A class of pyrimidine compounds containing polysubstitution, pharmaceutical composition, combination drug and application

Abstract

The application provides a kind of multi-substituted pyrimidine compound, pharmaceutical composition, combination drug and application, belongs to the technical field of biological medicine.The application provides the compound shown in formula I.The compound can be used as Rho-related protein kinase (ROCK) inhibitor, has selective inhibitory activity to Rho-related protein kinase 2 (ROCK2);Meanwhile, the compound has excellent physicochemical properties, including good solubility and physical and / or chemical stability;And have the favorable pharmacokinetic characteristics, including ideal bioavailability, suitable half-life and duration of action;In addition, the compound also has higher safety, including low toxicity, less side effects and wide therapeutic window.The compound of the application can be used as the selective inhibitor of Rho-related protein kinase 2 (ROCK2), for preventing or treating Rho-related protein kinase 2 (ROCK2) mediated diseases.Formula I

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of pyrimidine compounds containing multiple substitutions, pharmaceutical compositions, combination drugs, and their applications. Background Technology

[0002] Rho-associated protein kinase (ROCK) belongs to the AGC kinase family and is a serine / threonine protein kinase, which can be divided into two isoforms: ROCK1 and ROCK2. Human ROCK1 and ROCK2 proteins are approximately 160 kDa in size and contain an N-terminal serine / threonine kinase domain, a coiled-coil region, an embedded Rho-binding domain (RBD), a C-terminal pleckstrin homology domain (PH), and an embedded cysteine-rich region (CRD). The primary amino acid sequences of ROCK1 and ROCK2 proteins share up to 64% homology, the coiled-coil region shares up to 55% homology, and the kinase domain shares up to 92% homology. More importantly, the primary amino acid sequence homology at the ATP-binding pocket can even reach 100%, making the development of isoform-selective inhibitors of ROCK2 extremely difficult. Therefore, the object of certain embodiments of the present invention is to provide a selective ROCK2 inhibitor.

[0003] Rho is a small GTPase that, in its resting state, binds to guanosine-5'-diphosphate (GDP) to form a heterodimer. Upon stimulation, guanine nucleotide exchange factors (GEF) convert GDP to guanosine triphosphate (GTP). The Rho-GTP heterodimer binds to the RBD domain of the downstream rock, thereby activating it. The activated rock then phosphorylates its substrates, activating them as well. These substrates include myosin light chain (MLC), LIMK kinase 1 / 2, myristylated alanine-rich C-kinase (MARCKS), neurofilament protein (NF-L), collapsing response mediator protein 2 (CRMP2), adducin, and sodium / hydrogen exchanger. In addition to 1, NHE1, etc., ROCK can bind to the myosin-binding subunit of myosin light chain phosphatase (MLCP), phosphorylating it and inhibiting MLCP, thereby inhibiting the dephosphorylation of MLC. Activated ROCK can regulate the actin cytoskeleton, further regulating cell shape and various biological functions, such as the formation of stress fibers and focal adhesion complexes, apoptosis, growth and development, fibroblast proliferation, anti-airway and prostate smooth muscle cell proliferation, and cardiomyocyte proliferation. ROCK inhibitors can be used to treat a variety of diseases, including asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, kidney failure, neuropathy, osteoporosis, and idiopathic pulmonary fibrosis.

[0004] Over the past two decades, numerous ROCK inhibitors have been designed and synthesized from various scaffolds. Currently, a large number of ROCK inhibitors have entered clinical trials, including AR-13503 (NCT03835884), AT-13148 (NCT01585701), LX-7101 (NCT01528111), AR-12132, and Y-39983 (NCT00515424 and NCT00846989). In addition, several ROCK inhibitors are already on the market. In 1995, Japan approved the marketing of fasudil for the treatment of cerebral vasospasm; in 2014, Japan and China approved the marketing of rapasudil for the treatment of glaucoma and ocular hypertension; netarsudil combined with latanoprost to form Rocklatan eye drops was approved by the FDA in 2018 for the treatment of glaucoma; and belumosudil (KD-025) was approved by the FDA in 2021 for the treatment of graft-versus-host disease. In addition to its approved indications, KD-025 can be used to treat a variety of other conditions and has entered clinical trials, such as psoriasis (Phase 2, NCT02852967, NCT02317627, and NCT02106195), generalized scleroderma (Phase 2, NCT04680975 and NCT03919799), idiopathic pulmonary fibrosis (Phase 2, NCT02688647), cancer (Phase 1, NCT04550663 and NCT04270461), and liver failure (Phase 1, NCT04166942). Beijing Tide Pharmaceutical's small molecule compound TDI-01 and Redx Pharmaceutical's small molecule compound RXC-007, used to treat idiopathic pulmonary fibrosis, are also currently in Phase 1 clinical trials. Therefore, the search for new highly selective ROCK2 inhibitors for the treatment of various diseases still holds broad market potential. Summary of the Invention

[0005] The purpose of this invention is to provide a class of pyrimidine compounds containing multiple substitutions, pharmaceutical compositions, combination pharmaceuticals, and applications.

[0006] This invention provides compounds of Formula I, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, N-oxides, isotope-labeled compounds, metabolites, or prodrugs thereof: Formula I Among them, R 1 Selected from hydrogen, halogens, -COR a , by 0~3 R b The following groups are substituted: C1-6 Alkyl or C 1-6 Alkoxy; R a Selected from C 1-6 Alkyl, C 1-6 Alkoxy or halogen; R b Each was independently selected from C 1-6 Alkyl, C 1-6 Alkoxy or halogen; Ring A is or Among them, # end and Connected, the ## terminal is connected to L; X and Y are each independently selected from C or N, and not both of them are N; R 2 Each is independently selected from hydrogen, halogens, and 0 to 3 R atoms. c The following groups are substituted: C 1-6 Alkyl or C 1-6 Alkoxy; R c Each was independently selected from C 1-6 Alkyl, C 1-6 Alkoxy or halogen; a is selected from 1, 2, or 3; L is selected from -CO-, , , or ,in, The end is connected to ring A. End and R 3 Connected; L1 and L2 are each independently selected from none, C 1-6 Alkylene, -OC 1-6 Alkylene; R d Selected from hydrogen or C 1-6 alkyl; R 3 Selected from hydrogen, halogen, amino, cyano, nitro, C 2-6 alkenyl, C 2-6 Alkyne group, with 0-3 R groups e The following groups are substituted: C 1-6 Alkyl, C 1-6 Alkoxy, 3-10 member monocyclic saturated heterocyclic group, 3-10 member monocyclic saturated carbocyclic group, 6-10 member aryl, 5-14 member heteroaryl, 4-10 member spirocyclic carbocyclic group, 4-10 member spirocyclic bridged ring group or 3-10 member monocyclic saturated heterocyclic group with 6-10 member aryl; R e Each is independently selected from halogens, C 1-6 Alkyl, C 1-6Alkoxy, C 2-6 alkynyl group, C 2-6 alkenyl, hydroxyl, hydroxy-substituted C 1-6 Alkyl, oxo, and halogen-substituted C 1-6 Alkyl or -NR f R g ; R f R g Each is independently selected from hydrogen or C. 1-6 alkyl.

[0007] Furthermore, R 1 Selected from hydrogen or C 1-3 alkyl; Ring A is or Among them, # end and Connected, the ## terminal is connected to L; X and Y are each independently selected from C or N, and not both of them are N; R 2 Each is independently selected from hydrogen, halogens, and 0 to 3 R atoms. c The following groups are substituted: C 1-3 Alkyl or C 1-3 Alkoxy; R c Each was independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen; a is selected from 1, 2, or 3; L is selected from -CO-, , , or ,in, The end is connected to ring A. End and R 3 Connected; L1 and L2 are each independently selected from none, C 1-3 Alkylene, -OC 1-3 Alkylene; R d Selected from hydrogen or C 1-3 alkyl; R 3 Selected from hydrogen, halogen, amino, cyano, nitro, C 2-4 alkenyl, C 2-4 Alkyne group, with 0-3 R groups e The following groups are substituted: C 1-3 Alkyl, C 1-3 Alkoxy, 4-6 membered monocyclic saturated heterocyclic group, 3-6 membered monocyclic saturated carbocyclic group, phenyl, 4-8 membered spirocyclic carbocyclic group, 4-6 membered spirocyclic bridged ring group or 5-6 membered monocyclic saturated heterocyclic group phenyl; R e Each is independently selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, C 2-4 alkynyl group, C 2-4 alkenyl, hydroxyl, hydroxy-substituted C 1-3 Alkyl, oxo, and halogen-substituted C 1-3 Alkyl or -NR f R g ; R f R g Each is independently selected from hydrogen or C. 1-3 alkyl.

[0008] Furthermore, R 1 Selected from hydrogen or C 1-3 alkyl; Ring A is selected from , , or Among them, # end and Connect, ## terminal is connected to L; R 2 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl or C 1-3 Alkoxy; a is selected from 1 or 2; L is selected from -CO-, , , , , , , or ;in, The end is connected to ring A. End and R 3 Connected; R 3 Selected from halogens, with 0~3 R e The following groups are substituted: , , , , , , , , or ; R e Each is independently selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, C 2-4 alkynyl group, C 2-4alkenyl, hydroxyl, hydroxy-substituted C 1-3 Alkyl, oxo, and halogen-substituted C 1-3 Alkyl or -NR f R g ; R f R g Each is independently selected from C 1-3 alkyl.

[0009] Furthermore, the compound has one of the following structures: .

[0010] The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned compound, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, N-oxide, isotope-labeled compound, metabolite or prodrug thereof as the active ingredient.

[0011] The present invention also provides the use of the above-described compounds, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, N-oxides, isotope-labeled compounds, metabolites, or prodrugs, or the above-described pharmaceutical compositions, in the preparation of Rho-associated protein kinase (ROCK) inhibitors.

[0012] Furthermore, the Rho-associated protein kinase inhibitor is used for the prevention and / or treatment of diseases mediated by Rho-associated protein kinase 1 (ROCK1) and / or Rho-associated protein kinase 2 (ROCK2).

[0013] Furthermore, the diseases mediated by the Rho-associated protein kinase 2 are selected from fibrotic diseases, autoimmune diseases, inflammatory diseases, central nervous system diseases, cancer, metabolic diseases, genetic diseases, infectious diseases, ocular angiogenesis-related diseases, cardiovascular and circulatory system diseases, bone and joint diseases, skin diseases, and musculoskeletal system diseases.

[0014] Further, the Rho-associated protein kinase 2-mediated diseases are selected from one or more of the following: rheumatoid arthritis, psoriatic arthritis, osteoarthritis, gout, ankylosing spondylitis, polymyalgia rheumatica, relapsing rheumatism, juvenile idiopathic arthritis, reactive arthritis, sacroiliitis, Achilles tendinitis, bicephalic tendinitis, rotator cuff tendinitis, posterior tibial tendinitis, epicondylitis, plantar fasciitis, olecranon bursitis, prepatellar bursitis, calcaneal bursitis, trochanteric bursitis, pemphigoid bursitis, popliteal cyst, carpal tunnel syndrome, tarsal tunnel syndrome, impingement syndrome, medial synovial plica syndrome, patellofemoral pain syndrome, patellar chondromalacia, chondrodysplasia, acromegaly, adhesive bursitis, diffuse idiopathic osteophyte. Disorders of the joint (DISH), arthritis, exercise-induced ventricular septal syndrome, flat feet, hip dysplasia, overexertion syndrome, Ehlers-Danlos syndrome, osteogenesis imperfecta, osteomalacia, Paget's disease, osteochondrosis, multiple epiphyseal dysplasia, Scheuermann's osteochondritis, Osgood-Schlatter disease, Legg-Calve-Perthes disease, Fryber's disease, Kimberly's disease, slipped axillary spondylolisthesis, spinal stenosis, spondylolisthesis, Porter's disease, Blondt's disease, Titzer's syndrome, Pellegrini-Stieda syndrome, retrocalcaneal bursitis, systemic lupus erythematosus (SLE) (including discoid lupus erythematosus), Drug-induced lupus, subacute cutaneous lupus, Sjögren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease (MCTD), undifferentiated connective tissue syndrome (UCTS), overlap syndrome, cryoglobulinemia, mixed cryoglobulinemia, polyarteritis nodosa, allergic vasculitis, urticarial vasculitis, granulomatous arteritis, rheumatoid vasculitis, giant cell arteritis, Goyan's arteritis, Behçet's syndrome, Cogan's syndrome, CREST syndrome, Churg-Strauss syndrome, Goodpasture syndrome, Wegener's granulomatous disease, allergic purpura, Kawasaki disease, Siddenham's chorea, Sweet's syndrome, Lofgren's syndrome, Felty's syndrome. Kaplan syndrome, costochondritis syndrome, neonatal lupus, nodular panniculitis, pachydermal periosteal disease, reflex sympathetic dystrophy syndrome, idiopathic pulmonary fibrosis (IPF), systemic sclerosis (SSC), interstitial lung disease (ILD), primary biliary cirrhosis, primary biliary sclerosis, scleroderma, diabetic finger sclerosis, Dupuitren's contracture, myocardial fibrosis, fibromatosis-related lung, fibromatosis-related liver, fibromatosis-related kidney, intestinal stenosis, type 1 diabetes, type 2 diabetes, diabetic nephropathy, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hemochromatosis, Gaucher's disease, Fabry disease, mucopolysaccharidosis, okra, familial Mediterranean fever, Fabry's lipogranulomatosis.Lesch-Nyhan syndrome, Hüller syndrome, Wilson's disease, pseudoxanthoma elastica, cystine storage disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Guillain-Barré disease, myasthenia gravis, cognitive deficits caused by neuronal inflammation, liver cancer, bladder cancer, squamous cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, small cell lung cancer, head and neck cancer, breast cancer, colorectal cancer, peritoneal cancer, gastrointestinal cancer, esophageal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, pituitary cancer, astrocytoma, soft tissue sarcoma, pancreatic cancer. Adenocarcinoma, glioblastoma, cervical cancer, ovarian cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, malignant synovitis, hypertension, atherosclerosis, restenosis, stroke, heart failure, coronary artery spasm, cerebral vasospasm, peripheral circulatory disorders, peripheral artery occlusive disease, ischemia / reperfusion injury, pulmonary hypertension, angina pectoris, hemangioma, angiofibroma, Osler-Weber syndrome, glaucoma, ocular hypertension, retinopathy, retinopathy of prematurity, retrolental fibrosis, corneal transplant rejection-related neovascularization. Corneal neovascularization associated with refractive surgery complications, corneal neovascularization associated with contact lens complications, corneal neovascularization associated with pterygium, recurrent corneal neovascularization associated with pterygium, corneal ulceration, nonspecific ocular surface diseases, asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease (COPD), chronic synovitis, sarcoidosis, psoriasis, dermatitis, inflammatory bowel disease (IBD), autoimmune nephritis, acute pancreatitis, chronic pancreatitis, fungal infections, parasitic infections, viral infections, septic shock, Lyme disease, Whipple's disease, brucellosis, erectile dysfunction. Allogeneic rejection, allergic inflammation, delayed-type hypersensitivity reaction, artificial joint infection, sickle cell arthritis, foreign body synovitis, avascular necrosis, osteomyelitis, osteonecrosis, joint hemarthrosis, chondrocalcinosis, calcium pyrophosphate dihydrate deposition disease (CPPD), crystal deposition disease, Raynaud's phenomenon, Addison's disease, Graves' disease, Duchenne muscular dystrophy, myofascial pain syndrome, fibromyalgia, multicentric reticulocyte hyperplasia, neurogenic arthritis, relapsing polychondritis, rheumatic fever, shoulder-hand syndrome, Stickler syndrome, chondromal malformation, pigmented villonodular synovitis, piriformis syndrome.

[0015] The present invention also provides a combination drug, wherein the combination drug is a formulation prepared by adding pharmaceutically acceptable excipients to one or more of the following pharmaceutical active ingredients: anti-inflammatory agents, anti-fibrotic agents, chemotherapeutic agents, anticancer agents, immunosuppressants, antitumor vaccines, cytokine therapy, or tyrosine kinase inhibitors, using the above-mentioned compound, or its stereoisomer, or its tautomer, or its solvate, its prodrug, its isotope label, or its pharmaceutically acceptable salt as active ingredients: anti-inflammatory agents, antifibrotic agents, chemotherapeutic agents, anticancer agents, immunosuppressants, antitumor vaccines, cytokine therapy, or tyrosine kinase inhibitors.

[0016] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0017] Regarding the definition of terminology used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to that group or term throughout the specification; for terms not specifically defined in this invention, their meanings should be given based on the disclosure and context, meanings that those skilled in the art would assign to them. Wherein: "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0018] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a-b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1-6 Alkyl groups are straight-chain or branched alkyl groups containing 1, 2, 3, 4, 5 or 6 carbon atoms, and so on.

[0019] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of carbon atoms. For example, C 1-6 Alkyl groups are alkyl groups having 1 to 6 carbon atoms, that is, alkyl groups with 1, 2, 3, 4, 5, or 6 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl, etc.

[0020] "Alkenyl" refers to an aliphatic hydrocarbon group having at least one carbon-carbon double bond. Alkenyl groups can be straight-chain or branched.

[0021] "Alkyne" refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond. The alkyne group can be straight-chain or branched.

[0022] "Alkoxy" refers to the aforementioned alkyl group having a specific number of carbon atoms connected by an oxygen bridge, unless otherwise specified. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, and isopropoxy.

[0023] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0024] "Heterocyclic group" refers to a saturated ring or a non-aromatic unsaturated ring containing at least one heteroatom and having a single ring; where the heteroatom refers to a nitrogen atom, oxygen atom, sulfur atom, or boron atom. Including but not limited to: , , , .

[0025] "Aryl" refers to a monocyclic group consisting of all carbon atoms with a conjugated π-electron system, such as phenyl.

[0026] "Heteroaryl" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl.

[0027] "Saturated carbocyclic groups" refer to cyclic hydrocarbon groups composed of carbon atoms, where all carbon-carbon bonds are single bonds. They do not contain heteroatoms, are not aromatic, and the bond to the parent compound is located on a carbon atom in the ring. Examples include cyclopropyl, cyclopentyl, and cyclohexyl.

[0028] "Saturated heterocyclic groups" refer to cyclic groups containing one or more heteroatoms (including but not limited to oxygen, sulfur, and nitrogen) within the ring, and all bonds within the ring are single bonds. They are not aromatic, and the bond to the parent compound is located on a carbon atom or heteroatom in the ring. Examples include tetrahydrofuranyl, piperidinyl, tetrahydrothiopheneyl, pyrrolidinyl, and morpholinyl.

[0029] "Monocyclic" refers to a group containing only one ring structure, which can be a saturated ring, aromatic ring, heteroaromatic ring, or non-aromatic unsaturated ring. Monocyclic structures do not include multi-ring linkages such as fused rings, spiro rings, or bridged rings.

[0030] "Parallelized rings" refer to polycyclic rings with two rings sharing two adjacent ring atoms. This includes, but is not limited to: , .

[0031] A "bridged ring" refers to a saturated cyclic hydrocarbon in which any two rings share two non-directly connected carbon atoms. This includes, but is not limited to: , , , , , , , , , .

[0032] A "spirocycle" refers to a polycyclic ring consisting of two rings sharing a single atom. This includes, but is not limited to: , , .

[0033] The present invention has achieved the following beneficial effects: This invention provides a class of pyrimidine compounds containing multiple substitutions, which have a significant inhibitory effect on ROCK2 and excellent selectivity for ROCK2. Therefore, they can be used as ROCK2 inhibitors to prepare drugs for the prevention and treatment of ROCK-mediated diseases.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, but these embodiments do not constitute any limitation on the scope of the present invention. The raw materials and equipment used in the present invention are all known commercially available products and can be routinely purchased through commercial channels.

[0037] The structure of the target compound was determined by nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 Confirmation was performed by 1H NMR or mass spectrometry (MS). Chemical shifts (δ) were reported in parts per million (ppm). 1¹H NMR spectra were determined using a Bruker BioSpin GmbH 400 MHz NMR spectrometer. The solvents used were deuterated methanol (CD₃OD), deuterated chloroform (CDCl₃), or hexadeuterated dimethyl sulfoxide (DMSO-d₆), with tetramethylsilane (TMS) as the internal standard. LC-MS analysis was performed on a Shimadzu LC-MS-2020 system (Shimadzu Corporation, model: LC-MS-2020). Preparative high-performance liquid chromatography (HPLC) was performed using a Waters 2767 system equipped with a Waters Sunfire C18 column (19×250 mm, 10 µm). Thin-layer chromatography (TLC) used Huanghai brand HSGF 254 silica gel plates (5×20 cm); preparative TLC used Yantai-made GF 254 silica gel plates (0.4–0.5 nm). The reaction process was monitored by thin-layer chromatography (TLC) or LC-MS. The developing solvent systems used included dichloromethane and methanol, n-hexane and ethyl acetate, and petroleum ether and ethyl acetate. The developing solvent systems were adjusted appropriately based on the polarity differences of the compounds to be separated (e.g., adjusting the solvent volume ratio or adding additives such as triethylamine). The microwave reaction was performed in a Biotage Initiator. + The separation was carried out in a microwave reactor (400W, room temperature to ~300℃). Column chromatography separation used 200~300 mesh silica gel produced by Yucheng Chemical as the stationary phase. The eluent systems included dichloromethane and methanol and n-hexane and ethyl acetate. The eluent systems were adjusted according to the polarity of the compounds to be separated (such as adjusting the solvent volume ratio or adding additives such as triethylamine).

[0038] Unless otherwise specified in the examples, the reaction temperature is room temperature (20℃~30℃).

[0039] Example 1, (4-(4-((4-(1) H Preparation of pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)phenyl)(3,3-difluoroazacyclobutane-1-yl)methyl ketone (GLP010) first step 4-Bromoaniline (10.0 g, 58.1 mmol) and 1-Boc-pyrazole-4-boronic acid pinacol ester (22.2 g, 75.6 mmol) were dissolved in 1,4-dioxane:water (140:80 mL), and potassium carbonate (20.0 g, 145.3 mmol) and Pd(dppf)Cl2 (0.425 g, 0.58 mmol) were added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 90 °C for 12 hours. LC-MS analysis showed that the reaction was complete. The reaction solution was then cooled to room temperature, filtered, and the solvent was removed by vacuum rotary evaporation of the filtrate. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:2 to ethyl acetate) to give compound R1-SM1 (11.9 g, 84.5% yield). 1 H NMR (400 MHz, DMSO) δ 8.05 (s, 1H), 7.73 (s, 1H), 7.31 – 7.21 (m, 2H), 6.61 –6.51 (m, 2H), 5.34 (dd, J = 10.0, 2.2 Hz, 1H), 5.03 (s, 2H), 3.97 – 3.87 (m,1H), 3.61 (ddd, J = 11.5, 8.1, 5.8 Hz, 1H), 2.17 – 2.05 (m, 1H), 1.93 (qt, J =13.0, 2.5 Hz, 2H), 1.67 (ddt, J = 17.6, 14.7, 8.6 Hz, 1H), 1.52 (tq, J = 7.9, 3.8Hz, 2H). MS m / z (ESI): 244.1 [M+H] + .

[0040] Step 2 Compound R1-SM1 (10.0 g, 41.1 mmol) and 2,4-dichloropyrimidine (8.0 g, 53.4 mmol) were dissolved in isopropanol (50 mL), and N,N-diisopropylethylamine (18.0 mL, 102.8 mmol) was added. The reaction mixture was heated to 80 °C and reacted for 16 hours. After the reaction was confirmed to be complete by LC-MS, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with water and dried to obtain the target compound R1-SM2 (13.2 g, 90.5% yield). 1 H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 5.9 Hz, 1H), 7.79(d, J= 0.8 Hz, 1H), 7.74 (d, J = 0.9 Hz, 1H), 7.49 – 7.37 (m, 3H), 7.31 – 7.23(m, 2H), 6.52 (d, J = 5.9 Hz, 1H), 5.35 (dd, J = 8.8, 3.5 Hz, 1H), 4.02 (ddt, J =11.6, 3.9, 1.8 Hz, 1H), 3.66 (ddd, J = 13.6, 7.5, 3.0 Hz, 1H), 2.14 – 1.94 (m, 3H), 1.73 – 1.49 (m, 3H). MS m / z (ESI): 356.1 [M+H] + .

[0041] Step 3 Compound R1-SM2 (10.0 g, 28.1 mmol) and pinacol 4-methoxyformylphenylboronic acid (9.6 g, 36.5 mmol) were dissolved in 1,4-dioxane:water (70:40 mL), and potassium carbonate (9.7 g, 70.3 mmol) and Pd(dppf)Cl2 (0.205 g, 0.28 mmol) were added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 90 °C for 12 hours. LC-MS analysis showed that the reaction was complete. The reaction solution was then cooled to room temperature, filtered, and the solvent was removed by vacuum rotary evaporation of the filtrate. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:2 to ethyl acetate) to give compound R1-SM3 (10.3 g, 80.4% yield). 1 H NMR (400 MHz, DMSO) δ 9.78 (s, 1H), 8.54 – 8.45 (m, 2H), 8.43 (d, J = 5.9 Hz, 1H),8.30 (s, 1H), 8.14 – 8.05 (m, 2H), 7.93 (s, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.69– 7.61 (m, 2H), 6.78 (d, J = 5.9 Hz, 1H), 5.41 (dd, J= 10.0, 2.2 Hz, 1H), 3.98 –3.92 (m, 1H), 3.90 (s, 3H), 3.71 – 3.62 (m, 1H), 2.19 – 2.09 (m, 1H), 1.98 –1.91 (m, 2H), 1.76 – 1.64 (m, 1H), 1.56 (tt, J = 6.1, 3.8 Hz, 2H). MS m / z (ESI): 456.2 [M+H] + .

[0042] Step 4 Compound R1-SM3 (10.0 g, 22.0 mmol) was added to methanol:water (40:10 mL), followed by lithium hydroxide (2.6 g, 110.0 mmol). The reaction mixture was stirred at 60 °C for 2 hours. After LC-MS showed that the reaction was complete, the reaction mixture was cooled to room temperature, the pH was adjusted to 5-6 with HCl (3 M), and the mixture was filtered. The filter cake was washed with water and dried to obtain the target compound R1-SM4 (9.3 g, 95.6% yield). MS m / z (ESI): 442.5 [M+H] + .

[0043] Step 5 Compound R1-SM4 (200 mg, 0.45 mmol) and 3,3-difluorotrimethylimide hydrochloride (76.3 mg, 0.59 mmol) were dissolved in N,N-dimethylformamide (10 mL), and HATU (224 mg, 0.59 mmol) and diisopropylethylamine (116 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 3 h. Thin-layer chromatography (dichloromethane / methanol = 10:1) showed that the reaction was complete. Water (60 mL) was slowly added to the reaction solution, and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was added to dichloromethane (20 mL), followed by HCl / 1,4-dioxane (5 mL). The mixture was reacted at room temperature for 1 h. After the reaction was confirmed to be complete by LC-MS, the pH was adjusted to 9-10 with sodium hydroxide, and the mixture was extracted with dichloromethane. The organic phase was concentrated and purified by column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound GLP010 (66.4 mg, yellow solid, yield: 34.1%). 1 H NMR (400MHz, DMSO) δ 12.91 (s, 1H), 9.75 (s, 1H), 8.43 (dd, J= 7.0, 5.0 Hz, 3H), 8.16(s, 1H), 7.93 (s, 1H), 7.87 – 7.80 (m, 2H), 7.76 (d, J = 8.2 Hz, 2H), 7.68 –7.61 (m, 2H), 6.77 (d, J = 5.9 Hz, 1H), 4.68 (d, J = 122.8 Hz, 4H). MS m / z (ESI): 436.1 [M+H] + .

[0044] The compounds in Table 1 were prepared using a method similar to that described in Example 1.

[0045] .

[0046] Table 1 Compound Information Example 2 N -(4-(4-((4-(1 H Preparation of pyrazol-4-yl)phenyl)amino)pyrimidin-2-yl)-2-methylphenyl)-3,3-difluorocyclobutane-1-carboxamide (GLP120) first step Compound R1-SM2 (5.0 g, 14.1 mmol) and pinacol ester of 3-methyl-4-aminophenylboronic acid (4.3 g, 18.3 mmol) were dissolved in 1,4-dioxane:water (70:40 mL), and potassium carbonate (4.9 g, 35.3 mmol) and Pd(dppf)Cl2 (0.102 g, 0.14 mmol) were added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 90 °C for 12 hours. LC-MS analysis showed that the reaction was complete. The reaction solution was then cooled to room temperature, filtered, and the solvent was removed by vacuum rotary evaporation of the filtrate. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:2 to ethyl acetate) to give compound R1-SM5 (5.1 g, 84.5% yield). 1 H NMR (404MHz, Chloroform- d ) δ 9.41 (s, 1H), 8.99 (d, J = 4.1 Hz, 1H), 7.91 (d, J = 1.5 Hz,2H), 7.86 – 7.79 (m, 2H), 7.62 – 7.54 (m, 4H), 6.85 (d, J= 9.1 Hz, 1H), 6.78 (d, J = 4.0 Hz, 1H), 5.83 (td, J = 2.3, 0.6 Hz, 1H), 4.11 (s, 2H), 3.82 (ddd, J =10.5, 4.8, 3.3 Hz, 1H), 3.70 (ddd, J = 10.5, 4.8, 3.4 Hz, 1H), 2.33 (dddd, J =14.7, 7.9, 5.5, 2.4 Hz, 1H), 2.06 – 1.96 (m, 1H), 1.96 – 1.85 (m, 1H), 1.79 –1.68 (m, 1H), 1.67 – 1.57 (m, 2H).MS m / z (ESI): 427.2 [M+H] + .

[0047] Step 2 Compound R1-SM5 (200 mg, 0.47 mmol) and 3,3-difluorocyclobutanecarboxylic acid (83.0 mg, 0.61 mmol) were dissolved in N,N-dimethylformamide (10 mL), and HATU (232.2 mg, 0.61 mmol) and diisopropylethylamine (121.1 mg, 0.94 mmol) were added. The reaction was carried out at room temperature for 3 h. Thin-layer chromatography (dichloromethane / methanol = 10:1) showed that the reaction was complete. Water (60 mL) was slowly added to the reaction solution, and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was added to dichloromethane (20 mL), followed by HCl / 1,4-dioxane (5 mL). The mixture was reacted at room temperature for 1 h. After the reaction was confirmed to be complete by LC-MS, the pH was adjusted to 9-10 with sodium hydroxide, and the mixture was extracted with dichloromethane. The organic phase was concentrated and purified by column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound GLP120 (60.9 mg, yellow solid, yield: 28.2%). 1 H NMR (400 MHz, DMSO) δ12.88 (s, 1H), 9.68 (s, 1H), 9.55 (s, 1H), 8.36 (d, J = 5.8 Hz, 1H), 8.27 –8.13 (m, 2H), 8.04 (s, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.67 (dd, J = 16.4, 8.3 Hz, 3H), 6.70 (d,J = 5.9 Hz, 1H), 3.27 (h, J = 5.1 Hz, 1H), 2.83 (dt, J = 17.0, 8.7Hz, 4H). MS m / z (ESI): 461.2 [M+H] + .

[0048] The compounds in Table 2 were prepared using a method similar to that described in Example 2.

[0049] .

[0050] Table 2 Compound Information The following experimental examples demonstrate the beneficial effects of the present invention.

[0051] Experiment Example 1, Biological Test 1. Experimental Methods Using the Lance Ultra Assay method at Km ATP, the inhibitory activity and IC50 of compounds against two kinase targets, ROCK1 and ROCK2, were tested. 50 The specific method is as follows.

[0052] The initial concentrations for the compound kinase activity assay were 2 μM and 1 μM, respectively. A 100-fold concentrated stock solution was prepared in advance: 97 μL of pure DMSO and 3 μL of 10 mM compound solution were added to the second well of the 384-well Echo plate, and 99 μL of pure DMSO and 1 μL of 10 mM compound solution were added to the other well. A 3-fold serial dilution was performed starting from the two stock solutions, with a total of 6 concentration gradients set. 40 μL of pure DMSO was added to each of the two blank wells as the Max control well and the Min control well, respectively. Then, 200 nL of compound dilution was transferred to the 384-well assay plate using an Echo650 acoustic pipette. Prepare 1× kinase buffer (containing 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.01% Triton X-100, 0.01% BSA, and 2 mM DTT). Add kinase to the buffer to prepare an enzyme solution. Add 10 μL of the enzyme solution to each well of the detection plate. Add 1× buffer without kinase to the negative control wells. Centrifuge at 1000 rpm for 1 min and incubate at room temperature for 10 min. Dissolve the substrate, ATP, and other reagents in the kinase buffer to prepare a substrate solution. Add 10 μL of the substrate solution to each well. Centrifuge at 1000 rpm for 1 min and incubate at room temperature for 30 min to complete the enzymatic reaction. Next, prepare 2× antibody stop solution, add 20 μL of stop solution to each well, centrifuge at 1000 rpm for 1 min, incubate at room temperature for 60 min, and use an Envision 2104 multi-mode microplate reader to read the fluorescence values ​​at 665 nm and 615 nm respectively. Calculate the homogeneous time-resolved fluorescence signal ratio (665 nm / 615 nm) of the five replicates, and calculate the compound's inhibitory activity according to the formula: Inhibition rate % = (Max signal ratio - Sample signal ratio) / (Max signal ratio - Min signal ratio) × 100, where Min is the signal of the enzyme-free control group and Max is the signal of the DMSO control group. Finally, import the data into Excel, and use the XLFit 5.4.0.8 plugin to calculate the inhibitory activity using the formula Y = Bottom + (Top − Bottom) / (1 + (IC 50 / X)^HillSlope) was used to fit the dose-response curve.

[0053] Table 3 Kinase test conditions 2. Experimental Results Tables 4 and 5 show the ROCK1 / ROCK2 inhibitory activities of certain structural compounds determined by the above-described assays, based on the compound's IC50. 50 The value is classified as " " " " " "and" "." "Refers to IC" 50 Compounds with a value >20 μM; "Refers to IC" 50 Compounds with values ​​of 20 to 10 μM; "Refers to IC" 50 Compounds with values ​​of 10 to 1 μM; "Refers to IC" 50 Compounds with values ​​from 1 to 0.1 μM; "Refers to IC" 50 Compounds with values ​​from 0.1 to 0.01 μM; "Refers to IC" 50 Compounds with a value <0.01 μM.

[0054] Table 4. Kinase activity of compounds against ROCK1 / ROCK2 Table 5. Kinase activity of compounds against ROCK1 / ROCK2 The data above show that the tested compound has an IC50 response to ROCK2. 50 The value is significantly lower than the IC for ROCK1. 50 The value indicates that the compound of the present invention has good selectivity for ROCK2.

[0055] Experiment Example 2, Biological Test 1. Experimental Methods The inhibitory effect of the test compounds on the TGF-β1-induced fibrotic phenotype of rat renal fibroblasts NRK-49F was detected by quantitative real-time PCR (qPCR). Logarithmically growing NRK-49F cells were seeded in 6-well plates and cultured for 24 h until cell attachment. The culture medium was then replaced with serum-free DMEM for a simultaneous 12 h starvation period. The experiment included a blank control group, a TGF-β1 model group, a compound gradient administration group, and a positive control group. Each group had three biological replicates. The administration groups were pretreated with different concentrations of the test compounds for 2 h. The model and administration groups were continuously stimulated with a final concentration of 5 ng / mL TGF-β1 for 24 h. The blank group was treated with an equal volume of DMSO. After stimulation, the culture medium was discarded, and the cells were washed with pre-cooled PBS. Total RNA was extracted from the cells using the Trizol method, and RNA purity and concentration were detected using Nanodrop. Qualified samples were selected, and genomic DNA was removed and cDNA was synthesized by reverse transcription according to the kit instructions. Gapdh was used as an internal control gene, and SYBR Green DNA was used for analysis. The Green quantitative PCR system was used to amplify the fibrosis markers α-SMA and Col1a1 by qPCR. After amplification, melting curves were plotted to verify primer specificity, and the relative expression levels of mRNA of each fibrosis gene were calculated by the 2^-ΔΔCt method.

[0056] 3. Experimental Results Table 6 shows the ability of certain structural compounds to inhibit fibrosis as determined by the above-described assays. Based on their inhibitory ability, the compounds are classified as "..." " " " " "", "This refers to an inhibition rate value of <50%;" "This refers to an inhibition rate value > 50%;" "This refers to an inhibition rate value > 80%;" "This refers to an inhibition rate value > 100%;" "This refers to an inhibition rate value > 200%".

[0057] Table 6. Inhibitory activity of compounds against fibrosis As can be seen from the above data, multiple compounds exhibit significant inhibitory activity against TGF-β1-induced fibrosis at the cellular level, and are superior to marketed ROCK2 inhibitors Benzamide and Belumosudil. This indicates that the compounds of the present invention have a good therapeutic effect on ROCK2-mediated fibrotic diseases.

Claims

1. The compound of formula I, or a pharmaceutically acceptable salt thereof: Formula I in, R 1 Selected from hydrogen or C 1-3 alkyl; Ring A is selected from , or Among them, # end and Connect, ## terminal is connected to L; R 2 Each is independently selected from hydrogen, halogen, and C. 1-3 Alkyl or C 1-3 Alkoxy; a is selected from 1 or 2; When L is selected , , , , , , or At that time; where the * end is connected to ring A, and the ** end is connected to R 3 Connected; R 3 Selected from 0 to 3 R e The following groups are substituted: , , , , , , , , or ; When L is -CO-, R 3 Selected from 0 to 3 R e The following groups are substituted: or ; R e Each is independently selected from halogens, C 1-3 Alkyl, C 1-3 Alkoxy, C 2-4 alkynyl group, C 2-4 alkenyl, hydroxyl, hydroxy-substituted C 1-3 Alkyl, oxo, and halogen-substituted C 1-3 Alkyl or -NR f R g ; R f R g Each was independently selected from C 1-3 alkyl.

2. A compound, or a pharmaceutically acceptable salt thereof, characterized in that: The compound has one of the following structures: 。 3. A pharmaceutical composition, characterized in that: The pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to a compound as described in any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof as the active ingredient.

4. Use of the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of a Rho-related protein kinase inhibitor.

5. The use according to claim 4, characterized in that: The Rho-associated protein kinase inhibitor is used to treat diseases mediated by Rho-associated protein kinase 2.

6. The use according to claim 5, characterized in that: The Rho-associated protein kinase 2-mediated disease is a fibrotic disease.

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