A nerve-specific dual-targeting fluorescent probe for cholinergic neuron AChR and application thereof

Abstract

This invention relates to the field of organic small molecule fluorescent probe technology, specifically to a neuron-specific dual-targeting fluorescent probe targeting the AChR of cholinergic neurons and its applications. The fluorescent probe, whose structure follows general formula I, is characterized by its dual-targeting structure, making it particularly suitable for the identification and labeling of cholinergic neurons. Furthermore, this fluorescent probe exhibits significant advantages in multiple application scenarios, such as cell imaging, protein and organelle labeling, tumor-specific recognition, and photodynamic therapy, with its potential application value in neuroimaging being particularly prominent. This fluorescent probe is expected to advance the development of neurobiology, disease diagnosis, and treatment methods, and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, specifically relating to a neuron-specific dual-targeting fluorescent probe for cholinergic neurons AChR and its applications. Background Technology

[0002] The nervous system, composed of the central nervous system (CNS) and the peripheral nervous system (PNS), acquires information from the surrounding environment and coordinates tissues and organs by transmitting signals and receiving feedback between different parts of the body. It controls muscle activity and glandular secretion, enabling the organism to respond to stimuli or pressure. Nervous system diseases can be caused by aging, poisoning, trauma, and genetic problems, leading to disturbances of consciousness, sensory deficits, motor disorders, dystonia, and more. Over the past few decades, magnetic resonance imaging (MRI) has become an indispensable tool for diagnosing and evaluating CNS diseases such as encephalopathy, myelopathy, and cranial nerve lesions. However, due to the similarity in signal intensity between the PNS and surrounding tissues, PNS imaging currently relies on high-resolution ultrasound. Ultrasound-based nerve identification is based on a network of hypoechoic bands and hyperechoic lines. Pathologically, ultrasound performs well in diagnosing tumors, with its regular contours and clear boundaries, making it particularly useful for PNS tumors. However, high-resolution ultrasound is not suitable for visualizing nerves in deeper tissues and hyperechoic structures; it is only effective for superficial peripheral nerves. Therefore, nerve identification remains challenging, and there is still significant room for development in nerve identification technology.

[0003] Surgical treatment of disease is an integral part of healthcare. While the primary goal of surgery is to remove, reshape, and repair diseased tissue, the protection and preservation of vital anatomical structures such as nerves, blood vessels, ureters, and bile ducts are equally crucial for achieving optimal results. Thin or buried nerves are most likely to be damaged during surgery because they are difficult to distinguish. Iatrogenic nerve injury is also one of the most dreaded surgical complications. Experienced surgeons can easily identify major nerves in anatomically normal conditions, but previous trauma, radiation therapy, tumors, and previous surgeries can lead to fibrous tissue deposits and atypical surgical anatomical planes, making nerve delineation challenging. For example, some peripheral nerves, such as the delicate cavernous nerve responsible for male self-control and strength, are frequently injured during prostatectomy because they are so small that they are difficult to identify even under optical magnification. According to US statistics, surgery results in up to 600,000 nerve injuries annually, accounting for approximately 17% of all peripheral nerve injuries. Nerve damage during surgery (e.g., prostatectomy, colorectal surgery, and head and neck tumor resection) can lead to serious patient morbidity, including chronic pain or permanent paralysis, which limits patients’ quality of life and increases medical costs; therefore, it is crucial to identify important nerves during surgery.

[0004] Computed tomography (CT), magnetic resonance imaging (MRI), electromyography (EMG), ultrasound, and confocal microscopy can be used to assist in intraoperative nerve identification. However, this disrupts the normal surgical workflow, prolongs surgery / anesthesia time, and requires a large workspace, not to mention their high cost. Currently, intraoperative neurophysiological monitoring (IONM) is widely used in clinical practice, utilizing neurophysiological and cerebral hemodynamic methods to reflect changes in electrophysiological signals during nerve transmission during surgery. However, IONM only shows the functional integrity of specific nerves, not their location and changes, and cannot directly visualize important neural structures intraoperatively; therefore, even if a nerve location is identified, surgeons lack direct visual guidance to ensure the length or direction of nerve fibers. Therefore, real-time neural imaging technology that displays the anatomical details required for precise localization and treatment planning urgently needs development.

[0005] Fluorescence imaging boasts advantages such as wide coverage, high spatial resolution, strong tissue penetration, and fast optical imaging speed without ionizing radiation. It also reduces potential ambiguity during procedures. Over the past few decades, fluorescent probes have achieved tremendous success in in vivo imaging, bringing increasingly exciting developments to various fields such as biomedicine and bioanalysis. The development of fluorescence imaging has shifted from structural and functional fluorescence imaging to the use of more specific molecular fluorescent imaging agents. Extensive evidence suggests that many bioactive substances are crucial for physiological processes throughout the cell cycle, and many major diseases are associated with the abnormal expression of these substances. Based on biomarkers of different diseases or the specific structures of different tissues, corresponding fluorescent probes are designed. Illumination with fluorescent lamps enhances the visual differences between tissues, enabling direct intraoperative visualization of regions of interest at the surgical site. The past few years have witnessed an explosive growth in clinical trials of fluorescence imaging of the ureter, blood vessels, nerves, and biomarkers. Fluorescence-guided surgery (FGS) has been successfully integrated into clinical medicine with two FDA-approved near-infrared (NIR) contrast agents, indocyanine green (ICG) and methylene blue, and a large number of cancer-targeting molecular imaging agents are also being used in surgical practice. Simple tubular structures such as blood vessels, ureters, and bile ducts can be well visualized through perfusion with various dyes, demonstrating the promising future of FGS. FGS uses nerve-specific fluorescent contrast agents, allowing surgeons to visualize signals hidden beneath muscle and fat in real time during dissection, providing guidance for nerve-free surgical procedures and reducing the incidence of iatrogenic injury, related morbidity, surgical time, and relative costs.

[0006] A series of small organic molecule fluorescent probes targeting myelin have been developed for in vivo neural imaging. Styrene-pyridine (FM) fluorescent probes have been shown to label muscle spindle organs, taste receptors, and pain receptors, such as the dorsal root ganglion (DRG) and trigeminal ganglion. The stilbene derivative 4,4′-diamino-trans-stilbene (DAS) and the coumarin derivative 3-(4-aminophenyl)-2H-chromium-2-one (CMC) exhibit myelin specificity in brain tissue and show fluorescent signals. Divinylbenzene (DSB) fluorophores also exhibit myelin specificity and show fluorescent signals in brain tissue. However, due to the short wavelengths of these compounds, they are easily affected by the autofluorescence of other non-neural tissues, making these molecules less than optimal choices for guiding neural recognition in fluorescence imaging.

[0007] Bruno Stankoff first described BMB, a lipophilic Congo red derivative whose chemical structure endows it with fluorescent properties and the ability to specifically bind to myelin. It can cross the blood-brain barrier (BBB), meaning it can affect the central nervous system (CNS), highlighting all neural tissue after systemic administration. Research speculated that its binding to neural tissue sections was possible due to BMB's lipophilicity; however, BMB exhibits similar fluorescence spectra in nerve and adipose tissue, showing lower contrast between nerve and fat. Based on the BMB structure, phenylvinylbenzene (DSB) derivatives were designed, among which GE3082, GE3111, and GE3126 showed good imaging performance, crossing both the BNB and BBB, and producing fluorescence in myelinated nerves via intravenous injection. Immunohistochemical and biochemical analyses indicated that the binding target of GE3111, GE3082, and GE3126 is also MBP. Gibbs et al. proposed that the overall configuration of the tricyclic system dominates, influencing the neuron-specific characteristics; compounds with secondary configurations around the middle ring are most favorable for neuron-specific fluorescence. While GE3082 possesses similar physical characteristics to BMB, it exhibits spectral shift in adipose tissue, allowing for the differentiation of neuronal signals from those of adipose and muscle tissue. However, these molecules still exhibit high lipophilicity and charge, showing a high affinity for adipose tissue and a relatively low signal-to-noise ratio. Furthermore, due to their high lipophilicity, BMB and GEs require specialized intravenous formulations, but their unsuitability for clinical use is limited by the potential negative physiological and pharmacological effects of the solvent.

[0008] In recent years, Gibbs' team discovered that oxazine 4, a fluorophore of oxazine, exhibits strong release and high neurospecificity after systemic administration, targeting all neurons within a chromosome, with wavelengths located in the near-infrared window. Although not yet confirmed, the prototype of the oxazine scaffold is considered a fundamental unit for expressing neurospecificity, potentially providing a promising and flexible structure to contain neurospecific fluorescent agents. Based on the oxazine 1 and oxazine 4 scaffolds, Gibbs' team conducted targeted synthetic modifications to construct a broad library of oxazine fluorophores and screened four oxazine derivatives (LGW01-08, LGW05-75, LGW04-31, and LGW03-76) using in vivo neurofluorescence assays based on N / M, N / A, and SBR>1.5 as criteria. These derivatives possess excellent near-infrared spectral characteristics, including high neurospecificity, bright fluorescence signals, and structural diversity. Compared to DSB derivatives, oxazine fluorophores are more suitable for clinical use for the following reasons: (1) higher SBR after direct and systemic administration, (2) rapid and long-term neurovisualization, and (3) compatibility with various surgical procedures.

[0009] Current research shows promising initial results in illuminating nerves with fluorophores, but the fluorescence signals of nerves and surrounding tissues are similar, resulting in low contrast. Solving this problem is crucial for a significant step forward in clinical translation. Therefore, we need to find a highly specific nerve probe that achieves high contrast between nerve and surrounding tissue signals, selectively highlighting the nerve, and hopefully enabling its clinical application, making iatrogenic nerve injuries a thing of the past.

[0010] Cholinergic nerves are nerve fibers that release acetylcholine (ACh) from their nerve endings as a chemical transducer. Currently, all preganglionic fibers of the sympathetic and parasympathetic nervous systems, all motor nerves, all postganglionic fibers of the parasympathetic nervous system, and a very small number of postganglionic fibers of the sympathetic nervous system are known to be cholinergic nerves. Acetylcholine is synthesized from acetyl-CoA and choline under the catalysis of choline acetyltransferase. Once synthesized, it is transported from the cytoplasm to small vesicles near the nerve ending. When a nerve impulse reaches the nerve ending, ACh is released from the vesicles into the synaptic cleft through a slit. The released ACh binds to acetylcholine receptors (AChRs) on the presynaptic and postsynaptic membranes, exciting them and producing an effect. Most of the released ACh is reuptaken by the nerve ending, while a small portion is rapidly hydrolyzed by acetylcholinesterase (AChE) into choline and acetic acid, thus terminating its biological effect. Currently, cholinergic and anticholinergic drugs designed based on the above process exist. These drugs act on the AChR and are used to treat cholinergic neurological disorders. Therefore, the AChR is a good target for cholinergic neurons and can be used to design fluorescent imaging probes to improve the neuronal specificity of the probes.

[0011] In summary, we selected the oxazine derivatives screened by the Gibbs team from a broad library of oxazine fluorophores using in vivo neural fluorescence assays with N / M, N / A, and SBR>1.5 as the parent material. By modifying these derivatives with cholinergic recognition groups, we were able to achieve dual targeting of the oxazine derivatives and the recognition groups, specifically recognizing cholinergic nerves and distinguishing them from other tissues. Summary of the Invention

[0012] To overcome the problem of difficulty in distinguishing nerves from surrounding tissues in existing methods, this invention provides a nerve-specific dual-targeting fluorescent probe for cholinergic neurons (AChR) and its application. The technical solution of this application is as follows:

[0013] A neuron-specific dual-targeting fluorescent probe for cholinergic neurons AChR, the probe having a structure as shown in general formula I:

[0014]

[0015] In general formula I,

[0016] R1 is selected from hydrogen, alkyl groups having 1-18 carbons, carboxyl groups having 1-18 carbons, alkyl sulfonates having 1-18 carbons, alkyl sulfonates having 1-18 carbons, hydroxyalkyl groups having 1-18 carbons, amino groups having 1-18 carbons, and aldehyde groups having 1-18 carbons; more preferably, R1 is selected from hydrogen, alkyl groups having 1-6 carbons, carboxyl groups having 1-6 carbons, hydroxyalkyl groups having 1-6 carbons, and amino groups having 1-6 carbons; most preferably, hydrogen or methyl.

[0017] R2 and R3 are each independently selected from one of hydrogen, alkyl groups having 1-18 carbons, carboxyl groups having 1-18 carbons, alkyl sulfonates having 1-18 carbons, alkyl sulfonates having 1-18 carbons, hydroxyalkyl groups having 1-18 carbons, amino groups having 1-18 carbons, and aldehyde groups having 1-18 carbons; more preferably, R2 and R3 are each independently selected from one of hydrogen, alkyl groups having 1-6 carbons, carboxyl groups having 1-6 carbons, hydroxyalkyl groups having 1-6 carbons, and amino groups having 1-6 carbons; more preferably, methyl or ethyl; most preferably, methyl or ethyl.

[0018] R4 is selected from alkyl groups having 1-18 carbons and alkoxy groups having 1-18 carbons, more preferably from alkyl groups having 1-6 carbons and alkoxy groups having 1-6 carbons; most preferably from methoxy or ethoxy groups.

[0019] X1 and X2 are each independently selected from one of carbon, nitrogen, sulfur, and oxygen; more preferably from oxygen or nitrogen.

[0020] X3 is selected from one of carbon, nitrogen, and oxygen.

[0021] In the most preferred embodiment, the examples verified that when the structure of general formula I is such as XNN, it has the specific recognition ability of cholinergic nerves and can effectively distinguish the fluorescence signals of nerves from those of surrounding tissues.

[0022]

[0023] The second aspect of this application is to protect the method for preparing the dual-targeting fluorescent probe described above, comprising the following steps:

[0024] (1) At 0-5℃, reactant M and catalyst Z are first dissolved in organic solvent S, then reactant N-1 is dissolved in organic solvent S, and then slowly added dropwise to a solution containing M and catalyst Z, wherein the molar ratio of N-1:M:Z is 1:(1-3):(1-3), and the further preferred molar ratio is 1:2:3. Then the temperature is raised to room temperature, stirred for 2-4 hours, and concentrated to obtain crude intermediate product N-2.

[0025] (2) At 0-5℃, the crude intermediate product N-2 is first redissolved in organic solvent S and catalyst Z is added. Then reactant X is dissolved in organic solvent S and then slowly added dropwise to a solution containing N-2 and catalyst Z, wherein the molar ratio of X:N-2:Z is 1:(1-3):(1-3), and the further preferred molar ratio is 1:2:3. Then the temperature is raised to room temperature, stirred for 4-6 h, and then concentrated and purified to obtain a nerve-specific dual-target fluorescent probe with the structure of general formula I.

[0026]

[0027] Furthermore, in steps (1) and (2) above, the organic solvent S is selected from at least one of dichloromethane, chloroform, acetonitrile, toluene, and tetrahydrofuran;

[0028] Furthermore, in step (1) above, the reactant M is selected from at least one of triphosgene, benzyl p-nitrochloroformate, and p-nitrobenzene chloroformate;

[0029] Furthermore, in steps (1) and (2) above, the catalyst Z is selected from at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium acetate, potassium acetate, sodium acetate, potassium acetate, sodium sulfite, and triethylamine.

[0030] Furthermore, in steps (1) and (2) above, the room temperature is 20-30℃.

[0031] A third aspect of this application is to protect the application of the dual-targeting fluorescent probe described above, the scope of which includes the application of the dual-targeting fluorescent probe in neural imaging, cell imaging, protein labeling, organelle labeling, preparation of reagents for specific tumor recognition and photodynamic therapy.

[0032] Compared with the prior art, the beneficial effects of the present invention

[0033] 1. The XNN probe has well-defined spectral characteristics: Under physiological conditions (pH = 7.4 PBS + 30% DMSO), the XNN probe has ideal optical properties, with its ultraviolet absorption peak at 565 nm and fluorescence emission peak at 676 nm, demonstrating good photostability and sensitivity, which is beneficial for subsequent biological detection applications.

[0034] 2. Precise molecular docking simulation: Molecular docking results of XNN with M3 muscarinic acetylcholine receptor and α7 nicotinic receptor showed that the binding energies were all below -4 kcal / mol, demonstrating its good affinity with these two cholinergic receptors and potential dual-targeting effect.

[0035] 3. Excellent biological stability: After incubation with acetylcholinesterase at 37℃, the absorbance at the ultraviolet absorption peak of the probe changes little, proving that XNN is not easily hydrolyzed, thus ensuring its stability and durability when used in vivo.

[0036] 4. Low cytotoxicity: MTT assay results showed that the XNN probe exhibited low cytotoxicity to PC-12 cells (as a neural cell model) and 4T1 cells (as a control group) within a certain concentration range. Even at higher concentrations, it could still coexist with more than 85% of the cells, which enhanced its safety and practicality as a biomarker molecule.

[0037] 5. The XNN probe features a unique design. Experiments showed that the XNN probe exhibited significantly different uptake characteristics in PC-12 and 4T1 cells. The former showed effective uptake with increasing fluorescence intensity over time, while the latter failed to uptake it. This innovative characteristic demonstrates the potential of the XNN probe to distinguish nerve cells from other cell types, solving the problem of indistinguishable fluorescence signals between nerves and surrounding tissues. This has significant value for neurobiological research and the development of potential tissue-specific diagnostic or therapeutic approaches. Attached Figure Description

[0038] Figure 1 : Probe XNN structure diagram.

[0039] Figure 2 (a) Absorption and (b) Emission spectra of probe XNN.

[0040] Figure 3 Simulated diagram of probe XNN docking with M3 muscarinic acetylcholine receptor molecule.

[0041] Figure 4 Simulated diagram of probe XNN docking with α7 nicotinic receptor molecule.

[0042] Figure 5 Biological stability experiment of probe XNN.

[0043] Figure 6 : MTT assay of probe XNN in PC-12 cells under no-light conditions.

[0044] Figure 7 : Experiment on the uptake of probe XNN by PC-12 cells.

[0045] Figure 8 : 4T1 cell uptake of probe XNN. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] Example 1

[0049] Synthesis of intermediate and probe molecule XNN

[0050]

[0051] (1) Synthesis of compound 2

[0052] Compound 1 (500 mg, 3.03 mmol) was dissolved in ice-cold 6M HCl solution (5–7 mL). Then, while maintaining the solution temperature below 5°C, NaNO2 (209 mg, 3.03 mmol) was slowly added over approximately one hour. No brown NOx fumes were observed during this process. The reaction was stirred for another 2 hours. The precipitate was then filtered through a Büchner funnel and washed with a small amount of ice-cold 6M HCl solution to obtain a yellow solid. The product was placed in a funnel and air-dried overnight to give compound 2 (0.562 g, 78.1% yield) as a yellow solid, which could be used in the next step without further purification.

[0053] (2) Synthesis of compound 3

[0054] 3-Amino-4-methylphenol (127 mg, 1.03 mmol) was dissolved in an isopropanol:water (9:1, 2 mL) solution and dissolved in an 80°C water bath for 30 minutes. A suspension of 2 (200 mg, 1.043 mmol) and HClO4 (70%, 90 μL) in an isopropanol:water (9:1, 2 mL) solution was added in four portions to the above solution, and the reaction was carried out over approximately 1 hour, followed by stirring overnight. During this process, the color of the reaction mixture changed from light brown to green, and finally to dark blue. The dark blue solution was then rotary evaporated to remove the solvent, and the crude product was purified by silica gel column chromatography (using dichloromethane:methanol = 50:1 as eluent) to obtain pure product 3 (254 mg, 72%) as a dark green solid.

[0055] (3) Synthesis of compound 5 and XNN

[0056] Because compound 5 is unstable and easily decomposes, steps 3 and 4 were prepared consecutively during the synthesis to avoid its deterioration. NN dimethylethanolamine (90 mg, 1.01 mmol) was dissolved in anhydrous dichloromethane (8 mL), and triethylamine (143 mg, 1.41 mmol) was added as a catalyst. The temperature was maintained at 0 °C. Compound 4 (142 mg, 0.706 mmol) in anhydrous dichloromethane (4 mL) was added dropwise to the above solution over approximately half an hour. The reaction was then stirred for another two hours, during which the reaction solution changed from colorless to yellow. Then, compound 3 (270 mg, 0.706 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to the above reaction solution, and the reaction was stirred overnight. During this process, the color of the reaction mixture changed from blue to purple. The purple solution was rotary evaporated to remove the solvent, and the crude product was purified by neutral alumina column chromatography (dichloromethane:methanol = 120:1 as eluent) to obtain the pure product XNN (78 mg, 22%) as a dark purple solid. Design the probe XNN structure as follows: Figure 1 The dark portion represents the designed recognition group, while the gray portion is the oxazine matrix. The two parts bind together for dual-target neural recognition. The dark purple solid was verified by mass spectrometry as an XNN, with ESI-HRMS results of 397.2237 m / z C. 22 H 29 N4O3 + [M] + Calculated value: 397.2235. The dark purple solid was verified as XNN by NMR. NMR results: 1 H NMR (500MHz, DMSO-d6) δ9.22(s,1H), δ7.65(d,J=9.2Hz,1H), δ7.45(s,1H), δ7.31(s,1H), δ6.98(dd,J=9.2,2.2Hz,1H ), δ6.56(d,J=2.2Hz,1H), δ4.11(t,J=6.9Hz,2H), δ3.70(q,J=7.1Hz,4H), δ2.93–2.78(m,2H), δ1.32(t,J=7.0Hz,6H).

[0057] Example 2

[0058] Absorption and emission spectra of probe molecule XNN

[0059] The probe molecules were precisely weighed and prepared to a concentration of 4.0 × 10⁻⁶. -3The DMSO stock solution of M was stored in a refrigerator (-20℃) for later use. Its photophysical properties under physiological conditions (0.01M PBS, pH 7.4, 30% DMSO) were tested using a 1cm quartz cuvette. The UV-Vis absorption spectra were measured using a CARY 60UV-vis UV-Vis absorption spectrometer (Agilent Technologies, model NO. MY15230004); the fluorescence spectra were measured using a VARIAN CARY Eclipse fluorescence spectrometer (Agilent Technologies, model NO. MY16500004), with the instrument slit adjusted to 5nm to achieve the optimal signal-to-noise ratio. During testing, 15μL of the stock solution and 3mL of solvent were added to a quartz cuvette. The absorbance (A) of the solution in the wavelength range of 300–800nm ​​was measured using a UV spectrophotometer, and the emission spectrum was measured using a fluorescence spectrophotometer with an excitation wavelength of 565nm. The results are shown below. Figure 2 The probe XNN showed an ultraviolet absorption peak of 565 nm and a fluorescence emission peak of 676 nm in the test system (PBS with pH = 7.4 mixed with 30% DMSO).

[0060] Example 3

[0061] Molecular docking simulation

[0062] Molecular docking computation is a method that predicts intermolecular binding ability by simulating intermolecular interactions using computer simulations. Its basic principle is based on the structures of two molecules, then calculating to predict their interactions, which can include van der Waals forces, electrostatic interactions, hydrogen bonds, etc. The molecular docking process can be divided into four steps: preprocessing, building a computational model, executing the docking process, and result analysis. In the preprocessing step, a bimolecular configuration search is performed, that is, finding the lowest-energy conformation for each molecule for subsequent molecular docking calculations. In building the computational model, the three-dimensional structure of each molecule needs to be converted into a computationally viable structure, and both molecules are simultaneously input into the computer. During the docking process, a molecular docking algorithm is invoked to calculate the interactions between the two molecules and find the optimal pairing method. Finally, in the result analysis, the molecular docking results need to be evaluated to determine whether these molecules are suitable for binding and to determine the optimal binding mode. As shown in Table 1, the molecular docking results between probe XNN and the protein show that the binding energy of the probe with the M3 muscarinic acetylcholine receptor and the α7 nicotinic receptor is below -4 kcal / mol, proving that the binding effect is good.

[0063] Table 1. Molecular docking data between probe XNN and protein

[0064] Complex name Affinity (kcal / mol) M3 muscarinic acetylcholine receptor -7.7 α7 Nicotine receptor -7.5

[0065] To discover the interaction patterns between the designed compounds and their targets, molecular docking studies were conducted. This involved converting the designed compounds from 2D to 3D format using Openbabel; preparing the necessary receptor and ligand files for docking using Autodock Tools; performing docking using Autodock Vina; and visualizing the protein-ligand complex docking results using Chimera, analyzing various parameters, and plotting the data. Figure 3 , Figure 4 Molecular docking simulations of the probe XNN and the M3 muscarinic acetylcholine receptor revealed that, in addition to interacting with the oxazine parent compound, it also interacts with the recognition group via H bonds, exhibiting a dual-targeting effect on cholinergic neurons. Figure 6 Molecular docking simulations of the probe XNN and α7 nicotinic receptor revealed that, in addition to interacting with the oxazine parent, it also interacts with the recognition group via CH bonds, demonstrating a dual-targeting effect on cholinergic neurons.

[0066] Example 4

[0067] Probe biostability verification

[0068] Add acetylcholinesterase (exogenous) to a test system containing 12 μM probe XNN (PBS with 30% DMSO at pH 7.4), mix thoroughly, and place in a 37°C horizontal shaking incubator. Collect the absorbance (A) of the test system in the wavelength range of 350-750 nm every ten minutes using a UV-Vis absorption spectrometer. Determine the XNN concentration by observing the change in absorbance at the XNN absorption peak. The basic principle is Beer-Lambert law, based on the formula:

[0069] A = kbc

[0070] Where A represents absorbance, k is the molar absorptivity, c is the concentration of the absorbing substance, and b is the thickness of the absorbing layer. When a beam of parallel monochromatic light passes perpendicularly through a uniform, non-scattering absorbing substance, its absorbance A is directly proportional to the concentration c of the absorbing substance and the thickness b of the absorbing layer.

[0071] Test results are as follows Figure 5 The test system showed a small change in UV absorption over time, proving that the probe was not hydrolyzed by AChE.

[0072] Example 5

[0073] Cytotoxicity test

[0074] (1) Cell Culture

[0075] As is well known, nerve cells are non-regenerative, which is why it is essential to protect nerves from damage. Therefore, nerve cells are not cultured in scientific research. Highly differentiated rat adrenal pheochromocytoma cells (PC-12 high differentiation) are derived from transplantable male rat adrenal pheochromocytomas. These cells do not synthesize adrenaline but express nerve growth factor (NGF) receptors, which can induce a neural phenotype. Therefore, this cell line was chosen to represent nerve cells. The control cell line was mouse breast cancer cells (4T1).

[0076] Cell culture technology, also known as cell cloning technology, is a crucial step in the design and application of fluorescent probes, and has become one of the most fundamental and core technologies in fields such as biochemistry, medicine, and new drug development. Highly differentiated rat adrenal pheochromocytoma cells (PC-12 high differentiation) and mouse breast cancer cells (4T1) were sourced from cell culture facilities worldwide. Cells were cultured in RMPI 1640 medium, mixed with 10% fetal bovine serum (Invitrogen) to provide the necessary nutrients for cell growth, and 1% penicillin 100U / mL and streptomycin 0.1mg / mL (Hyclone) to maintain cell viability. The culture dishes were then placed in a cell culture incubator at 37°C with 5% CO2 until the cells adhered and reached the logarithmic growth phase before use.

[0077] (2) Cytotoxicity test

[0078] Cytotoxicity generally refers to cell-killing events caused by chemical substances. In practical applications, the cytotoxicity of fluorescent probes is an important indicator for evaluating their biological application prospects. A common method is the MTT assay. Its basic principle is that succinate dehydrogenase in the mitochondria of living cells can reduce thiazolyl blue (MTT) to form a purple crystalline substance called formazan, which is then deposited within the mitochondria. In dead cells, mitochondrial activity is inhibited, and the content of succinate dehydrogenase is reduced, resulting in a decreased ability to formazan. Therefore, with the same number of cells, the amount of formazan formed is directly proportional to cell activity.

[0079] The specific operating procedure is described as follows: 100 μL of PC-12 cell suspension was seeded into 96-well cell plates (1640 complete medium) at a density of 1×10⁵ / mL and placed in a cell culture incubator (37℃, 5% CO₂, constant temperature and humidity environment). After 12 h, when the cells adhered and grew to 70% of the wells, the medium was carefully removed and the cells were washed three times with PBS. Then, medium containing different concentrations (0, 1, 2, 4, 6, 10 μM) of XNN probe was added to each well. The cells were then placed in the cell culture incubator for another 12 h. The medium was removed and the cells were washed three times with PBS. 100 μL of freshly prepared MTT (5 mg / mL in 1640 medium) culture medium was added to each well. The cells were placed in the incubator again for 4 h. The MTT solution was then carefully removed and the blue-purple crystalline formazan was dissolved in 100 μL of dimethyl sulfoxide (DMSO). Finally, absorbance at 570 nm and 630 nm was measured using a Berthold Technologies multi-functional microplate reader. Cell viability was expressed as a percentage of control culture values, and the relative cell viability was calculated using formula 2.1 as follows:

[0080]

[0081] Wherein, ODdye570 and ODdye630 represent the absorbance values ​​of the dyed pores at 570 nm and 630 nm, respectively;

[0082] ODcontrol570 and ODcontro630 represent the absorbance values ​​at 570 nm and 630 nm for a hole with a dye concentration of 0, respectively.

[0083] from Figure 6 It can be seen that even with increased concentration, the dye can still coexist with more than 85% of cells, indicating that the probe has extremely low toxicity to cells and can be used as a molecular tool for detection in the biological field.

[0084] Example 6

[0085] Cellular uptake test

[0086] Passivable PC-12 and 4T1 cells were seeded in confocal microscopy dishes at approximately 3 × 10⁴ cells per dish, with 2 mL of 1640 complete culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) added. The cells were incubated at 37°C, 5% CO₂, and a constant temperature and humidity environment for at least 12 hours to allow for adhesion and morphological expansion. XNN (8 μM) was added to the culture dishes, and incubation continued for 6 hours. The fluorescence intensity of XNN in the cells was detected using LSCM at different time points to analyze the cell uptake of the compound. The excitation wavelength was 565 nm, and the detection wavelength was 650-700 nm. By studying the uptake of XNN by PC-12 and 4T1 cells, the aim was to determine whether binding to acetylcholine receptors affects the targeting effect of XNN and whether it can distinguish between neural, adipose, and muscle tissues. Results are as follows: Figure 7 , Figure 8 After the addition of XNN probe molecules, PC-12 cells showed intracellular fluorescence signals after 5 minutes. The intracellular fluorescence increased over time, reaching its peak intensity at 80 minutes, indicating that XNN could be taken up by PC-12 cells. In contrast, after the addition of XNN probe molecules, 4T1 cells showed accumulation of the probe's fluorescence signal outside the cells. This fluorescence signal remained outside the cells after 90 minutes, indicating that XNN could not enter the 4T1 cells and could not be taken up by them. This experiment strongly demonstrates the different uptake capabilities of XNN probe molecules in different cell types. This characteristic makes XNN a potential tool for distinguishing nerve cells from other cell types.

[0087] Several preferred embodiments and their corresponding details and principles have been described in detail in this disclosure. However, these contents are only for better illustrating the core concept and technical features of this invention and are not intended to limit the scope of protection of this invention. Under the premise of following the basic spirit and principles of this invention, any person skilled in the art can make various reasonable changes, adjustments and improvements according to the actual situation, such as, but not limited to, the selection of raw materials, optimization of manufacturing processes, adjustment of operating steps, changes in parameter ranges, and redesign of the overall structure or partial components without departing from the core idea of ​​this invention. Any equivalent substitutions, technical modifications, improvements and extensions made based on the disclosure of this invention, as long as they do not depart from the basic principles and essential characteristics of this invention, should be considered to fall within the scope of protection of this invention.

Claims

1. A neuron-specific dual-targeting fluorescent probe for cholinergic neurons AChR, the probe having a structure as shown in MNN: 。 2. The method for preparing the dual-targeting fluorescent probe as described in claim 1, comprising the following steps: 。 3. The application of the dual-targeting fluorescent probe as described in claim 1 in the preparation of reagents for neural imaging, cell imaging, protein labeling, and organelle labeling.

4. The application of the dual-targeting fluorescent probe as described in claim 1 in the preparation of reagents for specific tumor recognition and photodynamic therapy.

Images