Application of TBK1 inhibitor in preparation of medicine for autoimmune diseases

Abstract

The invention provides application of a compound (miindopturine) as shown in a formula I, or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug of the compound in preparation of a medicine. The medicine is used for inhibiting the activity of TBK1 kinase. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to the use of TBK1 inhibitors in preventing and / or treating autoimmune diseases. Background Art

[0002] Autoimmune diseases are a class of diseases caused by dysfunction of the immune system, characterized by the immune system mistakenly attacking its own healthy tissues, causing inflammation and tissue damage. Common autoimmune diseases include systemic lupus erythematosus (SLE), systemic sclerosis (SSc), ulcerative colitis (UC), Sjögren's syndrome (SjD), etc. Although the etiology of these diseases has not been fully clarified, more and more studies have shown that inflammatory response and abnormal activation of the immune system play a vital role in their pathogenesis. Although important progress has been made in the study of the pathogenesis, genetic factors and environmental triggers of autoimmune diseases in recent years, there is still a lack of radical treatment methods. Existing treatment methods mainly focus on immunosuppression and inflammation regulation to alleviate disease symptoms and delay disease progression. However, these treatment methods have many limitations, such as limited efficacy, obvious side effects, and large individual differences among patients, making it difficult to achieve precise treatment.

[0003] Interferon (IFN) is a type of cytokine secreted by immune cells and infected cells, which plays an important role in antiviral immunity, immune regulation and inflammatory response. IFN activates a series of downstream signal transduction pathways by binding to cell surface receptors and enhances immune response by activating various immune molecules. Although IFN plays an important role in normal immune response, its excessive or dysregulated activation is closely related to the occurrence and progression of many autoimmune diseases.

[0004] TBK1 (TANK-binding kinase 1) is an important hub of the IFN pathway, which can mediate the activation signals of multiple pattern recognition receptors and trigger the initiation of IFN signals by downstream IRF3 / 7. For example, TBK1 can be activated by cGAS-STING, which recognizes double-stranded DNA, as well as TLR3-TRIF, which recognizes double-stranded RNA and single-stranded DNA, and TLR4-TRAM, which recognizes lipopolysaccharide. TBK1 is not only involved in the activation of immune cells and the production of inflammatory factors, but is also closely related to the occurrence and development of various autoimmune diseases. When the TBK1 pathway is abnormally activated, it will lead to excessive inflammatory response, thereby promoting the progression of autoimmune diseases. The protein phosphorylation level of TBK1 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SjD), etc. will be abnormally increased. When the patient's peripheral blood mononuclear cells are treated with TBK1 inhibitors, the activation of IFN can be effectively inhibited.

[0005] Therefore, TBK1 is a potential therapeutic target for autoimmune diseases, and there is an urgent need to develop new TBK1 inhibitors for clinical use in the treatment of related diseases. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. In view of the problem that there is currently a lack of effective treatments for autoimmune diseases, in a first aspect of the present invention, the present invention proposes the use of a compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of a drug, wherein the drug is used to inhibit the activity of TBK1 kinase,

[0007]

[0008] The inventors of the present invention have discovered in experiments that the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, namely midostaurin, can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase, and IFN signals are related to immune regulation. Therefore, the inventors have verified through cell model and animal model experiments that midostaurin can effectively treat autoimmune diseases, which provides a new alternative for the treatment of autoimmune diseases.

[0009] In the second aspect of the present invention, the present invention provides the use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of drugs, wherein the drugs are used to prevent and / or treat TBK1-mediated related diseases.

[0010]

[0011] The inventors of the present invention have discovered in experiments that the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, namely midostaurin, can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase, and IFN signals are related to immune regulation. Therefore, the inventors have verified through cell model and animal model experiments that midostaurin can effectively treat autoimmune diseases, which provides a new alternative for the treatment of autoimmune diseases.

[0012] According to an embodiment of the present invention, the TBK1-mediated related disease is an autoimmune disease.

[0013] According to an embodiment of the present invention, the autoimmune disease includes at least one of systemic sclerosis (SSc), systemic lupus erythematosus (SLE), Aicardi-Goutieres syndrome (AGS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), STING-associated vasculopathy in infancy (SAVI), Niemann-Pick disease type C (NPC), Sjögren's syndrome (SjD), arthritis, pulmonary hemorrhage (DAH), pyrrhosis, chronic lymphocytic thyroiditis (CLT), hyperthyroidism, insulin-dependent diabetes mellitus (IDDM), myasthenia gravis (MG), ulcerative colitis (UC), Crohn's disease (CD), pernicious anemia with chronic atrophic gastritis (PAG), primary biliary cirrhosis (PBC), multiple sclerosis (MS), and acute idiopathic polyneuritis (AIP).

[0014] In the third aspect of the present invention, the present invention provides the use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of reagents for regulating the activation of IFN,

[0015]

[0016] As mentioned above, the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase. Therefore, after being prepared as a solvent, it can effectively regulate the activation of IFN.

[0017] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: a compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0018]

[0019] According to an embodiment of the present invention, the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can inhibit the activity of TBK1 kinase and treat TBK1-mediated related diseases. Thus, a pharmaceutical composition containing the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can effectively prevent and / or treat TBK1-mediated related diseases.

[0020] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or carrier.

[0021] According to an embodiment of the present invention, the pharmaceutical composition is an injection preparation or an oral preparation.

[0022] In the fifth aspect of the present invention, the present invention proposes use of the pharmaceutical composition described in the fourth aspect in the preparation of a drug for preventing and / or treating TBK1-mediated related diseases.

[0023] According to an embodiment of the present invention, the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can inhibit the activity of TBK1 kinase and treat TBK1-mediated related diseases. Thus, a pharmaceutical composition containing the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can effectively prevent and / or treat TBK1-mediated related diseases.

[0024] According to an embodiment of the present invention, the TBK1-mediated related disease is an autoimmune disease.

[0025] According to an embodiment of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, STING-related vascular disease in infancy, Niemann-Pick disease type C, Sjögren's syndrome, arthritis, pulmonary hemorrhage, herpes simplex, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Crohn's disease, pernicious anemia with chronic atrophic gastritis, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0028] Figure 1 The figure shows the inhibitory effect of midostaurin on TBK1 kinase activity detected by ADP-Glo ​​in Example 1 of the present invention;

[0029] Figure 2 It shows that the inhibitory effect of midostaurin on IFN signaling was detected using the THP1-ISRE-Lucia reporter cell line in Example 2 of the present invention;

[0030] Figure 3 The inhibitory effect of midostaurin on the expression level signal of IFN-related genes in Example 2 of the present invention is shown by using fluorescent quantitative PCR to detect;

[0031] Figure 4 It shows that the inhibitory effect of midostaurin on IFN signaling was detected using the gene knockdown THP1-ISRE-Lucia reporter cell line in Example 3 of the present invention;

[0032] Figure 5 The figure shows the therapeutic effect of midostaurin on the skin symptoms of systemic sclerosis mice detected by H&E staining and Masson staining in Example 4 of the present invention;

[0033] Figure 6 The figure shows the therapeutic effect of midostaurin on the dermal thickness of systemic sclerosis mice in Example 4 of the present invention;

[0034] Figure 7 The figure shows the therapeutic effect of midostaurin on the lung symptoms of systemic sclerosis mice detected by H&E staining in Example 4 of the present invention;

[0035] Figure 8 The figure shows the therapeutic effect of midostaurin on pulmonary fibrosis in systemic sclerosis mice in Example 4 of the present invention;

[0036] Fig. 9 The figure shows the therapeutic effect of midostaurin on the spleen index of systemic lupus erythematosus mice in Example 5 of the present invention;

[0037] Fig.10 The representative fluorescence staining results of midostaurin reducing the ANA level in systemic lupus erythematosus mice in Example 5 of the present invention are shown;

[0038] Fig.11 The figure shows the therapeutic effect of midostaurin on the ANA level of systemic lupus erythematosus mice in Example 5 of the present invention;

[0039] Fig.12 The abnormal IFN signal induced by the SAVI mutant in Example 6 of the present invention is shown;

[0040] Fig.13 The therapeutic effect of midostaurin on the SAVI mutant in Example 6 of the present invention is shown;

[0041] Fig.14 The figure shows the therapeutic effect of midostaurin on the stool characteristics of mice with ulcerative colitis in Example 7 of the present invention;

[0042] Fig.15 The figure shows the inhibitory effect of midostaurin on the number of leukocytes in ulcerative colitis mice in Example 7 of the present invention;

[0043] Fig.16 The figure shows the inhibitory effect of midostaurin on the number of neutrophils in ulcerative colitis mice in Example 7 of the present invention. DETAILED DESCRIPTION

[0044] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0045] The present invention is intended to cover all alternatives, modifications and equivalent technical solutions, which are all included in the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0046] It should be further appreciated that certain features of the invention, which for clarity are described in the context of multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0047] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0048] As used herein, the articles "a", "an", and "the" are intended to include "at least one" or "one or more" unless otherwise specified or clearly contradicted by context. Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated for use or use in the implementation of the described embodiment.

[0049] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0050] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0051] "Stereoisomers" refer to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0052] "Chiral" refers to a molecule that is non-superimposable on its mirror image; "achiral" refers to a molecule that is superimposable on its mirror image.

[0053] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0054] "Diastereoisomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high resolution analytical procedures such as electrophoresis and chromatography, for example HPLC.

[0055] The terms "racemate," "racemate" or "racemic mixture" refer to an equimolar mixture of two enantiomers devoid of optical activity.

[0056] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0057] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans isomers), atropisomers, and the like.

[0058] The term "geometric isomers" is also called "cis-trans isomers", which are isomers caused by the fact that double bonds (including olefin double bonds, C=N double bonds and N=N double bonds) or single bonds of ring carbon atoms cannot rotate freely.

[0059] The stereochemical definitions and conventions used in the present invention generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof, such as racemic mixtures, are also included within the scope of the present invention.

[0060] The term "nitrogen oxide" means that when a compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide. Special examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. Available oxidants, such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids), can be used to treat the corresponding amines to form N-oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), wherein, for example, in an inert solvent, such as dichloromethane, an amine compound is reacted with meta-chloroperbenzoic acid (MCPBA).

[0061] The term "metabolite" refers to a product obtained by the metabolism of a specific compound or its salt in vivo. The metabolite of a compound can be identified by techniques known in the art, and its activity can be characterized by experimental methods as described in the present invention. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting the compounds of the present invention with mammals for a period of time.

[0062] The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" of the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.

[0063] The term "salt" or "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., J. Pharmaceutical Sciences, 66, 1-19, 1977. Pharmaceutically acceptable salts formed by non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups. Pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts are also included. The term "pharmaceutically acceptable acid addition salt" refers to salts formed with inorganic or organic acids that can retain the biological effectiveness of free bases without other side effects. "Pharmaceutically acceptable base addition salt" refers to salts formed with inorganic or organic bases that can retain the biological effectiveness of free acids without other side effects. In addition to pharmaceutically acceptable salts, the present invention also contemplates other salts. They can act as intermediates in compound purification or in the preparation of other pharmaceutically acceptable salts or can be used for identification, characterization or purification of the compounds of the present invention.

[0064] The term "solvate" refers to an association formed by one or more solvent molecules and the compound of the present invention. The solvent can be water, acetic acid, ether, isopropyl ether, petroleum ether, ethyl formate, ethyl acetate, isopropyl acetate, n-propyl acetate, isobutyl acetate, n-butyl acetate, methyl tert-butyl ether (MTBE), n-heptane, a mixed solvent of ethanol and water in a volume ratio of 10:90 to 90:10, acetone, methyl isobutyl ketone, acetonitrile, benzene, chloroform, carbon tetrachloride, dichloromethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, n-butanol, tert-butanol , sec-butyl alcohol, N,N-dimethylacetamide, N,N-dimethylformamide, formamide, formic acid, n-hexane, cyclohexane, n-heptane, a mixed solvent of n-heptane and ethyl acetate in a volume ratio of 1:5 to 5:1, isopropanol, methanol, butanone, l-methyl-2-pyrrolidone, mesitylene, nitromethane, polyethylene glycol, n-propanol, isopropanol, 2-acetone, 4-methyl-2-pentanone, pyridine, tetrahydrofuran, methyl ethyl ketone, toluene, xylene, isopropylbenzene or a mixture thereof, etc.

[0065] The term "hydrate" refers to an association of one or more water molecules with a compound of the present invention.

[0066] The term "prodrug" as used in the present invention refers to a compound that is converted into a compound of formula (I) in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug compounds of the present invention may be esters. In the prior art, esters that can be used as prodrugs include phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For a complete discussion of prodrugs, please refer to the following literature: T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270, and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0067] As used herein, the term "administering to a patient the compound shown in the preceding formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs" refers to introducing a predetermined amount of a substance into a patient by a suitable manner. The compound shown in formula I of the present invention or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal administration, but the present invention is not limited to these exemplified modes of administration. However, due to oral administration, the active ingredient of the composition for oral administration should be coated or formulated to prevent it from being degraded in the stomach. In addition, the compound shown in formula I of the present invention can be administered using a specific device that delivers the active ingredient to the target cell.

[0068] The administration frequency and dosage of the drug of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the patient's age, sex, weight and severity of the disease and the type of drug as the active ingredient.

[0069] The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosing regimen. A therapeutically effective amount of a drug or compound does not need to cure a disease or condition, but will provide treatment for the disease or condition, such that the onset of the disease or condition in an individual is delayed, stopped or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or, for example, the disease or condition becomes less severe, or recovery is accelerated.

[0070] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein encompasses the treatment of a disease in mammals, particularly humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, mitigate or inhibit a disease in the individual, including but not limited to administering a drug or compound to an individual in need thereof.

[0071] Midostaurin (PKC412) is an oral multi-targeted kinase inhibitor that works by blocking several enzymes that promote cell growth. It can inhibit the activity of type III receptor tyrosine kinase encoded by the FLT3 gene, induce cell cycle arrest, and have apoptotic effects on AML primitive cells with mutant FLT3, wild-type FLT3, and mutant FLT3 receptor expression. In terms of FLT3 targets and indications, midostaurin is the first new drug for targeted therapy combined with chemotherapy for the treatment of acute myeloid leukemia.

[0072] In the present invention, the inventors found through a series of experimental studies that midostaurin (including stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs) can significantly inhibit the activity of TBK1 kinase. TBK1 (TANK-binding kinase 1) is a key kinase in the interferon (IFN) signaling pathway, and its abnormal activation is closely related to the pathogenesis of various autoimmune diseases. By inhibiting the activity of TBK1, midostaurin can further block the activation of IFN signals, thereby regulating immune responses.

[0073] Based on this discovery, the inventors further verified the potential of midostaurin in the treatment of autoimmune diseases through cell model and animal model experiments. The experimental results showed that midostaurin can not only effectively inhibit the activity of TBK1 kinase, but also significantly improve the pathological symptoms associated with autoimmune diseases, such as reducing tissue fibrosis and reducing the levels of inflammatory markers. These results show that midostaurin provides a new alternative for the treatment of autoimmune diseases and is expected to become a new therapeutic drug with broad application prospects. The present invention proposes a new use of midostaurin, a pharmaceutical composition and its use, which will be described in detail below.

[0074] use

[0075] In the first aspect of the present invention, the present invention provides the use of a compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of a drug, wherein the drug is used to inhibit the activity of TBK1 kinase,

[0076]

[0077] The inventors of the present invention have discovered in experiments that the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, namely midostaurin, can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase, and IFN signals are related to immune regulation. Therefore, the inventors have verified through cell model and animal model experiments that midostaurin can effectively treat autoimmune diseases, which provides a new alternative for the treatment of autoimmune diseases.

[0078] In the second aspect of the present invention, the present invention provides the use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of drugs, wherein the drugs are used to prevent and / or treat TBK1-mediated related diseases.

[0079]

[0080] The inventors of the present invention have discovered in experiments that the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, namely midostaurin, can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase, and IFN signals are related to immune regulation. Therefore, the inventors have verified through cell model and animal model experiments that midostaurin can effectively treat autoimmune diseases, which provides a new alternative for the treatment of autoimmune diseases.

[0081] In some embodiments of the present invention, the TBK1-mediated related disease is an autoimmune disease.

[0082] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis (SSc), systemic lupus erythematosus (SLE), Aicardi-Goutieres syndrome (AGS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), STING-associated vasculopathy in infancy (SAVI), Niemann-Pick disease type C (NPC), Sjögren's syndrome (SjD), arthritis, pulmonary hemorrhage (DAH), pyrrhosis, chronic lymphocytic thyroiditis (CLT), hyperthyroidism, insulin-dependent diabetes mellitus (IDDM), myasthenia gravis (MG), ulcerative colitis (UC), Crohn's disease (CD), pernicious anemia with chronic atrophic gastritis (PAG), primary biliary cirrhosis (PBC), multiple sclerosis (MS), and acute idiopathic polyneuritis (AIP).

[0083] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, STING-associated vasculopathy of infancy, and ulcerative colitis.

[0084] In the third aspect of the present invention, the present invention provides the use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of reagents for regulating the activation of IFN,

[0085]

[0086] As mentioned above, the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs can effectively inhibit the activity of TBK1 kinase, and can further inhibit the activation of IFN signals by inhibiting the activity of TBK1 kinase. Therefore, after being prepared as a solvent, it can effectively regulate the activation of IFN.

[0087] In some embodiments of the present invention, regulating the activation of IFN includes inhibiting the activation of IFN or promoting the activation of IFN.

[0088] Pharmaceutical composition

[0089] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: a compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0090]

[0091] According to an embodiment of the present invention, the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can inhibit the activity of TBK1 kinase and treat TBK1-mediated related diseases. Thus, a pharmaceutical composition containing the compound shown in the above formula I, or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug can effectively prevent and / or treat TBK1-mediated related diseases.

[0092] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0093] The term "pharmaceutically acceptable carrier" used in the present invention includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, adhesive, excipient, dispersant, lubricant, sweetener, flavoring agent, coloring agent, or a combination thereof, which are known to those skilled in the art (such as Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical composition is covered.

[0094] The term "pharmaceutically acceptable excipient" used in the present disclosure may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0095] In some embodiments of the present invention, the pharmaceutical composition is an injection preparation or an oral preparation.

[0096] In some embodiments of the present invention, the oral preparation is a tablet, a capsule, a pill, a granule, a decoction, an oral liquid, a drop pill or a syrup.

[0097] In some embodiments of the present invention, the pharmaceutical composition is an oral preparation. The shape of the oral preparation is not particularly limited, and can be any one of a circle, a capsule, a doughnut, a rectangle, and the like.

[0098] As solid preparations, for example, tablets, capsules, powders, granules, lozenges and the like can be mentioned.

[0099] The solid preparation may be coated with a coating agent and may have markings and letters for identification and further lines for separation. Coating is carried out under the condition of adding conventional coating media and film-forming agents (generally referred to as coating materials) familiar to those skilled in the art. Coating can be carried out using, for example, sugar coating bases, water-soluble film coating bases, enteric film coating bases, sustained-release film coating bases, etc. For sugar coating bases, a combination of sucrose and one or more selected from the following substances can be used: talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax, etc. For water-soluble film coating bases, for example, cellulose polymers such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, etc. can be used; synthetic polymers such as diethylaminoethyl polyvinyl acetal, aminoalkyl methacrylate copolymer E [Eudragit E (trade name)], polyvinyl pyrrolidone, etc.; polysaccharides such as pullulan, etc. For the enteric film coating matrix, for example, cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, cellulose acetate phthalate, etc. can be used; acrylic polymers such as methacrylic acid copolymer L [Eudragit L (trade name)], methacrylic acid copolymer LD [Eudragit L-30D55 ​​(trade name)], methacrylic acid copolymer S [Eudragit S (trade name)], etc.; naturally occurring substances such as shellac, etc. For the sustained-release film coating matrix, for example, cellulose polymers such as ethylcellulose, cellulose acetate, etc. can be used; acrylic polymers such as aminoalkyl methacrylate copolymer RS ​​[Eudragit RS (trade name)], ethyl acrylate-methyl methacrylate copolymer suspension [EudragitNE (trade name)], etc. can be used. Two or more of the above coating matrices can be mixed and used in a suitable ratio. Moreover, coating additives can also be used during coating. As coating additives, for example, light-shielding agents and / or colorants, such as titanium oxide, talc, iron oxide, etc.; plasticizers, such as polyethylene glycol, triethyl citrate, castor oil, polysorbate, etc.; organic acids, such as citric acid, tartaric acid, malic acid, ascorbic acid, etc. can be used.

[0100] Solid dosage forms can be formulated for immediate release (ie, rapid release) and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0101] When the solid preparation is a tablet, any pharmaceutically acceptable excipient commonly used in the preparation of solid preparations can be used. Tablets can be prepared by compression or molding, optionally using one or more physiologically acceptable / pharmaceutically acceptable excipients. Compressed tablets can also be prepared by compressing the active ingredient in a free-flowing form (e.g., powder or capsule) in a suitable machine, and the active ingredient is optionally mixed with a binder, lubricant, filler, solubilizer or disintegrant. Molded tablets can be prepared by molding a mixture of a wetted powdered compound and an inert liquid dispersion medium in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide a sustained release or controlled release of the active ingredient therein. The preparation of tablets is discussed in detail in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, NY, 1980.

[0102] When the solid preparation is a capsule, any conventional encapsulation is suitable, for example, using the above-mentioned carriers in a hard gelatin capsule. When the composition is in the form of a soft gelatin capsule, any physiologically acceptable / pharmaceutically acceptable excipient commonly used for preparing dispersions or suspensions can be considered, and the physiologically acceptable / pharmaceutically acceptable excipient is incorporated into the soft gelatin capsule.

[0103] The pharmaceutical preparation can be conveniently presented in a unit dosage form and can be prepared by any method known in the pharmaceutical field so that a unit dose can be given to a subject. Preferably, the pharmaceutical composition is in a unit dosage form, such as a solid preparation (such as a tablet, powder, dry suspension, granule or capsule) in a unit dosage form.

[0104] Uses of the pharmaceutical composition

[0105] In the fifth aspect of the present invention, the present invention proposes the use of the pharmaceutical composition described in the fourth aspect in the preparation of a drug for preventing and / or treating TBK1-mediated related diseases. As shown above, the compound shown in Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs can inhibit the activity of TBK1 kinase and treat TBK1-mediated related diseases. Thus, a pharmaceutical composition containing the compound shown in the above Formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, can effectively prevent and / or treat TBK1-mediated related diseases.

[0106] In some embodiments of the present invention, the TBK1-mediated related disease is an autoimmune disease.

[0107] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, STING-related vasculopathy in infancy, Niemann-Pick disease type C, Sjögren's syndrome, arthritis, pulmonary hemorrhage, herpes simplex, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Crohn's disease, pernicious anemia with chronic atrophic gastritis, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0108] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, STING-associated vasculopathy of infancy, and ulcerative colitis.

[0109] method

[0110] In the sixth aspect of the present invention, the present invention proposes a method for preventing and / or treating TBK1-mediated related diseases. According to an embodiment of the present invention, the method comprises: administering an effective amount of a compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof to a patient. As shown above, the compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof can inhibit the activity of TBK1 kinase and treat TBK1-mediated related diseases. Thus, the method of the present invention can effectively treat TBK1-mediated related diseases.

[0111] In some embodiments of the present invention, the TBK1-mediated related disease is an autoimmune disease.

[0112] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, STING-related vasculopathy in infancy, Niemann-Pick disease type C, Sjögren's syndrome, arthritis, pulmonary hemorrhage, herpes simplex, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Crohn's disease, pernicious anemia with chronic atrophic gastritis, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0113] In some embodiments of the present invention, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, STING-associated vasculopathy of infancy, and ulcerative colitis.

[0114] In the seventh aspect of the present invention, the present invention proposes a method for inhibiting the activity of TBK1 in cells in vitro. According to an embodiment of the present invention, the method comprises: contacting the cell with a compound shown in Formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof. As shown above, the compound shown in Formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof can inhibit the activity of TBK1 kinase. Thus, the method of the present invention can effectively inhibit the activity of TBK1 in cells.

[0115] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0116] Example 1: Inhibition of TBK1 kinase activity by midostaurin

[0117] Different concentrations of midostaurin (0.51nM, 1.52nM, 4.6nM, 13.7nM, 41.2nM, 123.5nM, 370.4nM, 1111nM, 3333nM, 10000nM) were added to a 384-well plate, and each concentration was repeated 3 wells. Subsequently, 9nM TBK1 protein, 0.1mg / mL MBP, and 10μM ATP were added to each well, and deionized water was added to each well to reach a volume of 5μL, and the reaction was carried out at 25℃ for 60 minutes. After the reaction, 5μL ADP-Glo ​​reagent was added to each well and incubated at 25℃ for 40 minutes. 10μL ADP-Glo ​​detection reagent was added to each well and incubated at 25℃ for 60 minutes, waiting for ADP to be converted into fluorescent signal. The fluorescence signal was detected using the chemiluminescence module (full channel) of the multifunctional microplate reader. The intensity of the fluorescence is proportional to the amount of ADP generated, reflecting the kinase activity level of TBK1.

[0118] The inhibition rate curves of different experimental groups were simulated and the IC of midostaurin for inhibiting TBK1 enzyme activity was calculated. 50 ,get Figure 1 The results showed that midostaurin inhibited TBK1 kinase activity with an IC 50It is 4.59nM, indicating that midostaurin can effectively inhibit the kinase activity of TBK1.

[0119] Example 2: Inhibition of IFN signaling by midostaurin

[0120] 2.1 Different concentrations of midostaurin (1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 750 nM, 1000 nM, 5000 nM, 10000 nM, 20000 nM) were added to the THP1-ISRE-Lucia reporter cell line, and 4 μM SR-717 was added to activate IFN signaling via the STING-TBK1 pathway. The cells were cultured at 6×10 5 The cells were plated at a density of 100 μL / mL in a 384-well plate (25 μL / well), and each condition was repeated in 4 wells, and then placed in a cell culture incubator at 37°C. After 24 hours, the Gaussia luciferase reporter gene detection kit was added to the cells at a volume ratio of 1:1, reacted for 5 minutes, and the fluorescence signal was detected using the chemiluminescence module (full channel) of the multi-function microplate reader.

[0121] according to Figure 2 The results showed that midostaurin showed a concentration-dependent inhibition trend of IFN signaling, and its IC value for inhibiting IFN signaling was calculated. 50 It is 279.8nM, indicating that midostaurin can effectively inhibit the activation of IFN signaling by inhibiting TBK1.

[0122] 2.2 HEK293T cells with good growth status were cultured at 0.5×10 5 The cells were plated in a 6-well plate at a density of 100 μg / mL. After the cells grew for 12 hours, 100 μg / mL LPS was added to activate the IFN signal via the TLR4-TRAF3-TBK1 pathway, and 100 nM midostaurin or an equal volume of DMSO was added for 24 hours. In addition, a group of cells without LPS and only DMSO was set up as a negative control group. After 24 hours, each group of cells was collected and RNA was extracted using an RNA extraction kit. 2 μg of RNA from each sample was reverse transcribed using a reverse transcription kit. The reverse transcription product was used as a template for fluorescent quantitative PCR to detect the mRNA expression levels of IFN-β and IFN signal downstream (ISG15, ISG56). The primers used are shown in Table 1:

[0123] Table 1 Fluorescence quantitative PCR primer sequence information (I)

[0124]

[0125] The results detected by fluorescence quantitative PCR were Figure 3As shown in the results, midostaurin can inhibit the gene expression of IFN-β, and also has a significant inhibitory effect on the expression of IFN signal downstream genes ISG15 and ISG56, indicating that midostaurin can inhibit IFN and its downstream by inhibiting TBK1.

[0126] Example 3: The ability of midostaurin to inhibit IFN signaling is independent of the original target

[0127] 3.1 Packaging of Lentivirus Containing shRNA Plasmid

[0128] One day in advance, HEK293T cells were plated in 6-well plates (number: 0.5×10 6 / well), and transfection was started the next day when the cell density reached 60% to 80%. During transfection, 4 μg of the target plasmid (pLKO.1-puro-shRNA), 3 μgpsPAX2, and 1 μg pMD2.G were added to 100 μL Opti-MEM, incubated at room temperature for 5 minutes, and then 8 μL Neofect was added. After standing for 30 minutes, the transfection complex was gently added to the supernatant of HEK293T. Among them, the target genes of the pLKO.1-puro-shRNA plasmid are FLT3, KIT, PRKCA, PRKCB, PRKCD, and PRKCG, which were purchased from Merck. The cell status of HEK293T was checked 8 to 12 hours after transfection, the culture medium was aspirated, and replaced with fresh culture medium without dual antibodies. The supernatant of the cells was collected into a centrifuge tube at 48 hours and 96 hours after transfection, and the cell debris was removed by centrifugation at 4500 rpm for 10 minutes. The supernatant obtained contained lentiviral particles.

[0129] 3.2 Infection of THP1-ISRE-Lucia cells with lentivirus

[0130] THP1-ISRE-Lucia cells were counted. 6 The cells were plated into a 6-well plate at a density of 100 μg / well (1 mL of antibiotic-free culture medium was added to each well), and then 1 mL of the lentiviral particles prepared in step 1 and 2 μL of 10 mg / mL polybrene were added to each well of cells. After gently shaking, the cells were placed in a cell culture incubator. 8 hours after infection, the culture medium of the cells was replaced with fresh antibiotic-free culture medium; 48 hours later, the culture medium was replaced with a complete culture medium containing 2.5 μg / mL puromycin for resistance screening. After continuous selection with 2.5 μg / mL puromycin for 2 to 3 weeks, the THP1-ISRE-Lucia cells were obtained and the gene knockdown was verified by fluorescent quantitative PCR. The primers used are shown in Table 2:

[0131] Table 2 Fluorescence quantitative PCR primer sequence information (II)

[0132]

[0133] 3.3 Testing IFN signaling

[0134] Different concentrations of midostaurin (156 nM, 625 nM, 2.5 μM) were added to different knockdown THP1-ISRE-Lucia cell lines (FLT3 knockdown, KIT knockdown, PRKCA knockdown, PRKCB knockdown, PRKCD knockdown, PRKCG knockdown, knockdown control), and 4 μM SR-717 was added to activate IFN signaling. The cells were cultured at 6×10 5 The cells were plated at a density of 100 μL / mL in a 384-well plate (25 μL / well), and each condition was repeated in 4 wells, and then placed in a cell culture incubator at 37°C. After 24 hours, the Gaussia luciferase reporter gene detection kit was added to the cells at a volume ratio of 1:1, reacted for 5 minutes, and the fluorescence signal was detected using the chemiluminescence module (full channel) of the multi-function microplate reader.

[0135] according to Figure 4 The results shown in the figure show that midostaurin has consistent inhibitory effects on IFN signaling in different knockdown THP1-ISRE-Lucia cell lines (FLT3 knockdown, KIT knockdown, PRKCA knockdown, PRKCB knockdown, PRKCD knockdown, PRKCG knockdown, and knockdown control), indicating that the ability of midostaurin to inhibit IFN signaling is independent of the original targets (FLT3, KIT, PKC-α, PKC-β, PKC-δ, and PKC-γ).

[0136] Example 4: Effect of midostaurin in a systemic sclerosis mouse model

[0137] Six-week-old BALB / C female mice were adaptively fed for 1 week. After 1 week, the dorsal skin of mice was depilated with a depilatory device and depilatory cream (area 2 cm × 2 cm), and the mice were randomly divided into a control group (4 mice), a model group (4 mice), and a midostaurin treatment group (4 mice). Starting from the second day of depilation, the mice in the control group were given subcutaneous injection of saline and solvent gavage, the mice in the model group were given subcutaneous injection of bleomycin and solvent gavage; the mice in the midostaurin treatment group were given subcutaneous injection of bleomycin and drug gavage. In the first 6 days, 100 μL of saline was injected into the dorsal skin of the control mice, and 100 μL of 0.3 mg / mL bleomycin was injected into the dorsal skin of the mice in the model group and the midostaurin treatment group. The dorsal skin was suspended for 3 days, and then 100 μL of saline or bleomycin was continuously injected for 14 days. The drugs and solvents for intragastric administration were prepared according to the method of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline, wherein the dose of midostaurin contained in the drug was 100 mg / kg / day, and the intragastric administration was also performed for 6 days, then suspended for 3 days, and then continued for 14 days. On the 26th day, all mice were euthanized, and the back skin and lung tissues of the mice were collected and fixed for H&E staining and Masson staining. When scoring pulmonary fibrosis, the Ashcroft scale was used as the evaluation basis, and the Ashcroft scale is shown in Table 3:

[0138] Table 3 Ashcroft Scale

[0139]

[0140]

[0141] according to Figure 5 and Figure 6 The results showed that the normal structure of subcutaneous tissues such as hair follicles, sweat glands, and capillaries in the mice treated with midostaurin was restored, and the symptoms of collagen accumulation and increased dermal thickness were alleviated. Figure 7 and 8 The results show that the alveolar structure and interstitial tissue structure of mice in the midostaurin treatment group returned to normal, and the symptoms of pulmonary fibrosis were effectively delayed. The above results indicate that midostaurin can be used to treat systemic sclerosis.

[0142] Example 5: Effect of midostaurin in a systemic lupus erythematosus mouse model

[0143] After 1 week of adaptive feeding, 6-8 week-old C57BL / 6 female mice were randomly divided into a control group (4 mice), a model group (4 mice) and a midostaurin treatment group (4 mice). The mice in the control group were smeared with vaseline inside and outside the ears with a cotton swab every day, and the mice in the model group and the midostaurin group were smeared with 5% imiquimod cream inside and outside the ears with a cotton swab every day. The control group and the model group were given solvent by gavage, and the midostaurin treatment group was given drug by gavage (100 mg / kg / day) for 30 consecutive days. The drug and solvent for gavage were prepared according to the method of 10% DMSO+40% PEG300+5% Tween-80+45% Saline, wherein the dose of midostaurin contained in the drug was 100 mg / kg / day. The weight and general condition of the mice were recorded every day. At the end point, the mice were euthanized by CO2 asphyxiation. The spleen of the mice was collected for weighing, and the serum of the mice was collected for the detection of antinuclear antibodies (ANA). When testing ANA, indirect immunofluorescence staining was used. Hep-2 cells were placed on a glass slide as a matrix, mouse serum was added for reaction, and then FITC-rat anti-mouse Kappa (H139-52.1) antibody was used for binding. Finally, observation and analysis were performed under a fluorescence microscope.

[0144] according to Fig. 9 The results show that the spleen index of mice in the midostaurin treatment group decreased significantly, indicating that midostaurin treatment can alleviate the inflammatory hyperplasia symptoms of the spleen in mice with systemic lupus erythematosus. The increase in ANA in serum is a typical symptom of SLE. Fig.10 and 11 The results show that the ANA titer of mice in the midostaurin treatment group decreased, indicating that midostaurin treatment can effectively reduce the level of ANA. The above results indicate that midostaurin can be used to treat systemic lupus erythematosus.

[0145] Example 6: Effect of midostaurin in the STING-associated vasculopathy of infancy (SAVI) cell model

[0146] HEK293T cells were transfected with pIFN-Luc and co-transfected with plasmids expressing wild-type STING (STING-WT) or SAVI mutants (STING-N154S, STING-R281E, STING-R284A). The next day, 2.5 × 10 5The cells were plated into a 384-well plate (25 μL / well) at a density of 100 μL / mL, and different concentrations of midostaurin (500 nM, 1000 nM, 5000 nM, 10000 nM) were added at the same time, and each condition was repeated 4 wells. After 24 hours of cell culture, the firefly luciferase reporter gene detection kit was added to the cells at a volume ratio of 1:1, and the reaction was carried out for 5 minutes. The fluorescence signal was detected using the chemiluminescence module (full channel) of the multi-function microplate reader.

[0147] according to Fig.12 The results show that the SAVI mutant can produce abnormally increased IFN signals compared to wild-type STING. Fig.13 The results showed that midostaurin (500nM, 1000nM, 5000nM, 10000nM) could effectively reduce the abnormal IFN signal caused by SAVI mutants, indicating that midostaurin can treat STING-associated juvenile-onset vasculopathy (SAVI).

[0148] Example 7: Effect of midostaurin in a mouse model of ulcerative colitis

[0149] C57BL / 6 male mice aged 7 to 8 weeks were adaptively fed for 1 week. After 1 week, the mice were randomly divided into a control group (4 mice), a model group (4 mice), and a midostaurin treatment group (4 mice). The control group was given normal water and solvent by gavage; the model group was given 2% (W / V) dextran sulfate sodium aqueous solution and solvent by gavage; the midostaurin treatment group was given 2% (W / V) dextran sulfate sodium aqueous solution and midostaurin by gavage. The drugs and solvents for gavage were prepared according to the method of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline, and the gavage dose was 100 mg / kg / day; normal water and 2% dextran sulfate sodium aqueous solution were replaced every two days. C57BL / 6 mice were allowed to drink water freely for 7 days, and the fecal viscosity of the mice was recorded every day. On the 8th day, the mice were euthanized. Blood samples of mice were collected for blood cell counting.

[0150] according to Fig.14 The results showed that the stool characteristics scores of mice in the midostaurin treatment group were significantly decreased, indicating that midostaurin treatment can improve the stool viscosity of mice with ulcerative colitis. Fig.15 and Fig.16 The results showed that the number of white blood cells and monocytes in the blood of mice treated with midostaurin decreased, indicating that midostaurin treatment can effectively reduce the inflammation level of mice with ulcerative colitis. The above results show that midostaurin can be used to treat ulcerative colitis.

[0151] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0152] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof in the preparation of a drug for inhibiting the activity of TBK1 kinase, 2. Use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of a drug for preventing and / or treating TBK1-mediated related diseases, 3. The use according to claim 2, characterized in that: The TBK1-mediated related diseases are autoimmune diseases.

4. The use according to claim 3, characterized in that The autoimmune diseases include at least one of systemic sclerosis, systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, STING-related vasculopathy in infancy, Niemann-Pick disease type C, Sjögren's syndrome, arthritis, pulmonary hemorrhage, pyometra, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Crohn's disease, pernicious anemia with chronic atrophic gastritis, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

5. Use of the compound of formula I, or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs in the preparation of an agent for regulating the activation of IFN, 6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: a compound of formula I, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof, 7. The pharmaceutical composition according to claim 6, characterized in that It further includes pharmaceutically acceptable excipients or carriers.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is an injection preparation or an oral preparation.

9. Use of the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a medicament for preventing and / or treating TBK1-mediated related diseases.

10. The use according to claim 9, characterized in that The TBK1-mediated related diseases are autoimmune diseases; Optionally, the autoimmune disease includes at least one of systemic sclerosis, systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, STING-related vasculopathy of infancy, Niemann-Pick disease type C, Sjögren's syndrome, arthritis, pulmonary hemorrhage, pyometra, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, Crohn's disease, pernicious anemia with chronic atrophic gastritis, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

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