TANK binding kinase 1 inhibitor and pharmaceutical application thereof

Abstract

The invention relates to a TANK binding kinase 1 inhibitor and pharmaceutical application thereof, and belongs to the technical field of biological medicine. The TANK binding kinase 1 inhibitor is a compound ITA-9. The invention also provides an application of the compound ITA-9 in preparation of a pharmaceutical composition for preventing and / or treating TBK1 activity-related diseases, wherein the TBK1 activity-related diseases comprise infectious diseases, autoimmune diseases, metabolic diseases or cancers and the like. The invention discloses an application of a compound ITA-9 as a TANK binding kinase 1 (TBK1) inhibitor in preparation of a pharmaceutical composition for preventing and / or treating TBK1 activity related diseases for the first time. The compound has the potential of being developed into TBK1 inhibitor drugs and drugs for treating TBK1-related diseases such as infectious diseases, autoimmune diseases, metabolic diseases and cancers, provides a new treatment drug for clinic, and has good clinical application value and wide application prospects.

Description

Technical Field

[0001] The present invention relates to a TANK binding kinase 1 inhibitor and its pharmaceutical use, belonging to the field of biopharmaceutical technology. Background Art

[0002] Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. Common autoimmune diseases include rheumatoid arthritis, psoriasis, Crohn's disease, systemic lupus erythematosus, multiple sclerosis, type I diabetes, allergic diseases, chronic obstructive pulmonary disease, asthma, leukemia, lymphoma diseases, etc.

[0003] IKKε and TBK1 are highly homologous Ser / Thr kinases that critically participate in the innate immune response by inducing type I interferons and other cytokines. These kinases are stimulated in response to viral / bacterial infections. The immune response to viral and bacterial infections involves the binding of antigens such as bacterial lipopolysaccharide (LPS), viral double-stranded RNS (dsRNA) to Toll-like receptors, followed by activation of the TBK1 pathway. Activated TBK1 and IKKε phosphorylate IRF3 and IRF7, which trigger the dimerization and nuclear translocation of those interferon regulatory transcription factors, ultimately inducing the signal transduction cascade leading to IFN production.

[0004] TANK binding kinase 1 (TBK1) is a serine / threonine kinase belonging to the non-classical IκB kinase (IKK) family. TBK1 is involved in regulating multiple signaling pathways and transcription factors such as interferon regulatory factor (IRF), nuclear factor κB (NF-κB), type I interferon (IFN-I), type II interferon (IFN-II) target genes, as well as STING-mediated cytosolic DNA detection, regulating antiviral defense, host-virus interaction, and playing an important role in the occurrence and development of diseases such as immunity, tumors, inflammation, and metabolism. TBK1 can also activate its substrate IRF3 and IRF7 transcription factors by directly phosphorylating specific sites, inducing their localization to the nucleus to promote the transcription of type I IFN genes. Excessive expression of type I IFN may cause damage to the body. And after the activation of different pattern recognition receptors mediated by viral infections, by activating the common key kinase TBK1, recruiting and activating IRF3, thereby inducing the production of type I interferon (IFN-α / β), initiating the antiviral immune response. The innate immunity mediated by TBK1 plays a key role in clearing viruses, which makes the research on small molecule inhibitors of TBK1 become a hot spot.

[0005] At present, the research and development of TBK1 inhibitors is still in its infancy. On the one hand, the biological functions and mechanisms of action of TBK1 have not been fully elucidated; on the other hand, there are few structural types of TBK1 inhibitors, and selective TBK1 inhibitors have not yet entered clinical research. Most of the existing TBK1 inhibitors have multiple targets. For example, BX795 (Amlexanox) developed by Takeda Pharmaceutical Company Limited was marketed as a PDK1 inhibitor, and later it was found that it showed comparable inhibitory activity against TBK1 and IKKε (PDK1 IC50 = 17 nM, TBK1 IC50 = 2.3 nM, IKKε IC50 = 9.5 nM). GSK8612 is a selective TBK1 inhibitor reported by GlaxoSmithKline in 2019. Although it has a 10-fold selectivity for the IKK kinase, its poor activity against the TBK1 kinase (TBK1 pIC50 = 6.8) has led to its failure to enter clinical research. Patent document WO2018154315A1 provides a TBK1 inhibitor. Although this inhibitor has good inhibitory activity against the TBK1 kinase (IC50 = 20 nM), it still has the problem of having multiple targets and targeting both TBK1 and IKKε (IKKε IC50 = 21 nM).

[0006] Therefore, it is still very necessary, challenging and essential to find highly efficient selective small molecule TBK1 inhibitors. Therefore, it is necessary to conduct in-depth structural optimization research on TBK1 inhibitors to obtain a class of TBK1 inhibitor compounds with high activity. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a novel TANK-binding kinase 1 inhibitor and its pharmaceutical use.

[0008] The technical solution of the present invention is as follows: A compound ITA-9, whose structural formula is shown as follows: .

[0009] The preparation method of the above compound ITA-9 is as follows: (1) Dissolve itaconic anhydride in dichloromethane, then add anhydrous methanol and sulfuric acid, and stir the reaction overnight at room temperature; then add dichloromethane to the reaction solution, and then basify the reaction solution with potassium carbonate, separate the aqueous phase to obtain the organic phase; then acidify the organic phase with hydrochloric acid, extract three times with dichloromethane, combine the organic phases after extraction, dry with anhydrous sodium sulfate, filter, evaporate under reduced pressure, and finally recrystallize the crude product in a mixed solution of ethyl acetate / n-hexane to obtain compound 1-2; Among them, the structure of compound 1-2 is shown as follows: ; (2) Dissolve compound 1-2 and methyl N 6 -(tert-butylcarbonyl)- L -lysine hydrochloride in dichloromethane, then add 2-(7-azabenzotriazole)- N , N , N' , N' -tetramethylurea hexafluorophosphate and triethylamine, stir at room temperature overnight. After the reaction is completed, add saturated brine, extract with dichloromethane, dry the organic phase over anhydrous sodium sulfate, filter, evaporate under reduced pressure, and purify by column chromatography to obtain the target compound ITA-9.

[0010] Preferably according to the present invention, in step (1), the ratio of itaconic anhydride, dichloromethane, anhydrous methanol and sulfuric acid is (0.45~0.55 g): (1.5~2.5 ml): (3~6 ml): (70~80 μl).

[0011] Preferably according to the present invention, in step (2), the ratio of compound 1-2, methyl N 6 -(tert-butylcarbonyl)- L -lysine hydrochloride, dichloromethane, 2-(7-azabenzotriazole)- N , N , N' , N' -tetramethylurea hexafluorophosphate and triethylamine is (0.1~0.2 g): (0.25~0.35 g): (3~6 ml): (0.35~0.45 g): (0.4~0.5 ml).

[0012] Use of the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof in the preparation of a TANK-binding kinase 1 (TBK1) inhibitor.

[0013] Use of the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof as a TANK-binding kinase 1 inhibitor in the preparation of a pharmaceutical composition for preventing and / or treating diseases related to TBK1 activity.

[0014] The diseases related to TBK1 activity described in the present invention refer to diseases that can be improved, prevented or treated by inhibiting TBK1 activity.

[0015] Preferably according to the present invention, the diseases related to TBK1 activity are infectious diseases, autoimmune diseases, metabolic diseases or cancers and other diseases.

[0016] More preferably, the infectious disease is a disease caused by infection with one or more of DNA viruses or RNA viruses.

[0017] More preferably, the DNA virus is herpes simplex virus 1 (HSV-1), and the RNA virus is vesicular stomatitis virus (VSV).

[0018] More preferably, the autoimmune diseases include AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and other diseases.

[0019] More preferably, the metabolic diseases include obesity, diabetes, non-alcoholic fatty liver disease and other diseases.

[0020] A pharmaceutical composition for treating diseases related to TBK1 activity, the pharmaceutical composition comprising the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0021] Preferably according to the present invention, the pharmaceutical composition is any pharmaceutically acceptable dosage form prepared with compound ITA-9 or a pharmaceutically acceptable salt thereof as the main active ingredient and a pharmaceutically acceptable excipient.

[0022] More preferably, the dosage form is tablets, capsules, granules, pills, liquid preparations, decoctions, suspensions, dispersions, syrups, suppositories, gels, aerosols, patches.

[0023] The beneficial effects of the present invention are as follows: 1. The present invention provides a novel compound ITA-9, which exhibits significantly improved TBK1 inhibitory activity compared with existing compounds of similar structure methylene succinic acid, and can be used as a TBK1 inhibitor.

[0024] 2. The present invention discloses for the first time the application of compound ITA-9 as a TANK-binding kinase 1 (TBK1) inhibitor in the preparation of a pharmaceutical composition for preventing and / or treating diseases related to TBK1 activity. This compound has the potential to be developed into a TBK1 inhibitor drug and a therapeutic drug for TBK1-related diseases, such as infectious diseases, autoimmune diseases, metabolic diseases and cancers, providing a new therapeutic drug for clinical use, with good clinical application value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a synthetic route diagram of compound ITA-9.

[0026] Figure 2 It is for compound ITA-9 to inhibit antiviral immune response; In the figure, A represents the content of IFN-β in the cell supernatant after VSV virus infection; B represents the content of IFN-β in the cell supernatant after HSV-1 virus infection; C represents the expression levels of p-TBK1, p-IRF3, and p-STAT1 in cells after VSV virus infection; D represents the expression levels of p-TBK1, p-IRF3, and p-STAT1 in cells after HSV-1 virus infection.

[0027] Figure 3 Compound ITA-9 targets and inhibits the activity of TBK1; In the figure, A shows mouse peritoneal macrophages pretreated with Ctrl or ITA-9, and the stability of TBK1 was detected by a cellular thermal shift assay; B shows the affinity between ITA-9 and TBK1 detected by surface plasmon resonance assay; C shows the covalent binding between ITA-9 and TBK1 detected by click chemistry experiment; D shows the molecular docking simulation of ITA-9 and TBK1.

[0028] Figure 4 Compound ITA-9 inhibits the excessive inflammatory response induced by poly(I:C); In the figure, A shows the content of IFN-β in mouse serum detected by ELISA; B shows the Cxcl10 mRNA expression levels detected by qPCR in mouse lung tissues; C shows hematoxylin and eosin staining of mouse lung tissue sections; D shows Sirius red staining of mouse lung tissue sections.

[0029] Figure 5 Compound ITA-9 inhibits the inflammatory response in a systemic lupus erythematosus (SLE) model; In the figure, A shows the content of Creatinine in mouse plasma detected by biochemical analysis; B shows the Cxcl10 and Isg15 mRNA expression levels detected by qPCR in mouse lung tissues; C shows hematoxylin and eosin staining of mouse lung tissue sections.

[0030] Figure 6 Compound ITA-9 inhibits the inflammatory response in an Aicardi-Goutières syndrome (AGS) model; In the figure, A shows the Cxcl10 、 Isg15 、 Isg54 、 Isg56 、 Ccl5 and Mx1 mRNA expression levels detected by qPCR in mouse lung tissues; B shows hematoxylin and eosin staining of mouse lung tissue sections. Specific embodiments

[0031] To better understand the essence of the present invention, the content of the present invention will be further described below in conjunction with embodiments, but it should not be regarded as a limitation to the present invention. The following further illustrates the present invention in conjunction with the specification drawings and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0032] The present invention provides an application of a novel TBK1 inhibitor ITA-9 in the preparation of a pharmaceutical composition for treating or preventing diseases related to TBK1 activity.

[0033] In the present invention, the drug includes ITA-9 or its pharmaceutically acceptable salts, as well as a pharmaceutically acceptable carrier.

[0034] In the present invention, the drug is any pharmaceutically acceptable dosage form prepared with ITA-9 or its pharmaceutically acceptable salts as the drug active ingredient and a pharmaceutically acceptable excipient.

[0035] In the present invention, the dosage forms are tablets, capsules, granules, pills, liquid preparations, decoctions, suspensions, dispersants, syrups, suppositories, gels, aerosols, patches.

[0036] The RNA virus is vesicular stomatitis virus (VSV), and the DNA virus is herpes simplex virus type I (HSV-1). Both are conventional experimental viruses and are available from biological companies.

[0037] The C57BL / 6 mice are available from Tonglihua Animal Technology Company. All animals are used at 6-8 weeks. Animal experiments are conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health and are approved by the Ethics Committee of the School of Basic Medicine, Shandong University.

[0038] Synthesis method of ITA-9: Use standard operations and purification methods known to those skilled in the art. Unless otherwise specified, raw materials are usually available from commercial sources. Commercially available solvents and reagents are generally used without further purification. Anhydrous solvents are treated by standard methods, and other reagents are commercially available analytical pure. Unless otherwise stated, all temperatures are expressed in °C (degrees Celsius), and room temperature or ambient temperature refers to 20-25 °C. The structure of the compound is determined by nuclear magnetic resonance spectroscopy (NMR). The nuclear magnetic resonance hydrogen spectrum shift (δ) is given in parts per million (ppm). The nuclear magnetic resonance hydrogen spectrum is measured with a Bruker-400MHz nuclear magnetic resonance spectrometer, using deuterated chloroform (Chloroform- d ), deuterated dimethyl sulfoxide (DMSO- d 6 ) as solvents and tetramethylsilane (TMS) as the internal standard.

[0039] The chromatography column generally uses silica gel with 200 - 300 mesh as the carrier.

[0040] Experimental methods for ELISA and qPCR analysis: Use an IFN-β ELISA detection kit (R&D Systems) to detect the content of IFN-β in cell supernatants and mouse sera according to the instruction manual. Use TRIzol reagent (Invitrogen) to extract total RNA from cells or tissues. Use HiScript III RT SuperMix (Vazyme) to reverse transcribe RNA into cDNA. Perform PCR analysis using SYBR Green (Vazyme). The data is normalized and calculated using β-actin as the internal reference gene.

[0041] Experimental method for Western Blot: Wash the cells with phosphate-buffered saline (PBS), lyse the cells with RIPA protein extraction reagent (Pierce, Thermo Fisher Scientific) added with protease inhibitors (phenylmethylsulfonyl fluoride, Beyotime) and phosphatase inhibitors (CWBIO), and then centrifuge at 12,000×g for 15 minutes at 4°C. Use a BCA protein assay kit to measure the protein concentration in the supernatant. Adjust the lysates to the same concentration and perform electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel. Then transfer the proteins to a PVDF membrane (Millipore) for immunoblotting. Incubate the membrane with the primary antibody dilution overnight. The next day, wash the membrane three times with TBST and then incubate it in the secondary antibody (Immunoway) dilution (1:4000) for 1 hour. Image the protein bands using enhanced chemiluminescence (NCM Biotech) according to the manufacturer's protocol.

[0042] Experimental method for cellular thermal shift: After pretreating macrophages with a solvent or 100 μM ITA-9 for 12 h, cool the cells on ice, wash them with PBS buffer containing protease inhibitors, and then transfer them to PCR tubes. Then heat the cells at different temperatures (40, 42.5, 45, 47.5, 50, 52.5, and 55°C) for 3 min respectively and immediately cool them in liquid nitrogen for 10 s. After repeating three times, centrifuge the cells at 12,000×g for 15 min at 4°C, add 5×SDS loading buffer to the supernatant and boil it, and perform Western Blot experiment. Quantitatively analyze the protein bands using ImageJ software.

[0043] Experimental method for surface plasmon resonance: The affinity was measured using a BIAcore T200 instrument. Specifically, the hTBK1 protein was covalently coupled to a CM7 sensor chip, and the response value was 20,000 RU. The binding measurement was carried out at 25 °C and a flow rate of 30 μl / min. ITA-9 was serially diluted with PBS solution containing 5% DMSO and 0.05% surfactant P20. The binding time of ITA-9 to the protein on the chip was 60 s, and the dissociation time was 120 s. The binding curve was exported using GraphpPad Prism 9 for analysis, and the affinity was calculated using BIA evaluation software.

[0044] Example 1. Synthesis of compound ITA-9 As Figure 1 shown, a method for synthesizing compound ITA-9 includes the following steps: (1) Dissolve itaconic anhydride (Compound 1-1, 0.50 g, 4.46 mmol) in 2 ml of dichloromethane (DCM), then add 5 ml of anhydrous methanol and 75 μl of sulfuric acid, and stir the reaction overnight at room temperature; after monitoring the reaction by thin-layer chromatography (TLC) until completion, add 30 ml of DCM to the reaction solution, then alkalize the reaction solution with 10% potassium carbonate (pH > 10), separate the aqueous phase to obtain the organic phase; acidify the separated organic phase with 1 M hydrochloric acid (pH < 5), then extract three times with DCM, combine the organic phases after extraction, dry with anhydrous sodium sulfate, filter, evaporate under reduced pressure, and finally recrystallize the crude product in a mixed solution of ethyl acetate / n-hexane to obtain Compound 1-2; Among them, the structure of Compound 1-2 is shown in the following formula: ; (2) Dissolve Compound 1-2 (0.15 g, 1.04 mmol) and methyl N 6 -(tert-butylcarbonyl)- L -lysine hydrochloride (methyl N 6 -( tert -butoxycarbonyl)- L -lysinate hydrochloride, 0.31 g, 1.04 mmol) in 5 ml of DCM, then add 2-(7-azabenzotriazol)- N , N , N' , N'-Tetramethylurea hexafluorophosphate (HATU, 0.40 g, 1.04 mmol) and triethylamine (0.46 ml, 2.60 mmol) were stirred at room temperature overnight; after monitoring the reaction by TLC until completion, 100 ml of saturated brine was added, and the mixture was extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the target compound ITA-9.

[0045] The spectral data of compound 1-2 and compound ITA-9 prepared in this example are as follows: Compound 1-2: 1 H NMR (400 MHz, Chloroform- d ) δ 6.48 (s, 1H), 5.85 (s, 1H), 3.72 (s, 3H), 3.36 (s, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 171.73, 171.19, 133.29, 131.14, 52.32, 37.19. ESI-MS: Calculated for C6H8O4 144.04, found: 167.00 [M+Na] + .

[0046] Compound ITA-9: 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.37 (d, J J = 7.5 Hz, 1H), 6.78 (t, J J = 5.7 Hz, 1H), 5.95 (s, 1H), 5.59 (s, 1H), 4.25 – 4.19 (m, 1H), 3.61 (s, 3H), 3.56 (s, 3H), 3.34 (s, 2H), 3.17 (d, J J = 5.2 Hz, 1H), 2.89 (q, J J = 6.1 Hz, 2H), 2.69 (s, 1H), 1.69 (tq, J J = 8.8, 5.4 Hz, 2H), 1.37 (s, 9H), 1.25 (q, J J = 5.5 Hz, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 173.12, 171.33, 167.33, 156.03, 137.66, 122.94, 77.80, 52.77, 52.22, 51.93, 38.71, 37.92, 30.69, 29.48, 28.72, 23.37. ESI-MS: Calculated for C18H30N2O7 386.21, found: 409.24 [M+Na] + 。

[0047] The above data indicate that the compound ITA-9 of the present invention has been successfully prepared, and its structural formula is shown as follows: 。

[0048] Example 2. Compound ITA-9 inhibits the antiviral immune response mediated by TBK1 1. Incubate the compound ITA-9 prepared in Example 1 at different concentrations (0, 6.25, 12.5, 25, 50, 100 μM) with mouse peritoneal macrophages for 2 h, and then infect them with VSV virus and HSV-1 virus respectively, and continue to culture the infected cells. Take the cell supernatant, and detect the content of IFN-β in the cell supernatant by ELISA experiment. The results are shown in Figure 2 A and B.

[0049] As can be seen from Figure 2 A and B, ITA-9 inhibits the secretion of IFN-β in a concentration-dependent manner, and the EC 50 values are 26.33 μM and 17.33 μM respectively.

[0050] 2. Divide mouse peritoneal macrophages into four groups. The first group is experimental group 1 (ITA-9 / VSV), incubate 50 μM of the compound ITA-9 prepared in Example 1 with mouse peritoneal macrophages for 2 h respectively, and then infect them with VSV virus, and continue to culture the infected cells for 0, 8, 12 h. The second group is control group 1 (Ctrl / VSV), which uses dimethyl sulfoxide (DMSO) to replace the compound ITA-9 in experimental group 1. The third group is experimental group 2 (ITA-9 / HSV-1), incubate 50 μM of the compound ITA-9 prepared in Example 1 with mouse peritoneal macrophages for 2 h respectively, and then infect them with HSV-1 virus, and continue to culture the infected cells for 0, 2, 4 h. The fourth group is control group 2 (Ctrl / VSV), which uses dimethyl sulfoxide (DMSO) to replace the compound ITA-9 in experimental group 2.

[0051] The expression levels of p-TBK1, p-IRF3, and p-STAT1 in the cell lysates of the above 4 groups were detected by Western Blot assay, and the results are shown in Figure 2 C and D.

[0052] As can be seen from Figure 2 C and D, during the infection of VSV virus and HSV-1 virus, ITA-9 significantly inhibited the expression levels of p-TBK1, p-IRF3, and p-STAT1.

[0053] 3. Mouse peritoneal macrophages were divided into two groups. The first group was the experimental group (ITA-9), and mouse peritoneal macrophages were pretreated with different concentrations of the compound ITA-9 prepared in Example 1 (0, 6.25, 12.5, 25, 50, 100 μM) for 12 h. The second group was the control group (Ctrl), in which dimethyl sulfoxide (DMSO) was used to replace the compound ITA-9 in the experimental group.

[0054] At different temperatures (40, 42.5, 45, 47.5, 50, 52.5, and 55 °C), the stability of TBK1 protein in the two groups of cells was detected by cellular thermal shift assay, and the results are shown in Figure 3 A.

[0055] As can be seen from Figure 3 A, after treatment with ITA-9, the stability of TBK1 protein in the cells was significantly improved, indicating that ITA-9 interacted with TBK1 protein.

[0056] Next, the affinity between hTBK1 protein and ITA-9 was detected in vitro by surface plasmon resonance assay, and the results are shown in Figure 3 B.

[0057] As can be seen from Figure 3 B, there was a strong affinity between hTBK1 protein and ITA-9 in vitro, with K D = 55.87 nM, which also indicated that ITA-9 bound to TBK1 in a non-enzymatic manner.

[0058] 4. TBK1-MYC plasmid was transfected into HEK293T cells and cultured for 6 h. Then, the cells were incubated with DMSO or 50 μM ITA-9 for 12 h, and then incubated with an alkynylated probe ITALK (150 μM) that could alkylate TBK1 for another 12 h. After click chemical reaction of the cell lysate, the expression level of TBK1-MYC was detected by Western Blot assay, with untreated HEK293T cells as the control, and the results are shown in Figure 3 C.

[0059] As can be seen fromFigure 3 As can be seen from C, ITA-9 can competitively inhibit the binding of ITALK to TBK1, indicating that ITA-9 covalently binds to TBK1.

[0060] 5. The molecular docking of ITA-9 and TBK1 was performed using the CovDock module in the Schrodinger 2021-2 software package, and the reaction type of covalent docking was set to Michael addition. The results are shown in Figure 3 Figure D.

[0061] All the above data results indicate that the compound ITA-9 covalently binds to TBK1, and alkylating TBK1 can effectively inhibit the antiviral immune response mediated by TBK1.

[0062] Example 3. Compound ITA-9 inhibits the excessive inflammatory response induced by poly(I:C) 1. C57BL / 6J mice were divided into three groups. The first group was the experimental group (ITA-9 + poly(I:C)). After intraperitoneally injecting ITA-9 (10 mg / kg) prepared in Example 1 into C57BL / 6J mice for 2 h, poly(I:C) (15 mg / kg) was continuously intraperitoneally injected, and cultured for 8 h. The second group was the control group (Ctrl + poly(I:C)). After intraperitoneally injecting an equal amount of control solvent Ctrl into C57BL / 6J mice for 2 h, poly(I:C) (15 mg / kg) was continuously intraperitoneally injected, and cultured for 8 h. The third group was the blank group (Ctrl + PBS). After intraperitoneally injecting Ctrl into C57BL / 6J mice for 2 h, an equal amount of PBS buffer was injected, and cultured for 8 h.

[0063] The sera of the three groups of mice were taken, and the content of IFN-β in the sera of the mice was detected by ELISA experiment. The results are shown in Figure 4 Figure A.

[0064] The three groups of mice were sacrificed, and the lung tissues of the mice were taken. RNA was extracted according to the conventional method, and the expression level of Cxcl10 mRNA in the lung tissues of the mice was detected by qPCR experiment. The results are shown in Figure 4 Figure B.

[0065] As can be seen from Figure 4 Figures A and B, compared with the control group mice, after poly(I:C) infection, the content of IFN-β in the sera of ITA-9-treated mice and the expression of Cxcl10 mRNA in the lung tissues were significantly decreased.

[0066] 2. Divide C57BL / 6J mice into three groups. The first group is the experimental group (ITA-9 + poly(I:C)). After injecting ITA-9 (10 mg / kg) prepared in Example 1 into the peritoneal cavity of C57BL / 6J mice for 2 hours, inject poly(I:C) (25 mg / kg) into the peritoneal cavity, culture for 12 hours, then inject ITA-9 (10 mg / kg) into the peritoneal cavity again, and continue to culture for 12 hours. The second group is the control group (Ctrl + poly(I:C)). After injecting an equal amount of control solvent Ctrl into the peritoneal cavity of C57BL / 6J mice for 2 hours, inject poly(I:C) (25 mg / kg) into the peritoneal cavity, culture for 12 hours, then inject Ctrl into the peritoneal cavity again, and continue to culture for 12 hours. The third group is the blank group (PBS). Inject an equal amount of control solvent Ctrl into the peritoneal cavity of C57BL / 6J mice, inject an equal amount of PBS buffer solution into the peritoneal cavity, culture for 12 hours, then inject Ctrl into the peritoneal cavity again, and continue to culture for 12 hours.

[0067] Euthanize the mice in the three groups, take the lung tissues of the mice, prepare mouse lung tissue sections according to the existing method, then stain the mouse lung tissue sections with hematoxylin and eosin, Sirius red staining, and observe. The results are as Figure 4 shown in C and D.

[0068] As can be seen from Figure 4 C and D, compared with the control group, after poly(I:C) infection, the infiltration of inflammatory cells and airway collagen deposition in ITA-9-treated mice were significantly reduced.

[0069] The above data results all indicate that the compound ITA-9 can effectively inhibit the excessive inflammatory response induced by poly(I:C).

[0070] Example 4. Compound ITA-9 inhibits the inflammatory response of systemic lupus erythematosus (SLE) Divide C57BL / 6J mice into three groups. The first group is the experimental group (TMPD + ITA-9), the second group is the control group (TMPD), and the third group is the blank group (Ctrl). Inject 0.5 ml / mouse of 2,6,10,14-tetramethylpentadecane (TMPD) into the peritoneal cavity of the mice in the first and second groups to construct an SLE model, and inject an equal amount of PBS buffer solution into the third group. After three weeks, inject ITA-9 (10 mg / kg) prepared in Example 1 into the peritoneal cavity of the mice in the first group, inject daily for one week; inject an equal amount of DMSO into the peritoneal cavity of the mice in the second and third groups, inject daily for one week.

[0071] Take the plasma of the mice in the three groups, and biochemically analyze the content of Creatinine in the plasma of the mice. The results are as Figure 5 shown in A.

[0072] Three groups of mice were sacrificed, and their lung tissues were taken. RNA was extracted according to the conventional method, and the expression levels of Cxcl10 and Isg15 mRNA in the mouse tissues were detected by qPCR experiment. The results are shown in Figure 5 Figure B.

[0073] Mouse lung tissues were taken, and mouse lung tissue sections were prepared according to the existing method. Then, the mouse lung tissue sections were stained with hematoxylin and eosin and observed. The results are shown in Figure 5 Figure C.

[0074] As can be seen from Figure 5 Figures A, B, and C, compared with the control group, after treatment with ITA-9 in the SLE model, the content of Creatinine in the mouse plasma and the expression levels of Cxcl10 and Isg15 mRNA in the lung tissues were significantly decreased, and the infiltration of inflammatory cells in the mouse lung tissues was significantly reduced.

[0075] All the above data results indicate that the compound ITA-9 can effectively inhibit the inflammatory response in systemic lupus erythematosus.

[0076] Example 5. Inhibition of the inflammatory response of Aicardi-Goutières syndrome by compound ITA-9 The AGS syndrome mouse model Trex1 gene-deficient mice ( Trex1 - / - ) were divided into two groups, Group 1 and Group 2; wild-type mice of the same age and sex were divided into Group 3. Group 1 was the experimental group ( Trex1 - / - + ITA-9), Group 2 was the control group ( Trex1 - / - ), and Group 3 was the blank group (WT). The mice in Group 1 were intraperitoneally injected with the ITA-9 prepared in Example 1 (10 mg / kg) every day for one week; the mice in Group 2 and Group 3 were intraperitoneally injected with an equal amount of the control solvent Ctrl every day for one week.

[0077] Three groups of mice were sacrificed, and their lung tissues were taken. RNA was extracted according to the conventional method, and the expression levels of Cxcl10 , Isg15 , Isg54 , Isg56 , Ccl5 and Mx1 mRNA in the mouse tissues were detected by qPCR experiment. The results are shown in Figure 6 Figure A.

[0078] Take mouse lung tissue, prepare mouse lung tissue sections according to the existing method, then stain the mouse lung tissue sections with hematoxylin and eosin, and observe. The results are as Figure 6 shown in B.

[0079] As can be seen from Figure 6 A and B, compared with the control group and the blank group, after treatment with ITA-9 in the AGS syndrome mouse model, the expression levels of Cxcl10 , Isg15 , Isg54 , Isg56 , Ccl5 and Mx1 mRNA in the mouse lung tissue were significantly decreased, and the infiltration of inflammatory cells in the mouse lung tissue was significantly reduced.

[0080] All the above data results indicate that the compound ITA-9 can effectively inhibit the inflammatory response in the AGS syndrome model.

[0081] In summary, the present invention verified through experiments that the compound ITA-9 has a significant inhibitory activity function on TBK1, indicating that the compound ITA-9 can be used as a TBK1 inhibitor. And it also verified that the compound ITA-9 can effectively inhibit the antiviral immune response mediated by TBK1 and the excessive inflammatory response induced by poly(I:C). In addition, the present invention also verified that ITA-9 can effectively inhibit the inflammatory response in mice with systemic lupus erythematosus model and AGS syndrome model. It shows that this compound has the potential to be developed into a TBK1 inhibitor drug and drugs for treating TBK1-related diseases, such as infectious diseases, autoimmune diseases, metabolic diseases and cancers, providing a new therapeutic drug for clinical use, with good clinical application value and broad application prospects.

Claims

1. A compound ITA-9, whose structural formula is shown below: 。 2. The method for preparing the compound ITA-9 according to claim 1, comprising the following steps: (1) Dissolve itaconic anhydride in dichloromethane, then add anhydrous methanol and sulfuric acid, and stir the reaction at room temperature overnight; then add dichloromethane to the reaction solution, then use potassium carbonate to alkalize the reaction solution, separate the aqueous phase, and obtain an organic phase; then use hydrochloric acid to acidify the organic phase, extract it three times with dichloromethane, combine the organic phases after extraction, dry it with anhydrous sodium sulfate, filter it, evaporate it under reduced pressure, and finally recrystallize the crude product in an ethyl acetate / n-hexane mixed solution to obtain compound 1-2; in, The structure of compound 1-2 is shown below: ； The ratio of itaconic anhydride, dichloromethane, anhydrous methanol and sulfuric acid is (0.45-0.55 g): (1.5-2.5 ml): (3-6 ml): (70-80 μl); (2) Compound 1-2 and methyl N 6 -(tert-Butylcarbonyl)- L -Lysine hydrochloride was dissolved in dichloromethane, and then 2-(7-azobenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate and triethylamine, stirred at room temperature overnight, after the reaction, added saturated brine, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the target compound ITA-9; Wherein, the compound 1-2, methyl N 6 -(tert-Butylcarbonyl)- L -Lysine hydrochloride, dichloromethane, 2-(7-azobenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate and triethylamine ratio is (0.1~0.2g):(0.25~0.35g):(3~6 ml):(0.35~0.45g):(0.4~0.5ml).

3. Use of the compound ITA-9 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a TANK binding kinase 1 inhibitor.

4. Use of the compound ITA-9 or a pharmaceutically acceptable salt thereof according to claim 1 as a TANK binding kinase 1 inhibitor in the preparation of a pharmaceutical composition for preventing and / or treating diseases associated with TBK1 activity.

5. The use according to claim 4, characterized in that The disease associated with TBK1 activity refers to a disease that can be improved, prevented or treated by inhibiting TBK1 activity; Further preferably, the disease associated with TBK1 activity is an infectious disease, an autoimmune disease, a metabolic disease or cancer.

6. The use according to claim 5, characterized in that The infectious disease is a disease caused by one or more infections of DNA viruses or RNA viruses; Further preferably, the DNA virus is herpes simplex virus 1 (HSV-1), and the RNA virus is vesicular stomatitis virus (VSV).

7. The use according to claim 5, characterized in that The autoimmune diseases are AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis.

8. The use according to claim 5, characterized in that The metabolic diseases are obesity, diabetes and non-alcoholic fatty liver disease.

9. A pharmaceutical composition for treating a disease associated with TBK1 activity, characterized in that: The pharmaceutical composition comprises the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; Further preferably, the pharmaceutical composition is any pharmaceutically acceptable dosage form prepared by using compound ITA-9 or a pharmaceutically acceptable salt thereof as the main active ingredient and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage forms are tablets, capsules, granules, pills, liquid preparations, decoctions, suspensions, dispersants, syrups, suppositories, gels, aerosols, and patches.

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