N-phenylpiperazine fluorescent probe molecule as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent molecular materials for biological imaging, in particular to an N-phenylpiperazine fluorescent probe molecule as well as a preparation method and application thereof. The probe molecule is a fluorescent ligand receptor combined type fluorescent sensor, takes N-(2-methoxyphenyl) piperazine as a targeting group and 4-dimethylamino-1, 8-naphthalimide as a fluorescent reporter group, has specific recognition capability on a dopamine D3 receptor, and is excellent in fluorescence performance, low in cytotoxicity, fast in cell uptake (10s) and remarkable in fluorescence enhancement; the method can be used for cell imaging of mouse hippocampal nerve HT-22 cells, achieves the purpose of visualization, and has potential development value and good application prospects. The fluorescent probe is short in synthesis route, simple and easy to operate and suitable for industrial popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent molecular materials for biological imaging, and in particular to N-phenylpiperazine fluorescent probe molecules and a preparation method and application thereof. Background Art

[0002] Dopamine is one of the key neurotransmitters in the human brain that maintains physiological functions. Fluctuations in dopamine concentrations can cause serious neurological diseases (such as Parkinson's disease, schizophrenia, restless legs syndrome, and addictive behaviors). All dopamine receptors belong to the large G protein-coupled receptor (GPCR) family, which is divided into two subfamilies: D1-like (D1R, D5R) and D2-like (D2R, D3R, and D4R). Some dopamine D3R-specific compounds can reduce opioid, cocaine, nicotine, and alcohol-seeking behaviors, suggesting that dopamine D3R may be an effective target for antipsychotic drugs and neuroleptics. Dopamine itself has an affinity for D3R that is 100 times that of D2 receptors (D2R). Therefore, specific identification of dopamine D3R is particularly important.

[0003] Fluorescent probes, with their low detection limits, simple operation, low cost, and instantaneous response, have attracted increasing attention from researchers. Currently, fluorescent probes primarily include organic small molecules, nano-quantum dots, rare earth complexes, and proteins. Small molecule fluorescent probes, characterized by rapidity, sensitivity, high throughput, and ease of automation, have been widely used in the biological and pharmacological detection of important biomolecules such as proteins and nucleic acids, and are of great significance for the exploration of disease mechanisms, clinical diagnosis, and drug screening. Small molecule fluorescent probes generally consist of two components: a pharmacophore and a fluorophore. The pharmacophore binds to the target biomolecule with high affinity through receptor-ligand interactions, while the fluorophore stimulates fluorescence to label the protein. Therefore, the performance of small molecule fluorescent probes is closely related to the specificity of the pharmacophore and, on the one hand, the stability, safety, and penetrance of the fluorophore.

[0004] Over the past few decades, fluorescence imaging of dopamine receptors has been widely reported. Monsma et al. coupled fluorophores to dopaminergic ligands in 1989. The dopamine D1 receptor selective antagonist SCH-23390 and the D2 selective antagonist NAPS were the basic frameworks for coupling BODIPY, rhodamine, and fluorescein. Competition binding experiments identified SCH-23390-BODIPY VI (K i =4.0 nM) and NAPS-rhodamine (K i=3.79nM). In recent years, only a few fluorescent probes targeting dopamine receptors have been reported. Conformational benzamide analogs that specifically bind to dopamine D3Rs and dopamine D2Rs exhibit good affinity and selectivity, possessing high research and application value and attracting considerable attention. In 2016, Tabor et al. synthesized fluorescent ligands with N-phenylpiperazine substructures for single-molecule fluorescence imaging (TIRF) studies of dopamine D2 and D3 receptors in living cells. Fluorescently labeled agonists (2-[N-phenylethyl-propyl]-amino-5-hydroxytetraethylamine scaffolds) have been used for fluorescence resonance energy transfer (FRET) ligand binding studies at the dopamine D2 receptor level. However, reports of small molecule fluorescent probes that specifically bind to the dopamine D3 receptor, particularly those capable of distinguishing between the two receptors, are rare. Furthermore, the fluorescent reporters used in these reports suffer from limitations such as unstable optical properties, susceptibility to photobleaching, poor lipophilicity, and poor cell penetration.

[0005] Studies have shown that a ligand structure composed of a primary pharmacophore (PP, 4-phenylpiperazine), a secondary pharmacophore (SP, arylamide), and a butyl linker exhibits specific binding to the dopamine D3 receptor and can distinguish between dopamine D2 and D3 receptors. However, reports on small-molecule fluorescent probes based on this structure are limited, and their application is limited by complex molecular structures and lengthy synthetic routes. Summary of the Invention

[0006] In order to overcome the defects of the prior art, one of the objectives of the present invention is to provide an N-phenylpiperazine fluorescent probe molecule that has specific recognition ability for dopamine D3 receptors, has stable fluorescence properties, low cytotoxicity, and excellent cell penetration ability.

[0007] A second object of the present invention is to provide a method for preparing an N-phenylpiperazine fluorescent probe molecule.

[0008] A third object of the present invention is to provide an application of the fluorescent probe molecule of the present invention in rapid detection of dopamine D3 receptor reagents, particularly as an imaging reagent for detecting dopamine D3 receptors in living cells.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] An N-phenylpiperazine fluorescent probe molecule, characterized in that: its chemical structure is as shown in Formula I or Formula II:

[0011]

[0012] wherein n=1-3; and R is selected from a C1-C5 straight chain alkyl group.

[0013] Preferably, n = 1, and R = CH3. The specific embodiments of the present invention are illustrated using the fluorescent probe molecule represented by Formula II. It is understood that the present invention is not limited to the examples of the specific embodiments. At least the fluorescent probe molecule represented by Formula I is expected to have equivalent properties to the fluorescent probe molecule represented by Formula II and is within the scope of protection of the present invention.

[0014] The N-phenylpiperazine fluorescent probe molecules can be used to prepare dopamine D3R receptor detection reagents. Preferably, they can be used as imaging reagents for detecting dopamine D3R receptors in living cells.

[0015] Specifically, the probe molecule of the present invention is a fluorescent receptor ligand-binding probe that can target and recognize dopamine D3 receptors; it can be used for cell imaging of mouse hippocampal neural HT-22 cells to achieve visualization.

[0016] The preparation method of the above-mentioned N-phenylpiperazine fluorescent probe molecule comprises the following steps:

[0017] 1) Using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material to synthesize the intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine or (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine;

[0018] 2) The intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine or (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine and the fluorescent reporter group 4-dimethylamino-1,8-naphthalimide are heated to reflux in an organic solvent, and the fluorescent probe molecule is obtained after separation and purification.

[0019] Optionally, in step 2), the organic solvent is anhydrous ethanol, the heating temperature is 80° C., and the reaction time is 3-6 hours; the separation and purification method is column chromatography, using 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably, 300 mesh silica gel is used, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 40:1.

[0020] Optionally, the method further includes preparing a fluorescent reporter group 4-dimethylamino-1,8-naphthalimide. The specific method is: using 4-bromo-1,8-naphthalene dicarboxylic anhydride as a raw material, carrying out a substitution reaction with 3-dimethylaminopropionitrile in an isopentanol system, and separating and purifying to obtain 4-(N,N-dimethylamino)-1,8-naphthalene dicarboxylic anhydride and a fluorescent reporter group.

[0021] The reaction time is 30-36 hours; the column chromatography adopts 200-600 mesh column silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably 300 mesh column silica gel, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 20:1.

[0022] Optionally, the specific method for preparing the intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine in step 1) includes using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material, performing a substitution reaction with 4-bromobutyronitrile in a dichloromethane or tetrahydrofuran solvent system, and then performing a reduction reaction with lithium aluminum tetrahydride (LiAlH4), and isolating and purifying to obtain 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine;

[0023] The reaction time is 3-4 hours; the column chromatography uses 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably 300 mesh silica gel, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 20:1.

[0024] Optionally, the specific method for preparing the intermediate product (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine in step 1) includes: using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material and performing a substitution reaction with phthalimide in which the double bond of 1,4-dibromo-2-butene is protected, and performing a hydrazinolysis reaction with hydrazine hydrate to obtain (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine;

[0025] The solvent for the substitution reaction is selected from N,N-dimethylformamide, dichloromethane, and ultra-dry methanol; the reaction time is 1-4 hours; the column chromatography uses 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably, 300 mesh silica gel is used, and the eluent is a petroleum ether / ethyl acetate composite eluent with a volume ratio of 1:3.

[0026] This invention uses 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material to prepare 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine or (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine, which then reacts with a naphthalimide fluorescent reporter group to produce a fluorescent ligand-receptor binding fluorescent sensor. Through probe synthesis, spectral characterization, live cell imaging, cellular uptake, and Western blotting studies, a highly efficient probe for specific recognition of dopamine D3 receptors was developed.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. The fluorescent probe molecule of the present invention exhibits excellent fluorescence properties in dichloromethane, dimethyl sulfoxide, and dodecanol, with absorption wavelengths between 405 and 426 nm. The probe exhibits fluorescence excitation wavelengths of 474 nm and emission wavelengths of 535 nm, high fluorescence intensity, strong photochemical stability, and good optical stability. The working concentration of the probe is only 10 μmol / L.

[0029] 2. The probe molecule prepared by the present invention has specific recognition ability for dopamine D3 receptors, excellent fluorescence performance, low cytotoxicity, fast cellular uptake (10s), and significant fluorescence enhancement. It can be used for cell imaging of mouse hippocampal neural HT-22 cells to achieve the purpose of visualization, and has potential development value and good application prospects.

[0030] 3. The synthesis process of the N-phenylpiperazine fluorescent probe described in the present invention only requires two to three steps, the raw materials are cheap and easily available, the reaction conditions are mild, the post-treatment is simple and easy to operate, the molecular structure is simple, and the yield is high, reaching more than 80%, which is suitable for industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the molecular structure characterization results of compound 5 prepared in Example 2 of the present invention; Figure (a) is 1 HNMR spectrum; Figure (b) is 13 CNMR spectrum; Figure (c) is HRMS spectrum;

[0032] Figure 2 Schematic diagram of the molecular structure characterization results of the probe molecule 6 prepared in Example 3 of the present invention; Figure (a) is 1 HNMR spectrum; Figure (b) is 13 CNMR spectrum; Figure (c) is HRMS spectrum;

[0033] Figure 3 Schematic diagram of the molecular structure characterization results of the probe molecule 7 prepared in Example 3 of the present invention; Figure (a) is 1 HNMR spectrum; Figure (b) is 13 CNMR spectrum; Figure (c) is HRMS spectrum;

[0034] Figure 4 Spectral characteristics of the probe molecule 7 of the present invention. (a) Ultraviolet absorption spectrum of the probe molecule 7 of the present invention; (b) Fluorescence excitation spectrum of the probe molecule 7 of the present invention; (c) Fluorescence emission spectrum of the probe molecule 7 of the present invention;

[0035] Figure 5 The cytotoxicity experiments of probe molecules 6 and 7 of the present invention on HT-22 cells. (a) Probe molecule 6; (b) Probe molecule 7;

[0036] Figure 6Live cell imaging experiments of HT-22 cells using probe molecules 6 and 7 of the present invention. (a) Live cell imaging experiment of HT-22 cells using probe molecule 7; (b) Live cell imaging experiment of HT-22 cells using probe molecule 6;

[0037] Figure 7 This is the cell uptake experiment of the probe molecule 7 of the present invention on HT-22 cells;

[0038] Figure 8 This is a Western blotting experiment of the probe molecule 7 of the present invention on HT-22 cells; (a) Western blotting experiment of the probe molecule 7 on dopamine D2 receptor; (b) Western blotting experiment of the probe molecule 7 on dopamine D3 receptor. DETAILED DESCRIPTION

[0039] Below in conjunction with specific embodiment, the present invention is further described, but embodiment does not limit the present invention in any form.Unless otherwise stated, the reagent, method and equipment adopted in the present invention are conventional reagents, methods and equipment in the art.Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0040] Example 1

[0041] The preparation of the fluorescent reporter group, the chemical reaction equation is shown in the following formula 1:

[0042]

[0043] The specific preparation process is as follows: 4-bromo-1,8-naphthalene dicarboxylic anhydride is used as the raw material, and a substitution reaction with 3-dimethylaminopropionitrile in an isoamyl alcohol system is performed to obtain an orange solid crude product. Column chromatography is performed using 300-mesh silica gel and a 20:1 dichloromethane / methanol eluent to obtain orange-yellow crystals of 4-(N,N-dimethylamino)-1,8-naphthalene dicarboxylic anhydride and a fluorescent reporter group.

[0044] The preparation of compound 2, the chemical reaction equation is shown in Formula 2 below:

[0045]

[0046] The specific preparation process is as follows: 1-(2-methoxyphenyl)-piperazine hydrochloride is used as a raw material, and a substitution reaction is carried out with 4-bromobutyronitrile in a dichloromethane solvent system to obtain compound 1. Compound 1 is then reduced with lithium aluminum tetrahydride (LiAlH4) in a tetrahydrofuran solvent system at 0°C for 4 hours. The reaction product is separated and purified by column chromatography using 300 mesh silica gel and a dichloromethane / methanol composite eluent with a volume ratio of 20:1 to obtain compound 2 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine.

[0047] Example 2

[0048] The preparation of compound 5, the chemical reaction equation is shown in formula 3 below:

[0049]

[0050] The specific preparation process is as follows: 1-(2-methoxyphenyl)-piperazine hydrochloride is used as a raw material, and a substitution reaction is carried out with phthalimide in which the double bond of 1,4-dibromo-2-butene is protected to prepare compound 4; then, compound 4 is reacted with hydrazine hydrate in an ultra-dry methanol system for 4 hours, and the reaction product is separated and purified by column chromatography using 300 mesh silica gel and a petroleum ether / ethyl acetate composite eluent with a volume ratio of 1:3 to obtain compound 5(E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine.

[0051] like Figure 1 The characterization results showed that compound 5 was successfully prepared with a yield of 89%. 1 H NMR (400MHz, DMSO) δ8.09-8.04(m,1H),7.84-7.80(m,1H),6.95-6.90(m,2H),6.86(dd,J=1.6Hz,J=4H z,2H),5.72-5.55(m,2H),3.76(s,3H),3.20(d,J=6Hz,1H),2.94(d,J=6.4Hz,5H),2.51-2.49(m,4H).

[0052] 13 C NMR(101MHz,DMSO)δ156.30,151.97,141.27,132.63,131.66,128.57,127.47 ,125.36,122.35,120.83,117.89,111.87,59.82,55.30,52.81,50.05,42.18.

[0053] HR-MS (m / z) calculation for C 15 H 23N3O,[M+H + ]262.1914,found 262.1910.

[0054] The naphthalene imide fluorescent reporter group used in the following examples was prepared by the following method:

[0055] The fluorescent reporter group 4-dimethylamino-1,8-naphthalimide is prepared by the following method: using 4-bromo-1,8-naphthalene dicarboxylic anhydride as a raw material, carrying out a substitution reaction with 3-dimethylaminopropionitrile in an isoamyl alcohol system for 32 hours, adopting a 300-mesh silica gel column and a dichloromethane / methanol composite eluent with a volume ratio of 20:1 to separate and purify 4-(N,N-dimethylamino)-1,8-naphthalene dicarboxylic anhydride, a fluorescent reporter group.

[0056] Example 3

[0057] The chemical reaction equations for the preparation of probe molecules 6 and 7 are shown in Formula 4 below:

[0058]

[0059] The specific preparation process is:

[0060] Compound 2 or 5 was reacted with a naphthalene imide fluorescent reporter in anhydrous ethanol and refluxed at 80°C for 6 hours. The reaction was complete as monitored by TLC. After completion, the product was concentrated under reduced pressure to yield a yellow oil. This was then separated by column chromatography using dichloromethane / methanol (40 / 1 volume ratio) to yield a yellow solid.

[0061] like Figure 2 The characterization results shown indicate that probe molecule 6 was successfully prepared with a yield of 88%. 1 H NMR (400MHz, CDCl3) δ8.57(d,J=6.4Hz,1H),8.48-8.42(m,2H),7.66(t,J=7.2Hz,1H),7.12(d,J=8.4Hz,1H),7.00-6.97(m,1H),6.93-6.91(m, 2H), 6.85 (d, J=8Hz, 1H), 4.21 (t, J=8Hz, 2H), 3.86 (s, 3H), 3.11 (s, 9H) ,2.71-2.62(m,4H),2.54-2.48(m,2H),1.81-1.66(m,4H),1.25(s,1H).

[0062] 13C NMR (101MHz, CDCl3) δ164.67,164.13,157.02,152.26,141.03,132.71,131.27,131.07,130.27,125.28,124.93,1 24.89,123.13,121.03,118.36,114.92,113.34,111.53,58.25,55.41,53.57,50.14,44.84,39.85,26.15,23.84.

[0063] HR-MS (m / z) calculation for C 34 N4O3,[M+H + ]487.2704,found 487.2708.

[0064] like Figure 3 The characterization results shown indicate that probe molecule 7 was successfully prepared with a yield of 82%. 1 H NMR (400MHz, CDCl3) δ8.57(d,J=7.2Hz,1H),8.49-8.42(m,2H),7.65(t,J=7.2Hz,1H),7.11(d,J=8.2Hz,1H),7.00-6.96(m,1H),6.94-6.87(m,2 H),6.84(d,J=7.8Hz,1H),5.90-5.78(m,2H),4.80(d,J=4.2Hz,2H),3.8 4(s,3H),3.12(s,2H),3.11(s,6H),3.08(d,J=5.4Hz,4H),2.67(s,4H).

[0065] 13 C NMR (101MHz, CDCl3) δ164.41,163.85,157.13,152.26,141.22,132.85,131.39,131.21,130.37,128.83,128.7 5,125.31,124.95,123.00,121.01,118.29,114.87,113.36,111.13,60.29,55.38,53.15,50.37,44.86,41.25.

[0066] HR-MS (m / z) calculation for C 29 H 32 N4O3,[M+H + ]485.2548,found 485.2555.

[0067] Example 4 Spectral properties of probe molecules

[0068] Preparation of probe solution: Weigh a certain amount of the probe molecule synthesized by the present invention and configure it into a 1 mmol / L stock solution with dimethyl sulfoxide (DMSO). The probe stock solution is then diluted to a working concentration of 10 μmol / L in acetone (ACE), acetonitrile (ACN), dichloromethane (DCM), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol (ETOH), methanol (MEOH), ethyl acetate (EA), and dodecanol solution. The ultraviolet absorption spectrum, fluorescence excitation spectrum, and emission spectrum of the probe molecule in different solutions are measured by UV / visible spectrophotometer and fluorescence spectrophotometer.

[0069] The spectral characteristics of probe molecule 7 are shown in Figure 2. Figure 4 shown.

[0070] Example 5 Cytotoxicity experiment of probe molecules on HT-22 cells

[0071] Preparation of probe solution: Weigh a certain amount of the probe synthesized by the present invention and configure it into a 20 mmol / L stock solution with dimethyl sulfoxide (DMSO). Preparation of diphenyltetrazolium bromide (MTT) solution: Weigh 7.14 mg of MTT and configure it into a 2.12 mmol / L stock solution with 10 mL of Dulbecco's Modified Eagle Medium (DMEM) (store in the dark). Dilute the probe stock solution to a working concentration of 0, 5, 10, 20, 40, and 60 μmol / L in Dulbecco's Modified Eagle Medium (DMEM) (store in the dark). HT-22 cells were seeded in a 96-well plate and incubated for 24 hours. The probe was then added to DMEM and placed in a 96-well plate. After the probe was incubated at 37°C for 48 hours, the DMEM stock solution of MTT was placed in a 96-well plate. After incubation for 4 hours, DMSO was added and the absorbance of each well was measured at 490 nm using a microplate reader.

[0072] The results of the cytotoxicity experiments of probe molecules 6 and 7 on HT-22 cells are shown in Figure 2. Figure 5 shown.

[0073] Example 6 Live cell imaging experiment of HT-22 cells using probe molecules

[0074] Preparation of probe solution: Weigh a certain amount of the synthesized probe of the present invention and prepare a 20 mmol / L stock solution with dimethyl sulfoxide (DMSO). Dilute the probe stock solution to a working concentration of 5 μM in phosphate-buffered saline (PBS) (store in dark). HT-22 cells were seeded in a six-well plate and incubated for 24 hours. The probe was then added to PBS and placed in the six-well plate. The plate was incubated at 37°C for 60 minutes. Cell fluorescence images were captured without washing.

[0075] The results of live cell imaging experiments of HT-22 cells with probe molecules 6 and 7 are shown in Figure 2. Figure 6 shown.

[0076] Example 7 Cellular uptake experiment of the probe molecule of the present invention on HT-22 cells

[0077] Preparation of probe solution: Weigh a certain amount of the probe synthesized in the present invention and configure it into a 20 mmol / L stock solution with dimethyl sulfoxide (DMSO). Dilute the probe stock solution to a working concentration of 5 μM in PBS solution (store in the dark). HT-22 cells were seeded in a 24-well plate and incubated for 24 hours. The probe was then added to PBS and placed in a 24-well plate. The plate was incubated at 37°C for 0 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 120 seconds, 240 seconds, 480 seconds, 960 seconds, 32 minutes, 64 minutes, and 128 minutes, respectively. The PBS containing the probe was discarded and fresh PBS was added to capture cell fluorescence images.

[0078] The results of the cell uptake experiment of probe molecule 7 on HT-22 cells are shown in Figure 2. Figure 7 shown.

[0079] Example 8 Western Blot Experiment of the Probe Molecules of the Present Invention on HT-22 Cells

[0080] Preparation of probe solution: Weigh a certain amount of the probe synthesized according to the present invention and prepare a 20 mmol / L stock solution with dimethyl sulfoxide (DMSO). Dilute the probe stock solution to working concentrations of 0, 2, 4, and 8 μM in Dulbecco's Modified Eagle Medium (DMEM) (store in dark). HT-22 cells were seeded in six-well plates and incubated for 24 hours. DMEM containing different working concentrations of the probe was then added to the six-well plates and incubated at 37°C for 24 hours. Total protein was extracted and the total protein concentration was determined using the BCA assay. 60 μg of total protein from each sample was subjected to polyacrylamide gel (SDS-PAGE) electrophoresis. After electrophoresis, the membrane was transferred to the membrane and blocked with 5% milk for 1 hour. The membrane was incubated with the primary antibody (1:1000) (ABclonal) at 4°C overnight, washed three times with 1× TBST, incubated with the secondary antibody (1:3000) for 1 hour, washed three times with 1× TBST, and developed. The grayscale ratio of each target protein to the internal reference GAPDH was used as the relative expression level.

[0081] Probe molecule 7 has specific recognition ability for dopamine D3 receptors, such as Figure 8 shown.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An N-phenylpiperazine fluorescent probe molecule, characterized in that: Its general chemical structure is shown in Formula I or Formula II: wherein n=1-3; and R is selected from a C1-C5 straight chain alkyl group.

2. The N-phenylpiperazine fluorescent probe molecule according to claim 1, characterized in that: n=1,R=CH3。 3. Use of the N-phenylpiperazine fluorescent probe molecule according to any one of claims 1 to 2 in the preparation of a dopamine D3R receptor detection reagent.

4. The use according to claim 3, characterized in that The N-phenylpiperazine fluorescent probe molecule is used as an imaging reagent for detecting dopamine D3R receptors in living cells.

5. A method for preparing an N-phenylpiperazine fluorescent probe molecule as claimed in claim 2, characterized in that: The following steps are included: 1) Using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material to synthesize the intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine or (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine; 2) The intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine or (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine and the fluorescent reporter group 4-dimethylamino-1,8-naphthalimide are heated to reflux in an organic solvent, and the fluorescent probe molecule is obtained after separation and purification.

6. The method for preparing the N-phenylpiperazine fluorescent probe molecule according to claim 5, wherein: In step 2), the organic solvent is anhydrous ethanol, the heating temperature is 80° C., and the reaction time is 3-6 hours. The separation and purification method is column chromatography, using 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably, 300 mesh silica gel is used, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 40:

1.

7. The method for preparing the N-phenylpiperazine fluorescent probe molecule according to claim 6, wherein: It also includes the preparation of the fluorescent reporter group 4-dimethylamino-1,8-naphthalimide. The specific method is: using 4-bromo-1,8-naphthalene dicarboxylic anhydride as a raw material, carrying out a substitution reaction with 3-dimethylaminopropionitrile in an isopentanol system, and separating and purifying to obtain 4-(N,N-dimethylamino)-1,8-naphthalene dicarboxylic anhydride and a fluorescent reporter group. The reaction time is 30-36 hours; the column chromatography adopts 200-600 mesh column silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably 300 mesh column silica gel, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 20:

1.

8. The method for preparing the N-phenylpiperazine fluorescent probe molecule according to claim 7, wherein: The specific method for preparing the intermediate product 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine in step 1) comprises: using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material, performing a substitution reaction with 4-bromobutyronitrile in a dichloromethane or tetrahydrofuran solvent system, and then performing a reduction reaction with lithium aluminum tetrahydride (LiAlH4), and isolating and purifying to obtain 4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine; The reaction time is 3-4 hours; the column chromatography uses 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably 300 mesh silica gel, and the eluent is a dichloromethane / methanol composite eluent with a volume ratio of 20:

1.

9. The method for preparing the N-phenylpiperazine fluorescent probe molecule according to claim 8, wherein: The specific method for preparing the intermediate product (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine in step 1) comprises: using 1-(2-methoxyphenyl)-piperazine hydrochloride as a raw material to carry out a substitution reaction with phthalimide in which the double bond of 1,4-dibromo-2-butene is protected, and then carrying out a hydrazinolysis reaction with hydrazine hydrate to obtain (E)-4-(4-(2-methoxyphenyl)piperazin-1-yl)but-2-en-1-amine; The solvent for the substitution reaction is selected from N,N-dimethylformamide, dichloromethane, and ultra-dry methanol; the reaction time is 1-4 hours; the column chromatography uses 200-600 mesh silica gel, and the eluent is one or more of ethyl acetate, petroleum ether, dichloromethane, and methanol; preferably, 300 mesh silica gel is used, and the eluent is a petroleum ether / ethyl acetate composite eluent with a volume ratio of 1:3.

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