Compounds used to inhibit the activity of AIM2 protein

Abstract

To provide the use of a compound or a medicinal derivative thereof in inhibiting AIM2 protein activity, in the technical field of protein inhibitory medicines.SOLUTION: Compared with traditional broad-spectrum immunomodulators, a compound has a strong targeting effect, and is more accurate, rapid, effective, safe and stable. Moreover, the compound has a strong binding force with human-derived AIM2 protein and mouse-derived protein, and binding constants are as high as KD=1.56E-6M and KD=5.12E-6M, respectively. Compared with traditional biological inhibitors, the compound has advantages of easy storage, stable activity, small molecular weight, lower production cost and easy absorption.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention belongs to the technical field of protein inhibitors, and is particularly used to inhibit the activity of AIM2 protein. compound This is regarding. [Background technology]

[0002] Psoriasis is a refractory and highly prevalent inflammatory immune disease worldwide. As of 2021, more than 6.5 million people in China alone suffer from the disease. However, the current level of medical care cannot completely treat it. Clinically, there are many treatments for psoriasis, many of which aim to alleviate inflammatory symptoms by broadly suppressing the immune system, including the use of steroids and immunosuppressants. There is also the use of physical therapy and herbal medicine. However, extensive symptomatic treatments are less effective and may cause disease recurrence or damage to other systems, which can bring psychological and economic burdens to patients. In recent years, precision medicine for psoriasis has to some extent made up for the shortfalls of traditional treatment methods. Many biological preparations have been developed, IL-17, TNF-α, IL-23 Efforts are being made to eradicate the psoriatic phenotype by targeting downstream effector molecules such as

[0003] Compared with conventional treatments, biologics can increase the cure rate of psoriasis patients to different degrees within a certain period of time, and the administration interval is long and they tend to be highly dependent. However, they have the same disadvantages as conventional treatments. All biologics for psoriasis treatment currently on the market are fundamentally symptomatic treatments that alleviate the phenotype by blocking the effects of downstream effector molecules, and if the use of biologics is discontinued for more than six months, the risk of psoriasis recurrence increases significantly. In addition, because the price of biologics is high and long-term maintenance treatment is required, if the effect decreases along the way, many biologics must be used in combination, which can cause a large economic burden on patients.

[0004] In summary, the drugs currently used to treat psoriasis are broad-spectrum immunomodulators and targeted drugs. 2They can be roughly divided into three main types. The long-term effect of a wide range of traditional immunomodulators is not ideal, and targeted drugs, represented by biologics, can make up for the shortcomings of traditional drug therapy to some extent, but they are expensive and have relatively high application standards. There are few other types of targeted drugs (natural small molecules or artificial compounds) for psoriasis on the market, most of which target the downstream effect pathway in the pathogenesis of psoriasis. Compared with the treatment that interferes with upstream factors, the long-term effect of this symptomatic treatment may be insufficient because the effect on IL-17, an important cellular factor in pathogenesis, and its upstream initiation pathway is relatively weak.

[0005] Therefore, it is necessary to develop new types of drugs that complement the advantages of traditional treatments and biologics, and various aspects such as drug efficacy, targeting, economy, safety, and adaptability must be considered simultaneously. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a method for inhibiting the activity of AIM2 protein. compound The objective of this invention is to provide a compound that can be used as a therapeutic agent for psoriasis, and can significantly suppress the production of IL-1β and CD3 in IMQ mice. + TCR d + ROR γT + The compound reduces the ratio and clearly ameliorates the inflammatory phenotype in IMQ mice. [ka] [Means for solving the problem]

[0007] To achieve the above objectives, the present invention adopts the following technical solutions: On the other hand, it is used to suppress the activity of AIM2 protein. compound to provide.

[0008] On the other hand, compound The present invention provides a method for producing a drug that inhibits the activity of AIM2 protein.

[0009] On the other hand, The present invention provides a pharmaceutical composition for use as a therapeutic agent for psoriasis. It comprises at least a compound according to formula I and / or a pharmaceutical carrier and / or diluent. Effect of the Invention

[0010] (1) Compounds of the Invention I (Compound library number is T6S1683 ), a small molecule with activity against the AIM2 protein, is used in the manufacture of medicines used to treat psoriasis. Compared with conventional broad-spectrum immunomodulators, it has stronger targeting action and is more precise, rapid, effective, safe, and stable. In addition, it has very strong binding ability with AIM2 human protein and mouse protein, with binding constants of 0.01 to 0.01, respectively. KD=1.56E -6 M and KD=5.12E -6 M reaches. (2) The compound of the present invention and its manufactured pharmaceutical product bind and inhibit targets upstream of the AIM2 pathway in psoriasis. Compared with conventional biological inhibitors (biological inhibitors act on the mid-downstream of the immune-inflammatory pathway of psoriasis and act directly on symptoms, but the effect is unstable in maintaining, and almost all biological inhibitors have a significant increase in recurrence rate after half a year), the inhibition level of inflammatory factors is higher, and the compound of the present invention has a longer-term effect than biological inhibitors, and can achieve significant effects. In addition, many of the conventional biological inhibitors are administered systemically, and are very likely to affect other systems, but the compound of the present invention is used locally, is easily absorbed, and many side effects can be avoided, so it is safe, widely used, and easy to use, so it has high compliance. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram of the crystal structure of AIM2. [Diagram 2]FIG. 1 is a schematic diagram of the virtual screening workflow. [Diagram 3] This shows the results of real-time quantitative PCR detection of IL-1β mRNA in HaCat cells. [Figure 4] Nuclear magnetic resonance spectrum of T6S1683. [Diagram 5] HPLC spectrum of T6S1683. [Figure 6] Schematic diagram of the interaction between T6S1683 and AIM2 protein. [Figure 7] This shows the affinity detection results between T6S1683 and AIM2 human protein. [Figure 8] This shows the affinity detection results between T6S1683 and AIM2 mouse protein. [Figure 9] IL-1β transcription levels in HaCat cells treated with different doses of T6S1683. [Figure 10] Morphological changes in HaCat cells treated with 10 uM T6S1683. [Figure 11] Expression status (gel electrophoresis) of AIM2 pathway proteins in HaCat cells treated with 10 uM T6S1683. [Figure 12] Expression status (ELISA) of AIM2 pathway proteins in HaCat cells treated with 10 uM T6S1683. [Figure 13] PASI scores for mice in each treatment group. [Figure 14] Phenotype of IMQ mice treated with 5 mg / kg of T6S1683. [Figure 15] Skin pathology phenotypes of mice in each treatment group. [Figure 16] CD3 + TCRd + RORγT + T lymphocyte ratios at different T6S1683 concentrations. [Figure 17] Percentage of CD3 + TCRd + RORγT + T lymphocytes in mice from each treatment group. [Figure 18]IL-1β protein expression in the skin of IMQ mice treated with 5 mg / kg of T6S1683. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The embodiments of the present invention are provided to better illustrate the present invention, and the content of the present invention is not limited to these embodiments. Therefore, those skilled in the art can essentially improve and adjust the implementation based on the content of the present invention, which are within the protection scope of the present invention.

[0013] The terms used herein are used only to describe certain embodiments and are not intended to limit the present disclosure. Unless the context clearly indicates otherwise, singular expressions include plural expressions. As used herein, terms such as "comprises", "has", "includes", etc. are used to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terms disclosed herein are not intended to exclude the possibility that other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. Also, " / " used herein can be interpreted as "and" or "or".

[0014] An embodiment of the present invention is used to inhibit the activity of AIM2 protein and is represented by formula I compound to provide. [ka]

[0015] CaMK2γ The amino acid sequence of a protein includes: MESKYKEILLLTGLDNITDEELDRFKFFLSDEFNIATGKLHTANRIQVATLMIQNAGAVSAVMKTIRIFQKLNYMLLAKRLQEEKEKVDKQYKSVTKPKPLSQAEMSPAASAAIRNDVAKQRAAPKVSPHVKPEQKQMVAQQESIREGFQKRCLPVMMVLKAKKPFTFETQE GKQEMFHATVATEKEFFFVKVFNTLLKDKFIPKRIIIIARYYRHSGFLEVNSASRVLDAESDQKVNVPLNIIRKAGETPKINTLQTQPLGTIVNGLFVVQKVTEKKKNILFDLSDNTGKMEVLGVRNEDTMKCKEGDKVRLTFFTLSKNGEKLQLTSGVHSTIKVIKAKKKT " is included.

[0016] Also of note, single-cell transcriptome sequencing of psoriasis patients and Micamot-induced psoriasis mice, ATACNumerous omics studies, including sequencing, transcriptomics, and genetics, have demonstrated that only the AIM2 inflammatory gene is significantly activated in psoriatic skin lesions and peripheral blood. -IL-1β- The IL-17 signal pathway contains multiple susceptibility genes for psoriasis, and it has been proven that the AIM2 pathway plays an important role in the development and exacerbation of psoriasis. Regulating the activity of the AIM2 pathway can control the psoriasis-like inflammatory phenotype in the Micamot mouse model. AIM2 is the upstream initiator of the pathway, and in psoriasis, blocking the ability of AIM2 protein to inhibit the activation of downstream proteins can effectively block the effect of the pathway. In principle, it has a more powerful and sustained therapeutic effect than simply removing the execution factor IL-17. Considering that inhibiting upstream molecules in conventional systems biology treatments may cause other system abnormalities that may affect related pathways, the present invention Biacore T200 Using the instrument, the compound affinity of a protein model was measured based on the amino group coupling method, and the compounds were sorted by affinity using a computer. 100 We screened several types of AIM2 small molecule inhibitors. Finally, we identified safer compounds with inhibitory effects. HaCat Screening in cell models The above formula I The compound represented by the formula:

[0017] The above Compound derivative products refers to a compound in which the compound structure is modified based on the mother structure while retaining the mother structure, and the modification of the compound structure can maintain the inhibitory effect on AIM2 protein activity, or enhance the activity of the compound, or improve the pharmacokinetic properties.

[0018] The present invention Specific examples of the above A compound used for inhibiting the activity of the AIM2 protein according to claim 1 In the above, the compound may be in the form of a salt of a pharmaceutical agent.

[0019] The present invention Specific examples of the above A compound used for inhibiting the activity of the AIM2 protein according to claim 1 In The aboveThe compounds may be in the form of an acid addition salt of the pharmaceutical agent, although other salt forms are of course not excluded.

[0020] In an embodiment of the present invention, compound The present invention provides an agent for use in the production of the above-mentioned compound and for suppressing the activity of AIM2 protein.

[0021] Compounds used to inhibit the activity of the AIM2 protein according to specific embodiments of the present invention In the above, the drug that inhibits the activity of AIM2 protein is intended to inhibit the activity of AIM2 protein in psoriatic tissue. The above Compound AIM2 for psoriasis -IL-1β- Suppresses AIM2 protein activity via the IL-17 signaling pathway and inhibits psoriasis of In addition, this compound primarily targets AIM2 and inhibits active caspase- 1 p20 and IL-1β The p17 content can be reduced, The above Compound AIM2 for psoriasis -IL-1β- It has been shown to affect only the IL-17 signaling pathway and not other systems, meaning it is specific for psoriasis and has low side effects.

[0022] Compounds used to inhibit the activity of the AIM2 protein according to specific embodiments of the present invention In The above The compounds are in the form of pharmaceutical salts.

[0023] Compounds used to inhibit the activity of the AIM2 protein according to specific embodiments of the present invention In The above The compounds are in the form of medicinal acid addition salts.

[0024] Compounds used to inhibit the activity of the AIM2 protein according to specific embodiments of the present invention Inhibits AIM2 protein activity in The above The dosage form of the drug may be a capsule, tablet, oral preparation, microcapsule preparation, injection, suppository, spray, or cream. Specifically, a person skilled in the art can select an appropriate dosage form according to the route of administration, the subject of administration, etc.

[0025] In a particular embodiment of the present invention, a pharmaceutical composition for use in the treatment of silver sludge is provided, the pharmaceutical composition comprising at least one of the formula I and, if necessary, a pharmaceutical carrier and / or diluent. An appropriate dosage form can be selected depending on the route and subject of administration. [ka]

[0026] In some specific embodiments, The above In the composition, The above The soluble carrier and / or diluent may be applied in any of the following forms: solution, colloidal solution, emulsion, suspension, gas dispersion, particulate dispersion, and solid dispersion.

[0027] In some specific embodiments, The above In the composition, The above formula I The compound represented by the formula (I) may be used in combination with other psoriasis drugs to obtain better therapeutic effects. For example, combination with a biological agent targeting the downstream molecule IL-17 or IL-23 can lower the minimum effective dose of the biological agent, reduce interference with a certain degree of the system, and obtain a more stable and safer effect than a single biological agent.

[0028] In some specific embodiments, The above In the composition, the amount of the active ingredient of the compound in the drug is 5 mg / kg is selected.

[0029] In the present invention, the activity of AIM2 protein of The compounds used for inhibition are Biacore T200 The device is used to measure the compound affinity of the protein model based on the amino group coupling method, and the small molecule inhibitor of AIM2 is selected according to the affinity by computer. 100 Select the pieces and finally HaCat The selection is made by selecting compounds that have an inhibitory effect in a cell model. Specifically, the selection includes the following steps:

[0030] (1) Selection of the crystal structure of AIM2 protein The amino acid sequence (SEQ ID NO: 1) of AIM2 protein (Interferon-inducible protein AIM2, UniProtKB: https: / / www.uniprot.org / uniprot / O14862) and the 3 Based on the three-dimensional structure, possible protein binding regions are screened. RCSBPDB AIM2 protein in the library 3 Dimensional crystal structure 5 Types found, table 1 As shown in the figure, the resolution is good. 3VD8 and 4O7Q It is. 3VD8 The crystal structure of 4O7Q Since it provides more information than the amino acid sequence of , it has been preferentially listed as a screenable crystal structure.

[0031] [Table 1]

[0032] (2) Selection of binding sites AIM2 PYD The structural domains are crucial for regulating downstream signaling pathways. PYD In the structural area α2 Amino acids have specificity and PYD The structural region is 6 It consists of two helices. PYD In the structural area α2 of D19, E20, D23, F27, F28 AIM2 is a ligand protein ASC of PYD and PYD-PYD It is the most important amino acid for interaction. Therefore, 3VD8 With reference to the crystal structure of D19, E20, D23, F27, F28A binding site composed of the above will be selected to perform virtual screening of new small molecule inhibitors of AIM2 protein.

[0033] (3) Determination of computational virtual screening procedures, selection and preparation of small molecule compound libraries and target proteins Computational virtual screening procedure: AIM2 D19, E20, D23, F27, F28 The binding site composed of the above was identified as a screening site for new small molecule inhibitors, and the AIM2 three-dimensional crystal structure (PDBID: 3VD8) was used as a reference ( Figure 1 The steps for planning a virtual screening are: Figure 2 As shown in.

[0034] Small molecule compound library: The small molecule database selected was Chemdiv. Selection and preparation of target protein: Based on the three-dimensional structure of AIM2 (3vd8.pdb), sybyl-X2.1 We processed the AIM2 protein using the "PrepareProteinStructure" module of . We selected and removed all water molecules with "RemoveSubstructures", then used "AnalyzeSelectedStructure" to analyze and modify the protein and add hydrogen atoms of the protein. Finally, sybyl Using the protomol module, PYD Domain alpha2 of D19, E20, D23, F27, F28 Select the inhibitor binding cavity file using the active site "Residue" generation method. 3vd8_H-R-0.50-0.sfxc This is the output file after processing of the target protein.

[0035] (4) Virtual screening calculations sybyl-X2.1 The “CompoundFiltering” module of was used to select compounds in the database that met the following rules: (a) Molecular weight 700 To be less than.(b) cLogP (ester water partition coefficient) -4 from 6 Within the range. (c) The number of hydrogen bond acceptors 15 It is less than or equal to. (d) The number of hydrogen bond donors 6 It is less than or equal to. (e) The number of rotatable bonds is 11 In this project, sybyl The “Compound Filtering” module of the drug design platform was used to initially screen the small molecule database. 5 In accordance with the overall principle based on three rules, 5 If screening is performed according to one rule, the number of compounds in the small molecule database will be greatly reduced, and the diversity of compounds will obviously be reduced. Therefore, in order to increase the diversity of compounds in the virtual screening stage, it is necessary to increase the number of lead compounds with a certain degree of biological activity. Therefore, in order to increase the diversity of compounds, in the virtual screening stage, 5 It is necessary to appropriately expand the parameter range of the "one rule". For example, the molecular weight should be 700 and clogP can be expanded to describe the hydrophilicity / hydrophobicity of a compound. Similarly, the range of hydrogen bond donors and acceptors and the number of rotatable bonds needs to be appropriately expanded.

[0036] (i) First virtual screening calculation Sybyl-X2.1 Screening was performed using the Surflex module of the software. Some docking parameters were modified to speed up the initial virtual screening and to obtain the top score. 1% Screen the molecules. Specifically, set "MaxconformationsperFragment" to the default 20 from 10 and change "Max number of rotatable bonds per molecule" to the default 100 from 50Cancel the default "Per-Dock Minimization" and "Post-Dock Minimization" options. Change "Maximum number of poses perligand" to the default 20 from 3 and the top of each ligand molecule 3 Only one molecular structure is retained, accelerating the docking speed and finally 1% The compound is obtained.

[0037] (ii) Second virtual screening calculation Sybyl-X2.1 Use the Surflex module of the software to perform a second screening. 1% Based on the molecule entered in, restore the default parameters (i.e., "MaxconformationsperFragment" is set to the default 20 , "Max number of rotatable bonds per molecule" is left as default; "Per-Dock Minimization" and "Post-Dock Minimization" options are selected as default to optimize the energy minimization before and after docking of compounds. In addition, the number of initial structures of each molecule is set to 4 Increase to top 500 Artificially screen target compounds.

[0038] (iii) Manual Filtering and Review Obtained in the second screening 500 Artificially screen compounds in targets and find AIM2 PYD Structural domain alpha 2 We took into consideration the fact that the nucleoids on AIM2 can have stable interactions (hydrogen bonds, hydrophobicity, and π-π stacking interactions) with the nucleoids. Arg24, Leu72, Asn73 It can form multiple hydrogen bond interactions with many amino acids such as , and at the same time, it can bind to binding sites where many hydrophobic amino acids are present. Phe27, Phe28, Ala36, Leu40, Leu72form a hydrophobic pocket. Therefore, the target compound should have many hydrogen bond donor (and acceptor) groups, aromatic ring structures and hydrophobic substituents. The structure of the compound should be relatively rigid and not have too many rotatable keys. Finally, from the Chemdiv library, 2 As shown in 44 This compound was selected as an AIM2 inhibitor.

[0039] [Table 2]

[0040] (5)HaCat Screening for compounds with inhibitory effects in cell models the aforesaid Selected by screening 100 Types of compounds HaCat The ability of the antibody to protect cells from OligoD A-T stimulation was evaluated. The evaluation criteria were: IL-1β mRNA Real-time fluorescence quantification PCR Compared to the positive control, 44 The most significant inhibitory effect among the small molecule inhibitors ( p<0.0001 ) are selected as ideal inhibitor candidates, and the compounds used in the present invention are I (or T6S1683 (also called).

[0041] (i) Experimental groups were positive control ( PC ) and negative control ( NC ) and PC The group Lip3000 (Lipofectamine TM 3000Transfection Reagent) and OligodA-T The package (OligodA-T: Lip3000: P3000 = 1:1:25, OligodA-T concentration is 1ug / mL) HaCat The cells were transfected. Each treatment group was Lip3000-OligodA-T The packaging and 10μM Small molecule inhibitors ofHaCat Co-transfect the cells. NC Groups added only the same amount of transfection reagent (Lip3000:P3000=1:1).

[0042] (ii) Each experimental group was placed in a cell culture chamber. 5% Included 37℃ in 24 Cultivation time, actual fluorescence amount PCR Collecting cells for testing; 46 Among the various small molecule inhibitors, the most significant inhibitory effect ( p<0.0001 ) were selected as candidates. 2 The second verification was performed and the result was Figure 3 (Note: T6S1683 is the screening number 4 is)

[0043] The small molecule selected by this method, demethoxylated ginger ( T6S1683 (also called 1,6-hexenedione, 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-, (1E,6E)-; the compound has a molecular weight of 338.36 and a structural formula of I It is shown as follows. [ka]

[0044] Below are selected small molecule compounds: I Details on the characteristics of. (1) small molecule compound T6S1683 The characteristics of the compound were evaluated by nuclear magnetic resonance. As shown in the figure, the characteristic hydrogen atoms of the aromatic ring were clearly observed. 2 The active hydrogens of the two phenolic hydroxyl groups show peaks and the clefts are clear, which is consistent with the structure of the compound. The structure of the compound is correct.

[0045] (2) small molecule compound T6S1683The characteristics were evaluated by high performance liquid chromatography (HPLC). Figure 5 As shown in the retention time 10.14 , the peak area ratio is 98.1% This indicates that the purity of the compound is very high.

[0046] Selected small molecule compounds I The synthetic route is as follows: [ka]

[0047] In the following examples, the statistical analysis of the data was carried out as follows: SPSS23.0 and RVersion 4.0.2 Data processing and analysis were performed using software. All tests were two-sided, with P values ​​of 0.01 and 0.001. 0.05 A value less than this is considered statistically significant.

[0048] In order to better understand the present invention, the contents of the present invention will be further described in combination with specific examples, but the contents of the present invention are not limited to the following examples.

[0049] Working Example 1 T6S1683 Interaction mode of AIM2 protein with In the examples of the present invention, the binding mode of T6S1683 and AIM2 was predicted and analyzed. Sybyl-X2.1 The Surflex module of the software was used to predict the binding mode and then the binding of the small molecule to the protein AIM2. Arg24, Leu72, Asn73 We artificially screened and reconfirmed whether it could form multiple hydrogen bonds with multiple amino acids such as. Figure 6 It is shown in T6S1683 teeth Arg24, Asp23, His41 Multiple hydrogen bond interactions form hydrogen bonds between the compound T6S1683 and cooperate in maintaining the binding of the AIM2 protein.

[0050] Example 2 T6S1683 and high affinity testing of AIM2 human protein In an embodiment of the present invention, T6S1683 The affinity of the human AIM2 protein to the IgG was measured. pET28A Using the vectors, human full-length AIM2 protein was expressed and purified. Then, surface plasmon resonance (SPR) was used to detect the CM5 The affinity capacity of the chip was measured using the amine coupling method. Figure 7 It is shown in T6S1683 specifically binds to human source AIM2 protein, with a binding constant of 1.56E -6 M It was shown to have a high binding capacity.

[0051] Example 3 T6S1683 Strong affinity test of AIM2 mouse protein with In an embodiment of the present invention, T6S1683 We examined the affinity of the mouse protein AIM2 to the IL-11. pET28A The mouse full-length AIM2 protein was expressed and purified using the vector. CM5 The affinity was measured using the amino group binding method of the chip. Figure 8 It is shown in T6S1683 specifically binds to mouse AIM2 protein, with a binding constant of 5.12E -6 M It has been shown that the binding capacity is very strong.

[0052] Working Example 4 HaCat In cell models T6S1683 Effective inhibition of AIM2 protein activity by (1) Positive control ( PC ), negative control ( NC ) and each processing group. PC In the group, Lip3000 (Lipofectamine TM3000Transfection Reagent) and OligodA-T Using the package (OligodA-T: Lip3000: P3000 = 1:1:25, OligodA-T concentration is 1ug / mL) HaCat Transfects cells. NC In each treatment group, add only the same amount of transfection reagent (Lip3000:P3000=1:1). Lip3000-OligodA-T Together in a package HaCat Cells are given a ladder dose ( 0.1uM, 1uM, 5uM, 10uM and 25uM )of T6S1683 Transfer the dye.

[0053] (2) the aforesaid Positive control group ( PC ), negative control group ( NC ) and each treatment group in a culture cell box. 5% CO Contains 2 37℃ in 24 After incubation for 2 h, cells were harvested and quantified for real-time fluorescence. PCR Tests were performed and different doses of the inhibitors HaCat The effects of AIM2 on the activation of the AIM2 pathway in cells were observed. Figure 9 As shown in Fig. 1, compared with the positive control group, 10μM of T6S1683 but 5 Within the three dosage gradations HaCat Cellular IL-β It was found to reduce transcription levels the most.

[0054] (3) at the same time, 100 2x microscope field of view 10μM The cell status of the gradient was observed, and the results were Figure 10 The cell state of this group was close to that of the negative control group, with a very small proportion of transparent pyroblastic cells, high cell density, and cells closely attached to the wall.

[0055] (2) and (3) From the results, 10μM of T6S1683 but HaCat This may be the optimal dose to suppress AIM2 activity in cells.

[0056] (4) after that, 10uM of T6S1683 Processed in HaCat The expression status of AIM2 downstream proteins in cells was examined, and the results were Figure 11 and Figure 12 As shown in. 10uM of T6S1683 It was confirmed that the expression of caspase-1 downstream of AIM2 was slightly decreased by the treatment (WB test showed that PMA-stimulated THP1 (The cells were used as a positive reference for the protein.) Then, activated IL-1β To detect the expression of ELISA A reagent kit was used. 10uM of T6S1683 but IL-1β It has been shown to have an inhibitory effect on the secretion of

[0057] Working Example 5 T6S1683 Testing that highly efficiently inhibits AIM2 protein activity in psoriasis animal models In the present embodiment, DMSO is used. T6S1683 The powder is dissolved and the different amounts of powder are weighed and diluted with β-cyclodextrin. The final concentration of DMSO is 5% The result is as follows. T6S1683 The dosage gradation of 0mg / kg, 1mg / kg, 2.5mg / kg, 5mg / kg and 10mg / kg And each mouse 20g It is calculated as 1 The daily dosage is 0mg / kg, 1mg / kg, 2.5mg / kg, 5mg / kg and 10mg / kg become.

[0058] 7 Weeks of age C57 / BL6 Mice were selected and bred and modeled in an SPF-level environment, and the experimental groups were as follows: (a) 0mg / kg of T6S1683 Used IMQ and applied to the skin; (b) 1mg / kg of T6S1683 Used IMQ and applied to the skin; (c) 2.5mg / kg ofT6S1683 Used IMQ and applied to the skin; (d) 5mg / kg of T6S1683 Used IMQ and applied to the skin; ( e) 10mg / kg of T6S1683 Used IMQ and applied to the skin; (f) 0mg / kg of T6S1683 Using Vaseline and applying to skin; (g) 1mg / kg of T6S1683 Using Vaseline and applying to skin; (h) 2.5mg / kg of T6S1683 Using Vaseline and applying to skin; (i) 5mg / kg T6S1683 Using Vaseline and applying to skin; (j) 10mg / kg of T6S1683 Use Vaseline and apply to skin. (Note: (a), (b), (c), (d) and (e) The mice in the group IMQ of 1 Per day 62.5mg / 1 Use one. (f), (g), (h), (i) and (j) Mice in the control group were given Vaseline. 1 Per day 62.5mg / 1 Use one.

[0059] 6 Mice were shaved in groups to remove hair from the back and the bare skin area was 4 Adjust it to be square centimeters, and set the start date of the experiment. Day0 , end date Day6 It was set as follows. Day0 from Day5 Every day until 500 Microliters of drug solution are applied evenly to the mouse skin and allowed to absorb. 500 μl of solution in the morning 150 μl, at noon 200 μl, at night 150 The mixture is applied in divided doses of 1 μl. Day2 from Day5 to, T6S1683 After applying the inhibitor, (a), (b), (c), (d), (e) Each mouse in the group 62.5mgof IMQ Apply the following. (f), (g), (h), (i), (j) Each mouse in the group 62.5 mg The mice's skin was evaluated daily and Day 6 The mice were euthanized and skin tissue was collected for a series of tests.

[0060] IMQ The PASI evaluation results, skin phenotype, and skin pathology phenotype of the model mice were as follows: Figure 13 , Figure 14 , Figure 15 As shown in. 3 One dose T6S1683 teeth, IMQ They reduced the inflammatory phenotype in mice to different degrees. 5 mg / kg The effect of reducing the dosage is most prominent, 1 mg / kg There was no difference in dose effect.

[0061] The above Skin lymphocytes from each group of mice ( CD3 + TCRd + RORγT + of T A proportion of psoriasis-related inflammatory cells (this type of cell is the main effector cell mediated by the AIM2 pathway and aggravates psoriasis by secreting large amounts of IL-17 cellular factors) were isolated and subjected to flow cytometry analysis. T6S1683 of 5 mg / kg Processed IMQ mouse( Figure 16 and Figure 17 )and, T6S1683 Do not use IMQ Compared to a mouse, CD3 + TCRd + RORγT + It was shown that the proportion of

[0062] The transcription levels of AIM2 pathway genes were then measured in mouse skin. 5 mg / kg In mice from the same group IL-1β It was statistically shown that gene expression levels were significantly decreased ( Figure 18 ).this is, 5 mg / kg of T6S1683 AIM2 downstream in the skin of mice treated with IL-1β This indicates that the amount of inflammatory cytokines was suppressed and their content decreased, alleviating inflammatory symptoms.

[0063] Finally, to explain, The above The examples are for illustrating the technical solutions of the present invention, but are not limiting. Even with reference to the best embodiments, it should be understood that a person skilled in the art can modify or replace the technical solutions of the present invention with equivalents. They shall be included in the claims of the present invention without departing from the purpose and scope of the technical solutions of the present invention.

Claims

1. A method for producing a drug for inhibiting AIM2 protein activity using a compound represented by the following formula I: [Case 1] Represented, The method for producing a drug for inhibiting AIM2 protein activity using a compound, wherein the compound is in the form of a pharmaceutical salt.

2. The method for preparing a drug for inhibiting AIM2 protein activity using the compound according to claim 1, wherein the compound is in the form of a pharmaceutical acid addition salt.

3. The method for preparing a drug for inhibiting AIM2 protein activity using the compound according to claim 1, characterized in that the drug for inhibiting AIM2 protein activity is in the form of a capsule, tablet, oral administration, microparticles, injection, plug, spray, or ointment.

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