A tank-binding kinase 1 inhibitor and pharmaceutical uses thereof

Abstract

The present application relates to a kind of TANK binding kinase 1 inhibitor and its pharmaceutical use, belong to biological medicine technical field.The TANK binding kinase 1 inhibitor is compound ITA-9.The present application further provides the application of compound ITA-9 in preparation of prevention and / or treatment with TBK1 activity related disease drug composition, and the disease related with TBK1 activity is infectious disease, autoimmune disease, metabolic disease or cancer and the like.The present application first discloses compound ITA-9 as TANK binding kinase 1 (TBK1) inhibitor, the application in preparation prevention and / or treatment with TBK1 activity related disease drug composition.The compound has the potential to be developed into TBK1 inhibitor drug and the treatment drug of TBK1 related disease, for example infectious disease, autoimmune disease, metabolic disease and cancer and the like, provide new treatment drug for clinic, with good clinical application value and broad application prospect.

Description

Technical Field

[0001] This invention relates to a TANK-binding kinase 1 inhibitor and its pharmaceutical use, belonging to the field of biomedical technology. Background Technology

[0002] Autoimmune diseases are diseases caused by damage to the body's own tissues due to an immune response to self-antigens. Common autoimmune diseases include rheumatoid arthritis, psoriasis, Crohn's disease, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, allergic diseases, chronic obstructive pulmonary disease, asthma, leukemia, lymphoma, etc.

[0003] IKKε and TBK1 are highly homologous Ser / Thr kinases that are crucial in the innate immune response by inducing type I interferon 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) and viral double-stranded RNS (dsRNA) to Toll-like receptors, subsequently activating the TBK1 pathway. Activated TBK1 and IKKε phosphorylate IRF3 and IRF7, which triggers dimerization and nuclear translocation of those interferon-regulated transcription factors, ultimately inducing a signaling 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 participates in the regulation of multiple signaling pathways and transcription factors, including interferon regulatory factor (IRF), nuclear factor κB (NF-κB), type I interferon (IFN-I), and type II interferon (IFN-II) target genes, as well as STING-mediated cytoplasmic DNA detection. It regulates antiviral defense and host-virus interactions, playing a crucial role in the occurrence and development of diseases such as those related to immunity, tumors, inflammation, and metabolism. TBK1 can also activate its substrates IRF3 and IRF7 transcription factors by directly phosphorylating specific sites, inducing them to localize to the nucleus to drive the transcription of type I IFN genes. Excessive expression of type I IFN may cause damage to the body. Furthermore, after different pattern recognition receptors are activated by viral infection, they recruit and activate IRF3 by activating the common key kinase TBK1, thereby inducing the production of type I interferon (IFN-α / β) and initiating an antiviral immune response. TBK1-mediated innate immunity plays a key role in clearing the virus, which makes the research on TBK1 small molecule inhibitors a hot topic.

[0005] The development of TBK1 inhibitors is still in its early stages. This is partly because the biological functions and mechanisms of action of TBK1 are not yet fully understood, and partly because there are few structural types of TBK1 inhibitors, with selective TBK1 inhibitors yet to enter clinical trials. Most existing TBK1 inhibitors have multiple targets. For example, BX795 (Amlexanox), developed by Takeda Pharmaceutical Company Limited, was marketed as a PDK1 inhibitor, but it was later found to exhibit comparable inhibitory activity against both TBK1 and IKKε (PDK1 IC50 = 17 nM, TBK1 IC50 = 2.3 nM, IKKε IC50 = 9.5 nM). GSK8612, a selective TBK1 inhibitor reported by GlaxoSmithKline in 2019, although exhibiting 10-fold selectivity for IKK kinases, has not yet entered clinical trials due to its poor activity against TBK1 kinases (TBK1 p IC50 = 6.8). Patent document WO2018154315A1 provides a TBK1 inhibitor. Although this inhibitor has good TBK1 kinase inhibitory activity (IC50 = 20 nM), it still has the problem of having multiple targets, simultaneously targeting TBK1 and IKKε (IKKε IC50 = 21 nM).

[0006] Therefore, the search for highly efficient and selective small-molecule inhibitors of TBK1 remains both necessary and challenging. Consequently, it is essential to conduct in-depth structural optimization studies on TBK1 inhibitors to obtain a class of highly active TBK1 inhibitor compounds. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a novel TANK-binding kinase 1 inhibitor and its pharmaceutical applications.

[0008] The technical solution of the present invention is as follows:

[0009] A compound called ITA-9 has the following structural formula:

[0010] .

[0011] The preparation method of the above compound ITA-9 is as follows:

[0012] (1) Itaconic anhydride was dissolved in dichloromethane, and then anhydrous methanol and sulfuric acid were added. The mixture was stirred overnight at room temperature. Then dichloromethane was added to the reaction solution, and the reaction solution was alkalized with potassium carbonate. The aqueous phase was separated to obtain the organic phase. The organic phase was then acidified with hydrochloric acid and extracted three times with dichloromethane. The organic phases were combined after extraction, dried with anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and finally the crude product was recrystallized in a mixed solution of ethyl acetate / n-hexane to obtain compounds 1-2.

[0013] The structures of compounds 1-2 are shown in the following formula:

[0014] ;

[0015] (2) Compounds 1-2 and methyl N 6 -(tert-butylcarbonyl)- L -Lysine hydrochloride was dissolved in dichloromethane, and then 2-(7-azobenzotriazole)- was added. N , N , N' , N' -Tetramethylurea hexafluorophosphate and triethylamine were stirred overnight at room temperature. After the reaction was completed, saturated brine was added, and the mixture was 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.

[0016] According to a preferred embodiment of the present invention, in step (1), the ratio of itaconic anhydride, dichloromethane, anhydrous methanol and sulfuric acid is (0.45~0.55g): (1.5~2.5ml): (3~6ml): (70~80μl).

[0017] According to a preferred embodiment of the present invention, in step (2), compounds 1-2 and methyl... N 6 -(tert-butylcarbonyl)- L -Lysine hydrochloride, dichloromethane, 2-(7-azobenzotriazole)- N , N , N' , N' The ratio of tetramethylurea hexafluorophosphate to triethylamine is (0.1~0.2g): (0.25~0.35g): (3~6 ml): (0.35~0.45g): (0.4~0.5ml).

[0018] The use of the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof in the preparation of TANK-binding kinase 1 (TBK1) inhibitors.

[0019] The above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof is used as a TANK-binding kinase 1 inhibitor in the preparation of pharmaceutical compositions for the prevention and / or treatment of diseases related to TBK1 activity.

[0020] The diseases related to TBK1 activity described in this invention refer to diseases that can be improved, prevented, or treated by inhibiting TBK1 activity.

[0021] According to a preferred embodiment of the present invention, the disease associated with TBK1 activity is an infectious disease, an autoimmune disease, a metabolic disease, or a cancer.

[0022] More preferably, the infectious disease is caused by one or more DNA viruses or RNA viruses.

[0023] More preferably, the DNA virus is herpes simplex virus 1 (HSV-1), and the RNA virus is vesicular stomatitis virus (VSV).

[0024] More preferably, the autoimmune disease is AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis, or multiple sclerosis.

[0025] More preferably, the metabolic disease is an obesity disease, diabetes, or non-alcoholic fatty liver disease.

[0026] A pharmaceutical composition for treating diseases associated with TBK1 activity, the pharmaceutical composition comprising the above-mentioned compound ITA-9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0027] According to a preferred embodiment of the present invention, the pharmaceutical composition is prepared as any pharmaceutically acceptable dosage form using compound ITA-9 or a pharmaceutically acceptable salt thereof as the main active ingredient, combined with pharmaceutically acceptable excipients.

[0028] More preferably, the dosage form is a tablet, capsule, granule, pill, liquid preparation, decoction, suspension, dispersant, syrup, suppository, gel, aerosol, or patch.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. This invention provides a novel compound, ITA-9, which exhibits significantly enhanced TBK1 inhibitory activity compared to existing methylene succinic acid compounds with similar structures, and can be used as a TBK1 inhibitor.

[0031] 2. This invention discloses for the first time the application of compound ITA-9 as a TANK-binding kinase 1 (TBK1) inhibitor in the preparation of pharmaceutical compositions for the prevention and / or treatment of diseases related to TBK1 activity. This compound has the potential to be developed into a class of TBK1 inhibitor drugs and a treatment for TBK1-related diseases, such as infectious diseases, autoimmune diseases, metabolic diseases, and cancer, providing new therapeutic agents for clinical use and possessing significant clinical application value and broad application prospects. Attached Figure Description

[0032] Figure 1This is the synthetic route diagram for compound ITA-9.

[0033] Figure 2 The compound ITA-9 inhibits antiviral immune responses;

[0034] In the figure, A represents the IFN-β content in the cell supernatant after VSV virus infection; B represents the IFN-β content 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; and D represents the expression levels of p-TBK1, p-IRF3, and p-STAT1 in cells after HSV-1 virus infection.

[0035] Figure 3 The compound ITA-9 targets and inhibits TBK1 activity;

[0036] In the figure, A represents mouse peritoneal macrophages pretreated with Ctrl or ITA-9, and the stability of TBK1 was detected by the cell thermal displacement experiment; B represents the surface plasmon resonance experiment to detect the affinity between ITA-9 and TBK1; C represents the click chemistry experiment to detect the covalent binding between ITA-9 and TBK1; and D represents the molecular simulated docking between ITA-9 and TBK1.

[0037] Figure 4 The compound ITA-9 inhibits the excessive inflammatory response induced by poly(I:C);

[0038] In the figure, A shows the IFN-β content in mouse serum detected by ELISA; B shows the IFN-β content in mouse lung tissue detected by qPCR. Cxcl10 mRNA expression level; C is hematoxylin and eosin staining of mouse lung tissue sections; D is Sirius red staining of mouse lung tissue sections.

[0039] Figure 5 The compound ITA-9 was used to inhibit the inflammatory response in a systemic lupus erythematosus (SLE) model.

[0040] In the figure, A shows the biochemical analysis of Creatinine levels in mouse plasma; B shows the qPCR detection of Creatinine levels in mouse lung tissue. Cxcl10 and Isg15 mRNA expression level; C represents hematoxylin and eosin staining of mouse lung tissue sections.

[0041] Figure 6 The compound ITA-9 was used to inhibit the inflammatory response in a model of Aicardi-Goutières syndrome (AGS).

[0042] In the figure, A represents the detection of [a specific substance] in mouse lung tissue by qPCR. Cxcl10 , Isg15 , Isg54 , Isg56 , Ccl5 and Mx1 mRNA expression level; B represents hematoxylin and eosin staining of mouse lung tissue sections. Detailed Implementation

[0043] To better understand the essence of this invention, the following embodiments further illustrate the content of this invention, but these should not be construed as limiting the invention. The invention is further described below with reference to the accompanying drawings and specific embodiments, but these embodiments do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0044] This invention provides the use of a novel TBK1 inhibitor, ITA-9, in the preparation of pharmaceutical compositions for the treatment or prevention of diseases related to TBK1 activity.

[0045] In this invention, the drug comprises ITA-9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0046] In this invention, the drug is any pharmaceutically acceptable dosage form made by using ITA-9 or its pharmaceutical salt as the active pharmaceutical ingredient and combining it with pharmaceutically acceptable excipients.

[0047] In this invention, the dosage form is tablet, capsule, granule, pill, liquid preparation, decoction, suspension, dispersant, syrup, suppository, gel, aerosol, or patch.

[0048] The RNA virus is vesicular stomatitis virus (VSV), and the DNA virus is herpes simplex virus type I (HSV-1). Both are routine laboratory viruses and are available from biotechnology companies.

[0049] The C57BL / 6 mice were available from Tonglihua Animal Technology Co., Ltd. All animals were used at 6–8 weeks of age. Animal experiments were conducted in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals" and approved by the Ethics Committee of the School of Basic Medical Sciences, Shandong University.

[0050] ITA-9 Synthesis Method: Standard operating and purification methods known to those skilled in the art were used. Unless otherwise specified, the starting materials were generally available from commercially available sources. Commercially available solvents and reagents were generally used without further purification; anhydrous solvents were treated using standard methods, and other reagents were commercially available of analytical grade. Unless otherwise specified, all temperatures are expressed in °C (degrees Celsius), and room temperature or ambient temperature refers to 20–25 °C. The structure of the compound was determined by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR shift (δ) is given in parts per million (ppm). 1H NMR spectra were determined using a Bruker 400MHz NMR spectrometer. Deuterated chloroform (Chloroform- d ), deuterated dimethyl sulfoxide (DMSO- d 6 ) was used as the solvent, and tetramethylsilane (TMS) was used as the internal standard.

[0051] Chromatography columns typically use 200-300 mesh silica gel as a carrier.

[0052] ELISA and qPCR assays: IFN-β levels in cell supernatants and mouse serum were measured using the IFN-β ELISA kit (R&D Systems) according to the instructions. Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme). PCR analysis was performed using SYBR Green (Vazyme). Data were normalized using β-actin as an internal reference gene.

[0053] Western Blot Procedure: Cells were washed with phosphate-buffered saline (PBS), lysed with RIPA protein extraction reagent (Pierce, Thermo Fisher Scientific) with protease inhibitors (phenylmethylsulfonyl fluoride, Beyotime) and phosphatase inhibitors (CWBIO), and then centrifuged at 12000×g for 15 min at 4°C. 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. Proteins were then transferred to a PVDF (Millipore) membrane for immunoblotting. The membrane was incubated overnight with primary antibody dilution buffer. The next day, the membrane was washed three times with TBST and then incubated for 1 h with secondary antibody (Immunoway) dilution buffer (1:4000). Protein bands were imaged using enhanced chemiluminescence (NCM Biotech) according to the manufacturer's protocol.

[0054] Experimental method for cell thermal shift: Macrophages were pretreated with solvent or 100 μM ITA-9 for 12 h, then cooled on ice, washed with PBS buffer containing protease inhibitors, and transferred to PCR tubes. Cells were then heated at different temperatures (40, 42.5, 45, 47.5, 50, 52.5, and 55 °C) for 3 min, and immediately cooled in liquid nitrogen for 10 s. This process was repeated three times. Cells were then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was added to 5 × SDS loading buffer and boiled before Western blotting. Protein bands were quantitatively analyzed using ImageJ software.

[0055] Experimental method for surface plasmon resonance: Affinity was measured using a BIAcore T200 instrument, specifically by covalently coupling the hTBK1 protein to a CM7 sensor chip, with a response value of 20,000 RU. Binding measurements were performed at 25 °C and a flow rate of 30 μl / min. ITA-9 was serially diluted with PBS containing 5% DMSO and 0.05% surfactant P20. The binding time to the protein on the chip was 60 s, and the dissociation time was 120 s. Binding curves were exported using GraphpPad Prism 9 for analysis, and affinity was calculated using BIA evaluation software.

[0056] Example 1: Synthesis of compound ITA-9

[0057] like Figure 1 As shown, a method for synthesizing compound ITA-9 includes the following steps:

[0058] (1) Itaconic anhydride (compound 1-1, 0.50 g, 4.46 mmol) was dissolved in 2 ml of dichloromethane (DCM), and then 5 ml of anhydrous methanol and 75 μl of sulfuric acid were added. The mixture was stirred overnight at room temperature. After the reaction was completed by thin-layer chromatography (TLC), 30 ml of DCM was added to the reaction solution. The reaction solution was then alkalized with 10% potassium carbonate (pH>10) to separate the aqueous phase and obtain the organic phase. The separated organic phase was acidified with 1 M hydrochloric acid (pH<5), and then extracted three times with DCM. The organic phases were combined after extraction, dried with anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and finally recrystallized the crude product in a mixed solution of ethyl acetate / n-hexane to obtain compound 1-2.

[0059] The structures of compounds 1-2 are shown in the following formula:

[0060] ;

[0061] (2) Compounds 1-2 (0.15 g, 1.04 mmol) and methyl N6 -(tert-butylcarbonyl)- L -Lysine hydrochloride (methyl N 6 -( tert -butoxycarbonyl)- L -lysinate hydrochloride (0.31 g, 1.04 mmol) was dissolved in 5 ml of DCM, and then 2-(7-azobenzotriazole)- was added. N , N , N' , N' - Tetramethylurea hexafluorophosphate (HATU, 0.40 g, 1.04 mmol) and triethylamine (0.46 ml, 2.60 mmol) were stirred overnight at room temperature. After the reaction was completed by TLC monitoring, 100 ml of saturated saline 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.

[0062] The spectral data of compounds 1-2 and ITA-9 prepared in this embodiment are as follows:

[0063] Compounds 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 C6H8O4144.04, found: 167.00 [M+Na] + .

[0064] Compound ITA-9: 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] + .

[0065] The above data demonstrates that the present invention successfully prepared compound ITA-9, the structural formula of which is shown below:

[0066] .

[0067] Example 2: Compound ITA-9 inhibits TBK1-mediated antiviral immune response.

[0068] 1. Mouse peritoneal macrophages were co-incubated with different concentrations of ITA-9 (0, 6.25, 12.5, 25, 50, 100 μM) prepared in Example 1 for 2 h. The cells were then infected with VSV virus and HSV-1 virus, respectively, and cultured further. The cell supernatant was collected, and the IFN-β content in the cell supernatant was detected by ELISA. The results are as follows: Figure 2 As shown in A and B.

[0069] Depend on Figure 2 A and B indicate that ITA-9 inhibits IFN-β secretion in a concentration-dependent manner, EC 50 The values ​​were 26.33 μM and 17.33 μM, respectively.

[0070] 2. Mouse peritoneal macrophages were divided into four groups. Group 1 (Experimental Group 1, ITA-9 / VSV) was incubated with 50 μM of ITA-9 (prepared in Example 1) for 2 hours, followed by infection with VSV virus. Cells were cultured for 0, 8, and 12 hours post-infection. Group 2 (Control Group 1, Ctrl / VSV) was used instead of ITA-9 in Experimental Group 1. Group 3 (Experimental Group 2, ITA-9 / HSV-1) was incubated with 50 μM of ITA-9 (prepared in Example 1) for 2 hours, followed by infection with HSV-1 virus. Cells were cultured for 0, 2, and 4 hours post-infection. Group 4 (Control Group 2, Ctrl / VSV) was used instead of ITA-9 in Experimental Group 2.

[0071] The expression levels of p-TBK1, p-IRF3, and p-STAT1 in the cell lysates of the four groups were detected by Western blotting. The results are as follows: Figure 2 As shown in C and D.

[0072] Depend on Figure 2 As shown in C and D, ITA-9 significantly inhibits the expression levels of p-TBK1, p-IRF3, and p-STAT1 during VSV and HSV-1 virus infection.

[0073] 3. Mouse peritoneal macrophages were divided into two groups. Group 1 was the experimental group (ITA-9), which was 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. Group 2 was the control group (Ctrl), which used dimethyl sulfoxide (DMSO) instead of the compound ITA-9 in the experimental group.

[0074] The stability of TBK1 protein in two groups of cells was detected by a cell thermal displacement assay at different temperatures (40, 42.5, 45, 47.5, 50, 52.5, and 55 °C). The results are as follows: Figure 3 As shown in Figure A.

[0075] Depend on Figure 3 As shown in A, the stability of TBK1 protein in cells was significantly improved after ITA-9 treatment, indicating that ITA-9 interacted with TBK1 protein.

[0076] Next, the affinity between hTBK1 protein and ITA-9 was detected in vitro using a surface plasmon resonance (SPR) assay, and the results are as follows: Figure 3 As shown in B.

[0077] Depend on Figure 3 As shown in B, hTBK1 protein and ITA-9 have a strong affinity for each other in vitro. D =55.87nM, which also indicates that ITA-9 binds to TBK1 in a non-enzymatic manner.

[0078] 4. TBK1-MYC plasmid was transfected into HEK293T cells and cultured for 6 h. Then, the cells were incubated for 12 h with either DMSO or 50 μM ITA-9, followed by incubation for another 12 h with ITALK (150 μM), an alkylation probe that can alkylate TBK1. Cell lysates were subjected to click chemistry, and the expression level of TBK1-MYC was detected by Western blotting. Untreated HEK293T cells were used as a control. Results are shown below. Figure 3 As shown in C.

[0079] Depend on Figure 3 C indicates that ITA-9 can competitively inhibit the binding of ITA-9 to TBK1, suggesting that ITA-9 and TBK1 undergo covalent bonding.

[0080] 5. Using the CovDock module in the Schrodinger 2021-2 software package, ITA-9 and TBK1 were molecularly docked. The reaction type for covalent docking was set to Michael addition. The results are as follows: Figure 3 As shown in D.

[0081] The above data all indicate that compound ITA-9 covalently binds to TBK1, and alkylation of TBK1 can effectively inhibit TBK1-mediated antiviral immune responses.

[0082] Example 3: Compound ITA-9 inhibits poly(I:C)-induced excessive inflammatory response.

[0083] 1. C57BL / 6J mice were divided into three groups. Group 1 was the experimental group (ITA-9 + poly(I:C)). C57BL / 6J mice were intraperitoneally injected with ITA-9 (10 mg / kg) prepared in Example 1 for 2 hours, followed by intraperitoneal injection of poly(I:C) (15 mg / kg), and cultured for 8 hours. Group 2 was the control group (Ctrl + poly(I:C)). C57BL / 6J mice were intraperitoneally injected with an equal volume of the control solvent Ctrl for 2 hours, followed by intraperitoneal injection of poly(I:C) (15 mg / kg), and cultured for 8 hours. Group 3 was the blank group (Ctrl + PBS). C57BL / 6J mice were intraperitoneally injected with Ctrl for 2 hours, followed by injection of an equal volume of PBS buffer, and cultured for 8 hours.

[0084] Serum from three groups of mice was collected, and the IFN-β content in the mouse serum was detected by ELISA. The results are as follows: Figure 4 As shown in Figure A.

[0085] Three groups of mice were sacrificed, and lung tissue was collected. RNA was extracted using standard methods, and its concentration in the mouse lung tissue was detected by qPCR. Cxcl10 mRNA expression levels, results as follows Figure 4 As shown in B.

[0086] Depend on Figure 4 A and B indicate that, compared to the control group, after poly(I:C) infection, ITA-9 treated mice had significantly higher levels of IFN-β in serum and lung tissue. Cxcl10 The expression of mRNA was significantly reduced.

[0087] 2. C57BL / 6J mice were divided into three groups. Group 1 was the experimental group (ITA-9 + poly(I:C)). C57BL / 6J mice were intraperitoneally injected with ITA-9 (10 mg / kg) prepared in Example 1 for 2 hours, followed by an intraperitoneal injection of poly(I:C) (25 mg / kg). After 12 hours of culture, they were again intraperitoneally injected with ITA-9 (10 mg / kg) and cultured for another 12 hours. Group 2 was the control group (Ctrl + poly(I:C)). C57BL / 6J mice were intraperitoneally injected with an equal volume of the control solvent Ctrl for 2 hours, followed by an intraperitoneal injection of poly(I:C) (25 mg / kg). After 12 hours of culture, they were again intraperitoneally injected with Ctrl and cultured for another 12 hours. Group 3 was the blank group (PBS). C57BL / 6J mice were intraperitoneally injected with an equal volume of the control solvent Ctrl, followed by an intraperitoneal injection of an equal volume of PBS buffer. After 12 hours of culture, they were again intraperitoneally injected with Ctrl and cultured for another 12 hours.

[0088] Three groups of mice were sacrificed, and their lung tissue was collected. Lung tissue sections were prepared using existing methods, and then stained with hematoxylin and eosin, and Sirius red. The results were observed. Figure 4 As shown in C and D.

[0089] Depend on Figure 4 As shown in C and D, compared with the control group, ITA-9-treated mice showed a significant reduction in inflammatory cell infiltration and airway collagen deposition after poly(I:C) infection.

[0090] The above data all indicate that compound ITA-9 can effectively inhibit poly(I:C)-induced excessive inflammatory response.

[0091] Example 4: Compound ITA-9 inhibits the inflammatory response in systemic lupus erythematosus (SLE).

[0092] C57BL / 6J mice were divided into three groups: Group 1 was the experimental group (TMPD+ITA-9), Group 2 was the control group (TMPD), and Group 3 was the blank group (Ctrl). SLE models were established by intraperitoneal injection of 0.5 ml / mouse of 2,6,10,14-tetramethylpentadecane (TMPD) into Groups 1 and 2, while Group 3 mice were injected with an equal volume of PBS buffer. Three weeks later, Group 1 mice were intraperitoneally injected with ITA-9 (10 mg / kg) prepared in Example 1, daily for one week; while Groups 2 and 3 mice were intraperitoneally injected with an equal volume of DMSO, daily for one week.

[0093] Plasma from three groups of mice was collected, and biochemical analysis was performed to detect the content of Creatinine in the mouse plasma. The results are as follows: Figure 5 As shown in Figure A.

[0094] Three groups of mice were sacrificed, and lung tissue was collected. RNA was extracted using standard methods, and its concentration in the mouse tissue was detected by qPCR. Cxcl10 and Isg15 mRNA expression levels, results as follows Figure 5 As shown in B.

[0095] Mouse lung tissue was collected and prepared into sections using existing methods. The sections were then stained with hematoxylin and eosin, and the results were observed. Figure 5 As shown in C.

[0096] Depend on Figure 5 A, B, and C indicate that, compared to the control group, ITA-9 treatment in the SLE model resulted in lower levels of Creatinine in mouse plasma and lung tissue. Cxcl10 and Isg15 The expression levels of mRNA were significantly reduced, and the infiltration of inflammatory cells in mouse lung tissue was significantly decreased.

[0097] The above data all indicate that compound ITA-9 can effectively inhibit the inflammatory response in systemic lupus erythematosus.

[0098] Example 5: The inhibitory effect of compound ITA-9 on the inflammatory response of AGS syndrome (Aicardi-Goutières syndrome)

[0099] AGS syndrome mouse model Trex1 Gene-deficient mice ( Trex1 - / - Mice were divided into two groups, Group 1 and Group 2; wild-type mice of the same age and sex were further divided into a third group. Group 1 was the experimental group ( Trex1 - / - + ITA-9), Group 2 was the control group ( Trex1 - / -Group 1 mice were injected intraperitoneally with ITA-9 (10 mg / kg) prepared in Example 1, daily for one week; and mice in Groups 2 and 3 were injected intraperitoneally with an equal volume of the control solvent Ctrl, daily for one week.

[0100] Three groups of mice were sacrificed, and lung tissue was collected. RNA was extracted using standard methods, and its concentration in the mouse tissue was detected by qPCR. Cxcl10 , Isg15 , Isg54 , Isg56 , Ccl5 and Mx1 mRNA expression levels, results as follows Figure 6 As shown in Figure A.

[0101] Mouse lung tissue was collected and prepared into sections using existing methods. The sections were then stained with hematoxylin and eosin, and the results were observed. Figure 6 As shown in B.

[0102] Depend on Figure 6 As shown in A and B, compared with the control group and the blank group, ITA-9 treatment in the AGS syndrome mouse model resulted in a decrease in lung tissue concentration. Cxcl10 , Isg15 , Isg54 , Isg56 , Ccl5 and Mx1 The expression levels of mRNA were significantly reduced, and the infiltration of inflammatory cells in mouse lung tissue was significantly decreased.

[0103] The above data all indicate that compound ITA-9 can effectively inhibit the inflammatory response in the AGS syndrome model.

[0104] In summary, this invention experimentally verified that compound ITA-9 possesses significant inhibitory activity against TBK1, indicating that it can be used as a TBK1 inhibitor. Furthermore, it verified that compound ITA-9 can effectively inhibit TBK1-mediated antiviral immune responses and poly(I:C)-induced excessive inflammatory responses. In addition, this invention also verified that ITA-9 can effectively inhibit inflammatory responses in mouse models of systemic lupus erythematosus and AGS syndrome. This demonstrates that this compound has the potential to be developed into a class of TBK1 inhibitors and a therapeutic agent for TBK1-related diseases, such as infectious diseases, autoimmune diseases, metabolic diseases, and cancer, providing new therapeutic drugs for clinical application and possessing good clinical application value and broad application prospects.

Claims

1. A compound ITA-9, the structural formula of which is shown below: 。 2. The method for preparing the compound ITA-9 according to claim 1, characterized in that, The steps are as follows: (1) Itaconic anhydride was dissolved in dichloromethane, and then anhydrous methanol and sulfuric acid were added. The mixture was stirred overnight at room temperature. Then dichloromethane was added to the reaction solution, and the reaction solution was alkalized with potassium carbonate. The aqueous phase was separated to obtain the organic phase. The organic phase was then acidified with hydrochloric acid and extracted three times with dichloromethane. The extracted organic phases were combined, dried with anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and finally the crude product was recrystallized in a mixed solution of ethyl acetate / n-hexane to obtain compounds 1-2. The structures of compounds 1-2 are shown in the following formula: ； The ratio of itaconic anhydride, dichloromethane, anhydrous methanol, and sulfuric acid is (0.45~0.55g): (1.5~2.5ml): (3~6ml): (70~80μl). (2) Compounds 1-2 and methyl N 6 -(tert-butylcarbonyl)- L -Lysine hydrochloride was dissolved in dichloromethane, and then 2-(7-azobenzotriazole)- was added. N , N , N' , N' -Tetramethylurea hexafluorophosphate and triethylamine were stirred overnight at room temperature. After the reaction was completed, saturated brine was added, and the mixture was 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. Among them, compounds 1-2 and methyl N 6 -(tert-butylcarbonyl)- L -Lysine hydrochloride, dichloromethane, 2-(7-azobenzotriazole)- N , N , N' , N' The ratio of tetramethylurea hexafluorophosphate to triethylamine is (0.1~0.2g): (0.25~0.35g): (3~6 ml): (0.35~0.45g): (0.4~0.5ml).

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

4. The use of the compound ITA-9 of claim 1 or a pharmaceutically acceptable salt thereof as a TANK-binding kinase 1 inhibitor in the preparation of pharmaceutical compositions for the prevention and / or treatment of diseases related to TBK1 activity.

5. The application as described in claim 4, characterized in that, The diseases associated with TBK1 activity are those that can be improved, prevented, or treated by inhibiting TBK1 activity.

6. The application as described in claim 5, characterized in that, The diseases associated with TBK1 activity are infectious diseases, autoimmune diseases, metabolic diseases, or cancer.

7. The application as described in claim 6, characterized in that, The infectious disease is caused by one or more of DNA viruses or RNA viruses.

8. The application as described in claim 7, characterized in that, The DNA virus is herpes simplex virus 1, and the RNA virus is vesicular stomatitis virus.

9. The application as described in claim 6, characterized in that, The autoimmune diseases mentioned are AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

10. The application as described in claim 6, characterized in that, The metabolic diseases mentioned are obesity, diabetes, and non-alcoholic fatty liver disease.

11. A pharmaceutical composition for treating diseases related to TBK1 activity, characterized in that, The pharmaceutical composition comprises the compound ITA-9 of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition for treating diseases related to TBK1 activity as described in claim 11, characterized in that, The pharmaceutical composition is any pharmaceutically acceptable dosage form made by using compound ITA-9 or a pharmaceutically acceptable salt thereof as the main active ingredient, combined with pharmaceutically acceptable excipients.

13. The pharmaceutical composition according to claim 12, characterized in that, The dosage forms include tablets, capsules, granules, pills, decoctions, suspensions, dispersants, syrups, suppositories, gels, aerosols, and patches.

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