A responsive pyridine near-infrared fluorescent probe targeting MOR and a synthesis method and application thereof

Abstract

The application discloses a kind of pyridine near-infrared fluorescent probe of response of targeting MOR and its synthesis method and application, the structure formula of the pyridine near-infrared fluorescent probe of response of targeting MOR as shown in following formula.The fluorescence ligand excitation emission wavelength of the present application is located in near-infrared region, is not susceptible to biological own fluorescence interference, with higher signal-to-noise ratio, can specifically recognize MOR, and after being combined with the hydrophobic region of MOR, considerable fluorescence signal is generated, and high sensitivity detection can be carried out to MOR;The stability of the fluorescence ligand is improved, the influence on receptor binding force is reduced, the targeting activity is not susceptible to be destroyed, and in-vivo and in-vitro experimental results show that the series of probes have excellent stability and targeting in vivo and in vitro, and are suitable for being used to carry out real-time monitoring to MOR on the cell membrane of living cell under confocal microscope.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of MOR targeted response pyridine near-infrared fluorescent probe and its synthesis method and application, belong to fluorescent molecular probe technical field. BACKGROUND

[0002] μ Opioid receptor (μ Opioid Receptor, MOR), belongs to G protein-coupled receptor (GPCRs) A family, mainly through the heterotrimeric G protein (Gi / o) of adenylyl cyclase to realize its physiological effect, mainly distributed brain stem, spinal cord and cerebral cortex and many regions of central nervous system. μ Opioid receptor can be combined with specific antagonists and agonists to produce specific opioid effects such as analgesic effect, reward system regulation, respiratory depression, digestive system regulation, immune regulation, etc. MOR is combined with specific endogenous or exogenous small molecules (buprenorphine, etc.) or short peptides (enkephalins, etc.) to activate downstream Gi / o signal pathway to produce analgesic effect, and early development Small molecule MOR ligand activates the β-arresting (β-arresting recruitment protein) pathway with respiratory depression, addiction and drug tolerance, etc. Side effects, which seriously limit the development of drugs acting on MOR receptor. In recent years, in order to reduce the adverse reactions of analgesic drugs, the development of MOR biased ligand acting on Gi / o pathway without activating β-arresting pathway has become an important research direction for developing new analgesic drugs.

[0003] In the research of MOR biased ligand, small molecule fluorescent ligand plays an important role. It is a kind of small molecule compound that can emit specific wavelength fluorescent signal by exciting light and has specific target. It is composed of fluorescent group for emitting fluorescent signal, targeting group for target recognition and connecting chain connecting the two. The design of fluorescent ligand needs to consider the physicochemical properties and pharmacological activity of the final probe to ensure that the affinity and selectivity of the fluorescent conjugate to the receptor will not be significantly reduced. By reasonably conjugating MOR agonists or antagonists with various fluorescent groups, fluorescent ligands that can specifically recognize MOR can be designed. This kind of fluorescent ligand shows low fluorescent signal when not combined with target and generally requires high stability and biocompatibility. These advantages enable them to be used to monitor the interaction between ligand and target receptor in living cells or in vivo to display the relevant information of target receptor pharmacology, physiology and dynamics. This kind of fluorescent ligand with high specificity, high sensitivity and fluorescence opening characteristics can be used to visualize and locate receptors in living cells or in vivo and even monitor complex biological processes in vivo, and is widely used in biological sensing and detection, drug screening and pharmacodynamic evaluation, etc.

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

[0005] Commercial fluorophores include fluorescein, rhodamine, coumarin, BODIPY, Alexa Fluor series dyes, Cy series (all flower cation) dyes, etc. However, the development of some of these dyes is limited by their chemical properties, such as poor water solubility, overlapping emission wavelength with biological tissue autofluorescence causing signal interference, poor stability, reduced binding ability of active groups to targets due to the addition of fluorescent groups, and poor drugability due to large molecular weight. For example, the fluorescent probe disclosed in patent (202210961498.2) has a maximum emission wavelength of 480 nm, and the fluorescent signal at this wavelength is interfered by 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 therefore can only serve as simple markers. After labeling is completed, the environment and non-specifically bound fluorescent ligands need to be washed. In addition, there are few applications of such fluorescent ligands for zebrafish in vivo imaging to explain the relevant information of the corresponding target biomolecules. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a targeted MOR responsive pyridine near-infrared fluorescent probe that is not affected by biological autofluorescence and a synthesis method and application thereof.

[0007] Technical scheme: In order to solve the above technical problems, the present application provides a targeted MOR responsive pyridine near-infrared fluorescent probe or its pharmaceutically acceptable salt, the structure of the probe is shown in the following formula:

[0008]

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

[0010] Preferably, the structure of the probe is as shown in the following formula:

[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 with a linkage site was synthesized by a reduction reaction;

[0014] (2) Using 2,4,6-trimethylpyrantetrafluoroborate and 4-(dimethylamino)cinnamaldehyde as raw materials, 4-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-2,6-dimethylpyridinium 2 was synthesized, and pyridine fluorophore 3 was synthesized through nucleophilic substitution reaction;

[0015] (3) Intermediate 1 with a linking site and pyridine fluorophore 3 with a linking chain generate final product (I) through a carboxyl-amino condensation reaction.

[0016] Specifically, the synthesis step (1) includes:

[0017]

[0018] Synthesis step (2) specifically includes:

[0019]

[0020] Synthesis step (3) specifically includes:

[0021]

[0022] The fluorescent ligand described in this invention utilizes an intramolecular charge transfer (ICT) mechanism to synthesize a small-molecule near-infrared fluorescent ligand with an environmentally responsive fluorescence activation mechanism by combining naltrexone, a pharmacophore specifically targeting MOR, with a pyridine fluorophore possessing excellent fluorescence properties. Based on the properties of this probe, it can be used as a tool molecule for the visualization of MOR.

[0023] This invention also provides the application of the aforementioned MOR-targeting responsive pyridine near-infrared fluorescent probe in aggregation-induced emission materials.

[0024] This invention also provides the application of the aforementioned MOR-targeting responsive pyridine near-infrared fluorescent probe in the preparation of reagents or drugs for the detection / diagnosis of μ-opioid receptor-mediated diseases.

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

[0026] The drug is a μ-opioid receptor agonist or antagonist.

[0027] The concentration of MOR ranged from 0.81 to 104.00 μg / ml.

[0028] Therefore, based on the shortcomings of various small molecule fluorescent ligands developed previously, structural modification of pyridine dyes to develop fluorescent ligands with good water solubility, strong stability, good drug-like properties, 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 biomolecular-related in vivo localization monitoring and visualization. The present invention aims to provide a method for synthesizing a responsive small-molecule near-infrared fluorescent ligand with targeted MOR and its application in live cells and zebrafish.

[0030] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. The fluorescent probe described in this invention plays an important role in the study of the binding kinetics of MOR and various ligands, helps to explain the active mechanism of related compounds, and provides lead compounds for the study of related diseases. It also has broad application prospects in the research field of diagnostic reagents for detecting μ-opioid receptors; 2. The pyridine small molecule fluorescent ligand (Ⅰ) developed in this 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, it utilizes the unique chemical properties of the pharmacophore and improves the water solubility of the pyridine fluorophore through a simple protonation step without changing the chemical structure of the ligand; 3. The excitation and emission wavelength of the fluorescent ligand is located in the near-infrared region, which is not easily affected by biological autofluorescence and has It has a high signal-to-noise ratio, can specifically identify MOR, and generates 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 and acceptor parts are relatively stable, and the fluorophore is small in size, thus improving the stability of the fluorescent ligand (I) and reducing the impact on the receptor binding force. The targeting activity is not easily destroyed. In vitro and in vivo experimental results 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 confocal microscopy; 5. Through zebrafish brain injection experiments, it was proved that this ligand can locate the MOR receptor in the zebrafish brain, which is of great value for determining the specific distribution of MOR in vivo and related dynamic information research. Attached Figure Description

[0031] Figure 1 The hydrogen NMR spectrum of intermediate 1;

[0032] Figure 2 Mass spectrum of intermediate 1;

[0033] Figure 3 The 1H NMR spectrum of the fluorescent ligand (I);

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

[0035] Figure 5 The absorption spectra of the fluorescent ligand (I) in different solvents are shown.

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

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

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

[0039] Figure 9 Confocal microscopy cell imaging of cells acting with fluorescent ligand (I);

[0040] Figure 10 Confocal microscopy image (a) of fluorescent ligand (I) acting on the brain of zebrafish and comparison of fluorescence signals (b). Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to 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), and 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 under vacuum, the pH was adjusted to 10 with 1 M NaOH, and the mixture was extracted with dichloromethane (5 mL × 3). The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under vacuum to obtain a milky white solid. The obtained solid was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30:1–10:1) to give 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 1H NMR spectrum of intermediate 1 is as follows Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown.

[0047] Step b:

[0048] Synthesis route:

[0049]

[0050] 4-(dimethylamino)cinnamaldehyde (700 mg, 4.7 mmol) and 2,4,6-trimethylpyranium tetrafluoroborate (1.18 g, 5.64 mmol, manufacturer: Leyan; catalog number: 137087) were dissolved in 20 mL of ethanol. The mixture was 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, yielding a blue-black solid mixture. This mixture was recrystallized in ethyl acetate and filtered. The filter cake was collected and dried under vacuum to give a metallic blue solid (1.09 g, 83.4%), i.e., 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. The mixture was 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. The solvent was removed by rotary evaporation to obtain a dark red solid powder. The obtained solid was purified by silica gel column chromatography (dichloromethane / methanol = 20:1-10:1) to obtain a deep red solid powder (569.7 mg, 78%), i.e., 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 of LDM under nitrogen atmosphere and stirred at room temperature for 24 hours. The solvent was then removed under vacuum. The resulting solid was purified by silica gel column chromatography (dichloromethane / methanol = 20:1-15:1) to give a deep red solid (10.4 mg, 26%), i.e., 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 a saturated solution of hydrogen chloride in ethyl acetate and stirred for 3 h. The solvent was removed under vacuum. After vacuum drying, a pale 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 = 6.4 Hz, 2H), 2.68-2.51 (m, 3H), 2.34 (d, J = 4.7 Hz, 1H), 2.30 (d, J = 8.7 Hz, 1H), 2.25 (d, J = 7.5 Hz, 2H), 2.01 (d, J = 4.6 Hz, 1H), 1.80-1.70 (m, 2H), 1.67-1.57 (m, 2H), 1.47-1.35 (m, 8H), 1.27 (d, J = 9.4 Hz, 7H), 0.86 (dd, J = 14.5, 6.3 Hz, 5H), 0.52 (dd, J = 7.3, 4.4 Hz, 2H), 0.13 (q, J = 4.8 Hz, 2H). The 1H NMR spectrum of fluorescent ligand (Ⅰ) is shown below. Figure 3 As shown, the mass spectrum is as follows Figure 4 As 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, respectively. 100 μl of each solution was placed in a 96-well microplate, and the absorption and fluorescence spectra were detected using a microplate reader. The results are as follows: Figure 5 As shown, the maximum excitation wavelength of the fluorescent probe (Ⅰ) in PBS is approximately 445 nm, in DMSO and ACN it is approximately 470 nm, and in ethanol and methanol it is approximately 490 nm. Figure 6 As shown, the fluorescent probe (Ⅰ) exhibits bimodal emission between 600 nm and 800 nm, with varying fluorescence intensity in different solvents. It shows strong fluorescence emission in DMSO, with the strongest emission peak at approximately 710 nm in both DMSO and acetonitrile, while the maximum emission peak is approximately 680 nm in PBS, methanol, and ethanol. It exhibits relatively low 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 660 nm under 445 nm excitation.

[0066] The plasmid containing human MOR (amino acid sequence: SEQ ID NO.1: MDSSAAPTNASNCTD) ALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCT LTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHF CIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEATAAPLPDYKDDDDK, article disclosing this plasmid: JIAY,XU L,WANG L,et al.A Light-up fluorescence probe for wash-free analysis of Mu-opioid receptor and ligand-binding events[J]. Analytica Chimica Acta, 2023, 1261:341-220. (Authors: Jia Yan, Xu Lili, Wang Lancheng, Yan Kun, Chen Jieru, Xu Pengcheng. Corresponding authors: Di Bin, Yan Fang, Hu Chi) The plasmid was cloned into the pcDNA3.1 vector (Changzhou Jiyu Biotechnology Co., Ltd.), transformed into Escherichia coli BL21(DE3) strain (Beyond BL21(DE3) glycerol strain (protein-inducible expression strain), catalog number: D0337), and then the plasmid was extracted and expressed.HEK 293T cells (Beyotime HEK293T (human embryonic kidney cells), catalog number: C6008) were transfected with the extracted plasmid, and the MOR protein expressed by the cells was extracted (amino acid sequence: SEQ ID). NO.2: MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGL PVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLPDYKDDDDK). After dissolving MOR and BSA, prepare protein solutions of equal concentration (MOR and BSA concentration both 104 μg / ml) using PBS. Dilute eight times at a 1 / 2 ratio. Place 50 μl of 1 μM fluorescent ligand (I) solution into a 96-well microplate. Add 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) to each well. After incubation for 5 minutes, measure the fluorescence spectrum of each concentration gradient. Figure 7 , Figure 8 As shown, the fluorescence signal increases with increasing MOR concentration, and the response signal of MOR is 3.2 times higher than that of BSA, proving that the fluorescent ligand (Ⅰ) can 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 this invention acts on HEK293T cells that overexpress MOR in the cell membrane.

[0068] HEK 293T cells were transfected with the pcDNA3.1-MOR-flag recombinant plasmid using a commercially available liposome nucleic acid transfection reagent (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 40802ES01). After culturing for 48 hours, MOR receptor protein was extracted from the cell membrane using a plasma membrane protein and cell component separation kit (Invent, catalog number SM-005) according to the manufacturer's instructions, thereby overexpressing MOR on the cell membrane. Overexpressing cells (6cm cell culture dish) were incubated with 2mL of fluorescent ligand (I) in PBS solution (50nM) at 37°C for 30 minutes, then placed under a confocal fluorescence microscope for excitation at 445nm and fluorescence emission signal at 660nm. Cells not expressing MOR were used as a blank control group, while the known strong binding MOR ligand naltrexone (Aladdin, catalog number: H2330623) and the μ-OPR agonist DAMGO (Maclean's, catalog number: D877395) were used as control groups for confocal microscopy imaging. Figure 9 As shown, the fluorescent ligand (I) produces a strong fluorescent signal on the cell membrane, while the control group shows almost no fluorescent signal. This demonstrates that the fluorescent ligand (I) specifically binds to the overexpressed MOR receptor on the cell membrane. When the probe molecule enters the hydrophobic structure of the MOR, the fluorescent signal is enhanced, resulting in a significantly higher fluorescence intensity on the cell membrane than other cell structures.

[0069] The above experimental examples demonstrate that the fluorescent ligand (Ⅰ) described in this invention can monitor MOR on the cell membrane in real time under physiological conditions. It has important value for the study of the relevant kinetics of MOR binding to 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 this invention was applied to the brain of zebrafish and the fluorescence signals were compared.

[0071] Zebrafish for imaging were incubated at 24 hpf (hours post-fertilization, hpf) with 50 ml of 0.003% 1-phenyl-2-thiourea. Zebrafish at 5 dpf (days post-fertilization, dpf) were anesthetized in 10 ml of 0.02% tricaine and positioned dorsally upwards, fixed to a slide with 1.5% low-melting-point agarose. Using a microinjector, 1 nL of 0.5 mM fluorescent ligand solution (P5N3) and 1 nL of a mixture of 0.5 mM fluorescent ligand and 2.5 mM Naltrexone (P5N3+Naltrexone) were injected into the rhomboid ventricles of the zebrafish, respectively. After 1 hour, the injected solutions dispersed in the zebrafish brain tissue. Z-stack scanning was performed under a laser confocal microscope, and the projection was set to maximum intensity. Figure 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] Furthermore, quantitative analysis of the fluorescence intensity in the zebrafish brain was performed. Data analysis using the Shapiro-Wilk method and the Bartlett method showed that the two groups of data conformed to a normal distribution and had homogeneity of variance. According to the t-test results of the two groups of data (n=6 / group), the fluorescent ligand group was significantly different from the group with the strong binding ligand Naltrexone (P=0.0024).

[0073] This fluorescent ligand has good tissue penetration, enabling real-time fluorescence visualization of MOR through in vivo imaging in zebrafish. Combined with high-resolution confocal imaging technology, it is of great significance for studying the mechanism of action of analgesic drugs and developing new analgesic drugs.

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. The application of the MOR-targeting responsive pyridine near-infrared fluorescent probe of claim 1 in aggregation-induced emission materials.

3. The use of the MOR-targeting responsive pyridine near-infrared fluorescent probe of claim 1 in the preparation of reagents or drugs for the detection / diagnosis of μ-opioid receptor-mediated diseases.

4. The use of the MOR-targeting responsive pyridine near-infrared fluorescent probe of claim 1 in the preparation of drugs for screening μ-opioid receptor-related diseases.

5. The application according to claim 4, characterized in that, The drug is a μ-opioid receptor agonist or antagonist.

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