CN119219647A

CN119219647A - A responsive pyridine near-infrared fluorescent probe targeting MOR and its synthesis method and application

Info

Publication number: CN119219647A
Application number: CN202410877604.8A
Authority: CN
Inventors: 胡驰; 蔺晓萱; 徐鹏程
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2024-07-02
Filing date: 2024-07-02
Publication date: 2024-12-31

Abstract

The invention discloses a response pyridine near infrared fluorescent probe for targeting MOR, a synthesis method and application thereof, wherein the structural formula of the response pyridine near infrared fluorescent probe for targeting MOR is shown as the following formula. The fluorescent ligand of the invention has excitation emission wavelength in near infrared region, is not easy to be interfered by biological autofluorescence, has higher signal to noise ratio, can specifically identify MOR, generates considerable fluorescent signal after being combined with the MOR hydrophobic region, can carry out high-sensitivity detection on MOR, improves the stability of the fluorescent ligand, reduces the influence on receptor binding force, and is not easy to damage the targeting activity, and the in-vitro and in-vivo experimental result shows that the series of probes have excellent stability and targeting property in-vivo and in-vitro, thereby being suitable for carrying out real-time monitoring on MOR on living cell membranes under a confocal microscope.

Description

Responsive pyridine near infrared fluorescent probe for targeting MOR and synthesis method and application thereof

Technical Field

The invention relates to a responsive pyridine near infrared fluorescent probe for targeting MOR, and a synthesis method and application thereof, and belongs to the technical field of fluorescent molecular probes.

Background

Mu opioid receptors (mu Opioid Receptor, MOR), belonging to the G-protein coupled receptor (GPCRs) A family, achieve their physiological effects mainly by inhibiting the heterotrimeric G-protein (Gi/o) of adenylate cyclase, mainly distributing the brain stem, spinal cord and cortex and many areas of the central nervous system. Mu opioid receptors can combine with specific antagonists and agonists to produce specific opioid effects such as analgesic effects, modulation of the reward system, respiratory depression, modulation of the digestive system, immune modulation, and the like. The MOR is combined with specific endogenous or exogenous small molecules (buprenorphine and the like) or short peptides (enkephalin and the like) so as to activate downstream Gi/o signal pathways to produce analgesic effects, and the small molecule MOR ligands developed in the early stage activate Gi/o mediated analgesic effect pathways and also activate beta-arresting (beta-repressor recruitment protein) pathways with side effects such as respiratory depression, addiction, drug tolerance and the like, thereby severely limiting drug development acting on MOR receptors. In recent years, in order to reduce adverse reactions of analgesic drugs, development of MOR-biasing ligands acting on the Gi/o pathway without activating the β -arresting pathway has become an important research direction for developing novel analgesic drugs.

In the course of the investigation of MOR-biased ligands, small molecule fluorescent ligands play an important role. This is a class of small molecule compounds that can emit fluorescent signals of specific wavelengths by excitation with excitation light and have specific targets, consisting of a fluorescent group that emits fluorescent signals, a targeting group that has a target recognition effect, and a connecting chain that connects the two. The physicochemical properties and pharmacological activity of the final probe need to be considered in the design of the fluorescent ligand to ensure that the affinity and selectivity of the fluorescent conjugate for the receptor is not significantly reduced. By reasonably conjugating the MOR agonist or antagonist to various fluorophores, fluorescent ligands can be designed that specifically recognize MOR. This class of fluorescent ligands exhibits lower fluorescent signals when not bound to the target and generally requires higher stability and biocompatibility. These advantages make them useful for monitoring interactions between living cells or ligands and target receptors in real time to display information about target receptor pharmacology, physiology, kinetics. The fluorescent ligand with high specificity, high sensitivity and fluorescence opening characteristic can be used for visually locating receptors in living cells or in vivo and even monitoring complex biological processes in vivo, and can be widely applied to the fields of biosensing and detection, drug screening, drug effect evaluation and the like.

Zebra fish is used as a novel model organism and has the unique imaging advantages of transparency in embryo period, small size and the like. Therefore, the specific fluorescent ligand with strong tissue penetrating power can be used for visual analysis of biological molecules in zebra fish, and can furthest improve the signal to noise ratio while reducing the damage of biological samples. In addition, zebra fish has a high degree of genetic homology to humans, the nervous system is highly similar to that of mammals, with corresponding brain regions such as the olfactory bulb, the telencephalon, the optic head, the metaencephalon, the midbrain, the brain extension, the hypothalamus, etc., and various neurotransmitters such as glutamate, gamma-aminobutyric acid, acetylcholine, dopamine, 5-hydroxytryptamine, etc. are characterized in the zebra fish brain. Zebra fish encoded mu-opioid receptor (ZFOR 2) has seven potential transmembrane domains, whose gene sequences also show a high degree of identity with human receptors. Zebra fish as an easy-to-operate model organism combines with a high-resolution in-vivo imaging technology, so that the zebra fish can be used as an ideal choice for fluorescent ligand in-vivo imaging to realize in-vivo imaging of biomolecule visualization.

Examples of commercial fluorophores are fluorescein, rhodamine, coumarin, BODIPY, alexa Fluor-series dyes, cy-series (holohydrin) dyes, and the like. However, some chemical properties of these dyes limit the development, such as poor water solubility, signal interference caused by overlapping of emission wavelength and autofluorescence of biological tissue, poor stability, poor drug formation caused by reduced binding capacity of active group and target due to addition of fluorescent group, and large molecular weight, for example, the maximum emission wavelength of the fluorescent probe disclosed in patent (202210961498.2) is 480nm, and the fluorescent signal at the wavelength is interfered by autofluorescence of biological tissue to generate false positive signals, which is unfavorable for in vivo imaging. In addition, most of the current MOR fluorescent ligands do not have a lighting mechanism, so that the MOR fluorescent ligand can only play a simple marking function, and the fluorescent ligands which are in the environment and are not specifically combined need to be washed after the marking is completed. Furthermore, there is currently little application of such fluorescent ligands to zebra fish live imaging to interpret information about the corresponding target biomolecules.

Disclosure of Invention

The invention aims to solve the technical problem of providing a response pyridine near infrared fluorescent probe targeting MOR without being influenced by biological autofluorescence, and a synthesis method and application thereof.

The technical scheme is that in order to solve the technical problems, the invention provides a response pyridine near infrared fluorescent probe targeting MOR or pharmaceutically acceptable salt thereof, and the structural formula of the probe is shown as follows:

Wherein n=3, 4, 5, 6, 7.

Preferably, the structural formula of the probe is as follows:

The invention also provides a method for preparing the response pyridine near infrared fluorescent probe for targeting MOR, which comprises the following steps:

(1) Synthesizing a racemization intermediate 1 with a linking site by taking naltrexone as a raw material through a reduction reaction;

(2) 2,4, 6-trimethyl pyran tetrafluoroborate, 4- (dimethylamino) cinnamaldehyde is used as a raw material to synthesize 4- ((1E, 3E) -4- (4- (dimethylamino) phenyl) butyl-1, 3-diene-1-yl) -2, 6-dimethyl pyridinium 2, and pyridine fluorophore 3 is synthesized through nucleophilic substitution reaction;

(3) Intermediate 1 having a linking site and pyridine fluorophore 3 having a linking chain are reacted by condensation reaction of carboxyl group with amino group to give the final product (I).

Wherein the synthesizing step (1) specifically comprises:

The synthesis step (2) specifically comprises:

The synthesis step (3) specifically comprises:

The fluorescent ligand disclosed by the invention combines a pharmacophore naltrexone with a specific targeting MOR with a pyridine fluorophore with good fluorescence property through a reasonable synthesis route based on an intramolecular charge transfer mechanism (ICT), and a small-molecule near-infrared fluorescent ligand with an environment-responsive fluorescence opening mechanism and specific targeting MOR is constructed. The visual study of MOR was performed on the basis of the nature of the probe with it as a tool molecule.

The invention also provides application of the response pyridine near infrared fluorescent probe targeting MOR in aggregation-induced emission materials.

The invention also provides application of the response pyridine near infrared fluorescent probe for targeting MOR in preparation of reagents or medicines for detecting/diagnosing related diseases mediated by mu opioid receptors.

The invention also provides application of the response pyridine near infrared fluorescent probe for targeting MOR in preparing medicines for screening mu opioid receptor related diseases.

Wherein the drug is a mu opioid receptor agonist or antagonist.

Wherein the concentration of MOR is 0.81-104.00 μg/ml.

Therefore, based on the defects of various small molecular fluorescent ligands developed before, structural modification of pyridine dyes is performed to develop fluorescent ligands with good water solubility, strong stability, good drug property and high specificity and high sensitivity on characteristics, which are always important directions in the research field of medical diagnostic reagents.

Synthetic fluorescent ligands are widely used for in vivo localization monitoring and visualization of biological molecule related events. The invention aims to provide a synthesis method of a responsive small molecule near infrared fluorescent ligand with a targeted MOR and application of the ligand in living cells and zebra fishes.

Compared with the prior art, the fluorescent probe has the following remarkable advantages that 1, the fluorescent probe has an important effect on the research on the binding kinetics of MOR and various ligands, is beneficial to explaining the active action mechanism of related compounds, provides a lead compound for the research of related diseases, and has wide application prospect in the research field of detecting mu opioid receptor diagnostic reagents; 2, the pyridine micromolecule fluorescent ligand (I) developed by the invention has small molecular weight, low toxicity, large Stokes shift, large molar extinction coefficient, simple synthesis step and low cost, compared with most of the prior fluorescent ligands, the pyridine micromolecule fluorescent ligand (I) has the advantages of improving the water solubility of the pyridine fluorophor under the condition of not changing the chemical structure of the ligand by utilizing the special chemical property of a pharmacophore through a simple protonation step, 3, the excitation emission wavelength of the fluorescent ligand is positioned in a near infrared region, is not easy to be interfered by biological autofluorescence, has higher signal to noise ratio, can specifically identify MOR, generates a considerable fluorescent signal after being combined with a hydrophobic region of the MOR, can avoid washing operation, can detect the MOR with high sensitivity, 4, introduces a simple and stable fluorophore structure, improves the stability of the fluorescent ligand (I) by virtue of a relatively stable electron donor part and a receptor, reduces the influence on the receptor, is not easy to be destroyed by targeting activity binding force, shows that the series of probes have excellent stability in vivo and in vitro and in vivo and in vitro are suitable for monitoring a zebra cell membrane by injecting a living cell membrane in a zebra cell membrane 5, the ligand is proved to be capable of locating the MOR receptor of the brain of the zebra fish, and has important value in determining the specific distribution of MOR in living bodies and relevant dynamic information research.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of intermediate 1;

FIG. 2 is a mass spectrum of intermediate 1;

FIG. 3 is a nuclear magnetic resonance spectrum of fluorescent ligand (I);

FIG. 4 is a mass spectrum of fluorescent ligand (I);

FIG. 5 is an absorption spectrum of fluorescent ligand (I) in different solvents;

FIG. 6 is a graph showing fluorescence spectra of fluorescent ligand (I) in different solvents;

FIG. 7 is a graph of fluorescence spectra of fluorescent ligand (I) interacting with MOR;

FIG. 8 is a graph comparing fluorescent signals of fluorescent ligand (I) interaction with BSA and MOR;

FIG. 9 is confocal microscopy cell imaging of fluorescent ligand (I) on cells;

FIG. 10 is a confocal microscopy image (a) of fluorescent ligand (I) acting on the brain of zebra fish and a fluorescent signal contrast plot (b).

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Example 1 Synthesis of Small molecule near-infrared fluorescent ligand (I)

Step a:

the synthetic route is as follows:

Naltrexone (0.12 g,0.35 mmol), ammonium acetate (0.27 g,3.5 mmol), sodium cyanoborohydride (0.033 g,0.53 mmol) was dissolved in 3mL of methanol under nitrogen protection, and stirred at room temperature for 24 hours. The solvent was removed in vacuo, 1M NaOH was adjusted to pH 10, dichloromethane extracted (5 mL. Times.3), the organic phase was taken, dried over anhydrous magnesium sulfate, filtered and the solvent removed in vacuo to give a milky solid. The obtained solid is separated and purified by a silica gel column chromatography (petroleum ether/ethyl acetate=30:1-10:1), and a nuclear magnetic resonance hydrogen spectrum of the intermediate 1 of the white solid 1(0.056g,47％).MS(ESI,m/z,C₂₀H₂₆N₂O₃,[M+H]⁺):calcd.,342.2;found 342.2. is shown in a figure 1, and a mass spectrum is shown in a figure 2.

Step b:

the synthetic route is as follows:

4- (dimethylamino) cinnamaldehyde (700 mg,4.7 mmol) and 2,4, 6-trimethylpyrylium tetrafluoroborate (1.18 g,5.64mmol, manufacturer: leyan; product number: 137087) were dissolved in 20mL ethanol, warmed to 80℃under nitrogen protection, stirred, refluxed for 1 hour, cooled to room temperature, filtered, the filtrate was collected, the solvent was removed by rotary evaporation to give a blue-black solid mixture, recrystallized in ethyl acetate, filtered. The cake was collected and dried in vacuo to give a metallic lustrous blue solid (1.09 g, 83.4%), intermediate 2.MS (ESI, m/z, C ₁₉H₂₂NO,[M+H]⁺) calcd, 280.17;found 280.17.

Step c:

the synthetic route is as follows:

Intermediate 2 (560 mg,2 mmol) and gamma-aminobutyric acid (258 mg,2.5 mmol) were dissolved in 10mL ethanol, warmed to 40 ℃ under nitrogen protection, stirred, refluxed for 5 hours, cooled to room temperature, filtered, the filtrate was collected, the solvent was removed by rotary evaporation to obtain a black red solid powder, and 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,C₂₃H₂₉N₂O₂,[M+H]⁺):calcd.,365.22;found 365.22.¹H 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).

Step d:

the synthetic route is as follows:

Intermediate 3 (21 mg,0.058 mmol), white solid 1 (20 mg,0.058 mmol), p-dimethylaminopyridine (DMAP, 8.5mg,0.07 mmol) and hydroxybenzotriazole (HOBt, 9.4mg,0.07 mmol) were dissolved in 2mL DMF under nitrogen and 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%), which was an intermediate 4.MS(ESI,m/z,C₄₃H₅₃N₄O₄,[M+H]⁺;[(M+2H)/2]⁺):calcd.,689.41;found 689.41;found 345.22.

Step e:

the synthetic route is as follows:

Intermediate 4 (10.4 mg,0.015 mmol) was placed in 2mL of saturated ethyl acetate in hydrogen chloride and the reaction stirred for 3h. The solvent was removed in vacuo. And vacuum drying to obtain yellowish solid, i.e. nuclear magnetic hydrogen spectrum of fluorescent ligand (Ⅰ)(9.1mg,83.6％).MS(ESI,m/z,C₄₃H₅₃N₄O₄,[M+H]⁺;[(M+2H)/2]⁺):calcd.,689.41;found 689.41;found345.22.¹H 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). fluorescent ligand (I) is shown in figure 3, and mass spectrum is shown in figure 4.

Example 2 absorption spectra and fluorescence emission spectra of small molecule near infrared fluorescent ligand (I) in PBS, methanol (MeOH), ethanol (EtOH), DMSO, acetonitrile (ACN)

The fluorescent ligand (I) is respectively prepared into 1.04mg/mL solution in PBS, methanol, ethanol, DMSO and acetonitrile, 100 μl of each solution is placed in a 96-well ELISA plate, and detection of absorption spectrum and fluorescence spectrum is carried out by an ELISA instrument. As a result, as shown in FIG. 5, the maximum excitation wavelength of the fluorescent probe (I) in PBS was about 445nm, the maximum excitation wavelength in DMSO and ACN was about 470nm, and the maximum excitation wavelength in ethanol and methanol was about 490nm. As shown in FIG. 6, the fluorescent probe (I) exhibits bimodal emission at the most emission wavelength between 600nm and 800nm, and the fluorescence intensity varies in different solvents. Exhibits strong fluorescence emission in DMSO, with a maximum emission peak of about 710nm in DMSO and acetonitrile, and a maximum emission peak of about 680nm in PBS, methanol, ethanol. Wherein lower fluorescence emission is exhibited in PBS.

Example 3 fluorescence response Spectrum of fluorescent ligand (I) acting on MOR and comparison of fluorescent signals of fluorescent ligand (I) at 660nm with MOR, BSA, respectively, under 445nm excitation

Plasmids containing human MOR (amino acid sequence ：SEQ ID NO.1：MDSSAAPTNASNCTD ALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLPDYKDDDDK,, paper ：JIAY,XU L,WANG L,et al.A light-up fluorescence probe for wash-free analysis of Mu-opi oid receptor and ligand-binding events[J].Analytica Chimica Acta,2023,1261:341220., authors of which were published: gu Yan, xu Lili, wang Lancheng, kun, chen Jieru, xu Pengcheng. Communication authors: di, yan Fang, hu Chi) were cloned into pcDNA3.1 vector (Changzhou-keyu Biotechnology Co., ltd.) and transformed into E.coli BL21 (DE 3) strain (Biyunsan BL21 (DE 3) glycerol bacterium (protein-inducible expression strain), cat# D0337) and the plasmids were extracted and expressed. HEK293T cells (Biyun HEK293T (human embryonic kidney cells), cat# C6008) were transfected with the plasmids after extraction, MOR proteins expressed by the cells (amino acid sequence ：SEQ ID NO.2：MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQLENLEAETAPLPDYKDDDDK).MOR and BSA were dissolved and prepared into protein solutions of the same concentration (concentration of MOR and BSA were 104. Mu.g/ml) with PBS, diluted 8 times in a 1/2 ratio, 50. Mu.l of 1. Mu.M fluorescent ligand (I) solution was placed in 96-well ELISA plates, 50. Mu.l of each concentration of MOR and BSA solution (104.00. Mu.g/ml, 52.00. Mu.g/ml, 26.00. Mu.g/ml, 13.00. Mu.g/ml, 6.50. Mu.g/ml, 3.25. Mu.g/ml, 1.63. Mu.g/ml) were added to each well, and fluorescence spectra of each concentration gradient were measured after 5 minutes, as shown in FIGS. 7 and 8, the fluorescence signal was enhanced with increasing MOR concentration, and the response signal was shown to be far higher than that the fluorescence signal of the ligand combined with fluorescence signal of 3.660 nm (specific binding at 660 nm).

Example 4 HEK 293T cells overexpressing MOR by the Small molecule near-infrared fluorescent ligand (I) described in the present invention

The normal growth of HEK 293T cells was subjected to pcDNA3.1-MOR-flag recombinant plasmid transfection using a commercial liposome nucleic acid transfection reagent (product No. 40802ES01, shanghai) and MOR receptor protein on the cell membrane was extracted according to the instructions using plasma membrane protein and cell fraction separation kit (Invent, product No. SM-005) after 48 hours of culture, allowing MOR to be overexpressed on the cell membrane. Cells after overexpression (6 cm cell culture dish) were incubated with 2mL of PBS solution of fluorescent ligand (I) (50 nM) at 37℃for 30 minutes, then placed under a confocal fluorescence microscope, excited at 445nM excitation wavelength, and fluorescence emission signal at 660nM was detected, and cells that did not express MOR were used as a blank group, while cells were confocal microscopy imaged with MOR ligand naltrexone (Naltrexone, allatin, cat# H2330623) and mu-OPR agonist DAMGO (microphone, cat# D877395) known to have strong binding capacity as a control group. As shown in FIG. 9, the fluorescent ligand (I) produced a strong fluorescent signal on the cell membrane, whereas the control group showed little fluorescent signal, demonstrating that the fluorescent ligand (I) of the invention specifically binds to the MOR receptor overexpressed on the cell membrane, and when the probe molecule enters into the MOR hydrophobic structure, the fluorescent signal is enhanced, resulting in significantly higher intensity of fluorescent signal on the cell membrane than other structures of the cell.

The above experimental examples show that the fluorescent ligand (I) can monitor the MOR on the cell membrane in real time under physiological conditions, has important value for the related dynamics research of the combination of the MOR and the specific ligand, and has potential for developing clinical diagnostic reagents.

Example 5 Small molecule near infrared fluorescent ligand (I) described in the present invention acts on the brain of Zebra fish and performs fluorescent signal contrast

Zebra fish required for imaging were incubated with 50ml, 0.003% 1-phenyl-2-thiourea at 24hpf (hours post fertilization, hpf), 5dpf (days post fertilization, dpf) were anesthetized in 10ml, 0.02% tricaine, and adjusted with the back side facing up, and fixed on slides with 1.5% low melting agarose. 1nL, 0.5mM fluorescent ligand solution (P5N 3) and 1nL, 0.5mM fluorescent ligand and 2.5mM Naltrexone mixed solution (P5N3+ Naltrexone) are respectively injected into the diamond-shaped ventricles of the zebra fish by using a microinjection instrument, the injected solution is dispersed in the brain tissue of the zebra fish after 1h, and the Z-stack layer scanning is carried out under a laser confocal microscope and then the projection is set to the maximum intensity. As shown in fig. 10, opioid receptors showed a broad distribution in the zebra fish nervous system, significantly reducing the fluorescent signal generated by the fluorescent ligand upon addition of Naltrexone.

In addition, the quantitative analysis of the fluorescence intensity of the zebra fish brain, the data analysis by the shape-Wilk method and the Bartlett method showed that the two sets of data were in normal distribution, with variance alignment, and the fluorescent ligand set was significantly different (p=0.0024) from the group to which the strong binding ligand Naltrexone was added according to the t-test result (n=6/set) of the two sets of data.

The fluorescent ligand has good tissue penetrability, can realize real-time fluorescence visualization of MOR by performing living body imaging in zebra fish, and has important significance for researching the action mechanism of analgesic drugs and developing novel analgesic drugs by combining a high-resolution confocal imaging technology.

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 as shown below:

2. A method for preparing the responsive pyridine near-infrared fluorescent probe targeting MOR according to claim 1, characterized in that it comprises the following steps:

(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.