Application of indole base compound in preparation of medicine for treating or preventing intestinal diseases

Abstract

The invention relates to application of an indole base compound in preparation of a medicine for treating or preventing intestinal diseases. The intestinal diseases are related diseases caused by abnormity of a high-level serotonin trigger signal channel. According to the invention, active molecules for specific regulation and control of serotonin signals of blood and intestinal tracts are screened, and the indoline can lower intestinal inflammation damage and promote intestinal mucosal barrier; the anti-tumor immunity can also be promoted. And the medicine has the characteristic of high safety while playing a treatment role.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to application of an indole alkaloid compound in preparation of a drug for treating or preventing intestinal diseases. BACKGROUND

[0002] Serotonin (5-HT) is an important neurotransmitter and hormone, which plays a key role in the regulation of the gastrointestinal system. Recent studies have shown that dysfunction of the intestinal serotonin signaling pathway is a common pathophysiological mechanism of various gastrointestinal diseases. About 90% of the body's serotonin is synthesized and secreted by enterochromaffin cells (EC cells), which release serotonin to regulate intestinal peristalsis, secretion function and inflammatory response by sensing chemical and physical stimuli in the intestinal lumen.

[0003] 5-serotonin hyperfunction plays a key role in intestinal inflammatory diseases: (1) inflammatory bowel disease (IBD): including Crohn's disease and ulcerative colitis, the serum and tissue levels of serotonin in patients are significantly increased, which is closely related to the activity and severity of the disease; (2) irritable bowel syndrome (IBS): especially in patients with diarrhea-predominant IBS, the synthesis of intestinal serotonin is increased, leading to intestinal hypersensitivity and motor dysfunction; Serotonin hyperfunction plays an important role in tumor diseases: serotonin is involved in tumor cell proliferation and angiogenesis, and is an important driver of tumor progression. HT is closely related to tumors, and intestinal cancer, small cell lung cancer, and pancreatic neuroendocrine tumors have the ability to synthesize and secrete 5-HT. These tumor cells express tryptophan hydroxylase (TPH) to convert tryptophan to 5-HT and release it into the tumor microenvironment and worsen the disease. 1) Immunosuppression: 5-HT can up-regulate immune suppressor molecules (such as PD-L1) on the surface of tumor cells, while down-regulating the expression of major histocompatibility complex class I (MHC-I), allowing tumor cells to escape recognition and attack by the body's immune system and create an immunosuppressive tumor microenvironment. 2) Inducing neutrophil extracellular trap (NETs) formation: during the process of neuroendocrine tumor liver metastasis, 5-HT can induce neutrophils to release NETs, which can promote tumor cell adhesion, migration, and provide protection for tumor cells, promoting tumor metastasis.

[0004] Currently, there is a lack of therapeutic drugs for down-regulating the level of serotonin in peripheral blood and intestinal tissue in clinic. Traditional anti-inflammatory drugs such as mesalazine mainly play a role through anti-inflammatory and antioxidant mechanisms, but have no direct regulation on the serotonin signaling pathway, and long-term use has the risk of liver and kidney toxicity. Serotonin receptor antagonists such as alosetron have certain anti-diarrhea effect, but are invalid for inflammatory bowel disease (IBD), and have serious cardiovascular side effects, which limits their clinical application. Therefore, it has important clinical value to develop safe and effective serotonin modulators. SUMMARY

[0005] Based on the above technical problems, the present application provides a class of indole alkaloid compounds with specific structural characteristics for regulating serotonin and its signaling pathway, and treating and preventing related diseases. The present application also relates to the preparation method, pharmaceutical composition, administration regimen, combination therapy strategy and related diagnostic detection method of these compounds, which provides a systematic technical solution for intestinal serotonin dysfunction related diseases.

[0006] The first inventive point of the present application is the use of an indole alkaloid compound in the preparation of a drug for treating or preventing intestinal diseases.

[0007] Further, the intestinal disease is a disease related to high level of serotonin as a marker and abnormality of its signaling pathway.

[0008] Further, the intestinal disease includes inflammatory bowel disease, functional gastrointestinal disease, gastrointestinal tumor, intestinal barrier dysfunction related disease and other diseases with elevated serotonin as a biomarker. Preferably, the inflammatory bowel disease includes Crohn's disease, ulcerative colitis and microscopic colitis. Preferably, the functional gastrointestinal disease includes irritable bowel syndrome, functional diarrhea and functional constipation. Preferably, the gastrointestinal tumor includes colon cancer, rectal cancer and gastrointestinal neuroendocrine tumor.

[0009] Further, the indole alkaloid compound has the structure shown in general formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, precursor compound thereof, (I) wherein: R1 is selected from H, OH, OCH3, OC2H5, halogen, amido group; R2 is selected from H, CH3, C2H5, NH2, NHCH3, N(CH3)2, N(CH3) 3+ ; R3 is selected from H, CH3, C2H5.

[0010] Further, the indole alkaloid compound reduces blood serotonin levels to achieve treatment of intestinal diseases; reduces the serotonin activation and release of inflammatory factors, and upregulates one or more mechanisms of intestinal tight junction protein expression to inhibit inflammatory bowel disease; weaken the inhibitory effect of serotonin on anti-tumor immunity, thereby increasing the killing ability of lymphocytes in the tumor microenvironment and NK / macrophage secretion of granzyme and perforin, and reducing PDL1 expression to promote anti-tumor immunity.

[0011] Further, the indole alkaloid compound includes at least one of the following compounds:

[0012] Preferably, the indole alkaloid compound includes at least one of compounds C3, C4, C5, C6, C8, or C9; More preferably, the indole alkaloid compound includes one of , .

[0013] Further, the preparation method of the indole alkaloid compound includes the following steps: Taking tryptamine or its derivative as a starting material, functional group modification is carried out by acid, base, and salt catalysis, and optionally functional group introduction or removal, so as to obtain the indole alkaloid compound or its pharmaceutically acceptable salt.

[0014] The second invention point of the present application is a drug combination, which includes an effective amount of the above-mentioned indole alkaloid compound and an effective amount of one or more other therapeutic agents, and the weight ratio of the indole alkaloid compound to the other therapeutic agents is 1:(1-500); The other therapeutic agents include anti-inflammatory drugs, immunosuppressants, biological agents, probiotics, prebiotics, or intestinal barrier protectants; Preferably, the anti-inflammatory drugs include mesalazine, sulfasalazine, balsalazide sulfasalazine; Preferably, the immunosuppressants include azathioprine, methotrexate, cyclosporine; Preferably, the biological agents include ustekinumab O drug, pembrolizumab K drug, infliximab, adalimumab, vedolizumab.

[0015] Further, the anti-inflammatory drug is mesalazine; preferably, the weight ratio of the indole alkaloid compound to mesalazine is 1:(10-300).

[0016] The third invention of the present application is a pharmaceutical preparation comprising an effective amount of the above-mentioned indole base compound or a pharmaceutically acceptable salt thereof, wherein the content of the indole base compound in the pharmaceutical preparation is 0.01%-99% of the total weight, and a pharmaceutically acceptable carrier or excipient; Preferably, the administration route comprises oral administration, rectal administration, nasal administration, intravenous injection or intramuscular injection; the administration dose is 0.1-100 mg / kg body weight / day; Preferably, the dosage form thereof is selected from oral preparations, enteric preparations, sustained-release preparations, colon-targeted release preparations, injection preparations or inhalants; Preferably, the oral preparations comprise tablets, capsules, granules, oral liquids, buccal tablets; The enteric preparations comprise enteric tablets, enteric capsules and suppositories; The sustained-release preparations comprise sustained-release tablets, sustained-release capsules and sustained-release suppositories; The colon-targeted release preparations comprise colon-targeted release preparations; The injection preparations comprise injection solutions and lyophilized powder injections; The inhalants comprise sprays and powder inhalation.

[0017] Compared with the prior art, the present application has the following advantages: (1) Targeted precision: specific regulation of the intestinal serotonin signaling pathway significantly reduces the potential risk of central nervous system side effects while exerting therapeutic effects; (2) Mechanism synergy: multiple pharmacological mechanisms such as pro-inflammatory factor inhibition, serotonin receptor antagonism, transporter function regulation and intestinal barrier repair work together to produce synergistic therapeutic effects; (3) Wide application: suitable for various gastrointestinal diseases related to abnormal serotonin signaling, including inflammatory bowel disease, irritable bowel syndrome and colorectal cancer, etc., with a wide treatment spectrum; (4) Treatment optimization: when combined with existing clinical drugs (such as mesalazine), it shows significant synergistic effect, providing the possibility of reducing the dosage of existing drugs and alleviating their adverse reactions; (5) Diagnosis and treatment in one: detection of related biomarkers such as serotonin can guide patient stratification and medication regimen adjustment, providing a basis for precision treatment based on biomarkers. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 (A) Effect of compounds C1-C8 on diarrhea in mouse irritable bowel syndrome (n=6); (B) Effect of compounds C1-C8 on blood 5HT concentration in mice (n=6); Figure 2(A) Schematic diagram of animal experiment. Acute colitis was induced by adding 3% DSS to drinking water for 7 consecutive days, followed by 3 days of drinking plain water. Mice were orally administered C8 (15 or 30 mg / kg) or mesalazine (200 mg / kg) daily from day 1 to day 9. The experimental animals were sacrificed on day 10. (B) Representative images of the colon at sacrifice in each group. (C) Quantitative analysis of colon length in each group (n=8 per group). (D, E) Specific changes in mouse body weight (D) and inflammation score (E) were measured daily over 10 days. (F) Representative pathological images of colon tissue sections stained with H&E. Scale bar, 100 μm; data results are expressed as mean ± SEM, and statistical significance was analyzed using the Tukey multiple comparison test, (C)-(E). ##P<0.01 compared with the control group; #P<0.05 compared with the control group. **P<0.01, compared with the model group; *P<0.05, compared with the model group; Figure 3 C8 reduces pro-inflammatory cytokines in DSS-induced ulcerative colitis. Serum TNF-α and IL-1β levels were compared among the sham-operated group, DSS group, DSS + mesalazine (200 mg / kg) group, and DSS + C8 (15-30 mg / kg) group (n=6-8). Data are expressed as mean ± standard error (SEM). P-values ​​were determined using Tukey's test. ##P<0.01 vs. sham-operated group; **P<0.01, *P<0.05 vs. DSS group.

[0019] Figure 4 Compound C8 inhibited TNBS-induced Crohn's disease. (A) Schematic diagram of animal experiments. Mice were presensitized with 1% TNBS on day 1 and induced with 2.5% TNBS on day 7. C8 (15 or 30 mg / kg) was administered orally daily from day 7 to day 9. Mice were sacrificed on day 10. (B) Representative sections of colon tissue stained with H&E from each group (top: scale bar 200 μm; bottom: enlarged inset scale bar 50 μm). (C) Representative images of the colon taken at the time of sacrifice from each group. (D) Quantitative analysis of colon length (n=5-7 per group). (E) Rate of change in body weight during the experiment (%). Data are expressed as mean ± standard error. P values ​​were determined using Tukey's multiple comparison test (D) and (E). ##P<0.01 vs. sham-operated group; **P<0.01, *P<0.05 vs. TNBS group; Figure 5Compound C8 restored the intestinal epithelial barrier integrity in mice with ulcerative colitis and Crohn's disease. (A, B) Quantitative PCR analysis of mRNA encoding colitis-associated tight junction proteins in the colon of DSS-induced (A) or TNBS-induced (B) mice treated with different doses of C8 or solvent for 10 days. Results were normalized based on Gapdh expression. (C, D) Representative immunofluorescence staining of CLDN1, OCLN, and ZO1 (red) in the colonic tissue of DSS-induced (C) or TNBS-induced (D) mice 10 days after drug or solvent treatment, with reverse staining using DAPI (blue). Scale bar, 100 μm. Data are expressed as mean ± standard error. P values ​​were determined by Tukey multiple comparison test (A) and (B). ##P<0.01, #P<0.05 compared with sham-operated group; **P<0.01, *P<0.05 compared with DSS or TNBS group. ns, no statistical significance; Figure 6 Efficacy evaluation of compound C8 in vivo against colorectal cancer: A, B: Tumor photographs and tumor tissue quality after sampling; C: Changes in mouse body weight after administration; D: Changes in mouse tumor tissue volume after administration. Note: Experimental results are expressed as Mean±SEM, *P<0.05, **P<0.01; Figure 7 Effect of compound C8 on the infiltration of macrophages (Mφ) in colorectal cancer tumor tissue.

[0020] Figure 8 Compound C8 (10, 20 mg / kg) inhibits granzyme B in colorectal cancer tumor tissue. gzmb ), perforin ( prf1 ) and PD-L1 ( pdl1 The effect on gene expression. Note: Experimental results are expressed as Mean±SD, *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 9 The effect of different concentrations of C8 on the in vitro proliferation of colon cancer cells was detected by MTT assay (n=5).

[0022] Figure 10 C8 molecules promote the killing effect of splenic lymphocytes on MC-38 colon cancer cells in vitro (n=3). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0025] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0026] Example 1 This embodiment provides the application of indole base compounds in the preparation of drugs for treating or preventing intestinal diseases.

[0027] As a further preferred embodiment, the intestinal disease is a disease related to abnormalities in the serotonin signaling pathway.

[0028] Preferably, the intestinal diseases include inflammatory bowel disease, functional gastrointestinal disorders, gastrointestinal tumors, diseases related to intestinal barrier dysfunction, and other diseases where elevated serotonin is a biomarker; Preferably, the inflammatory bowel disease is Crohn's disease, ulcerative colitis, or microcolitis; Preferably, the functional gastrointestinal disorders are irritable bowel syndrome, functional diarrhea, and functional constipation; preferably, the gastrointestinal tumors are colon cancer, rectal cancer, and gastrointestinal neuroendocrine tumors.

[0029] As a further preferred embodiment, the indole base compound has the structure shown in general formula (I) or a pharmaceutically acceptable salt, solvent compound, polymorph, or precursor compound thereof. (I) Wherein: R1 is selected from H, OH, OCH3, OC2H5, halogens, and amide groups; R2 is selected from H and CH3. C2H5, NH2, NHCH3, N(CH3)2, N(CH3) 3+ R3 is selected from H, CH3, and C2H5.

[0030] As a further preferred embodiment, The indole base compounds achieve the treatment of intestinal diseases by lowering blood serotonin levels; One or more mechanisms by which serotonin reduces the activation and release of inflammatory factors and upregulates the expression of intestinal tight junction proteins can inhibit inflammatory bowel disease. Weakening the inhibitory effect of serotonin against tumor immunity, thereby enhancing lymphocyte killing ability and NK / macrophage secretion of granzymes and perforin in the tumor microenvironment, and reducing PDL1 expression through one or more mechanisms to promote anti-tumor immunity.

[0031] The serotonin receptors include serotonin receptor 3 (5-HT3) and serotonin receptor 4 (5-HT4). The tight junction proteins include Claudin-1, Occludin, and ZO-1. The pro-inflammatory cytokines include IL-1β, TNF-α, and IL-6.

[0032] As a further preferred embodiment, the indole base compound includes at least one of the following compounds:

[0033] Compound C1: 2-(5-methoxy-1H-indol-3-yl)ethylamine; Compound C2: 2-(5-fluoro-1H-indol-3-yl)ethylamine; Compound C3: 3-(2-aminoethyl)-1H-indole-5-carboxamide; Compound C4: 3-(2-aminopropyl)-1H-indole-5-phenol; Compound C5: 3-(2-(methylamino)ethyl)-1H-indole-5-phenol; Compound C6: 3-(2-(dimethylamino)ethyl)-1H-indole-5-phenol; Compound C7: 2-(5-methoxy-1H-indol-3-yl)-N,N-dimethylethylamine; Compound C8: 2-(5-hydroxy-1H-indol-3-yl)-N,N,N-trimethylethylamine; Compound C9: 2-(5-methoxy-1H-indol-3-yl)-N,N,N-trimethylethylamine; Preferably, the indole base compound includes at least one of compounds C3, C4, C5, C6, C8, or C9; More preferably, indole base compounds include , One of them.

[0034] Example 2 This embodiment provides a method for preparing the indole base compound described in Example 1, comprising the following steps: I. Preparation method of C8 The structural formula of compound 2-(5-hydroxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride is shown below:

[0035] Step 1-1: Synthesis of compound 2-(5-(benzyloxy)-1H-indol-3-yl)-N,N-dimethylethylamine Weigh 1.4792 g (5.90 mmol) of 4-benzyloxyphenylhydrazine hydrochloride (CAS No. 51145-58-5) into an appropriate amount of water and stir at room temperature for 10 min to ensure complete dissolution. Slowly add 400 μL of concentrated sulfuric acid to the system. Dissolve 1.516 ml (7.08 mmol) of 4-dimethylaminobutyraldehyde diethanolate (CAS No. 1116-77-4) in acetonitrile and add the mixed solution dropwise to a flask. React under N2 conditions for 20 h. Monitor the starting material using TLC during the reaction. After the starting material has reacted completely, add excess 10% NaOH solution to the reaction system, extract the organic phase with ethyl acetate, and dry with anhydrous sodium sulfate. Reduced pressure distillation, silica gel mixing, column chromatography separation, and dichloromethane:methanol = 20:1 eluent yielded 1.1355 g of compound 2-(5-(benzyloxy)-1H-indol-3-yl)-N,N-dimethylethylamine, a brownish-red oily substance with a yield of 78%.

[0036] 1 H NMR (500 MHz, DMSO- d 6) δ 10.71 (d, J = 2.7 Hz, 1H), 7.48 (dd, J =6.4, 1.6 Hz, 2H), 7.40-7.37 (m, 2H), 7.32 (dd, J = 7.6, 1.8 Hz, 1H), 7.25 (dd, J = 8.6, 2.1 Hz, 1H), 7.11 (d, J = 2.4 Hz, 2H), 6.81 (dd, J = 8.7, 2.4 Hz, 1H), 5.11(s,2H), 2.81 (dd, J = 9.1, 6.7 Hz, 2H), 2.58-2.54 (m, 2H), 2.27 (d, J = 2.0 Hz, 6H). Steps 1-2: Synthesis of compound 2-(5-(hydroxy)-1H-indol-3-yl)-N,N-dimethylethylamine At room temperature, 1.050 g (3.55 mmol) of 2-(5-(benzyloxy)-1H-indol-3-yl)-N,N-dimethylethylamine and 10% palladium on carbon (1.2465 g, 11.715 mmol) were weighed into a round-bottom flask and dissolved completely in a mixed solution (MeOH:THF=8:1). The reaction was carried out overnight for 6 h under H2 atmosphere. After the reaction was complete, the palladium on carbon was removed by diatomaceous earth filtration, washed with ethyl acetate, dried over anhydrous sodium sulfate, distilled under reduced pressure, mixed with silica gel, and separated by column chromatography with dichloromethane and methanol as eluents (20:1). 0.6377 g of 2-(5-(hydroxy)-1H-indol-3-yl)-N,N-dimethylethylamine was obtained as a brownish-yellow oil with a yield of 88%.

[0037] 1 H NMR (500 MHz, DMSO- d 6) δ 10.70 (d, J = 2.6 Hz, 1H), 8.74 (s, 1H)7.15(d, J = 8.6 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 6.64 (dd, J = 8.6, 2.3Hz, 1H), 3.23 -3.20 (m, 2H), 3.04- 3.01 (m, 2H), 2.78 (s, 6H). Steps 1-3: Synthesis of compound 2-(5-hydroxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride Weigh 554.4 mg (2.714 mmol) of 2-(5-hydroxy-1H-indol-3-yl)-N,N-dimethylethylamine into an appropriate amount of methanol, add 1.690 ml (27.141 mmol) of iodomethane to the system, and mix thoroughly. React in the dark for 48 h. After the reaction of the starting material was complete as monitored by TLC, the system solution was evaporated to dryness, and excess AgCl and methanol were added. The reaction was continued in the dark for 24 h. Impurities were filtered out. The mixture was then distilled under reduced pressure, mixed with silica gel, and separated by column chromatography using dichloromethane:methanol = 10:1 as the eluent to give 435.59 mg of 2-(5-hydroxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride (5-OH-TMT), a pale yellow solid, with a yield of 63%.

[0038] 1H NMR (500 MHz, DMSO-d6) δ 9.11 (d, J = 7.0 Hz, 1H), 8.44 (s, 1H), 7.25 – 7.17 (m, 2H), 7.06 (s, 1H), 6.80 (dd, J = 7.8, 2.1 Hz, 1H), 3.67 (t, J= 8.3 Hz, 2H), 3.28 (td, J = 8.3, 0.8 Hz, 2H), 3.21 (s, 9H). Steps 1-4: Preparation of C8 compound crystals 1) Dissolution stage: Place the crude 5-OH-TMT in an Erlenmeyer flask, add ethanol at a ratio of approximately 1g solid to 10-15mL solvent, heat to 60-70°C in a water bath or oil bath while stirring. If the solid is not completely dissolved, add ethanol in small amounts several times until it is completely dissolved.

[0039] 2) Hot filtration: Use a preheated funnel to perform suction filtration or gravity filtration while the solution is hot to remove insoluble and mechanical impurities. Maintain the solution temperature to prevent premature crystallization.

[0040] 3) Crystallization: Allow the hot filtrate to cool naturally to room temperature. Induce crystallization by rubbing the bottle wall with a glass rod. If crystallization is slow, further cool in an ice bath (0-5°C) and let stand for 30 minutes to several hours to allow sufficient crystals to separate. Collect the crystals by suction filtration or gravity filtration, and wash them 2-3 times with a small amount of cold ethanol (3-5 mL each time). Transfer the crystals to a watch glass or weighing bottle and allow them to air dry in a fume hood.

[0041] The X-ray powder diffraction pattern of the C8 crystal form includes the following peaks: 17.45, 17.79, 19.59, 22.43, 24.30, 26.05 and 30.22 º2θ.

[0042] II. Preparation method of C9 The structural formula of compound 2-(5-methoxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride is shown below:

[0043] Step 2-1: Synthesis of compound 2-(5-methoxy-1H-indol-3-yl)-N,N-dimethylethane-1-amine Weigh 4-methoxyphenylhydrazine hydrochloride (CAS No. 19501-58-7, 1.0303 g, 5.90 mmol) into an appropriate amount of water and stir at room temperature for 10 min to ensure complete dissolution. Slowly add 400 μL of concentrated sulfuric acid to the system. Then dissolve 4-dimethylaminobutyral diethanol acetal (CAS No. 1116-77-4, 1.516 ml, 7.08 mmol) in acetonitrile and add the mixed solution dropwise to a flask. React under N2 conditions for 20 h. Monitor the starting material using TLC during the reaction. After the starting material has reacted completely, add excess 10% NaOH solution to the reaction system, extract the organic phase with ethyl acetate, and dry with anhydrous sodium sulfate. Reduced pressure distillation, silica gel mixing, column chromatography separation, and dichloromethane:methanol = 30:1 eluent yielded 1.1334 g of compound 2-(5-methoxy-1H-indol-3-yl)-N,N-dimethylethyl-1-amine, a brownish-brown oily substance with a yield of 88%.

[0044] 1H NMR (500 MHz, DMSO-d6) δ 10.70 – 10.66 (m, 1H), 7.26 (d, J = 8.7Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.74 (dd, J =8.7, 2.4 Hz, 1H), 3.77 (s, 3H), 2.85 – 2.78 (m, 2H), 2.55 (dd, J = 9.0, 6.8Hz, 2H), 2.25 (s, 6H). Step 2-2: Synthesis of compound 2-(5-methoxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride Weigh 592.5 mg (2.714 mmol) of 2-(5-methoxy-1H-indol-3-yl)-N,N-dimethylethane-1-amine into an appropriate amount of methanol. Add 1.690 ml (27.141 mmol) of iodomethane to the system and mix thoroughly. Incubate in the dark for 48 h. After the reaction of the starting material is complete as monitored by TLC, evaporate the system solution to dryness, add excess AgCl and methanol, and incubate in the dark for 24 h. Filter out impurities. Distill under reduced pressure, mix with silica gel, and separate by column chromatography using dichloromethane:methanol = 10:1 as the eluent to obtain 402.92 mg of compound 2-(5-methoxy-1H-indol-3-yl)-N,N,N-trimethylethane-1-ammonium chloride (5-MeO-TMT), a pale yellow solid, with a yield of 55%.

[0045] 1H NMR (500 MHz, DMSO-d6) δ 10.98 (d, J = 2.6 Hz, 1H), 7.27 (d, J =8.7 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.76 (dd, J= 8.7, 2.4 Hz, 1H), 3.80 (s, 3H), 3.62 – 3.57 (m, 2H), 3.21 (s, 9H), 3.16 –3.11 (m, 2H). Steps 2-3: Preparation of C9 compound crystals Solvent evaporation method: Weigh an appropriate amount of 5-MeO-TMT and place it in a glass test tube. Add water or methanol at a ratio of approximately 1g solid to 10-15mL solvent. Stir thoroughly until no more dissolves and the mixture reaches saturation. After the solid has completely precipitated, transfer the supernatant to a new glass test tube. Then, place each sample at room temperature and slowly evaporate for 7 to 10 days. The rapid evaporation time is 3 to 4 hours.

[0046] Three crystals with different crystal forms were successfully obtained by evaporation using water and methanol as solvents. When water was used as the solvent and evaporated slowly, pale yellow flaky crystals were observed. When methanol was used as the solvent, pale yellow lumpy and granular crystals were observed with both rapid and slow evaporation methods, respectively. PXRD characterization identified these as two different crystal forms, named crystal form I (water as evaporation solvent, Form I) and crystal form II (methanol as evaporation solvent, Form II). Differential scanning calorimetry (DSC) analysis showed that crystal form I underwent an endothermic event at 185.35 °C, while crystal form II underwent endothermic events at 166.05 °C and 185.98 °C, indicating a transition to a stable crystal form. Furthermore, thermogravimetric analysis showed no weight loss in either crystal form, indicating that neither crystal form is a solvent compound. In this example, two crystal forms of the bufotenine derivative C9 (5-MeO-TMT) were obtained using a solvent method, and characterization by PXRD, DSC, and TGA ultimately determined crystal form I to be the dominant crystal form. The preparation method is not limited to this embodiment. Polymorphic preparation methods can also be used, including solvent crystallization, melt cooling crystallization, sublimation crystallization, grinding crystallization, spray drying and solvent removal.

[0047] The crystal forms of C9 include C9 crystal form I and C9 crystal form II; The X-ray powder diffraction pattern of the C9 crystal form I includes the following peaks: 14.5, 18.1, and 22.2º2θ; The C9 crystal form II includes C9 crystal form II-A and C9 crystal form II-A; The X-ray powder diffraction pattern of the C9 crystal form II-A includes the following peaks: 17.17, 20.1, 21.05, 21.71, 24.5 and 25.57 º2θ; The X-ray powder diffraction pattern of the C9 crystal form II-B includes the following peaks: 17.09, 20.02, 20.96, 21.63, 24.42 and 25.48º2θ.

[0048] In addition, it should be noted that C9 is a prodrug molecule of C8. The intraperitoneal injection dose of C9 (5-MeO-TMT) was 15 mg / kg, administered for 3 consecutive days. One hour after administration on the third day, the mice were euthanized, and tissues were taken for mass spectrometry analysis. The results are shown in Table 1 below. The prototype molecule C9 and the demethylated product C8 were identified. Their molecular weight, secondary fragments, and retention time were consistent with those of C8.

[0049] Table 1. Identification of C9 metabolites in vivo

[0050] Example 3 An example provides a pharmaceutical combination comprising an effective amount of the indole alkaloid compound of Example 1, and an effective amount of one or more other therapeutic agents, wherein the indole alkaloid compound and the other therapeutic agent are in a weight ratio of 1:(1-500); Preferably, the other therapeutic agents include anti-inflammatory drugs, immunosuppressants, biologics, probiotics, prebiotics, or intestinal barrier protectants; Preferably, the anti-inflammatory drug includes mesalazine, sulfasalazine, and balsalazine; Preferably, the immunosuppressants include azathioprine, methotrexate, and cyclosporine; Preferably, the biological agent includes volumab (O), pembrolizumab (K), infliximab, adalimumab, and vedolizumab.

[0051] As a further preferred embodiment, the anti-inflammatory drug is mesalazine; preferably, the weight ratio of the indole base compound to mesalazine is 1:(10-300). This exhibits a synergistic therapeutic effect and can reduce the dosage of mesalazine by 30-60%.

[0052] This embodiment also provides a pharmaceutical formulation comprising an effective amount of an indole base compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6, and a pharmaceutically acceptable carrier or excipient, wherein the indole base compound comprises 0.01%-99% of the total weight in the pharmaceutical formulation; Preferably, the route of administration includes oral, rectal, nasal, intravenous, or intramuscular injection; the dosage is 0.1-100 mg / kg body weight / day; Preferably, the dosage for experimental animals is 0.1-50 mg / kg body weight / day, more preferably 1-30 mg / kg body weight / day. The dosage for humans is 0.01-10 mg / kg body weight / day, more preferably 0.05-3 mg / kg body weight / day.

[0053] Preferably, the administration frequency is 1-4 times daily, preferably once daily.

[0054] Preferably, the course of treatment is 2-4 weeks in the acute phase and 3-6 months in the maintenance phase.

[0055] Preferably, the dosage form is selected from oral preparations, enteric-coated preparations, sustained-release preparations, colon-targeted release preparations, injectable preparations, or inhalers; Preferably, the oral preparation includes tablets, capsules, granules, oral liquids, and lozenges; The enteric-coated formulations include enteric-coated tablets and enteric-coated capsules; The sustained-release formulation includes sustained-release tablets and sustained-release capsules; The targeted colon-release formulation includes a colon-targeted release formulation; The injectable preparations include injection solutions and lyophilized powder injections. The inhalant includes sprays and powders.

[0056] Example 4 This embodiment provides a method for detecting a patient's serotonin level to assist in treatment decision-making, including: (a) Detection of serotonin concentration in serum or plasma; (b) Detect the level of 5-HIAA (5-hydroxyindoleacetic acid) in urine; (c) Detection of serotonin synthase TPH1 expression in intestinal tissue; (d) When the serotonin level in any of (a), (b), or (c) above exceeds 1.5 times the normal value, treatment with the indole base compounds described in this invention is recommended.

[0057] This embodiment also provides a method for diagnosing diseases related to hyperserotonin levels in the gut, including: (1) Measure the patient's serum serotonin level. (2) Assess intestinal barrier function markers: serum D-lactic acid, endotoxin, and fatty acid-binding protein; (3) Detection of inflammatory markers: CRP, procalcitonin, and cytokine profile; (4) Optionally assess intestinal barrier function markers and / or inflammatory markers, and use the combined score to determine the severity of the disease and treatment plan.

[0058] Example 5: Efficacy evaluation of the compound of the present invention on an irritable bowel syndrome model This embodiment established a serotonin-induced mouse model of irritable bowel syndrome (IBS) and evaluated the therapeutic effects of different indole compounds. C57BL / 6 mice were intraperitoneally injected with 5-HT (5 mg / kg), and 30 minutes later, the test compounds (30 mg / kg) were administered by gavage. The results showed that the order of compound activity was: C8≈C9>C3≈C4>C5>C6, among which C8 and C9 significantly reduced the diarrhea index (P<0.01) and serum 5-HT levels (P<0.01).

[0059] Experimental methods: (1) Diarrhea Experiment: C57 mice were randomly divided into a control group, a model group, different indole molecule treatment groups (30 mg / kg), and a mesalazine group (200 mg / kg). Chronic water avoidance stress (WAS) was used to model the diarrhea. Mice were placed in a 25°C water environment with a self-made box containing an upright platform (11cm x 5cm). The water surface was 1cm above the platform. The mice were subjected to stress for 2 hours daily for 10 consecutive days. The tested molecules were administered via gavage daily. Diarrhea was recorded after the last stress treatment. Diarrhea Index: Clean filter paper was placed at the bottom of the cage. The defecation of each mouse was observed, and the filter paper was changed every 2 hours. The defecation of the mice was recorded in detail for a total of 6 hours. The diameter of the loose stool was measured by the same researcher. If the filter paper stain was approximately circular, the diameter was measured; if the shape was irregular, the longest length and the approximate diameter of the circle were measured, and the average of the two was taken as the result; if the fecal boundaries were unclear and the number of pellets was unknown, it was considered as one pellet. Calculate the diarrhea index for each group of mice: Diarrhea index = loose stool rate × average loose stool grade, loose stool rate = number of loose stools / total number of stools, average loose stool grade = sum of loose stool grades / number of loose stools. Based on the diameter of the filter paper stain, <1cm is grade 1, 1cm-2cm is grade 2, 2cm-3cm is grade 3, and >3cm is grade 4. (2) 5HT Detection Experiment: C57 mice were randomly divided into a control group, a model group, different indole molecular treatment groups, and a mesalazine group (200 mg / kg). The control group was injected intraperitoneally with physiological saline, while the model group and drug treatment groups were injected intraperitoneally with 5-HT hydrochloride (5 mg / kg), once daily for 5 consecutive days. Half an hour after the injection of 5-HT hydrochloride, different treatment drugs (30 mg / kg) were administered by gavage. One hour after the last administration, blood samples were collected from the orbital rim to determine the concentration of serotonin 5-HT in the blood by mass spectrometry. 50 μL of whole blood was collected from the mice, 150 μL of methanol solution containing internal standard was added, vortexed, and centrifuged at high speed to remove proteins before mass spectrometry analysis.

[0060] Liquid chromatography-mass spectrometry (LC-MS) detection conditions: Samples were analyzed using UPLC-QTRAP-MS (AB 5500) with the following operating parameters: Column: XBridge C18 column (100 mm × 3.5 mm, 3.5 μm); Mobile phase: Phase A was acetonitrile, and Phase B was 0.1% formic acid in water; Gradient elution program: 0–0.5 min (0–95% B); 0.5–4.5 min (95–20% B); 4.5–5 min (20% B); 5–5.6 min (20–95% B); 5.6–6.1 min (0% B); Column temperature: 35℃; Flow rate: 0.5 mL / min; Injection volume: 5 μL. A QTRAP 5500 mass spectrometer was used in positive ion scanning mode with the following instrument parameters: CPU = 10 psi; GS1 = 30 psi; IS = -4500 V; TEMP = -525 V. 5-HT hydrochloride: parent ion 177.0 m / z, daughter ion 160.0 m / z, declustering voltage 80 V, collision energy 12 eV.

[0061] Experimental results: such as Figure 1 The results show the effects of gavage administration of different indole molecules (30 mg / kg) on ​​diarrhea caused by irritable bowel syndrome (IBS) in mice. After modeling, the diarrhea index of normal mice was 0, while that of the model group was 0.34 ± 0.17. Screening revealed that the following molecules effectively reduced the diarrhea index in mice: C8≈C9>C3≈C4>C5>C6. Compared with the model group, C3, C4, C5, C8, and C9 showed significant pharmacological effects in inhibiting stress-induced diarrhea (*P < 0.05, **P < 0.01); however, C1, C2, and C7 did not exhibit this pharmacological effect. Furthermore, under these test conditions, mesalazine did not show any anti-stress diarrhea effect.

[0062] Liquid chromatography-mass spectrometry analysis revealed that the order of decrease in peripheral blood 5HT levels was: C8≈C9>C3≈C4>C5>C6.

[0063] Example 6: Evaluation of the efficacy of the present invention in a model of ulcerative colitis Acute colitis was induced in C57BL / 6 mice with 3% DSS for 7 consecutive days, while the mice were treated with compound C8 (15 or 30 mg / kg / day). The results showed that: (1) the colon length was significantly shortened (recovered to 85% of normal); (2) the disease activity index (DAI) score was reduced (by 60%); (3) pathological examination showed that the integrity of the intestinal structure was protected and the infiltration of inflammatory cells was significantly reduced; (4) the expression of tight junction proteins Claudin-1, Occludin, and ZO-1 was upregulated (2-3 times).

[0064] Experimental Methods: C57BL / 6 mice (male, 6-8 weeks old) were housed together for one week prior to the experiment. A 12-hour light-dark cycle was used, with constant temperature control (22±2°C), and free access to standard feed and water. All animal experiments were conducted strictly in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals".

[0065] Dextran sulfate sodium (DSS)-induced colitis: Acute colitis was induced in mice by adding 3% (w / v) dextran sulfate (molecular weight 40-50 kDa; MPBiomedicals) to their drinking water for 7 consecutive days, followed by a 3-day recovery period with regular drinking water. On day 10, male C57BL / 6 mice were sacrificed, and the colon was rapidly removed. Colon length was measured from the cecotocolonic junction to the anal verge as a macroscopic indicator of inflammation. The distal colonic segment was then harvested for histological analysis (H&E staining and immunofluorescence).

[0066] The Disease Activity Index (DAI) is a comprehensive scoring system based on three indicators: weight change, fecal characteristics, and bleeding. It is primarily used for evaluation in animal models of inflammatory bowel diseases such as ulcerative colitis (UC). The scoring criteria assign each indicator a score from 0 to 4, and the average of the three indicators is taken as the DAI value, directly reflecting the severity of intestinal inflammation and the effectiveness of treatment.

[0067] Weight rating: graded by daily weight loss percentage, from 0 (no weight loss) to 4 (weight loss >20%). Stool characteristics: Normal stool (0 points), semi-loose stool (2 points), loose stool (4 points) Bleeding severity: No bleeding (0 points), positive occult blood (2 points), gross hematochezia (4 points) First, an acute colitis model was established in C57BL / 6 mice using sodium dextran sulfate (DSS). The treatment regimen was as follows: Figure 2 As shown in Figure A, acute colitis was induced in mice by adding 3% DSS to their drinking water for 7 consecutive days, followed by 3 days of drinking plain water. Mice were orally administered compound C8 (15 or 30 mg / kg) or mesalazine 200 mg / kg daily from day 1 to day 9. The experimental animals were sacrificed on day 10.

[0068] Experimental results: such as Figure 2 As shown, mice treated with DSS developed severe colitis, and their colons were significantly shortened, visibly so, compared to the healthy control group (Sham). Oral administration of compound C8 significantly alleviated this colonic shortening, indicating its protective effect on colonic structural integrity. Figure 2As shown in BC. Consistent with experimental observations, mice in the DSS group exhibited rapid and sustained weight loss, accompanied by correspondingly higher Disease Activity Index (DAI) scores—an index that comprehensively reflects weight loss, fecal characteristics, and rectal bleeding. Compound C8 significantly reduced weight loss, as shown in BC. Figure 2 As shown in D, it suppressed diarrhea scores as shown in Figure D. Figure 2 As shown in Figure E, HE pathological staining revealed severe histopathological damage in the DSS group, characterized by extensive destruction of mucosal structure, disappearance of crypts, depletion of goblet cells, and extensive infiltration of inflammatory cells into the lamina propria. In contrast, the compound C8 group significantly maintained the integrity of the colonic structure, preserved crypt structure, restored the number of goblet cells, and significantly reduced immune cell infiltration. Figure 2 As shown in F.

[0069] C8 administration significantly inhibited the levels of serum inflammatory factors TNFα and IL-1 in mice with DSS-induced colitis, as shown in... Figure 3 As shown.

[0070] The positive control drug mesalazine showed beneficial therapeutic effects, but considering all the above indicators, the anti-colitis efficacy of compound C8 at 30 mg / kg was significantly better than that of mesalazine at 200 mg / kg. Therefore, compound C8 has significant pharmacological effects in inhibiting colitis and protecting the intestines.

[0071] Example 7: Pharmacodynamic evaluation of the compounds of the present invention in a Crohn's disease model. In the treatment of Crohn's disease-like colitis induced by rectal administration of TNBS, the C8 compound treatment group showed a significant protective effect: significant improvement in intestinal pathological damage, a 50% reduction in weight loss, and significant recovery of intestinal length shortening.

[0072] Experimental methods: 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced a mouse model of Crohn's disease. Colitis was induced using the previously described two-step method. On day 1, 200 µL of an acetone-olive oil mixture (1:1 volume ratio) containing 1% TNBS was applied to the dorsal skin of shaved male C57BL / 6 mice to sensitize the epidermis. On day 7, after anesthesia with isoflurane, mice were rectally instilled with 100 µL of a 2.5% TNBS solution dissolved in 50% ethanol via a 3.5-gauge catheter. Following induction, mice were randomly assigned to groups as described above. Figure 4 Mice in the sham-operated group and the TNBS carrier group were administered 0.9% saline via oral gavage daily from day 7 to day 9. The treatment group received compound C8 via oral gavage at 15 mg / kg or 30 mg / kg during the same period. Mice were sacrificed on day 10, and colon tissue was collected and analyzed.

[0073] Experimental results: such as Figure 4As shown, the effect of compound C8 in a T-cell-dependent TNBS-induced Crohn's disease model was evaluated. Histopathological analysis of H&E-stained colon sections showed that compound C8 treatment significantly protected tissue structure. Mice in the TNBS group exhibited severe Crohn's-like lesions in the colon—manifested as extensive destruction of mucosal structure, disappearance of crypts, and dense inflammatory infiltration; while the pathological features of mice in the treatment group were significantly improved, with significantly more intact colonic mucosal structure, reduced crypt damage, and significantly decreased immune cell infiltration. Figure 4 B). Observational examination of the resected colon confirmed these histological findings. The colon of TNBS-treated mice showed significant inflammation, manifested as edematous thickening and significant shortening of the parietal layer; TNBS-treated mice also exhibited persistent and significant weight loss. In contrast, oral administration of compound C8 effectively reversed intestinal damage and significantly maintained colon length. Figure 4 CD significantly reduced the degree of weight loss in mice. Figure 4 E). These findings confirm that compound C8 has a potent protective effect on intestinal tissue and its potential application in the treatment of Crohn's disease.

[0074] Example 8: Barrier protection effect of the compounds of the present invention against DSS and TNBS-induced intestinal damage. In this embodiment, we found that the anti-inflammatory drug mesalazine does not have a protective effect on the gut; however, compound C8 has a significant effect on enhancing the intestinal barrier and promoting the expression of intestinal cell junction proteins.

[0075] Experimental methods: Quantitative real-time PCR detection: Total RNA was extracted from frozen mouse colon tissue using RNA Isolater total RNA extraction reagent. 1 μg of total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vistar Biotechnology Co., Ltd., China). qRT-PCR was performed using AceQ universal SYBR qPCR premix on a CFX100 real-time quantitative PCR detection system (Bio-Rad, Hercules, USA). The thermal cycling conditions were: 95℃ pre-denaturation for 2.5 min, followed by 35 cycles (95℃ denaturation for 15 s, 60℃ extension for 30 s). Melting curve analysis was used to verify the specificity of the amplified fragments. Relative mRNA expression levels were measured using a 2-1... -ΔΔCt The calculation was performed using the internal reference gene Gapdh (mouse) or ACTIN (human), and normalized accordingly.

[0076] Immunohistochemical analysis: Formalin-fixed and paraffin-embedded mouse colon tissue was sectioned. Morphological evaluation was performed using hematoxylin and eosin (H&E) staining. In the immunofluorescence (IF) experiment, sections were heat-induced antigen retrieval with sodium citrate buffer, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum. The sections were then incubated overnight at 4°C with the following primary antibodies (all diluted 1:100): anti-CLDN1 (Abmart, PA2349F), anti-OCLN (Abmart, TD7504F), and anti-ZO1 (Abmart, TA5145F). Detection was performed using Alexa Fluor-labeled secondary antibodies (1:100 dilution). Detection was also performed using Alexa Fluor-labeled secondary antibodies (1:200; Absin), with cell nuclei reverse-stained with DAPI.

[0077] Experimental results: such as Figure 5 As shown, intestinal epithelial barrier dysfunction is a key pathophysiological feature of inflammatory bowel disease. Therefore, we investigated whether the therapeutic effect of compound C8 involves the maintenance of barrier function. We first assessed the gene expression encoding key tight junction (TJ) proteins. Quantitative RT-PCR analysis showed that colonic expression of Cldn1 (tight junction protein-1), Ocln (occlusion protein), and Tjp1 (tight junction protein 1, also known as ZO-1) was significantly downregulated in both DSS and TNBS-induced colitis. Notably, in both DSS and TNBS-induced colitis, treatment with compound C8 significantly restored the expression of all three TJ genes in both models. Figure 5 AB). To validate these findings at the protein level, we performed immunofluorescence staining for CLDN1, OCLN, and ZO-1. In colon sections from control colitis mice, the expression of these tight junction proteins was significantly reduced, and the linear arrangement at the apical intercellular junctions of epithelial cells was severely disrupted. C8 restored their normal localization at the colonic epithelial cell junctions (AB). Figure 5 CD) strengthens the intestinal epithelial barrier function.

[0078] In stark contrast, mesalazine, a recognized positive control drug with anti-inflammatory activity, showed no effect on gene expression of the key tight junction (TJ) protein in the intestine of the aforementioned model. Therefore, mesalazine does not have the efficacy of promoting intestinal epithelial barrier function. This comparison clearly demonstrates the unique efficacy and clinical application value of compound C8 in restoring and promoting intestinal epithelial barrier function by regulating the excessive release of 5HT.

[0079] Example 9: Combined use of the compound of the present invention with mesalazine Due to the high risk of liver damage associated with long-term use of mesalazine, clinical practice often seeks to reduce the dosage through combination therapy with multiple drugs. This example presents a novel drug combination—bufotenine quaternary ammonium salt + mesalazine.

[0080] The combined effects of compounds C8 and C9 with mesalazine were evaluated in a DSS colitis model. The experimental design included: monotherapy groups (mesalazine 120 mg / kg, C8 10 mg / kg, C8 20 mg / kg, C9 10 mg / kg, C9 20 mg / kg) and combination therapy groups (C8 + mesalazine, C9 + mesalazine). Results showed a significant synergistic effect with the combination therapy. In the C8 + mesalazine combination group: the DAI inhibition rate increased from 18.2% and 35.3% in the monotherapy group to 56.1%.

[0081] In the C9 + mesalazine combination group, the DAI inhibition rate increased from 18.2% and 47.1% in the monotherapy group to 64.7%.

[0082] Synergy index (CI) calculations show that the CI value of combined drug use is 0.6-0.8, indicating a moderate to strong synergistic effect.

[0083] Experimental method: Same as in Example 8.

[0084] Experimental Results: After gavage administration of sub-effective doses of mesalazine (120 mg / kg) and compound C8 (10-20 mg / kg), the DAI score reached its peak on day 9 post-modeling. The DAI score inhibition rates in the mesalazine and compound C8 (5-OH-TMT) monotherapy groups were 18.2%, 23.8%, and 35.3%, respectively. However, when the above doses of mesalazine were combined with compound C8, the DAI score inhibition rates significantly increased to 48.3% and 56.1% compared to pre-administration levels (P < 0.05), indicating a more significant anti-colitis effect in the combination group. This suggests that compound C8 has a better anti-ulcerative colitis effect than mesalazine at lower doses; and that compound C8 and mesalazine have a synergistic effect.

[0085] Administering mesalazine (120 mg / kg) and compound C9 (10.8-21.6 mg / kg) by gavage resulted in peak DAI scores on day 9 post-modeling. In the mesalazine and compound C9 monotherapy groups, the DAI score inhibition rates were 18.2%, 29.5%, and 47.1%, respectively. However, when the above-mentioned doses of mesalazine and compound C9 were used in combination, the DAI score inhibition rates significantly increased to 48.0% (P < 0.05) and 64.7% (P < 0.01) compared to pre-administration levels, indicating a more significant anti-colitis effect in the combination group. This suggests that compound C9 has a better anti-ulcerative colitis effect than mesalazine at lower doses; and that compound C9 has a synergistic effect when used in combination with mesalazine. Specific results are shown in Table 2.

[0086] Table 2. Effects of compounds C8 and C9 in combination with mesalazine on DAI scores in DDS-induced colitis in mice (n=6)

[0087] Note: ##P<0.01, indicating a highly significant difference between the model group and the normal group. **P<0.01, indicating a highly significant difference between the model group and the normal group. △△P<0.01, indicating a significant difference between the combined treatment group and the toad tryptamine quaternary ammonium salt group.

[0088] Example 10: Effect of the compounds of the present invention on the MC38 colon cancer xenograft model Chronic colitis (especially inflammatory bowel diseases such as ulcerative colitis and Crohn's disease) is a high-risk factor for colorectal cancer. The gradual process from inflammation to carcinogenesis usually takes 5-10 years. During repeated erosion, ulceration, and repair of the intestinal mucosa, mild, moderate, and severe dysplasia occurs, eventually developing into cancer. Studies have shown that in colorectal cancer, serotonin regulates cancer cell proliferation and angiogenesis, ultimately leading to tumor progression. Clinically, abnormally elevated serum serotonin levels in colorectal cancer patients indicate that serotonin is an important biomarker for colorectal cancer progression. The human colorectal cancer cell line HT29 also shows high expression of serotonin receptors. Lowering blood serotonin levels is a novel strategy for intervening in colorectal cancer.

[0089] In this embodiment, a xenograft tumor model was established by subcutaneously inoculating MC38 colon cancer cells into C57BL / 6 mice. The treatment groups with compound C8 (10 and 20 mg / kg, intraperitoneal injection, once daily for 14 days) showed significant anti-tumor effects: tumor volume decreased by 40-55%, and tumor weight decreased by 35-48%. C8 highly expressed perforin and granzyme B in lymphocytes, NK cells, and macrophages, indicating enhanced immune killing function.

[0090] Experimental methods: (1) MC38 mouse colon cancer transplant model Logarithmic growth phase MC38 mouse colon cancer cells were collected, washed with PBS to remove impurities, and digested with trypsin to obtain a cell suspension. 10 μL of the cell suspension was transferred to a hemocytometer for cell counting, and the cell suspension concentration was adjusted to 102. 7 single-cell suspension, 200 μL / mouse, kept on ice. Mice that had been acclimatized for one week had their right axillary hair removed, and were subcutaneously inoculated with the adjusted density of single-cell suspension. After successful modeling, mice were randomly divided into three groups: a model group (PBS group), a medium-dose group (10 mg / kg) of compound C8, and a high-dose group (20 mg / kg), with five mice in each group. The first administration was given when the tumor volume was approximately 50 mm³. The model group received an equal volume of PBS solution via intraperitoneal injection once daily; the medium-dose group received 10 mg / kg via intraperitoneal injection once daily; and the high-dose group received 20 mg / kg via intraperitoneal injection once daily, for two consecutive weeks. The long axis (a) and short axis (b) of the tumor were measured daily starting from the day of administration (Day 1). The tumor volume was calculated using the formula: V = 0.5ab². All mice were euthanized on Day 15, and tumors were collected, weighed, and photographed.

[0091] (2) Analysis of immune infiltration indicators in tumor tissue After euthanasia of mice, tumor tissue was dissected and homogenized. An appropriate amount of digestive enzyme working solution was added and thoroughly mixed. The digested single-cell suspension was filtered through a 70 μm cell sieve, and RPMI 1640 medium containing 10% fetal bovine serum was added to terminate digestion. The cells were centrifuged, and the supernatant was discarded. The cells were washed twice with flow cytometry buffer, and the corresponding antibody was added at a 1:100 μL ratio. After thorough mixing, the cells were incubated at 4°C in the dark for 30 min. The cells were then washed again with flow cytometry buffer, transferred to flow cytometry tubes, and analyzed.

[0092] Tumor tissue infiltrated by F4 / 80 macrophages was detected by immunofluorescence. The tissue was immersed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. The sections were baked on a staining rack for 1 h, then sequentially immersed in xylene for 30 min, 95% ethanol, 75% ethanol, 50% ethanol, and ultrapure water for 10 min. 0.5% Triton X-100 was added, and the tissue was incubated at room temperature for 30 min to permeate the cells and facilitate nuclear staining. 5% goat serum was added to the tissue, and the tissue was blocked at room temperature for 2 h. Antibody was added to the tissue, and the tissue was incubated overnight. After washing with PBS, 0.01% DAPI solution was added to the tissue, and the tissue was incubated and washed. An anti-fluorescence quencher was added, the tissue was mounted, stored in the dark, and then analyzed by immunofluorescence.

[0093] Approximately 50 mg of tumor tissue from each animal was collected in an Eppendorf tube for q-PCR gene expression detection. 1 mL of Trizol solution was added, and RNA concentration was extracted and measured. Reverse transcription was performed according to the kit's settings and procedures. After reverse transcription, cDNA was extracted and stored at -20 °C for later use. Granulase B was then added... gzmb ), perforin ( prf1 ) and PD-L1 ( pdl1 Gene primers, q-PCR amplification.

[0094] (3) In vitro proliferation experiment of colon cancer cells Cells in the logarithmic growth phase were collected, washed with PBS to remove impurities, and digested with trypsin to obtain a cell suspension. 10 μL of the cell suspension was transferred to a hemocytometer for cell counting, and the cell concentration was adjusted. The cells were then evenly seeded into 96-well plates, with 5000 cells per well. The 96-well plates were incubated overnight to allow cell adhesion. C8 stock solution was sequentially diluted to twelve concentrations: 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 μM. Each concentration was added to five replicates in each well. The 96-well plates containing the drug were incubated for 72 h. After incubation, the plates were removed, and 20 μL of MTT solution was added to each well, with the plates incubated for another 4 h. Take out the 96-well plate, add 200 μL of DMSO solution to each well, shake at 300 rpm for 10 min, and measure the absorbance using a microplate reader.

[0095] (4) In vitro killing experiment of spleen lymphocytes against fluorescently tagged MC-38 2.0 × 10⁵ cells / mL of logarithmically growing MC-38 cells were seeded into 12-well plates, and 1.6 μL of fluorescently tagged lentiviral vector was added and cultured for 24 h. Meanwhile, mouse spleens were crushed into a cell suspension, and erythrocytes were lysed and washed for later use. Ten stable transgenic MC-38 cells with fluorescent tags were obtained by trypsin digestion. 6 Cells / mL, 100 μL of cell suspension was transferred to a six-well plate, and 500 μL of 300 μM C8 assay molecule was added to the wells. Incubation was carried out overnight. Lymphocytes treated with concanavalin A were then collected and cultured for 10... 7 Cells were cultured at a ratio of MC-38 cells to spleen lymphocytes of 1:10, with three replicates. The cells were seeded into six-well MC-38 plates and cultured for 48 h. After culture, the cells were resuspended in PBS and transferred to flow cytometry tubes, stored on ice, and analyzed.

[0096] (5) Statistical analysis The statistical differences between experimental groups were compared using GraphPad Prism 9 software with Student's t-test analysis. A p-value < 0.05 was considered statistically significant.

[0097] Experimental results: such as Figure 6 As shown, during the administration process, the body weight of mice in all groups showed an increasing trend, with the model group (Model) showing a larger increase due to rapid tumor growth, while the body weight changes in the medium-dose (10 mg / kg) and high-dose (20 mg / kg) groups were smaller. After administration, the tumor volume of mice showed an increasing trend, with the growth in the administration groups tending to slow down. Starting from the sixth day, the tumor volume growth in the high-dose group (20 mg / kg) was slower than that in the model group (Model), showing a statistically significant difference (p value < 0.05). The tumor volume growth in the medium-dose group (10 mg / kg) was also slower than that in the model group (Model), showing a statistically significant difference starting from the sixth day (p value < 0.05). After administration, the body weight of the medium-dose group, high-dose group, and model group was smaller than that of the model group, showing a statistically significant difference (p value < 0.05), indicating that C8 can significantly inhibit the growth of colon cancer xenografts in mice.

[0098] like Figure 7 As shown, immunofluorescence results also confirmed that macrophage infiltration in tumor tissue increased after drug administration. Therefore, C8 may inhibit tumor cell proliferation by promoting immune cell infiltration into tumor tissue.

[0099] like Figure 8 As shown in the q-PCR results, compared with the blank group (con), the expression of perforin and granzyme B from lymphocytes or other killer cells in tumor tissue was significantly increased in the C8-treated group in a dose-dependent manner. This indicates that C8 can promote the secretion of granzyme and perforin by cytotoxic T cells and NK cells, exert cytotoxic effects, and inhibit tumor proliferation.

[0100] Stimulation by serotonin (5HT) leads to enhanced PD-L1 expression in tumor cells. Literature has confirmed that attenuation of peripheral 5-HT derived from platelets can inhibit tumor growth and enhance the efficacy of immune checkpoint inhibitory therapy (Attenuation of peripheral serotonin inhibits tumor growth and enhances immune checkpoint blockade therapy in murine tumor models. Sci Transl Med. 2021 Sep 15;13(611):eabc8188). In this embodiment, as... Figure 8As shown in the q-PCR results, the expression of the PD-L1 gene in tumor tissue was significantly reduced in the C8-treated group compared to the control group (con), and this reduction was dose-dependent. This indicates that C8 exerts a unique anti-tumor mechanism, which is achieved by reducing peripheral blood serotonin (5HT) levels. Given that reducing peripheral 5-HT levels is a highly valuable problem in the field of anti-tumor immunotherapy, this invention screens promising C8 and C9 molecules from a variety of candidate molecules for use in tumor immunotherapy, particularly for the regulation of PD-L1 immune checkpoint molecules. This mechanism also supports the combined application of C8 or C9 molecules with PD-1 antibodies.

[0101] like Figure 9 As shown, C8 (0.01-1000 μM) did not directly inhibit the in vitro proliferation of three types of colon cancer cells: MC38, SW480, and SW620. Compared with the control group, there was no significant difference in tumor cell survival rate among the C8 concentration groups. This indicates that the properties of C8 are completely different from those of bufotalin, which has direct tumor-killing activity derived from toad venom.

[0102] like Figure 10 As shown, flow cytometry results indicated that MC38 cells pretreated with C8 (50-100 μM) followed by co-culture with mouse spleen lymphocytes (20:1 ratio of mouse spleen lymphocytes to MC38 cells) significantly reduced the proportion of MC38 cells compared to the control group. This further demonstrates that C8 can promote the killing effect of lymphocytes on colon cancer cells.

[0103] Example 11 Acute Toxicity Test Experimental Methods: Following the "Guidelines for Acute Toxicity of Chemical Drugs," each group of animals was administered the drug at a dose of 20 mL / kg via gavage. Animals were randomly divided into 5 groups, with 6 animals in each group (half male and half female): 200 mg / kg (C8 group), 400 mg / kg (C8 group), 800 mg / kg (C8 group), 1600 mg / kg (C8 group), and a blank control group. The blank control group received an equal volume of physiological saline.

[0104] Each group received a single dose via gavage, while the model group received an equal volume of physiological saline. After gavage administration, all groups were fed normally. Observations were taken every 15 minutes for the first 2 hours after administration; every 30 minutes for the first 2-4 hours; every hour for the first 4-8 hours; and every 4 hours for the first 8-24 hours. From the second day after administration, observations were taken daily, and mouse weight was recorded. Close monitoring of movement, food and water intake was conducted, along with any abnormal muscle movements, external responses, pupillary changes, abnormal secretions, abnormal urination and defecation, bulging eyes, drooping eyelids, abnormal breathing, changes in coat color, and any toxic reactions or mortality that may occur within 14 days.

[0105] Experimental Results: Mice were orally administered compound C8 and observed for 14 days. No hallucinations or other significant behavioral abnormalities were observed at 800 mg / kg, classifying it as low toxicity. No significant toxic reactions were observed at the effective dose, indicating a wide safety window. As shown in Table 3, one hour after administration, two mice in the 1600 mg / kg group experienced convulsive death, indicating acute toxicity at this dose. The remaining mice survived normally for 14 days, demonstrating that C8 has a wide therapeutic index (lethal dose / effective dose) and good safety profile.

[0106] Table 3. Survival status of mice 14 days after drug administration (n=6)

[0107] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of an indole base compound in the preparation of a medicament for treating or preventing intestinal diseases.

2. Use according to claim 1, characterized in that, The intestinal diseases are diseases caused by high levels of serotonin as a marker and related diseases caused by abnormalities in the signaling pathways thereof.

3. Use according to claim 2, characterized in that, The intestinal diseases include inflammatory bowel disease, functional gastrointestinal disease, gastrointestinal tumor, intestinal barrier dysfunction related disease and other diseases with elevated blood serotonin as a biomarker. Preferably, the inflammatory bowel disease includes Crohn's disease, ulcerative colitis, microscopic colitis. Preferably, the functional gastrointestinal disease includes irritable bowel syndrome, functional diarrhea, functional constipation. Preferably, the gastrointestinal tumor includes colon cancer, rectal cancer, gastrointestinal neuroendocrine tumor.

4. Use according to claim 1, characterized in that, The indole base compound has the structure shown in general formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, precursor compound thereof, （Ⅰ） wherein: R1 is selected from H, OH, OCH3, OC2H5, halogen, amide group; R2 is selected from H, CH3, C2H5, NH2, NHCH3, N(CH3)2, N(CH3) 3+ ; R3is selected from H, CH3, C2H5.

5. The use according to claim 1, characterized in that, The indole base compound reduces blood serotonin levels to achieve treatment of intestinal diseases. One or more mechanisms of reducing the activation and release of inflammatory factors by serotonin and up-regulating the expression of intestinal tight junction proteins to inhibit inflammatory bowel disease. One or more mechanisms of weakening the inhibitory effect of serotonin on anti-tumor immunity to improve the killing ability of lymphocytes and NK / macrophage secretion of granzyme and perforin in the tumor microenvironment and reduce PDL1 expression to promote anti-tumor immunity.

6. Use according to claim 4, characterized in that, The indole base compound includes at least one of the following compounds: Preferably, the indole base compound includes at least one of compounds C3, C4, C5, C6, C8 or C9. More preferably the indole base compound comprises one of , .

7. Use according to claim 6, characterized in that, The preparation method of the indole base compound includes the following steps: Using tryptamine or its derivative as a starting material, functional group modification is carried out by acid, base and salt catalysis, and optional functional group introduction or removal to obtain an indole base compound or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical combination, characterized in that, It includes an effective amount of the indole base compound of any one of claims 1-7 and an effective amount of one or more other therapeutic agents, and the weight ratio of the indole base compound to the other therapeutic agent is 1:(1-500); The other therapeutic agent includes an anti-inflammatory drug, an immunosuppressant, a biological agent, a probiotic, a prebiotic or an intestinal barrier protective agent; Preferably, the anti-inflammatory drug includes mesalazine, sulfasalazine, balsalazide sulfasalazine; Preferably, the immunosuppressant includes azathioprine, methotrexate, cyclosporine; Preferably, the biological agent includes vedolizumab, pabozilumab, infliximab, adalimumab, vedolizumab.

9. The pharmaceutical combination according to claim 8, characterized in that, The anti-inflammatory drug is mesalazine; preferably, the weight ratio of the indole base compound to mesalazine is 1:(10-300).

10. A pharmaceutical preparation, characterized in that, It includes an effective amount of the indole base compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient, and the content of the indole base compound in the pharmaceutical preparation is 0.01%-99% of the total weight. Preferably, the administration route comprises oral, rectal, nasal, intravenous injection or intramuscular injection; the administration dose is 0.1-100 mg / kg body weight / day; Preferably, the dosage form thereof is selected from oral preparation, enteric preparation, sustained release preparation, colon-targeted release preparation, injection preparation or inhalant; Preferably, the oral preparation comprises tablet, capsule, granule, oral liquid, buccal tablet; The enteric preparation comprises enteric tablet, enteric capsule, suppository; The sustained release preparation comprises sustained release tablet, sustained release capsule, sustained release suppository; The colon-targeted release preparation comprises colon-targeted release preparation; The injection preparation comprises injection liquid, freeze-dried powder injection; The inhalant comprises spray, powder inhalation.

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