Compound ITA-5 as a TBK1 inhibitor and a synthetic method and application thereof

Abstract

The present application relates to a kind of compound ITA-5 as TBK1 inhibitor and its synthesis method and application, belong to biological medicine technical field.The TBK1 inhibitor is compound ITA-5.The present application also provides the application of compound ITA-5 in the preparation of the drug composition for preventing and / or treating the disease related to TBK1 activity, and the disease related to TBK1 activity is infectious disease, autoimmune disease, metabolic disease or cancer and the like.The present application first discloses the application of compound ITA-5 as TBK1 inhibitor inhibitor in the preparation of the drug composition for preventing and / or treating the disease related to TBK1 activity.The compound has the potential to be developed into TBK1 inhibitor drug and the treatment drug of TBK1 related disease, such as infectious disease, autoimmune disease, metabolic disease and cancer and the like, provides new treatment drug for clinic, has good clinical application value and broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound ITA-5 as a TBK1 inhibitor and a synthesis method and application thereof, and belongs to the technical field of biological medicine. BACKGROUND

[0002] TANK binding kinase 1 (TBK1) is a serine / threonine kinase belonging to the non-canonical IKB kinase (IKK) family. TBK1 is involved in the regulation of interferon regulatory factor (IRF), nuclear factor kappa B (NF-kappa B), type I interferon (IFN-I), type II interferon (IFN-II) target genes, and other signaling pathways and transcription factors, as well as STING-mediated cytoplasmic DNA detection, regulating antiviral defense, host-virus interaction, and playing an important role in the occurrence and development of immune, tumor, inflammation, metabolism and other diseases.

[0003] The innate immune system is the first line of defense against viral infection. DNA recognition receptors cGAS (Cyclic GMP-AMP synthase) and RNA recognition receptors 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. Different pattern recognition receptors (PRRs) are activated after being mediated by various viral infections, and all of them pass through the common key kinase TBK1 to induce the activation of transcription factor IRF3, and ultimately cause the expression of type I interferon (IFN-alpha / beta) to start the antiviral innate immune response. Appropriate type I interferon response helps the host to timely clear the invading virus in the early stage; however, abnormal innate immune response such as excessive inflammatory response can induce cytokine storm, and further cause host tissue damage, and even lead to organ failure. Therefore, the normal activation of TBK1 plays a key role in the process of antiviral immune response, which makes the research of TBK1 small molecule inhibitors a hot spot.

[0004] With the in-depth understanding of the tertiary structure of TBK1 kinase and its biological function, TBK1 has become a potential target for the treatment of inflammatory diseases, autoimmune diseases, immunodeficiency diseases, neurodegenerative diseases, metabolic diseases, and cancer. Although TBK1 is very crucial, the research of specific inhibitors thereof is still in the early stage, and therefore, it is still very urgent to find high-efficiency and safe TBK1 small molecule inhibitors. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a compound ITA-5 as a TBK1 inhibitor and a synthesis method and application thereof.

[0006] The technical scheme of the present application is as follows:

[0007] A compound ITA-5, the structural formula of which is shown in the following formula:

[0008] .

[0009] The preparation method of the compound ITA-5 is as follows:

[0010] (1) itaconic anhydride is dissolved in dichloromethane, then anhydrous methanol and sulfuric acid are added, and the reaction is stirred at room temperature overnight; then dichloromethane is added to the reaction solution, followed by alkali treatment of the reaction solution using potassium carbonate, separation of the aqueous phase to obtain the organic phase; then the organic phase is acidified using hydrochloric acid, extracted three times with dichloromethane, and the combined organic phase is dried using anhydrous sodium sulfate, filtered, and evaporated under reduced pressure; finally, the crude product is recrystallized in an ethyl acetate / n-hexane mixed solution to obtain the compound ITA-1;

[0011] The structure of the compound ITA-1 is shown in the following formula:

[0012] ;

[0013] (2) the compound ITA-1 and L -phenylalanine methyl ester hydrochloride are dissolved in dichloromethane, then 2-(7-azobenzenetriazole)- N,N,N',N -tetramethylurea hexafluorophosphate and triethylamine are added, and the reaction is stirred at room temperature overnight; after the reaction is completed, saturated brine is added, extracted with dichloromethane, and the organic phase is dried with anhydrous sodium sulfate, filtered, and evaporated under reduced pressure; after column chromatography purification, the target compound ITA-5 is obtained.

[0014] According to the present application, 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).

[0015] According to the present application, in step (2), the ratio of the compound ITA-1, L -phenylalanine methyl ester hydrochloride, dichloromethane, 2-(7-azobenzenetriazole)- 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).

[0016] The compound ITA-5 or a pharmaceutically acceptable salt thereof has the application in preparing a TBK1 inhibitor.

[0017] Use of the above-mentioned compound ITA-5 or a pharmaceutically acceptable salt thereof as a TBK1 inhibitor in the manufacture of a pharmaceutical composition for preventing and / or treating a disease associated with TBK1 activity.

[0018] The disease associated with TBK1 activity according to the present application refers to a disease that is improved, prevented or treated by inhibiting the activity of TBK1.

[0019] According to the present application, preferably, the disease associated with TBK1 activity is an infectious disease, an autoimmune disease, a metabolic disease or a cancer.

[0020] Further preferably, the infectious disease is a disease caused by one or more infections of DNA viruses or RNA viruses.

[0021] More preferably, the DNA virus is herpes simplex virus 1 (HSV-1) and the RNA virus is vesicular stomatitis virus (VSV).

[0022] Further preferably, the autoimmune disease is AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and the like.

[0023] Further preferably, the metabolic disease is obesity, diabetes, non-alcoholic fatty liver disease and the like.

[0024] A pharmaceutical composition for treating a disease associated with TBK1 activity, the pharmaceutical composition comprising the above-mentioned compound ITA-5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0025] According to the present application, preferably, the pharmaceutical composition is any one of pharmaceutically acceptable dosage forms prepared by using the compound ITA-5 or a pharmaceutically acceptable salt thereof as a main active ingredient, together with pharmaceutically acceptable excipients.

[0026] Further preferably, the dosage form is a tablet, a capsule, a granule, a pill, a liquid preparation, a decoction, a suspension, a dispersion, a syrup, a suppository, a gel, an aerosol, a patch and the like.

[0027] The present application has the following advantages:

[0028] 1. The present application provides a novel compound ITA-5, which exhibits significantly improved TBK1 inhibitory activity compared to existing methylene succinic acid compounds with similar structures, and can be used as a TBK1 inhibitor.

[0029] 2. The application discloses a compound ITA-5 as a TANK-binding kinase 1 (TBK1) inhibitor for the first time, and application of the compound in preparation of a medicine composition for preventing and / or treating diseases related to TBK1 activity. The compound has the potential to be developed into a TBK1 inhibitor and a therapeutic drug for diseases related to TBK1, such as infectious diseases, autoimmune diseases, metabolic diseases and cancers, and provides a new therapeutic drug for clinic, and has good clinical application value and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A synthesis route of the compound ITA-5.

[0031] Figure 2 Effect of the compound ITA-5 on macrophage activity.

[0032] Figure 3 Inhibition of the compound ITA-5 on RNA virus VSV-mediated antiviral immune response;

[0033] In the figure, A is ELISA detection of IFN-β secretion and calculation of EC 50 ; B is Western Blot detection of p-TBK1, p-IRF3 and p-STAT1 protein levels; and C is immunofluorescence detection of TBK1 aggregation around the nucleus.

[0034] Figure 4 Inhibition of the compound ITA-5 on DNA virus HSV-1-mediated antiviral immune response;

[0035] In the figure, A is ELISA detection of IFN-β secretion and calculation of EC 50 ; B is Western Blot detection of p-TBK1, p-IRF3 and p-STAT1 protein levels; and C is immunofluorescence detection of TBK1 aggregation around the nucleus.

[0036] Figure 5 Verification of the compound ITA-5 targeting TBK1;

[0037] In the figure, A is molecular docking of ITA-5 and TBK1; B is surface plasmon resonance experiment for detecting the affinity of ITA-5 and TBK1; and C is cell thermal shift experiment for detecting the stability of TBK1 by pre-incubating mouse peritoneal macrophages with Ctrl or ITA-5.

[0038] Figure 6 Inhibition of ITA-5 on poly(I:C)-induced excessive inflammatory response;

[0039] In the figure, A is ELISA detection of mouse serum IFN-β secretion; B is RT-PCR detection of mouse lung tissueCxcl10 mRNA expression; C, D are liver and kidney injury indicators in mouse plasma; E is hematoxylin and eosin staining of mouse lung tissue sections.

[0040] Figure 7 To inhibit the inflammatory response in the systemic lupus erythematosus (SLE) model by the compound ITA-5;

[0041] In the figure, A is the biochemical analysis of the content of kidney injury indicator Creatinine in mouse plasma; B is hematoxylin and eosin staining of mouse lung tissue sections; C is qPCR detection of the expression level of Cxcl10 and Isg15 mRNA in mouse lung tissue.

[0042] Figure 8 To inhibit the inflammatory response in the Aicardi-Goutières syndrome (AGS) model by the compound ITA-5;

[0043] In the figure, A is hematoxylin and eosin staining of mouse lung tissue sections; B is qPCR detection of the expression level of Cxcl10 , Isg15 mRNA in mouse lung tissue. DETAILED DESCRIPTION

[0044] In order to better understand the essence of the present application, the content of the present application will be further described below in combination with examples, but it cannot be regarded as a limitation to the present application. The present application will be further described below in combination with the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0045] Example 1, synthesis of compound ITA-5

[0046] As shown in Figure 1 , a method for synthesizing compound ITA-5 includes the following steps:

[0047] (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 at room temperature overnight. After monitoring the end of the reaction by thin layer chromatography (TLC), add 30 ml of DCM to the reaction solution, then alkalinize 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 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 mixture of ethyl acetate / n-hexane to obtain compound ITA-1;

[0048] The structure of compound ITA-1 is shown in the following formula:

[0049] ;

[0050] (2) Compound ITA-1 (0.15 g, 1.04 mmol) and L -Phenylalanine methyl ester hydrochloride ( L 2-(7-Azobenzotriazole)- phenylalanine methyl ester hydrochloride (0.19 g, 0.87 mmol) was dissolved in 5 ml of DCM, and then 2-(7-azobenzotriazole)- 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 (monitored by TLC), then 100 ml of saturated saline was added, and the mixture was extracted with DCM. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the target compound ITA-5.

[0051] The spectral data of compounds ITA-1 and ITA-5 prepared in this embodiment are as follows:

[0052] 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 C6H8O4144.04, found: 167.00[M+Na] + .

[0053] 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] + 。

[0054] The above data show that the compound ITA-5 is successfully prepared, and the chemical structural formula is as follows:

[0055] .

[0056] Example 2, Effect of Compound ITA-5 on Macrophage Activity

[0057] 1. Experimental materials

[0058] The CellTiter-Glo® 2.0 Cell Viability Assay (Promega).

[0059] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice of 6-11 weeks old were used, both male and female. The mice were bred in the SPF barrier environment of the Model Animal Research Center of Shandong University, under the conditions of temperature 20-26℃, humidity 40-70%, and were bred according to the day-night cycle (every 12 h). All animal experiments were performed 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 Medical Sciences of Shandong University.

[0060] 2. Experimental method

[0061] The C57BL / 6 mice were injected intraperitoneally with 6% starch solution, and 3 days later, the peritoneal cavity was repeatedly washed with high-sugar DMEM solution. The washing solution was collected into a 50 mL centrifuge tube, centrifuged at room temperature at 1000 rpm for 5 min, and the supernatant was discarded to obtain mouse primary peritoneal macrophages (PMs).

[0062] The PMs were resuspended with complete medium, counted, and inoculated into a 384-well plate. Different concentrations of compound ITA-5 (0, 5, 10, 50, 100, 150, 200, and 250 μM) were used for preincubation for 2 h, respectively. After the preincubation was completed, 25 μL of CellTiter-Glo®2.0 Reagent was added to each well, and the reaction was carried out at room temperature for 30 min. The mixture was shaken for 2 min, and the incubation was continued at room temperature for 10 min. The fluorescence was detected by a microplate reader, and the cell activity was determined. The results are shown in Figure 2 .

[0063] 3. Experimental results

[0064] As can be seen from Figure 2 , different concentrations (0, 5, 10, 50, 100, 150, 200, and 250 μM) of compound ITA-5 do not affect the activity of mouse primary peritoneal macrophages (PMs).

[0065] Example 3: Inhibition of RNA virus VSV-mediated antiviral immune response by compound ITA-5

[0066] 1. Experimental materials

[0067] The RNA virus is a vesicular stomatitis virus (VSV), which is a conventional experimental virus and is commercially available from Bio Company.

[0068] The Mouse IFN-β ELISA kit was purchased from R&D Systems;

[0069] The antibody anti-Phospho-TBK1 was purchased from Abeam;

[0070] The anti-Phospho-IRF3, anti-Phospho-STAT1, and anti-TBK1 were purchased from CST;

[0071] The anti-Actin was purchased from Proteintech;

[0072] The mouse fibroblasts (L929) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0073] C57BL / 6 mice were purchased from Beijing Vitrolife Biotech Co., Ltd. and the mouse primary peritoneal macrophages (PMs) were induced and obtained by intraperitoneal injection of 6% starch solution.

[0074] 2. Experimental method

[0075] ①, the mouse peritoneal macrophages were co-incubated with different concentrations of the compound ITA-5 prepared in Example 1 (0, 6.25, 12.5, 25, 50, 100 μM) for 2 h, then infected with VSV virus, and the infected cells were further cultured.

[0076] The supernatants of the three groups of cells were taken, and the content of IFN-β in the cell supernatant was detected by ELISA experiment, and the results are shown in Figure 3 A.

[0077] ELISA detection: the content of IFN-β in the cell supernatant was detected using an IFN-β ELISA detection kit (R&D Systems) according to the instructions.

[0078] ②, the mouse peritoneal macrophages were divided into two groups. The first group was pre-incubated with the 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, 12 h, which was the experimental group (ITA-5 / VSV). The second group was pre-incubated with an equal amount of dimethyl sulfoxide (DMSO) for 2 h, then infected with VSV virus, and the infected cells were further cultured for 0, 8, 12 h, which was the control group (Ctrl / VSV).

[0079] The expression levels of p-TBK1, p-IRF3 and p-STAT1 proteins in the two groups of cells were detected by Western Blot experiment, and the results are shown in Figure 3 B.

[0080] Western Blot Analysis: Cells were washed with phosphate-buffered saline (PBS), lysed with RIPA protein extraction reagent (Pierce, Thermo Fisher Scientific) containing 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.

[0081] ③ Mouse fibroblasts (L929) were divided into four groups. Group 1 (ITA-5 / VSV) consisted of L929 cells pre-incubated with 50 μM ITA-5 for 2 hours, then infected with VSV virus. Group 2 (Ctrl / VSV) consisted of L929 cells pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 hours, then infected with VSV virus. Group 3 (ITA-5 / Mock) consisted of L929 cells pre-incubated with 50 μM ITA-5 for 2 hours. Group 4 (Ctrl / Mock) consisted of L929 cells pre-incubated with an equal volume of dimethyl sulfoxide (DMSO) for 2 hours.

[0082] The accumulation of TBK1 in the perinuclear region of four groups of cells was detected by immunofluorescence assay. The results are as follows: Figure 3 As shown in C.

[0083] Immunofluorescence method: The four groups of cells treated above 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 overnight at 4°C with anti-TBK1, washed three times with PBS buffer, added the fluorescent secondary antibody Alexa Fluor 488, incubated at room temperature for 1 h, washed three times with PBS buffer, added DAPI to stain the nuclei, washed three times with PBS buffer, and then fixed the slides onto glass slides with anti-fluorescence quenching mounting solution. The cells were analyzed using a Zeiss LSM980 confocal laser microscope.

[0084] 3、Experimental results

[0085] As can be seen from Figure 3 A, ITA-5 significantly inhibited the secretion of IFN-β induced by VSV infection, and the inhibition was in a concentration-dependent manner.

[0086] As can be seen from Figure 3 B, ITA-5 significantly inhibited the protein expression levels of p-TBK1, p-IRF3 and p-STAT1 induced by VSV infection.

[0087] As can be seen from Figure 3 C, ITA-5 inhibited the aggregation of TBK1 to the perinuclear.

[0088] The above data results all show that the compound ITA-5 inhibits the RNA virus VSV-mediated antiviral immune response.

[0089] Example 4, ITA-5 inhibits DNA virus HSV-1-mediated antiviral immune response

[0090] 1、Experimental materials

[0091] The DNA virus is herpes simplex virus type I (HSV-1), which is a conventional experimental virus and is commercially available from Bio Company.

[0092] The mouse IFN-β ELISA kit is purchased from R&D Systems;

[0093] The antibody anti-Phospho-TBK1 is purchased from Abeam;

[0094] The anti-Phospho-IRF3, anti-Phospho-STAT1 and anti-TBK1 are purchased from CST;

[0095] The anti-Actin is purchased from Proteintech;

[0096] The mouse fibroblasts (L929) are purchased from the Cell Bank of the Chinese Academy of Sciences.

[0097] The C57BL / 6 mice are purchased from Beijing VitoLiu Biotechnology Co., Ltd., and the mouse primary peritoneal macrophages (PMs) are induced and obtained by intraperitoneal injection of 6% starch solution.

[0098] 2、Experimental methods

[0099] ①, different concentrations of the compound ITA-5 prepared in Example 1 (0, 6.25, 12.5, 25, 50, 100 μM) and mouse peritoneal macrophages were co-incubated for 2 h, then infected with HSV-1 virus, and the infected cells were further cultured.

[0100] The supernatant of three groups of cells was taken, and the content of IFN-β in the cell supernatant was detected by ELISA experiment, and the results are shown in Figure 4 A.

[0101] The ELISA detection method is the same as that in Example 3.

[0102] 2. The mouse peritoneal macrophages were divided into two groups. The first group was pre-incubated with compound ITA-5 at a concentration of 50 μM for 2 h, and then infected with HSV-1 virus, and the infected cells were cultured for 0, 2, and 4 h, which was the experimental group (ITA-5 / HSV-1). The second group was pre-incubated with an equal amount of dimethyl sulfoxide (DMSO) for 2 h, and then infected with VSV virus, and the infected cells were cultured for 0, 2, and 4 h, which was the control group (Ctrl / HSV-1).

[0103] The expression levels of p-TBK1, p-IRF3, and p-STAT1 proteins in the two groups of cells were detected by Western Blot experiment, and the results are shown in Figure 4 B.

[0104] The Western Blot detection method is the same as that in Example 3.

[0105] 3. The mouse peritoneal macrophages were divided into two groups. The first group was pre-incubated with compound ITA-5 at a concentration of 50 μM for 2 h, and then infected with HSV-1 virus, and the infected cells were cultured for 0, 2, and 4 h, which was the experimental group (ITA-5 / HSV-1). The second group was pre-incubated with an equal amount of dimethyl sulfoxide (DMSO) for 2 h, and then infected with VSV virus, and the infected cells were cultured for 0, 2, and 4 h, which was the control group (Ctrl / HSV-1).

[0106] The aggregation of TBK1 in the perinuclear region of the four groups of cells was detected by immunofluorescence method experiment, and the results are shown in Figure 4 C.

[0107] The immunofluorescence method detection method is the same as that in Example 3.

[0108] 3. Experimental results

[0109] From Figure 4As shown in A, ITA-5 significantly inhibited the secretion of IFN-β induced by HSV-1 infection in a concentration-dependent manner.

[0110] As shown in B, ITA-5 significantly inhibited the protein expression levels of p-TBK1, p-IRF3 and p-STAT1 induced by HSV-1 infection. Figure 4

[0111] As shown in C, ITA-5 inhibited the aggregation of TBK1 to the perinuclear. Figure 4

[0112] The above data results all show that the compound ITA-5 inhibits the antiviral immune response mediated by the DNA virus HSV-1.

[0113] Example 5, verification of ITA-5 targeting TBK1

[0114] 1. Experimental materials

[0115] hTBK1 purified protein was purchased from Detaibio (Nanjing, China); anti-TBK1 was purchased from CST.

[0116] C57BL / 6 mice were purchased from Beijing Vitorlaihua Biotechnology Co., Ltd., and mouse primary peritoneal macrophages (PMs) were induced and obtained by intraperitoneal injection of 6% starch solution.

[0117] 2. Experimental methods

[0118] ①, ITA-5 and TBK1 were subjected to molecular docking using the CovDock module in the Schrodinger 2021-2 software package, wherein the reaction type of covalent docking was set to Michael addition, and the results are shown in Figure 5 A.

[0119] ②, the affinity between hTBK1 protein and ITA-5 was detected in vitro by surface plasmon resonance experiment, and the results are shown in Figure 5 B.

[0120] Surface plasmon resonance experimental method: BIAcore T200 instrument was used to measure the affinity. hTBK1 protein was covalently coupled with 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-5 was gradiently diluted with PBS solution containing 5% DMSO and 0.05% surfactant P20, and the binding time of protein on the chip was 60 s and the dissociation time was 120 s. The binding curve was analyzed using GraphpPad Prism 9, and the affinity was calculated using BIAevaluation software. ​​

[0121] ③ Mouse peritoneal macrophages were divided into two groups. Group 1 (ITA-5) consisted of mouse peritoneal macrophages pretreated with 50 μM of compound ITA-5 for 12 h. Group 2 (Ctrl) consisted of mouse peritoneal macrophages pretreated with an equal amount of dimethyl sulfoxide (DMSO) for 12 h.

[0122] 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 5 As shown in C.

[0123] Experimental method for cell thermal shift: Macrophages were pre-incubated for 12 h with control solvent or ITA-5. After cell collection, they were washed with PBS, then resuspended in PBS containing protease inhibitors and aliquoted into multiple PCR tubes. Each PCR tube was heated at a specified temperature (40, 42.5, 45, 47.5, 50, 52.5, or 55 °C) for 3 min, and immediately cooled in liquid nitrogen for 10 s. This was repeated three times. After centrifugation at 12,000 g for 15 min at 4 °C, the supernatant was collected, and the mixture was boiled with loading buffer and analyzed by Western blotting. Protein bands were quantitatively analyzed using ImageJ software.

[0124] 3. Experimental Results

[0125] Depend on Figure 5 As shown in A, ITA-5 and TBK1 proteins form a relatively stable covalent bond.

[0126] Depend on Figure 5 B indicates that there is a strong affinity between TBK1 and ITA-5, K D =81.75 nM.

[0127] Depend on Figure 5 As shown in C, ITA-5 treatment enhanced the stability of the TBK1 protein.

[0128] The above data all indicate that compound ITA-5 can directly target TBK1 to exert its effect.

[0129] Example 6: ITA-5 inhibits poly(I:C)-induced excessive inflammatory response;

[0130] 1. Experimental Materials

[0131] C57BL / 6 mice were purchased from Beijing Vital River Biotechnology Co., Ltd., using 6-8 week old female mice.

[0132] The poly(I:C) was purchased from InvivoGen.

[0133] 2. Experimental method

[0134] ①, C57BL / 6J mice were divided into three groups, the first group was the experimental group (ITA-5 + poly (I:C)), C57BL / 6J mice were injected intraperitoneally with ITA-5 prepared in Example 1 (20 mg / kg) 2 h later, and then injected intraperitoneally with poly (I:C) (15 mg / kg) and cultured for 8 h. The second group was the control group (Ctrl + poly (I:C)), and the C57BL / 6J mice were injected intraperitoneally with the same amount of control solvent Ctrl 2 h later, and then injected intraperitoneally with poly (I:C) (15 mg / kg) and cultured for 8 h. The third group was the blank group (Ctrl + PBS), and the C57BL / 6J mice were injected intraperitoneally with Ctrl 2 h later, and then injected with the same amount of PBS buffer and cultured for 8 h.

[0135] The serum of the three groups of mice was taken, and the content of IFN-β in the serum of the mice was detected by ELISA experiment, and the results are shown in Figure 6 A.

[0136] The three groups of mice were sacrificed, the lung tissues of the mice were taken, and the RNA was extracted according to the conventional method, and the expression level of mRNA in the lung tissues of the mice was detected by RT-PCR experiment, and the results are shown in Cxcl10 B. Figure 6

[0137] ②, C57BL / 6J mice were divided into three groups, the first group was the experimental group (ITA-5 + poly (I:C)), C57BL / 6J mice were injected intraperitoneally with ITA-5 prepared in Example 1 (20 mg / kg) 2 h later, and then injected intraperitoneally with poly (I:C) (25 mg / kg) and cultured for 12 h, and then injected intraperitoneally with ITA-5 (20 mg / kg) again and cultured for 12 h. The second group was the control group (Ctrl + poly (I:C)), and the C57BL / 6J mice were injected intraperitoneally with the same amount of control solvent Ctrl 2 h later, and then injected intraperitoneally with poly (I:C) (25 mg / kg) and cultured for 12 h, and then injected intraperitoneally with the control solvent Ctrl again and cultured for 12 h. The third group was the blank group (Ctrl + PBS), and the C57BL / 6J mice were injected intraperitoneally with the control solvent Ctrl, and then injected intraperitoneally with the same amount of PBS buffer and cultured for 12 h, and then injected intraperitoneally with the control solvent Ctrl again and cultured for 12 h.

[0138] The three groups of mice were sacrificed, the eyeball blood of the mice was collected, and then creatinine (Creatinine) and urea (Urea) were used as kidney injury indicators, glutamic-pyruvic transaminase (ALT), total protein (Total Protein) and globulin (Globulin) were used as liver injury indicators, and the eyeball blood of the mice was biochemically analyzed, and the results are shown in​Figure 6 C and D.

[0139] After the three groups of mice were sacrificed, the mouse lung tissues were collected at the same time, the 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, and the results are as follows Figure 6 E.

[0140] 3、Experimental results

[0141] As can be seen from Figure 6 A, compared with the control group of mice, the secretion of IFN-β in the serum of ITA-5 group of mice was significantly reduced.

[0142] As can be seen from Figure 6 B, the expression of Cxcl10 mRNA in the lung tissue of ITA-5 group of mice was significantly lower than that of the control mice.

[0143] As can be seen from Figure 6 C and D, ITA-5 inhibits the indicators of kidney injury and liver injury in the plasma of mice.

[0144] As can be seen from Figure 6 E, ITA-5 attenuates the inflammatory cell infiltration of mouse lung tissue induced by poly(I:C) infection.

[0145] The above data results all show that the compound ITA-5 can effectively inhibit the excessive inflammatory response induced by poly(I:C).

[0146] Example 4, Compound ITA-5 inhibits the inflammatory response of systemic lupus erythematosus (SLE)

[0147] 1、Experimental materials

[0148] C57BL / 6 mice were purchased from Beijing VitoLiuHua Biotechnology Co., Ltd., and 6-week-old female mice were used;

[0149] TMPD was purchased from Sigma.

[0150] 2、Experimental method

[0151] The C57BL / 6J mice were divided into three groups, the first group was the experimental group (TMPD+ITA-5), the C57BL / 6J mice were injected with 0.5ml 2,6,10,14-tetramethylpentadecane (TMPD) to construct the SLE model, and after three weeks, the mice were injected with ITA-5 (20mg / kg) prepared in Example 1 once a day for one week. The second group was the control group (TMPD), the C57BL / 6J mice were injected with 0.5ml TMPD to construct the SLE model, and after three weeks, the mice were injected with the same amount of control solvent once a day for one week. The third group was the blank group (Ctrl), the C57BL / 6J mice were injected with 0.5ml PBS, and after three weeks, the mice were injected with the same amount of control solvent once a day for one week.

[0152] The plasma of the three groups of mice was taken, and the content of Creatinine in the plasma of the mice was detected by biochemical analysis, and the results are shown in Figure 7 A.

[0153] After the three groups of mice were sacrificed, the lung tissues of the mice were collected, and the lung tissue sections of the mice were prepared according to the existing method, then the lung tissue sections of the mice were stained with hematoxylin and eosin, and observed, and the results are shown in Figure 7 B.

[0154] The three groups of mice were sacrificed, and the lung tissues of the mice were taken, and the RNA was extracted according to the conventional method, and the expression levels of Cxcl10 and Isg15 mRNA in the lung tissues of the mice were detected by RT-PCR experiment, and the results are shown in Figure 7 C.

[0155] 3, Experimental results

[0156] As shown in Figure 7 A, ITA-5 inhibited the renal injury indicator Creatinine in the plasma of the mice.

[0157] As shown in Figure 7 B, ITA-5 reduced the inflammatory cell infiltration in the lung tissues of the TMPD-induced SLE model mice.

[0158] As shown in Figure 7 C, the expression of Cxcl10 and Isg15 mRNA in the lung tissues of the mice in the ITA-5 group was significantly lower than that in the control mice.

[0159] The above data results all show that the compound ITA-5 can effectively inhibit the inflammatory response in systemic lupus erythematosus.

[0160] Example 5, compound ITA-5 inhibits inflammatory response of AGS syndrome (Aicardi-Goutières syndrome)

[0161] 1. Experimental materials

[0162] Trex1 Gene-deficient mice were purchased from Jackson Laboratory and bred in the SPF barrier environment of the Model Animal Research Center of Shandong University; C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and all 4-week-old female mice were used;

[0163] 2. Experimental methods

[0164] AGS syndrome mouse model was established Trex1 The gene-deficient mice were divided into two groups, group 1 and group 2; wild-type mice of the same age and gender were divided into group 3. Group 1 was the experimental group (ITA-5 was prepared according to Example 1, and 20 mg / kg of ITA-5 was injected intraperitoneally to the mice at the age of 4 weeks once a day for one week. Trex1 - / - + ITA-5), Trex1 - / - At the age of 4 weeks, the mice were injected intraperitoneally with ITA-5 prepared in Example 1 (20 mg / kg) once a day for one week. Group 2 was the control group (Ctrl was injected intraperitoneally to the mice at the age of 4 weeks once a day for one week. Trex1 - / - ), Trex1 - / - At the age of 4 weeks, the mice were injected intraperitoneally with an equal amount of control solvent Ctrl once a day for one week. Group 3 was the blank group (WT), and the WT mice were injected intraperitoneally with an equal amount of control solvent Ctrl once a day for one week at the age of 4 weeks.

[0165] After the three groups of mice were sacrificed, the lung tissues of the mice were collected at the same time, and the 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, and the results are shown in Figure 8 A.

[0166] The three groups of mice were sacrificed, and the mouse lung tissues were taken, and the RNA was extracted according to the conventional method, and the expression levels of Cxcl10 and Isg15 mRNA in the mouse lung tissues were detected by RT-PCR experiment, and the results are shown in Figure 8 B.

[0167] 3. Experimental results

[0168] As shown in Figure 8 A, ITA-5 attenuated the inflammatory cell infiltration in the lung tissues of mice (AGS syndrome model). Trex1 - / -

[0169] As shown in Figure 8 ​B can be known that the ITA-5 group Trex1 - / - In the lung tissue of mice Cxcl10 and Isg15 The mRNA expression is obviously lower than that of the control mice.

[0170] The above data results all show that the compound ITA-5 can effectively inhibit the inflammatory response in the AGS syndrome model.

[0171] In conclusion, the present application verifies that the compound ITA-5 has significant inhibitory activity on TBK1, and the compound ITA-5 can be used as a TBK1 inhibitor. It is also verified that the compound ITA-5 can effectively inhibit the anti-virus immune response mediated by TBK1 and the excessive inflammatory response induced by poly(I:C). In addition, the present application also verifies that ITA-5 can effectively inhibit the inflammatory response of the systemic lupus erythematosus model mice and the AGS syndrome model mice. It shows that the compound has the potential to develop into a TBK1 inhibitor drug and a therapeutic drug for diseases related to TBK1, such as infectious diseases, autoimmune diseases, metabolic diseases and cancer, and provides a new therapeutic drug for clinic, and has good clinical application value and broad application prospect.

Claims

1. The use of compound ITA-5 or a pharmaceutically acceptable salt thereof in the preparation of TBK1 inhibitors, characterized in that, The structural formula of the compound ITA-5 is shown below: 。 2. The use of compound ITA-5 or a pharmaceutically acceptable salt thereof as a TBK1 inhibitor in the preparation of pharmaceutical compositions for the prevention and / or treatment of diseases related to TBK1 activity, characterized in that, The structural formula of the compound ITA-5 is shown below: 。 3. The application as described in claim 1 or 2, characterized in that, The preparation method of the compound ITA-5 includes the following steps: (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 compound ITA-1. The structure of compound ITA-1 is 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) Compound ITA-1 and L -Phenylalanine methyl ester 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-5. Among them, the compound ITA-1, L -Phenylalanine methyl ester hydrochloride, dichloromethane, 2-(7-azobenzotriazole)- N,N,N',N' The ratio of tetramethylurea hexafluorophosphate to triethylamine is (0.1~0.2g): (0.15~0.25g): (3~6 ml): (0.3~0.5g): (0.4~0.5ml).

4. The application as described in claim 2, characterized in that, The diseases associated with TBK1 activity are those that can be improved, prevented, or treated by inhibiting TBK1 activity.

5. The application as described in claim 4, characterized in that, The diseases associated with TBK1 activity are infectious diseases, autoimmune diseases, metabolic diseases, or cancer.

6. The application as described in claim 5, characterized in that, The infectious disease is caused by one or more of DNA viruses or RNA viruses.

7. The application as described in claim 6, characterized in that, The DNA virus is herpes simplex virus 1, and the RNA virus is vesicular stomatitis virus.

8. The application as described in claim 5, characterized in that, The autoimmune diseases mentioned are AGS syndrome, systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

9. The application as described in claim 5, characterized in that, The metabolic diseases mentioned are obesity, diabetes, and non-alcoholic fatty liver disease.

10. A pharmaceutical composition for treating diseases related to TBK1 activity, characterized in that, The pharmaceutical composition comprises the compound ITA-5 of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, characterized in that, The pharmaceutical composition is any pharmaceutically acceptable dosage form made by using compound ITA-5 or a pharmaceutically acceptable salt thereof as the main active ingredient, combined with pharmaceutically acceptable excipients.

12. The pharmaceutical composition of claim 10, characterized in that, The dosage forms include tablets, capsules, granules, pills, decoctions, suspensions, dispersants, syrups, suppositories, gels, aerosols, and patches.

Images