Dual-responsive fluorescent probe for simultaneous imaging of myelin and abeta aggregates in ad brain and preparation method and application thereof

Abstract

The application discloses a dual-response fluorescent probe for simultaneously imaging AD brain myelin and A beta aggregate as well as a preparation method and application thereof, and the probe structure is shown in any one of the following structures. The application prepares a series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structure for central nervous system and peripheral nervous system myelin response, wherein the probe NQPI6 can specifically simultaneously image myelin integrity and A beta aggregation in mouse brain tissue. The probe can perform detailed three-dimensional imaging on the myelin structure rich in lipids, can be effectively combined with A beta plaque, and can generate strong red fluorescence signals. The dual-functional fluorescent probe provides a powerful chemical tool for synchronous imaging of myelin and A beta plaque, and provides a promising tool and method for studying the interaction between A beta plaque formation and demyelination in the AD progress process.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a dual-response fluorescent probe that simultaneously images myelin sheath and Aβ aggregates in the AD brain, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive impairment and memory loss. Due to the complexity of its pathogenesis, effective intervention remains a significant challenge. Initially, attention deficit disorder (AD) was thought to primarily affect the cerebral cortex, with prominent pathological changes in the gray matter, such as senile plaques formed by β-amyloid (Aβ) deposition and neurofibrillary tangles (NFTs) caused by tau protein hyperphosphorylation. However, recent studies have shown that alterations in the structure of the myelin sheath (produced by oligodendrocytes) in the central nervous system are closely related to AD, leading to changes in the white matter (WM) structure in AD patients. Histological and functional imaging studies have revealed severe disruption of the brain white matter microstructure not only in late-stage AD patients but also in patients with early symptoms (including preclinical symptoms, subjective cognitive impairment (SCI), and mild cognitive impairment (MCI)). The observed WM changes are primarily demyelination and axonal damage, resulting in a reduction in nerve fiber bundles and WM volume, particularly affecting the integrity of the myelin sheath.

[0003] Myelin, a lipid-rich structure, encapsulates nerve axons and plays a crucial role in insulating nerve fibers, maintaining axonal action potentials, and facilitating rapid and precise electrical signal transmission. These key characteristics have given the vertebrate nervous system an evolutionary advantage. Given the critical role of myelin in regulating axonal conduction velocity and integrity, myelin sheath lesions are likely one of the causes of overall cognitive decline in Alzheimer's disease (AD) patients; simultaneously, myelin degeneration and demyelination are also important warning signs of AD progression. Comparative analysis of brain tissue from patients without dementia and AD patients showed a significant reduction in total protein content and myelin-related proteins (such as myelin basic protein (MBP), myelin lipoprotein (PLP), cyclic nucleotide phosphorylase (CNP), and cholesterol), indicating myelin loss in the pathological process of AD. Some studies suggest that the abnormal accumulation of Aβ plaques triggers a series of abnormal cytoskeletal changes, leading to dendritic spine loss, atrophic axon formation, and ultimately neuronal dysfunction. In an AD mouse model, in addition to changes in oligodendrocyte characteristics near amyloid plaques, focal demyelination was observed near Aβ plaques, indicating that Aβ pathology has downstream effects in the late stages of the disease. In the mouse AD model, age-related myelin structural defects promote the accumulation of Aβ-generating mechanisms within axonal swelling, thereby promoting the cleavage of cortical amyloid precursor proteins and ultimately forming Aβ plaques. Therefore, loss of myelin integrity may be an upstream risk factor for neuronal Aβ deposition. Thus, studying the interaction between demyelination, WM damage, and Aβ plaque formation is crucial for understanding the pathogenesis of attention deficit disorder (ADHD) and has significant clinical implications for its prevention, diagnosis, and treatment.

[0004] Simultaneous detection of Aβ and myelin can provide insights into the complex interactions between these key biomarkers. While two single-response fluorescent probes can be used for imaging separately, this approach is overly complex, as the spatial and temporal distributions of the probes may differ and they may interfere with each other. Currently, however, no single probe or chemical tool can simultaneously detect and image these markers in brain tissue. Summary of the Invention

[0005] Objective of the Invention: To address the problems existing in the prior art, this invention provides a dual-response fluorescent probe for simultaneously imaging myelin and Aβ aggregates in the AD brain, along with its preparation method and applications. The probes described in this invention are all amphiphilic fluorescent probes with a pyridinium salt structure, capable of selectively staining myelin regions of the central and peripheral nervous systems. They can be used for three-dimensional (3D) myelin imaging of mouse brain tissue, exhibiting good tissue penetration and high specificity. The representative probe NQPI6 can also simultaneously and specifically label Aβ aggregates, thereby achieving dual-function imaging of Aβ plaques and myelin in mouse brain slices. This reveals the close relationship between myelin damage and Aβ plaque distribution in different brain regions and developmental stages of AD model mice, effectively solving the current predicament of lacking probes or chemical tools that can simultaneously detect and image Aβ and myelin in AD brain tissue. This provides a new perspective for understanding the complex relationship between myelin degradation and Aβ pathology.

[0006] The present invention also provides a method for preparing and applying the dual-response fluorescent probe that simultaneously images myelin sheath and Aβ aggregates in the AD brain.

[0007] Technical solution: To achieve the above objective, the present invention provides a myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure, wherein the structure of the probe is shown in any of the following embodiments:

[0008]

[0009] The R is selected from any of the following:

[0010]

[0011] Preferably, the present invention provides a myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure and various control probes, the structures of which are shown below:

[0012]

[0013] Preferably, the present invention proposes a series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structures that target the myelin sheath response of the central and peripheral nervous systems, wherein probe NQPI6 can simultaneously and specifically label Aβ plaques.

[0014] The Chinese name of the structural formula NPPI is 4-(4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-methylpyridine-1-onium iodide.

[0015] The Chinese name of the structural formula PAP-1 is 2-(3-(2-(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)vinyl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile.

[0016] The Chinese name for the structural formula PAP-2 is 2-(5,5-dimethyl-3-(2-(4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)vinyl)cyclohexyl-2-en-1-yl)malononitrile.

[0017] The Chinese name of the structural formula NQPI1 is 4-(2-(6-(9H-carbazo-9-yl)quinoline-2-yl)vinyl)-1-methylpyridine-1-onium iodide.

[0018] The Chinese name for the structural formula NQPI2 is 4-(2-(6-(bis(4-methoxyphenyl)amino)quinoline-2-yl)vinyl)-1-methylpyridine-1-onium iodide.

[0019] The Chinese name for the structural formula NQPI3 is 4-(2-(6-(10H-phenthiazin-10-yl)quinoline-2-yl)vinyl)-1-methylpyridine-1-onium iodide.

[0020] The Chinese name for the structural formula NQPI4 is 4-(2-(6-(dimethylamino)quinoline-2-yl)vinyl)-1-methylpyridine-1-onium iodide.

[0021] The Chinese name for the structural formula NQPI5 is 4-(2-(6-(azacyclobutane-1-yl)quinoline-2-yl)vinyl)-1-methylpyridine-1-onium iodide.

[0022] The Chinese name for the structural formula NQPI6 is 1-methyl-4-(2-(6-(pyrrolidine-1-yl)quinolin-2-yl)vinyl)pyridine-1-onium iodide.

[0023] The method for preparing the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure according to the present invention includes the following steps:

[0024] N,N-dimethyl-p-toluidine and (E)-2-butenal were added to HCl solution and stirred at room temperature. An organic solvent was added, and the mixture was heated and stirred. After the reaction, the mixture was extracted, washed, dried, and concentrated under reduced pressure to obtain a crude product. The crude product was purified to obtain a yellow-brown solid compound 1.

[0025] SeO2 was dissolved in 1,4-dioxane / H2O, heated and stirred, then compound 1 was added, heated and stirred. After the reaction was completed, the reactants were cooled to room temperature, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified to obtain a yellow solid compound 2.

[0026] Compound 2 and 1,4-dimethylpyridine iodide were dissolved in an organic solvent. Piperidine was added dropwise to the mixture, and the mixture was heated and stirred. After the reaction was complete, the reactants were cooled to room temperature and concentrated under reduced pressure to give a crude product. The crude product was dissolved and recrystallized to give a red solid, NQPI4.

[0027] The reaction formula is shown below:

[0028]

[0029] The method for preparing the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure according to the present invention includes the following steps:

[0030] Cesium carbonate, 2,2'-bis(diphenylphosphine)-1,1'-binaphthylene, and palladium(II) acetate were dissolved in an organic solvent. Then, 6-bromo-2-methylquinoline and different secondary amines were added. The mixture was heated and stirred, and after cooling to room temperature, the solution was filtered. The residue obtained by extraction and vacuum concentration was purified to obtain the desired compound 1R. Among them, the secondary amine required for the preparation of NQPI1 was carbazole; the secondary amine required for the preparation of NQPI2 was 4,4'-dimethoxydiphenylamine; the secondary amine required for the preparation of NQPI3 was phenthiazide; the secondary amine required for the preparation of NQPI5 was cyclobutane; and the secondary amine required for the preparation of NQPI6 was tetrahydropyrrole.

[0031] SeO2 was dissolved in 1,4-dioxane / H2O, heated and stirred, and then compound 1R was added. The mixture was heated and stirred. After the reaction was completed, the reactants were cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified to obtain the corresponding aldehyde 2R.

[0032] Compound 2R and 1,4-dimethylpyridine iodide were dissolved in anhydrous ethanol. Piperidine was added dropwise to the mixture and the mixture was heated and stirred. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain crude products. The crude products were dissolved and recrystallized to obtain red solids NQPI1, 2, 3, 5 and 6.

[0033] The reaction formula is shown below:

[0034]

[0035] Preferably, the method for preparing the myelin probe NQPI1-6 of the present invention includes the following steps:

[0036] To a solution of N,N-dimethyl-p-toluidine (10 mmol, 1.36 g) in 22 mL of 6N HCl, (E)-2-butenal (20 mmol, 0.7 g) was added, and the mixture was stirred at room temperature for 1 h. Then, 10 mL of toluene was added, and the reaction mixture was stirred at 110 °C for 12 h. After extraction three times with dichloromethane, the organic phase was washed 2–3 times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 40:1, v / v) to give a yellow-brown solid compound 1 (1.0 g, 72%).

[0037] Cesium carbonate (3.4 g, 10.4 mmol), 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl (340 mg, 0.52 mmol), and palladium(II) acetate (58 mg, 0.26 mmol) were dissolved in toluene (10 mL). Then, 6-bromo-2-methylquinoline (1.76 g, 8 mmol) and various secondary amines (RH, 9.6 mmol) were added. The reaction mixture was further refluxed at 110 °C for 8 h. After cooling to room temperature, the solution was filtered through diatomaceous earth, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the desired compound 1R (yield approximately 54%).

[0038] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 ml: 1 ml, v / v) and heated at 60 °C for 30 min. Then, compound 1 or compound 1R (10 mmol) was added, and the mixture was stirred at 80 °C for another 4 h. After the reaction was complete, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth, and the filter residue was washed several times with a small amount of dichloromethane to remove the precipitate. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to give the corresponding compound aldehyde 2 or aldehyde 2R as a yellow solid (yield approximately 76%).

[0039] Compound 2R (5 mmol) and 1,4-dimethylpyridine iodide (117 mg, 5 mmol) were dissolved in anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture, and the mixture was stirred at 80 °C for 8 h. The crude product was dissolved in a small amount of anhydrous ethanol, and recrystallized by adding diethyl ether to give a red solid NQPI1-6 (yield approximately 60%).

[0040] The application of the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure described in this invention in the preparation of drugs or reagents for detecting myelin.

[0041] The myelin sheath refers to the myelin sheath of the central nervous system and the peripheral nervous system.

[0042] The myelin sheaths referred to here are the myelin sheaths of different brain regions in brain slices, the myelin sheaths of spinal cord slices, the myelin sheaths of sciatic nerve slices, and the myelin sheaths within the right occipital lobe of the brain.

[0043] Furthermore, the NQPI1-6 probes mark myelin sheaths in different brain regions of mouse brain slices, including the NQPI6 probe marking myelin sheaths in different brain regions of mouse brain slices, mouse spinal cord slices, mouse sciatic nerve slices, and human right occipital lobe slices, and the NQPI6 probe performing three-dimensional mapping imaging of mouse brain tissue myelin sheaths.

[0044] Furthermore, the probe detects myelin sheath for analyzing myelin sheath density and mapping the trajectory of myelin sheath nerve fiber bundles.

[0045] The application of the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure described in this invention in the preparation of drugs or reagents for simultaneous detection and imaging of myelin and Aβ aggregates in the AD brain.

[0046] The amphiphilic fluorescent probe is used in the preparation of drugs or reagents for imaging myelin sheath integrity and Aβ aggregation in brain tissue.

[0047] The amphiphilic fluorescent probe is used in the preparation of drugs or reagents for three-dimensional mapping and imaging of myelin sheaths in brain tissue and for labeling Aβ plaques in brain slices.

[0048] Furthermore, the application of the probe NQPI6, which is dual-responsive to myelin and Aβ aggregates in the AD brain, in detecting Aβ aggregates in the AD brain; the labeling of Aβ plaques in brain slices of 5xFAD mice by probe NQPI6; and the simultaneous dual-responsive imaging of myelin and Aβ plaques in brain slices of 5xFAD mice and their age-matched WT mice by probe NQPI6.

[0049] The application of the amphiphilic fluorescent probe described in this invention in the preparation of reagents or tools capable of simultaneously imaging myelin and Aβ plaques.

[0050] This invention discloses the preparation methods and applications of a series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structures that respond to myelin sheaths in both the central and peripheral nervous systems. Probe NQPI6 can simultaneously detect and image myelin sheaths and Aβ aggregates in the brain of Alzheimer's disease (AD). Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline and memory loss. While β-amyloid (Aβ) plaques in gray matter are a recognized pathological feature, significant white matter damage, including myelin sheath destruction and axonal injury, can also be observed in Alzheimer's patients. However, due to the lack of robust imaging tools, the intrinsic link between myelin sheath damage and Aβ plaque deposition during AD development remains unclear. This invention demonstrates a series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structures that respond to myelin sheaths in both the central and peripheral nervous systems. Among them, the novel dual-responsive fluorescent probe NQPI6 specifically and simultaneously images myelin sheath integrity and Aβ aggregation in mouse brain tissue. NQPI6 emits a unique green fluorescent signal with a high signal-to-noise ratio, enabling detailed three-dimensional imaging of lipid-rich myelin sheath structures. Importantly, this probe also binds effectively to Aβ plaques, producing a strong red fluorescent signal. Simultaneous imaging with NQPI6 allows for the detection of varying degrees of demyelination in different brain regions of AD model mice of different ages, which correlates with the distribution of Aβ plaques. This bifunctional fluorescent probe provides a powerful chemical tool for the simultaneous imaging of myelin and Aβ plaques, offering a promising product and method for studying the interaction between Aβ plaque formation and demyelination during AD progression.

[0051] This invention develops a bifunctional fluorescent probe capable of simultaneously detecting Aβ and myelin. Based on a fragment assembly strategy of quinoline molecules, this invention designs and develops a series of amphiphilic two-photon fluorescent probes with pyridinium salt structures. These probes can selectively stain myelin regions of the central and peripheral nervous systems with a high signal-to-noise ratio, and can be used for three-dimensional (3D) myelin imaging of mouse brain tissue, exhibiting good tissue penetration and high specificity. Through probe screening, the representative probe NQPI6 was found to simultaneously and specifically label Aβ aggregation, thereby achieving bifunctional imaging of Aβ plaques and myelin in mouse brain slices. Using NQPI6, this invention reveals the close relationship between myelin damage and Aβ plaque distribution in different brain regions and developmental stages of AD model mice, providing a new perspective for understanding the complex relationship between myelin degradation and Aβ pathology. The innovatively designed bifunctional probe of this invention will contribute to a better understanding of the pathogenesis of AD.

[0052] The design principle of the fluorescent probes NQPI1-6: The probes of this invention mimic the amphiphilic structure of phospholipids in the membrane, balancing hydrophobic donor and hydrophilic acceptor molecules. Hydrophilic and positively charged pyridinium salt molecules face the lipid bilayer surface and interact with negatively charged phospholipid phosphate groups through strong electrostatic interactions, thereby enhancing the probe's plasma membrane targeting. Furthermore, a quinoline ring with good fluorescence properties is added as a fluorophore. To optimize these dyes for the complex brain environment, various cyclic aliphatic amines are introduced as electron donors to adjust the probe properties, including lipophilicity and fluorescence characteristics. A series of novel myelin-targeting fluorescent probes, named NQPIs, were designed and synthesized. This invention develops a novel dual-response fluorescent probe that can simultaneously image myelin integrity and Aβ aggregates in the AD brain, with a low signal-to-noise ratio and good selectivity.

[0053] This invention proposes a novel two-photon probe structure that exhibits dual response to both myelin sheath and Aβ plaques, enabling two-photon imaging in brain slice myelin sheath detection. Figure 1 The importance of amphiphilic structures in myelin detection was verified by modifying functional groups. Simultaneously, various cyclic aliphatic amines were introduced as electron donors to replace N,N′-dimethylamine, thereby modulating probe properties, including lipophilicity and fluorescence. Through this iterative process, NQPI6 with tetrahydropyrrole as the electron donor was determined to be the optimal probe.

[0054] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0055] This invention constructs a bifunctional molecular probe capable of simultaneously imaging myelin and Aβ plaques. The probe efficiently labels myelin fibers and facilitates deep imaging of the mouse central nervous system (CNS) and peripheral nervous system (PNS). The resulting images exhibit a high signal-to-noise ratio, providing a valuable chemical tool for analyzing myelin density and mapping myelin nerve fiber bundles. The environmentally sensitive probe NQPI6 can also effectively identify Aβ plaques in different brain regions in an Alzheimer's disease (AD) mouse model. Utilizing dual-channel simultaneous imaging technology, NQPI6 can simultaneously observe Aβ plaques and myelin in different brain slices from Alzheimer's disease model mice, revealing a direct correlation between cortical and hippocampal demyelination and the presence of Aβ plaques. The probe constructed in this invention not only provides a new tool and strategy for studying the intricate relationship between Aβ plaque formation and demyelination during the pathological development of AD, but will also promote the development of innovative diagnostic and therapeutic strategies targeting both myelin and Aβ plaques.

[0056] The present invention provides a novel, simple, and cost-effective method for preparing and synthesizing fluorescent probes for NQPI, which is suitable for industrial production due to its low raw material utilization rate. Attached Figure Description

[0057] Figure 1 The chemical structures of PAP-1, PAP-2, NPPI, and NQPI1-6 listed in this invention, and the sagittal whole brain sections of 6-week-old C57BL / 6J mice stained with the probes, and their fluorescence intensity ratios.

[0058] Figure 2 The cytotoxicity MTT assay for NQPI6 listed in this invention;

[0059] Figure 3 In Figure A, confocal fluorescence images of SH-SY5Y cells with different cholesterol concentrations co-incubated with NQPI6 as listed in this invention are shown; in Figure B, the relative ratio of fluorescence intensity of SH-SY5Y cells with different cholesterol concentrations after incubation with NQPI6 as listed in this invention are shown.

[0060] Figure 4 Images A and B in the middle are representative images of double staining of the cerebellum (CB), cortex, and corpus callosum (CC) of the control group and Akt cTKO mice at P14 with NQPI6 and Plp1 antibodies; Images C and D are representative images of staining of the cerebellum (CB), cortex, and corpus callosum (CC) of the control group and Akt cTKO mice at P14 with NQPI6 and Mbp antibodies.

[0061] Figure 5 Image A shows LFB staining of sciatic nerve fiber sections; Image B shows a confocal image of sciatic nerve fiber sections of the peripheral nervous system of NQPI6 and 6-week-old C57BL / 6J mice as described in this invention after incubation.

[0062] Figure 6 This is a confocal image of spinal cord sections from the peripheral nervous system of NQPI6 mice and 6-week-old C57BL / 6J mice after incubation, as described in this invention.

[0063] Figure 7 Image A shows an LFB staining of a human left occipital sagittal section adjacent to an NQPI6-stained section; Image B shows a confocal image of the NQPI6 and the human left occipital sagittal section after incubation.

[0064] Figure 8 A stitched image of sagittal whole brain sections stained with NQPI6 from 6-week-old C57BL / 6J mice;

[0065] Figure 9 Image A shows 3D two-photon fluorescence images of different depths of the striatum in a sagittal mouse brain section stained with NQPI6 as described in this invention; Image B shows 3D two-photon fluorescence images of different depths of the striatum in a transverse mouse brain section stained with NQPI6 as described in this invention.

[0066] Figure 10A shows the fluorescence spectrum of NQPI6 as described in this invention after reacting with Aβ aggregates in PBS buffer; B shows the linear fit between the fluorescence intensity at 640 nm and the concentration of Aβ aggregates after NQPI6 as described in this invention reacts with different concentrations of Aβ aggregates; C shows the fluorescence intensity at 640 nm and the fluorescence intensity after adding 5 μM Aβ... 1-42 A graph showing the relationship between NQPI6 concentration in PBS solution of aggregates; D is a schematic diagram showing the in vitro selectivity of NQPIs for different substances as described in this invention;

[0067] Figure 11 The NQPI and Aβ listed in this invention 1-42 A schematic diagram of molecular docking in the cryo-electron microscopy structure of the fiber (PDB ID: 5OQV);

[0068] Figure 12 Fluorescence imaging of paraffin sections of 5xFAD mouse cortex after double staining with specific β-amyloid antibody and NQPI6;

[0069] Figure 13 The images show confocal microscopy images and myelin channel fluorescence intensity values ​​of different brain regions after incubation of sagittal whole brain sections of NQPI listed in this invention with 6-month-old 5xFAD mice and wild-type WT mice of the same age; Figure 13 The images in A and C are confocal imaging results, showing changes in tissue structure. A is the cortex, B is the corpus callosum, and C is the hippocampus. Figure 13 EG is a Figure 13 Data analysis of fluorescence intensity in myelin loss in middle AC;

[0070] Figure 14 Immunofluorescence imaging of antibody-labeled Aβ protein and surrounding myelin in 5xFAD mice and age-matched wild-type control groups. Detailed Implementation

[0071] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0072] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0073] Example 1

[0074] A method for preparing a probe NPPI, the preparation process of which is as follows:

[0075]

[0076] 4-Dimethylaminocinnamaldehyde (875 mg, 5 mmol) and 1,4-dimethylpyridine iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture, and the mixture was stirred at 80 °C for 8 h. The crude product was dissolved in a small amount of anhydrous ethanol, and recrystallized by adding diethyl ether to give a red solid NPPI (1.30 mg, 3.3 mmol, 66.3%). 1 H NMR (600MHz, DMSO-d6) δ8.69(d,J=6.8Hz,2H),8.02(d,J=6.9Hz,2H),7.79(dt,J=15.3,5.2Hz ,1H),7.47(d,J=8.9Hz,2H),7.04–6.97(m,2H),6.76–6.70(m,3H),4.18(s,3H),2.99(s,6H). 13 CNMR(151MHz,DMSO-d6)δ153.18,151.55,144.95,143.71,142.48,129.56,123.99,123.73,123.54,122.88,112.49,46.97,40.51.MS(ESI):C 18 H 21 N2 + [M] + ,calcd 265.17,found 265.1700.

[0077] Example 2

[0078] A method for preparing the probe NQPI4, the preparation process of which is as follows:

[0079]

[0080] N,N-dimethyl-p-toluidine (10 mmol, 1.36 g) and 2-butenal (20 mmol, 0.7 g) were added to a 22 mL solution of 6N HCl and stirred at room temperature for 1 h. Then, 10 mL of toluene was added, and the reaction mixture was stirred at 110 °C for 12 h. After extraction three times with dichloromethane, the organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 40:1, v / v) to give a yellow-brown solid compound 1 (1.0 g, 72%).

[0081] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 mL: 1 mL, v / v) and heated at 60 °C for 30 min. Then, compound 1 (10 mmol) was added, and the mixture was stirred at 80 °C for another 4 h. After the reaction was complete, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth, and the residue was washed three times with a small amount of dichloromethane. The filtrates and washings were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (PE:EA = 60:1, v / v) to give a yellow solid compound 2 (76%).

[0082] Compound 2 (5 mmol) and 1,4-dimethylpyridine iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture, and the mixture was stirred at 80 °C for 8 h. After the reaction was complete, the reactants were cooled to room temperature and concentrated under reduced pressure to give a crude product. The crude product was dissolved in a small amount of anhydrous ethanol, and recrystallized dropwise with diethyl ether to give a red solid NQPI4 (60%). 1 H NMR (600MHz, Methanol-d4) δ8.74(d,J=6.5Hz,2H),8.19(d,J=6.5Hz,2H),8.12(d,J=8.6Hz,1H),7.94–7.86(m,2H),7.8 0(d,J=8.6Hz,1H),7.75(d,J=16.2Hz,1H),7.50(dd,J=9.4,2.9Hz,1H),6.92(d,J=2.8Hz,1H),4.33(s,3H),3.13(s,6H). 13 C NMR(151MHz,Methanol-d4)δ153.12,149.65,148.62,144.84,141.64,140.31,134.7 8,130.34,129.15,125.42,124.04,120.50,119.99,104.05,46.55,39.23.MS(ESI):C 19 H 20 N + [M] + ,calcd 290.17,found 290.1651.

[0083] Example 3

[0084] A method for preparing probes NQPI1, 2, 3, 5 and 6, the preparation process of which is as follows:

[0085]

[0086] Cesium carbonate (3.4 g, 10.4 mmol), 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl (340 mg, 0.52 mmol), and palladium(II) acetate (58 mg, 0.26 mmol) were dissolved in toluene (10 mL). Then, 6-bromo-2-methylquinoline (1.76 g, 8 mmol) and various secondary amines (RH, 9.6 mmol) were added. The reaction mixture was further refluxed at 110 °C for 8 h. After cooling to room temperature, the solution was filtered through diatomaceous earth, extracted with ethyl acetate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE:EA = 20:1, v / v) to give the desired compound 1R (yield approximately 54%). The secondary amine required for the preparation of NQPI1 is carbazole; the secondary amine required for the preparation of NQPI2 is 4,4'-dimethoxydiphenylamine; the secondary amine required for the preparation of NQPI3 is phenthiazide; the secondary amine required for the preparation of NQPI5 is cyclobutane; and the secondary amine required for the preparation of NQPI6 is tetrahydropyrrole.

[0087] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 ml: 1 ml, v / v) and heated at 60 °C for 30 min. Then, compound 1R (10 mmol) was added, and the mixture was stirred at 80 °C for another 4 h. After the reaction was complete, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth, and the residue was washed several times with a small amount of dichloromethane to remove the precipitate. The filtrate and washings were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (PE:EA = 40:1, v / v) to give the corresponding aldehyde 2R as a yellow solid (yield approximately 76%).

[0088] Compound 2R (5 mmol) and 1,4-dimethylpyridine iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture, and the mixture was stirred at 80 °C for 8 h. After the reaction was complete, the reactants were cooled to room temperature and concentrated under reduced pressure to give the crude product. The crude product was dissolved in a small amount of anhydrous ethanol and recrystallized dropwise with diethyl ether to give red solids NQPI1, 2, 3, 5 and 6 (yiels of 71%, 63%, 60%, 69% and 72%, respectively).

[0089] NQPI1: 1H NMR(600MHz,DMSO-d6)δ8.98(d,J=6.5Hz,2H),8.65(d,J=8.5Hz,1H),8.46(d,J=6.9Hz,2H),8.39(d,J=2.5Hz,1H),8.32(dd,J=11.9,8.3Hz,3H),8.26(d,J=16.2Hz,1H),8.13–8.04(m,3H),7.56(dd,J=8.2,0.9Hz,2H),7.49(d,J=15.3Hz,2H),7.41–7.32(m,2H),4.32(s,3H). 13 C NMR(151MHz,DMSO-d6)δ154.43,151.81,146.70,145.62,140.30,139.76,137.81,135.97,131.56,129.53,129.10,128.94,127.02,124.99,124.91,123.42,122.13,121.13,121.08,110.17,47.72.MS(ESI):C 29 H 22 N3 + [M] + ,calcd412.18,found 412.1833.

[0090] NQPI2: 1 H NMR(600MHz,DMSO-d6)δ8.91(d,J=7.2Hz,2H),8.36(d,J=7.1Hz,2H),8.16–8.08(m,2H),7.92–7.83(m,2H),7.77(d,J=8.6Hz,1H),7.31(d,J=6.5Hz,1H),7.16(d,J=9.0Hz,4H),7.03(s,1H),6.99(d,J=9.1Hz,4H),4.28(s,3H),3.78(s,6H). 13 C NMR(151MHz,DMSO-d6)δ157.02,152.33,150.73,147.96,145.70,143.75,140.69,139.72,135.42,130.44,129.67,127.98,126.78,124.81,124.59,122.04,115.70,111.89,55.79,55.41,47.53.MS(ESI):C 31 H 28 N3O2 + [M] +,calcd 474.22,found 474.2166.

[0091] NQPI3: 1 H NMR(600MHz,Chloroform-d)δ9.19(d,J=5.9Hz,2H),8.30(dd,J=13.9,7.6Hz,3H),8.24–8.12(m,3H),8.03(d,J=8.6Hz,1H),7.75–7.63(m,1H),7.63–7.46(m,1H),7.32(d,J=7.7Hz,2H),7.16(t,J=7.7Hz,2H),7.09(t,J=7.5Hz,2H),6.92(d,J=8.1Hz,2H),4.68(s,3H). 13 C NMR(151MHz,Chloroform-d)δ

[0092] 167.71,153.31,145.92,140.55,139.01,137.85,137.71,132.24,129.88,129.64,126.99,126.63,126.33,124.37,123.99,123.88,121.38,120.76,120.61,119.93,116.16,109.69.MS(ESI):C 29 H 22 N3S + [M] + ,calcd 444.15,found 444.1509.

[0093] NQPI5: 1 H NMR(600MHz,DMSO-d6)δ8.88(d,J=6.7Hz,2H),8.34(d,J=6.8Hz,2H),8.15(d,J=8.6Hz,1H),8.09(d,J=16.2Hz,1H),7.88–7.82(m,2H),7.78(d,J=8.6Hz,1H),7.10(dd,J=9.1,2.5Hz,1H),6.67(d,J=2.5Hz,1H),4.27(s,3H),4.01(t,J=7.3Hz,4H),2.43–2.36(m,2H). 13 C NMR(151MHz,DMSO-d6)δ

[0094] 152.49,150.60,149.20,145.64,142.56,140.97,134.49,130.57,129.93,126.02 ,124.46,121.96,119.13,103.29,52.29,47.47,40.53,16.59.MS(ESI):C20H20N3+

[0095] [M]+,calcd 302.17,found 302.1663.

[0096] NQPI6: 1 H NMR (600MHz, DMSO-d6) δ8.87(d,J=6.8Hz,2H),8.33(d,J=6.8Hz,2H),8.13(d,J=8.6Hz,1H),8.08(d,J=16.2Hz,1H),7.88–7.79(m,2H ),7.76(d,J=8.6Hz,1H),7.33(dd,J=9.3,2.6Hz,1H),6.77(d,J=2.5Hz,1H),4.27(s,3H),3.41(d,J=13.0Hz,4H),2.06–1.99(m,4H). 13 C NMR(151MHz,DMSO-d6)δ152.54,148.44,146.79,145.54,141.90,141.09,134.23,130.60,130 .40,125.46,124.33,121.92,120.56,103.61,48.01,47.44,40.52,25.54.MS(ESI):C21H22N3+

[0097] [M]+,calcd 316.18,found 316.1805.

[0098] Example 4

[0099] A method for preparing a probe PAP, the preparation process is as follows:

[0100]

[0101] Isophorone (3.8 g, 27.6 mmol) and malononitrile (1.82 g, 27.6 mmol) were dissolved in anhydrous ethanol (150 mL). Piperidine (23 mg, 0.276 mmol) was added dropwise to the mixture, and the solution was stirred at 60 °C for 12 h, monitored by TLC. After the reaction mixture cooled to room temperature, the reaction solution was slowly poured into water (200 mL), and the precipitate was filtered off. The residue was dissolved in anhydrous ethanol at 60 °C to give a saturated crude product ethanol solution. Petroleum ether was added dropwise until a stable solid precipitate formed in the solution. The solution was cooled to room temperature, filtered, and the residue was collected to give 2-(3,5,5-trimethylcyclohexyl-2-enyl)malononitrile, a brown solid compound a, in 80% yield.

[0102] Under argon protection, compound a (1.38 g, 10 mmol) and p-bromobenzaldehyde (1.85 g, 10 mmol) were dissolved in anhydrous acetonitrile (100 mL), and then piperidine (8.3 mg, 1 mmol) was added to the mixture. The reaction was stirred at 80 °C for 6 h and monitored by TLC. After the reaction was complete, the reaction solution was diluted with 100 mL of ethyl acetate at room temperature and washed with water and saturated sodium chloride, respectively. Finally, the crude product was dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether:ethyl acetate = 2:1, V / V) to give yellow powder b in 50% yield.

[0103] Compound b (1.0 mmol), 4-(4-methyl-1-piperazinyl)phenylboronic acid (1.3 mmol), and tetrakis(triphenylphosphine)palladium (0) (0.1 mmol) were placed in a flask and stirred under argon protection. 30 mL of 1,4-dioxane and 10 mL of 2M sodium carbonate aqueous solution were added to the flask, and the mixture was heated to 110 °C and refluxed with stirring for 12 h. After cooling to room temperature, the aqueous phase was removed. The separated organic phase was washed with water and saturated brine, and then dried over anhydrous magnesium sulfate. After leaching the magnesium sulfate, the organic phase was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1, v / v) to give a red powder PAP-1 in 30% yield. 1 H NMR (400MHz, CDCl3) δ7.63-7.56(m,6H),7.11-6.98(m,4H),6.87(s,1H),3.37(t,J=11 .6Hz,4H),2.72(t,J=7.6Hz,4H),2.63(s,2H),2.51(s,2H),2.46(s,3H),1.11(s,6H). 13C NMR (101MHz, CDCl3) δ154.04,150.94,136.90,133.68,128.49,128.12,127. 62,126.76,123.36,115.90,113.64,55.02,48.60,46.18,43.02.MS(ESI):C 30 H 32 N4[M+H] + ,calcd449.27,found 449.264.

[0104] Compound b (1.0 mmol), 4-(diphenylamino)phenylboronic acid (1.3 mmol), and tetra(triphenylphosphine)palladium (0) (0.1 mmol) were placed in a flask and stirred under argon atmosphere. 30 mL of 1,4-dioxane and 10 mL of 2M sodium carbonate aqueous solution were added to the flask, and the mixture was heated to 110 °C and refluxed with stirring for 12 h. After cooling to room temperature, the aqueous phase was removed. The separated organic phase was washed with water and saturated brine, and then dried over anhydrous magnesium sulfate. After leaching the magnesium sulfate, the organic phase was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1, v / v) to give a brown solid PAP-2 in 20% yield. 1 H NMR (400MHz, CDCl3) δ7.66-7.51 (m, 7H), 7.32 (D, J = 7.9Hz, 3H), 7.19 -7.16(m,6H),7.11-7.07(m,4H),6.89(s,1H),2.64(s,2H),2.53(s,2H),1.13(s,6H). 13 C NMR (101MHz, CDCl3)δ

[0105] 169.27,153.97,147.80,147.46,141.92,136.78,133.47,129.37,128.73, 128.14,127.59,126.98,124.68,123.48,123.26,112.84,43.03.MS(ESI):C 37 H 31 N3[M+H] + ,calcd 518.26,found 518.264.

[0106] Example 5

[0107] Probe synthesis and preliminary evaluation

[0108] The final structures of probes NPPI, NQPI1-6, and PAP1-2 prepared in Examples 1-4 were obtained through... 1H and 13 CNMR spectroscopy and mass spectrometry provided full confirmation. To demonstrate whether the probe could be used for myelin imaging, the effectiveness of NQPI in myelin imaging of sagittal brain slices from six-week-old C57BL / 6J mice was investigated.

[0109] To prepare tissue sections, mice were deeply anesthetized with isoflurane and euthanized by cervical dislocation. The mouse brains were dissected, and the brain tissue was embedded in OCT at the optimal cutting temperature and sectioned using a cryomicrotome (Leica CM1950). Each brain slice was approximately 25 μm thick and adhered to a bonding glass slide. After incubation with different probes (10 μM, DMSO) for 30 min and rinsing three times with PBS, the tissue sections were post-fixed in paraformaldehyde (PFA) for 15 min, incubated with DAPI for 15 min, and rinsed three times with PBS. Observation was then performed using a confocal fluorescence microscope (Leica TCS SP8 MP). DAPI channel, excitation wavelength: 405nm, emission wavelength: 410-500nm; PAP-1 channel, excitation wavelength: 561nm, emission wavelength: 590-690nm; PAP-2 channel, excitation wavelength: 561nm, emission wavelength: 590-690nm; NPPI channel, excitation wavelength: 488nm, emission wavelength: 500-690nm; NQPI1 channel, excitation wavelength: 405nm, emission wavelength: 610-700nm; NQPI2 channel, excitation wavelength: 561nm, emission wavelength: 610-700nm; NQPI3 channel, excitation wavelength: 405nm, emission wavelength: 610-700nm; NQPI4 channel, excitation wavelength: 476nm, emission wavelength: 500-580nm; NQPI5 channel, excitation wavelength: 488nm, emission wavelength: 510-610nm. NQPI 6-channel, excitation wavelength: 488nm, emission wavelength: 530-565nm. Image editing is performed using ImageJ. (Example: ...) Figure 1 As shown in Figure AC, compared to PAP-1 and PAP-2, the pyridinium salt-containing probe NPPI exhibited a strong fluorescent signal along myelinated nerve fibers in the striatum (STR), corpus callosum (CC), and cerebellum (CB) of mouse brain slices. Given that myelin is more abundant in white matter than in gray matter, fluorescence intensity ratio (FIR) was used as a parameter to evaluate the specificity of the probe binding to myelin in the brain and spinal cord. Figure 1 The results showed that the fluorescence intensity of NPPI was significantly higher than that of PAP-1 and PAP-2, indicating that the NPPI fluorophore containing the pyridinium salt terminal group has a stronger binding affinity to myelin.

[0110] The design of NPPI mimics the amphiphilic structure of phospholipids in the membrane. The hydrophilic and positively charged pyridinium salt molecules face the lipid bilayer surface and interact with the negatively charged phospholipid phosphate groups through strong electrostatic interactions, thereby enhancing the plasma membrane targeting of NPPI. Following the NPPI structure, positively charged pyridinium salt molecules are introduced as hydrophilic groups in the NQPI series of myelin probes. Furthermore, the lipophilic structure of the probes in this invention incorporates a quinoline ring with good fluorescence properties as a fluorophore. To optimize these dyes for the complex brain environment, various cyclic aliphatic amines are introduced as electron donors to replace N,N′-dimethylamine, thereby modulating the probe properties, including lipophilicity and fluorescence. Comparison using fluorescence imaging of brain slices revealed that NQPI1-6 all have a certain labeling ability for myelin regions in brain slices, demonstrating that the six probes constructed in this invention can detect and image myelin in brain tissue. Among them, NQPI6, using tetrahydropyrrole as an electron donor, exhibits good selectivity and an extremely high signal-to-noise ratio. Figure 1 ).

[0111] Example 6

[0112] Cytotoxicity assessment was performed prior to fluorescence imaging, using a standard MTT assay, to evaluate probe cytotoxicity in SH-SY5Y cells. SH-SY5Y cells were cultured at 7 x 10⁶ cells per well. 3 Cells were cultured at a density of [number] cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 12 hours to achieve 80% confluence. Then, cells were treated with different concentrations of NQPI6 (0.01, 0.1, 0.5, 1, 2.5, 5, 10, 20, 50 μM) and cultured for another 24 hours. Readings were then taken at 570 nm (reference wavelength 630 nm) using an Infinite M200 Pro microplate reader (Tecan, Switzerland). Figure 2 As shown, after culturing with 10 μM NQPI6 for 24 h, the survival rate of SH-SY5Y cells remained above 75%, indicating that the probe has low cytotoxicity and biocompatibility.

[0113] Example 7

[0114] Cell membrane cholesterol imaging

[0115] The NQPI6 probe was used to detect changes in cholesterol content in the membranes of live cells. SH-SY5Y cells were cultured at 7 x 10⁶ cells per well. 5Cells were cultured at a density of 10 mcg in 12-well plates and incubated at 37°C in a 5% CO2 incubator for 12 h. Cholesterol consumption and replenishment were determined using the method described in the literature (Two-Photon Fluorescent Turn-On Probe for Lipid Rafts in Live Cell and Tissue. Journal of the American Chemical Society, 2008, 130(13): p.4246-4247.). The cholesterol depletion method involved incubating live cells with 10 mM methyl-β-cyclodextrin (M-β-CD) at 37°C for 30 min, washing three times with PBS, and then incubating with NQPI6 (10 μM) in fresh culture medium for 30 min, followed by washing three times with PBS to obtain the cholesterol-consuming group. Cholesterol-depleted cells were incubated with 50 μM cholesterol at 37°C for 1 h in the presence of a cholesterol / M-β-CD (1 / 10) mixture. The cells were then refilled with PBS, washed three times, and incubated with NQPI6 (10 μM) in fresh culture medium for 30 min, followed by three PBS washes to obtain the cholesterol-replenished group. Microscopic imaging was performed using a confocal fluorescence microscope (Leica TCS SP8 MP) with an excitation wavelength of 488 nm and a collection wavelength range of 530-565 nm.

[0116] like Figure 3 As shown, after M-β-CD-mediated cholesterol depletion, the green fluorescence of the probe became almost imperceptible. However, upon co-incubation with a cholesterol solution, cellular cholesterol was replenished, and the probe fluorescence significantly increased, with the fluorescence signal primarily localized to the cell membrane. This phenomenon can be attributed to the increased cholesterol levels in the plasma membrane, which may reduce the cell permeability of NQPI6, thereby enhancing its localization within the plasma membrane. These results highlight the effectiveness of NQPI6 in detecting and observing different cholesterol levels in the plasma membrane of living cells, demonstrating its ability to label myelin (a cholesterol-rich membrane structure).

[0117] Example 8

[0118] Myelin colocalization imaging

[0119] To further verify the ability of NQPI6 to map the myelin sheath of the central nervous system (CNS), NQPI6 staining and immunohistochemical detection of myelin components Plp1 or Mbp were performed on normal control mice and Akt1 / Akt2 / Akt3 triple conditional knockout (Akt cTKO) mice. Akt dysfunction is associated with white matter diseases, including corpus callosum agenesis. Oligodendrocyte lineage cell-specific Akt1 / Akt2 / Akt3 triple conditional knockout mice showed defects in white matter development in the CNS, loss of mature oligodendrocytes, loss of myelin formation, and unchanged apoptotic cell death (Akt Regulates Sox10 Expression to Control Oligodendrocyte Differentiation via Phosphorylating FoxO1. J Neurosci, 2021, 41(39): p. 8163-8180.).

[0120] Whole brains of mice on day 14 postnatal were dissected and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. After dehydration, the tissues were embedded in paraffin. 10 μm sections were prepared using a microtome. These sections were then deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen recovery treatment with boiling sodium citrate buffer for 20 min. The sections were then incubated with NQPI6 (10 μM) at room temperature (RT) for 30 min and washed with 1x PBS. After blocking with 5% BSA at room temperature (RT) for 30 min, the sections were stained overnight with primary antibody at 4°C, followed by incubation with secondary antibody at room temperature (RT) for 30 min. Cell nuclei were stained with DAPI (1 mg / ml). Images were analyzed using a fluorescence microscopy system (BX53, Olympus). The antibodies used are as follows: Mbp (Catalog#MAB386, Millipore, 1:100), Plp1 (Catalog#MAB388, Millipore, 1:1000), Alexa... 488AffiniPure Donkey Anti-Mouse IgG(Catalog#715-545-150,JacksonImmunoResearch Labs,1:500)and Alexa 488AffiniPure Goat Anti-Rat IgG (Catalog #112-545-003, Jackson ImmunoResearch Labs, 1:500).

[0121] Co-staining results showed that NQPI6 fluorescence was detected in the Plp1+ myelin sheath of the cerebellar white matter (CB) and corpus callosum (CC) of control mice; however, no obvious signal was detected in the cortical gray matter. Figure 4 (A and B). Conversely, no fluorescence signal of NQPI6 or Plp1 was observed in the CB, CC, or cortex of AktcTKO mice. Consistent with control mice, NQPI6 fluorescence was also detected in the Mbp+ myelin sheath of the CB and CC white matter, but no obvious fluorescence signal was detected in the cortical gray matter. Figure 4 (C and D). Akt cTKO mice also showed no fluorescence signal of NQPI6 or Mbp. Colocalization results indicated that myelin in white matter could be specifically stained by NQPI6.

[0122] Example 9

[0123] Mouse sciatic nerve imaging

[0124] The sciatic nerve fibers in the PNS are enveloped by Schwann cells. Therefore, this multilayered, membrane-rich myelin sheath is a plausible target for NQPI6. To test this possibility, mouse sciatic nerve fibers were stained with NQPI6.

[0125] Six-week-old C57BL / 6J mice were anesthetized with isoflurane and under deep anesthesia, resulting in cervical dislocation. After shaving the hip area, a 3 mm incision was made in the skin above the trochanter, and the conjunctival tissue was incised on the right side of the hip. A small notch was made in the conjunctival tissue. The muscle fascia was pushed apart with fine forceps to excise a small portion of the nerve. The tissue was embedded by OCT at the optimal cutting temperature and sectioned using a cryomicrotome (Leica CM1950). Each section was approximately 10 μm thick and adhered to an adhesive glass slide. After incubation with probe NQPI6 (10 μM) for 30 minutes and rinsing three times with PBS, the 10 μm sciatic nerve section was post-fixed in paraformaldehyde (PFA) for 15 minutes, incubated with DAPI for 15 minutes, and rinsed three times with PBS before observation using a confocal fluorescence microscope (Zeiss LSM 880). For the DAPI channel, excitation: 405 nm, emission: 410-500 nm. The probe channel was excited at 488 nm and emitted at 530-565 nm. Images were edited using ImageJ. Adjacent tissue sections were stained with LFB (Luxol Fast Blue) myelin and photographed using a Pannoramic MIDI (Pannoramic Method) technique by Savill Biotechnology Co., Ltd. LFB is a copper-phthalocyanine dye commonly used for staining the brain, spinal cord, and peripheral nerves; nerve myelin appears bright blue. Figure 5 As shown, ( Figure 5 Fluorescence imaging region of NQPI6 in sciatic nerve slices (B) Figure 5The bright blue areas stained with LFB in the middle A) are consistent, indicating that NQPI6 can label sciatic nerve fibers.

[0126] Example 10

[0127] Mouse spinal cord myelin imaging

[0128] Six-week-old C57BL / 6J mice were deeply anesthetized by intraperitoneal injection of chlorpromazine (15 mg / kg). The chest and abdomen were sprayed with 75% ethanol, followed by a midline longitudinal incision in the abdominal cavity. The chest cavity was opened to expose the heart; the heart was held with forceps, and a 21-gauge needle was inserted into the left ventricle. An incision was made in the right atrium, and a cryotherapy device (free of calcium) was used. 2+ and Mg 2+ Mice were slowly perfused with ACSF (containing 95% O2 / 5% CO2) through the left ventricle. After perfusion, the mice were decapitated, the spine was removed and rapidly immersed in normal, ice-cold ACSF. Small incisions were made on both sides of one end of the cervical vertebra, the ventral vertebral body was removed, the dorsal processes on both sides were cut, the spinal canal was opened, the nerves were severed, and the spinal cord was exposed. The spinal cord was removed and immersed in frozen normal ACSF saturated with 95% O2 / 5% CO2, and the remaining spinal nerve roots near the spinal cord were cut. At the optimal cutting temperature, the tissue was embedded by OCT and sectioned using a cryomicrotome (Leica CM1950). Each section was approximately 10 μm thick and adhered to an adhesive glass slide. After incubation with NQPI6 (10 μM) probe for 30 minutes and rinsing three times with PBS, 10 μm mouse spinal cord sections were fixed in paraformaldehyde (PFA) for 15 minutes, incubated with DAPI for 15 minutes, and rinsed three times with PBS. Observation was then performed using a confocal fluorescence microscope (Leica TCS SP8 MP). For the DAPI channel, excitation was 405 nm, and emission was 410-500 nm. The probe channel had an excitation wavelength of 488 nm and emission of 530-565 nm. Images were edited using ImageJ. Figure 6 As shown, obvious fluorescence appeared in the white matter area of ​​the spinal cord slices after incubation with NQPI6, indicating that NQPI6 specifically stains the myelin sheath of the spinal cord white matter.

[0129] Example 11

[0130] Clinical tissue imaging

[0131] Clinical human left occipital lobe sections were obtained from Liaocheng People's Hospital (2024277). After being inserted into a slide holder, the slides were baked in a 60℃ oven for 1.5 hours. The sections were deparaffinized twice with xylene, 10 minutes each time. The sections were rehydrated with ethanol of decreasing concentration: anhydrous ethanol twice, 5 minutes each; 95% ethanol for 5 minutes; 70% ethanol for 5 minutes; and 50% ethanol for 5 minutes each. After simple rinsing with distilled water, the sections were incubated with probe NQPI6 (10 μM) for 30 minutes and rinsed three times with PBS before observation using a confocal fluorescence microscope (Leica TCS SP8MP). The probe channel excitation wavelength was 488 nm, and the emission wavelength was 530-565 nm. Images were edited using ImageJ. Adjacent sections from the same tissue were stained with LFB myelin and photographed using a Pannoramic MIDI scanner (Saiwell Biotechnology Co., Ltd.). Figure 7 As shown, ( Figure 7 The fluorescence imaging region of NQPI6 in sagittal sections of the left occipital lobe in clinical humans is related to (B) NQPI6. Figure 7 The bright blue area stained with LFB in the middle A) is consistent, indicating that NQPI6 can image the myelin sheath in sagittal sections of the left occipital lobe in clinical humans.

[0132] Example 12

[0133] 3D drawing of brain tissue myelin sheath

[0134] Compared with single-photon fluorescent probes, two-photon fluorescent probes have significant advantages such as lower excitation energy, less photodamage, and deeper tissue penetration, and are attracting increasing attention in biomedical research. To further evaluate the superiority of NQPI6 in myelin fiber fluorescence imaging, two-photon imaging was performed on NQPI6-stained mouse brain slices.

[0135] Six-week-old C57BL / 6J mice were anesthetized with isoflurane, and their cervical spines were dislocated under deep anesthesia. The whole brain of the mice was extracted using a scalpel, forceps, and fine scissors. The brain was placed in a container containing an appropriate amount of liquid nitrogen for 10-15 seconds until the tissue turned white. It was then removed, wrapped in aluminum foil, labeled, and placed at -80°C until ready for sectioning. At the optimal cutting temperature, the tissue was embedded using OCT and sectioned using a cryomicrotome (Leica CM1950). Each section was approximately 100 μm thick and adhered to an adhesive glass slide. After incubation with NQPI6 probe (10 μM) for 30 minutes and rinsing three times with PBS, the 100 μm whole brain sections were post-fixed in paraformaldehyde (PFA) for 15 minutes. The brain tissue sections incubated with NQPI6 were observed and imaged at different depths using a two-photon confocal fluorescence microscope (Leica TCS SP8MP), scanning every 5 μm for a total depth of 80 μm. The probe channel was excited at 920 nm and emitted at 530-565 nm. 3D modeling was performed using Leica TCS SP8 MP software.

[0136] Under 920 nm excitation, NQPI6-stained mouse brain sections showed strong fluorescence signals in the sagittal brain sections in the CB, STR, and CC regions. NQPI6 clearly labeled and localized myelin axonal fibers in the white matter. Figure 8 ).like Figure 9 As shown, in the 3D reconstructed images, the probe can resolve individual nerve tracts with a high signal-to-noise ratio in the caudate nucleus thalamus (CPu) at a depth of 80 μm. These results collectively confirm that NQPI6 is a reliable myelin probe capable of labeling myelin fibers in different regions, providing a new tool and strategy for high signal-to-noise ratio 3D depth imaging of the brain's myelin sheath.

[0137] Example 13

[0138] NQPI6 response to in vitro Aβ fibers

[0139] Given that one potential application of highly environmentally sensitive probes is to develop them into tools for detecting amyloid plaques (AD-specific biomarkers), the potential of probe NQPI6 to become a probe for the dual detection of myelin sheath and amyloid plaques was further explored.

[0140] Aβ 1-42The polymer was prepared using a previously reported method (In Vivo Brain Imaging of Amyloid-β Aggregates in Alzheimer's Disease with a Near-Infrared Fluorescent Probe. ACSSensors, 2021. 6(3): p. 863-870. 3D dynamic tracking Aβ plaques in live brains using vinyl-bridged dyes with two-photon excitation / NIR emission and large Stokes shifts. Biosensors and Bioelectronics, 2023. 238: p. 115563.). 0.2 mg of Aβ 1-42 Lyophilized powder (P9001, Beyotime) was dissolved in 44 μL of pre-cooled hexafluoroisopropanol (HFIP) to prepare Aβ. 1-42 Storage solution. After sealing with a film, shake the mixture and incubate at room temperature for 1 hour, then remove the film and add HFIP / Aβ. 1-42 Leave at room temperature overnight (12 h). After HFIP evaporates, a transparent Aβ is formed. 1-42 Peptide membrane. Then, Aβ 1-42 The peptide membrane was redissolved in 8.8 μL of DMSO to obtain a 5 mM concentration of Aβ. 1-42 Peptide stock solution. Dilute the stock solution with pre-chilled PBS (10 mM, containing 1 mM EDTA) and centrifuge at 16,000 × g for 10 min to obtain 50 μM Aβ. 1-42 The monomer was stored at 193K. Finally, Aβ was... 1-42 Aβ monomers were incubated at 310 K for different times (0-72 h) to prepare Aβ in different aggregation states. 1-42 Peptide.

[0141] In vitro, the probe NQPI6 (10 μM) was observed to react with Aβ obtained after incubation in PBS for 72 hours. 1-42 After incubation of the aggregates, the fluorescence intensity at 640 nm increased in a concentration-dependent manner. Figure 10 (In AB). The Kd value is as high as 1.266 μM ( Figure 10 C), showing the relationship with Aβ 1-42 The aggregates exhibit good binding affinity. Furthermore, to assess whether NQPI6 is affected by other interfering species in vivo, the probe was bound to different metal ions (K+, K ... + Zn 2+ Cu2+ Mg 2+ Ca 2+ Na + Fe 2+ Fe 3+ Incubation with amino acids (Lys, His, Val, Ala, Cys, Ile, 50 μM), GSH (50 mM), and representative proteins (BSA, HSA, 50 μg / mL) was performed. Figure 10 In the study of Aβ fibers (D), these effects were found to be almost negligible. These results indicate that NQPI6 is an ideal molecular tool for the specific detection of Aβ fibers.

[0142] Example 14

[0143] Molecular docking research

[0144] To investigate the binding mode of NQPI6 to Aβ aggregates, Aβ was used. 1-42Cryo-electron microscopy (PDB ID: 5OQV) structures of the fibers were used for molecular docking studies. The crystal structure of the Aβ protein used for docking studies was stored in the RCSB protein database (RCSB PDB ID: 5OQV). Covalent docking of NQPI6 with the Aβ protein binding pocket was performed using Schrodinger software suite 2015 (Docking covalent inhibitors: a parameter-free approach to pose prediction and scoring. J Chem Inf Model, 2014, 54(7): p. 1932-40.). All parameters for the docking studies were set to default. Furthermore, the binding free energy between the docking posture and the protein receptor was calculated using the MM-GBSA (Molecular mechanics with generalized Born and surface area solvation) model in Schrodinger package Prime 3.9 (A hierarchical approach to all-atom protein loop prediction. Proteins, 2004, 55(2): p.351-67. On the role of the crystal environment in determining protein side-chain conformations. J MolBiol, 2002, 320(3): p.597-608.). The first identified binding site consisted of a tunnel along the fiber axis, composed of the side chains of Val36, Asp1, and Ile41. It was observed that NQPI6 along Aβ... 1-42 The fiber inserts into the tunnel, forming a fiber axis, with a docking score of -7.250 kcal / mol. Figure 11 (A). Similarly, the second binding site was identified as a tunnel along the fiber axis, consisting of side chains of Phe19, Asn27, Gly29, and Ile31. NQPI6 along Aβ 1-42 The fiber inserted into this tunnel also forms a fiber shaft, with a docking fraction of -10.346 kcal / mol. Figure 11 (B). The third binding site is located on the groove along the fiber axis on the adjacent exposed surfaces of Phe20 and Glu22. NQPI6 can attach along the fiber axis into the groove formed by the exposed surfaces of adjacent amino acid residues, resulting in a docking fraction of -4.773 kcal / mol. Figure 11(C) The final binding site is located in a groove along the fiber axis on the adjacent exposed surfaces of Lys16 and His14. NQPI6 can attach along the fiber axis into the grooves formed by the exposed surfaces of adjacent amino acid residues, resulting in a docking fraction of -2.763 kcal / mol (D) in 11. Notably, the intermolecular forces between the probe and the protein fiber axis tunnel provide higher binding affinity and a more stable planar structure compared to probes attached to the protein surface.

[0145] Example 15

[0146] Aβ patch colocalization imaging

[0147] Given that NQPI6 has an in vitro effect on Aβ 1-42 The aggregates reacted well, and their imaging capabilities in brain tissue sections from 5x FAD model mice were further investigated to explore their potential for simultaneous imaging of Aβ plaques and myelin. The brains of transgenic mice (5x FAD, 6 months old, male) were dissected and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. The tissues were dehydrated and embedded in paraffin. 10 μm sections were prepared using a microtome. These sections were then deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen recovery treatment with boiling sodium citrate buffer for 20 min. The sections were then incubated with NQPI6 (10 μM) at room temperature (RT) for 30 min and washed with 1x PBS. After blocking with 5% BSA at room temperature (RT) for 30 min, the sections were stained with primary antibody at 4°C overnight, followed by incubation with secondary antibody at room temperature (RT) for 30 min. Cell nuclei were stained with DAPI (1 mg / ml). Images were analyzed using a fluorescence microscopy system (BX53, Olympus). The antibodies used were β-Amyloid (Catalog#8243, Cell Signaling Technology, 1:500) and Alexa Fluor 488 Affini Pure Goat Anti-Rabbit IgG (Cat#111-545-003, Jackson Immuno Research Labs, 1:500).

[0148] like Figure 12 As shown, brain slices from 5xFAD mice exhibited a significant red fluorescent signal after incubation with NQPI6. Co-localization studies of amyloid plaque-specific antibodies and NQPI6 confirmed that NQPI6 can specifically stain Aβ plaques in brain slices from 5x FAD model mice.

[0149] Example 16

[0150] Simultaneous imaging of Aβ plaques and myelin pathology in brain slices of 5x FAD mice

[0151] Myelin sheath structure changes with age, but this is more pronounced in Alzheimer's disease (AD). Dense plaque cores often have the greatest detrimental effect on surrounding nerve fibers, and focal demyelination can be observed in human cases. Given the excellent dual labeling ability of the probe NQPI6 for myelin sheath and Aβ plaques, this probe was used to investigate pathological changes associated with Aβ plaques in different brain regions of AD mice, including changes in the number and morphology of myelin sheaths, to further investigate the correlation between myelin sheath and Aβ plaques in the pathogenesis of AD. Sagittal sections were prepared from 5x FAD mice and age-matched wild-type mice. Transgenic mice (5x FAD, 1, 3, and 6 months old, male) and age-matched wild-type mice (WT, 1, 3, and 6 months old, male) were euthanized by cervical dislocation under deep anesthesia with isoflurane. The whole brain was dissected and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. After tissue dehydration, the tissues were embedded in paraffin. 10 μm sections were prepared using a microtome. These sections were then deparaffinized and rehydrated. The slices were incubated with NQPI6 (10 μM) at room temperature (RT) for 30 min and washed with 1x PBS. Detailed fluorescence imaging and analysis of different brain regions in these slices were performed using confocal fluorescence microscopy (Leica TCS SP8MP). DAPI channel: Ex, 405 nm; Em, 420-480 nm. NQPI6 channel 1: Ex, 488 nm; Em, 530-565 nm. NQPI6 channel 2: Ex, 561 nm; Em, 690-730 nm.

[0152] By comparing imaging results ( Figure 13The study found that the fluorescence signals in the cerebral cortex, hippocampus, and corpus callosum of NQPI6-stained 5xFAD mice differed from those in age-matched WT mice. Aβ deposition appeared to induce focal demyelination, with maximal demyelination occurring at the core of the plaque. No myelin fibers penetrated the amyloid plaque, and demyelination decreased with increasing distance from the plaque core. Given the crucial role of myelin in regulating the speed and integrity of axonal transmission, myelin lesions are likely to significantly contribute to the systemic cognitive decline characteristic of AD. To further verify whether myelin degeneration is a significant feature of AD, the number and morphological changes of myelin in different brain regions of 5xFAD mice were investigated, with age-matched WT mice as controls. Compared to WT mice, the brain tissue structure and composition of the cerebral cortex, hippocampus, and corpus callosum in AD model mice were significantly altered. In AD, the myelin sheath of the hippocampus is severely affected and exhibits morphological atrophy, indicating widespread myelin loss in brain regions crucial for memory and other cognitive functions during AD pathology. Furthermore, enhancing myelin regeneration alleviates AD-related cognitive impairment. These results suggest that demyelination is closely associated with the development of cognitive dysfunction in 5x FAD model mice.

[0153] Example 17

[0154] Immunofluorescence imaging of 5xFAD brain tissue sections

[0155] Brains of transgenic mice (5xFAD, 6 months old, male) and age-matched wild-type mice (WT, 6 months old, male) were dissected and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. After dehydration, the tissues were embedded in paraffin. 10 μm sections were prepared using a microtome. These sections were then deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen recovery treatment with boiling sodium citrate buffer for 20 min. After blocking in 5% BSA for 30 min, staining was performed overnight at 4°C with primary antibodies (Mbp and β-Amyloid), followed by incubation at RT for 30 min with secondary antibodies (Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG). Cell nuclei were counterstained with DAPI (1 mg / mL). Images were analyzed using a fluorescence microscopy system (BX53, Olympus). The antibodies used were Mbp (Catalog#MAB386, Millipore, 1:100), β-Amyloid (Catalog#8243, CellSignaling Technology, 1:500), Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (Cat#711-585-152, Jackson ImmunoResearch Labs, 1:500) and Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (Catalog#112-545-003, Jackson ImmunoResearch Labs, 1:500).

[0156] like Figure 14 As shown, the same phenomenon observed in brain slices incubated with MBP and Aβ antibodies was also observed in brain slices incubated with NQPI6. There was no myelin sheath around the plaques, and demyelination was more pronounced in the cerebral cortex and hippocampus of 5x FAD mice than in the corpus callosum. This indicates that the NQPI6 probe can accurately image the myelin sheath and Aβ plaques in the brains of AD mice, and that NQPI6 incubation is more convenient than antibody incubation.

[0157] In summary, this invention demonstrates the practicality of probes such as NQPI6 as a powerful tool. It can simultaneously detect Aβ plaques and myelin sheaths in the brain through dual channels, offering greater precision and convenience. This capability is invaluable for elucidating the complex relationship between demyelination and Aβ plaque formation, and is of great significance for future research in the prevention, diagnosis, and treatment of attention deficit disorder.

Claims

1. A myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure, characterized by, The structure of the probe is shown as follows: The R is selected from any one of the following:

2. A method for preparing the myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure according to claim 1, characterized by, The method comprises the following steps: Cesium carbonate, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene and palladium(II) acetate are dissolved in toluene, then 6-bromo-2-methylquinoline and different secondary amines are added, the reaction is carried out under reflux, and after the reaction, the solution is cooled to room temperature, extracted, concentrated under reduced pressure, and the obtained residue is purified to obtain the required compound 1R; the required secondary amine for preparing NQPI5 is heteroazacyclobutane; and the required secondary amine for preparing NQPI6 is tetrahydropyrrole; SeO2 is dissolved in 1,4-dioxane, heated and stirred, then compound 1R is added, the mixture is heated and stirred, after the reaction is completed, the reaction is cooled to room temperature, filtered, and the filtrate is concentrated under reduced pressure to obtain a crude product, which is purified to obtain the corresponding aldehyde 2R; Compound 2R and 1,4-dimethylpyridine iodide are dissolved in anhydrous ethanol, piperidine is added dropwise to the mixture, and heated and stirred, after the reaction is completed, the reaction is cooled to room temperature, concentrated under reduced pressure to obtain a crude product, which is dissolved and recrystallized to obtain red solid NQPI5 and NQPI6; The reaction formula is shown as follows: The R is selected from any one of the following:

3. Use of the myelin-responsive pyridinium salt structure-containing amphiphilic fluorescent probe in the preparation of a reagent for detecting myelin.

4. Use according to claim 3, characterized in that, The myelin is central nervous system myelin or peripheral nervous system myelin.

5. The use according to claim 3, characterized in that, The myelin is brain section different brain region myelin, spinal cord section myelin, sciatic nerve section myelin or brain right occipital lobe section inner myelin.

6. The use according to claim 4, characterized in that, The probe for detecting myelin is used for analyzing myelin density and drawing myelin nerve fiber bundle trajectory.

7. Use of the myelin-responsive pyridinium salt structure-containing amphiphilic fluorescent probe in the preparation of a reagent for simultaneously detecting and imaging myelin and Aβ aggregates in the brain of AD.

8. Use according to claim 7, characterized in that, The use of the amphiphilic fluorescent probe in the preparation of a reagent for imaging the integrity of myelin and Aβ aggregation in brain tissue.

9. Use according to claim 7, characterized in that, The use of the amphiphilic fluorescent probe in the preparation of a reagent for three-dimensional mapping imaging of brain tissue myelin and labeling of brain section Aβ plaques.

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