Muscarinic acetylcholine receptor 3 fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of synthetic chemistry, and provides a muscarinic acetylcholine receptor 3 fluorescent probe as well as a preparation method and application thereof. The chemical structural formula of the fluorescent probe is shown in the specification, and the fluorescent probe can be used for M3 receptor tracing in cells or organisms. The probe provided by the invention has a good fluorescent property and can be influenced by the polarity and viscosity of an external environment; and the probe has good biocompatibility, and the IC50 of the probe is 43 times of the concentration of a working solution, so that the probe can meet the requirements of cell fluorescence imaging. In the aspect of cell fluorescence imaging, the probe can realize selective fluorescence labeling of the M3 receptor in the cell, washing is not needed in the dyeing process, and the operation is simple and convenient, so that visual research on positioning of the M3 receptor in the cell is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic chemistry, and in particular relates to a fluorescent probe for detecting muscarinic acetylcholine receptor 3 and an application thereof. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Cholinergic receptors refer to receptors that can bind to acetylcholine and are divided into two major categories: one is the cholinergic receptors that can specifically bind to muscarine and be excited, called muscarinic acetylcholine receptors (mAChRs, referred to as M receptors); the other is the cholinergic receptors that can specifically bind to nicotine and be excited, called nicotinic receptors (nAChRs, referred to as N receptors). M receptors are important cholinergic receptors in the body and are an important member of the GPCRs family. They play an extremely important role in various physiological processes. According to different pharmacological effects, M receptors are currently divided into five subtypes: M1, M2, M3, M4 and M5. Among them, M1, M3 and M5 are mainly coupled to G q / 11 Proteins activate phospholipase C, M2 and M4 mainly couple to G i / o Proteins inhibit adenylate cyclase and regulate ion channels. Studies have shown that the M1 subtype receptor is involved in regulating central nervous system (CNS) functions, including cognition; M1 subtype receptors are primarily distributed in the heart and are further associated with the regulation of gastrointestinal muscles; M1 subtype receptors are widely distributed in the lungs and bladder; M4 and M5 subtype receptors are primarily distributed in the central nervous system, and the M4 receptor has been found to be associated with the treatment of Alzheimer's disease, schizophrenia, and Parkinson's disease.

[0004] At the molecular level, the M3 receptor differs from other M receptors primarily by its unusually large third intracellular loop, which enables it to interact with a unique set of ligands. The M3 receptor is widely expressed in a variety of peripheral and central tissues, possessing crucial peripheral and central physiological functions and long serving as a model system for understanding the function of GPCRs. The M3 receptor also mediates a variety of important physiological functions, including smooth muscle contraction, glandular secretion, and regulation of various cholinergic processes in the brain.

[0005] Central M3 receptors are involved in regulating food intake, learning and memory, and normal development of the anterior pituitary gland. They may be potential drug targets for the treatment of human growth disorders. Central M3 receptors also promote bone formation. M3 receptors on β cells play a crucial role in maintaining systemic glucose homeostasis. Enhancing M3 receptor activity on β cells may aid in the treatment of type 2 diabetes. M3 receptors in the heart play a crucial role in cardiac development, maintenance of cardiac function, and the pathogenesis of heart diseases, such as cardiac ischemia, pathological cardiac hypertrophy, cardiac arrhythmias, and heart failure. M3 receptors are downregulated during cardiac aging, and activation of M3 receptors can delay cardiac aging by inhibiting caspase-1 / IL-1βs. M3 receptors also participate in LPS-induced lung inflammation and fibrosis by mediating the NF-κB signaling pathway. Other studies have shown that M3 receptors play a crucial role in cancer cell proliferation and invasion. Studies in intestinal tumor models have found that M3 receptor deficiency is associated with a significant reduction in tumor number and volume, demonstrating the potential of subtype-selective M3 receptor antagonists for clinical treatment of certain tumor forms. Literature reports indicate that M3 receptors are widely expressed in gastrointestinal tumors and have been shown to be highly expressed in cholangiocarcinoma tissue. M3 receptor expression is closely associated with differentiation and lymph node metastasis, impacting patient survival. M3 receptor stimulation is also closely associated with tumor growth in prostate cancer, where M3 receptors are overexpressed.

[0006] In summary, the M3 receptor is associated with a variety of physiological functions and potential pathophysiological functions, making it a potential target for the treatment of numerous diseases. However, the current lack of highly selective ligands for the M3 receptor poses significant challenges for in-depth investigation of its physiological and pathological functions and the treatment of related diseases. Currently, studying the distribution and function of the M3 receptor in vivo using traditional pharmacological approaches remains challenging. This is because the tissue and subtype selectivity of M3 receptor antagonists and agonists still require further optimization. Furthermore, the three-dimensional structure of the M3 receptor remains unknown, making it difficult to interpret complex experimental phenomena at the receptor structural level. In 2022, Heinrich et al. first reported M3 fluorescent probes based on cytochrome 5 and tetramethylrhodamine coupled to biphenylcarbamate. However, the relatively large fluorophores attached to these M3 ligands hindered precise visualization of M3 receptor function. This makes the use of modern techniques such as fluorescence labeling, flow cytometry, and in vivo imaging to study the pharmacology and pathology of the M3 receptor challenging. Summary of the Invention

[0007] In view of the problem that there is a lack of small molecule fluorescent probes for M3 receptors in the prior art, the present invention provides a fluorescent probe for detecting muscarinic acetylcholine receptor 3 (M3) with good specificity, high sensitivity and resistance to photobleaching.

[0008] Another object of the present invention is to provide an application of the above fluorescent probe in real-time fluorescence visualization positioning and quantitative analysis of M3 receptors.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] A fluorescent probe for detecting muscarinic acetylcholine receptor 3 (M3), the chemical structure of which is shown in formula (I): Formula (I).

[0011] The preparation method of the fluorescent probe comprises the following steps: (1) and N-Boc-bromoethylamine in a solvent under alkaline conditions, and separated and purified to obtain ; (2) The product is reacted with an acid in a solvent, and the product is directly reacted with 4-chloro-7-nitrobenzofuran in a solvent under alkaline conditions without purification, and the target product is separated and purified to obtain the target product.

[0012] In step (1), the alkaline condition is provided by potassium carbonate.

[0013] In step (2), the acid is hydrochloric acid.

[0014] In step (1), the separation and purification step is as follows: after the reaction, the system is subjected to SiO2 column chromatography, and gradient elution is performed using petroleum ether and ethyl acetate in a volume ratio of 3:1, 2:1, 1:1, 1:2, and 1:4 to obtain the target compound eluate, and remove the solvent.

[0015] In step (4), the separation and purification step is as follows: after the reaction, the system is subjected to SiO2 column chromatography, and a gradient elution is performed using petroleum ether and ethyl acetate in a volume ratio of 3:1, 2:1, 1:1, 1:2, and 1:3 to obtain the target compound eluate, and remove the solvent.

[0016] The present invention also provides an application of the fluorescent probe in detecting M3 receptors in cells or organisms and an M3 receptor tracing reagent prepared by using the fluorescent probe.

[0017] The present invention has the following advantages: The M3S4 probe provided by the present invention has good fluorescence properties and can be affected by the polarity and viscosity of the external environment; and has good biocompatibility. 50This is 43 times the concentration of the working solution, indicating that the probe can meet the requirements for cell fluorescence imaging. In terms of cell fluorescence imaging, the M3S4 probe can achieve selective fluorescent labeling of M3 receptors in cells. The staining process does not require washing, and the operation is simple and convenient, thus enabling the visualization of the localization of M3 receptors in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the H NMR spectrum of the target compound M3S5; Figure 2 is the carbon NMR spectrum of the target compound M3S5; Figure 3 This is the high-resolution mass spectrum of the target compound M3S5; Figure 4 is the UV-visible absorption spectrum of M3S5 at different concentrations; Figure 5 The excitation spectra (left) and emission spectra (right) of M3S5 at different concentrations are shown; Figure 6 The excitation spectra (left) and emission spectra (right) of M3S5 in different buffers are shown; Figure 7 The excitation spectra (left) and emission spectra (right) of M3S5 in different solvents are shown; Figure 8 The excitation spectra (left) and emission spectra (right) of M3S5 at different viscosities are shown; Figure 9 It is the cytotoxicity of the M3S5 fluorescent probe; Figure 10 M3S5 is a fluorescent label for M3 receptor. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0020] Example 1 Synthesis of fluorescent probe (1) Synthesis of intermediate M3S1 Piperidine derivative 2 (209.5 mg, 0.973 mmol, 0.95 eq) and pyridine (121.6 mg, 1.54 mmol, 1.5 eq) were dissolved in anhydrous dichloromethane (50 mL). A dichloromethane solution of isocyanate 1 (200 mg, 1.02 mmol, 1 eq) was added dropwise at room temperature over 30 min. The reaction was completed upon completion of the addition. Dichloromethane (15 mL) and ultrapure water (10 mL) were added to the system. The layers were separated, and the aqueous phase was washed with dichloromethane (10 mL*3). The organic phase was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica column chromatography (petroleum ether:ethyl acetate = 6:1, 5:1, 4:1, 3:1), and concentrated to obtain 326 mg (yield 77.51%) of the target product, M3S1.

[0021] (2) Synthesis of intermediate M3S2 M3S1 (200 mg, 0.487 mmol, 1 eq) was added to DCM (20 mL), and the system turned blue. Upon addition of ethanolic HCl solution (254 μL, 2.43 mmol, 5 eq), the reaction system turned yellow. After stirring at room temperature overnight, a white precipitate precipitated. TLC monitoring (PE:EA=3:1, RF=0.6) showed that the starting material disappeared. The pH was adjusted to 9 with ammonia water. The layers were separated, and the organic phase was washed with ultrapure water (10 mL*2), dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by preparative liquid chromatography on a C18 column with a mobile phase of acetonitrile:water (85:15) at a flow rate of 5 mL / min and a detection wavelength of 254 nm to obtain 124.3 mg (yield 82.20%) of a white oil, namely M3S2.

[0022] (3) Synthesis of intermediate M3S3 M3S2 (100 mg, 0.322 mmol, 1 eq), N-Boc-bromoethylamine (144.4 mg, 0.644 mmol, 2 eq), and potassium carbonate (133.5 mg, 0.966, 3 eq) were added to an acetonitrile solution (10 mL) and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, SiO2 column chromatography was performed using petroleum ether / ethyl acetate as eluent (3:1, 2:1, 1:1, 1:2, 1:4) to obtain 113.3 mg (yield 77.6%) of a colorless oil, namely M3S3.

[0023] (4) Synthesis of target compound M3S4 M3S3 (101.4 mg, 0.224 mmol, 1 eq) was placed in DCM (20 mL), and HCl in ethanol (116 μL, 1.12 mmol, 5 eq) was added. The mixture was stirred at room temperature overnight. The disappearance of the starting material was monitored by TLC. The solvent was evaporated to dryness to obtain a residue. 40 mg of the residue was taken, 4-chloro-7-nitrobenzofuran (33.8 mg, 0.169 mmol, 1.5 eq) and potassium carbonate (31.2 mg, 0.226 mmol, 2 eq) were added thereto and poured into anhydrous ethanol (10 mL). The mixture was heated under reflux. After 8 h, the reaction was monitored by TLC (PE; EA = 3:1, RF = 0.7). The starting material disappeared. The reaction system was directly mixed with SiO2 and eluted with column chromatography using petroleum ether / ethyl acetate (3:1, 2:1, 1:1, 1:2, 1:3) to obtain 35.2 mg (yield 60.27%) of a colorless oil, namely the target compound M3S4. Its hydrogen spectrum, carbon spectrum and high-resolution mass spectrum are as follows Figure 1-3 As shown: 1 H NMR (600 MHz, CD3OD) δ 8.52(d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.41 (t, J = 7.1 Hz, 2H), 7.35 (dd, J = 18.6,7.9 Hz, 4H), 7.29 (d, J = 7.3 Hz, 1H), 7.24 (dd, J = 18.1, 10.9 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H), 3.90 (s, 2H), 3.04 (d, J = 10.6 Hz, 2H), 2.76 (t, J = 6.2 Hz,2H), 2.12 (t, J = 11.4 Hz, 2H), 1.71 – 1.59 (m, 3H). 13 C NMR (151 MHz, DMSO- d6) δ155.07 (1C), 145.63 (1C), 139.60 (1C), 138.44 (1C), 135.21 (1C), 130.76 (1C), 129.20 (2C), 128.73 (4C), 128.34 (2C), 127.53 (2C), 126.50 (1C), 121.12 (1C), 99.83 (1C), 68.63 (1C), 56.10 (1C), 53.23 (3C), 35.56 (1C), 28.68 (2C). ESI-HRMS: m / z calcd. for [C 27 H 29 N6O5] + 517.2199, found 517.2201 [M+H] + ; m / z calcd.for [C 54 H 57 N 12 O 10 ] + 1033.4321, found 1033.4331 [2M+H] + . Example 2 Fluorescence properties of fluorescent probes 1. UV-Vis spectroscopy 1 mg of the probe compound was weighed and dissolved in DMSO to prepare a concentrated stock solution with a concentration of 10 mM. Before testing, the solution was diluted with PBS buffer solution (pH 7.4) to concentrations of 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM, respectively. The UV-visible absorption spectra were obtained using an Agilent Cary 3500 UV-visible spectrophotometer.

[0024] UV-visible absorption spectra of target compounds at different concentrations are shown in Figure 2. Figure 4 As shown, it has three absorption peaks, among which the maximum absorption wavelength in the near-ultraviolet region is 230 nm, the maximum absorption wavelength in the π-π* absorption band is 335 nm, and the maximum absorption wavelength in the n-π* absorption band is 468 nm.

[0025] 2. Fluorescence spectroscopy The probe compound concentrated stock solution (10 mM, DMSO) was diluted with PBS buffer solution (pH 7.4) to concentrations of 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, and 5 μM, respectively. Fluorescence excitation and emission spectra were obtained by scanning with a fluorescence spectrophotometer (Agilent Cary Eclipse).

[0026] M3S4 fluorescence spectrum Figure 5 As shown, it has three excitation and emission peaks. The peak at the n-π* absorption band is beneficial for fluorescence imaging experiments, and its absorption intensity is greater than that of the π-π* absorption band, indicating that the n-π* absorption band has a good fluorescence quantum yield. M3S4 has a maximum excitation wavelength of 462 nm and a maximum emission wavelength of 530 nm, which are within the wavelength range of green light. The Stokes shift is 68 nm, and a large Stokes shift helps reduce interference from the excitation wavelength on fluorescence imaging. The fluorescence intensity gradually increases with increasing concentration, reaching a maximum at 30 μM. As the concentration continues to increase, the fluorescence intensity decreases, indicating that fluorescence quenching occurs at high concentrations of M3S4.

[0027] Example 3 Selectivity of fluorescent probes under different environmental conditions The concentrated stock solution of the probe compound (10 mM, DMSO) was diluted to a concentration of 50 µM using the following different solutions, and the fluorescence emission and excitation spectra were scanned using a fluorescence spectrophotometer (Agilent Cary Eclipse): (1) Buffer solutions: PBS (pH=7.40), HEPES (pH=7.40), and Tris-HCl (pH=7.40) buffers; (2) Solvents of different polarity: PBS (pH 7.40), water, DMSO, methanol, acetonitrile, and ethanol; (3) Different viscosities: different ratios of glycerol-water ( v / v ) mixed solution, as the volume ratio of glycerol increases from 0-100%, the viscosity gradually increases.

[0028] Fluorescence excitation and emission spectra of M3S4 in different buffer solutions are shown in Figure 2. Figure 6 As shown in the figure: the fluorescence performance of PBS buffer is better than that of HEPES and Trisbase buffer. PBS buffer will be used as the solvent for M3S5 in the future.

[0029] Compared with aqueous solution and PBS buffer, the fluorescence enhancement of probe M3S4 in organic solvent was obvious ( Figure 7 In addition, the fluorescence intensity of the probe M3S4 increases with the increase of solution viscosity. When the viscosity increases to 100%, the probe compound shows a fluorescence enhancement of about 9.64 times ( Figure 8 The above results indicate that the fluorescence intensity of the probe compound M3S4 changes with the change of environmental polarity and viscosity, and the fluorescence intensity is enhanced in an environment with low polarity and high viscosity.

[0030] Example 4 Toxicity of fluorescent probe to cells The cytotoxicity of the M3S4 probe was detected by CCK-8 assay. HKE293T cells were revived and incubated at 37 o C and 5% CO2 incubator. All cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS). After three passages, the CCK-8 assay was performed when the cells were stable. (1) After cell digestion and centrifugation, the cell concentration was adjusted to 8×10 4 / mL, and then 100 μL per well was inoculated into a 96-well clear cell culture plate; (2) After culturing in the incubator for 24 h, the probe compound solution was diluted with DMEM without FBS to different final concentrations (starting at 200 μM, diluted 2-fold, and a total of 8 concentration points were set), and directly added to the above 96-well cell plate (100 μL / well); three parallel wells were set for each concentration, and repeated three times. The positive control compound was phenylephrine, the blank control group was the well with only culture medium added, and the negative control group was the well with only cells added; (3) After culturing in the incubator for 24 h, remove the old culture medium and add CCK-8 dye diluted with DMEM at 37 o After incubation at C for 2 h, the absorbance OD at 450 nm was measured using a microplate reader (BioTek Synergy H1), and the data were processed and the IC 50 value.

[0031] The results are as follows Figure 9 As shown in the results, M3S4 exhibited micromolar cytotoxicity to HEK293T cells, indicating low cytotoxicity.

[0032] Example 5 Imaging of M3 Receptors by Fluorescent Probes in Living Cells HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 5% CO2, 37°C incubator. Plasmids containing the M3 sequence were transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). P3000 (5 μL) and Lipo 3000 (7.5 μL) diluted in Opti-MEM were added to 2.5 μg of plasmid, and a 1.5×10 5The cells were cultured in glass-bottomed dishes for 24 h. After 24 h, the culture medium was discarded and M3S4 (1 μM) was dissolved in Tris-HCl (pH = 7.4). After incubation with M3S4 for 30 min, the cells were imaged using a laser confocal microscope (60× oil objective lens, Zeiss). The atropine group was incubated with M3S5 and then given atropine (10 μM) for 30 min. The cells were then imaged using a laser confocal microscope (60× oil objective lens, Zeiss).

[0033] M3S4 was used to image the fluorescence localization of M3 receptor in HEK293T cells transiently transfected with M3 plasmid. Figure 10 As shown: After incubating the probe compound with cells for 10 minutes, fluorescence imaging was performed directly using an inverted fluorescence microscope without washing. The results showed that after the cells were exposed to the probe M3S5, the cell membrane was fluorescently stained, and this staining was attenuated by a 10-fold dose of the M3 receptor antagonist atropine, without the need for washing. This indicates that the probe compound's environmentally sensitive fluorescence switch can be selectively activated by the M3 receptor and can achieve fluorescent localization staining of the M3 receptor.

[0034] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A fluorescent probe for detecting muscarinic acetylcholine receptor 3, whose chemical structure is: 。 2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) and N-Boc-bromoethylamine in a solvent under alkaline conditions, and separated and purified to obtain ; (2) The product is reacted with an acid in a solvent, and the product is directly reacted with 4-chloro-7-nitrobenzofuran in a solvent under alkaline conditions without purification, and the target product is separated and purified.

3. The preparation method according to claim 2, characterized in that In step (1), the alkaline condition is provided by potassium carbonate; in step (2), the acid is hydrochloric acid.

4. The preparation method according to claim 2, characterized in that In step (1), the separation and purification step is as follows: after the reaction, the system is subjected to SiO2 column chromatography, and a gradient elution is performed using petroleum ether and ethyl acetate in a volume ratio of 3:1, 2:1, 1:1, 1:2, and 1:4 to obtain an eluate of the target compound, and the solvent is removed; In step (4), the separation and purification step is as follows: after the reaction, the system is subjected to SiO2 column chromatography, and a gradient elution is performed using petroleum ether and ethyl acetate in a volume ratio of 3:1, 2:1, 1:1, 1:2, and 1:3 to obtain the target compound eluate, and remove the solvent.

5. Use of the fluorescent probe according to claim 1 in detecting muscarinic acetylcholine receptor 3 in cells or organisms.

6. A muscarinic acetylcholine receptor 3 tracing reagent prepared by the fluorescent probe according to claim 1.

Images