An anti-inflammatory small molecule compound, its synthesis method and application

Abstract

The present invention relates to an anti-inflammatory small molecule compound, its synthesis method and applications. The structural formula of the anti-inflammatory small molecule compound is as follows. The anti-inflammatory small molecule compound of the present invention has a significant therapeutic effect on inflammatory diseases (such as acute colitis), has a significant anti-inflammatory effect, and has good drug-likeness. At the animal level, it has no obvious toxicity to mice and has a certain protective effect on the kidneys, with good safety, and can be used to prepare drugs for treating inflammatory diseases (such as acute colitis). #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, relates to small molecule drug compounds, and specifically relates to a novel anti-inflammatory small molecule compound, its synthesis method and application. Background Art

[0002] Inflammatory bowel disease (IBD) is an idiopathic intestinal inflammatory disease involving the ileum, rectum, and colon, including ulcerative colitis (UC) and Crohn's disease (CD). Its pathogenesis involves immune system disorders, gut microbiota imbalance, genetic susceptibility, and environmental factors, etc. An overreaction of the immune system to gut microbes or food components can lead to chronic inflammation, while acute colitis can be caused by infections, drugs, ischemia, or radiotherapy, etc., and the mechanisms are usually related to overactivation of the immune response and impaired gut barrier, etc. Ulcerative colitis (UC) is a chronic, relapsing, immune-induced gastrointestinal disease that increases the risk of colon cancer in patients and has become a public health problem. The exact cause of UC remains unclear. However, the overactivation of the mucosal immune system, especially the innate immunity, along with a large number of immune cell recruitment and cell inflammatory factors, such as the secretion of interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor (TNF)-α in the colon, will further promote the onset and exacerbation of colitis.

[0003] Currently, the drugs for treating inflammatory bowel disease (IBD) mainly include anti-inflammatory drugs (such as 5-aminosalicylic acid (5-ASA) drugs) and steroid drugs (such as prednisone and beclomethasone). Although they are effective in treating mild to moderate inflammatory bowel disease (IBD), they also have some disadvantages. 5-ASA drugs have limited effects in some patients, especially poor treatment for Crohn's disease, and long-term use may cause side effects such as liver and kidney function damage. Steroid drugs can rapidly relieve acute inflammation, but due to their serious side effects that may occur with long-term use, such as osteoporosis, diabetes, weight gain, gastrointestinal bleeding, and immunosuppression, etc., they are not suitable for long-term dependence and cannot fundamentally change the disease process.

[0004] Therefore, it is necessary to develop more safe and effective anti-inflammatory drugs for the treatment of inflammatory bowel disease. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a novel anti-inflammatory small molecule compound for treating inflammatory diseases, such as colitis.

[0006] The technical solutions for achieving the above invention purpose are as follows.

[0007] In the first aspect, the present invention provides compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and the structural formula of the compound ZYZ311 is as follows:

[0008]

[0009] In the second aspect, the present invention provides the use of compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of an anti-inflammatory drug.

[0010] In the third aspect, the present invention provides the use of compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a drug for preventing and / or treating inflammatory diseases.

[0011] Among them, the inflammatory disease is preferably inflammatory bowel disease, more preferably colitis, and more preferably acute colitis or ulcerative colitis.

[0012] In the fourth aspect, the present invention provides a preparation method of compound ZYZ311, comprising the following steps:

[0013] (1) Thymol reacts with ethyl chloroacetate to obtain 2-(5-isopropyl-2-methylphenoxy)ethyl acetate;

[0014] (2) 2-(5-isopropyl-2-methylphenoxy)ethyl acetate reacts with hydrazine hydrate to obtain 2-(5-isopropyl-2-methylphenoxy)acetohydrazide;

[0015] (3) 2-(5-isopropyl-2-methylphenoxy)acetohydrazide reacts with 4-pyridinecarboxaldehyde to obtain a compound with the structure shown in formula (I);

[0016] The reaction formula is as follows:

[0017]

[0018] The present invention has the following beneficial effects:

[0019] The present invention synthesizes a novel anti-inflammatory small molecule compound by a simple and efficient method. This compound has a significant therapeutic effect on inflammatory diseases (such as acute colitis). It can inhibit the expression of nitric oxide (NO) and inflammatory factors (such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), etc.) in the supernatant of inflammatory cells, and reduce the protein expression levels of inflammation-related proteins p-NF-κB / NF-κB, COX-2, IL-6, TNF-α. It can improve the colon length and histological score of mice with acute colitis, inhibit the release of inflammatory factors such as IL-1β, IL-6, TNF-α, etc., reduce the expression level of the orphan receptor F4 / 80 (a marker of mature mouse macrophages) involved in cell adhesion, up-regulate the expression level of the tight junction protein ZO-1 involved in proliferation, differentiation, metastasis and material transport, and inhibit the expression of myeloperoxidase (MPO, also known as peroxidase, a marker of neutrophil activation), showing a significant anti-inflammatory effect.

[0020] Moreover, the anti-inflammatory small molecule compound of the present invention has good drug-likeness. At the animal level, it has no obvious toxicity to mice and has a certain protective effect on the kidneys, with good safety, and can be used to prepare drugs for treating inflammatory diseases (such as acute colitis). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the mass spectrometry for molecular weight characterization of compound ZYZ311.

[0022] Figure 2 It is a schematic diagram of the nuclear magnetic resonance hydrogen spectrum for structural confirmation of compound ZYZ311.

[0023] Figure 3 It is a schematic diagram of the nuclear magnetic resonance carbon spectrum for structural confirmation of compound ZYZ311.

[0024] Figure 4 It is a schematic diagram of the expression of nitric oxide (NO) and inflammatory factor IL-6 in the supernatant of the LPS-induced Raw264.7 cell inflammation model inhibited by compound ZYZ311.

[0025] Figure 5 It is a schematic diagram showing that compound ZYZ311 inhibits the protein expression of inflammation-related proteins p-NF-κB / NF-κB, COX-2, IL-6 in the LPS-induced Raw264.7 cell inflammation model by protein immunoblotting experiment.

[0026] Figure 6 It is a schematic diagram of the analysis curve for the drug-likeness study of compound ZYZ311.

[0027] Figure 7 It is a graph of the animal toxicity experiment results of compound ZYZ311.

[0028] Figure 8 Schematic diagram of body weight of C57BL / 6 mice with dextran sulfate sodium salt (DSS)-induced acute colitis model and kidney weight / body weight of mice.

[0029] Figure 9 Schematic diagram of compound ZYZ311 improving the colon length of C57BL / 6 mice with dextran sulfate sodium salt (DSS)-induced acute colitis model.

[0030] Figure 10 Schematic diagram of compound ZYZ311 improving the histological score of C57BL / 6 mice with dextran sulfate sodium salt (DSS)-induced acute colitis model.

[0031] Figure 11 Schematic diagram of compound ZYZ311 reducing the expression level of orphan receptor F4 / 80 (a marker of mature mouse macrophages) involved in cell adhesion in mice with dextran sulfate sodium salt (DSS)-induced acute colitis model.

[0032] Figure 12 Schematic diagram of compound ZYZ311 increasing the expression level of tight junction protein ZO-1 involved in proliferation, differentiation, metastasis and material transport in mice with dextran sulfate sodium salt (DSS)-induced acute colitis model.

[0033] Figure 13 Schematic diagram of compound ZYZ311 reducing the expression level of myeloperoxidase (MPO, also known as peroxidase, a marker of neutrophil activation) in mice with dextran sulfate sodium salt (DSS)-induced acute colitis model. Detailed implementation mode

[0034] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0035] The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention are only for the purpose of describing specific embodiments and are not intended to limit this invention. The term "and / or" used in this invention includes any and all combinations of one or more of the related listed items.

[0037] In addition, as used in this invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0038] In some embodiments of the present invention, there is involved a compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof. The structural formula of the compound ZYZ311 is as follows:

[0039]

[0040] The compound ZYZ311 of the present invention has a significant therapeutic effect on inflammatory diseases (such as acute colitis). It can inhibit the expression of nitric oxide (NO) and inflammatory factors (such as interleukin 6 (IL-6) and tumor necrosis factor (TNF-α), etc.) in the supernatant of inflammatory cells, and reduce the protein expression levels of inflammation-related proteins p-NF-κB / NF-κB, COX-2, IL-6, TNF-α; it can improve the colon length and histological score of mice with acute colitis, inhibit the release of inflammatory factors such as IL-1β, IL-6, TNF-α, etc., reduce the expression level of the orphan receptor F4 / 80 (a marker of mature mouse macrophages) involved in cell adhesion, up-regulate the expression level of the tight junction protein ZO-1 involved in proliferation, differentiation, metastasis, and material transport, and inhibit the expression of myeloperoxidase (MPO, also known as peroxidase, a marker of neutrophil activation), and has a significant anti-inflammatory effect. Moreover, the compound ZYZ311 of the present invention has good drug-likeness. At the animal level, it has no obvious toxicity to mice and has a certain protective effect on the kidneys, and has good safety.

[0041] Based on this, in some embodiments of the present invention, there is involved the use of the compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of anti-inflammatory drugs.

[0042] Some embodiments of the present invention also relate to the use of compound ZYZ311 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a medicament for preventing and / or treating inflammatory diseases.

[0043] In some of these embodiments, the inflammatory disease is inflammatory bowel disease.

[0044] In some of these embodiments, the inflammatory bowel disease is colitis.

[0045] In some of these embodiments, the colitis is acute colitis or ulcerative colitis.

[0046] Some embodiments of the present invention also relate to a method for preparing compound ZYZ311, comprising the following steps:

[0047] (1) Thymol reacts with ethyl chloroacetate to obtain ethyl 2-(5-isopropyl-2-methylphenoxy)acetate;

[0048] (2) Ethyl 2-(5-isopropyl-2-methylphenoxy)acetate reacts with hydrazine hydrate to obtain 2-(5-isopropyl-2-methylphenoxy)acetohydrazide;

[0049] (3) 2-(5-isopropyl-2-methylphenoxy)acetohydrazide reacts with 4-pyridinecarboxaldehyde to obtain a compound having the structure shown in formula (I);

[0050] The reaction formula is as follows:

[0051]

[0052] In some of the preferred embodiments, the reaction in step (1) is carried out under the action of a base, and the base is preferably potassium carbonate, and the molar ratio of the base to thymol is preferably 1-1.5:1.

[0053] In some of the preferred embodiments, the reaction in step (1) is carried out in an organic solvent, and the organic solvent is preferably acetone.

[0054] In some of the preferred embodiments, the reaction in step (1) is carried out under the protection of an inert gas.

[0055] In some of the preferred embodiments, the molar ratio of thymol to ethyl chloroacetate in step (1) is 1:1-1.5.

[0056] In some of the preferred embodiments, the temperature of the reaction in step (1) is 50°C - 70°C, and the time is 2 - 6 hours, preferably 3 - 5 hours.

[0057] In some of the preferred embodiments, the reaction in step (2) is carried out in an organic solvent, and the organic solvent is preferably ethanol.

[0058] In some of the preferred embodiments, the reaction in step (2) is carried out under the protection of an inert gas.

[0059] In some of the preferred embodiments, the molar ratio of 2-(5-isopropyl-2-methylphenoxy)ethyl ester to hydrazine hydrate in step (2) is 1:1.2 - 1.5.

[0060] In some of the preferred embodiments, the temperature of the reaction in step (2) is 70°C - 90°C and the time is 2 - 4 hours.

[0061] In some of the preferred embodiments, the reaction in step (3) is carried out in an organic solvent, and the organic solvent is preferably ethanol.

[0062] In some of the preferred embodiments, the reaction in step (3) is carried out under the protection of an inert gas.

[0063] In some of the preferred embodiments, the molar ratio of 2-(5-isopropyl-2-methylphenoxy)acetylhydrazine to 4-pyridinecarboxaldehyde in step (3) is 1:2 - 4.

[0064] In some of the preferred embodiments, the temperature of the reaction in step (3) is 70°C - 90°C and the time is 1 - 3 hours.

[0065] The present invention will be further described in detail below with reference to specific embodiments.

[0066] Example 1 Synthesis of an anti-inflammatory small molecule compound

[0067]

[0068] Step 1: Use a pipette to measure carvacrol (2.75 mL, 20 mmol) and place it in a 50 mL round-bottom flask. Add acetone (20 mL), stir to make it uniform, add potassium carbonate (3.0 g, 22 mmol), and then slowly dropwise add ethyl chloroacetate (2.4 mL, 22 mmol) under stirring. Fill the reaction system with nitrogen, and then react at 60°C for 4 hours. Monitor the reaction progress by thin-layer chromatography (TLC). After the reaction is completed, cool to room temperature, then concentrate the reaction mixture under reduced pressure until dry, extract with ethyl acetate, wash with water, collect the organic layer, dry with anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain a pale yellow oily liquid, which is 2-(5-isopropyl-2-methylphenoxy)ethyl ester and can be used for the next step without further purification.

[0069] Step 2: Weigh 2-(5-isopropyl-2-methylphenoxy)ethyl ester (3.54 g, 15 mmol) with an electronic analytical balance and place it in a 50 mL round-bottom flask. Add ethanol (20 mL), stir to dissolve it, then slowly drop hydrazine hydrate (1 mL, 20 mmol) dropwise under stirring. Charge nitrogen into the reaction system, and react the resulting reaction mixture at 80 °C for 3 hours. Monitor the reaction progress by TLC. After the reaction is completed, concentrate under reduced pressure to form a suspension, pour it into crushed ice, let it stand, a large amount of precipitate will precipitate out. Filter the precipitate, wash it with cold water, dry it, and then recrystallize it with ethanol to obtain a white powder, which is 2-(5-isopropyl-2-methylphenoxy)acetylhydrazine (3.33 g, 92%).

[0070] Step 3: Weigh 2-(5-isopropyl-2-methylphenoxy)acetylhydrazine (222 mg, 1 mmol) with an electronic analytical balance and place it in a 25 mL round-bottom flask. Add ethanol (10 mL), stir to dissolve it, then slowly drop 4-pyridinecarboxaldehyde (300 μL, 3 mmol) dropwise under stirring. Charge nitrogen into the reaction system, and react the resulting reaction mixture at 80 °C for 2 h. Monitor the reaction progress by TLC. After the reaction is completed, add 2 - 3 drops of acetic acid and mix well, concentrate under reduced pressure to form a suspension, pour it into crushed ice, let it stand, a large amount of precipitate will precipitate out. Filter the precipitate, wash it with cold water, dry it, and then recrystallize it with ethanol to obtain an anti-inflammatory small molecule compound named ZYZ311, 280 mg, with a yield of 90%.

[0071] Nuclear magnetic resonance spectrum: 1 H NMR (500 MHz, CDCl3) δ 9.91 (s, 1H), 8.65 (s, 2H), 8.29 (s, 1H), 7.66–7.38 (m, 2H), 7.10 (dd, J = 14.4, 7.7 Hz, 1H), 6.91–6.76 (m, 1H), 6.70 (s, 1H), 5.18 (s, 1H), 4.70 (s, 1H), 2.29 (s, 3H), 1.21 (dd, J = 15.3, 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 171.71, 165.37, 156.52, 155.19, 150.40, 148.79, 147.99, 146.94, 142.91, 140.76, 131.15, 130.93, 124.76, 123.89, 121.50, 121.02, 120.29, 119.37, 110.77, 110.48, 67.72, 66.22, 34.06, 24.14, 24.09, 16.09, 15.99.

[0072] Its molecular weight characterization is as Figure 1As shown, the chemical structure characterization is as Figure 2 and Figure 3 shown.

[0073] Example 2 detected the effects of ZYZ311 on the expression of nitric oxide (NO) and inflammatory factor IL-6 in the supernatant of the LPS-induced Raw264.7 cell inflammation model

[0074] Method: Raw264.7 cells were cultured to the logarithmic growth phase at 37°C and 5% CO2. When the cell density reached about 80-90%, the cells were pretreated with ZYZ311 (20, 40, 60, 80, 100, 120, 140, 160, 180, 200 μM) and carvacrol (200 μM) for 1 h, and then stimulated with LPS for 24 h. The supernatant was collected, and the NO level in the supernatant was detected using Griess reagent, and the secretion level of IL-6 inflammatory factor was detected by ELISA.

[0075] The results are as Figure 4 shown. ZYZ311 was able to down-regulate the secretion of nitric oxide (NO) and inflammatory factor IL-6 in the supernatant of the LPS-induced Raw264.7 cell inflammation model, while carvacrol had no obvious effect at a concentration of 200 μM.

[0076] Example 3 Protein extraction and Western blot were used to investigate the effects of ZYZ311 on the expression of inflammation-related proteins in the LPS-induced Raw264.7 cell inflammation model

[0077] Method: Raw264.7 cells were cultured at 37 °C and 5% CO2 until the logarithmic growth phase. When the cell density reached about 80 - 90%, the cells were pretreated with ZYZ311 (120 μM) for 1 h, and then stimulated with LPS for different times (0, 0.5, 1, 2, 4, 6 h). The cells were collected and lysed on ice for 1 h in Ripa lysis buffer. Then the lysate was centrifuged at 4 °C for 10 minutes (13,000 rpm). The protein concentration in the supernatant was detected by BCA protein assay (Thermo, Waltham, MA). Equal amounts of proteins were separated by 12% or 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) using a semi-dry transfer system (Bio-rad, Hercules, CA). Proteins were detected using specific antibodies against β-actin, pp65, p65, COX-2, and IL-6 overnight at 4 °C, and then incubated with an HRP-conjugated secondary antibody for 1 h at 37 °C. All antibodies were diluted in PBST containing 1% BSA. Enhanced chemiluminescence reagent (Beyotime, Jiangsu, China) was used to detect HRP on the immunoblot. The signals were analyzed using an ECL chemiluminescence detection system (Tanon, Nanjing, China).

[0078] The results, as Figure 5 shown, ZYZ311 could inhibit the protein expression of inflammation-related proteins p-NF-κB / NF-κB, COX-2, and IL-6 in the LPS-induced Raw264.7 cell inflammation model.

[0079] Example 4 ADMET Prediction of Anti-inflammatory Small Molecule Compound ZYZ311

[0080] Method: Use Discovery Studio software to calculate the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity of drugs) properties, namely aqueous solubility at 25 degrees Celsius; Blood brain barrier penetration (BBB); Cytochrome P4502D6 inhibition; hepatotoxicity; human intestinal absorption (HIA); plasma protein binding. Through Small Molecules|Calculate Molecular Properties|ADMET Descriptors in Discovery Studio, with the parameter selection set to the default settings, the ADMET properties of ZYZ311 will be calculated.

[0081] The results are as Figure 6 shown. This ADMET plot is a two-dimensional graph of ADMET_PSA_2D vs ADMET_AlogP98, which respectively represent the 95% and 99% confidence intervals of the blood-brain barrier permeability (BBB) model, and the 95% and 99% confidence intervals of the human intestinal absorption (HIA) model. The experimental results show that ZYZ311 has good ADMET properties and good drug-likeness.

[0082] Example 5 Animal Toxicity Experiment of ZYZ311

[0083] Method: Randomly select 20 C57BL / 6J mice at 6 weeks of age and weighing 22 - 26 g in good condition, and randomly divide them into a blank group and a drug administration group (n = 10). Administer the maximum dose of 2 g / kg / once by gavage and continuously observe for 14 days. As Figure 7 shown, the experimental results show that none of the mice died within 14 days after drug administration, and there were no abnormalities in the food intake, water intake, spontaneous activities, etc. of the mice. The LD50 of ZYZ311 > 2 g / kg.

[0084] Example 6 Establishment and Histological Analysis of a Dextran Sulfate Sodium Salt (DSS)-Induced Acute Colitis Model in C57BL / 6 Mice

[0085] Method: Induce acute colitis by administering DSS in drinking water. The specific experimental steps are as follows:

[0086] Sixty male C57BL / 6J mice were randomly assigned to a control group, a DSS treatment group, ZYZ311 treatment groups (50, 100, 200 mg / kg), and a 5-ASA treatment group (40 mg / kg) (n = 10). Among them, ZYZ311 and 5-ASA were dissolved in an aqueous solution of 0.5% carboxymethyl cellulose sodium (CMC-Na) to obtain test drug solutions of ZYZ311 at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5-ASA at 0.4 mg / mL for gavage administration; the control group and the DSS treatment group were given the same dose of 0.5% CMC-Na aqueous solution.

[0087] Six days after gavage administration, the mice began to drink drinking water containing 3% (w / v) DSS for 6 days. Gavage was performed once a day and the body weight of the mice was recorded.

[0088] Six days after giving DSS drinking water, the mice were euthanized by cervical dislocation, and the colon and kidneys of each mouse were removed, and the colon length and kidney weight / body weight of the mouse were statistically analyzed. Part of the colon was fixed in 10% buffered formalin and embedded in paraffin. The sections were stained with H&E according to the standard protocol. The histological evaluation grading of the H&E-stained colon sections was as follows: 0, no signs of inflammation; 1, low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte infiltration, moderate fibrosis, high vascular density, thickening of the colon wall, moderate loss of goblet cells, focal crypt loss; 4, transmural infiltration, massive loss of goblet cells, extensive fibrosis, and diffuse loss of crypts.

[0089] As Figure 8 shown, on the 6th day, the body weight of the mice in the model group decreased sharply, and the body weight of the mice in the ZYZ311 administration group did not change significantly, demonstrating that ZYZ311 can alleviate the weight loss caused by colitis.

[0090] As Figure 9 shown, compared with the normal control group, the colon in the DSS model group was significantly shortened, while ZYZ311 could effectively increase the colon length.

[0091] As Figure 10 shown, ZYZ311 could effectively reduce inflammatory infiltration, erosion and deformation of crypts, loss of glandular epithelial cells, and reduce the histological score.

[0092] Example 7 Immunohistochemical Detection

[0093] In this example, immunohistochemistry was used to detect the expression of the orphan receptor F4 / 80 (a marker of mature mouse macrophages) involved in cell adhesion, the tight junction protein ZO-1 involved in proliferation, differentiation, metastasis and material transport, and myeloperoxidase (MPO, also known as peroxidase, a marker of neutrophil activation).

[0094] Method: The colon tissue treated in Example 6 was fixed in 4% paraformaldehyde, and then dehydrated, cleared, infiltrated with wax and sectioned. The paraffin-embedded slides were deparaffinized, rehydrated and washed in 1% PBS-Tween. Then it was treated with 3% hydrogen peroxide and blocked with 10% goat serum at 37°C for 1 hour. The slides were incubated with the primary antibody in PBS containing 1% BSA (1:50) at 37°C for 1 hour. Biotinylated anti-rabbit secondary antibody was added and incubated at room temperature for 1 h. Streptavidin-HRP was added, and after 40 minutes, the sections were stained with DAB substrate and counterstained with hematoxylin.

[0095] As Figures 11 - 13 shown, ZYZ311 can reduce the expression of F4 / 80 and MPO and increase the expression of ZO-1.

[0096] The above results prove that the small molecule compound ZYZ311 of the present invention has a significant therapeutic effect on inflammatory diseases such as acute colitis, is safe and effective, and can be used to prepare drugs for treating acute colitis.

[0097] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of compound ZYZ311 or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug, characterized in that, The structural formula of the compound ZYZ311 is as follows:

2. Use of the compound ZYZ311 or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of a drug for preventing and / or treating inflammatory diseases.

3. The application according to claim 2, wherein The inflammatory disease is inflammatory bowel disease.

4. The application according to claim 3, wherein The inflammatory disease is colitis.

5. The application according to claim 4, wherein The colitis is acute colitis or ulcerative colitis.

6. The application according to any one of claims 1-5, characterized in that, The compound ZYZ311 or a pharmaceutically acceptable salt thereof can inhibit the expression of nitric oxide, inflammatory factors IL-6 and TNF-α in inflammatory cells.

7. The application according to any one of claims 1-5, characterized in that, The compound ZYZ311 or a pharmaceutically acceptable salt thereof can inhibit the protein expression of inflammation-related proteins p-NF-κB / NF-κB, COX-2, IL-6 and TNF-α in inflammatory cells.

8. The application according to any one of claims 1-5, characterized in that, The compound ZYZ311 or a pharmaceutically acceptable salt thereof can inhibit the expression of orphan receptor F4 / 80 and myeloperoxidase.

9. The application according to any one of claims 1-5, characterized in that, The compound ZYZ311 or a pharmaceutically acceptable salt thereof can increase the expression of tight junction protein ZO-1.

10. A method for preparing the compound ZYZ311 described in claim 1, characterized in that, It includes the following steps: (1) Thymol reacts with ethyl chloroacetate to obtain 2-(5-isopropyl-2-methylphenoxy)ethyl acetate; (2) 2-(5-isopropyl-2-methylphenoxy)ethyl acetate reacts with hydrazine hydrate to obtain 2-(5-isopropyl-2-methylphenoxy)acetohydrazide; (3) 2-(5-isopropyl-2-methylphenoxy)acetohydrazide reacts with 4-pyridinecarboxaldehyde to obtain a compound with the structure shown in formula (I); The reaction formula is as follows:

11. The method for preparing compound ZYZ311 according to claim 10, characterized in that, The reaction in step (1) is carried out under the action of a base; The reaction in step (1) is carried out in an organic solvent; The reaction in step (1) is carried out under the protection of an inert gas; The molar ratio of thymol to ethyl chloroacetate in step (1) is 1:1 - 1.5; The temperature of the reaction in step (1) is 50°C - 70°C, and the time is 2 - 6 hours.

12. The method for preparing compound ZYZ311 according to claim 11, characterized in that, The base is potassium carbonate.

13. The preparation method of compound ZYZ311 according to claim 11, characterized in that, The molar ratio of the base to thymol is 1 - 1.5:

1.

14. The preparation method of compound ZYZ311 according to claim 11, characterized in that, The reaction in step (1) is carried out in an organic solvent, and the organic solvent is acetone.

15. The preparation method of compound ZYZ311 according to claim 10, characterized in that, The reaction in step (2) is carried out in an organic solvent; The reaction in step (2) is carried out under the protection of an inert gas; The molar ratio of 2-(5-isopropyl-2-methylphenoxy)ethyl acetate to hydrazine hydrate in step (2) is 1:1.2 - 1.5; The temperature of the reaction in step (2) is 70°C - 90°C, and the time is 2 - 4 hours.

16. The preparation method of compound ZYZ311 according to claim 15, characterized in that, The reaction in step (2) is carried out in an organic solvent, and the organic solvent is ethanol.

17. The preparation method of compound ZYZ311 according to any one of claims 10-16, characterized in that, The reaction in step (3) is carried out in an organic solvent; The reaction in step (3) is carried out under the protection of an inert gas; The molar ratio of 2-(5-isopropyl-2-methylphenoxy)acetohydrazide to 4-pyridinecarboxaldehyde in step (3) is 1:2 - 4; The temperature of the reaction in step (3) is 70°C - 90°C, and the time is 1 hour - 3 hours.

18. The preparation method of compound ZYZ311 according to claim 17, characterized in that, The reaction in step (3) is carried out in an organic solvent, and the organic solvent is ethanol.

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