A responsive all-cyanine small molecule near-infrared fluorescent probe targeting MOR, its synthesis method and application

Abstract

The present invention discloses a responsive full-cyanine small molecule near-infrared fluorescent probe targeting MOR, its synthesis method and application. The fluorescent probe is a compound with the structural formula shown in (I) or a pharmaceutically acceptable salt thereof. The fluorescent probe constructed by the present invention is based on a full-cyanine fluorescent dye and the MOR antagonist naltrexone, with low synthesis cost and simple operation. It can specifically identify MOR and produce a considerable fluorescent signal after binding to the hydrophobic region of MOR, avoiding washing operations and detecting MOR with high sensitivity. The fluorescent probe described in the present invention has a stable skeleton structure and is suitable for real-time monitoring of MOR on the cell membrane of living cells under a confocal microscope. The fluorescent probe described in the present invention plays an important role in the study of the binding kinetics of MOR and various ligands, and can also be used for screening ligands that specifically bind to MOR receptors. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a responsive full-cyanine small molecule near-infrared fluorescent probe targeting MOR, a synthesis method and application thereof, and belongs to the technical field of fluorescent probes. Background Art

[0002] The μ-opioid receptor (MOR) belongs to the A family of G protein-coupled receptors (GPCRs). These receptors, arranged longitudinally within the cell membrane's phospholipid bilayer and containing seven transmembrane helical bundles, are the primary targets of exogenous opioid peptides (e.g., morphine) and endogenous opioid peptides (e.g., enkephalins). They are primarily distributed in numerous regions of the central nervous system, including the brainstem, spinal cord, and cerebral cortex. μ-opioid receptors can bind to specific antagonists and agonists to produce specific opioid effects, such as analgesia, reward system regulation, respiratory depression, digestive system regulation, and immune regulation. As the primary target of morphine-like substances, studies of the physicochemical properties and kinetics of MORs have significant implications for drug control efforts and the improvement of national laws and regulations governing psychotropic substances. Therefore, the development of fluorescent probes that can specifically monitor MORs has been a key area of ​​research related to MORs.

[0003] As an opioid receptor antagonist, naltrexone can bind to MOR through a specific binding pocket with high binding affinity, producing an antagonistic effect, inhibiting the binding of agonists to MOR, thereby weakening or reversing the dependence caused by opioid agonists. Due to its stable properties and long-lasting efficacy, it has been used in pharmacological studies related to MOR.

[0004] Responsive small molecule fluorescent probes are a class of small molecule compounds that can emit fluorescent signals of specific wavelengths upon excitation and have specific targets. These small molecule compounds generally consist of three components: a fluorescent group that emits fluorescent signals, a targeting group that recognizes the target, and a linker chain connecting the two. These fluorescent probes exhibit a low fluorescent signal when not bound to their target and are generally required to have high stability and biocompatibility. They can be used for biomolecule detection and quantitative analysis, cell imaging and localization, biosensing and detection, and drug screening and efficacy evaluation. Cy dyes have advantages such as small molecular weight, low toxicity, a wide wavelength tunable range, a large molar extinction coefficient, and excellent imaging effects, making them widely used in the field of fluorescent probes.

[0005] Currently commercial fluorescent dyes include fluorescein, rhodamine, coumarin, BODIPY, Alexa Fluor series dyes, and Cy series (all-cyanine) dyes. However, the chemical properties of these dyes have limited their development, such as poor water solubility, signal interference caused by overlap of emission wavelengths with biological tissue autofluorescence, poor stability, and reduced binding ability of active groups to targets due to the addition of fluorescent groups. Summary of the Invention

[0006] Objectives of the invention: The first objective of the present invention is to provide a highly specific and sensitive MOR-targeting responsive all-cyanine small molecule near-infrared fluorescent probe. The second objective of the present invention is to provide a synthesis method and application of the above fluorescent probe.

[0007] Technical solution: The present invention provides a responsive all-cyanine small molecule near-infrared fluorescent probe targeting MOR, wherein the fluorescent probe is a compound represented by the structural formula (I) or a pharmaceutically acceptable salt thereof:

[0008]

[0009] The present invention also provides a method for synthesizing the responsive all-cyanine small molecule near-infrared fluorescent probe targeting MOR, comprising the following steps:

[0010] (1) Using 4-hydrazinobenzenesulfonic acid, 3-methyl-2-butanone and iodoethane as raw materials to synthesize 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate, i.e., intermediate 1-1; synthesizing a full-cyanine dye 1-2 through a condensation reaction; and synthesizing a full-cyanine dye intermediate 1-3 having a connecting chain through a substitution reaction of the full-cyanine dye 1-2 and 5-aminopentanoic acid;

[0011] (2) Using naltrexone as a raw material, the carbonyl group was reduced by sodium cyanoborohydride to obtain the intermediate 2-1 with a linking site;

[0012] (3) The intermediate 2-1 with a connection site prepared in step (2) and the full cyanine dye intermediate 1-3 with a connection chain prepared in step (1) are subjected to a condensation reaction between the carboxyl group and the amino group to generate the final product (I).

[0013] Wherein, step (1) specifically includes:

[0014]

[0015] Wherein, step (2) specifically includes:

[0016]

[0017] Wherein, step (3) specifically includes:

[0018]

[0019] Wherein, the eluent used for separation and purification in step (2) is petroleum ether / ethyl acetate 30:1-10:1.

[0020] The present invention also provides the use of the fluorescent probe in preparing aggregation-induced luminescence materials.

[0021] The present invention also provides the use of the fluorescent probe in preparing a detection reagent or a diagnostic reagent for diseases related to μ opioid receptors.

[0022] Wherein, the reagent includes an antagonist or an agonist.

[0023] The present invention also provides the use of the fluorescent probe in combination with μ opioid receptors in the preparation of drugs for inhibiting pain, regulating the reward system, regulating the digestive system or regulating the immune system.

[0024] The fluorescent probe described in this invention is based on the aggregation-induced mechanism (AIE) and twisted intramolecular charge transfer (TICT) mechanism. Through a rational synthetic route, the pharmacophore naltrexone, which specifically targets MOR, is combined with a pancyanine dye with excellent fluorescent properties. This results in a small-molecule near-infrared fluorescent probe that specifically targets MOR and has a rational fluorescence onset mechanism. Based on the properties of this probe, it is used as a tool molecule for MOR visualization studies.

[0025] Beneficial Effects: Compared with existing technologies, the present invention offers the following significant advantages: The fluorescent probe constructed in the present invention is based on a cyanine fluorescent dye and the MOR antagonist naltrexone, is inexpensive to synthesize, and is simple to operate. It specifically recognizes MOR and produces a significant fluorescent signal upon binding to the hydrophobic region of MOR, eliminating the need for washing and enabling highly sensitive detection of MOR. The excitation and emission wavelengths of the fluorescent probe described in the present invention are in the near-infrared region, making it less susceptible to interference from biological autofluorescence and exhibiting a high signal-to-noise ratio. The fluorescent probe described in the present invention has been structurally optimized, with a sulfonate group introduced at the 5-position of the indole to balance the electrical properties of the probe molecule. This modification prevents cellular uptake, improves water solubility, and reduces nonspecific adsorption. These improvements make the probe suitable for studies of MOR pharmacology under physiological conditions. The fluorescent probe described in the present invention has a stable backbone structure and is suitable for real-time monitoring of MOR on the cell membrane of living cells using confocal microscopy. The fluorescent probe described in the present invention plays an important role in studying the binding kinetics of MOR with various ligands and can also be used to screen ligands that specifically bind to MOR receptors. The research field of detecting μ opioid receptor diagnostic reagents has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is the H NMR spectrum of the fluorescent probe (I) prepared in Example 1;

[0027] Figure 2 This is the mass spectrum of the fluorescent probe (I) prepared in Example 1;

[0028] Figure 3 The fluorescence spectra of different concentrations of fluorescent probe (I) in PBS in Example 2 are shown;

[0029] Figure 4 3 is a comparison diagram of the fluorescence signals of the interaction between the fluorescent probe (I) and BSA and MOR in Example 3;

[0030] Figure 5 This is confocal microscopy cell imaging after the fluorescent probe (I) in Example 4 acts on HEK 293T cells that overexpress MOR on the cell membrane. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below with reference to the accompanying drawings. The chemical substances involved in the synthesis steps are all commercially available commodities.

[0032] Example 1: Synthesis steps of the small molecule near-infrared fluorescent probe (I) described in the present invention

[0033]

[0034] Step a: 4-Hydrazinobenzenesulfonic acid (5.00 g, 26.57 mmol) and 3-methyl-2-butanone (8.38 ml, 39.85 mmol) were dissolved in 15 ml of acetic acid. The mixture was heated to 123°C under nitrogen and stirred at reflux for 4 hours. The reaction system was cooled to room temperature and filtered. Excess ethyl acetate was added to the filtrate and filtered. The resulting filter cake was dissolved in methanol (35 ml) and a solution of potassium hydroxide (1.4 g, 24.56 mmol) in isopropanol (35 ml) was added. The mixture was stirred at room temperature for 15 hours, filtered, and the filter cake was washed with ethyl acetate and dried in vacuo to obtain a light brown solid (4.09 g, 64.32%). MS (ESI, m / z, C 11 H 13 NO3S,[M+H] + ):calcd.,238.1;found 238.1.

[0035]

[0036] Step b: The product obtained in step a (4.00 g, 16.72 mmol) was dissolved in acetonitrile (30 ml), iodoethane (3.91 g, 25.08 mmol) was added, and the mixture was heated to 85°C under nitrogen and stirred at reflux for 24 hours. The reaction system was cooled to room temperature, excess acetone was added, filtered, and the filter cake was washed with acetone and dried under vacuum to obtain a dark red solid (3.51 g, 78.52%), namely, intermediate 1-1 (1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate). MS (ESI, m / z, C 13 H 17 NO3S,[M+H] + ):calcd.,267.1;found 267.1.

[0037]

[0038] Step c: Dissolve the product 1-1 (2 g, 7.48 mmol) obtained in step b, 2-chloro-1-formyl-3-hydroxymethylcyclohexene (0.65 g, 3.74 mmol), and sodium acetate (0.613 g, 7.48 mmol) in 15 ml of acetic anhydride. Under nitrogen protection, heat to 75°C and stir for 5 hours. The reaction system is cooled to room temperature, and an excess of anhydrous ether is added with vigorous stirring. The system is filtered, the filter cake is washed with ether, and vacuum dried to obtain a dark green solid. The obtained solid is separated and purified by silica gel column chromatography (dichloromethane / methanol = 20:1-10:1). The solvent is removed in vacuo, and the product is freeze-dried to obtain a metallic green solid 1-2 (0.88 g, 35%). MS (ESI, m / z, C 34 H 39 ClN2O6S2,[M+H] + ):calcd.,671.2;found 671.2.

[0039]

[0040] Step d: Dissolve the product 1-2 (0.45 g, 0.67 mmol) obtained in step c and 5-aminopentanoic acid (0.12 g, 1.01 mmol) in 5 ml of N,N-dimethylformamide (DMF), heat to 85°C under nitrogen protection, and stir to react for 30 minutes. The reaction system is cooled to room temperature and the solvent is removed in vacuo. The obtained solid is separated and purified by silica gel column chromatography (dichloromethane / methanol = 10:1-5:1), the solvent is removed in vacuo, and after lyophilization, a metallic dark blue solid 1-3 (0.43 g, 86%) is obtained. MS (ESI, m / z, C 39 H 49 N3O8S2,[M-2H] - / 2):calcd.,374.6;found 374.6.

[0041]

[0042] Step e: 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 in vacuo, the pH was adjusted to 10 with 1 M NaOH, and the mixture was extracted with dichloromethane (5 mL × 3). The organic phase was taken and dried over anhydrous magnesium sulfate, 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 2-1 (0.056 g, 47%) as a white solid. MS (ESI, m / z, C 20 H 26 N2O3,[M+H] + ):calcd.,342.2;found 342.2.

[0043]

[0044] Step f: 1-3 (0.025 g, 0.033 mmol), 1-2 (0.017 g, 0.049 mmol), 4-dimethylaminopyridine (DMAP) (0.006 g, 0.049 mmol), and HOBt1-hydroxybenzotriazole (HOBt) (0.007 g, 0.049 mmol) were dissolved in 2 ml of DMF under nitrogen, stirred at room temperature for 36 hours, and the solvent was removed in vacuo. The resulting solid was separated and purified by silica gel column chromatography (dichloromethane / methanol = 20:1-8:1) to obtain a dark blue solid, which is the MOR-targeting responsive all-cyanine small molecule near-infrared fluorescent probe (I) of the present invention (0.023 g, 65%). MS (ESI, m / z, C 59 H 73 N5O 10 S2,[MH] - ):calcd.,1074.4; found 1074.4.

[0045] 1H NMR (300MHz, Methanol-d4) δ8.46 (d, J=14.7Hz, 3H), 7.90 (dd, J=5.9, 3.2Hz, 1H) ,7.79(dd,J=5.7,3.1Hz,1H),7.62(d,J=7.4Hz,3H),7.51(d,J=7.0Hz,4H),7.45 –7.37(m,2H),6.35(d,J=14.8Hz,3H),4.74(s,6H),4.53(s,1H),4.33(t,J=7.4H z,6H),3.43(s,8H),3.37(s,4H),2.40–2.31(m,8H),1.78(s,17H),1.62(s,6H).

[0046] Example 2: Fluorescence spectrum of fluorescent probe (I) in PBS

[0047] Different volumes (20, 40, 60, 80, 100, 120, 140, 160 μL) of the fluorescent probe (I) (50 μM) prepared in Example 1 were added to different centrifuge tubes, and then the volume was supplemented to 200 μL with PBS and mixed evenly. The fluorescence signal value at an excitation wavelength of 640 nm and an emission wavelength of 670-800 nm was measured. The results are shown in FIG. Figure 3 As shown in the figure, it can be seen that the maximum emission wavelength of the fluorescent probe (Ⅰ) is at 740 nm, and as the concentration of the probe increases, the fluorescence intensity also increases, indicating that the prepared probe has the potential to be used in high-resolution near-infrared imaging of biological tissues.

[0048] Example 3: Comparison of fluorescence signals of the interaction between fluorescent probe (I) and BSA and MOR The MOR gene OPRM1 (NP_001138751.1) was downloaded from the Gene Expression Omnibus of the National Center for Biotechnology Information (NCBI). The plasmid containing human MOR (ordered from Changzhou Jiyu Biotechnology Co., Ltd.) was cloned into the pcDNA3.1 vector, which was transformed into the Escherichia coli BL21 (DE3) strain and then the plasmid was extracted and expressed.

[0049] The extracted MOR was dissolved using a non-denaturing protein dissolving solution (Ingwin Biotechnology Co., Ltd.) and prepared into a 50 μM protein stock solution, which was diluted to 10 μM with PBS before use; BSA (Biyuntian Biotechnology Co., Ltd.) was also prepared into a 10 μM protein solution. 50 μL of each of the MOR solution and the BSA solution were placed in a well of a 96-well ELISA plate, 50 μL of PBS was added to another well, and 50 μL of PBS solution containing the fluorescent probe (I) prepared in Example 1 was added to each of the three wells (the concentration of the fluorescent probe was 5 μM). A multifunctional microplate reader was used to collect the fluorescence signal, and the fluorescence emission signal with an excitation wavelength of 640 nm and a detection wavelength of 740 nm was recorded. Each well was repeated three times. A histogram was drawn with the three wells as the horizontal axis and the fluorescence intensity (FI, Fluorescence Intensity) with an emission wavelength of 740 nm as the vertical axis. The results are shown in the figure. Figure 4 As shown, the fluorescent probe of the invention responds to both BSA and MOR, and the fluorescence intensity of the response to MOR is 3.6 times that of BSA, indicating that the fluorescent probe of the invention can specifically recognize MOR and is not easily interfered by other proteins. It also shows that the fluorescent probe of the invention has great potential as a tool molecule to study MOR-ligand binding-related events.

[0050] Example 4: Fluorescent probe (I) acts on HEK 293T cells overexpressing MOR on the cell membrane

[0051] A commercial DNA transfection kit (Beyotime Lipo8000 TM Normally grown HEK293T cells were transfected with plasmids using a transfection reagent (e.g., 5-mercaptoethanol) to overexpress MOR on the cell membrane. The overexpressed cells (6 cm cell culture dish) were incubated with 2 mL of PBS solution (1 μM) containing the fluorescent probe (I) prepared in Example 1 under cell culture conditions for 30 minutes, and then placed under a confocal fluorescence microscope. The cells were excited at a wavelength of 640 nm, and the fluorescence emission signal at a wavelength of 750 nm was detected, and the cells were imaged. The results are shown in Figure 2. Figure 5 As shown, the fluorescent probe of the invention specifically binds to the MOR receptor overexpressed on the cell membrane. When the probe molecule enters the hydrophobic structure of MOR, the fluorescent signal is enhanced, resulting in the fluorescent signal intensity on the cell membrane being significantly higher than that in other structures of the cell.

[0052] The above experimental examples show that the fluorescent probe of the present invention can monitor MOR receptors on the cell membrane in real time under physiological conditions, has great potential for qualitative and quantitative studies of the binding of MOR and ligands, and also has considerable application prospects for the screening and affinity determination of MOR ligands.

Claims

1. A responsive all-cyanine small molecule near-infrared fluorescent probe targeting MOR, characterized in that: The fluorescent probe is a compound represented by the structural formula (I) or a pharmaceutically acceptable salt thereof:

2. A method for synthesizing the MOR-targeting responsive all-cyanine small molecule near-infrared fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Using 4-hydrazinobenzenesulfonic acid, 3-methyl-2-butanone and iodoethane as raw materials to synthesize 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate, i.e., intermediate 1-1; synthesizing a full-cyanine dye 1-2 through a condensation reaction; and synthesizing a full-cyanine dye intermediate 1-3 having a connecting chain through a substitution reaction of the full-cyanine dye 1-2 and 5-aminopentanoic acid; The structural formula of the intermediate product 1-1 is The structural formula of the full-flower cyanine dye 1-2 is The structural formula of the cyanine dye intermediate 1-3 is (2) Using naltrexone as a raw material, the carbonyl group was reduced by sodium cyanoborohydride to obtain the intermediate 2-1 with a linking site; (3) reacting the intermediate 2-1 having a connection site prepared in step (2) with the full cyanine dye intermediate 1-3 having a connection chain prepared in step (1) through a condensation reaction between the carboxyl group and the amino group to produce the final product (I); The structural formula of the intermediate 2-1 is 3. The synthesis method according to claim 2, characterized in that Step (1) specifically includes:

4. The synthesis method according to claim 2, characterized in that Step (2) specifically includes:

5. The synthesis method according to claim 2, characterized in that Step (3) specifically includes:

6. The synthesis method according to claim 2, characterized in that The eluent used for separation and purification in step (2) is petroleum ether / ethyl acetate 30:1-10:

1.

7. Use of the fluorescent probe according to claim 1 in the preparation of a detection reagent or a diagnostic reagent for diseases related to μ opioid receptors.

8. The use according to claim 7, characterized in that Such agents include antagonists or agonists.

9. Use of the fluorescent probe according to claim 1 in combination with μ opioid receptors in the preparation of drugs for inhibiting pain, regulating the reward system, regulating the digestive system or regulating the immune system.

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