Responsive pyridine near-infrared fluorescent probe for targeting MOR as well as synthesis method and application of responsive pyridine near-infrared fluorescent probe

Abstract

The invention discloses an MOR-targeted responsive pyridine near-infrared fluorescent probe as well as a synthesis method and application thereof. The structural formula of the MOR-targeted responsive pyridine near-infrared fluorescent probe is shown in the specification. The excitation emission wavelength of the fluorescent ligand is located in a near-infrared region, the fluorescent ligand is not prone to interference of biological fluorescence, the signal-to-noise ratio is high, MOR can be specifically recognized, a considerable fluorescence signal is generated after the fluorescent ligand is combined with an MOR hydrophobic region, and high-sensitivity detection can be conducted on MOR; compared with the prior art, the probe has the advantages that the stability of the fluorescent ligand is improved, the influence on the binding force of a receptor is reduced, the targeting activity is not easily damaged, and in-vivo and in-vitro experimental results show that the series of probes have excellent stability and targeting property in vivo and in vitro, and are suitable for real-time monitoring of MOR on a living cell membrane under a confocal microscope. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a responsive pyridine near-infrared fluorescent probe targeting MOR and a synthesis method and application thereof, belonging to the technical field of fluorescent molecular probes. Background Art

[0002] μOpioid receptor (MOR), belonging to the A family of G protein-coupled receptors (GPCRs), mainly achieves its physiological effects by inhibiting the heterotrimeric G protein (Gi / o) of adenylate cyclase. It is mainly distributed in the brainstem, spinal cord, cerebral cortex and many areas of the central nervous system. μOpioid receptors can bind to specific antagonists and agonists to produce specific opioid effects such as analgesia, regulation of the reward system, respiratory depression, regulation of the digestive system, and immune regulation. MOR binds to specific endogenous or exogenous small molecules (buprenorphine, etc.) or short peptides (enkephalin, etc.) to activate the downstream Gi / o signaling pathway to produce analgesia. Most of the small molecule MOR ligands developed in the early stage activated the Gi / o-mediated analgesia pathway while also activating the β-arresting (β-arrestin recruitment protein) pathway with side effects such as respiratory depression, addiction and drug tolerance, which seriously limited the development of drugs acting on MOR receptors. In recent years, in order to reduce the adverse reactions of analgesics, the development of MOR-biased ligands that act on the Gi / o pathway without activating the β-arresting pathway has become an important research direction for the development of new analgesics.

[0003] In the research process of MOR biased ligands, small molecule fluorescent ligands have played an important role. This is a class of small molecule compounds that can emit fluorescence signals of specific wavelengths and have specific targets through excitation light. They are composed of fluorescent groups that emit fluorescence signals, targeting groups that have target recognition functions, and connecting chains connecting the two. The design of fluorescent ligands needs to consider the physicochemical properties and pharmacological activities of the final probe to ensure that the affinity and selectivity of the fluorescent conjugate for the receptor will not be significantly reduced. By rationally conjugating MOR agonists or antagonists with various fluorophores, fluorescent ligands that can specifically recognize MOR can be designed. This type of fluorescent ligand exhibits a low fluorescence signal when not bound to the target and generally requires high stability and biocompatibility. These advantages make them useful for real-time monitoring of the interaction between ligands and target receptors in living cells or in vivo to display relevant information on the pharmacology, physiology, and kinetics of target receptors. This type of fluorescent ligand with high specificity, high sensitivity, and fluorescence turn-on characteristics can visualize the location of receptors in living cells or in vivo and even monitor complex biological processes in vivo, and is widely used in the fields of biosensing and detection, drug screening, and drug efficacy evaluation.

[0004] As a new model organism, zebrafish has unique imaging advantages such as embryonic transparency and small size. Therefore, specific fluorescent ligands with strong tissue penetration can be used for visualization analysis of biomolecules in zebrafish, which can maximize the signal-to-noise ratio while reducing damage to biological samples. In addition, zebrafish have a high degree of genetic homology with humans, and their nervous system is highly similar to that of mammals, with corresponding brain regions such as olfactory bulb, telencephalon, optic tectum, diencephalon, midbrain, medulla oblongata, hypothalamus, etc., and a variety of neurotransmitters such as glutamate, γ-aminobutyric acid, acetylcholine, dopamine, 5-hydroxytryptamine, etc. have been characterized in the zebrafish brain. The μ-opioid receptor (ZFOR2) encoded by zebrafish has seven potential transmembrane domains, and its gene sequence also shows a high degree of identity with the human receptor. As an easy-to-operate model organism combined with high-resolution in vivo imaging technology, zebrafish can be used as an ideal choice for in vivo imaging of fluorescent ligands to achieve in vivo imaging of biomolecule visualization.

[0005] Commercial fluorophores include fluorescein, rhodamine, coumarin, BODIPY, Alexa Fluor series dyes, Cy series (all-cyanine) dyes, etc. However, some chemical properties of these dyes limit their development, such as poor water solubility, signal interference caused by the overlap of emission wavelength and biological tissue autofluorescence, poor stability, reduced binding ability of active groups to targets due to the addition of fluorescent groups, and poor drugability caused by large molecular weight. For example, the maximum emission wavelength of the fluorescent probe disclosed in the patent (202210961498.2) is 480nm, and the fluorescence signal at this wavelength is interfered by the biological autofluorescence to produce false positive signals, which is not conducive to in vivo imaging. In addition, most of the current MOR fluorescent ligands do not have a lighting mechanism and can only play a simple labeling function. After labeling, the fluorescent ligands in the environment and non-specific binding need to be washed. In addition, there are currently few applications of such fluorescent ligands for zebrafish in vivo imaging to explain relevant information about the corresponding target biological molecules. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a responsive pyridine near-infrared fluorescent probe targeting MOR that is not affected by the biological autofluorescence, as well as a synthesis method and application thereof.

[0007] Technical solution: To solve the above technical problems, the present invention provides a responsive pyridine near-infrared fluorescent probe targeting MOR or a pharmaceutically acceptable salt thereof, the structural formula of the probe is shown below:

[0008]

[0009] Among them, n=3, 4, 5, 6, 7.

[0010] Preferably, the structural formula of the probe is as shown below:

[0011]

[0012] The present invention also provides a method for preparing the responsive pyridine near-infrared fluorescent probe targeting MOR, comprising the following steps:

[0013] (1) Using naltrexone as a raw material, a racemic intermediate 1 having a linking site was synthesized by reduction reaction;

[0014] (2) 2,4,6-trimethylpyran tetrafluoroborate and 4-(dimethylamino)cinnamaldehyde were used as raw materials to synthesize 4-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridinium 2, and pyridine fluorophore 3 was synthesized by nucleophilic substitution reaction;

[0015] (3) The intermediate 1 having a connection site and the pyridine fluorophore 3 having a connection chain undergo a condensation reaction between the carboxyl group and the amino group to generate the final product (I).

[0016] Wherein, the synthesis step (1) specifically comprises:

[0017]

[0018] The synthesis step (2) specifically comprises:

[0019]

[0020] The synthesis step (3) specifically comprises:

[0021]

[0022] The fluorescent ligand of the present invention combines the pharmacophore naltrexone that specifically targets MOR with a pyridine fluorophore with good fluorescence properties through a reasonable synthesis route based on the intramolecular charge transfer mechanism (ICT), thereby constructing a small molecule near-infrared fluorescent ligand that specifically targets MOR with an environmentally responsive fluorescence on mechanism. According to the properties of the probe, it is used as a tool molecule to conduct visualization research on MOR.

[0023] The present invention also provides application of the responsive pyridine near-infrared fluorescent probe targeting MOR in aggregation-induced luminescence materials.

[0024] The present invention also provides the use of the responsive pyridine near-infrared fluorescent probe targeting MOR in the preparation of reagents or drugs for detecting / diagnosing diseases related to μ opioid receptors.

[0025] The present invention also provides the use of the responsive pyridine near-infrared fluorescent probe targeting MOR in the preparation of drugs for screening μ opioid receptor-related diseases.

[0026] Wherein, the drug is a μ opioid receptor agonist or antagonist.

[0027] Among them, the concentration of MOR was 0.81-104.00 μg / ml.

[0028] Therefore, based on the defects of various small molecule fluorescent ligands developed previously, structural modification of pyridine dyes to develop fluorescent ligands with good water solubility, strong stability, good drugability, and high specificity and sensitivity has always been an important direction in the field of medical diagnostic reagent research.

[0029] Synthetic fluorescent ligands are widely used for in vivo localization monitoring and visualization of biomolecules. The present invention aims to provide a method for synthesizing a responsive small molecule near-infrared fluorescent ligand targeting MOR and its application in living cells and zebrafish.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The fluorescent probe of the present invention plays an important role in the study of the binding kinetics of MOR and various ligands, helps to explain the active action mechanism of related compounds, and provides lead compounds for the study of related diseases, and has broad application prospects in the research field of detecting μ opioid receptor diagnostic reagents; 2. The pyridine small molecule fluorescent ligand (I) developed by the present invention has a small molecular weight, low toxicity, large Stokes shift, large molar extinction coefficient, simple synthesis steps, and low cost. Compared with most previous fluorescent ligands, the unique chemical properties of the pharmacophore are utilized, and the water solubility of the pyridine fluorophore is improved through a simple protonation step without changing the chemical structure of the ligand; 3. The excitation and emission wavelengths of the fluorescent ligand are in the near-infrared region, which is not easily interfered by the biological autofluorescence, and has It has a high signal-to-noise ratio, can specifically identify MOR, and produces a considerable fluorescence signal after binding to the hydrophobic region of MOR, avoiding washing operations and enabling high-sensitivity detection of MOR; 4. A simple and stable fluorophore structure is introduced. Due to its simple conjugated structure, its electron donor part and receptor are relatively stable, and the volume of the fluorophore is small, so the stability of the fluorescent ligand (I) is improved, the effect on receptor binding force is reduced, and the targeting activity is not easily destroyed. The results of in vivo and in vitro experiments show that this series of probes has excellent stability and targeting in vivo and in vitro, and is suitable for real-time monitoring of MOR on the cell membrane of living cells under a confocal microscope; 5. Through zebrafish brain injection experiments, it is proved that the ligand can locate the MOR receptor in the zebrafish brain, which is of great value for determining the specific distribution of MOR in living bodies and related dynamic information research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the H NMR spectrum of intermediate 1;

[0032] Figure 2 is the mass spectrum of intermediate 1;

[0033] Figure 3 is the H NMR spectrum of the fluorescent ligand (I);

[0034] Figure 4 is the mass spectrum of fluorescent ligand (I);

[0035] Figure 5 is the absorption spectrum of fluorescent ligand (I) in different solvents;

[0036] Figure 6 is the fluorescence spectra of the fluorescent ligand (I) in different solvents;

[0037] Figure 7 is the fluorescence spectrum of the interaction between the fluorescent ligand (I) and MOR;

[0038] Figure 8 The fluorescence signal comparison diagram of the interaction between the fluorescent ligand (I) and BSA and MOR;

[0039] Fig. 9 Confocal microscopy cell imaging of fluorescent ligand (I) acting on cells;

[0040] Fig.10 Confocal microscopy imaging of the fluorescent ligand (I) acting on the zebrafish brain (a) and fluorescence signal comparison (b). DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0042] Example 1: Synthesis of small molecule near-infrared fluorescent ligand (I)

[0043] Step a:

[0044] Synthesis route:

[0045]

[0046] Naltrexone (0.12 g, 0.35 mmol), ammonium acetate (0.27 g, 3.5 mmol), sodium cyanoborohydride (0.033 g, 0.53 mmol) were dissolved in 3 mL of methanol under nitrogen protection and stirred at room temperature for 24 hours. The solvent was removed in vacuo, the pH was adjusted to 10 with 1 M NaOH, extracted with dichloromethane (5 mL × 3), the organic phase was taken, anhydrous magnesium sulfate was added to dry the organic phase, filtered, and the solvent was removed in vacuo to obtain a milky white solid. The obtained solid was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30:1-10:1) to obtain a white solid 1 (0.056 g, 47%). MS (ESI, m / z, C 20 H 26 N2O3,[M+H] + ):calcd.,342.2; found 342.2. The H NMR spectrum of intermediate 1 is as follows Figure 1 As shown in the mass spectrum Figure 2 shown.

[0047] Step b:

[0048] Synthesis route:

[0049]

[0050] 4-(Dimethylamino)cinnamaldehyde (700 mg, 4.7 mmol) and 2,4,6-trimethylpyrylium tetrafluoroborate (1.18 g, 5.64 mmol, manufacturer: Leyan; item number: 137087) were dissolved in 20 mL of ethanol, heated to 80 ° C under nitrogen protection, stirred, and refluxed for 1 hour. The reaction system was cooled to room temperature, filtered, and the filtrate was collected. The solvent was removed by rotary evaporation to obtain a blue-black solid mixture, which was recrystallized in ethyl acetate and filtered. The filter cake was collected and vacuum dried to obtain a metallic blue solid (1.09 g, 83.4%), which is intermediate 2. MS (ESI, m / z, C 19 H 22 NO,[M+H] + ):calcd.,280.17; found280.17.

[0051] Step c:

[0052] Synthesis route:

[0053]

[0054] Intermediate 2 (560 mg, 2 mmol) and γ-aminobutyric acid (258 mg, 2.5 mmol) were dissolved in 10 mL of ethanol, heated to 40°C under nitrogen protection, stirred, and refluxed for 5 hours. The reaction system was cooled to room temperature, filtered, and the filtrate was collected and the solvent was removed by rotary evaporation to obtain a black-red solid powder. The obtained solid was separated and purified by silica gel column chromatography (dichloromethane / methanol = 20:1-10:1) to obtain a dark red solid powder (569.7 mg, 78%), namely intermediate 3. MS (ESI, m / z, C 23 H 29 N2O2,[M+H] + ):calcd.,365.22; found 365.22. 1 H NMR (300MHz, DMSO-d6) δ12.41(s,1H),7.86(s,2H),7.72(d,J=8.0Hz,1H),7.47(d,J=8.4Hz,2H),7.09-6.91(m,2H) ,6.73(d,J=8.5Hz,2H),6.59(d,J=15.3Hz,1H),4.40-4.29(m,2H),3.37(t,J=5.6Hz,4H),2.99(s,6H),2.79(s,6H).

[0055] Step d:

[0056] Synthesis route:

[0057]

[0058] Intermediate 3 (21 mg, 0.058 mmol), white solid 1 (20 mg, 0.058 mmol), p-dimethylaminopyridine (DMAP, 8.5 mg, 0.07 mmol) and hydroxybenzotriazole (HOBt, 9.4 mg, 0.07 mmol) were dissolved in 2 mL DMF under nitrogen, stirred at room temperature for 24 hours, and the solvent was removed in vacuo. The obtained solid was separated and purified by silica gel column chromatography (dichloromethane / methanol = 20:1-15:1) to obtain a dark red solid (10.4 mg, 26%), namely intermediate 4. MS (ESI, m / z, C 43 H 53 N4O4,[M+H] + ; [(M+2H) / 2] + ):calcd.,689.41; found 689.41; found 345.22.

[0059] Step e:

[0060] Synthesis route:

[0061]

[0062] Intermediate 4 (10.4 mg, 0.015 mmol) was placed in 2 mL of ethyl acetate saturated solution of hydrogen chloride and stirred for 3 h. The solvent was removed in vacuo. After vacuum drying, a light yellow solid was obtained, namely the fluorescent ligand (I) (9.1 mg, 83.6%). MS (ESI, m / z, C 43 H 53 N4O4,[M+H] + ; [(M+2H) / 2] + ):calcd.,689.41; found 689.41; found345.22. 1 H NMR(300MHz,Chloroform-d)δ7.38(d,J=11.7Hz,1H),7.00-6.86(m,1H),6.71(td,J=12.1,6.8Hz,2H),6.51(d,J=8.1Hz,1H),4.64-4.53(m,1H),4.36- 4.24(m,1H),4.22-4.12(m,1H),3.64(dt,J=13.2,6.6Hz,1H),3.47(s,1H), 3.10(t,J=7.3Hz,2H),3.04(d,J=3.6Hz,2H),2.98(d,J=7.7Hz,1H),2.76(d, J=6.4Hz,2H),2.68-2.51(m,3H),2.34(d,J=4.7Hz,1H),2.30(d,J=8.7Hz,1H),2.25(d,J=7.5Hz,2H),2.01(d,J=4.6Hz,1H),1.80-1.70(m,2H),1.67-1.57(m,2H),1.47-1.35(m,8H),1.27(d,J=9.4Hz,7H),0.86(dd,J=14.5,6.3Hz,5H),0.52(dd,J=7.3,4.4Hz,2H),0.13(q,J=4.8Hz,2H). The H NMR spectrum of the fluorescent ligand (Ⅰ) is shown in Figure 3 As shown in the mass spectrum Figure 4 shown.

[0063] Example 2: Absorption and fluorescence emission spectra of small molecule near-infrared fluorescent ligand (I) in PBS, methanol (MeOH), ethanol (EtOH), DMSO, and acetonitrile (ACN)

[0064] The aforementioned fluorescent ligand (I) was prepared into 1.04 mg / mL solutions in PBS, methanol, ethanol, DMSO, and acetonitrile, and 100 μl of each solution was placed in a 96-well ELISA plate. The absorption and fluorescence spectra were detected using an ELISA reader. Figure 5 As shown, the maximum excitation wavelength of the fluorescent probe (I) in PBS is about 445 nm, the maximum excitation wavelength in DMSO and ACN is about 470 nm, and the maximum excitation wavelength in ethanol and methanol is about 490 nm. Figure 6 As shown, the maximum emission wavelength of the fluorescent probe (I) is between 600nm and 800nm, with a double peak emission, and the fluorescence intensity is different in different solvents. It exhibits strong fluorescence emission in DMSO, the strongest emission peak in DMSO and acetonitrile is about 710nm, and the maximum emission peak in PBS, methanol, and ethanol is about 680nm. Among them, it exhibits lower fluorescence emission in PBS.

[0065] Example 3: Fluorescence response spectrum of fluorescent ligand (I) acting on MOR and comparison of fluorescence signals of fluorescent ligand (I) acting on MOR and BSA at 660nm under 445nm excitation

[0066] The plasmid containing human MOR (amino acid sequence: SEQ ID NO.1: MDSSAAPTNASNCTD ALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCT LTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHF CIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLPDYKDDDDK, article disclosing this plasmid: JIAY,XU L,WANG L,et al.A light-up fluorescence probefor wash-free analysis of Mu-opioid receptor and ligand-binding events[J].Analytica Chimica Acta,2023,1261:341220.Article author: Jia Yan, Xu Lili, Wang Lancheng, Yan Kun, Chen Jieru, Xu Pengcheng. Corresponding author: Di Bin, Yan Fang, Hu Chi) was cloned into pcDNA3.1 vector (Changzhou Jiyu Biotechnology Co., Ltd.), which was transformed into Escherichia coli BL21 (DE3) strain (Biyuntian BL21 (DE3) glycerol bacteria (protein inducible expression strain), catalog number: D0337) and then the plasmid was extracted and expressed.The extracted plasmid was used to transfect HEK 293T cells (Biyuntian HEK293T (human embryonic kidney cells), Catalog No.: C6008), and the MOR protein expressed by the cells was extracted (amino acid sequence: SEQ ID NO.2: MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGL PVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLPDYKDDDDK). After MOR and BSA were dissolved, they were prepared into protein solutions of the same concentration using PBS (the concentrations of MOR and BSA were both 104 μg / ml), diluted 8 times at a ratio of 1 / 2, 50 μl of 1 μM fluorescent ligand (Ⅰ) solution was placed in a 96-well ELISA plate, and 50 μl of MOR and BSA solutions of various concentrations (104.00 μg / ml, 52.00 μg / ml, 26.00 μg / ml, 13.00 μg / ml, 6.50 μg / ml, 3.25 μg / ml, 1.63 μg / ml, 0.81 μg / ml) were added to each well, and the fluorescence spectrum of each concentration gradient was measured after incubation for 5 minutes. Figure 7 , Figure 8 As shown, with the increase of MOR concentration, the fluorescence signal increased, and the response signal of MOR was much higher than that of BSA by 3.2 times, which proved that the fluorescent ligand (I) could specifically bind to MOR and generate a strong fluorescence signal at 660 nm.

[0067] Example 4: The small molecule near-infrared fluorescent ligand (I) described in the present invention acts on HEK293T cells overexpressing MOR on the cell membrane

[0068] Normally grown HEK 293T cells were transfected with the pcDNA3.1-MOR-flag recombinant plasmid using a commercial liposome nucleic acid transfection reagent (Yisheng Biotechnology (Shanghai) Co., Ltd., Catalog No. 40802ES01). After culturing for 48 hours, the MOR receptor protein on the cell membrane was extracted using a plasma membrane protein and cell component separation kit (Invent, Catalog No. SM-005) according to the instructions to overexpress MOR on the cell membrane. The overexpressed cells (6 cm cell culture dish) were incubated with 2 mL of PBS solution (50 nM) of fluorescent ligand (I) at 37 ° C for 30 minutes, and then placed under a confocal fluorescence microscope, excited at 445 nm, and the fluorescence emission signal at a wavelength of 660 nm was detected. Cells that did not express MOR were used as blank groups, and the MOR ligand naltrexone (Naltrexone, Aladdin, catalog number: H2330623) and μ-OPR agonist DAMGO (McLean, catalog number: D877395) known to have strong binding force were used as control groups for confocal microscopy imaging of cells. Fig. 9 As shown, the fluorescent ligand (I) produces a strong fluorescent signal on the cell membrane, while the control group has almost no fluorescent signal, proving that the fluorescent ligand (I) of the invention specifically binds to the MOR receptor overexpressed on the cell membrane. When the probe molecule enters the MOR hydrophobic structure, the fluorescent signal is enhanced, resulting in the fluorescent signal intensity on the cell membrane being significantly higher than that of other cell structures.

[0069] The above experimental examples show that the fluorescent ligand (I) of the present invention can monitor MOR on the cell membrane in real time under physiological conditions, which is of great value for the study of the related kinetics of the binding of MOR with specific ligands and has the potential to be developed into a clinical diagnostic reagent.

[0070] Example 5: The small molecule near-infrared fluorescent ligand (I) described in the present invention acts on the zebrafish brain and compares the fluorescence signals

[0071] The zebrafish required for imaging were incubated with 50 ml of 0.003% 1-phenyl-2-thiourea at 24 hpf (hours post fertilization, hpf), and the 5 dpf (days post fertilization, dpf) zebrafish were anesthetized in 10 ml of 0.02% tricaine, adjusted to have the dorsal side facing up, and fixed on a glass slide with 1.5% low-melting point agarose. A microinjector was used to inject 1 nL of 0.5 mM fluorescent ligand solution (P5N3) and 1 nL of 0.5 mM fluorescent ligand and 2.5 mM Naltrexone mixed solution (P5N3+Naltrexone) into the rhomboid ventricle of the zebrafish, respectively. After 1 hour, the injected solution was dispersed in the zebrafish brain tissue, and the Z-stack layer was scanned under a laser confocal microscope and set to maximum intensity projection. Fig.10 As shown, opioid receptors are widely distributed in the zebrafish nervous system, and the addition of Naltrexone significantly reduced the fluorescence signal generated by the fluorescent ligand.

[0072] In addition, the fluorescence intensity of the zebrafish brain was quantified and analyzed. Data analysis using the Shapiro-Wilk method and the Bartlett method showed that the two groups of data were normally distributed and had homogeneous variances. According to the t-test results of the two groups of data (n=6 / group), there was a significant difference between the fluorescent ligand group and the group with the strong binding ligand Naltrexone (P=0.0024).

[0073] This fluorescent ligand has good tissue penetrability. Through in vivo imaging in zebrafish, it can achieve real-time fluorescence visualization of MOR. Combined with high-resolution confocal imaging technology, it is of great significance for the study of the mechanism of action of analgesics and the development of new analgesics.

Claims

1. A responsive pyridine near-infrared fluorescent probe targeting MOR or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the probe is shown below:

2. A method for preparing the responsive pyridine near-infrared fluorescent probe targeting MOR according to claim 1, characterized in that: The following steps are involved: (1) Using naltrexone as a raw material, a racemic intermediate 1 having a linking site was synthesized by reduction reaction; (2) 2,4,6-trimethylpyran tetrafluoroborate and 4-(dimethylamino)cinnamaldehyde were used as raw materials to synthesize 4-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridinium 2, and pyridine fluorophore 3 was synthesized by nucleophilic substitution reaction; (3) The intermediate 1 having a connection site and the pyridine fluorophore 3 having a connection chain undergo a condensation reaction between the carboxyl group and the amino group to generate the final product (I).

3. Application of the responsive pyridine near-infrared fluorescent probe targeting MOR according to claim 1 in aggregation-induced emission materials.

4. Use of the responsive pyridine near-infrared fluorescent probe targeting MOR according to claim 1 in the preparation of reagents or drugs for detecting / diagnosing diseases related to μ opioid receptors.

5. Use of the responsive pyridine near-infrared fluorescent probe targeting MOR according to claim 1 in the preparation of drugs for screening diseases related to μ opioid receptors.

6. The use according to claim 5, characterized in that: The drug is a μ opioid receptor agonist or antagonist.

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