5-HT near-infrared fluorescent probe and preparation method and application thereof

Abstract

The present application relates to the technical field of 5-HT detection, in particular to a 5-HT near-infrared fluorescent probe and a preparation method and application thereof. The present application synthesizes a novel 5-HT near-infrared fluorescent probe, which has a longer emission wavelength (760 nm) and stronger tissue penetration, can effectively detect the level change of 5-HT in an IBD model, and can monitor the 5-HT level in IBD-related pathology in real time.

Description

Technical Field

[0001] This invention relates to the field of 5-HT detection technology, specifically to a 5-HT near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] Serotonin (5-HT) is a biologically significant monoamine neurotransmitter that plays a crucial role in the physiological and pathological processes of mammals. Under normal physiological conditions, 5-HT is mainly synthesized by enterochromaffin cells (EC cells) in the intestine using dietary tryptophan as a raw material, catalyzed by tryptophan hydroxylase 1 (TPH1), and regulates intestinal peristalsis, mucosal barrier function, and immune homeostasis. However, dysregulation of 5-HT levels can trigger intestinal inflammation by abnormally activating immune pathways, damaging the mucosal barrier, and disrupting the balance of gut microbiota, leading to cellular dysfunction and tissue damage.

[0003] The pathogenesis of intestinal dysfunction and inflammatory bowel disease (IBD, including ulcerative colitis (UC) and Crohn's disease (CD) has been confirmed to be closely related to 5-HT. Pathological examination shows that IBD patients have increased intestinal EC cells, elevated TPH1 expression, and upregulated serum and intestinal tissue 5-HT levels, suggesting enhanced 5-HT production in the disease state. 5-HT can bind to intestinal immune and epithelial cell surface receptors (such as 5-HT7R), activate related signaling pathways, promote the release of pro-inflammatory factors, inhibit epithelial cell autophagy, and exacerbate mucosal damage. IBD pathology is complex, and highly specific detection of endogenous 5-HT is crucial for understanding its pathological process. Developing a real-time detection method for 5-HT during the occurrence and development of IBD will help deepen our understanding of its pathophysiological mechanisms.

[0004] Fluorescent probes, with their high sensitivity, rapid response, non-invasiveness, and real-time visualization, have become powerful tools in the fields of biological detection and disease diagnosis. Currently, various fluorescent probes have been developed for monitoring 5-HT in biological tissues. However, most existing probes operate within the visible light range, and short-wavelength light has limited penetration into tissues, generally only enabling 5-HT imaging within cells, hindering effective in vivo fluorescence imaging. Furthermore, near-infrared fluorescent probes that can specifically detect 5-HT in IBD models are still lacking. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of existing technologies that cannot achieve in vivo detection / imaging due to short wavelengths, and provides a 5-HT near-infrared fluorescent probe, its preparation method and application to overcome the above-mentioned shortcomings.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a 5-HT near-infrared fluorescent probe, the structure of which is shown in formula (1): (1).

[0007] Hemicyanine fluorophores possess advantages such as strong photostability, high resistance to interference, and low toxicity, and are commonly used in fluorescence imaging of deep tissues in living organisms. Based on this, this invention constructs a novel fluorescent probe specifically for detecting 5-HT, using 3-mercaptopropionate as a recognition group. When the recognition group is attached, the near-infrared fluorescence of the probe is quenched due to disruption of the intramolecular charge transfer (ICT) system of the fluorophore. Upon reaction with 5-HT, the recognition group undergoes a cyclization reaction with 5-HT (see...). Figure 1 This allows the fluorescence signal to be restored.

[0008] Furthermore, experimental results from this invention demonstrate that the probe exhibits excellent reactivity to 5-HT, enabling specific imaging of endogenous 5-HT in living cells or in vivo systems. Simultaneously, the probe's near-infrared emission characteristics grant it deeper tissue penetration and lower background interference in bioimaging, which is significant for improving imaging sensitivity and accuracy.

[0009] In summary, this invention synthesizes a novel 5-HT near-infrared fluorescent probe with a longer emission wavelength (760 nm) and stronger tissue penetration, which can effectively detect changes in 5-HT levels in IBD models and monitor 5-HT levels in IBD-related pathologies in real time.

[0010] This invention provides a method for preparing a 5-HT near-infrared fluorescent probe, comprising: IR-780 iodide and 3-hydroxythiophenol undergoing a nucleophilic substitution reaction in anhydrous potassium carbonate to obtain an intermediate; the intermediate and 3-mercaptopropionic acid are reacted with dichloromethane as a solvent, followed by the addition of 4-dimethylaminopyridine and dicyclohexylcarbodiimide, and the reaction is carried out under light-protected stirring to obtain the 5-HT near-infrared fluorescent probe.

[0011] The method provided in this invention prepares a 5-HT near-infrared fluorescent probe. An intermediate is first synthesized using IR-780 iodide and 3-hydroxythiophenol. Then, the intermediate, 3-mercaptopropionic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide are reacted in dichloromethane to obtain a 5-HT near-infrared fluorescent probe in the form of a blue solid. The reaction formula is as follows: Figure 2 As shown.

[0012] Preferably, the molar ratio of the IR-780 iodide, 3-hydroxythiophenol, and anhydrous potassium carbonate is 1:(2~4):(2~4). And / or, during the nucleophilic substitution reaction, anhydrous potassium carbonate is mixed with acetonitrile as an auxiliary agent for the reaction of IR-780 iodide with 3-hydroxythiophenol; And / or, the nucleophilic substitution reaction is performed at 50-60°C for 4-6 h; And / or, after the nucleophilic substitution reaction is completed, the intermediate is obtained by vacuum concentration and purification.

[0013] Preferably, the molar ratio of the intermediate, 3-mercaptopropionic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:(1~2):(0.1~0.3):(1~3). And / or, the conditions for the stirring reaction are stirring at 20~25°C for at least 12 h; And / or, after the stirring reaction is completed, the 5-HT near-infrared fluorescent probe is obtained by vacuum concentration and purification.

[0014] Preferably, the purification is the purification of the crude product obtained after vacuum concentration / reduced pressure concentration by column chromatography; and / or, the elution buffer for purification is dichloromethane and methanol, with dichloromethane and methanol eluting at a volume ratio gradient of 100:1 to 10:1.

[0015] This invention provides the application of a 5-HT near-infrared fluorescent probe in the in vivo detection and / or imaging of 5-HT.

[0016] Preferably, the in vivo includes: cells, tissues and organs; and / or, the detection is a quantitative detection; and / or, the detection limit is ≤0.25 μM.

[0017] This 5-HT near-infrared fluorescent probe possesses advantages such as fast response speed, specificity and selectivity, low susceptibility to background interference, and high sensitivity (detection limit ≤ 0.25 μM). The response intensity of the 5-HT near-infrared fluorescent probe increases with increasing 5-HT concentration, making it suitable for the detection, quantitative detection, and imaging of 5-HT. Furthermore, experiments have demonstrated that this 5-HT near-infrared fluorescent probe can visualize and track dynamic changes in 5-HT levels in living cells, and can image changes in exogenous 5-HT levels in live mice, as well as endogenous 5-HT levels in live IBD mice; proving that this 5-HT near-infrared fluorescent probe can be used for detection in cells, tissues, and organs.

[0018] This invention provides the application of a 5-HT near-infrared fluorescent probe in the preparation of kits and / or bioimaging tracers for the detection of intestinal dysfunction and inflammatory bowel disease.

[0019] The present invention provides a kit for detecting 5-HT, the kit comprising a 5-HT near-infrared fluorescent probe.

[0020] The present invention provides a bioimaging tracer for dynamic monitoring of 5-HT, the bioimaging tracer comprising a 5-HT near-infrared fluorescent probe.

[0021] Therefore, the present invention has the following beneficial effects: (1) The present invention constructs a novel 5-HT near-infrared fluorescent probe, which is specifically used to detect 5-HT and can be applied to the diagnosis and research of various intestinal dysfunctions and inflammatory bowel diseases, including ulcerative colitis and Crohn's disease.

[0022] (2) The fluorescent probe developed in this invention has an emission wavelength of 760 nm, which is significantly higher than other near-infrared fluorescent probes based on hemicyanine structure, so it is suitable for biological imaging.

[0023] (3) The fluorescent probe developed in this invention is stable under physiological conditions, has excellent optical stability, and has high sensitivity and high selectivity for 5-HT, enabling it to identify 5-HT in complex environments; at the same time, it has a rapid response capability to 5-HT, thus enabling real-time monitoring of changes in 5-HT levels.

[0024] (4) This invention successfully monitored the changes in 5-HT levels in the IBD model using probes, and successfully realized the visualization of 5-HT in the in vivo inflammatory bowel disease model mice. It has great potential in real-time tracking of 5-HT-related pathological states and provides an effective tool for the diagnosis of 5-HT and the research of related drugs. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the mechanism of 5-HT detection by the fluorescent probe of the present invention; Figure 2 This is a synthesis route diagram of the fluorescent probe in Example 1 of the present invention; Figure 3 This is the mass spectrum of the fluorescent probe in Example 1 of the present invention; Figure 4 The fluorescent probe in Example 1 of this invention 1 H NMR spectrum; Figure 5 The fluorescent probe in Example 1 of this invention 13 C NMR spectrum; Figure 6 The figures show the optical experimental results of the 5-HT near-infrared fluorescent probe. In the figures, a is the absorption spectrum of the probe and 5-HT, b is the emission spectrum of the probe and 5-HT, c is the linear relationship between the probe and 5-HT at 760 nm, d is the time kinetic results of the reaction between the probe and different concentrations of 5-HT, e is the selective experiment results of the probe and other substances, and f is the anti-interference experiment results of the probe and 5-HT in the presence of other substances. Figure 7 The image shows the stability test results of the 5-HT near-infrared fluorescent probe. Figure 8 The figure shows the cytotoxicity results of the 5-HT near-infrared fluorescent probe in HCT-116 cells; Figure 9 The image shows the imaging results of 5-HT in HCT-116 cells pretreated with 5-HT by the 5-HT near-infrared fluorescent probe. In the image, a is the imaging result and b is the quantification result of control a. Figure 10 The image shows the imaging results of 5-HT in HCT-116 cells pretreated with nicotinamide, phenethylhydrazine, and 5-hydroxytryptophan by the 5-HT near-infrared fluorescent probe. In the image, a is the imaging result and b is the quantification result of control a. Figure 11 The image shows the immunofluorescence results of 5-HT in HCT-116 cells after pretreatment with nicotinamide, phenethylhydrazine and 5-hydroxytryptophan. In the image, a is the immunofluorescence result and b is the quantitative result of control a. Figure 12 The images show the imaging results of 5-HT in HCT-116 cells pretreated with different concentrations of LPS by the 5-HT near-infrared fluorescent probe. In the images, a is the imaging result and b is the quantification result of control a. Figure 13 The image shows the immunofluorescence results of 5-HT in HCT-116 cells after treatment with different concentrations of LPS using the 5-HT near-infrared fluorescent probe. In the image, a is the immunofluorescence result and b is the quantitative result of control a. Figure 14 Imaging images of 5-HT in normal mice, IBD model mice, and mice treated with 5-aminosalicylic acid using a 5-HT near-infrared fluorescent probe. Figure 15 For comparison Figure 14 The quantification results of the imaging results. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0027] In this section, HPLC-MS analysis was performed on a Waters LCMS system, and NMR spectroscopy was performed (…). 1 H and 13C) Data were recorded on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as an internal control; UV-Vis absorption spectra were obtained using a PerkinElmer Lambda (UV / VIS) spectrophotometer, and fluorescence emission spectra were collected using a PerkinElmer Fluorescence Spectrometer (FL 6500). Absorption and fluorescence test results were recorded at room temperature and in physiological saline; cell fluorescence imaging was performed using a Leica confocal microscope, blue channel: λex = 405 nm, λem = 425-475 nm; red channel: λex = 625 nm, λem = 730-780 nm, scale bar: 20 µm; data were analyzed using one-way ANOVA, and the means of the experimental groups were compared with the means of the control group (n=3; (p < 0.001).

[0028] The raw materials were sourced as follows: all chemicals and solvents used were analytical grade; IR-780 iodide was purchased from Sigma, CAS 207399-07-3; 3-hydroxythiophenol was purchased from Maclean, CAS 40248-84-8; acetonitrile was purchased from Norsch, CAS 75-05-8; dichloromethane was purchased from Shanghai Testing; methanol was purchased from Shanghai Testing; 3-mercaptopropionic acid was purchased from Aladdin, CAS 107-96-0; 4-dimethylaminopyridine, DMAP, was purchased from Aladdin, CAS 1122-58-3; dicyclohexylcarbodiimide, DCC, was purchased from Aladdin, CAS 538-75-0.

[0029] Unless otherwise specified, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0030] The 6-8 week old C57BL / 6J mice used were purchased from Wuhan Shubaili Biotechnology Co., Ltd., and all animal experiments were conducted in accordance with the institutional animal use and care regulations approved by the Ethics Committee of Jianghan University.

[0031] Example 1 After adding 5 mL of acetonitrile solution to a single-necked pear-shaped flask, K₂CO₃ (110 mg, 0.78 mmol) and 3-hydroxythiophenol (90 mg, 0.78 mmol) were added to obtain a mixed solution. The resulting mixed solution was stirred at room temperature for 10 min under nitrogen protection. IR 780 iodide (200 mg, 0.31 mmol) was then added to the above mixed solution. Finally, the mixture was reacted in an oil bath at 50 °C for 4 h, followed by vacuum concentration to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1~10:1, v / v) to obtain a blue powder intermediate (67.3 mg, yield 52.6%).

[0032] The intermediate (85.6 mg, 0.2 mmol) and 3-mercaptopropionic acid (37.17 mg, 0.3 mmol) were weighed and reacted with dichloromethane as solvent. Then, 4-dimethylaminopyridine (DMAP, 6.11 mg, 0.05 mmol) and dicyclohexylcarbodiimide (DCC, 61 mg, 0.4 mmol) were added. The reaction mixture was stirred at room temperature in the dark for 12 h. After the reaction was complete, the mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography, eluted (using dichloromethane:methanol = 100:1~10:1, v / v), and rotary evaporated to obtain a blue solid product (42.9 mg, yield 34.9%), which was the target 5-HT near-infrared fluorescent probe, denoted as Cs-OH.

[0033] The obtained 5-HT near-infrared fluorescent probe was subjected to HR-MS (ESI) testing, and the results are as follows: Figure 3 As shown. Analysis shows that C 31 H 34 NO2S2 + Calculated value: 516.20; Measured value: 516.2014. The results demonstrate that a near-infrared fluorescent probe based on hemicyanine was synthesized.

[0034] And NMR testing was performed, and the results were... Figures 4-5 As shown. 1H NMR (400 MHz, DMSO) δ 8.27-8.23 (d,1H), 7.80-7.47 (m, 5H), 7.39-7.38 (s, 1H), 7.15-7.14 (m, 2H), 6.97-6.94 (m,1H), 6.72-6.69 (d, 1H), 4.43-4.41 (t, 2H), 2.97-2.94(t, 2H), 2.90-2.84 (t,2H), 2.75-2.70 (t, 2H), 2.69-2.64 (t, 2H), 1.78-1.72 (m, 5H), 1.19-1.27 (s,6H), 1.0-0.94 (t, 3H). 13 C NMR (101 MHz, DMSO) δ 177.61, 173.10, 152.95,144.63, 142.69, 141.97, 136.45, 135.45, 131.45, 129.41, 127.67, 126.43,123.31, 121.79, 117.74, 113.90, 111.16, 106.35, 50.89, 46.68, 43.24, 43.02,33.07, 32.10, 29.49, 28,18, 25.10, 24.21, 24.07, 14.42, 11.47.

[0035] All the above results confirm that the synthesized product was as follows. Figure 2 The final product shown.

[0036] Application Example 1 This example demonstrates the probe's ability to detect 5-HT through optical experiments. All experiments were conducted in physiological saline with an excitation wavelength of 720 nm and slits of 5 nm / 10 nm.

[0037] (1) Absorption spectroscopy detection.

[0038] The absorption spectra of the probe (10 μM) and 5-HT (0-40 μM) are as follows: Figure 6 As shown in Figure a, the probe itself has an absorption peak at 650 nm; however, after the addition of 5-HT, a new absorption peak appears at 730 nm.

[0039] (2) Fluorescence spectroscopy detection.

[0040] The emission spectra of the probe (10 μM) and 5-HT (0-40 μM) are as follows: Figure 6As shown in b, the fluorescence was significantly enhanced after adding 5-HT to the probe solution, indicating that the probe has excellent fluorescence response to 5-HT detection.

[0041] To evaluate the response time, a fluorescence time-dependent experiment was also performed in this case. Figure 6 The results showed that after adding 5-HT to the 10 μM probe solution, the fluorescence of the probe reached saturation within 20 minutes, indicating that the probe has the ability to monitor 5-HT in real time.

[0042] (3) Specific detection.

[0043] This example uses common metal ions, anions, ROS, 5-HT structural analogs, and various other substances for selectivity experiments to determine the probe's specificity for 5-HT in complex environments. The selectivity results of the probe (10 μM) against 40 μM of other substances are shown below. Figure 6 As shown in e, where 1-25 are: Blank, KI, CuSO4, NaCl, Zn(NO3)2, NaNO2, KNO3, CaCl2, AlCl3, FeCl3, NH4Cl, FeSO4, H2O2, F - The following compounds were used: Aniline, Cysteine, Glycine, Threonine, Serine, Epinephrine, Norepinephrine, Tryptophan, NaHCO3, Melatonin, and 5-HT. The results showed that only 5-HT reacted with the probe and exhibited a significant fluorescence change.

[0044] Furthermore, the anti-interference experimental results of the probe (10 μM) and 5-HT (40 μM) in the presence of other substances (40 μM) are as follows: Figure 6 As shown in f, where 1-24 represent: KI, CuSO4, NaCl, Zn(NO3)2, NaNO2, KNO3, CaCl2, AlCl3, FeCl3, NH4Cl, FeSO4, H2O2, F - The study included Aniline, Cysteine, Glycine, Threonine, Serine, Epinephrine, Norepinephrine, Tryptophan, NaHCO3, Melatonin, and 5-HT. The results showed that the probe's specificity for 5-HT was unaffected in the presence of other interfering substances.

[0045] The results of the selectivity and anti-interference experiments show that the probe can identify 5-HT in complex environments, thus promising to accurately identify 5-HT in biological environments.

[0046] (4) Quantitative feasibility analysis.

[0047] This example assesses the probe's ability to quantify 5-HT concentration by detecting the fluorescence intensity after the probe reacts with different concentrations of 5-HT. The results are as follows: Figure 6 As shown in Figure c, the emission intensity at 760 nm increases with increasing 5-HT concentration, and there is a strong linear relationship between fluorescence intensity and 5-HT concentration. The detection limit of the probe for 5-HT was calculated to be 0.25 μM using the standard formula LOD = 3σ / S (σ refers to the standard deviation of the blank sample signal, and S refers to the slope of the calibration curve).

[0048] (5) Optical stability test.

[0049] This example tested the stability of the probe at different pH ranges and its reaction stability with 5-HT. The results are as follows: Figure 7 As shown, the probe maintains good stability in the pH range of 5.0–8.0, with or without 5-HT. These results indicate that the probe's stability makes it suitable for in vivo application.

[0050] Application Example 2 This example uses human colon cancer cells (HCT-116) to demonstrate the probe's ability to image 5-HT within cells.

[0051] The HCT-116 cells used in this example were cultured in complete medium containing 1% penicillin-streptomycin, 10% fetal bovine serum, and 90% DMEM. All cell plates and culture dishes were placed in a humidified incubator at 37°C, 5% CO2, and 95% O2.

[0052] Before imaging experiments, the cytotoxicity of the probe in HCT-116 cells was assessed using the MTT assay. The procedure was as follows: cultured HCT-116 cells were placed in 96-well cell culture plates. After 24 hours, the cells were treated with different concentrations of the probe (0-25 µM) for 4 hours. Then, the old culture medium was removed, and DMSO (150 µL per well) was added. After shaking for 5-10 minutes, the absorbance was measured at 570 nm using a microplate reader. The results showed that the probe had low cytotoxicity; even after treatment with 25 μM probe, more than 85% of HCT-116 cells remained viable, indicating that the probe has good biocompatibility. Figure 8 ).

[0053] The exogenous 5-HT in HCT-116 cells was monitored using a probe. Specifically, after culturing HCT-116 cells, a probe (10 μM) was added and incubated for 30 min, followed by the addition of 0-50 µM exogenous 5-HT and incubation for another 20 min. The results are as follows: Figure 9 As shown, the fluorescence intensity in HCT-116 cells increased with increasing 5-HT concentration. The red fluorescence of the highest concentration group was about twice that of the control group, indicating that the probe can effectively detect exogenous 5-HT in cells.

[0054] Using nicotinamide, phenelzine, and 5-hydroxytryptophan (L-5-HTP) as endogenous 5-HT inducers, this study also monitored endogenous 5-HT in HCT-116 cells using a probe. Specifically, 200 μM of different inducers were added to cultured HCT-116 cells, followed by the addition of a 10 µM probe and incubation for 30 minutes. The results are as follows: Figures 10-11 As shown, the probe can effectively detect endogenous 5-HT in cells. When treated with an endogenous inducer, the fluorescence intensity is significantly enhanced, and the increase in 5-HT is then verified again by immunofluorescence.

[0055] The probe can effectively detect not only the increase of intracellular 5-HT, but also the decrease. This example uses DL-4-chlorophenylalanine (a 5-hydroxytryptophan-specific inhibitor, DL-4-CPA) as a 5-HT production inhibitor to demonstrate the probe's ability to detect decreased intracellular 5-HT levels. The specific procedure was as follows: after incubating cultured HCT-116 cells with 5-hydroxytryptophan, DL-4-chlorophenylalanine (1 mM) was added, followed by the probe (10 µM), and then incubation for another 30 minutes. The results are as follows... Figures 10-11 As shown, the red fluorescence of HCT-116 cells treated with DL-4-chlorophenylalanine was significantly reduced, indicating that the probe could effectively detect the reduction of intracellular 5-HT.

[0056] The above results confirm that the probe is suitable for monitoring fluctuations in intracellular 5-HT levels.

[0057] Application Example 3 This example demonstrates the probe's ability to dynamically and in real-time monitor changes in 5-HT levels during inflammation by establishing an inflammatory cell model.

[0058] First, HCT-116 cells were treated with LPS to establish an inflammatory cell model.

[0059] Each group of cells was harvested and incubated with the probe (10 µM) for 30 minutes. Results are as follows: Figures 12-13As shown: compared with the control group, the red fluorescence of the LPS group was enhanced, indicating that the probe effectively detected the increase in 5-HT levels in the inflammatory cell model.

[0060] In addition, the reduction of 5-HT in inflammatory model cells was detected using the antioxidant NAC (N-Acetyl-L-Cysteine). The results are as follows: Figures 12-13 As shown, the fluorescence intensity of NAC-treated inflammatory model cells was weaker than that of the LPS group. This indicates that the probe can detect the decrease in 5-HT levels in NAC-pretreated inflammatory cells, further confirming the probe's ability to dynamically monitor 5-HT in inflammatory cell models.

[0061] The above results validate the probe's ability to monitor 5-HT dynamics under different conditions, demonstrating its strong application potential in IBD research.

[0062] Application Example 4 This example validates the probe's ability to monitor 5-HT fluctuations in animal models, specifically imaging 5-HT in an IBD mouse model.

[0063] In vivo imaging was performed using an IVIS imaging system (λex=710 nm, λem=760 nm) after intravenous injection of probes (0.5 mg / kg, 200 µL) into C57BL / 6J mice, IBD model mice induced by 3% DSS drinking water, and mice that had undergone 3% DSS modeling followed by 14 days of gavage with 5-aminosalicylic acid (5-ASA). Results are as follows: Figures 14-15 As shown: IBD mice showed a stronger fluorescent signal on their abdomen, reflecting the increased 5-HT level in IBD mice.

[0064] The above results confirm that the probe can track fluctuations in 5-HT levels in the body.

[0065] In summary, the probe provided by this invention can selectively and sensitively detect 5-HT, has a rapid response capability to 5-HT, and has been successfully applied to 5-HT imaging in cell and animal models. It can accurately reflect the regulated 5-HT level in cell and animal models, proving its application value in real-time monitoring of 5-HT in pathological conditions such as IBD.

Claims

1. A 5-HT near-infrared fluorescent probe, characterized in that, Its structural formula (1) is shown below: （1）。 2. The method for preparing the 5-HT near-infrared fluorescent probe as described in claim 1, characterized in that, include: IR-780 iodide and 3-hydroxybenzylthiophenol undergo a nucleophilic substitution reaction in anhydrous potassium carbonate to obtain an intermediate; the intermediate reacts with 3-mercaptopropionic acid in dichloromethane as solvent, followed by the addition of 4-dimethylaminopyridine and dicyclohexylcarbodiimide, and the reaction is carried out under light-shielded stirring to obtain a 5-HT near-infrared fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the IR-780 iodide, 3-hydroxythiophenol and anhydrous potassium carbonate is 1:(2~4):(2~4). And / or, during the nucleophilic substitution reaction, anhydrous potassium carbonate is mixed with acetonitrile as an auxiliary agent for the reaction of IR-780 iodide with 3-hydroxythiophenol; And / or, the nucleophilic substitution reaction is performed at 50-60°C for 4-6 h; And / or, after the nucleophilic substitution reaction is completed, the intermediate is obtained by vacuum concentration and purification.

4. The preparation method according to claim 2, characterized in that, The molar ratio of the intermediate, 3-mercaptopropionic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:(1~2):(0.1~0.3):(1~3). And / or, the conditions for the stirring reaction are stirring at 20~25°C for at least 12 h; And / or, after the stirring reaction is completed, the 5-HT near-infrared fluorescent probe is obtained by vacuum concentration and purification.

5. The preparation method according to claim 3 or 4, characterized in that, The purification is achieved by column chromatography of the crude product obtained after vacuum concentration / reduced pressure concentration; and / or, the elution buffer is dichloromethane and methanol, with dichloromethane and methanol eluted in a volume ratio gradient of 100:1 to 10:

1.

6. The application of the 5-HT near-infrared fluorescent probe as described in claim 1 or the 5-HT near-infrared fluorescent probe prepared by any of the preparation methods described in claims 2 to 5 in the in vivo detection and / or imaging of 5-HT.

7. The application as described in claim 6, characterized in that, The in vivo includes: cells, tissues and organs; and / or, the detection is a quantitative detection; and / or, the detection limit of the detection is ≤0.25 μM.

8. The use of the 5-HT near-infrared fluorescent probe as described in claim 1 or the 5-HT near-infrared fluorescent probe prepared by any of the preparation methods described in claims 2 to 5 in the preparation of kits for the detection of intestinal dysfunction and inflammatory bowel disease and / or bioimaging tracers.

9. A kit for detecting 5-HT, characterized in that, The kit includes the 5-HT near-infrared fluorescent probe as described in claim 1 or the 5-HT near-infrared fluorescent probe prepared by any of the preparation methods described in claims 2 to 5.

10. A bioimaging tracer for dynamic monitoring of 5-HT, characterized in that, The bioimaging tracer includes the 5-HT near-infrared fluorescent probe as described in claim 1 or the 5-HT near-infrared fluorescent probe prepared by any of the preparation methods described in claims 2 to 5.

Images