Application of covalent intestinal adhesion TGR5 agonist in inflammatory bowel disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a covalent intestinal adhesion TGR5 agonist and application of the covalent intestinal adhesion TGR5 agonist in inflammatory bowel diseases. The invention aims to provide a TGR5 agonist capable of forming a covalent bond with intestinal mucoprotein, and the TGR5 agonist can agonize the activity of TGR5 and reduce the toxic and side effects on gallbladder; meanwhile, expression of inflammatory factors can be inhibited, expression of proinflammatory factors can be reduced, and the inflammatory level of ulcerative colitis can be relieved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of a class of covalent intestinal adhesive TGR5 agonists of maleimide fragments in the preparation of drugs for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a general term for a class of chronic inflammatory diseases of the intestine. It has a continuous spectrum and is mainly divided into two categories according to the macroscopic symptoms and microscopic affected parts, namely Crohn's disease (CD) and ulcerative colitis (UC). Although IBD is a global disease, the incidence rate in various countries is not balanced, and the incidence rate in European and American countries is higher than that in Asian countries. In North America, the incidence rate of Crohn's disease is 6.3 / 100,000 to 23.8 / 100,000; in Asia, the incidence rate of Crohn's disease is about 0.54 / 100,000. In recent years, with the rapid development of my country's economy and society, the continuous increase in population, and the high-calorie diet intake of some residents, the incidence rate of inflammatory bowel disease has increased year by year, and it has gradually become a common digestive system disease that affects the quality of life index of residents. The symptoms of patients with inflammatory bowel disease are mainly intermittent abdominal pain, diarrhea and bloody diarrhea; in addition, there are some extraintestinal manifestations, such as lesions in joints, liver, gallbladder and skin. The course of the disease is prolonged and difficult to cure. Long-term recurrence will increase the risk of intestinal cancer. The etiology of inflammatory bowel disease is unknown. It is currently believed to be closely related to imbalanced intestinal immune responses, increased intestinal permeability, and metabolic disorders of the bacterial flora. Classic IBD treatment drugs are mainly 5-aminosalicylic acid drugs, glucocorticoids, immunosuppressants, biological targeted agents, etc. The therapeutic purpose of the above drugs is to reduce intestinal inflammatory responses. They can only induce remission and delay recurrence, but cannot achieve clinical cure. New drugs that can repair the intestinal epithelial barrier and regulate colonic flora metabolism are still to be developed.

[0003] TGR5, also known as GPBAR1, is a G protein-coupled receptor whose ligands include natural primary and secondary bile acids, synthetic steroids, and non-steroidal compounds. After the ligand binds to the TGR5 receptor protein, it activates the G protein, which activates the downstream adenylate cyclase, thereby increasing the concentration of intracellular cAMP, and then activates protein kinase A (PKA), which can phosphorylate a variety of PKA downstream proteins and exert a variety of biological effects. Current studies have shown that TGR5 is involved in glucose and lipid metabolism and bile acid circulation, and also has biological activity in regulating immune responses. Inflammatory cells such as macrophages express TGR5. After being activated, TGR5 can play an immunomodulatory role by reducing the expression levels of inflammatory factors and cell chemokines in immune cells. Studies have shown that the TGR5 agonist INT-777 can reduce the activation of NLRP3 inflammasomes in microglia through the TGR5 / cAMP / PKA signaling pathway, and downregulate the expression of proinflammatory factors to alleviate brain neuroinflammation. INT-777 relieves acute pancreatitis in mice mainly by reducing the production of intracellular ROS and lowering the expression of proinflammatory cytokines (IL-1β, IL-6 and TNFα). TGR5 agonists can also inhibit the apoptosis of intestinal epithelial cells in vitro through the cAMP / PKA / c-FLIP / JNK signaling pathway, thereby alleviating intestinal barrier disorders.

[0004] At present, the systemic TGR5 agonists under development are prone to strong toxic side effects. For example, TGR5 is highly expressed in the gallbladder. After being activated, it will increase the intracellular cAMP level and cause smooth muscle relaxation, thereby dilating the gallbladder and increasing the volume of the gallbladder pool. It can also reverse the gallbladder contraction caused by the secretion of cholecystokinin CCK under normal feeding conditions. The obstruction of bile excretion will increase the risk of gallstone formation. In addition, TGR5 agonists can activate K_Ca1.1 (a calcium-dependent potassium ion channel protein) to significantly reduce peripheral vascular tension and blood pressure, and have a certain impact on the body's cardiovascular system. The toxic side effects of TGR5 agonists have hindered the further development and clinical use of this type of drug. In order to reduce systemic side effects, non-systemic TGR5 agonists have also become an important research direction for this type of drug. This strategy aims to reduce the exposure of the drug in the body, target TGR5 in the intestine, and effectively reduce the toxic side effects of the drug in the systemic circulation. The main design strategies for the design and development of non-systemic TGR5 agonists include increasing molecular weight, improving molecular polarity, enhancing lipid solubility or soft drug strategies. Most of these molecules violate the five principles of drug-like properties. Due to solubility or lipid solubility issues, they often have better in vitro efficacy but poor in vivo efficacy. Therefore, further improving the in vivo efficacy of non-systemic TGR5 agonists has also become a key issue in the development of this type of drug.

[0005] In the early stage, we designed and synthesized a new type of intestinal restricted TGR5 agonist, and studied the application of this type of agonist in the treatment of diabetes (CN 116063284 A). This type of compound has good agonist activity on TGR5, high hypoglycemic activity in vivo, and poor membrane permeability, which effectively reduces the toxic side effects on the gallbladder. On this basis, we further expanded the structural type and explored the therapeutic effect of this type of molecule in inflammatory bowel disease. Summary of the invention

[0006] The purpose of the present invention is to provide a covalent intestinal adhesive TGR5 agonist, which activates intestinal epithelial TGR5 receptors in the intestine and exerts anti-ulcerative colitis activity in vivo; at the same time, this type of molecule can form a covalent bond with intestinal mucin, reduce the drug's permeability in the intestine, and increase the drug's retention time in the intestine.

[0007] The compound having anti-ulcerative colitis activity and containing a covalently bound fragment to mucin of the present invention has a general structural formula as shown in (I), wherein the maleimide fragment is connected to the TGR5 agonist via different types of connecting chains L.

[0008]

[0009] The connecting chain L is an alkyl chain or a PEG chain.

[0010] The alkyl chain Its length n is 2-10, preferably 6.

[0011] The PEG chain Its length m is 1-4, preferably 1-2.

[0012] Wherein, the TGR5 agonist is selected from:

[0013]

[0014] Preferably:

[0015]

[0016] The covalent intestinal adhesive TGR5 agonist, the preferred compound is:

[0017]

[0018] The synthesis route of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment of the present invention is as follows:

[0019] Step 1, synthesis of intermediate 14: Tetrachloronicotinic acid (compound 12) is used as a raw material, first reacted with dichlorothionyl to form an acyl chloride compound, and then reacted with N-cyclopropyl-tetrahydroquinoline (compound 13) to generate intermediate 14. The specific synthesis route is as shown in Scheme 1

[0020]

[0021] Route 1: Synthesis of intermediate 14. Reagents and conditions: (a) SOCl 2 ,DMF,80℃;Triethylamine,CH2Cl2,rt

[0022] Step 2, synthesis of final products H2, H3, and H5: 4-bromo-2,5-dichlorophenol (intermediate 15) is used as a raw material, and undergoes a nucleophilic substitution reaction with ethyl acrylate to obtain intermediate 16. Intermediate 16 is reduced with palladium carbon to obtain intermediate 17, which is then subjected to a substitution reaction with intermediate 14 to obtain intermediate 18. The ester bond is hydrolyzed in a NaOH solution to obtain intermediate 19. Intermediate 19 reacts with linkers of different lengths to obtain intermediates 20-22. Intermediates 23-25 ​​are subjected to a de-Boc reaction in a hydrochloric acid methanol solution to obtain intermediates 23-25, which are subjected to a condensation reaction with maleimide to obtain final products H2, H3, and H5. The specific synthesis route is as in Route 2.

[0023]

[0024] Route 2: Synthesis of final products H2, H3, and H5. Reagents and conditions: (a) Triethylamine, Palladiumacetate, DMF, 160°C; (b) Pd / C, H 2 ,EtOH,70℃;(c)K 2 CO 3 ,DMF,160℃;(d)NaOH,MeOH / H 2 O; (e) HATU, DIEA, DMF, 60℃; (f) MeOH / HCl; (g) HATU, DIEA, DMF, 60℃.

[0025] The invention discloses an application of the covalent intestinal adhesive TGR5 agonist containing maleimide fragment in the preparation of anti-inflammatory drugs.

[0026] The invention discloses an application of the covalent intestinal adhesive TGR5 agonist containing maleimide fragment in the preparation of a drug for treating inflammatory bowel disease.

[0027] The invention discloses an application of the covalent intestinal adhesive TGR5 agonist containing maleimide fragment in the preparation of an anti-ulcerative colitis drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effects of compound H2 on the body weight and DAI scores of mice; (a) The figure shows the experimental timetable, (b) The figure shows the DAI score curve of mice; (c) The figure shows the daily weight loss ratio curve of mice. *Compared with the DSS group, p<0.05, **Compared with the DSS group, p<0.01, ##Compared with the control group, p<0.01, ###Compared with the control group, p<0.001. The experimental subjects were male C57BL / 6J mice, n≥4 / group.

[0029] Figure 2 Effects of compound H2 on colon length and spleen weight of mice; (a) and (c) are visual and numerical statistical diagrams of colon length of male C57BL / 6J mice, respectively; (b) and (d) are visual and numerical statistical diagrams of spleen weight of mice, respectively, * compared with DSS group, p<0.05, # compared with control group, P<0.05. The experimental subjects were male C57BL / 6J mice, n≥4 / group.

[0030] Figure 3 Effect of compound H2 on mouse colon tissue morphology; (a) is the result of HE staining of colon tissue, magnification is 200×, (b) is the colon tissue pathology score, ** compared with DSS group, p<0.01, ## compared with control group, p<0.01, ### compared with control group, p<0.001. The experimental subjects were male C57BL / 6J mice, n≥4 / group.

[0031] Figure 4 Effect of compound H2 on the expression level of inflammatory factors in mouse colon tissue; * compared with the DSS group, p<0.05, # compared with the control group, p<0.05, ## compared with the control group, p<0.01, the experimental subjects were male C57BL / 6J mice, n≥4 / group.

[0032] Figure 5 Effect of compound H2 on intestinal permeability in mice; the in vivo imaging developer was FITC-dextran, and mice were anesthetized by gas 1 hour after intragastric administration, and the skin was cut open to expose the intestine. The fluorescence distribution intensity is shown on the right scale. The experimental subjects were male C57BL / 6J mice, n = 3 / group.

[0033] Figure 6 Effect of compound H2 on the expression of ZO-1 in mouse colon tissue; (a) The figure shows the protein bands and internal reference bands of ZO-1 in mouse colon tissue; (b) The figure shows the grayscale analysis of Western Blot experimental results, * compared with the DSS group, p<0.05, ## compared with the control group, p<0.01. The experimental subjects were male C57BL / 6J mice, n≥4 / group. DETAILED DESCRIPTION

[0034] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0035] Example 1

[0036] Preparation of intermediate 14

[0037] Tetrachloronicotinic acid (10.14 g, 64.36 mmol) was dissolved in dichlorothionyl, and an appropriate amount of DMF was added dropwise. The mixture was refluxed at 80°C for 3.5 h. After the reaction was completed, the dichlorothionyl was dried by rotary evaporation to obtain a brown powdery solid, which was dissolved in dichloromethane and poured into a dropping funnel. N-cyclopropyl-tetrahydroquinoline (11.21 g, 64.33 mmol) was dissolved in dichloromethane, and triethylamine was added dropwise. The above solution was slowly added dropwise under ice bath conditions, and the mixture was reacted at room temperature for 6 h. The mixture was extracted, dried, and the solvent was dried by rotary evaporation. The crude product was purified by column chromatography to obtain 7.05 g of a white powdery solid, which was intermediate 14, with a yield of 34.9%.

[0038] Preparation of intermediate 16

[0039] Take 4-bromo-2,5-dichlorophenol (intermediate 15) (10.00 g, 41.34 mmol) and dissolve it in DMF. Then add triethylamine (11.48 mL, 82.59 mmol), palladium acetate (742.60 mg, 3.30 mmol), tri(o-methylphenyl)phosphine (2.014 g, 6.61 mmol) to the sealed tube in this order, and finally add ethyl acrylate (8.8 mL, 80.95 mmol). React at 160°C for 30 h. After the reaction is completed, extract, dry, and spin-dry the solvent to obtain a black oily solid. Purify it by column chromatography to obtain 6.74 g of a white powdery product, which is intermediate 16, with a yield of 62.4%.

[0040] Preparation of intermediate 17

[0041] The intermediate 16 (2.00 g, 7.66 mmol) was dissolved in anhydrous methanol, and wet palladium carbon (240.00 mg, 12% w / w) was added. The mixture was reacted at room temperature for 6 h under H2 gas environment. After the reaction was completed, methanol was added to the reaction solution and boiled. The mixture was filtered with diatomaceous earth and the solvent was dried to obtain a light yellow oily liquid. The product was purified by column chromatography to obtain 1.73 g of white powdery solid, namely the intermediate 17, with a yield of 85.8%.

[0042] Preparation of intermediate 18

[0043] Intermediate 14 (2.00 g, 6.37 mmol) and intermediate 17 (1.67 g, 6.37 mmol) were weighed into a sealed tube. DMF was used as solvent. Anhydrous potassium carbonate (880.8 mg, 6.17 mmol), a small amount of copper powder and cuprous chloride were added to the sealed tube in sequence. The reaction was carried out at 160° C. for 10 h. After the reaction was completed by TLC, the mixture was extracted with ethyl acetate and anhydrous Na 2 SO 4 The mixture was dried, filtered, and the solvent was evaporated to dryness to obtain a black oily liquid. The crude product was purified by column chromatography to obtain 789.10 mg of a light yellow powdery solid, namely, intermediate 18, with a yield of 22.9%. 1 H-NMR (400 MHz, DMSO-d 6 )δ8.78(s,1H),8.43(d,J=5.7Hz,1H),7.62(s,1H),7.00(t,J=7.8Hz,1H), 6.87(d,J=8.2Hz,1H),6.59–6.44(m,2H),6.36(s,1H),5.58(s,1H),4.73(s ,1H),4.07(q,J=7.1Hz,2H),3.40(s,2H),2.91(q,J=5.9,4.0Hz,2H),2.62( t,J=7.6Hz,3H),2.21(s,1H),1.22–1.13(m,4H),0.62(s,2H),0.47(s,1H).

[0044] Preparation of intermediate 19

[0045] The intermediate 18 (900.00 mg, 1.66 mmol) was dissolved in anhydrous methanol solution, and 5 mL of 2N sodium hydroxide was added dropwise to hydrolyze the compound. The reaction was allowed to react at room temperature for 3 h. After the reaction was completed, 10% HCl solution was added dropwise to adjust the pH value of the reaction solution to weak acidity. Methanol was removed by distillation under reduced pressure, and the mixture was extracted with ethyl acetate. The solvent was dried and dried to obtain 820 mg of a light yellow powdery solid, namely the intermediate 19, with a yield of 96.4%.

[0046] Preparation of Intermediates 20-22

[0047] The intermediate 19 (1 times amount) was dissolved in 20 mL of NN dimethylformamide, EDCI and HOBt were added, and the mixture was stirred at 0°C for 1 h. Then, linkers of different lengths (1.1 times amount) and DIEA (2 times amount) were added, mixed and added dropwise to the reaction bottle, and the mixture was reacted at room temperature for 10 h. After the reaction, the mixture was extracted with ethyl acetate, dried, and the solvent was spin-dried to obtain a crude product, which was purified to obtain a light yellow oily liquid, namely the intermediate 20-22.

[0048] Preparation of Intermediates 23-25

[0049] Intermediate 20, 21 or 22 was dissolved in ethyl acetate, 1-2 mL of 2.5 M ethyl acetate-hydrochloric acid solution was added, and the mixture was reacted at room temperature for 3 h. After the reaction was completed, a brown-red granular filter cake was obtained by suction filtration. The filter cake was dried to obtain intermediate 23, 24 or 25, respectively.

[0050] Preparation of final products H2, H3 and H5

[0051] Maleimidic acid (1 times amount) was dissolved in 10 mL DMF, EDCI and HOBt were added, and the mixture was stirred at 0°C for 1 h. Intermediate 23, 24 or 25 (1.1 times amount) was dissolved in a small amount of DMF, DIEA was added, and the mixture was added dropwise to the reaction bottle after mixing. The mixture was reacted at room temperature for 8 h, and the mixture was extracted with ethyl acetate. After drying, the solvent was filtered and dried, and the purified product was prepared to obtain the final product H2, H3 or H5, respectively.

[0052] Compound H2 is a reddish brown powder. 1 H-NMR (400 MHz, DMSO-d 6 )δ8.78(s,1H),8.43(s,1H),7.84(t,J=5.6Hz,1H),7.70(d,J=5.9Hz,1H),7.54(s,1H),7.00(s,2H), 6.88(s,1H),6.51(s,2H),6.36(s,1H),5.58(s,1H),4.72(s,1H),3.37(d,J=7.1Hz,2H),3.34(s,2H), 3.05–2.94(m,4H),2.87(s,2H),2.70–2.65(m,1H),2.41–2.31(m,3H),2.21(s,1H),2.00(t,J=7.3Hz, 2H),1.51–1.41(m,5H),1.34(s,5H),1.26–1.19(m,5H),1.19–1.12(m,1H),0.64(s,2H),0.47(s,1H).

[0053] Compound H3 is a yellow powder. 1 H-NMR (400 MHz, DMSO-d 6)δ8.78(s,1H),8.43(d,J=5.7Hz,1H),8.07–7.09(m,1H),7.88–7.83(m,2H),7.55(s,1H),7.00(d, J=7.7Hz,1H),6.86(d,J=8.4Hz,1H),6.56–6.47(m,2H),6.36(d,J=7.8Hz,1H),5.56(s,1H),4.73(s ,1H),2.89(s,3H),2.73(s,3H),2.40(t,J=7.5Hz,2H),2.32(t,J=7.5Hz,1H),2.22(d,J=8.4Hz,1H ),2.09–1.99(m,5H),1.76(s,1H),1.54–1.41(m,6H),1.32–1.14(m,5H),0.63(s,3H),0.46(s,1H).

[0054] Compound H5 is a yellow powder. 1 H-NMR (400 MHz, DMSO-d 6 )δ8.77(s,1H),8.46–8.40(m,1H),8.02(t,J=5.4Hz,1H),7.85(d,J=5.9Hz,2H),7.55(s,1H),6.99(q, J=6.7,6.2Hz,1H),6.93–6.81(m,1H),6.52(t,J=9.3Hz,2H),6.35(s,1H),5.58(s,1H),4.72(s,1H),3. 23–3.11(m,8H),2.88(d,J=10.1Hz,3H),2.73(s,1H),2.40(d,J=7.8Hz,2H),2.35–2.27(m,1H),2.24–2 .17(m,1H),2.09–1.96(m,4H),1.55–1.37(m,8.1Hz,4H),1.27–1.21(m,8H),0.63(s,2H),0.46(s,1H).

[0055] Pharmacological activity studies:

[0056] In vitro biological evaluation of compounds H2, H3 and H5

[0057] The TGR5-293T stable cell line has a rich content of TGR5 in the cell membrane. After the compound stimulates TGR5, it increases the intracellular cAMP content through the TGR5-cAMP pathway. The natural cAMP produced by the cells competes with the d2-labeled cAMP in the HTRF-cAMP kit to bind to the monoclonal cAMP europium hole-labeled antibody and produce a fluorescent signal. The fluorescence ratio (665nm / 620nm) is inversely proportional to the cAMP concentration in the sample. The cAMP level of mTGR5-293T and hTGR5-293T cells was detected by HTRF technology to verify the agonist activity of the target compound on TGR5. The experimental results show that the three compounds H2, H3 and H5 all have good TGR5 agonist activity, as shown in Table 1, among which compound H2 has the best activity, and hTGR5 EC 50 The value was 7.1 nM, and the mTGR5 EC 50 The value is 15.3nM.

[0058] Table 1 In vitro activity data of H2, H3 and H5

[0059]

[0060]

[0061] Anti-ulcerative colitis activity of compound H2 in C57BL / 6J mice

[0062] Male C57BL / 6J mice were randomly divided into 4 groups: control group, DSS group, DSS+H2 group, and DSS+5-ASA group, with 10 mice in each group. On day 0, all mice drank sterile water. From day 1 to day 8, the drinking water of mice in the control group was sterile water, and the drinking water of mice in the other groups was replaced with 3% DSS solution. From day 0 to day 8, mice in the DSS+H2 group were gavaged with 50 mg / kg of H2 every day, and mice in the DSS+5-ASA group were gavaged with 100 mg / kg of 5-ASA every day. The mice in the control group and DSS group were gavaged with the same volume of solvent. Fresh feces of mice in each group were collected on day 7, and the experiment ended after gavage on day 8.

[0063] Effects of compound H2 on body weight and DAI scores of mice

[0064] The experimental results are as follows Figure 1As shown, the weight of mice was basically stable from day 0 to day 4. From day 5, the weight of mice in the modeling group began to show a significant downward trend. By day 8, when the experiment ended, the weight of mice in the control group decreased by an average of 3.79%, the weight of mice in the DSS group decreased by an average of 23.14%, the weight of mice in the DSS+H2 group decreased by an average of 14.11%, and the weight of mice in the DSS+5-ASA group decreased by an average of 16.83%. The weight of mice in the control group remained stable. The DAI index values ​​of mice were basically stable from day 0 to day 3. The mice in the DSS group did not show obvious symptoms of colitis. From day 4, the DSS group began to show symptoms of diarrhea and bloody stools, and the DAI index began to rise. The DAI index of mice in the DSS+H2 group and DSS+5-ASA group began to rise from day 5. Compared with the DSS group, the values ​​were slightly lower and the rising rate was slow, indicating that the symptoms of colitis in mice were alleviated and the disease progression slowed down after H2 administration. Compound H2 had similar therapeutic effects as the positive control drug 5-ASA.

[0065] Effects of compound H2 on colon and spleen tissues of mice

[0066] DSS enters the intestine and destroys the intestinal mucosal barrier, increases intestinal permeability, and induces an inflammatory response in the intestine. The colon tissue will become congested, edematous, and spasmodic. Figure 2 As shown, the colon length of the mice in the control group was 7.02±0.64cm, which was normal to the naked eye; the colon length of the mice in the DSS group was 4.96±0.63cm, which was shortened, congested and hardened, and the toughness of the intestinal tissue was reduced and easy to break; the colon lengths of the mice in the DSS+H2 group and the DSS+5-ASA group were 6.38±1.06cm and 6.24±0.60cm, respectively, which were still spasmodic and shortened compared with the control group, but were alleviated to varying degrees compared with the DSS group. This shows that compound H2 can effectively reduce the degree of inflammation in the colon of mice with ulcerative colitis, and its activity is comparable to that of 5-ASA.

[0067] Mice with ulcerative colitis will have enlarged spleens, which may be related to intestinal infection and inflammatory stimulation. Figure 2 As shown, the spleen weight of mice in the DSS group was 0.113±0.015g, the spleen weight of mice in the control group was 0.073±0.015g, the spleen weight of mice in the DSS+H2 group and the DSS+5-ASA group was 0.075±0.007g and 0.081±0.012g, respectively. Although the spleen weight of mice in the DSS+H2 group and the DSS+5-ASA group was greater than that in the control group, it was less severe than that in the DSS group, indicating that compound H2 can alleviate the phenomenon of spleen enlargement in mice with ulcerative colitis.

[0068] Effects of compound H2 on intestinal tissue morphology in mice

[0069] Colonic tissue was observed under a microscope and pathological scores were performed according to the extent of the lesioned mucosa. Figure 3 As shown in the figure, the colon tissue of the mice in the control group was clearly differentiated, the cells were neatly arranged, the mucosal layer had a typical crypt structure, and a large number of goblet cells were visible; the intestinal epithelium of the mice in the DSS group was disordered, the cells were swollen and broken, the crypt structure of the mucosal layer was atrophied, moved upward, or even disappeared, and the mucosal layer and submucosal layer were infiltrated with neutrophils and lymphocytes; the colon tissue of the mice in the DSS+H2 group and the DSS+5-ASA group had crypts moving upward in a small range of the intestinal epithelium, most of the mucosal layer still maintained a normal structure, and inflammatory cell infiltration was visible on the local mucosal basal side. The microscopic intestinal histopathological manifestations of the mice in the DSS+H2 group and the DSS+5-ASA group were improved compared with those in the DSS group, indicating that compound H2 can alleviate the inflammatory pathological changes in the colon tissue of mice with ulcerative colitis.

[0070] Effects of compound H2 on the expression levels of inflammatory factors in mouse colon tissue

[0071] In order to study whether compound H2 can affect the expression level of inflammatory factors in the colon tissue of mice with ulcerative colitis, we crushed the intestinal tissue of mice to extract total RNA and detected the gene expression level of inflammatory factors by qRT-PCR technology. Figure 4 As shown, the mRNA expression levels of IL-2, IL-6, COX2, and TNF-α in the colon tissue of mice in DSS group were higher than those in control group, the mRNA expression level of IL-10 showed no significant change compared with that in control group, and the mRNA expression level of FOXO1 was lower than that in control group; the mRNA expression levels of IL-2, IL-6, FOXO1, COX2, and TNF-α in the colon tissue of mice in DSS+H2 group were lower than those in DSS group, while the mRNA expression levels of IL-10 and FOXO1 were higher than those in DSS group, indicating that the administration of compound H2 can reduce the expression levels of pro-inflammatory factors IL-2, IL-6, COX2, and TNF-α in the colon tissue of mice, and can increase the expression levels of anti-inflammatory factors IL-10 and FOXO1.

[0072] Effects of compound H2 on intestinal permeability in mice

[0073] The occurrence of ulcerative colitis is not only accompanied by disorders of the intestinal immune response, but also by the destruction of the colonic epithelial barrier and increased intestinal permeability. After DSS-induced ulcerative colitis in mice, the intestinal permeability of the mice increased. In order to study whether compound H2 can improve the intestinal permeability of mice with ulcerative colitis, this study used fluorescent in vivo imaging technology to study the changes in intestinal permeability of mice. The higher the intestinal permeability of mice, the higher the transmittance of the FITC-dextran fluorescent agent, the wider the distribution of fluorescence captured on the mouse in vivo imaging scanner, and the stronger the distribution intensity. The experimental results are as follows Figure 5 As shown, the intestinal permeability of mice in the DSS group was increased compared with that of mice in the control group. Although the intestinal permeability of mice in the DSS+H2 group and the DSS+5-ASA group also tended to increase compared with that of the control group, it was alleviated compared with that of mice in the DSS group, indicating that compound H2 can alleviate the increased intestinal permeability of mice with ulcerative colitis.

[0074] Effect of compound H2 on the expression of tight junction protein ZO-1 in mice

[0075] In order to study whether compound H2 can regulate the expression of tight junction protein ZO-1 between intestinal epithelial cells in mice with ulcerative colitis, this study used Western Blot experiment to quantitatively analyze the expression of ZO-1 in mouse colon tissue. Figure 6 As shown in the figure, the expression of ZO-1 in the colon tissue of mice in the DSS group was lower than that in the control group, indicating that the intestinal epithelial barrier function was impaired. The expression of ZO-1 in the colon tissue of mice in the DSS+H2 group was higher than that in the DSS group, indicating that compound H2 can alleviate the phenomenon of decreased expression of tight junction protein ZO-1 between intestinal epithelial cells in mice with ulcerative colitis.

Claims

1. Use of a covalent intestinal adhesive TGR5 agonist containing a maleimide fragment in the preparation of an anti-inflammatory drug, characterized in that: The maleimide fragment of the TGR5 agonist is connected to the TGR5 agonist via different types of connecting chains L, and its general structural formula is shown in (I), 2. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 1 in the preparation of an anti-inflammatory drug, characterized in that: The agonist is selected from:

3. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 2 in the preparation of an anti-inflammatory drug, characterized in that: The agonist is preferably:

4. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 1 in the preparation of an anti-inflammatory drug, characterized in that: The connecting chain L is an alkyl chain or a PEG chain.

5. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 4 in the preparation of an anti-inflammatory drug, characterized in that: The alkyl chain is wherein n is 2-10, preferably 6.

6. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 4 in the preparation of an anti-inflammatory drug, characterized in that: The PEG chain is wherein m is 1-4, preferably 1-2.

7. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment in the preparation of an anti-inflammatory drug according to claim 3, characterized in that: The preferred compounds are:

8. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment according to any one of claims 1 to 7 in the preparation of anti-inflammatory drugs, specifically in the preparation of anti-inflammatory bowel disease drugs.

9. Use of the covalent intestinal adhesive TGR5 agonist containing a maleimide fragment as claimed in claim 8 in the preparation of an anti-inflammatory drug, wherein the inflammatory bowel disease is ulcerative colitis.

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