Application of piperazine amide derivative in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention discloses an application of a piperazine amide derivative in preparation of a medicine for treating inflammatory bowel disease. In a DSS-induced mouse inflammatory bowel disease model, by oral administration of the piperazine amide derivative ID230301A-1, ID240507A-1 or RD240404, body weight loss, hematochezia and diarrhea caused by modeling can be remarkably relieved, and colon shortening, intestinal wall thinning and brittleness increase caused by modeling can be improved. According to the present invention, the immunoblotting analysis is performed on the colon tissue of the laboratory mouse, and the result shows that the ID230301A-1, the ID240507A-1 or the RD240404 can regulate and control the expression of the STAT3 signal-related protein; the medicine can be used for preparing medicines for treating inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a piperazineamide derivative in the preparation of a drug for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is an autoimmune disease. Common symptoms are diarrhea and bloody stools, and long-term inflammation may affect organs throughout the body. The specific mechanism of IBD is still unclear. It is generally believed that genetic and environmental factors cause changes in the patient's digestive tract microbial composition and damage to the integrity of the intestinal barrier. At the same time, patients have immune system disorders, an imbalance between effector cells and regulatory cells in the intestinal mucosa, and mistakenly attack their own healthy cells, leading to persistent inflammation. Because IBD is a chronic disease that is difficult to cure, and the peak age of onset is 14-49 years old, it may affect the patient's life, so it is becoming a health problem that cannot be ignored.

[0003] Modern scientific research suggests that the etiology of IBD is based on factors such as genes, gut microbiota, and lifestyle. Large-scale genetic association analyses have uncovered numerous genes associated with IBD. These genes are generally involved in immune system regulation, the intestinal mucosal barrier, and the gut microbiome. For example, genes such as NOD2, IL23R, ATG16L1, and IRGM have been found to be closely associated with the risk of IBD. The human leukocyte antigen (HLA) gene family has also been implicated in the development of IBD. Specific HLA genotypes have been linked to susceptibility to IBD, particularly CD. Although IBD is not caused by a single gene mutation in most cases, some rare single-gene genetic disorders, such as IL-10 and IL-10 receptor deficiencies, have also been linked to the onset of IBD. These genetic disorders can lead to immune system dysfunction, thereby increasing the risk of IBD. With accelerating urbanization, the proportion of high-fat, high-sugar, processed foods, and foods containing polyunsaturated fatty acids in residents' daily diets has increased. This may lead to alterations in the gut microbiome and dysregulation of the gut immune system, further increasing the risk of IBD. In contrast, a diet rich in dietary fiber, fruits and vegetables, and omega-3 fatty acids, as well as fish and nuts, may help reduce the risk of IBD. Chemicals and pollutants in the environment may adversely affect the intestinal immune system, thereby increasing the risk of IBD. Invasion of the body by certain specific microorganisms will cause or worsen IBD. For example, the relationship between intestinal infection and the onset and recurrence of IBD has been confirmed by research. Nicotine intake can increase the incidence of IBD and CD, increasing the prevalence. Therefore, smoking may have different effects on the condition and progression of IBD patients. Some drugs, such as nonsteroidal anti-inflammatory drugs and antibiotics, may be associated with the onset or worsening of IBD. Psychological stress and pressure may lead to immune system disorders, thereby affecting intestinal immune function and increasing the risk of IBD.

[0004] Currently, IBD cannot be completely cured, so current treatment strategies and goals are aimed at alleviating symptoms, controlling inflammation, preventing complications, improving quality of life, and minimizing relapses. Symptom relief: Through medication and lifestyle adjustments, symptoms such as diarrhea, abdominal pain, and bloody stools are alleviated, improving quality of life. Anti-inflammatory drugs, immunomodulators, and biologics are used, while monitoring the patient's response and choosing safe and effective treatment regimens to minimize the adverse effects of medication side effects. Intestinal inflammation is controlled to minimize tissue damage. Early diagnosis, regular monitoring, and proactive treatment are used to prevent IBD-related complications such as intestinal strictures, perforation, and colon cancer. After the acute and chronic phases of remission, continued medication and dietary and lifestyle adjustments are used to alleviate symptoms, enhance immunity, prolong remission, and reduce relapses. Comprehensive interventions such as psychological support, nutritional counseling, and exercise are used to help patients adjust their mindset and improve their quality of life. Treatment is personalized based on genetic testing, medical imaging, and biomarkers. Summary of the Invention

[0005] In response to the problems of poor efficacy, severe side effects and easy recurrence of existing drugs for treating inflammatory bowel disease, the present invention provides a piperazinamide derivative or a biologically acceptable salt thereof with known pharmacokinetic and toxicological effects, few side effects, low price and good development and application prospects for use in the preparation of drugs for treating inflammatory bowel disease.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions:

[0007] Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a drug for treating inflammatory bowel disease, wherein the structure of the piperazinamide derivative is as follows:

[0008]

[0009] Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a STAT3 protein inhibitor, wherein the structure of the piperazinamide derivative is as follows:

[0010]

[0011] Furthermore, the drug is a drug prepared from one or a mixture of any proportion of ID230301A-1, ID240507A-1, RD240404 and biologically acceptable salts thereof in the form of an injection, tablet, powder injection, granule, capsule, oral solution, ointment, or cream.

[0012] The oral liquid is prepared by dissolving a piperazinamide derivative or a biologically acceptable salt thereof in a mixed solvent consisting of an organic solvent, a viscosity enhancer and an emulsifier to form a solution with a concentration of 1 to 10 mg / mL.

[0013] In the mixed solvent, the organic solvent accounts for 2% to 5% by volume; the emulsifier accounts for 10% to 15%; and the viscosity enhancer accounts for 80% to 88%; the organic solvent is DMSO, the viscosity enhancer is a 0.1wt% to 1wt% sodium carboxymethyl cellulose aqueous solution, and the emulsifier is Cremophor EL.

[0014] Furthermore, the inflammatory bowel disease refers to acute enteritis and chronic enteritis.

[0015] Furthermore, the piperazinamide derivative or a biologically acceptable salt thereof is administered alone or in combination with other pharmaceutically acceptable drugs.

[0016] Furthermore, the biologically acceptable salt refers to a biologically acceptable salt formed by ID230301A-1, ID240507A-1 or RD240404 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

[0017] The effects of ID230301A-1, ID240507A-1 and RD240404 were observed in an animal model of inflammatory bowel disease, and it was found that ID230301A-1, ID240507A-1 and RD240404 effectively inhibited the clinical symptoms of inflammatory bowel disease.

[0018] The classic animal model of inflammatory bowel disease (IBD) uses DSS (5g / kg) orally to induce enteropathy. This model was used to investigate the therapeutic effects of ID230301A-1, ID240507A-1, and RD240404 on IBD. Results showed that ID230301A-1, ID240507A-1, and RD240404 effectively inhibited DSS-induced enteropathy symptoms and colonic changes.

[0019] Immunoblotting analysis was performed on colon tissues of mice in an animal model to explore the molecular and signaling mechanisms by which ID230301A-1, ID240507A-1, and RD240404 inhibit inflammatory bowel disease.

[0020] The results showed that DSS induction could cause significant enrichment of STAT3 signals in mouse colon tissue, while ID230301A-1, ID240507A-1 and RD240404 could significantly downregulate the expression of STAT3 signals.

[0021] The above studies found that ID230301A-1, ID240507A-1 and RD240404 have excellent therapeutic activity against inflammatory bowel disease. This activity is closely related to the inhibition of STAT3 signaling pathway activation by ID230301A-1, ID240507A-1 and RD240404. Therefore, they can be used as active ingredients to prepare drugs for the treatment of inflammatory bowel disease. In order to further improve the efficacy, ID230301A-1, ID240507A-1, RD240404 can also be compounded with other active ingredients to prepare drugs for the treatment of inflammatory bowel disease. When preparing these drugs, pharmaceutically acceptable carriers can also be added.

[0022] ID230301A-1, ID240507A-1, and RD240404 are STAT3 protein inhibitors. Extensive research data indicates that STAT3 plays a crucial role in the pathogenesis of tumors and autoimmune diseases. Although no STAT3 protein inhibitors are currently marketed, their development is booming. Numerous clinical trial data indicate that STAT3 protein inhibitors have significant therapeutic potential in the treatment of tumors and autoimmune diseases such as psoriasis and ulcerative colitis. This study found that ID230301A-1, ID240507A-1, and RD240404 significantly reduced model-induced weight loss, hematochezia, and diarrhea, and improved model-induced colon shortening, intestinal wall thinning, and increased fragility. Therefore, ID230301A-1, ID240507A-1, and RD240404 downregulate inflammatory cytokine-induced STAT3 expression in intestinal epithelial cells, inhibiting the secretion of a range of inflammatory factors and thereby suppressing the inflammatory response in IBD. Preclinical studies have shown that ID230301A-1, ID240507A-1, and RD240404 are more effective than tofacitinib in treating IBD, and have fewer toxic side effects with long-term use, demonstrating good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 To observe the colon morphology of the therapeutic effects of ID230301A-1 and ID240507A-1 on the acute model of inflammatory bowel disease in mice;

[0024] Figure 2 To observe the colon morphology of the therapeutic effect of RD240404 on the acute model of inflammatory bowel disease in mice;

[0025] Figure 3 To observe the colon morphology of the therapeutic effect of ID230301A-1 on the chronic inflammatory bowel disease mouse model;

[0026] Figure 4 Colon histological observation of a chronic inflammatory bowel disease mouse model treated with ID230301A-1;

[0027] Figure 5 Body weight changes in acute inflammatory bowel disease mouse models treated with ID230301A-1 and ID240507A-1;

[0028] Figure 6 The body weight changes of acute inflammatory bowel disease mouse model treated with RD240404;

[0029] Figure 7 To investigate the body weight changes in a chronic inflammatory bowel disease mouse model treated with ID230301A-1;

[0030] Figure 8 Changes in DAI scores in the acute inflammatory bowel disease mouse model treated with ID230301A-1 and ID240507A-1;

[0031] Figure 9 The changes in DAI scores in the acute inflammatory bowel disease mouse model treated with RD240404;

[0032] Figure 10 To investigate the changes in DAI scores in a chronic inflammatory bowel disease mouse model treated with ID230301A-1;

[0033] Figure 11 The immunoblotting results of colon tissues of mice in the ID230301A-1 and ID240507A-1 acute inflammatory bowel disease models are shown;

[0034] Figure 12 The immunoblotting results of colon tissues of mice in each group in the RD240404 acute inflammatory bowel disease model are shown;

[0035] Figures 1 to 12 In the mean, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0036] In order to make the technical purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention is further described below in conjunction with specific embodiments. However, the implementation is intended to explain the present invention and should not be understood as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the field or the product instructions shall be followed.

[0037] For details on the preparation of ID230301A-1, please refer to patent application CN2023106821186.

[0038] The structural formula of ID240507A-1 is as follows:

[0039]

[0040] The name of compound ID240507A-1 is (1-methyl-6-(5-((4-(trifluoromethyl)phenyl)ethynyl)pyrazin-2-yl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone,

[0041] Its synthetic route is as follows:

[0042]

[0043] Step 1. (6-bromo-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 3)

[0044] Compound 1 (2.0 g, 10.21 mmol, 1.0 eq), compound 2 (2.92 g, 10.63 mmol, 1.0 eq), HBTU (4.85 g, 12.57 mmol, 1.2 eq), and DIEA (4.15 g, 31.38 mmol, 3.0 eq) were dissolved in 30 mL of DMF and stirred at room temperature for 3 hours. The reaction was monitored by TLC for completion. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL*3). The organic phase was dried and spin-dried, then purified by slurrying with 15 mL of ethyl acetate and filtered. The solid was collected and dried in an 80°C oven to obtain 4.03 g of compound 3 as a white solid, with a yield of 82.3%.

[0045] 1 H NMR(CDCl3,300MHz)δ:8.25(d,J=8Hz,1H),7.78(d,J=8Hz,1H),7.53(m,2H),7.22(d,J=8Hz,1H),7.18(d,J=8H z,2H),6.86(d,J=8Hz,1H),4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),2.85(s,3H),3.04-2.96(m,5H).

[0046] Step 2. (1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0047] Compound 3 (1.0 g, 1.96 mmol, 1.0 eq), compound 4 (0.995 g, 3.92 mmol, 2.0 eq), AcOK (0.58 g, 5.88 mmol, 3.0 eq), and Pd(dppf)Cl2 (71 mg, 0.098 mmol, 0.05 eq) were dissolved in 30 mL of DMF and stirred at 100°C for 3 hours. Completion of the reaction was monitored by TLC. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL x 3). The organic phase was dried and spin-dried, and then filtered through a column with petroleum ether / ethyl acetate (10 / 1 to 1 / 1) to obtain 0.57 g of compound 5 as a white solid, with a yield of 52.3%.

[0048] Step 3. (6-(5-bromopyrazin-2-yl)-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 7)

[0049] Compound 5 (0.5 g, 0.90 mmol, 1.0 eq), compound 6 (0.235 g, 0.99 mmol, 1.1 eq), Na2CO3 (0.142 g, 1.35 mmol, 1.5 eq), and Pd(dppf)Cl2 (33 mg, 0.045 mmol, 0.05 eq) were dissolved in 20 mL of DMSO and 5 mL of water. The mixture was stirred at 80°C for 3 hours and the reaction was monitored for completion by TLC. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL x 3). The organic phase was dried and spin-dried, and then filtered through a column with PE / EA (8 / 1 to 1 / 1) to obtain 0.35 g of compound 7 as a white solid, with a yield of 66.0%.

[0050] 1H NMR(CDCl3,300MHz)δ:8.604-8.601(d,J=1.2Hz,2H),8.494-8.491(d,J=1.2Hz,2H),8.25(d,J=8Hz,1H),7.78(d,J=8Hz,1H),7.22(d,J =8Hz,1H),7.18(d,J=8Hz,2H),6.86(d,J=8Hz,1H),4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),2.85(s,3H),3.04-2.96(m,5H).

[0051] Step 4. (1-methyl-6-(pyridin-3-ylethynyl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone(ID240507A-1)

[0052] Compound 7 (0.30 g, 0.50 mmol, 1.0 eq), compound 8 (0.13 g, 0.76 mmol, 1.5 eq), Pd(PPh3)2Cl2 (3.58 mg, 0.05 mmol, 0.01 eq), CuI (7.46 mg, 0.15 mmol, 0.03 eq), and triethylamine (850 mg, 2.50 mmol, 5.0 eq) were dissolved in 30 mL of DMF under nitrogen atmosphere. The mixture was stirred at 80°C for 12 hours. TLC monitoring showed complete reaction of the starting materials with the formation of new spots. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL x 3). The organic phase was dried and spun down to dryness. The mixture was purified by slurrying with 25 mL of ethyl acetate and filtered. The solid was collected and dried in an oven at 80°C to obtain 290 mg of Compound ID240507A-1 as a yellow solid in an 84.0% yield.

[0053] 1 H NMR(CDCl3,400MHz)δ:8.605-8.602(d,J=1.2Hz,2H),8.496-8.493(d,J=1 .2Hz,2H),7.856-7.808(m,4H),7.673-7.652(m,1H),7.482-7.477(m,1H) ,7.297-7.275(m,1H),7.041-6.975(m,2H),6.90(s,1H),4.777-4.711(q, J=8.8Hz,4H),3.729(s,3H),3.657(brs,4H),3.478(s,2H),2.421(s,2H).

[0054] The preparation process of RD240404 is detailed in patent application CN2024107072131. The hydrogen spectrum data of RD240404 are as follows 1 HNMR(DMSO-d6,400MHz)δ:8.52(d,J=5.90Hz,2H),8.43(s,1H),8.19(m,2H),7.34-7.21(d,J=8.30Hz,1H),8 .01(d,J=8.44Hz,1H),7.63(d,J=8.32Hz,1H),7.59(m,1H),7.52(m,1H),7.46(d,J=8.56Hz,2H),7.31(d,J= 5.57Hz,2H),7.03(d,J=8.44Hz,1H),6.98(d,J=8.56Hz,2H),6.91(t,J=6.02Hz,1H),5.19(s,2H),4.36(d,J =5.92Hz,2H),4.09(d,J=5.66Hz,2H),3.31(m,4H),2.43(m,4H),2.15(t,J=5.66Hz,2H),1.87-1.70(m,15H).

[0055] Example 1: Observation of colon morphology and length of the therapeutic effects of ID230301A-1 and ID240507A-1 on acute inflammatory bowel disease mouse model

[0056] The experimental animals were 6-week-old female C57BL6 mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and housed in IVC cages. After the mice adapted for 1 week, they were randomly divided into four groups: a normal mouse blank control group (n=6) was gavaged with normal saline once a day, 0.1 mL each time; a model control group (n=5) was gavaged with DSS (dextran sulfate sodium salt) once a day, 5.0 g / kg each time. The solvent used for the DSS solution was pure water. DSS was dissolved in pure water and ultrasonicated for 0.5 h. The DSS concentration was 500 mg / mL; an experimental group (n=5) was gavaged with DSS once a day, 5.0 g / kg each time, and at the same time, ID230301A-1 or ID240507A-1 was gavaged once a day, 40 mg / kg each time. The preparation process of the oral solution of ID230301A-1 was as follows: 2% DMSO, 10% Cremophor EL-35 and 88% CMCNa aqueous solution were mixed evenly to obtain a mixed solvent, the concentration of the CMCNa aqueous solution was 0.5wt%, ID230301A-1 or ID240507A-1 was dissolved in the above mixed solvent, and ultrasonication was performed for 2 hours. The prepared concentration of ID230301A-1 or ID240507A-1 was 8 mg / mL; in the positive control drug tofacitinib group (n=5), DSS was gavage once a day at 5.0 g / kg each time, and tofacitinib was gavage once a day at 40 mg / kg each time. The solvent used for the tofacitinib solution was a 0.5wt% CMCNa aqueous solution. Tofacitinib was dissolved in the CMCNa aqueous solution and ultrasonication was performed for 2 hours. The prepared concentration of tofacitinib was 8 mg / mL.

[0057] This experiment used a simultaneous modeling and drug administration approach for an 8-day experimental period. DSS was administered by gavage at a concentration of 5.0 g / kg on days 0-5 of the modeling phase, and water was given on days 6-8. The blank group had free access to water throughout the entire process, while the drug-treated group received gavage starting on day 0. Mice were weighed daily and sacrificed on day 8. Colon samples were collected and measured for length. Samples were then placed in cryovials and frozen at -80°C for subsequent experiments such as western blotting.

[0058] The results are as follows Figure 1 As shown in the figure, compared with the blank control group, the colon length of DSS-induced mice in the model group was shortened, the intestinal wall became thinner, and the feces in the intestine were obviously unformed; compared with the model group, the colon length of ID230301A-1 and ID240507A-1 groups was significantly longer, and the feces in the intestine were almost formed, indicating that they had better drug efficacy; the colon length of the positive drug group was between the DSS model group and the drug-treated group, and the feces in the colon were formed, indicating that they had certain drug efficacy, but the drug efficacy was not as good as that of ID230301A-1 and ID240507A-1.

[0059] Example 2: Observation of colon morphology and length of the therapeutic effect of RD240404 on an acute model of inflammatory bowel disease in mice

[0060] The experimental animals were 6-week-old female C57BL6 mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and housed in IVC cages. After acclimation for one week, the mice were randomly divided into four groups: a normal mouse blank control group (n=6) was gavaged with normal saline once a day, 0.1 mL each time; a model control group (n=6) was gavaged with DSS once a day, 5.0 g / kg each time; and an experimental group (n=6) was gavaged with DSS once a day, 5.0 g / kg each time. The preparation method was similar to that in Example 1. At the same time, RD240404 was gavaged once a day, 40 mg / kg each time. The preparation process of RD240404 oral solution was as follows: 2% DMSO, 10% Cremophor, and 10% Cremophor were added to the mixture by volume. EL-35 and 88% CMCNa aqueous solution were evenly mixed to obtain a mixed solvent, the concentration of the CMCNa aqueous solution was 0.5 wt %, RD240404 was dissolved in the above mixed solvent, and ultrasonicated for 2 h. The prepared concentration of RD240404 was 8 mg / mL; in the positive control drug tofacitinib group (n=6), DSS was gavage once a day at 5.0 g / kg each time, and tofacitinib was gavage once a day at 40 mg / kg each time. The preparation method was referred to Example 1.

[0061] This experiment used a simultaneous modeling and drug administration approach for an 8-day experimental period. DSS was administered by gavage at a concentration of 5.0 g / kg on days 0-5 of the modeling phase, and water was given on days 6-8. The blank group had free access to water throughout the entire process, while the drug-treated group received gavage starting on day 0. Mice were weighed daily and sacrificed on day 8. Colon samples were collected and measured for length. Samples were then placed in cryovials and frozen at -80°C for subsequent experiments such as western blotting.

[0062] The results are as follows Figure 2 As shown in the figure, compared with the blank control group, the colon length of DSS-induced mice in the model group was shortened, the intestinal wall became thinner, and the feces in the intestine were obviously unformed; compared with the model group, the colon length of the RD240404 group was significantly longer, and the feces in the intestine were almost formed, indicating that it had a good drug efficacy; the colon length of the positive drug group was between the DSS model group and the drug-treated group, and the feces in the colon were formed, indicating that it had a certain drug efficacy, but the drug efficacy was not as good as RD240404.

[0063] Example 3: Observation of colon morphology and length of the therapeutic effect of ID230301A-1 on a chronic inflammatory bowel disease mouse model

[0064] The experimental animals were 6-week-old female C57BL6 mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and kept in IVC cages. After the mice adapted for 1 week, they were randomly divided into four groups: a normal mouse blank control group (n=10) was gavaged with normal saline once a day, 0.1 mL each time; a model control group (n=10) was gavaged with DSS (dextran sulfate sodium salt) once a day, 5.0 g / kg each time; an experimental group 1 (n=10) was gavaged with DSS once a day, 5.0 g / kg each time, and the preparation method was referred to Example 1. At the same time, ID230301A-1 was gavaged once a day, 10 mg / kg each time; an experimental group 2 (n=10) was gavaged with DSS once a day, 5.0 g / kg each time, and the preparation method was referred to Example 1. The patients were gavaged with ID230301A-1 once a day at 20 mg / kg each time, and the preparation method was as described in Example 1. The experimental group 3 (n=10) was gavaged with DSS once a day at 5.0 g / kg each time, and ID230301A-1 was gavaged once a day at 40 mg / kg each time, and the preparation method was as described in Example 1. The positive control drug tofacitinib group (n=5) was gavaged with DSS once a day at 5.0 g / kg each time, and tofacitinib was gavaged twice a day at 10 mg / kg each time, and the preparation method was as described in Example 1.

[0065] This study used a 42-day modeling and simultaneous drug administration approach. The experimental period was 42 days: Days 0–5: DSS solution, Days 6–14: pure water; Days 15–21: DSS solution, Days 22–27: pure water, Days 28–34: DSS solution, and Days 35–42: pure water. Modeling was repeated for three consecutive cycles. Drug intervention began on Day 15, with the day of modeling defined as Day 0. Mice were weighed daily. Around Day 42, mice were sacrificed and their colons were harvested. Colon length was measured, and samples were collected and frozen in cryovials at -80°C for subsequent experiments such as western blotting.

[0066] like Figure 3 Results showed that compared to the colons of mice in the blank control group, the colons of mice in the model group had a darker appearance, thinner and more brittle colon walls, and significantly shorter colon length, consistent with enteritis. Compared to the model group, the colon length of the ID230301A-1 group was significantly longer, and nearly formed feces were visible in the intestine, indicating good drug efficacy. The colon length of the positive drug group was between the DSS model group and the drug-treated group, and the feces in the colon were formed, indicating some drug efficacy, but not as good as the medium-dose ID230301A-1 group, and close to that of the low-dose group.

[0067] Example 4: ID230301A-1 reduces the infiltration and increase of inflammatory cells in the intestinal tissues of mice with chronic enteritis induced by DSS.

[0068] Fixed mouse colon tissue was dehydrated, paraffin-impregnated, paraffin-embedded, sectioned, mounted on slides, and baked at 60°C for 3 hours. Sections were then dewaxed and hydrated by sequentially placing in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and then washed in distilled water for 5 minutes. Sections were stained with Harris hematoxylin for 3 minutes, washed in tap water, differentiated in 1% hydrochloric acid-alcohol for several seconds, rinsed in tap water, blued with 0.6% ammonia, and rinsed in running water. Sections were then stained in eosin solution for 3 minutes. Sections were then dehydrated and transparentized in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. Sections were removed from the xylene solution, air-dried, and mounted with neutral gum. Observation and image acquisition and analysis were performed under an Olympus pathology microscope. The results are detailed in Figure 4 .

[0069] Judging from the tissue sections, the number of glands in the model group was significantly reduced, and there was obvious inflammatory cell infiltration. The gland structure and number of the ID230301A-1 treated group were better, and there were fewer inflammatory cells. Among them, the 20mg / kg and 40mg / kg groups had the best treatment effects, and the positive drug tofacitinib had a certain effect.

[0070] Example 5: ID230301A-1, ID240507A-1, and RD240404 alleviate weight loss in mice with acute and chronic DSS-induced enteritis models

[0071] Figure 5 Results showed that compared to the normal (blank control) group of mice, the model control group experienced a significant weight loss under continuous DSS stimulation, demonstrating successful modeling. The positive drug control group experienced less weight loss than the DSS model group, but still showed a significant difference from the blank control group. The ID230301A-1 and ID240507A-1 groups showed the best results, with a slight weight loss followed by a rebound. By the end of the experiment, there was no significant difference in weight between the control group and the ID230301A-1 and ID240507A-1 groups, demonstrating the highest efficacy.

[0072] Figure 6 Results showed that compared to the normal (blank control) group, mice in the model control group experienced a significant weight loss under continuous DSS stimulation, demonstrating successful modeling. The positive drug control group experienced less weight loss than the DSS model group, but still showed a significant difference from the blank control group. The RD240404 group showed the greatest effect, with a slight weight loss followed by a rebound. By the end of the trial, there was no significant difference in weight between the control group and the RD240404 group, demonstrating the highest efficacy.

[0073] Figure 7 Results showed that over the three cycles of induction, the average weight of mice in the model groups decreased and then recovered compared to the blank control group. Compared to the model group, the rate of weight loss in the ID230301A-1-20mg / kg and ID230301A-1-40mg / kg groups was slower. The positive control group experienced less weight loss than the DSS model group, but still showed significant differences compared to the blank control group. The ID230301A-1-20mg / kg and ID230301A-1-40mg / kg groups showed the best results, with a slight weight loss followed by a recovery.

[0074] Example 6: ID230301A-1, ID240507A-1 and RD240404 significantly reduced the DAI scores of mice in the acute and chronic models of DSS-induced enteritis

[0075] The DAI scoring standard was used to score the apparent symptoms of each group, and the enteritis score was calculated based on the weight change (0-4), stool characteristics (0-4), and the severity of occult blood in stool (0-4). The results are detailed in Figure 5 and Figure 6 .

[0076] Figure 8 Results showed that mice in the DSS model group developed loose stools and bloody stools starting on day 1, followed by weight loss and a gradual increase in DAI scores, reaching their maximums on days 7 and 8. The DAI scores in the positive control group also showed an upward trend, but were generally lower than those in the DSS model group. The DAI scores in the ID230301A-1 and ID240507A-1 groups peaked on days 4 and 6, respectively, and then continued to decline, with no significant differences between the groups at the end of the trial. ID230301A-1 and ID240507A-1 were superior to tofacitinib in alleviating various symptoms and DAI scores.

[0077] Figure 9 Results showed that mice in the DSS model group developed loose stools and bloody stools starting on day 1, followed by weight loss and a gradual increase in DAI scores, reaching their maximum values ​​on days 7 and 8. The DAI scores in the positive drug control group also showed an upward trend, but were generally lower than those in the DSS model group. The DAI scores in the RD240404 group also showed an upward trend, but were generally lower than those in both the DSS model group and the positive drug group. RD240404 was more effective than tofacitinib in alleviating various symptoms and DAI scores.

[0078] Figure 10Results showed that over the three induction cycles, the DAI scores of mice in the model group increased and then recovered compared to the blank group. Compared to the model group, the DAI scores of mice in the ID230301A-1-20mg / kg and ID230301A-1-40mg / kg groups were significantly reduced over the three induction cycles, and defecation and occult blood in stool also improved significantly compared to the model group. ID230301A-1 was more effective than tofacitinib in alleviating various symptoms and DAI scores.

[0079] Example 7: Analysis of immunoblotting results of colon tissues in mice with acute inflammatory bowel disease model

[0080] The frozen colon tissues of the mice in each group of Example 1 and Example 2 were obtained, fully ground and homogenized, and then lysed with RIPA lysis buffer. Protein was collected, quantified, and subjected to Western blot analysis.

[0081] 1. Steps for determining protein concentration (BCA method):

[0082] A. Dilute protein standards (Table 1) in 1× phosphate buffered saline (PBS):

[0083] Table 1. Preparation system of protein standards.

[0084]

[0085] B. Preparation of BCA working solution: Calculate the total amount of BCA reagent A and B working solution required based on the number of standards and samples to be tested. Prepare the working solution at a 50:1 volume ratio of BCA reagent A to B. Vortex and mix thoroughly before use.

[0086] C. Add 25 μL each of the protein standard solution and the sample supernatant diluted 10-fold in phosphate-buffered saline (PBS) to a new 96-well plate. Then, add 200 μL of the previously prepared BCA working solution to each well and mix thoroughly. Be careful not to create bubbles by pipetting. Securely cover the 96-well plate and incubate at 37°C for 30 minutes.

[0087] D. Remove the 96-well plate and return it to room temperature for 3-5 minutes. Measure the absorbance at 562 nm on a microplate reader, create a standard curve, and calculate the protein content of 1 μL of each sample in preparation for protein loading.

[0088] 2. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)

[0089] (1) Fix the gel plate and prepare 10% SDS-PAGE separation gel (Table 2).

[0090] Prepare separation gel according to Table 2 below: 10 mL

[0091] Table 2 Separation gel formula information.

[0092]

[0093] (2) Add the mixed separation gel to two gel plates, adding it to a position 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let it stand for 30-45 minutes.

[0094] (3) After separation and gelation, pour out the remaining anhydrous ethanol and absorb the remaining anhydrous ethanol with filter paper.

[0095] (4) Prepare 5 mL of 5% concentrated gel according to Table 3

[0096] Table 3 Stacking gel recipe information.

[0097]

[0098] (5) Slowly add the prepared concentrated gel to the gel plate to avoid bubbles, insert the sample comb, and let it stand for 30-45 minutes.

[0099] (6) Take out the protein sample, heat it in a water bath at 100°C for 5 min, and centrifuge it at 10,000 rpm for 10 min.

[0100] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, pull out the sample comb, and add the processed protein samples into the sample tank in order.

[0101] (8) Electrophoresis at 80V for 40min.

[0102] (9) Change the voltage to 120 V and run the electrophoresis for about 1.5 hours until the bromophenol blue runs out of the colloid;

[0103] 3. Western-blot transfer

[0104] (1) Rinse the SDS-PAGE gel after electrophoresis in TBST buffer once, and soak the protein gel in transfer buffer.

[0105] (2) Soak a layer of sponge pad in membrane transfer buffer and clamp it onto the membrane transfer apparatus with tweezers. Place the sponge pad, three layers of filter paper, protein glue, polyvinylidene fluoride (PVDF) membrane, three layers of filter paper, and sponge pad in the order described above. Align them, pick them up, and place them onto the membrane transfer apparatus. During operation, both the filter paper and sponge pad must be soaked in transfer buffer. If there are bubbles between each layer, use a glass test tube to gently roll them out.

[0106] (3) Turn on the membrane transfer apparatus and transfer the membrane at 300 mA for 75 minutes.

[0107] (4) Remove the membrane, place it in TBST buffer, and rinse it three times on a 60 rpm horizontal shaker for 8 minutes each time.

[0108] (5) Block with 20 mL of 5% bovine serum albumin (BSA) blocking solution on a 60 rpm horizontal shaker at room temperature for 2 h.

[0109] (6) Incubate with 3 mL of antibody incubation solution containing 3 μL of primary antibody (1:1000) at 4°C on a horizontal shaker at 60 rpm overnight.

[0110] (7) Wash the PVDF membrane three times with 10 mL of TBST and shake at 60 rpm at room temperature for 10 min each time.

[0111] (8) Incubate the PVDF membrane with 20 mL of antibody incubation solution containing 2 μL of secondary antibody at room temperature on a horizontal shaker at 60 rpm for 2 h.

[0112] (9) Wash the PVDF membrane three times with 10 mL of TBST and shake at 60 rpm at room temperature for 10 min each time.

[0113] (10) Take 1 mL each of chemiluminescent substrate reagent solution A and solution B, mix well, and apply to the filter membrane.

[0114] (11) Use filter paper to absorb the liquid on the membrane and use a developer to develop the color.

[0115] like Figure 11 Western blotting revealed elevated p-STAT (Y705) protein expression in the colon of mice in the model group. Treatment with ID230301A-1 and ID240507A-1 decreased p-STAT (Y705) protein expression. This suggests that ID230301A-1 and ID240507A-1 may alleviate the inflammatory response by inhibiting STAT3 signaling, thereby suppressing the formation and maintenance of the DSS-induced inflammatory signaling network.

[0116] like Figure 12 Western blotting revealed elevated p-STAT (Y705) protein expression in the colon of mice in the model group. RD240404 treatment reduced p-STAT (Y705) protein expression. This result suggests that RD240404 may alleviate the inflammatory response by inhibiting STAT3 signaling, thereby suppressing the formation and maintenance of the DSS-induced inflammatory signaling network.

[0117] The above results show that ID230301A-1, ID240507A-1 and RD240404 have a good effect of inducing the relief of inflammatory response and show good application prospects in the treatment of inflammatory bowel disease.

Claims

1. Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a drug for treating inflammatory bowel disease, characterized in that: The piperazinamide derivative is at least one of the following structures:

2. Use of a piperazinamide derivative or a biologically acceptable salt thereof in the preparation of a STAT3 protein inhibitor, characterized in that: The structure of the piperazinamide derivative is as follows:

3. The use according to claim 1 or 2, characterized in that The drug is a drug prepared from one or a mixture of any proportion of ID230301A-1, ID240507A-1, RD240404 and biologically acceptable salts thereof into an injection, tablet, powder injection, granule, capsule, oral solution, ointment or cream.

4. The use according to claim 1 or 2, characterized in that The oral liquid is prepared by dissolving a piperazinamide derivative or a biologically acceptable salt thereof in a mixed solvent consisting of an organic solvent, a viscosity enhancer and an emulsifier to form a solution with a concentration of 1 to 10 mg / mL.

5. The use according to claim 4, characterized in that In the mixed solvent, the organic solvent accounts for 2% to 5% by volume; the emulsifier accounts for 10% to 15%; and the viscosity enhancer accounts for 80% to 88%; the organic solvent is DMSO, the viscosity enhancer is a 0.1wt% to 1wt% sodium carboxymethyl cellulose aqueous solution, and the emulsifier is Cremophor EL.

6. The use according to claim 1, characterized in that The inflammatory bowel disease refers to acute enteritis and chronic enteritis.

7. The use according to claim 1, characterized in that The piperazinamide derivative or its biologically acceptable salt is administered alone or in combination with other pharmaceutically acceptable drugs.

8. The use according to claim 1 or 2, characterized in that The biologically acceptable salt refers to a biologically acceptable salt formed by ID230301A-1, ID240507A-1 or RD240404 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

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