Compound ITA-5 serving as TBK1 inhibitor as well as synthesis method and application of compound ITA-5

Abstract

The invention relates to a compound ITA-5 serving as a TBK1 inhibitor as well as a synthesis method and application of the compound ITA-5, and belongs to the technical field of biological medicines. The TBK1 inhibitor is a compound ITA-5. The invention also provides an application of the compound ITA-5 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 the application of the compound ITA-5 serving as a TBK1 inhibitor in preparation of the 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 compound ITA-5 as a TBK1 inhibitor, its synthesis method and applications, belonging to the technical field of biomedicine. Background Art

[0002] TANK-binding kinase 1 (TBK1) is a serine / threonine kinase and belongs 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, tumor, inflammation, and metabolism.

[0003] The innate immune system is the first line of defense for the host to resist virus infection. DNA recognition receptor cGAS (Cyclic GMP-AMP synthase) and RNA recognition receptor RLRs can recognize DNA viruses (such as Herpes simplex virus, HSV) or RNA viruses (such as Vesicular stomatitis virus, VSV), thereby activating the host antiviral innate immune cascade reaction. After activation of various virus infections mediated by different pattern recognition receptors (PRRs), they all pass through the common key kinase TBK1 to induce the activation of transcription factor IRF3, and ultimately lead to the expression of type I interferon (IFN-α / β), starting the antiviral innate immune response. An appropriate type I interferon response helps the host to promptly clear the invading virus in the early stage; however, abnormal innate immune responses such as excessive inflammatory responses can induce cytokine storms, which in turn cause host tissue damage and even organ failure. Therefore, the normal activation of TBK1 plays a key role in the antiviral immune response process, which makes the research on small molecule inhibitors of TBK1 become a hot spot.

[0004] With the in-depth understanding of the tertiary structure and biological functions of TBK1 kinase, TBK1 has become a potential target for the treatment of inflammatory diseases, autoimmune diseases, immunodeficiency diseases, neurodegenerative diseases, metabolic diseases, and cancers. Although the role of TBK1 is crucial, the research on its specific inhibitors is still in the early stage. Therefore, it is still very urgent to find highly efficient and safe small molecule inhibitors of TBK1. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a compound ITA-5 as a TBK1 inhibitor, its synthesis method and applications.

[0006] The technical solution of the present invention is as follows: A compound ITA-5, whose structural formula is shown as follows: .

[0007] The preparation method of the above compound ITA-5 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 alkalize the reaction solution with potassium carbonate, separate the aqueous phase to obtain an 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 ITA-1; Among them, the structure of compound ITA-1 is shown as follows: ; (2) Dissolve compound ITA-1 and L -phenylalanine methyl ester hydrochloride in dichloromethane, then add 2-(7-azabenzotriazole)- N,N,N',N' -tetramethylurea hexafluorophosphate and triethylamine, stir overnight at room temperature, after the reaction is completed, add saturated brine, extract with dichloromethane, dry the organic phase with anhydrous sodium sulfate, filter, evaporate under reduced pressure, and after purification by column chromatography, obtain the target compound ITA-5.

[0008] 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).

[0009] Preferably according to the present invention, in step (2), the ratio of compound ITA-1, L -phenylalanine methyl ester hydrochloride, dichloromethane, 2-(7-azabenzotriazole)- N,N,N',N' -tetramethylurea hexafluorophosphate and triethylamine is (0.1~0.2 g):(0.15~0.25 g):(3~6 ml):(0.3~0.5 g):(0.4~0.5 ml).

[0010] The application of the above compound ITA-5 or its pharmaceutically acceptable salt in the preparation of a TBK1 inhibitor.

[0011] The application of the above compound ITA-5 or its pharmaceutically acceptable salt as a TBK1 inhibitor in the preparation of a pharmaceutical composition for preventing and / or treating diseases related to TBK1 activity.

[0012] 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.

[0013] Preferably according to the present invention, the diseases related to TBK1 activity are infectious diseases, autoimmune diseases, metabolic diseases or cancers and other diseases.

[0014] More preferably, the infectious disease is a disease caused by infection with one or more of DNA viruses or RNA viruses.

[0015] Even more preferably, the DNA virus is herpes simplex virus 1 (HSV-1), and the RNA virus is vesicular stomatitis virus (VSV).

[0016] More preferably, the autoimmune diseases are AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis and other diseases.

[0017] More preferably, the metabolic diseases are obesity, diabetes and non-alcoholic fatty liver disease and other diseases.

[0018] A pharmaceutical composition for treating diseases related to TBK1 activity, the pharmaceutical composition comprising the above-mentioned compound ITA-5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0019] Preferably according to the present invention, the pharmaceutical composition is any pharmaceutically acceptable dosage form prepared with compound ITA-5 or a pharmaceutically acceptable salt thereof as the main active ingredient and a pharmaceutically acceptable excipient.

[0020] More preferably, the dosage form is tablets, capsules, granules, pills, liquid preparations, decoction extracts, suspensions, dispersants, syrups, suppositories, gels, aerosols, patches, etc.

[0021] The beneficial effects of the present invention are as follows: 1. The present invention provides a novel compound ITA-5, which exhibits significantly improved TBK1 inhibitory activity compared with existing methylene succinic acid compounds with similar structures and can be used as a TBK1 inhibitor.

[0022] 2. The present invention firstly discloses the application of compound ITA-5 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 drug of the TBK1 inhibitor class and a therapeutic drug for diseases related to TBK1, 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

[0023] Figure 1 It is a synthetic route diagram of compound ITA-5.

[0024] Figure 2 It is the effect of compound ITA-5 on macrophage activity.

[0025] Figure 3 It is that compound ITA-5 inhibits the antiviral immune response mediated by the RNA virus VSV; In the figure, A is to detect the secretion of IFN-β by ELISA and calculate the EC 50 ; B is to detect the protein levels of p-TBK1, p-IRF3, and p-STAT1 by Western Blot; C is to detect the aggregation of TBK1 around the nucleus by immunofluorescence.

[0026] Figure 4 It is that compound ITA-5 inhibits the antiviral immune response mediated by the DNA virus HSV-1; In the figure, A is to detect the secretion of IFN-β by ELISA and calculate the EC 50 ; B is to detect the protein levels of p-TBK1, p-IRF3, and p-STAT1 by Western Blot; C is to detect the aggregation of TBK1 around the nucleus by immunofluorescence.

[0027] Figure 5 It is the verification that compound ITA-5 targets TBK1; In the figure, A is the molecular docking of ITA-5 and TBK1; B is to detect the affinity of ITA-5 and TBK1 by surface plasmon resonance experiment; C is to pre-incubate mouse peritoneal macrophages with Ctrl or ITA-5, and detect the stability of TBK1 by cellular thermal shift assay.

[0028] Figure 6 It is that ITA-5 inhibits the excessive inflammatory response induced by poly(I:C); In the figure, A is to detect the secretion of IFN-β in mouse serum by ELISA; B is to detect in mouse lung tissue by RT-PCR Cxcl10mRNA expression; C and D are liver and kidney injury indicators in mouse plasma; E is hematoxylin and eosin staining of mouse lung tissue sections.

[0029] Figure 7 Compound ITA-5 inhibits the inflammatory response in a systemic lupus erythematosus (SLE) model; In the figure, A is the content of the kidney injury indicator Creatinine detected by biochemical analysis in mouse plasma; B is hematoxylin and eosin staining of mouse lung tissue sections; C is qPCR detection of Cxcl10 and Isg15 the mRNA expression level in mouse lung tissue.

[0030] Figure 8 Compound ITA-5 inhibits the inflammatory response in an Aicardi-Goutières syndrome (AGS) model; In the figure, A is hematoxylin and eosin staining of mouse lung tissue sections; B is qPCR detection of Cxcl10 、 Isg15 the mRNA expression level in mouse lung tissue. Detailed implementation mode

[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 cannot be regarded as a limitation of the present invention. The following further illustrates the present invention in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0032] Example 1: Synthesis of Compound ITA-5 As Figure 1 shown, a method for synthesizing compound ITA-5 includes the following steps: (1) Dissolve itaconic anhydride (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 it ends, add 30 ml of DCM to the reaction solution, then alkalize the solution with 10% potassium carbonate (pH>10), separate the aqueous phase, acidify the separated organic phase with 1M hydrochloric acid (pH<5), then extract it 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 ITA-1; Among them, the structure of compound ITA-1 is shown in the following formula: ; (2) Dissolve compound ITA-1 (0.15 g, 1.04 mmol) and L -phenylalanine methyl ester hydrochloride ( L -phenylalanine methyl ester hydrochloride, 0.19 g, 0.87 mmol) in 5 ml of DCM, then add 2-(7-azabenzotriazol)- N,N,N',N' -tetramethyluronium hexafluorophosphate (HATU, 0.40 g, 1.04 mmol) and triethylamine (0.46 ml, 2.60 mmol). After stirring overnight at room temperature (monitored by TLC), add 100 ml of saturated brine, extract with DCM, separate the organic phase, dry with anhydrous sodium sulfate, filter, evaporate under reduced pressure, and purify by column chromatography to obtain the target compound ITA-5.

[0033] The spectral data of compound ITA-1 and compound ITA-5 prepared in this example are as follows: Compound ITA-1: 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] + .

[0034] Compound ITA-5: 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.37 (d, J = 7.5 Hz, 1H), 6.78 (t, 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= 5.2 Hz, 1H), 2.89 (q, J = 6.1 Hz,2H), 2.69 (s, 1H), 1.69 (tq, J = 8.8, 5.4 Hz, 2H), 1.37 (s, 9H), 1.25 (q, J = 5.5Hz, 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] + 。

[0035] The above data indicate that the compound ITA-5 was successfully prepared in the present invention, and its chemical structural formula is as follows: 。

[0036] Example 2. Effect of Compound ITA-5 on Macrophage Activity 1. Experimental Materials The CellTiter-Glo® 2.0 Cell Viability Assay (Promega)。

[0037] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice aged 6 - 11 weeks, both male and female, were used. The mice were housed in the SPF barrier environment of the Model Animal Research Center of Shandong University, under the conditions of temperature 20 - 26 °C and humidity 40 - 70%, and were raised according to the day-night cycle (every 12 h). All animal experiments were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health and were approved by the Ethics Committee of the School of Basic Medicine of Shandong University.

[0038] 2. Experimental Methods C57BL / 6 mice were intraperitoneally injected with 6% starch solution. After 3 days, the peritoneal cavity was repeatedly rinsed with high-glucose DMEM solution, and the rinsing fluid was collected into a 50 mL centrifuge tube. Centrifugation was carried out at room temperature and 1000 rpm for 5 min, and the supernatant was discarded to obtain primary peritoneal macrophages (PMs) of mice.

[0039] Resuspend PMs with complete medium, count the cells, inoculate the cells into a 384-well plate, and pre-incubate them with different concentrations of compound ITA-5 (0, 5, 10, 50, 100, 150, 200, 250 μM) for 2 h. After the pre-incubation, add 25 μL of CellTiter-Glo® 2.0 Reagent to each well, react at room temperature for 30 min, mix well on a shaker for 2 min, continue to incubate at room temperature for 10 min, detect the fluorescence with a microplate reader, and measure the cell viability. The results are as Figure 2 shown.

[0040] 3. Experimental Results As Figure 2 can be seen, compound ITA-5 at different concentrations (0, 5, 10, 50, 100, 150, 200, 250 μM) does not affect the viability of mouse primary peritoneal macrophages (PMs).

[0041] Example 3. Compound ITA-5 Inhibits the Antiviral Immune Response Mediated by RNA Virus VSV 1. Experimental Materials The RNA virus is vesicular stomatitis virus (VSV), which is a conventional experimental virus and is available from biological companies.

[0042] Mouse IFN-β ELISA kit is purchased from R&D Systems; The antibody anti-Phospho-TBK1 is purchased from Abcam; anti-Phospho-IRF3, anti-Phospho-STAT1 and anti-TBK1 are purchased from CST; anti-Actin is purchased from Proteintech; Mouse fibroblasts (L929) are purchased from the Cell Bank of the Chinese Academy of Sciences.

[0043] C57BL / 6 mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mouse primary peritoneal macrophages (PMs) are induced and obtained by intraperitoneal injection of 6% starch solution.

[0044] 2. Experimental Methods ①. Co-incubate mouse peritoneal macrophages with different concentrations of compound ITA-5 (0, 6.25, 12.5, 25, 50, 100 μM) prepared in Example 1 for 2 h, then infect with VSV virus, and continue to culture the infected cells.

[0045] Take the supernatants of three groups of cells, and detect the content of IFN-β in the cell supernatants by ELISA experiment. The results are as Figure 3 shown in

[0046] ELISA assay: The content of IFN-β in the cell supernatant was detected using an IFN-β ELISA kit (R&D Systems) according to the instruction manual.

[0047] ② Divide mouse peritoneal macrophages into two groups. For the first group, mouse peritoneal macrophages were pre-incubated with compound ITA-5 at a concentration of 50 μM for 2 h, then infected with VSV virus, and the infected cells were further cultured for 0, 8, and 12 h after infection, serving as the experimental group (ITA-5 / VSV). For the second group, mouse peritoneal macrophages were pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 h, then infected with VSV virus, and the infected cells were further cultured for 0, 8, and 12 h after infection, serving as the control group (Ctrl / VSV).

[0048] The expression levels of p-TBK1, p-IRF3, and p-STAT1 proteins in the two groups of cells were detected by Western Blot assay, and the results are shown as Figure 3 shown in B.

[0049] Western Blot detection: Cells were washed with phosphate-buffered saline (PBS), lysed with RIPA protein extraction reagent (Pierce, Thermo Fisher Scientific) supplemented with protease inhibitor (phenylmethylsulfonyl fluoride, Beyotime) and phosphatase inhibitor (CWBIO), and then centrifuged at 12,000 × g for 15 minutes at 4 °C. The protein concentration in the supernatant was determined using a BCA protein assay kit. The lysates were adjusted to the same concentration and electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel. Then the proteins were transferred to a PVDF membrane (Millipore) for immunoblotting. The membrane was incubated with the primary antibody dilution overnight. The next day, the membrane was washed three times with TBST and then incubated in the secondary antibody (Immunoway) dilution (1:4000) for 1 h. Protein bands were imaged using enhanced chemiluminescence (NCM Biotech) according to the manufacturer's protocol.

[0050] ③. The mouse fibroblast cells (L929) were divided into 4 groups. In the first group, L929 cells were pre-incubated with the compound ITA-5 at a concentration of 50 μM for 2 h, then infected with the VSV virus, and the infected cells were continuously cultured, which was the experimental group (ITA-5 / VSV). In the second group, L929 cells were pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 h, then infected with the VSV virus, and the infected cells were continuously cultured, which was the control group (Ctrl / VSV). In the third group, L929 cells were pre-incubated with the compound ITA-5 at a concentration of 50 μM for 2 h, and the cells were continuously cultured, which was the positive blank group (ITA-5 / Mock). In the fourth group, L929 cells were pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 h, and the cells were continuously cultured, which was the negative blank group (Ctrl / Mock).

[0051] The aggregation of TBK1 around the nucleus in the 4 groups of cells was detected by immunofluorescence assay, and the results were as Figure 3 shown in

[0052] Immunofluorescence assay: The 4 groups of cells after the above treatments were fixed with immunofluorescence fixative for 10 min, washed three times with PBS buffer, permeabilized with PBS buffer containing 0.5% Triton-X100, washed three times with PBS buffer, blocked with 3% BSA for 1 h, incubated with anti-TBK1 overnight at 4 °C, washed three times with PBS buffer, added with fluorescent secondary antibody AlexaFluor 488, incubated at room temperature for 1 h, washed three times with PBS buffer, added DAPI to stain the nucleus, and after washing three times with PBS buffer, the coverslips were fixed on the glass slides with anti-fluorescence quenching mounting medium, and the cells were analyzed using a Zeiss LSM980 confocal laser microscope.

[0053] 3. Experimental results As Figure 3 shown in

[0054] A, ITA-5 significantly inhibited the secretion of IFN-β induced by VSV infection in a concentration-dependent manner. Figure 3 As

[0055] shown in Figure 3 B, ITA-5 significantly inhibited the protein expression levels of p-TBK1, p-IRF3 and p-STAT1 induced by VSV infection.

[0056] As

[0057] shown in 1. Experimental materials The DNA virus is herpes simplex virus type I (HSV-1), which is a virus commonly used in laboratory experiments and is available for sale in biological companies.

[0058] The Mouse IFN-β ELISA kit was purchased from R&D Systems; The antibody anti-Phospho-TBK1 was purchased from Abcam; anti-Phospho-IRF3, anti-Phospho-STAT1 and anti-TBK1 were purchased from CST; anti-Actin was purchased from Proteintech; Mouse fibroblasts (L929) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0059] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and primary peritoneal macrophages (PMs) of mice were induced and obtained by intraperitoneal injection of 6% starch solution.

[0060] 2. Experimental methods ① Incubate the compound ITA-5 prepared in Example 1 at different concentrations (0, 6.25, 12.5, 25, 50, 100 μM) with mouse peritoneal macrophages for 2 h, then infect with HSV-1 virus, and continue to culture the infected cells.

[0061] Take the supernatant of three groups of cells, and detect the content of IFN-β in the cell supernatant by ELISA experiment. The results are as Figure 4 shown in A.

[0062] The ELISA detection method is the same as that in Example 3.

[0063] ② Divide mouse peritoneal macrophages into two groups. The first group is to pre-incubate mouse peritoneal macrophages with the compound ITA-5 at a concentration of 50 μM for 2 h, then infect with HSV-1 virus, and continue to culture the infected cells for 0, 2, 4 h, which is the experimental group (ITA-5 / HSV-1). The second group is to pre-incubate mouse peritoneal macrophages with an equal amount of dimethyl sulfoxide (DMSO) for 2 h, then infect with VSV virus, and continue to culture the infected cells for 0, 2, 4 h, which is the control group (Ctrl / HSV-1).

[0064] Detect the expression levels of p-TBK1, p-IRF3 and p-STAT1 proteins in the two groups of cells by Western Blot experiment. The results are as Figure 4 shown in B.

[0065] The Western Blot detection method is the same as that in Example 3.

[0066] ③ Divide mouse fibroblasts (L929) into 4 groups. For the first group, L929 cells are pre-incubated with compound ITA-5 at a concentration of 50 μM for 2 h, then infected with HSV-1 virus, and the infected cells are continuously cultured, serving as the experimental group (ITA-5 / HSV-1). For the second group, L929 cells are pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 h, then infected with HSV-1 virus, and the infected cells are continuously cultured, serving as the control group (Ctrl / HSV-1). For the third group, L929 cells are pre-incubated with compound ITA-5 at a concentration of 50 μM for 2 h, and the cells are continuously cultured, serving as the positive blank group (ITA-5 / Mock). For the fourth group, L929 cells are pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 h, and the cells are continuously cultured, serving as the negative blank group (Ctrl / Mock).

[0067] Detect the aggregation of TBK1 around the nucleus in the 4 groups of cells by immunofluorescence assay, and the results are as Figure 4 shown in C.

[0068] The detection method by immunofluorescence assay is the same as that in Example 3.

[0069] 3. Experimental results As Figure 4 shown in A, ITA-5 significantly inhibits the secretion of IFN-β induced by HSV-1 infection in a concentration-dependent manner.

[0070] As Figure 4 shown in B, ITA-5 significantly inhibits the protein expression levels of p-TBK1, p-IRF3 and p-STAT1 induced by HSV-1 infection.

[0071] As Figure 4 shown in C, ITA-5 inhibits the aggregation of TBK1 around the nucleus.

[0072] The above data results all indicate that compound ITA-5 inhibits the antiviral immune response mediated by DNA virus HSV-1.

[0073] Example 5. Verification of ITA-5 targeting TBK1 1. Experimental materials Purified hTBK1 protein is purchased from Detaibio (Nanjing, China); anti-TBK1 is purchased from CST.

[0074] C57BL / 6 mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and primary mouse peritoneal macrophages (PMs) are induced and obtained by intraperitoneal injection of 6% starch solution.

[0075] 2. Experimental methods ①. The molecular docking of ITA-5 with TBK1 was performed using the CovDock module in the Schrodinger 2021-2 software package. The reaction type of covalent docking was set to Michael addition, and the results are as Figure 5 shown in

[0076] A. Figure 5 ②. The affinity between hTBK1 protein and ITA-5 was detected in vitro by surface plasmon resonance experiment, and the results are as

[0077] shown in

[0078] B.

[0079] The experimental method of surface plasmon resonance: The affinity was measured using a BIAcore T200 instrument. The hTBK1 protein was covalently coupled to a CM7 sensor chip with a response value of 20,000 RU. The binding measurement was carried out at 25 °C and a flow rate of 30 μl / min. ITA-5 was serially diluted with PBS solution containing 5% DMSO and 0.05% surfactant P20. The binding time of ITA-5 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 BIAevaluation software. Figure 5 ③. Mouse peritoneal macrophages were divided into two groups. In the first group, mouse peritoneal macrophages were pretreated with compound ITA-5 at a concentration of 50 μM for 12 h, which was the experimental group (ITA-5). In the second group, mouse peritoneal macrophages were pretreated with an equal volume of dimethyl sulfoxide (DMSO) for 12 h, which was the control group (Ctrl).

[0080] The stability of TBK1 protein in the two groups of cells was detected by cell thermal shift experiment at different temperatures (40, 42.5, 45, 47.5, 50, 52.5, and 55 °C), and the results are as

[0081] shown in by Figure 5As can be seen from A, ITA-5 and TBK1 protein form a relatively stable covalent bond.

[0082] As can be seen from Figure 5 B, there is a strong affinity between TBK1 and ITA-5, and K D = 81.75 nM.

[0083] As can be seen from Figure 5 C, the treatment with ITA-5 enhanced the stability of TBK1 protein.

[0084] The above data results all indicate that the compound ITA-5 can directly target TBK1 to play a role.

[0085] Example 6: ITA-5 inhibits the excessive inflammatory response induced by poly(I:C); 1. Experimental materials C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and 6-8-week-old female mice were used; poly(I:C) was purchased from InvivoGen.

[0086] 2. Experimental methods ①. Divide C57BL / 6J mice into three groups. The first group is the experimental group (ITA-5 + poly(I:C)). After intraperitoneally injecting ITA-5 (20 mg / kg) prepared in Example 1 into C57BL / 6J mice for 2 h, continue to intraperitoneally inject poly(I:C) (15 mg / kg) and culture for 8 h. The second group is the control group (Ctrl + poly(I:C)). After intraperitoneally injecting an equal amount of control solvent Ctrl into C57BL / 6J mice for 2 h, continue to intraperitoneally inject poly(I:C) (15 mg / kg) and culture for 8 h. The third group is the blank group (Ctrl + PBS). After intraperitoneally injecting Ctrl into C57BL / 6J mice for 2 h, inject an equal amount of PBS buffer and culture for 8 h.

[0087] Take the sera of the three groups of mice and detect the content of IFN-β in the mouse sera through ELISA experiment. The results are as Figure 6 shown in A.

[0088] Euthanize the three groups of mice, take the lung tissues of the mice, extract RNA according to the conventional method, and detect the Cxcl10 mRNA expression level in the mouse lung tissues through RT-PCR experiment. The results are as Figure 6 shown in B.

[0089] ②. Divide C57BL / 6J mice into three groups. The first group is the experimental group (ITA-5 + poly(I:C)). After injecting ITA-5 (20 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-5 (20 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 the control solvent Ctrl again, and continue to culture for 12 hours. The third group is the blank group (Ctrl + PBS). Inject the 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 the control solvent Ctrl again, and continue to culture for 12 hours.

[0090] Euthanize the mice in the three groups, collect the blood from the mouse eyeballs, and then take creatinine and urea as indicators of kidney injury, and alanine aminotransferase (ALT), total protein, and globulin as indicators of liver injury to perform biochemical analysis on the blood from the mouse eyeballs. The results are as Figure 6 shown in C and D.

[0091] After euthanizing the mice in the three groups, collect the lung tissues of the mice at the same time. 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 E.

[0092] 3. Experimental results As Figure 6 shown in A, compared with the control group mice, the secretion of IFN-β in the serum of ITA-5 group mice was significantly reduced.

[0093] As Figure 6 shown in B, the Cxcl10 mRNA expression in the lung tissues of ITA-5 group mice was significantly lower than that of the control mice.

[0094] As Figure 6 shown in C and D, ITA-5 inhibited the kidney injury indicators and liver injury indicators in the plasma of mice.

[0095] As Figure 6 shown in E, ITA-5 attenuated the infiltration of inflammatory cells in the lung tissues of mice induced by poly(I:C) infection.

[0096] The above data results all indicate that the compound ITA-5 can effectively inhibit the excessive inflammatory response induced by poly(I:C).

[0097] Example 4: Compound ITA-5 inhibits the inflammatory response in systemic lupus erythematosus (SLE) 1. Experimental materials C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and 6-week-old female mice were used; TMPD was purchased from Sigma.

[0098] 2. Experimental methods C57BL / 6J mice were divided into three groups. The first group was the experimental group (TMPD + ITA-5). To establish an SLE model, 0.5 ml of 2,6,10,14-tetramethylpentadecane (TMPD) was intraperitoneally injected into C57BL / 6J mice. Three weeks later, ITA-5 prepared in Example 1 (20 mg / kg) was intraperitoneally injected once a day for one week. The second group was the control group (TMPD). To establish an SLE model, 0.5 ml of TMPD was intraperitoneally injected into C57BL / 6J mice. Three weeks later, an equal volume of the control solvent was intraperitoneally injected once a day for one week. The third group was the blank group (Ctrl). 0.5 ml of PBS was intraperitoneally injected into C57BL / 6J mice. Three weeks later, an equal volume of the control solvent was intraperitoneally injected once a day for one week.

[0099] Plasma was collected from the three groups of mice, and the content of Creatinine in the plasma of the mice was detected by biochemical analysis. The results are shown in Figure 7 A as follows.

[0100] After sacrificing the three groups of mice, the lung tissues of the mice were collected simultaneously. Mouse lung tissue sections were prepared according to the existing method, and then the mouse lung tissue sections were stained with hematoxylin and eosin and observed. The results are shown in Figure 7 B as follows.

[0101] After sacrificing the three groups of mice, the lung tissues of the mice were taken, and RNA was extracted according to the conventional method. The expression levels of Cxcl10 and Isg15 mRNA in the mouse lung tissues were detected by RT-PCR experiments. The results are shown in Figure 7 C as follows.

[0102] 3. Experimental results As can be seen from Figure 7 A, ITA-5 inhibits the renal injury index Creatinine in the plasma of mice.

[0103] As can be seen from Figure 7 B, ITA-5 reduces the infiltration of inflammatory cells in the lung tissues of the mouse systemic lupus erythematosus model induced by TMPD.

[0104] As can be seen from Figure 7It can be seen from C that the Cxcl10 and Isg15 mRNA expressions in the lung tissues of the ITA-5 group mice were significantly lower than those of the control mice.

[0105] The above data results all indicate that the compound ITA-5 can effectively inhibit the inflammatory response in systemic lupus erythematosus.

[0106] Example 5. Inhibition of the inflammatory response of Aicardi-Goutières syndrome by the compound ITA-5 1. Experimental materials Trex1 The gene-deficient mice were purchased from Jackson Laboratory and were raised and bred in the SPF barrier environment of the Model Animal Research Center of Shandong University; the C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and all were 4-week-old female mice; 2. Experimental methods The AGS syndrome mouse model Trex1 The gene-deficient mice were divided into two groups, Group 1 and Group 2; the wild-type mice of the same age and sex were divided into Group 3. Group 1 was the experimental group ( Trex1 - / - + ITA-5), Trex1 - / - When the mice were 4 weeks old, they were intraperitoneally injected with the ITA-5 prepared in Example 1 (20 mg / kg) once a day for one week. Group 2 was the control group ( Trex1 - / - ), Trex1 - / - When the mice were 4 weeks old, they were intraperitoneally injected with an equal amount of the control solvent Ctrl once a day for one week. Group 3 was the blank group (WT), and when the WT mice were 4 weeks old, they were intraperitoneally injected with an equal amount of the control solvent Ctrl once a day for one week.

[0107] After sacrificing the three groups of mice, the lung tissues of the mice were collected simultaneously, and the mouse lung tissue sections were prepared according to the existing method, and then the mouse lung tissue sections were stained with hematoxylin and eosin and observed. The results are as Figure 8 shown in A.

[0108] After sacrificing the three groups of mice, the lung tissues of the mice were taken, and RNA was extracted according to the conventional method. The expression levels of Cxcl10 and Isg15 mRNA in the mouse lung tissues were detected by RT-PCR experiment. The results are as Figure 8 shown in B.

[0109] 3. Experimental results From Figure 8 A, it can be seen that ITA-5 weakens Trex1- / - Inflammatory cell infiltration in lung tissue of mice (AGS syndrome model).

[0110] As Figure 8 shown by Trex1 - / - in the lung tissue of mice Cxcl10 and Isg15 mRNA expression was significantly lower than that of control mice.

[0111] The above data results all indicate that compound ITA-5 can effectively inhibit the inflammatory response in the AGS syndrome model.

[0112] In summary, the present invention verified through experiments that compound ITA-5 has a significant inhibitory activity function on TBK1, indicating that compound ITA-5 can be used as a TBK1 inhibitor. And it also verified that compound ITA-5 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-5 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 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.

Claims

1. A compound ITA-5, whose structural formula is shown below: 。 2. The preparation method of the compound ITA-5 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 ITA-1; in, The structure of compound ITA-1 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 ITA-1 and L -phenylalanine methyl ester 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-5; Wherein, the compound ITA-1, L -phenylalanine methyl ester hydrochloride, dichloromethane, 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethyluronium hexafluorophosphate and triethylamine ratio is (0.1~0.2g):(0.15~0.25g):(3~6 ml):(0.3~0.5g):(0.4~0.5ml).

3. Use of the compound ITA-5 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a TBK1 inhibitor.

4. Use of the compound ITA-5 or a pharmaceutically acceptable salt thereof according to claim 1 as a TBK1 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-5 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-5 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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