quantified using LC in conjunction with mass spectral peak identification and quantification by comparison to standards of known concentration. Solubility values ​​for test and control compounds were calculated as follows:

number

[0511] The data obtained is shown in Table 7 below.

[0512] TIFF2026502949000159.tif84170

[0513] Example 29: 24-hour pharmacokinetic profile in mice The pharmacokinetics of the compound was evaluated by the following method when it was repeatedly administered orally at 20 mg / kg to B6SJLF1 / J mice over a 24-hour period.

[0514] TIFF2026502949000160.tif50170

[0515] TIFF2026502949000161.tif52170

[0516] Feed and water were provided ad libitum unless otherwise noted. Samples were analyzed by the manufacturer for concentrations of selected heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. Litter was also analyzed by the manufacturer prior to certification to ensure acceptable levels of heavy metals, aflatoxins, bacteria, yeast, mold, and organophosphates were met. No contaminants were found in the feed, water, or litter at levels that would affect the achievement of the study objectives.

[0517] TIFF2026502949000162.tif66170

[0518] TIFF2026502949000163.tif195170

[0519] PO (oral) administration: Groups 2, 3, 4, 6, and 8: The test substances were dissolved in a minimum amount of DMSO (Groups 2, 4, 6, and 8: 40 μL–50 μL; Group 3: 100 μL), and 0.5% methylcellulose was added to the final volume, after which the solution was subjected to sonication for 2 min.

[0520] Group 7: Test substances were dissolved in 100 μL of DMSO, saline was added to the final volume, and then the solution was subjected to sonication for 2 minutes.

[0521] IV (intravenous) administration: Group 1 and Group 5: Test substances were dissolved in NMP (N-methylpyrrolidone) to a final concentration of 5% NMP. Solutol HS-15 was added to a final concentration of 5% Solutol HS-15, and PEG-400 was added to a final concentration of 30% PEG-400. The solution was then diluted to the final volume with saline.

[0522] Administration Group 1, Group 5: Test substance was administered as a single slow bolus intravenous injection into the tail vein using a 27G needle.

[0523] Groups 2, 4, 6, 7, and 8: The test substance was administered as a single dose directly into the stomach by oral gavage.

[0524] Group 3: Due to the volume administered, the test substance was administered as two single doses with a break of approximately 1-2 minutes between each dose.

[0525] sampling Blood and cerebrospinal fluid (CSF) samples were collected as shown in Table 12.

[0526] TIFF2026502949000164.tif38170

[0527] Approximately 150 μL of blood was collected from each mouse by tail nick at the first time point, followed by a maximum volume by cardiac puncture at the second (terminal) time point. Plasma was separated by spinning within 30 minutes of collection and stored frozen at -20°C until shipment.

[0528] CSF was collected prior to terminal blood collection. Animals were fully anesthetized with a ketamine / xylazine cocktail, and CSF was collected by making an incision through the occipital skin and skull, separating the muscles at the back of the neck to access the cisterna magna, and inserting a glass microneedle with an angled tip. The glass microneedle was connected via polyethylene tubing to a syringe placed on a foot-pedal-operated syringe pump. The syringe applied controlled negative pressure to aspirate CSF. Approximately 5 μL to 12 μL of CSF was collected from anesthetized mice prior to cardiac puncture for plasma sampling. The data obtained are shown in Tables 13, 14, 15, 16, and 17 below.

[0529] TIFF2026502949000165.tif92170

[0530] TIFF2026502949000166.tif29170

[0531] TIFF2026502949000167.tif105170

[0532] TIFF2026502949000168.tif29170

[0533] Example 30: 7-day mouse pharmacokinetics summary The pharmacokinetics of Compound 1 was evaluated in B6SJLF1 / J mice following repeated oral administration of 20 mg / kg over a 7-day period. Parameters evaluated included mortality, cageside observations, physical examination, and body weight over the 7-day period. Blood and cerebrospinal fluid (CSF) samples were collected at the designated time points, and plasma and CSF samples were submitted to KCAS Bioanalytical Services for analysis.

[0534] TIFF2026502949000169.tif43170

[0535] Animals were weighed upon arrival and allowed to acclimate to the laboratory environment for at least 24 hours before the first administration. During this period, animals were identified with a permanent marker according to Xyzagen's procedures, recorded, and used throughout the study. Prior to the start of the in-life phase, each animal underwent a physical examination and was weighed prior to administration of the test substance.

[0536] TIFF2026502949000170.tif51170

[0537] Feed and water were provided ad libitum unless otherwise noted. Samples were analyzed by the manufacturer for concentrations of certain heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. Litter was also analyzed by the manufacturer prior to certification to ensure acceptable levels of heavy metals, aflatoxins, bacteria, yeast, mold, and organophosphates were met. No contaminants were found in the feed, water, or litter at levels that would affect the achievement of the study objectives.

[0538] TIFF2026502949000171.tif60170

[0539] Dosage Formulation and Administration On the day of administration, Compound 1 was dissolved in a minimal amount of DMSO (50 μL per 3.0 mg of Compound 1) and then mixed with a 0.5% (w / v) aqueous methylcellulose vehicle. The resulting formulation was vortexed and sonicated for 3 to 5 minutes to ensure homogeneity. The daily formulation was also vortexed throughout the administration procedure.

[0540] TIFF2026502949000172.tif70170

[0541] Animals were dosed once daily by oral gavage at the designated dose volume, which was based on the most recently recorded body weight.

[0542] sampling Blood and CSF samples were collected as outlined in the table below.

[0543] TIFF2026502949000173.tif43170

[0544] TIFF2026502949000174.tif199170

[0545] Blood was collected via tail incision at the time points specified above. A total volume of 100-150 μl was collected at the pre-dose time point, followed by a maximum volume via cardiac puncture at the final post-dose time point. Blood was collected into K2EDTA tubes and kept on wet ice until processing. Within 30 minutes of collection, blood was spun to separate the plasma.

[0546] CSF was collected from Groups 1 and 2 (animals 31–35) before terminal blood collection. Animals were fully anesthetized with a ketamine / xylazine cocktail, and CSF was collected by making an incision through the occipital skin and skull, separating the muscles at the back of the neck to access the cisterna magna, and inserting a glass microneedle with an angled tip. The glass microneedle was connected via polyethylene tubing to a syringe placed on a foot-pedal-operated syringe pump. The syringe applied controlled negative pressure to aspirate CSF. Approximately 5–12 μL of CSF was collected from anesthetized mice before cardiac puncture for plasma samples.

[0547] Plasma and CSF samples were stored at -20°C. No animals died or experienced adverse events in the 20 mg / kg oral (PO) once daily (QD) study. No accumulation was observed, with mean CSF levels at 1 hour post-dose of 183 ng / ml and 105 ng / ml on days 1 and 7, respectively. Plasma dosing was found to be below the limit of detection at 24 hours.

[0548] Example 31: 24-hour rat pharmacokinetics summary A pharmacokinetic (PK) study was conducted in rats using a procedure similar to that described in Example 30.

[0549] Male Sprague-Dawley rats, 7-9 weeks of age on average and weighing approximately 200-300 g, were divided into groups of three. Rats were then administered the test substance either intravenously at a dose of 3 mg / kg or orally at a dose of 10 mg / kg. The intravenous test substance was formulated in saline with 20% DMA, 20% PEG 400, and 20% Kolliphor HS15. The oral test substance was formulated in an aqueous solution of 1% DMSO and 0.5% methylcellulose. The intravenous solution was prepared at a concentration of 1.5 mg / mL, and the oral solution was prepared at a concentration of 1 mg / mL. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours.

[0550] The data obtained is presented in the table below.

[0551] TIFF2026502949000175.tif65170

[0552] TIFF2026502949000176.tif65170

[0553] TIFF2026502949000177.tif65170

[0554] TIFF2026502949000178.tif73170

[0555] TIFF2026502949000179.tif47170

[0556] TIFF2026502949000180.tif64170

[0557] TIFF2026502949000181.tif65170

[0558] TIFF2026502949000182.tif65170

[0559] All publications and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0560] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the invention, as defined in the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed within the scope of this application.

Claims

1. formula: 【Chemistry 1】 (In the formula, R １ and R ２ are independently hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ selected from haloalkyl and halogen; R ３ is hydrogen, C １ ~C ４ Alkyl, and C １ ~C ４ haloalkyl; R ４ and R ５ are independently hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ selected from haloalkyl and halogen; R ６ is hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ Haloalkyl, halogen, C １ ~C ４ Alkyl-OR ７ , and OR ７ is selected from R ７ is hydrogen, C １ ~C ４ Alkyl, and C １ ~C ４ haloalkyl) or a pharmaceutically acceptable salt thereof.

2. The compound has the formula: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. The compound has the formula: 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

4. R ５ The compound according to any one of claims 1 to 3, wherein is hydrogen.

5. R ５ The compound according to any one of claims 1 to 3, wherein is a halogen.

6. R ５ The compound of any one of claims 1 to 3, wherein is fluoro.

7. R ５ The compound of any one of claims 1 to 3, wherein is chloro.

8. R ５ The compound according to any one of claims 1 to 3, wherein is methyl.

9. R ５ The compound according to any one of claims 1 to 3, wherein is ethyl.

10. The compound has the formula: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

11. The compound has the formula: 【Transformation 5】 or a pharmaceutically acceptable salt thereof.

12. R ６ 12. The compound of claim 10 or 11, wherein is hydrogen.

13. R ６ 12. The compound of claim 10 or 11, wherein is OMe.

14. R ６ 12. The compound of claim 10 or 11, wherein is halogen.

15. R ６ 12. The compound of claim 10 or 11, wherein is fluoro.

16. R ６ 12. The compound of claim 10 or 11, wherein is chloro.

17. R ６ 12. The compound of claim 10 or 11, wherein is methyl.

18. R ６ 12. The compound of claim 10 or 11, wherein is ethyl.

19. The compound has the formula: 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

20. The compound has the formula: 【Transformation 7】 or a pharmaceutically acceptable salt thereof.

21. R ７ 21. The compound of claim 19 or 20, wherein is methyl.

22. R ７ 21. The compound of claim 19 or 20, wherein is hydrogen.

23. R １ The compound of any one of claims 1 to 22, wherein is hydrogen.

24. R １ The compound of any one of claims 1 to 22, wherein is halogen.

25. R １ The compound of any one of claims 1 to 22, wherein is methyl.

26. R ２ The compound of any one of claims 1 to 25, wherein is hydrogen.

27. R ２ The compound of any one of claims 1 to 25, wherein is halogen.

28. R ２ The compound of any one of claims 1 to 25, wherein is methyl.

29. R ２ is C １ ~C ２ The compound of any one of claims 1 to 25, which is haloalkyl.

30. R ３ The compound of any one of claims 1 to 29, wherein is hydrogen.

31. R ３ The compound of any one of claims 1 to 29, wherein is methyl.

32. R ４ The compound of any one of claims 1 to 31, wherein is hydrogen.

33. R ４ The compound of any one of claims 1 to 31, wherein is halogen.

34. R ４ The compound of any one of claims 1 to 31, wherein is fluoro.

35. R ４ The compound of any one of claims 1 to 31, wherein is chloro.

36. R ４ is C １ ~C ２ The compound of any one of claims 1 to 31, which is haloalkyl.

37. structure: 【Transformation 8】 or a pharmaceutically acceptable salt thereof.

38. structure: 【Chemistry 9】 or a pharmaceutically acceptable salt thereof.

39. structure: 【Chemistry 10】 or a pharmaceutically acceptable salt thereof.

40. structure: 【Chemistry 11】 or a pharmaceutically acceptable salt thereof.

41. structure: 【Chemistry 12】 or a pharmaceutically acceptable salt thereof.

42. structure: 【Chemistry 13】 or a pharmaceutically acceptable salt thereof.

43. structure: 【Chemistry 14】 or a pharmaceutically acceptable salt thereof.

44. structure: 【Chemistry 15】 or a pharmaceutically acceptable salt thereof.

45. structure: 【Chemistry 16】 or a pharmaceutically acceptable salt thereof.

46. structure: 【Chemistry 17】 or a pharmaceutically acceptable salt thereof.

47. structure: [Chemistry 18] or a pharmaceutically acceptable salt thereof.

48. structure: 【Chemistry 19】 or a pharmaceutically acceptable salt thereof.

49. structure: 【Chemistry 20】 or a pharmaceutically acceptable salt thereof.

50. structure: 【Chemistry 21】 or a pharmaceutically acceptable salt thereof.

51. formula: 【Chemistry 22】 (In the formula, R １ and R ２ are independently hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ selected from haloalkyl and halogen; R ４ and R ５ are independently hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ haloalkyl, and halogen or a pharmaceutically acceptable salt thereof.

52. R ２ 52. The compound of claim 51, wherein is hydrogen.

53. R ２ 52. The compound of claim 51 , wherein is F.

54. R ２ 52. The compound of claim 51, wherein is Cl.

55. R ２ 52. The compound of claim 51, wherein is methyl.

56. R ２ is C １ ~C ２ 52. The compound of claim 51, which is haloalkyl.

57. formula: 【Chemistry 23】 (In the formula, R １ is hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ selected from haloalkyl and halogen; R ４ and R ５ are independently hydrogen, C １ ~C ４ Alkyl, C １ ~C ４ haloalkyl, and halogen or a pharmaceutically acceptable salt thereof.

58. R １ 58. The compound of any one of claims 51 to 57, wherein is hydrogen.

59. R １ The compound of any one of claims 51 to 57, wherein is F.

60. R １ The compound of any one of claims 51 to 57, wherein is Cl.

61. R １ 58. The compound of any one of claims 51 to 57, wherein is methyl.

62. R １ is C １ ~C ２ 58. The compound of any one of claims 51 to 57, which is haloalkyl.

63. R ４ 63. The compound of any one of claims 51 to 62, wherein is hydrogen.

64. R ４ 63. The compound of any one of claims 51 to 62, wherein is halogen.

65. R ４ 63. The compound of any one of claims 51 to 62, wherein is fluoro.

66. R ４ 63. The compound of any one of claims 51 to 62, wherein is chloro.

67. R ４ is C １ ~C ２ 63. The compound of any one of claims 51 to 62, which is haloalkyl.

68. R ５ 68. The compound of any one of claims 51 to 67, wherein is hydrogen.

69. R ５ 68. The compound of any one of claims 51 to 67, wherein is halogen.

70. R ５ 68. The compound of any one of claims 51 to 67, wherein is fluoro.

71. R ５ 68. The compound of any one of claims 51 to 67, wherein is chloro.

72. R ５ is C １ ~C ２ 68. The compound of any one of claims 51 to 67, which is haloalkyl.

73. 73. A pharmaceutical composition comprising a compound according to any one of claims 1 to 72, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

74. 74. The pharmaceutical composition of claim 73, suitable for oral administration.

75. 74. The pharmaceutical composition of claim 73, suitable for parenteral administration.

76. 74. The pharmaceutical composition of claim 73, suitable for intravenous administration.

77. 73. A method of treating a disorder mediated by ROCK1 or ROCK2, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1 to 72, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition.

78. 78. The method of claim 77, wherein the subject is a human.

79. 79. The method of claim 78, wherein the disorder is a neurodegenerative disorder.

80. 80. The method of claim 79, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis.

81. 79. The method of claim 78, wherein the neurodegenerative disorder is Parkinson's disease.

82. 73. Use of a compound according to any one of claims 1 to 72, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition, in the manufacture of a medicament for the treatment of a disorder mediated by ROCK1 or ROCK2.

83. 73. Use of a compound according to any one of claims 1 to 72, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition, in the treatment of a disorder mediated by ROCK1 or ROCK2.

84. 84. The use of claim 82 or 83, wherein the disorder is a neurodegenerative disorder.

85. 85. The use of claim 84, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis.

86. 84. The use of claim 82 or 83, wherein the neurodegenerative disorder is Parkinson's disease.

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