Heterocyclic compound as RIPK1 inhibitor

Abstract

The present invention relates to a compound of formula (I), a pharmaceutical composition containing same, a preparation method therefor, and a use thereof as a receptor-interacting protein kinase 1 (RIPK1) inhibitor. The present invention further relates to a use of the compound or composition containing same for treatment or prevention of related diseases or disorders mediated by RIPK1.

Description

Heterocyclic compounds as RIPK1 inhibitors Technical Field

[0001] The present invention relates to heterocyclic compounds, pharmaceutical compositions containing the same, and their use as inhibitors of receptor-interacting protein kinase 1 (RIPK1). More specifically, the present invention provides novel heterocyclic compounds as RIPK1 inhibitors, pharmaceutical compositions containing such compounds, and methods of using such compounds to treat or prevent diseases or conditions mediated by RIPK1. The present invention also relates to methods for preparing such compounds. Background Art

[0002] Receptor-interacting protein kinase 1 (RIPK1) is a serine / threonine protein kinase involved in innate immune signaling. RIPK1 is a 76 kDa protein composed of an N-terminal kinase domain, a C-terminal death domain, and an intermediate domain containing a receptor-interacting protein homotypic interaction motif (RHIM). The C-terminal death domain mediates homodimerization and heterodimerization with other death domain-containing proteins, while the N-terminal kinase domain mediates trans-autophosphorylation to promote self-activation.

[0003] RIPK1 has a dual immunomodulatory effect. On the one hand, it can act as a scaffold to promote the activation of MAPK and NF-κB signaling pathways, thereby promoting inflammatory responses, cell survival, and inhibiting cell apoptosis. On the other hand, abnormal regulation of RIPK1 activity will cause cell necrosis. RIPK1 is a major regulator of NF-κB signaling transduction and cellular determinants of death responses. NF-κB signaling responds to a wide range of inflammatory and pro-death stimuli in human diseases (Degterev, A., et.al. Proc. Natl Acad. Sci. USA, 2019, 116(20), 9714-9722).

[0004] RIPK1 is widely expressed in various cell types, with the highest expression in adipose, endothelial, and perivascular cell clusters. It is also expressed in immune cell clusters (dendritic cells, macrophages, and T cells). Studies have found that RIPK1 kinase activation is present in pathological samples of autoimmune diseases and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). Anti-tumor necrosis factor-α (TNF-α) drugs have achieved significant clinical success in the treatment of human peripheral inflammatory diseases such as rheumatoid arthritis, colitis, and psoriasis. However, because tumor necrosis factor receptor 2 (TNFR2) mediates nerve regeneration, the treatment of central nervous system diseases is unsafe. RIPK1 inhibitors can safely improve the harmful TNF-α response in the central nervous system without affecting TNFR2. Therefore, RIPK1 inhibitors have the potential to become alternative drugs to TNF-α antibodies to make up for the shortcomings of TNF-α antibodies.

[0005] Studies have shown that necrostatin-1 (Nec-1), a small molecule inhibitor of RIPK1 known in the art, can effectively block programmed cell death (Degterev et al. Nat. Chem. Biol. 2005; 1: 112-119), demonstrating promising therapeutic effects in a variety of inflammatory diseases. The brain-penetrant RIPK1 inhibitor DNL-788 is being used in amyotrophic lateral sclerosis, among other conditions. These trials have laid the foundation for advancing the clinical application of RIPK1 inhibitors.

[0006] Therefore, the development of highly active novel RIPK1 inhibitors is of great significance to this field, especially highly selective RIPK1 inhibitors that can penetrate the blood-brain barrier, providing possible therapeutic drugs for targeting neuroinflammation and cell death that lead to various neurological diseases, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis, as well as acute neurological diseases such as stroke and traumatic brain injury. Summary of the Invention

[0007] The present invention relates to a compound of formula (I), an isomer, a prodrug, a solvate, a stable isotope derivative or a pharmaceutically acceptable salt thereof,

[0008] in:

[0009] A is a 6-8 membered saturated heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S;

[0010] R 1is selected from halogenated C1-C8 alkyl, C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclyl, wherein the C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclyl is unsubstituted or replaced by one or more R x substituted, wherein said R x Each is independently selected from halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is unsubstituted or substituted with one or more halogen or cyano;

[0011] B is selected from the group consisting of one or more substituents R 2 Substituted C5-C10 cycloalkyl, said R 2 are each independently selected from halogen and cyano.

[0012] Further preferably, the present invention relates to a compound of formula (I) as described above, an isomer, a prodrug, a solvate, a stable isotopic derivative or a pharmaceutically acceptable salt thereof, which has formula (II):

[0013] in:

[0014] R 1 is selected from halogenated C1-C8 alkyl, C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclyl, wherein the C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclyl is unsubstituted or replaced by one or more R x substituted, wherein said R x Each is independently selected from halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is unsubstituted or substituted with one or more halogen or cyano;

[0015] B is selected from the group consisting of one or more substituents R 2 Substituted C5-C10 cycloalkyl, said R 2 are each independently selected from halogen and cyano.

[0016] Still further preferably, the present invention relates to a compound of formula (II) as described above, an isomer, a prodrug, a solvate, a stable isotope derivative or a pharmaceutically acceptable salt thereof, wherein:

[0017] R 1 is selected from halogenated C1-C6 alkyl, C6-C10 aryl, 5-8 membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, C3-C8 cycloalkyl, 4-8 membered saturated heterocyclic group containing 1-2 heteroatoms selected from N and O, wherein the C6-C10 aryl, 5-8 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered saturated heterocyclic group is unsubstituted or replaced by 1-2 Rx substituted, wherein said R x Each is independently selected from fluorine, chlorine, bromine, and C1-C4 alkyl; wherein the C1-C4 alkyl is unsubstituted or substituted with 1-3 substituents selected from fluorine, chlorine, bromine, and cyano;

[0018] B is selected from the group consisting of R 2 Substituted C6-C10 cycloalkyl, the R 2 are each independently selected from halogen and cyano.

[0019] More preferably, the present invention relates to a compound of formula (II) as described above, an isomer, a prodrug, a solvate, a stable isotope derivative or a pharmaceutically acceptable salt thereof, wherein:

[0020] R 1 is selected from halogenated C1-C4 alkyl, phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, C3-C6 cycloalkyl, 4-6 membered saturated heterocyclic group containing 1 heteroatom selected from N and O, wherein the phenyl, 5-6 membered heteroaryl, C3-C6 cycloalkyl, 4-6 membered saturated heterocyclic group is unsubstituted or replaced by 1-2 R x substituted; wherein said R x Each is independently selected from fluorine, chlorine, C1-C4 alkyl, and the C1-C4 alkyl is unsubstituted or substituted with 1-3 substituents selected from fluorine and cyano;

[0021] B is selected from the group consisting of 2 Substituted C6-C10 cycloalkyl, the R 2 Selected from fluorine, chlorine and cyano.

[0022] Still more preferably, the present invention relates to a compound of formula (II) as described above, an isomer, a prodrug, a solvate, a stable isotope derivative or a pharmaceutically acceptable salt thereof, wherein:

[0023] R 1 is selected from C1-C4 alkyl substituted by 1-3 fluorine groups, 5-membered heteroaryl groups containing 1-2 heteroatoms selected from N, O and S, and 4-6-membered saturated heterocyclic groups containing 1 O heteroatom, wherein the 5-membered heteroaryl groups and 4-6-membered saturated heterocyclic groups are unsubstituted or substituted by 1-2 R x substituted, wherein said R x Each is independently selected from fluorine, methyl, and methyl substituted by 1-3 fluorine groups;

[0024] B is selected from where R 2 Selected from fluorine and cyano.

[0025] Also preferably, the present invention relates to a compound of formula (II) as described above, an isomer, a prodrug, a solvate, a stable isotopic derivative or a pharmaceutically acceptable salt thereof, wherein:

[0026] R 1 Selected from

[0027] B is selected from

[0028] Most preferably, the present invention relates to a compound of formula (II) as described above, an isomer, a prodrug, a solvate, a stable isotopic derivative or a pharmaceutically acceptable salt thereof, which is selected from:

[0029] The present invention further relates to a pharmaceutical composition comprising a compound, isomer, prodrug, solvate, stable isotope derivative or pharmaceutically acceptable salt according to any embodiment of the present invention, optionally one or more other RIPK1 inhibitors, and one or more pharmaceutically acceptable carriers.

[0030] The present invention also relates to the use of the compound, isomer, prodrug, solvate, stable isotope derivative or pharmaceutically acceptable salt according to any embodiment of the present invention or the pharmaceutical composition according to the present invention in the preparation of a medicament for use as an inhibitor of RIPK1.

[0031] The present invention also relates to the use of a compound, isomer, prodrug, solvate, stable isotope derivative or pharmaceutically acceptable salt according to any embodiment of the present invention or a pharmaceutical composition according to the present invention in the preparation of a medicament for treating or preventing a disease or condition mediated by RIPK1.

[0032] The present invention also relates to the use of a compound, isomer, prodrug, solvate, stable isotope derivative or pharmaceutically acceptable salt according to any embodiment of the present invention in the preparation of a medicament for treating or preventing RIPK1-mediated related diseases or conditions, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute nervous system diseases, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, non-alcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary disease, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, cancer (e.g., pancreatic cancer), bacterial infection, hematological malignancies, solid organ malignancies, etc.

[0033] The present invention also relates to the use of the pharmaceutical composition according to the present invention in the preparation of a drug, wherein the drug is used to treat or prevent related diseases or conditions mediated by RIPK1, such as: Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute nervous system diseases, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, non-alcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary disease, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, cancer (such as pancreatic cancer), bacterial infection, blood malignancies, solid organ malignancies, etc.

[0034] The present invention also relates to a method for treating or preventing a disease or condition mediated by RIPK1, comprising administering to a patient in need thereof a therapeutically effective amount of a compound, isomer, prodrug, solvate, stable isotope derivative, or pharmaceutically acceptable salt of any embodiment of the present invention, or a pharmaceutical composition of the present invention. The disease or condition is, for example, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute neurological disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, non-alcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary disease, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, cancer (e.g., pancreatic cancer), bacterial infection, hematological malignancies, solid organ malignancies, and the like.

[0035] Another aspect of the present invention relates to a compound according to any embodiment of the present invention, or an isomer, prodrug, solvate, stable isotopic derivative, or pharmaceutically acceptable salt thereof, for use in treating or preventing a disease or condition mediated by RIPK1, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute neurological disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, nonalcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary disease, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, cancer (e.g., pancreatic cancer), bacterial infection, hematological malignancies, solid organ malignancies, and the like.

[0036] Another aspect of the present invention relates to a pharmaceutical composition comprising a compound, isomer, prodrug, solvate, stable isotopic derivative or pharmaceutically acceptable salt according to any embodiment of the present invention, optionally one or more other RIPK1 inhibitors, and one or more pharmaceutically acceptable carriers, diluents and excipients, for use in treating or preventing RIPK1-mediated diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute neurological diseases, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, non-alcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary disease, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, cancer (e.g., pancreatic cancer), bacterial infection, hematological malignancies, solid organ malignancies, and the like.

[0037] According to the present invention, the drug can be in any pharmaceutical dosage form, including but not limited to tablets, capsules, solutions, lyophilized preparations, and injections.

[0038] The pharmaceutical preparation of the present invention can be administered in a dosage unit form containing a predetermined amount of active ingredient per dosage unit. This unit can contain, for example, 0.5 mg to 1 gram, preferably 1 mg to 700 mg, particularly preferably 5 mg to 300 mg of the compound of the present invention, according to the disease to be treated, the method of administration, and the age, weight, and condition of the patient, or the pharmaceutical preparation can be administered in a dosage unit form containing a predetermined amount of active ingredient per dosage unit. Preferred dosage unit formulations are those containing the active ingredient of the daily dose or divided dose or its corresponding fraction as indicated above. In addition, methods known in the pharmaceutical field can be used to prepare this type of pharmaceutical preparation.

[0039] The pharmaceutical formulations of the present invention may be suitable for administration by any desired suitable method, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) administration. Such formulations may be prepared using all methods known in the pharmaceutical art, for example, by combining the active ingredient with one or more excipients or one or more adjuvants.

[0040] Preparation method

[0041] The present invention also provides methods for preparing the compounds of the present invention.

[0042] R 1 With R 2 As defined above, X is chlorine, bromine or iodine;

[0043] first step:

[0044] Compounds (I) and (II) are dissolved in a solvent (such as N,N-diisopropylethylamine), and under the protection of an inert gas (such as nitrogen or argon), a catalyst (such as cuprous iodide and bis(triphenylphosphine)palladium dichloride) is added. The system is evacuated, and the atmosphere is replaced with an inert gas (such as nitrogen or argon) three times. The reaction is carried out in an oil bath (60-130° C.) for 2-20 hours to obtain compound (III);

[0045] Step 2:

[0046] Dissolve compound (III) in a solvent (such as dichloromethane or dioxane), add an acid (such as hydrochloric acid or trifluoroacetic acid), and stir at room temperature for 1 to 5 hours to obtain compound (IV);

[0047] Step 3:

[0048] Dissolve compound (IV), compound (V), a condensing agent (such as 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), and a base (such as triethylamine) in a solvent (such as N,N-dimethylformamide), and stir at room temperature for 2 to 16 hours to obtain compound (VI);

[0049] Step 4:

[0050] Dissolve compound (VI) in a solvent (such as methanol), add a base (such as potassium carbonate), and stir at room temperature for 0.5 to 6 hours to obtain compound (VII);

[0051] Step 5:

[0052] Compounds (VII) and (VIII) are dissolved in tetrahydrofuran, and under the protection of an inert gas (such as nitrogen or argon), a base (such as triethylamine), a ligand (such as 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl), and a catalyst (such as cuprous iodide and bis(triphenylphosphine)palladium dichloride or cuprous iodide and tetrakis(triphenylphosphine)palladium) are added. The system is evacuated and replaced with an inert gas (such as nitrogen or argon) three times, and the reaction is heated in an oil bath at 60-90° C. for 2-20 hours to obtain compound (IX); DETAILED DESCRIPTION

[0053] definition

[0054] Unless otherwise stated, the following terms used in the specification and claims have the following meanings: Groups not specifically defined herein have their generally accepted meanings in the art as known to those skilled in the art.

[0055] The expression "Cx-Cy" used in the present invention represents the range of carbon atoms, wherein x and y are both integers, for example, C3-C8 cycloalkyl represents a cycloalkyl group having 3-8 carbon atoms.

[0056] In the present invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 20 carbon atoms, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, and various branched chain isomers thereof. Alkyl groups may be optionally substituted or unsubstituted.

[0057] In the present invention, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group containing 3 to 12 ring atoms, for example, 3 to 12, 5 to 10, 3 to 10, 3 to 8, or 3 to 6 ring atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2,2,1]heptane, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted.

[0058] In the present invention, the term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group comprising 3 to 20 ring atoms, for example, 3 to 16, 3 to 12, 3 to 10, 3 to 8 or 4 to 6 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 10 ring atoms, of which 1 to 4 are heteroatoms, more preferably the heterocyclyl ring comprises 3 to 8 ring atoms, more preferably 4 to 6 ring atoms, most preferably a 4-membered ring, a 5-membered ring or a 6-membered ring; wherein 1 to 4 are heteroatoms, more preferably 1 to 3 are heteroatoms, and most preferably 1 to 2 are heteroatoms. Non-limiting examples of heterocyclyl groups include oxetanyl, oxanyl, azetidinyl, morpholinyl, 2-morpholinyl, dihydropyrazolyl, etc. A heterocyclyl group may be optionally substituted or unsubstituted.

[0059] In the present invention, the term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group, a polycyclic (i.e., rings with adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, most preferably phenyl. Aryl groups can be substituted or unsubstituted.

[0060] In the present invention, the term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms include oxygen, sulfur and nitrogen. Preferably, the heteroaryl group comprises 1 to 3 heteroatoms, and the heteroatoms contained include at least one nitrogen atom. Preferably, the heteroaryl group is 5 to 10-membered. More preferably, the heteroaryl group is 5-membered or 6-membered. Preferred heteroaryls include pyrazolyl, imidazolyl, triazolyl (including 1,2,3-triazolyl, 1,2,4-triazolyl, etc.), thiazolyl, pyrazinyl, oxazolyl, isoxazolyl, pyridyl, etc. The heteroaryl group may be optionally substituted or unsubstituted.

[0061] In the present invention, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0062] In the present invention, the term "cyano" refers to -CN.

[0063] In the present invention, "optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0064] The substituents include but are not limited to the various groups described above.

[0065] The compounds claimed in the present invention include not only the compounds themselves but also optical isomers of the compounds or pharmaceutically acceptable salts thereof.

[0066] The term "pharmaceutical composition" as used herein refers to a mixture containing one or more compounds described herein, their optical isomers, or pharmaceutically acceptable salts thereof, and other chemical components. Other components include pharmaceutically acceptable carriers, diluents, and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0067] When used in the specification, the term "comprising" includes "consisting of".

[0068] The "room temperature" mentioned in the present invention refers to 15-30°C.

[0069] The "pharmaceutically acceptable salts" of the present invention are discussed in Berge, et al., "Pharmaceutically acceptable salts", J. Pharm. Sci., 1977, 66, 1-19, and are obvious to pharmaceutical chemists. The salts are substantially non-toxic and can provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion.

[0070] The pharmaceutically acceptable salts of the present invention can be synthesized by general chemical methods.

[0071] Generally, salts can be prepared by reacting a free base or acid with an equal chemical equivalent or excess of an acid (inorganic or organic) or base in a suitable solvent or solvent combination.

[0072] The term "prodrug" as used herein refers to a compound that is converted into the original active compound after metabolism in the body. Typically, a prodrug is inactive or less active than the active parent compound, but may provide convenient handling, administration, or improved metabolic properties.

[0073] The "isomers" of the present invention refer to tautomers, meso- and racemates, enantiomers, diastereomers, and mixtures thereof of the compounds of formula (I) of the present invention. All of these isomers, including stereoisomers such as optical isomers and geometric isomers, are encompassed by the present invention. The geometric isomers include cis- and trans-isomers.

[0074] The term "solvate" as used herein refers to an association of one or more solvent molecules with a compound of the present invention or a salt thereof. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, ethyl acetate, acetic acid, and the like.

[0075] The present invention includes any polymorphs and any hydrates or other solvates of the compound or its salt.

[0076] In the present invention, the term "patient" generally refers to mammals, especially humans.

[0077] In the present invention, the term "therapeutically effective amount" refers to an amount of the compound of the present invention that can effectively treat or prevent diseases mediated by RIPK1.

[0078] Example

[0079] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0080] The structures of all compounds of the present invention can be determined by nuclear magnetic resonance ( 1 H NMR) and / or mass spectrometry (MS).

[0081] 1 H NMR chemical shifts (δ) are reported in PPM (parts per million). NMR was performed on a Bruker AVANCE III-400 MHz spectrometer. Suitable solvents were selected from deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d 6 ), and tetramethylsilane (TMS) was used as the internal standard.

[0082] Low-resolution mass spectra (MS) were determined on an Agilent 1260 HPLC / 6120 mass spectrometer using an Agilent ZORBAX XDB-C18, 4.6×50 mm, 3.5 μm.

[0083] Gradient elution conditions: 0 min: 95% solvent A1 and 5% solvent B1, 1-2 min: 5% solvent A1 and 95% solvent B1; 2.01-2.50 min: 95% solvent A1 and 5% solvent B1. Percentages are the volume percentage of a particular solvent relative to the total solvent volume. Solvent A1: 0.01% formic acid in water; Solvent B1: 0.01% formic acid in acetonitrile. Percentages are the volume percentage of the solute relative to the total solvent volume.

[0084] Thin layer silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. Column chromatography generally uses Yantai Huanghai 100-200 or 200-300 mesh silica gel as a carrier.

[0085] Preparative liquid chromatography (prep-HPLC) was performed using a Waters SQD2 mass spectrometer-guided high-pressure liquid chromatography separator, XBridge-C18; 30×150 mm preparative column, 5 μm;

[0086] Method 1: acetonitrile-water (0.2% formic acid), flow rate 25 mL / min; Method 2: acetonitrile-water (0.8% ammonium bicarbonate), flow rate 25 mL / min;

[0087] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Shanghai Bid Pharmaceutical, Shanghai Aladdin Chemical, Shanghai Myrel Chemical, J&K Chemical, Anaiji Chemical, etc.

[0088] Unless otherwise specified in the examples, all solvents used in the reactions were anhydrous solvents, wherein commercially available tetrahydrofuran was used as an anhydrous tetrahydrofuran, sodium block was used as a dehydrating agent, and benzophenone was used as an indicator. The reaction was refluxed under argon protection until the solution turned blue-purple, and then collected by distillation and stored at room temperature under argon protection. Other anhydrous solvents were purchased from Anage Chemical and J&K Chemical. Unless otherwise specified, the transfer and use of all anhydrous solvents were carried out under argon protection.

[0089] Unless otherwise specified in the examples, all reactions were carried out under an argon or nitrogen atmosphere.

[0090] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0091] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0092] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0093] Unless otherwise specified in the examples, the reaction temperature is room temperature, which ranges from 15°C to 30°C.

[0094] The reaction progress in the examples was monitored by thin layer chromatography (TLC), and the developing solvent systems used in the reactions were A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compounds.

[0095] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include: A: dichloromethane and methanol; B: petroleum ether and ethyl acetate. The volume ratio of the solvents is adjusted according to the polarity of the compound and can also be adjusted by adding a small amount of triethylamine or an acidic or alkaline reagent.

[0096] The reagents used in the biological experiment of the present invention are as follows: acetonitrile (chromatographically pure) was purchased from Fisher-chemical, item number: A998-4L; dimethylacetamide (DMA) was purchased from Vetec, item number: V900211-500ML; DMSO (chromatographically pure) was purchased from Sigma-Aldrich, item number: D5879-1L; Solutol was purchased from Beijing Coupling Technology, item number: Solutol HS 15; methylcellulose MC was purchased from Sigma, item number: M6385-100G; terfenadine was purchased from Sigma, item number: MKBX6318V; Saline was purchased from Shandong Hualu Pharmaceutical, item number: H37022749; water was purchased from Watsons, item number: GB19298; warfarin was purchased from Dr. Ehrenstorfer GmbH, item number 30719; potassium dihydrogen phosphate was purchased from Vetec, USA, item number: WXBC3341V; disodium hydrogen phosphate was purchased from Vetec, USA, item number WXBC3348V.

[0097] The instruments used in the biological experiments of the present invention are: AB SCIEX Triple Quad 5500 and AB SCIEX Triple Quad 4500 liquid chromatography-mass spectrometry (products of AB SCIEX, USA), including Triple Quad 5500 and Triple Quad 4500 triple quadrupole tandem mass spectrometers, equipped with ESI source and Analyst 1.6.3 data processing system; the liquid phase part is AB SCIEX liquid phase, equipped with a high-pressure infusion pump, automatic sampler, and column oven; electronic balance: Sartorius, model: BT25S; centrifuge: Beckman, model: Allegra X-12R; refrigerators: Haier Group, China, models: HYC-198, BCD-248TMPM; homogenizer: Ding Haoyuan Company, model: TL2020; vortex mixer: Labnet Company, USA, model: VX-200; pipette dispensers (120 μL, 300 μL), purchased from Eppendorf; single pipettes (10 μL, 100 μL, 200 μL, 1000 μL), purchased from Eppendorf;

[0098] Intermediate 1

[0099] 4-(9-ethynyl-2,3,4,5-tetrahydropyridinyl[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0100] first step

[0101] 9-((Trimethylsilyl)ethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -tert-Butyl 4(5H)-formate 1a

[0102] Compound 9-bromo-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate (4.14 g, 12.60 mmol, synthesis reference WO2016075239) was dissolved in N, N-diisopropylethylamine (50 mL), and cuprous iodide (0.24 g, 1.26 mmol), bis(triphenylphosphine)palladium dichloride (0.88 g, 1.26 mmol) and trimethylsilyl acetylene (6.18 g, 63.00 mmol) were added sequentially at room temperature. The mixture was reacted at 80 ° C for 16 hours under nitrogen protection. The reaction solution was cooled to room temperature and filtered through celite. Water (30 mL) was added to the filtrate and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolvated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target product 9-((trimethylsilyl)ethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -tert-Butyl 4(5H)-formate 1a (3.49 g), yield: 80%.

[0103] MS m / z (ESI): 347 [M+1];

[0104] 1 H NMR (400MHz, CDCl3) δ 8.50 (s, 1H), 8.33-8.26 (m, 1H), 4.58-4.46 (m, 2H), 4.30 (t, J=4.0Hz, 2H), 3.86 (t, J=4.0Hz, 2H), 1.41 (s, 9H), 0.27 (s, 9H).

[0105] Step 2

[0106] 9-((Trimethylsilyl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine 1b

[0107] 9-((trimethylsilyl)ethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-Butyl-4(5H)-formate 1a (0.10 g, 0.004 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (2 mL) and stirred at room temperature for 30 minutes. Most of the solvent was removed by rotary evaporation under reduced pressure, and the mixture was neutralized by adding saturated aqueous sodium bicarbonate (20 mL). The mixture was then extracted with dichloromethane (20 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to yield the desired product, 9-((trimethylsilyl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine. 1b (80 mg, crude). MS m / z (ESI): 247 [M+1].

[0108] Step 3

[0109] 4-(9-((Trimethylsilyl)-ethynyl)-2,3,4,5-tetrahydropyridin[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 1c

[0110] 9-((trimethylsilyl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine 1b (10 mg, 0.04 mmol) and 4-cyanobicyclo[2.2.1]heptane-1-carboxylic acid (19 mg, 0.12 mmol, synthesis reference: US20150133428) were dissolved in N,N-dimethylformamide (1.5 mL). 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (30 mg, 0.08 mmol) and triethylamine (12 mg, 0.012 mmol) were added and stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate (5 mL) and washed with saturated brine (10 mL x 3). The separated organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 50:1) to obtain the target product 4-(9-((trimethylsilyl)ethynyl)-2,3,4,5-tetrahydropyridine[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 1c (12 mg), yield 99%.

[0111] MS m / z (ESI): 394 [M+1];

[0112] 1H NMR (400MHz, CDCl3) δ8.39 (s, 1H), 8.23 ​​(s, 1H), 4.62-4.54 (m, 2H), 4.38-4.23 (m, 2H), 3 .88-3.87(m, 2H), 2.07-2.00(m, 4H), 1.83-1.79(m, 4H), 1.65-1.58(m, 2H), 0.15(s, 9H).

[0113] Step 4

[0114] 4-(9-ethynyl-2,3,4,5-tetrahydropyridinyl[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile (Intermediate 1)

[0115] 4-(9-((trimethylsilyl)ethynyl)-2,3,4,5-tetrahydropyridinyl[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 1c (12 mg, 0.03 mmol) was dissolved in methanol (1.5 mL), potassium carbonate (21 mg, 0.15 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was filtered through celite. The organic phase was concentrated under reduced pressure to obtain the target product 4-(9-ethynyl-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile (Intermediate 1) (10.0 mg, crude).

[0116] MS m / z (ESI): 322 [M+1];

[0117] 1 H NMR (400MHz, CDCl3) δ8.47 (s, 1H), 8.33 (s, 1H), 4.73-4.58 (m, 2H), 4.49-4.28 (m, 2H), 3 .93-3.92(m, 2H), 3.32(s, 1H), 2.12-2.15(m, 4H), 1.98-1.84(m, 4H), 1.77-1.74(m, 2H).

[0118] Example 1

[0119] 4-(9-((1-(difluoromethyl)-1H-pyrazol-4-yl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0120] 4-(9-ethynyl-2,3,4,5-tetrahydropyridinyl[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile (Intermediate 1) (27 mg, 0.08 mmol), 1-(difluoromethyl)-4-iodo-1H-pyrazole (Synthesis Reference: WO2020043008 A1) (42 mg, 0.16 mmol) were dissolved in tetrahydrofuran (2 mL) and N,N-diisopropylethylamine (2 mL). Cuprous iodide (1 mg, 0.008 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (12 mg, 0.024 mmol) and tetrakistriphenylphosphine palladium (6 mg, 0.008 mmol) were added and stirred at 60 ° C overnight under nitrogen protection. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=50:1) to give the target compound 4-(9-((1-(difluoromethyl)-1H-pyrazol-4-yl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 1 (10 mg), yield 37%.

[0121] MS m / z (ESI): 438 [M+1];

[0122] 1 H NMR (400MHz, CDCl3) δ8.47 (s, 1H), 8.35 (s, 1H), 7.98 (s, 1H), 7.74 (s, 1H), 7.20 (t, J=60.0Hz, 1H), 4.70(s, 2H), 4.44-4.43(m, 2H), 3.97-3.95(m, 2H), 2.16-2.06(m, 2H), 1.87-1.18(m, 8H).

[0123] The synthetic steps of Examples 2 to 4 refer to Example 1.

[0124] Example 2

[0125] 4-(9-((3-fluoro-1-methyl-1H-pyrazol-4-yl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0126] MS m / z (ESI): 420 [M+1];

[0127] 1H NMR (400MHz, CDCl3) δ8.46 (s, 1H), 8.30 (s, 1H), 7.44 (s, 1H), 4.87-4.77 (m, 2H), 4.51-4.39 (m, 2H), 4.05-4.01(m, 2H), 3.82(s, 3H), 2.26-2.12(m, 4H), 2.03-1.92(m, 4H), 1.88-1.79(m, 2H).

[0128] Example 3

[0129] 4-(9-((5-fluoro-1-methyl-1H-pyrazol-4-yl)ethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0130] MS m / z (ESI): 420 [M+1];

[0131] 1 H NMR (400MHz, CDCl3) δ8.51 (s, 1H), 8.32 (s, 1H), 7.46 (s, 1H), 5.29-5.10 (m, 2H), 4.51-4.34 (m, 2H), 4.04-4.00(m, 2H), 3.82(s, 3H), 2.26-2.12(m, 4H), 2.03-1.92(m, 4H), 1.88-1.79(m, 2H).

[0132] Example 4

[0133] 4-(9-(1-methylpyrazol-4-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0134] MS m / z (ESI): 402[M+1];

[0135] 1 H NMR (400MHz, CDCl3) δ8.48 (s, 1H), 8.27 (s, 1H), 7.59 (s, 1H), 7.52 (s, 1H), 4.73-4.66 (m, 2H ), 4.43-4.34(m, 2H), 3.95-3.93(m, 2H), 3.85(s, 3H), 2.14-2.0(m, 6H), 1.84-1.77(m, 4H).

[0136] Example 5

[0137] 4-(9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0138] first step

[0139] 9-(3-((2-(tert-Butyldimethylsilyl)oxy)prop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -4(5H)-tert-Butylformate 5-1

[0140] 9-Bromo-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate (0.90 g, 2.74 mmol, synthesis reference WO2016075239) and tert-butyldimethyl(prop-2-yn-1-oxy)silane (1.86 g, 10.91 mmol) were dissolved in N,N-dimethylacetamide (6 mL) and N,N-diisopropylethylamine (6 mL), and cuprous iodide (0.14 g, 0.73 mmol) and tetrakis(triphenylphosphine)palladium (0.63 g, 0.55 mmol) were added. The reaction solution was purged with nitrogen twice and then stirred at 80°C for 8 hours. The reaction solution was cooled to room temperature and filtered through celite. Water (30 mL) was added to the filtrate, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolventized under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (petroleum ether: ethyl acetate = 8:1) to give 9-(3-((2-(tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -tert-Butyl 4(5H)-formate 5-1 (0.90 g), yield 78%. MS m / z (ESI): 419 [M+1];

[0141] Step 2

[0142] 9-(3-Hydroxyprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -4(5H)-tert-Butylformate 5-2

[0143] 9-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate 5-1 (0.12 g, 0.29 mmol) was dissolved in tetrahydrofuran (4 mL). Tetrabutylammonium fluoride (0.15 mL, 0.15 mmol, 1 M solution in tetrahydrofuran) was added dropwise in an ice-water bath. The mixture was allowed to react in an ice-water bath for 20 minutes. Water (15 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated brine (15 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolvated under reduced pressure to obtain the crude product. The target compound, 9-(3-hydroxyprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine, was obtained by thin-layer preparative chromatography (petroleum ether:ethyl acetate = 20:1). -tert-Butyl 4(5H)-formate 5-2 (70 mg), yield 36%.

[0144] MS m / z (ESI): 305 [M+1];

[0145] 1 H NMR (400MHz, CDCl3) δ8.53 (s, 1H), 8.35-8.29 (m, 1H), 4.67-4.40 (m, 4H), 4.31 (t, J=4.8Hz, 2H), 3.86 (t, J=4.8Hz, 2H), 1.41 (s, 9H).

[0146] Step 3

[0147] 9-(3-Carbonylprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -4(5H)-tert-Butylformate 5-3

[0148] 9-(3-hydroxyprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate 5-2 (0.28 g, 0.86 mmol) was dissolved in dry dichloromethane (10 mL). The reaction solution was cooled to 0°C in an ice-water bath, and Dess-Martin periodinane (0.73 g, 1.72 mmol) was added. The reaction was allowed to react at room temperature for 30 minutes. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate (15 mL) and extracted with dichloromethane (10 mL x 3). The combined organic phases were washed with saturated brine (15 mL x 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolvated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer preparative chromatography (petroleum ether:ethyl acetate = 20:1) to yield the target compound, 9-(3-carbonylprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine. -tert-Butyl 4(5H)-formate 5-3 (0.12 g), yield 46%.

[0149] MS m / z (ESI): 303 [M+1];

[0150] 1 H NMR (400MHz, CDCl3) δ9.46 (s, 1H), 8.61 (s, 1H), 8.35-8.29 (m, 1H), 4.56-4.43 (m, 2H), 4.28 (t, J=4.8Hz, 2H), 3.87 (t, J=4.8Hz, 2H), 1.42 (s, 9H).

[0151] Step 4

[0152] 9-(3,3-difluoroprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -4(5H)-tert-Butylformate 5-4

[0153] Under nitrogen protection, 9-(3-carbonylprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate 5-3 (0.12 g, 0.40 mmol) was dissolved in dry dichloromethane (5 mL). The mixture was cooled to 0°C, and diethylaminosulfur trifluoride (0.19 g, 1.20 mmol) diluted in dichloromethane (2 mL) was slowly added dropwise to the reaction mixture. The mixture was allowed to react at 0°C for 1 hour. The reaction was quenched with saturated aqueous sodium bicarbonate (10 mL) and extracted with dichloromethane (10 mL x 3). The organic phases were combined and washed with saturated brine (15 mL x 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolvated under reduced pressure to obtain the crude product. The crude product was purified by thin-layer preparative chromatography (petroleum ether:ethyl acetate = 20:1) to yield the target compound, 9-(3,3-difluoroprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine. -tert-Butyl 4(5H)-formate 5-4 (66 mg), yield 51%.

[0154] MS m / z (ESI): 325 [M+1];

[0155] 1 H NMR (400MHz, CDCl3) δ8.55 (s, 1H), 8.35-8.29 (m, 1H), 6.45 (t, J=56.4Hz, 1H), 4.56-4.43 (m, 2H), 4.28 (t, J=4.8Hz, 2H), 3.87 (t, J=4.8Hz, 2H), 1.42 (s, 9H).

[0156] Step 5

[0157] 9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine Trifluoroacetate 5-5

[0158] 9-(3,3-difluoroprop-1-yn-1-yl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate 5-4 (66 mg, 0.20 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.0 mL) was added at room temperature. The mixture was stirred at room temperature for 1 hour, concentrated and dried to give 9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine. Crude trifluoroacetic acid salt of 5-5 (70 mg). MS m / z (ESI): 225 [M+1].

[0159] Step 6

[0160] 4-(9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 5

[0161] 9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine Trifluoroacetate 5-5 (70 mg, crude) and 4-cyanobicyclo[2.2.1]heptane-1-carboxylic acid (0.04 g, 0.20 mmol) were dissolved in N,N-dimethylformamide (5 mL). 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.15 g, 0.40 mmol) and triethylamine (81 mg, 0.80 mmol) were added at room temperature and stirred at room temperature for 0.5 hours. The reaction was quenched with saturated brine (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined and washed with saturated brine (15 mL x 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and desolvated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column (dichloromethane: methanol = 20: 1) to give 4-(9-(3,3-difluoroprop-1-yn-1-yl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 5 (16 mg), yield 21.5%.

[0162] MS m / z (ESI): 372 [M+1];

[0163] 1 H NMR (400MHz, CDCl3) δ8.55-8.24 (br, 2H), 6.44 (t, J=56.4Hz, 1H), 4.73-6.60 (m, 2H), 4.50 -4.29 (m, 2H), 4.05-3.85 (m, 2H), 2.15-2.02 (m, 4H), 1.93-1.82 (m, 4H), 1.81-1.76 (m, 2H).

[0164] Example 6

[0165] 4-(9-(oxetan-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile

[0166] first step

[0167] 9-(Oxetane-3-ylethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine -4(5H)-tert-Butyl formate 6-1

[0168] 9-Bromo-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate (20 mg, 0.06 mmol) and 3-ethynyloxetane (0.06 g, 0.6 mmol) were dissolved in N,N-diisopropylethylamine (1 mL). Cuprous iodide (1 mg, 0.008 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (1 mg, 0.006 mmol), and bis(triphenylphosphine)dichloropalladium (4 mg, 0.006 mmol) were added. The reaction solution was sparged with nitrogen for 10 minutes and then stirred at 80°C overnight. The mixture was cooled to room temperature and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain the target product, 9-(oxetane-3-ylethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine. -tert-Butyl 4(5H)-formate 6-1 (20 mg), yield: 98%.

[0169] MS m / z (ESI): 331[M+1];

[0170] 1H NMR (400MHz, CDCl3) δ8.41 (s, 1H), 8.19 (s, 1H), 4.86-4.74 (m, 4H), 4.46-4.39 (m, 2H), 4.31-4.22 (m, 2H), 4.11-4.02 (m, 1H), 3.81-3.74 (m, 2H), 1.35 (s, 9H).

[0171] Step 2

[0172] 9-(Oxetane-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine 6-2

[0173] 9-(Oxetane-3-ylethynyl)-2,3-dihydropyrido[3,4-f][1,4]oxazepine Tert-butyl-4(5H)-formate 6-1 (0.02 g, 0.06 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (0.5 mL) and stirred at room temperature for 30 minutes. Most of the solvent was removed by rotary evaporation under reduced pressure, saturated sodium bicarbonate aqueous solution (20 mL) was added, and the mixture was extracted with dichloromethane (20 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the mixture was concentrated under reduced pressure to obtain the target product 9-(oxetane-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine 6-2 (30mg, crude product).

[0174] MS m / z (ESI): 231[M+1];

[0175] 1 H NMR (400MHz, CDCl3) δ8.46 (s, 1H), 8.23 ​​(s, 1H), 4.90-4.82 (m, 4H), 4.31-4.21 (m, 2H), 4.16-4.10(m, 1H), 4.03-3.96(m, 2H), 3.76-3.72(m, 1H), 3.29-3.27(m, 2H).

[0176] Step 3

[0177] 4-(9-(oxetan-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 6

[0178] 9-(Oxetane-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine 6-2 (30 mg, 0.10 mmol) and 4-cyanobicyclo[2.2.1]heptane-1-carboxylic acid (42 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (2.0 mL). 2-(7-Azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.98 g, 0.20 mmol) and triethylamine (1.0 mL) were added and stirred at room temperature for 1 hour. The mixture was then diluted with ethyl acetate (5 mL) and washed with brine (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried by spin drying. Purification by silica gel column chromatography (dichloromethane:methanol = 20:1) afforded the desired product, 4-(9-(oxetan-3-ylethynyl)-2,3,4,5-tetrahydropyrido[3,4-f][1,4]oxazepine -4-carbonyl)bicyclo[2.2.1]heptane-1-carbonitrile 6 (10 mg), yield: 45%.

[0179] MS m / z (ESI): 378 [M+1];

[0180] 1 H NMR (400MHz, CDCl3) δ8.40 (s, 1H), 8.27 (s, 1H), 4.84-4.82 (m, 2H), 4.80-4.73 (m, 2H), 4.40-4.27 (m, 1H), 4.09-4.00(m, 2H), 3.93-3.91(m, 2H), 3.78-3.76(m, 2H), 2.11-2.04(m, 4H), 1.88-1.82(m, 6H).

[0181] Biological experiments

[0182] Example 7: RIPK1 kinase inhibitory activity detection

[0183] Experimental method: This experiment uses the ADP-Glo ​​kinase activity assay to test the inhibitory effect of the compound on RIPK1 kinase activity and to obtain the half-maximal inhibitory concentration (IC) of the compound on RIPK1 kinase activity. 50First, dilute the enzyme reaction buffer. Dilute the enzyme reaction buffer stock solution (5x) from the ADP-Glo ​​Kinase Assay Kit purchased from Promega with deionized water and add DTT and 5mM MnCl2 to a final concentration of 1mM. Compounds were serially diluted 4-fold with DMSO and then diluted 40-fold with enzyme reaction buffer. Recombinant human RIPK1 protein was purchased from SignalChem, and recombinant monkey RIPK1 protein was purchased from Abcam. To a 384-well plate, 4 μL of compound solution and 2 μL of RIPK1 kinase solution diluted in reaction buffer were added. The final concentrations of human and monkey RIPK1 were 1 ng / μL and 4 ng / mL, respectively. The plates were mixed well and incubated at room temperature for 30 minutes. Then, 2 μL of ATP solution diluted in reaction buffer was added. After incubation at room temperature for 2 hours, 5 μL of ADP-Glo ​​was added to each well and the plates were incubated at room temperature for another 40 minutes. Finally, 10 μL of the kinase detection reagent in the kit was added to each well and the plates were incubated at room temperature for another 30 minutes. The chemiluminescent signal in each well was detected using an Envision multi-function microplate reader (Perkin Elmer, Waltham, MA).

[0184] Example 8: Experiments on TNF-α-induced p-RIPK1 activation in different species

[0185] Experimental method: This experiment uses the ultrasensitive electrochemiluminescence method of Meso Scale Discovery (MSD) to test the inhibitory effect of the compound on the level of p-RIPK1 and to obtain the half-maximal inhibitory concentration (IC) of the compound on the level of p-RIPK1. 50 .

[0186] Peripheral blood mononuclear cells (PBMC) from healthy humans and cynomolgus monkeys were purchased from Shanghai Aoneng Biotechnology Co., Ltd. Rat and mouse bone marrow mesenchymal stem cells (BMDM) were isolated from the femur and tibia of rats or mice. The compound was serially diluted 5-fold to 8 concentration points, with the highest final concentration being 5000 nM. 0.5-1×10 5 Cells were cultured in plates containing either PBMC or BMDM. TNF-α, Z-VAD-FMK, and SM-164 were added and incubated for 3 hours at 37°C in a 5% CO2 incubator. DMSO treatment served as a control. The cells were then washed with PBS and lysed with cell lysis buffer. Lysed on ice for 30 minutes, the cells were transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 minutes. The cell lysate was collected and protein quantified using the BCA assay.

[0187] p-RIPK levels in cell lysates were measured using the Meso Scale Discovery (MSD) assay. Biotinylated RIPK1 capture antibody was diluted in PBS and added to MSD 96-well streptavidin plates overnight at 4°C. Plates were blocked with MSD blocking buffer for 2 hours and then incubated with 20–40 μL of lysate at room temperature for 2 hours. A Phospho-RIPK1 antibody (Cell Signaling #31122) diluted in blocking buffer was added as a detection antibody and incubated at room temperature for 1 hour. A SULFO-conjugated goat anti-rabbit antibody (MSD, R32AB-1) was diluted 500–1000-fold in blocking buffer and incubated at room temperature for another 45 minutes. Then, 100 μL of a 2-fold diluted detection buffer (R92TC-3) was added. After incubation, the electrochemical signal was read on an MSD Meso Sector Imager S600.

[0188] Table 1: RIPK1 enzyme activity detection results

[0189] (The reference compound is Example 29 in patent US11203600B2)

[0190] The structure of Example 29 compound in patent US11203600B2 is as follows:

[0191] From the data in Table 1, the enzyme activity of the compounds of the present application, especially compound 1, has little species difference. Unlike Example 29 (US11203600B2), some compounds in the present application still maintain high activity in rodents.

[0192] Example 9: Programmed Necrosis of I2.1 Cells

[0193] I2.1 cells are Jurkat cells with FADD gene knockout, suitable for studying programmed cell necrosis under TNF-α-induced conditions.

[0194] Experimental Methods: I2.1 cells (purchased from ATCC, catalog number CRL-2572; cultured in 1640 medium at 37°C in a cell culture incubator with 5% CO2) were plated in 384-well plates. Compounds were serially diluted four-fold to a final starting concentration of 500 nM. 40 ng / mL TNF-α and the test drug were co-incubated with the cells for 24 hours. Example 29 (US11203600B2) was used as a positive control, and DMSO was used in control wells.

[0195] Cell viability was measured using the Cell-TiterGlo kit based on changes in ATP levels. The values ​​for cell viability are expressed as a percentage of cell viability in drug-treated wells compared to control wells, with higher values ​​representing greater cell viability (drug-induced viability calculation = drug-treated wells / control wells * 100%).

[0196] Example 10: Programmed Necrosis of HT-29 Cells

[0197] Experimental methods: HT-29 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP30001L) were cultured in a cell culture incubator at 37°C with 5% CO2 in 1640 medium. The cells were plated in 384-well white plates. The compounds were serially diluted 4-fold to a final starting concentration of 500 nM. 100 ng / mL TNF-α, Q-VD-Oph (10 μM), SM-164 (1 μM) and the test drugs were incubated with the cells for 48 hours. Example 29 (US11203600B2) was used as a positive control, and DMSO was used in the control wells.

[0198] Cell viability was determined using the Cell-TiterGlo kit based on ATP-based viability assay. The values ​​for cell viability assays represent the percentage of cell viability in drug-treated wells compared to control wells, with higher values ​​representing greater cell viability (drug-treated well / control well * 100%).

[0199] Example 11: Programmed Necrosis of L-929 Cells

[0200] Experimental Methods: L-929 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60878) were cultured in a cell culture incubator at 37°C with 5% CO2 in MEM medium and plated in 384-well white plates. Compounds were serially diluted 4-fold to a final starting concentration of 10,000 nM. 40 ng / mL TNF-α, Q-VD-Oph (10 μM), and the test drug were co-incubated with the cells for 24 hours. Example 29 was used as a positive control, and DMSO was used in the control wells. Cell viability was determined using the Cell-TiterGlo kit based on ATP viability assay. Cell viability assays represent the percentage of cell viability in drug-treated wells compared to control wells, with higher values ​​indicating stronger cell viability (drug activity calculation = drug-treated wells / control wells * 100%).

[0201] Table 2: Cell activity test results of the compounds of the present invention

[0202] The data in Table 2 further show that some compounds of the present application still maintain high inhibitory activity in rodent L929 cells, which is consistent with the low species variability in their enzyme activity, and their activity against HT29 cells and 12.1 cells is significantly better than that of Example 29 (US11203600B2).

[0203] Example 12: Pharmacokinetic study and blood-brain barrier permeability determination in SD rats

[0204] The concentrations in plasma, brain tissue, and CSF samples were determined by LC-MS / MS to obtain the ratio of the CSF concentration to the plasma concentration, K. p，CSF , the ratio of CSF concentration to free plasma sample concentration K puu， CSF , the ratio of brain tissue to plasma exposure K p,Brain and the ratio of free brain tissue to free plasma exposure, K puu,Brain , thereby quickly screening out compounds with blood-brain penetrability. At the same time, Phoenix WinNolin was used to calculate plasma pharmacokinetic parameters to screen out compounds with good pharmacokinetic properties.

[0205] Experimental conditions and process:

[0206] 1. Dispensing medicine

[0207] Weigh an appropriate amount of the test sample into a weighing bottle, add 5% DMA / 10% Solutol / 85% Saline in sequence, and stir at room temperature to form a homogeneous solution for oral administration.

[0208] 2. Medication

[0209] Twenty-four male Sprague-Dawley Sprague-Dawley rats (N = 3 / time point) were weighed and prepared for each group. The dose was 5 mg / kg in a 10 mL / kg volume, administered via oral gavage. Plasma and brain tissue were collected at 0.25, 0.5, 1, 2, 3, 5, 8, and 24 hours after administration, and cerebrospinal fluid (CSF) was collected at 1 and 5 hours.

[0210] 3. LC-MS / MS method

[0211] According to the precise molecular weight of the compound, the corresponding parent ion and fragment ion are found, and the appropriate mass spectrometry conditions and liquid phase methods are optimized.

[0212] 4. Preparation of standard curve and quality control samples

[0213] Preparation of working solution: Accurately weigh an appropriate amount of test sample and dissolve it in DMSO to prepare a 2 mg / mL standard stock solution. Using the standard stock solution, add acetonitrile and water (50:50, v / v) to prepare a series of test sample standard curves and quality control working solutions.

[0214] Preparation of plasma standard curve and quality control: Take 20 μL of SD rat blank plasma, add 2 μL of test sample standard curve and quality control working solution of series concentration to prepare standard curve plasma samples and quality control plasma samples.

[0215] Preparation of brain tissue homogenate standard curve and quality control: Take 20 μL of SD rat brain tissue blank homogenate, add 2 μL of test sample standard curve and quality control working solution of serial concentrations to prepare standard curve brain tissue samples and quality control brain tissue samples.

[0216] Preparation of cerebrospinal fluid (CSF) standard curve and quality control: Take 5 μL of blank CSF from SD rats, add 5 μL of the test sample standard curve and quality control working solution to prepare the standard curve CSF sample and quality control CSF sample.

[0217] 5. Sample Analysis Process

[0218] Plasma: Add 20 μL of plasma sample to 2 μL of acetonitrile and water (50:50, v / v) in a 1.1 mL tube. Add 200 μL of internal standard solution (5 ng / mL terfenadine). Vortex for 1 minute. Centrifuge at 3000 rpm for 10 minutes at 4°C. Dilute the supernatant with water, vortex to mix, and inject for analysis. Prepare the standard curve and quality control samples in the same manner when determining sample concentrations.

[0219] Brain tissue samples: Weigh and record the weight of a tissue sample in a 2 mL centrifuge tube. Add purified water at a 1:3 ratio (g:mL) and homogenize at 1800 rpm for 10 minutes. Place 20 μL of the homogenate in a 1.1 mL tube and add 200 μL of the internal standard solution (5 ng / mL terfenadine). Vortex for 1 minute and centrifuge at 3000 rpm for 20 minutes at 4°C. Dilute the supernatant with water, vortex to mix, and then inject the sample for analysis. Perform the same standard curve and quality control samples for sample concentration determination.

[0220] Cerebrospinal Fluid (CSF): 5 μL of CSF sample was added to 5 μL of acetonitrile and water (50:50, v / v). The sample was placed in a 1.1 mL tube. 200 μL of internal standard solution (5 ng / mL terfenadine) was added. The tube was vortexed for 1 minute. Centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was diluted with water, vortexed to mix thoroughly, and then injected for analysis. The standard curve and quality control samples were prepared in the same manner when determining sample concentrations.

[0221] 6. Sample testing

[0222] Samples were analyzed using an appropriate LC-MS / MS method.

[0223] 7. Data Processing

[0224] The main computerized systems used in this experiment are:

[0225] Microsoft Office Excel: 2010, data entry, calculation, statistical data;

[0226] Analysis 1.6.3, data collection and processing;

[0227] Phoenix WinNolin 8.3.1, calculation of pharmacokinetic parameters

[0228] GraphPad Prism 6.0, drug concentration-time curves

[0229] Table 3: Pharmacokinetic results of the RIPK1 inhibitors of the present invention in SD rats

[0230] From the pharmacokinetic data in Table 3, it can be seen that the exposure per unit time of compound 1 is much better than that of Example 29 (US11203600B2), and the half-life in plasma is t 1 / 2 Longer.

[0231] Table 4: Brain permeability assay results of the RIPK1 inhibitors of the present disclosure in SD rats

[0232] From the data in Table 4, it can be seen that compound 1 of the present application exhibits good blood-brain barrier penetration and good cerebrospinal fluid distribution, and can be used to treat central nervous system diseases.

[0233] Example 13: Pharmacokinetic Study in Beagle Dogs

[0234] The concentrations of the compounds in beagle dog plasma were determined by LC-MS / MS, and the pharmacokinetic parameters of the plasma were calculated using Phoenix WinNolin to screen out compounds with better pharmacokinetic properties.

[0235] Experimental conditions and process:

[0236] 1. Dispensing medicine

[0237] Weigh an appropriate amount of the test sample into a weighing bottle, sequentially add 10% DMA / 10% PEG400 / 80% Saline, and stir at room temperature until a homogeneous solution is obtained for intravenous administration. Weigh an appropriate amount of the test sample into a weighing bottle, sequentially add PEG-400 / 0.5% MC (v / v, 4 / 6), and stir at room temperature until a homogeneous solution is obtained for oral administration.

[0238] 2. Medication

[0239] Three male beagle dogs were weighed and prepared for each group. A dose of 1 mg / kg was administered intravenously in a volume of 1 mL / kg. Plasma was collected at 0.083 h, 0.25 h, 1 h, 2 h, 4 h, 8 h, and 24 h after dosing.

[0240] Three male beagle dogs were weighed and prepared for each group. The dose was 3 mg / kg in a 5 mL / kg volume, administered via gavage. Plasma was collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing.

[0241] 3. LC-MS / MS method

[0242] According to the precise molecular weight of the compound, the corresponding parent ion and fragment ion are found, and the appropriate mass spectrometry conditions and liquid phase methods are optimized.

[0243] 4. Preparation of standard curve and quality control samples

[0244] Preparation of working solution: Accurately weigh an appropriate amount of test sample and dissolve it in DMSO to prepare a 2 mg / mL standard stock solution. Using the standard stock solution, add acetonitrile and water (50:50, v / v) to prepare a series of test sample standard curves and quality control working solutions.

[0245] Preparation of plasma standard curve and quality control: Take 20 μL of beagle dog blank plasma, add 2 μL of test sample standard curve and quality control working solution of series concentration to prepare standard curve plasma samples and quality control plasma samples.

[0246] 5. Sample Analysis Process

[0247] Plasma: Add 20 μL of plasma sample to 2 μL of acetonitrile and water (50:50, v / v) in a 1.1 mL tube. Add 200 μL of internal standard solution (5 ng / mL terfenadine). Vortex for 1 minute. Centrifuge at 3000 rpm for 10 minutes at 4°C. Dilute the supernatant with water, vortex to mix, and inject for analysis. Prepare the standard curve and quality control samples in the same manner when determining sample concentrations.

[0248] 6. Sample testing

[0249] Samples were analyzed using an appropriate LC-MS / MS method.

[0250] 7. Data Processing

[0251] The main computerized systems used in this experiment are:

[0252] Microsoft Office Excel: 2010, data entry, calculation, statistical data;

[0253] Analysis 1.6.3, data collection and processing;

[0254] Phoenix WinNolin 8.3.1, calculation of pharmacokinetic parameters

[0255] GraphPad Prism 6.0, drug concentration-time curves

[0256] Table 5: Pharmacokinetic results of the RIPK1 inhibitors of the present invention in beagle dogs

[0257] From the pharmacokinetic data in Tables 3 and 5, it can be seen that the pharmacokinetic properties of Compound 1 in different species, especially in pit bulls, are much better than those of Example 29 (US11203600B2).

[0258] Example 14: Determination of plasma protein binding rate

[0259] Plasma protein binding was determined using 96-well equilibrium dialysis devices (RED Device Inserts). The binding of the test article to plasma proteins from different species was determined by incubating the test article at different concentrations of 0.100, 0.500, and 2.50 μM. The binding rate after 5 hours of incubation was determined.

[0260] Experimental conditions and process:

[0261] 1. Preparation of various reaction solutions

[0262] Accurately weigh a certain amount of test sample and reference substance (warfarin) and prepare a stock solution of a certain concentration with DMSO. Dilute the test sample and reference substance stock solutions with DMSO to prepare working solutions with concentrations of 250 μM, 50 μM, and 10 μM, respectively. Mix 5 μL of each test solution with 495 μL of blank plasma of each species. The concentrations of the test sample and reference substance in the resulting plasma are 2.50 μM, 0.500 μM, and 0.100 μM, respectively.

[0263] 2. Plasma protein binding rate determination

[0264] 200 μL of each plasma reaction solution was added to the sample chamber (side A) of the balanced dialysis tubing. 350 μL of 100 mM phosphate buffer (pH 7.4) was added to the dialysate chamber (side B) of the balanced dialysis tubing. This was repeated in duplicate (n=2). The substrate plate was then placed in a 37°C constant-temperature shaker with horizontal agitation at 140 rpm. After 5 hours, 20 μL of each sample was sampled from the sample chamber (side A) and the buffer chamber (side B).

[0265] 20 μL of phosphate buffer was added to 20 μL of sample taken out from the sample chamber (side A), and vortexed at 1000 rpm for 1 minute. Then 400 μL of stop solution (10 ng / mL terfenadine) was added to the sample on side A.

[0266] Add 20 μL of plasma of the corresponding species to the 20 μL sample removed from the dialysate chamber (side B). Vortex at 1000 rpm for 1 minute, then add 400 μL of stop solution (10 ng / mL terfenadine) to the samples on both sides of the B chamber.

[0267] After refrigeration at 4°C for 30 minutes, centrifuge at 3000 rpm for 15 minutes at 4°C. The supernatant was diluted with water, vortexed, and analyzed by LC-MS / MS.

[0268] 3. LC-MS / MS method

[0269] According to the precise molecular weight of the compound, the corresponding parent ion and fragment ion are found, and the appropriate mass spectrometry conditions and liquid phase methods are optimized.

[0270] 4. Data Analysis

[0271] The plasma protein binding rate is calculated by the following formula:

[0272] Among them C D and C R are the peak area ratios of the test sample and internal standard measured in the supply chamber and the receiving chamber after incubation, respectively, f b and f u are the protein-bound and free percentages, respectively.

[0273] The recovery rate is calculated by the following formula:

[0274] Among them C I The peak area ratio of the test sample and the internal standard measured for the 0 hr sample.

[0275] Table 6: Plasma protein binding rate determination results of the RIPK1 inhibitors of the present invention

[0276] As can be seen from Table 6, 20-30% of the compound 1 of the present application is in a free state in the plasma of various species and can enter the central nervous system. Combining the data in Tables 3 and 4, it can be considered that compound 1 has good brain penetration and can be used to treat central nervous system diseases.

[0277] Based on the above experiments, the cell activity of compound 1 of the present application in different cell lines is 53 to 64 times that of reference compound Example 29 (US1120360082). The exposure of compound 1 of the present application in PO experiments of SD rats and beagle dogs at the same dose is 1.9 times and 51 times that of reference compound Example 29 (US11203600B2), respectively. It is expected that the efficacy of compound 1 of the present application is far superior to that of reference compound Example 29 (US11203600B2) in the treatment of various diseases mediated by RIPK1.

[0278] The above experimental results demonstrate that the compounds of the present invention effectively inhibit RIPK1 activity, with minimal species variability, good plasma protein binding, and brain penetrance. They are potentially useful in treating or preventing RIPK1-mediated diseases and conditions, particularly central nervous system disorders such as rheumatoid arthritis, ulcerative colitis, psoriasis, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis.

Claims

1. A compound of formula (I), its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, Wherein: A is a 6- to 8-membered saturated heterocycle containing 1 to 3 heteroatoms selected from N, O, and S; R 1 selected from halo C1-C8 alkyl, C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclic group, wherein the C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclic group is unsubstituted or substituted by one or more R x wherein the R x are each independently selected from halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is unsubstituted or substituted by one or more halogen or cyano; B is selected from C5-C10 cycloalkyl optionally substituted by one or more substituents R 2 wherein the R 2 are each independently selected from halogen and cyano.

2. The compound according to claim 1, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, which have the formula (II): Wherein: R 1 selected from halo C1-C8 alkyl, C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclic group, wherein the C6-C10 aryl, 5-10 membered heteroaryl, C3-C8 cycloalkyl, 4-8 membered heterocyclic group are unsubstituted or substituted by one or more R x wherein the R x are each independently selected from halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is unsubstituted or substituted by one or more halogen or cyano; B is selected from C5-C10 cycloalkyl optionally substituted by one or more substituents R 2 wherein said R 2 are each independently selected from halogen and cyano.

3. The compound according to claim 2, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, wherein: R 1 Selected from halo C1-C6 alkyl, C6-C10 aryl, 5-8-membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, C3-C8 cycloalkyl, 4-8-membered saturated heterocyclic group containing 1-2 heteroatoms selected from N and O, wherein the C6-C10 aryl, 5-8-membered heteroaryl, C3-C8 cycloalkyl, 4-8-membered saturated heterocyclic group are unsubstituted or substituted by 1-2 R x wherein the R x are each independently selected from fluorine, chlorine, bromine, C1-C4 alkyl; wherein the C1-C4 alkyl is unsubstituted or substituted by 1-3 substituents selected from fluorine, chlorine, bromine, cyano; B is selected from C6-C10 cycloalkyl optionally substituted with 1-3 substituents R 2 wherein each R 2 is independently selected from halogen and cyano.

4. The compound according to claim 2, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, wherein: R 1 selected from halo C1-C4 alkyl, phenyl, 5-6-membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, C3-C6 cycloalkyl, 4-6-membered saturated heterocyclic group containing 1 heteroatom selected from N and O, wherein the phenyl, 5-6-membered heteroaryl, C3-C6 cycloalkyl, 4-6-membered saturated heterocyclic group are unsubstituted or substituted by 1-2 R x ; wherein said R x are each independently selected from fluorine, chlorine, C1-C4 alkyl, and the C1-C4 alkyl is unsubstituted or substituted by 1-3 substituents selected from fluorine and cyano; B is selected from C6-C10 cycloalkyl optionally substituted with 1 substituent R 2 wherein R 2 is selected from fluorine, chlorine and cyano.

5. The compound according to claim 2, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, wherein: R 1 selected from C1-C4 alkyl substituted by 1-3 fluorines, 5-membered heteroaryl containing 1-2 heteroatoms selected from N, O and S, 4-6 membered saturated heterocyclic group containing 1 O heteroatom, wherein the 5-membered heteroaryl and 4-6 membered saturated heterocyclic group are unsubstituted or substituted by 1-2 R x wherein the R x are each independently selected from fluorine, methyl, methyl substituted by 1-2 fluorines; B is selected from wherein R 2 is selected from fluorine and cyano groups.

6. The compound according to claim 2, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, wherein: R 1 Selected from B is selected from 7. The compound according to claim 1, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, which are selected from:

8. A pharmaceutical composition comprising the compound according to any one of claims 1-7, its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of the compound according to any one of claims 1-7 or its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts or the pharmaceutical composition according to claim 8 in the preparation of a drug for use as a RIPK1 inhibitor.

10. Use of the compound according to any one of claims 1-7 or its isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts or the pharmaceutical composition according to claim 8 in the preparation of a drug for the treatment or prevention of RIPK1-mediated related diseases or disorders.

11. The use according to claim 10, wherein the RIPK1-mediated related diseases or disorders are selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, acute neurological diseases, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, non-alcoholic steatohepatitis, alcoholic steatohepatitis, autoimmune hepatitis, autoimmune hepatobiliary diseases, systemic inflammatory response syndrome, cerebrovascular accident, Huntington's disease, pancreatic cancer, bacterial infection, hematological malignancies, solid organ malignancies.

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