Synthetic method of < 19 > F magnetic resonance probe and application of < 19 > F magnetic resonance probe in beta-amyloid protein detection

Abstract

The invention provides a synthesis method of a < 19 > F magnetic resonance probe and application of the < 19 > F magnetic resonance probe in beta-amyloid protein detection, and belongs to the technical field of biological medicine. Three < 19 > F magnetic resonance probes, namely a probe AD-A beta-1, a probe AD-A beta-2 and a probe AD-A beta-3, are synthesized and have similar D-pi-A structures, dimethylamino serves as an electron donating group and has the capacity of recognizing and combining A beta fibers, and trifluoromethyl serves as an electron withdrawing group and can provide F signals to help < 19 > F MRI. In addition, compared with AD-Abeta-1, AD-Abeta-2 and AD-Abeta-3 have the advantages that sulfonic groups are added, so that the polarity of the probe is increased, and the hydrophilicity of the probe is improved. The AIE technology and the 19F MRI technology are combined, the defects that a fluorescent probe is low in penetrability and MRI sensitivity are low are overcome, and the sensitivity and spatial resolution of A beta deposition detection are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more particularly to a 19 synthesis method of an F magnetic resonance probe and its application in the detection of β-amyloid protein. Background Art

[0002] With the rapid expansion of the aging population, the prevalence of Alzheimer's disease (AD) in the elderly has been steadily increasing, and the incidence rate in young people is also rising. The most prominent neuropathological feature of Alzheimer's disease is the formation of β-amyloid (Aβ) plaques, which is caused by the aggregation of Aβ protein. The aggregation of Aβ is crucial for its toxicity, and its accumulation in the brain is a key step leading to neuronal death and the progression of Alzheimer's disease. In addition, Aβ aggregation can accelerate other pathological processes of AD, such as the formation of neurofibrillary tangles (NFTs), the overactivation of microglia, and oxidative stress (OS). Detecting Aβ aggregation in the brain at the early stage of Alzheimer's disease is crucial for diagnosing and monitoring the disease progression, understanding the complex disease process, and evaluating the efficacy of potential Alzheimer's disease treatment methods.

[0003] Optical imaging technology has the advantages of real-time, strong in-situ detection ability, low biotoxicity, high sensitivity, etc., and has broad application prospects in detecting Aβ deposition and diagnosing AD. Therefore, the current molecular probes for Aβ are mainly optical imaging probes [7]. Aggregation-induced emission (AIE) probes have been widely used in the field of bioimaging due to their excellent photostability and large Stokes shift [8]. AIE probes are ideal probes for detecting Aβ deposition because their fluorescence emission is related to aggregation. However, most of the reported fluorescent probes have been studied in small animals (such as mice and zebrafish). Their limited fluorescence penetration limits imaging in biological tissues, highly restricting their application in large animals (such as primates) and deep tissue imaging. Therefore, combining fluorescence and other imaging technologies to achieve synergistic multimodal imaging has become a current research hotspot.

[0004] Multimodal imaging probes, such as fluorescence / magnetic resonance imaging (MRI) and fluorescence / radionuclide imaging, represent promising advancements in imaging capabilities. These probes have become powerful tools for improving detection sensitivity, penetration depth, and accuracy, thereby providing more comprehensive and precise information for the diagnosis and treatment of diseases, which is of considerable significance in clinical practice. Fluorescence / 1H MRI is an imaging technique widely used in clinical and experimental settings, with many advantages such as non-ionizing radiation, high resolution, multi-planar and multi-parametric. The development of 19F MRI based on 1H MRI has enhanced the penetration ability of biological tissues [16,17]. Given that elemental fluorine mainly exists in teeth and bones in the human body, 19F MRI has significantly lower background signals compared to 1H MRI and can perform specific imaging of target substances. In addition, the 19F nucleus is characterized by a wide chemical shift (>350 ppm) and higher sensitivity to relaxation changes, thus providing higher resolution than 1H MRI. AIE fluorescence imaging combined with 19F MRI may be an effective strategy for the early diagnosis of AD. Summary of the Invention

[0005] The object of the present invention is to provide a 19 Synthesis method of 19F magnetic resonance probe and its application in the detection of β-amyloid protein to improve the sensitivity and spatial resolution of detecting Aβ deposition.

[0006] The present invention is realized as follows:

[0007] Provide a 19 Synthesis method of 19F magnetic resonance probe, and the synthesis method specifically includes the following steps:

[0008] 1) Synthesis of compound 1-1:

[0009] Add p-bromobenzyl cyanide, p-dimethylaminobenzaldehyde, potassium tert-butoxide, and ethanol into a reaction vessel respectively, react for 2 h, filter to collect the solid to obtain the crude product, wash the obtained crude product with anhydrous ethanol, and dry to obtain the yellow solid compound 1-1;

[0010] 2) Synthesis of probe AD-Aβ-1:

[0011] Add the above-obtained compound 1-1, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate into a reaction vessel, add toluene and water, under N2 protection, add tetrakis(triphenylphosphine)palladium into the above reaction system, heat to reflux, react for 16 h, cool and wash with water, extract the aqueous phase with ethyl acetate, combine the organic phases, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product of AD-Aβ-1, and purify the obtained crude product of AD-Aβ-1 by silica gel column chromatography to obtain the probe AD-Aβ-1.

[0012] Further, in step 1), the mass ratio of p-bromobenzyl cyanide, p-dimethylaminobenzaldehyde, and potassium tert-butoxide is 1.8:1.4:1; in step 2), the mass ratio of compound 1-1, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate is 1:1.2:1.1.

[0013] Provide a 19 Synthesis method of an F magnetic resonance probe, and the synthesis method specifically includes the following steps:

[0014] 1) Synthesis of compound 2-1:

[0015] Take the obtained compound 1-1, 2-hydroxy-4-trifluoromethylphenylboronic acid, and potassium carbonate and place them in a reaction vessel, add toluene and water, under N2 protection, add tetrakis(triphenylphosphine)palladium to the above reaction system, heat to reflux, react for 16 h, cool and wash with water, extract the aqueous phase with ethyl acetate, combine the organic phases, use saturated sodium chloride and anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product of compound 2-1, and purify the obtained crude product of compound 2-1 by silica gel column chromatography to obtain compound 2-1;

[0016] 2) Synthesis of probe AD-Aβ-2:

[0017] Take the obtained compound 2-1, 1,3-propanesultone, and sodium hydroxide and place them in a reaction vessel, add tetrahydrofuran, stir and react for 12 h, concentrate under reduced pressure to obtain a crude product of AD-Aβ-2, and purify the obtained crude product of AD-Aβ-2 by silica gel column chromatography to obtain probe AD-Aβ-2.

[0018] Further, in step 1), the mass ratio of compound 1-1, 2-hydroxy-4-trifluoromethylphenylboronic acid, and potassium carbonate is 2.7:1.5:1; in step 2), the mass ratio of compound 2-1, 1,3-propanesultone, and sodium hydroxide is 1.7:1.4:1.

[0019] Provide a 19 Synthesis method of an F magnetic resonance probe, characterized in that the synthesis method specifically includes the following steps:

[0020] 1) Synthesis of compound 3-1:

[0021] Take p-bromobenzyl cyanide, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate and add them to a reaction vessel respectively, and add toluene and water. Under N2 protection, add tetrakis(triphenylphosphine)palladium to the reaction system, heat to reflux at 90 °C, react for 16 h, cool and extract with ethyl acetate and water respectively, collect the organic phase, and dry it with anhydrous sodium sulfate. After the organic phase is dried, purify it by silica gel column chromatography to obtain compound 3-1;

[0022] 3) Synthesis of Compound 3-2:

[0023] Add 4-dimethylaminophenylboronic acid and 2-bromo-5-methoxybenzaldehyde into a reaction vessel, then add 1,2-dimethoxyethane, absolute ethanol and aqueous sodium carbonate solution. Under N2 protection, add tetrakis(triphenylphosphine)palladium into the reaction system, heat to reflux for 16 h, cool and wash with water. Extract the aqueous phase with ethyl acetate, combine the organic phases, dry with saturated sodium chloride and anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product of Compound 3-2. Purify the obtained crude product of Compound 3-2 by silica gel column chromatography to obtain Compound 3-2;

[0024] 3) Synthesis of Compound 3-3:

[0025] Add Compound 3-1, Compound 3-2 and potassium tert-butoxide into a reaction vessel, then add absolute ethanol, react for 12 h, filter and collect the solid, wash with absolute ethanol, and dry to obtain Compound 3-3;

[0026] 4) Synthesis of Compound 3-4:

[0027] Add Compound 3-3 into a reaction vessel, add dichloromethane to dissolve it completely, place the reaction vessel in an ice-water bath, dropwise add boron tribromide, react for 12 h, add absolute ethanol to quench the reaction system, wash the system with cold water, extract the aqueous phase with ethyl acetate, combine the organic phases, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product of Compound 3-4. Purify the obtained crude product of Compound 3-4 by silica gel column chromatography to obtain Compound 3-4;

[0028] 5) Synthesis of Probe AD-Aβ-3:

[0029] Add Compound 3-4, 1,3-propanesultone and sodium hydroxide into a reaction vessel, then add tetrahydrofuran, stir and react for 12 h, then concentrate under reduced pressure to obtain the crude product of Compound AD-Aβ-3. Purify the obtained crude product of AD-Aβ-3 by silica gel column chromatography to obtain Probe AD-Aβ-3.

[0030] Furthermore, the mass ratio of bromophenylacetonitrile, 3,5-bis(trifluoromethyl)phenylboronic acid and potassium carbonate in step 1) is 1.1∶1.6∶1; the mass ratio of 4-dimethylaminophenylboronic acid and 2-bromo-5-methoxybenzaldehyde in step 2) is 1∶1; the mass ratio of Compound 3-1, Compound 3-2 and potassium tert-butoxide in step 3) is 2.9∶2.4∶1; the mass ratio of Compound 3-4, 1,3-propanesultone and sodium hydroxide in step 5) is 2.3∶1.5∶1.

[0031] The present invention also provides one obtained according to any of the above synthesis methods 19Application of F Magnetic Resonance Probe in the Detection of β-Amyloid Protein

[0032] The present invention has the following beneficial effects:

[0033] (1) This patent designed and synthesized three probes, AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3, with AIE effect and 19 F MRI ability. The three probes have similar "D-π-A" structures, including: dimethylamino: as an electron-donating group and having the ability to recognize and bind to Aβ fibrils; trifluoromethyl: as an electron-withdrawing group and capable of providing F signals to assist in 19 FMRI. Compared with AD-Aβ-1, AD-Aβ-2 and AD-Aβ-3 have an additional sulfonic acid group, which increases the polarity of the probe and thus improves the hydrophilicity of the probe.

[0034] (2) This patent combines AIE and 19 F MRI technologies, making up for the deficiencies of low penetrability of fluorescence probes and low sensitivity of MRI, and improving the sensitivity and spatial resolution for detecting Aβ deposition. This patent synthesizes three multimodal fluorescence / 19 F MRI probes through a simple synthesis method, and uses fluorescence imaging and MRI dual imaging means to diagnose Aβ deposition, giving full play to the characteristics of multimodal imaging. Brief Description of the Drawings

[0035] Figure 1 is a schematic diagram of the synthesis route of probe AD-Aβ-1.

[0036] Figure 2 is a schematic diagram of the synthesis route of probe AD-Aβ-2.

[0037] Figure 3 is a schematic diagram of the synthesis route of probe AD-Aβ-3.

[0038] Figure 4 is the characterization result diagram of probe AD-Aβ-1 in Example 1.

[0039] Figure 5 is the characterization result diagram of probe AD-Aβ-2 in Example 1.

[0040] Figure 6 is the characterization result diagram of probe AD-Aβ-3 in Example 1.

[0041] Figure 7 is the fluorescence emission spectrum diagram of the three probes in common solvents in Example 2.

[0042] Figure 8 is the ultraviolet absorption spectrum and fluorescence emission spectrum diagram of the three probes in good solvents in Example 2.

[0043] Figure 9 It is a test diagram of the AIE effect of the three probes in the "poor solvent - good solvent" mixed solution in Example 3.

[0044] Figure 10 It is a test diagram of the stability of the three probes under different pH conditions in Example 4.

[0045] Figure 11 It is the nuclear magnetic resonance fluorine spectrum of the three probes in Example 5 and the 19 F magnetic resonance imaging map in the mixed solvent.

[0046] Figure 12 It is a test result diagram of the specific recognition of Aβ fibers by the three probes in Example 6.

[0047] Figure 13 It is a fluorescence response result diagram of the three probes to different concentrations of Aβ fibers in Example 6.

[0048] Figure 14 It is a test result diagram of the change in fluorescence intensity of the three probes within 60 minutes in Example 6.

[0049] Figure 15 It is a test result diagram of the cytotoxicity of the three probes in Example 7. Detailed implementation manners

[0050] The present invention will be further described below in combination with comparative examples and examples. The following implementation manners are only for illustration and do not limit the protection scope of the present invention in any form.

[0051] Example 1 Synthesis of three probes

[0052] (1) Synthesis of probe AD - Aβ - 1

[0053] The schematic synthesis route of AD - Aβ - 1 is as Figure 1 shown.

[0054] Synthesis of compound 1 - 1: Respectively add p - bromobenzyl cyanide (2.00 g, 10.20 mmol), p - dimethylaminobenzaldehyde (1.60 g, 10.72 mmol), potassium tert - butoxide (1.12 g, 10.20 mmol) and ethanol (50 mL) into a 100 mL round - bottom flask, and react at room temperature for 2 h. After the reaction is completed, filter to collect the solid to obtain the crude product, and wash the crude product with anhydrous ethanol 3 times and dry it at room temperature to obtain the yellow solid compound 1 - 1.

[0055] Synthesis of compound AD-Aβ-1: Add compound 1-1 (500 mg, 1.53 mmol), 3,5-bis(trifluoromethyl)phenylboronic acid (590 mg, 2.30 mmol) and potassium carbonate (528.70 mg, 3.83 mmol) into a 100 mL round-bottom flask, and then add toluene (20 mL) and water (6 mL). Under N2 protection, add tetrakis(triphenylphosphine)palladium (176.80 mg, 0.15 mmol) into the above reaction system. Heat the reaction system to reflux for 16 h. After the system cools to room temperature, wash it with water, and then extract the aqueous phase with ethyl acetate (20 mL) three times. Combine the organic phases, wash them with saturated sodium chloride (NaCl), and dry them with anhydrous sodium sulfate (Na2SO4). After filtration, concentrate the solution under reduced pressure to obtain the crude product of AD-Aβ-1. Purify the crude product of AD-Aβ-1 by silica gel column chromatography (eluent V 二氯甲烷 :V 石油醚 = 2:1), collect the corresponding fractions to obtain the target product AD-Aβ-1.

[0056] (2) Synthesis of probe AD-Aβ-2

[0057] The schematic diagram of the synthesis route of AD-Aβ-2 is as Figure 2 shown.

[0058] Synthesis of compound 2-1: Take compound 1-1 (600 mg, 1.52 mmol), 2-hydroxy-4-trifluoromethylphenylboronic acid (329 mg, 1.60 mmol) and K2CO3 (221 mg, 1.60 mmol) and place them in a 100 mL round-bottom flask. Add toluene (25 mL) and water (9 mL) to the flask. Under N2 protection, add tetrakis(triphenylphosphine)palladium (175 mg, 0.18 mmol) into the above reaction system, heat the system to reflux, and react for 16 h. After the system cools to room temperature. Wash it with water, and then extract the aqueous phase with ethyl acetate (20 mL) three times. Combine the organic phases, wash them with saturated NaCl, and dry them with anhydrous Na2SO4. After filtration, concentrate the solution under reduced pressure to obtain the crude product of compound 2-1. Purify the crude product of compound 2-1 by silica gel column chromatography (eluent V 石油醚 :V 乙酸乙酯 = 4:1), collect the corresponding fractions to obtain compound 2-1.

[0059] Synthesis of compound AD-Aβ-2: Take compound 2-1 (100 mg, 0.24 mmol), 1,3-propanesultone (80 mg, 0.72 mmol) and NaOH (57.60 mg, 1.44 mmol) and place them in a 50 mL round-bottom flask, add THF (20 mL), and stir at room temperature for 12 h. After the reaction is completed, concentrate the solution under reduced pressure to obtain the crude product of AD-Aβ-2. Purify the crude product of AD-Aβ-2 by silica gel column chromatography (eluent V 二氯甲烷:V 甲醇 = 8:1), and the target product AD-Aβ-2 was obtained by collection.

[0060] (3) Synthesis of probe AD-Aβ-3

[0061] The schematic diagram of the synthesis route of AD-Aβ-3 is as Figure 3 shown.

[0062] Synthesis of compound 3-1: Respectively take p-bromobenzyl cyanide (1 g, 5.10 mmol), 3,5-bis(trifluoromethyl)phenylboronic acid (1.38 g, 5.36 mmol), and potassium carbonate (0.88 g, 6.38 mmol) and add them to a 100 mL round-bottom flask. Then add 30 mL of toluene and 9 mL of water. Under the protection of N2, add tetrakis(triphenylphosphine)palladium (0.59 g, 0.51 mmol) to the reaction system, and heat under reflux at 90 °C for 16 h. After the reaction is completed, cool the system to room temperature, extract 3 - 4 times with ethyl acetate and water respectively, collect the organic phase, dry it with anhydrous sodium sulfate, then rotary evaporate the organic phase to dryness, and purify it by silica gel column chromatography (eluent V 二氯甲烷 :V 石油醚 = 1:1), and collect the fractions to obtain white solid 3-1.

[0063] Synthesis of compound 3-2: Add 4-dimethylaminophenylboronic acid (1 g, 6.12 mmol) and 2-bromo-5-methoxybenzaldehyde (1.01 g, 4.66 mmol) to a 100 mL round-bottom flask, and then add 1,2-dimethoxyethane (40 mL), absolute ethanol (4 mL), and aqueous sodium carbonate solution (2 M, 4 mL). Under the protection of N2, add tetrakis(triphenylphosphine)palladium (0.16 g, 0.14 mmol) to the reaction system, heat to reflux, and react for 16 h. Wait for the system to cool to room temperature. After washing with water, the aqueous phase is extracted with ethyl acetate (20 mL × 3 times). Combine the organic phases, wash with saturated NaCl, and dry with anhydrous Na2SO4. After filtration, concentrate under reduced pressure to obtain the crude product of compound 3-2. The crude product of compound 3-2 is purified by silica gel column chromatography (eluent V CH2Cl2 :V 石油醚 = 3:2), and collect the fractions to obtain the target product compound 3-2.

[0064] Synthesis of compound 3-3: Add compound 3-1 (1.25 g, 3.80 mmol), compound 3-2 (1.02 g, 4.04 mmol), and potassium tert-butoxide (425 mg, 3.80 mmol) to a 250 mL round-bottom flask, then add absolute ethanol (40 mL), and react at room temperature for 12 h. Filter and collect the solid, wash it with absolute ethanol, and dry to obtain compound 3-3.

[0065] Synthesis of Compound 3-4: Compound 3-3 (1.89 g, 3.33 mmol) was added to a 250 ml round-bottom flask, and CH2Cl2 (120 mL) was added to dissolve it completely. The round-bottom flask was placed in an ice-water bath, and boron tribromide (10 mL, 104 mmol) was added dropwise, and the reaction was carried out for 12 h. Anhydrous ethanol (10 mL) was added to quench the reaction system, and the system was cooled to room temperature. After washing with water, the aqueous phase was extracted 3 times with ethyl acetate (20 mL). The organic phases were combined, washed with saturated NaCl, and dried over anhydrous Na2SO4. After filtration, the mixture was concentrated under reduced pressure to obtain the crude product of compound 3-4. The crude product of compound 3-4 was separated and purified by silica gel column chromatography (eluent V CH2Cl2 :V 石油醚 = 2:1), and the corresponding fractions were collected to obtain compound 3-4.

[0066] Synthesis of Compound AD-Aβ-3: Compound 3-4 (1.67 g, 3.08 mmol), 1,3-propane sultone (1.13 g, 9.24 mmol) and NaOH (0.74 g, 18.48 mmol) were added to a 250 mL round-bottom flask, and then THF (100 mL) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the crude product of compound AD-Aβ-3. The crude product of AD-Aβ-3 was separated and purified by silica gel column chromatography (eluent V 二氯甲烷 :V 甲醇 = 6:1), and the corresponding fractions were collected to obtain the target product AD-Aβ-3.

[0067] The three probes were characterized by nuclear magnetic resonance spectrometer (NMR), infrared spectrometer (IR), gas chromatography-mass spectrometry (GC-MS) and other methods. The results are shown in Figures 4 to 6 . Among them Figure 4 A, 4B, and 4C are the 1H NMR, 13C NMR, IR spectrum and mass spectrum of probe AD-Aβ-1, respectively; Figure 5 A, 5B, and 5C are the 1H NMR, 13C NMR, IR spectrum and mass spectrum of probe AD-Aβ-2, respectively; Figure 6 A, 6B, and 6C are the 1H NMR, 13C NMR, IR spectrum and mass spectrum of probe AD-Aβ-3, respectively.

[0068] Example 2 Screening of Good Solvents for Three Probes and Testing of Their Absorption and Emission Wavelengths

[0069] The three probes were respectively added to common solvents such as N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and THF, and their ultraviolet absorption spectra and fluorescence emission spectra (n = 3) were detected. The test results are shown in Figure 7 .Figure 8 Among them Figure 7 A, 4B, and 4C are the fluorescence emission spectra of probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3 in common solvents, respectively; Figure 8 A is the absorption spectrum and emission spectrum of probe AD-Aβ-1 in THF; Figure 8 B is the UV absorption spectrum and fluorescence emission spectrum of probe AD-Aβ-2 in DMSO; Figure 8 C is the UV absorption spectrum and fluorescence emission spectrum of probe AD-Aβ-3 in DMSO.

[0070] The test results show that the good solvent for probe AD-Aβ-1 is THF, and its absorption spectrum and emission spectrum in THF are shown in Figure 8 A; the good solvent for probe AD-Aβ-2 is DMSO, and its absorption spectrum and emission spectrum in DMSO are shown in Figure 8 B; the good solvent for probe AD-Aβ-3 is DMSO, and its absorption spectrum and emission spectrum in DMSO are shown in Figure 8 C.

[0071] Example 3: Test of the AIE effect of three probes

[0072] The AIE effect refers to the phenomenon that molecules emit stronger fluorescence in the aggregated state. The good solvent and poor solvent of the probe were selected respectively, and mixed solutions of "poor solvent - good solvent" with the volume fraction of the poor solution being 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% were prepared, with a volume of 5 mL. The mother liquor of the three probes was added to the mixed solution of "poor solvent - good solvent", and its UV absorption spectrum and fluorescence emission spectrum were detected (n = 3). The test results are shown in Figure 9 Among them, Figure 9 A, 9B, and 9C correspond to the AIE effects of probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3, respectively.

[0073] The test results show that the higher the content of the poor solvent in the mixed solution, the stronger the fluorescence intensity, indicating that the probe has the AIE effect.

[0074] Example 4: Stability test of three probes under different pH conditions

[0075] Take 4 mg of sodium hydroxide (NaOH), add it to 1 L of distilled water to prepare an aqueous solution with a pH value of 11. Concentrated hydrochloric acid was added dropwise to the above solution to prepare aqueous solutions with pH values of 10, 9, 8, 7, 6, 5, 4, and 3. The three probes were added to the aqueous solutions with different pH values, and their fluorescence emission spectra were detected (n = 3). The test results are shown in Figure 10Among them, 10A, 10B, and 10C respectively correspond to the stability results of probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3 under different pH conditions. The test results show that the three probes have good stability in the range of pH 5-10.

[0076] Example 5 of the three probes 19 F MRI test

[0077] Respectively add the three probes into the mixed solution of "poor solvent - good solvent", and use a Bruker 9.4T small animal nuclear magnetic resonance imaging instrument to perform imaging detection on the solution system. The results are shown in Figure 11 . Among them Figure 11 A, 11B, and 11C respectively correspond to the nuclear magnetic resonance fluorine spectra of probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3; 11D, 11E, and 11F respectively correspond to the 19 F magnetic resonance imaging maps of probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3 in the mixed solvent.

[0078] The test results show that the three probes have the ability of fluorine imaging in the good solvent, and at the same time, AD-Aβ-2 can also perform fluorine imaging under the condition that the proportion of the poor solvent (H2O) is 20%.

[0079] Example 6 Specific recognition ability and stability test of three probes

[0080] Incubate Aβ fibers:

[0081] Weigh 1 mg of Aβ 42 (0.22×10 -6 mol) is dissolved in 220 μL of hexafluoroisopropanol (HFIP) to prepare a 1 mM Aβ 42 solution. Let it stand at room temperature for 60 min and at 4 °C for 10 min, then place the solution in a vacuum drying oven and remove the HIFP solution under negative pressure. Add 2200 μL of phosphate buffer saline (PBS) to prepare a 100 μM Aβ 42 monomer solution. Transfer the Aβ 42 monomer solution to a chromatographic injection vial, place it in a thermostatic mixer, and incubate for 7 days (37 °C, 250 rpm) to obtain Aβ 42 fibers.

[0082] Thioflavine T (ThT) can specifically bind to the β-sheet structure of Aβ fibers and produce a new emission peak near the wavelength of 482 nm. Therefore, use ThT to detect Aβ 42Structural state. Dissolve 1.3 mg of ThT in 100 mL of PBS (c = 40 μM), and add 1 μL of the ThT solution to 100 μL of the unincubated Aβ 42 monomer solution and 100 μL of the incubated Aβ 42 solution. The test concentration of ThT is 0.4 μM, and its fluorescence emission spectrum is detected (n = 3).

[0083] Test for probe specific recognition ability:

[0084] Weigh 400 mg of horseradish peroxidase (HRP), 120 mg of cytochrome C (Cyto C), 1.49 mg of L-methionine (Met), 1.21 mg of L-cysteine (Cys), 3.07 mg of glutathione (GSH), 1.31 mg of isoleucine (IIe), 1.15 mg of proline (Pro), 1.81 mg of tyrosine (Tyr), 1.65 mg of phenylalanine (phe), 1.31 mg of leucine (Leu), 1.47 mg of glutamic acid (Glu), 1.80 mg of glucose (Glc), 1.80 mg of galactose (Gal), and 1.80 mg of mannose (Man) and dissolve them in 2 mL of PBS (c = 5 mM). Take the above substances (enzymes, amino acids, and sugars), Aβ monomer (Aβm), and Aβ fiber (Aβf) solutions respectively, add the solutions of the three probes, and detect their fluorescence emission spectra (n = 3). The test results are shown in Figure 12 . Among them Figure 12 A, 12B, and 12C respectively correspond to the specific recognition results of the probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3 for the above substances. The test results show that the three probes have specific recognition ability for Aβ fibers.

[0085] Fluorescence responses of the three probes to Aβ fibers at different concentrations:

[0086] Prepare Aβ fiber solutions with different concentrations respectively, then add the PBS solutions of the three probes, and detect their fluorescence emission spectra (n = 3). The test results are shown in Figure 13 . Among them Figure 13 A, 13B, and 13C respectively correspond to the fluorescence response results of the probes AD-Aβ-1, AD-Aβ-2, and AD-Aβ-3 to Aβ fibers at different concentrations.

[0087] As Figure 13 shown in A, within the concentration range of 0 - 50 μM of Aβ fibers, the fluorescence of the probe AD-Aβ-1 increases with the increase of the Aβ fiber concentration; as Figure 13 shown in B, within the concentration range of 0 - 25 μM of Aβ fibers, the fluorescence of the probe AD-Aβ-2 increases with the increase of the Aβ fiber concentration; as Figure 13As shown in C, within the concentration range of 0 - 30 μM of Aβ fibers, the fluorescence intensity of the probe AD - Aβ - 3 increased with the increase in the concentration of Aβ fibers.

[0088] Stability test:

[0089] The fluorescence intensity changes of the three probes were measured within 60 min, and the results are shown in Figure 14 . Among them, 14A, 14B, and 14C are the fluorescence intensity ratios of the probes AD - Aβ - 1, AD - Aβ - 2, and AD - Aβ - 3 within 60 min, respectively. The results show that the fluorescence intensities of the three probes are relatively stable within 60 minutes.

[0090] Example 7 Cytotoxicity test of three probes

[0091] The experiments were set up with a background color group, a blank group, a control group, and an experimental group.

[0092] Background color group: Only add basal medium; Blank group: Add cells and basal medium; Control group: Add cells, basal medium, and 0.5% (v / v) DMSO; Experimental group: Add cells, medium with different concentrations of the probe, and 0.5% (v / v) DMSO. SH - SY5Y neurocytes were seeded in a 96 - well plate (seeding density: 5×10 3 ~10×10 3 cells / well, 100 μL of basal medium), and placed in an incubator (37 °C, 5% CO2) for 24 h. Discard the liquid in the wells, add the medium containing the probe, with 5 replicates for each concentration, and culture for 24 h. Under light - protected conditions, add 10 μL of MTT working solution to each well and culture for 4 h. Under light - protected conditions, discard the liquid in the wells, and add 150 μL of DMSO to each well. Oscillate for 10 min to completely dissolve the crystals, and measure the absorbance (Optical density, OD) of each well at 570 nm using an enzyme - linked immunosorbent assay (ELISA) reader. The test results are shown in Figure 15 . Among them, Figure 15 A, 15B, and 15C correspond to the cytotoxicity results of the probes AD - Aβ - 1, AD - Aβ - 2, and AD - Aβ - 3, respectively. The results show that the three probes have no cytotoxicity to SH - SY5Y neurocytes.

Claims

1. A 19 synthesis method of an F magnetic resonance probe, characterized in that The synthesis method specifically includes the following steps: 1) Synthesis of compound 1-1: p-Bromophenylacetonitrile, p-dimethylaminobenzaldehyde, potassium tert-butoxide and ethanol were respectively added to a reaction vessel, and the reaction was carried out for 2 h. The solid was collected by filtration to obtain the crude product, and the obtained crude product was washed with absolute ethanol and dried to obtain the yellow solid compound 1-1; 2) Synthesis of probe AD-Aβ-1: The obtained compound 1-1, 3,5-bis(trifluoromethyl)phenylboronic acid and potassium carbonate were added to a reaction vessel, toluene and water were added. Under the protection of N2, tetrakis(triphenylphosphine)palladium was added to the above reaction system, the temperature was raised to reflux, and the reaction was carried out for 16 h. After cooling, it was washed with water, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product of AD-Aβ-1. The obtained crude product of AD-Aβ-1 was separated and purified by silica gel column to obtain the probe AD-Aβ-1.

2. According to claim 1 19 A method for synthesizing an F magnetic resonance probe, characterized in that In step 1), the mass ratio of p-bromophenylacetonitrile, p-dimethylaminobenzaldehyde, and potassium tert-butoxide is 1.8∶1.4∶1; in step 2), the mass ratio of compound 1-1, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate is 1∶1.2∶1.

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3. A 19 synthesis method of an F magnetic resonance probe, characterized in that The synthesis method specifically includes the following steps: 1) Synthesis of compound 2-1: The obtained compound 1-1, 2-hydroxy-4-trifluoromethylphenylboronic acid and potassium carbonate were placed in a reaction vessel, toluene and water were added. Under the protection of N2, tetrakis(triphenylphosphine)palladium was added to the above reaction system, the temperature was raised to reflux, and the reaction was carried out for 16 h. After cooling, it was washed with water, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride and anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product of compound 2-1. The obtained crude product of compound 2-1 was separated and purified by silica gel column to obtain compound 2-1; 2) Synthesis of probe AD-Aβ-2: The obtained compound 2-1, 1,3-propanesultone and sodium hydroxide were placed in a reaction vessel, tetrahydrofuran was added, and the reaction was stirred for 12 h. It was concentrated under reduced pressure to obtain the crude product of AD-Aβ-2. The obtained crude product of AD-Aβ-2 was separated and purified by silica gel column to obtain the probe AD-Aβ-2.

4. According to claim 3 19 A method for synthesizing an F magnetic resonance probe, characterized in that In step 1), the mass ratio of compound 1-1, 2-hydroxy-4-trifluoromethylphenylboronic acid, and potassium carbonate is 2.7∶1.5∶1; in step 2), the mass ratio of compound 2-1, 1,3-propanesultone, and sodium hydroxide is 1.7∶1.4∶1.

5. A 19 synthesis method of an F magnetic resonance probe, characterized in that The synthesis method specifically includes the following steps: 1) Synthesis of compound 3-1: p-Bromophenylacetonitrile, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate were respectively taken and added to a reaction vessel, toluene and water were added. Under the protection of N2, tetrakis(triphenylphosphine)palladium was added to the reaction system, and it was heated to reflux at 90 °C for 16 h. After cooling, it was extracted with ethyl acetate and water respectively, the organic phase was collected, and dried over anhydrous sodium sulfate. After the organic phase was dried, it was separated and purified by silica gel column to obtain compound 3-1; 3) Synthesis of compound 3-2: 4-Dimethylaminophenylboronic acid and 2-bromo-5-methoxybenzaldehyde were added to a reaction vessel, followed by the addition of 1,2-dimethoxyethane, absolute ethanol, and an aqueous sodium carbonate solution. Under N2 protection, tetrakis(triphenylphosphine)palladium was added to the reaction system. The temperature was raised to reflux for 16 h. After cooling and washing with water, the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over saturated sodium chloride and anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product of compound 3-2. The obtained crude product of compound 3-2 was separated and purified by silica gel column chromatography to obtain compound 3-2; 3) Synthesis of compound 3-3: Compound 3-1, compound 3-2, and potassium tert-butoxide were added to a reaction vessel, and then absolute ethanol was added. The reaction was carried out for 12 h. The solid was collected by filtration, washed with absolute ethanol, and dried to obtain compound 3-3; 4) Synthesis of compound 3-4: Compound 3-3 was added to a reaction vessel and dissolved completely in dichloromethane. The reaction vessel was placed in an ice-water bath, and boron tribromide was added dropwise. The reaction was carried out for 12 h. Absolute ethanol was added to quench the reaction system. After washing with cold water, the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product of compound 3-4. The obtained crude product of compound 3-4 was separated and purified by silica gel column chromatography to obtain compound 3-4; 5) Synthesis of probe AD-Aβ-3: Compound 3-4, 1,3-propanesultone, and sodium hydroxide were added to a reaction vessel, and then tetrahydrofuran was added. After stirring for 12 h, the mixture was concentrated under reduced pressure to obtain the crude product of compound AD-Aβ-3. The obtained crude product of AD-Aβ-3 was separated and purified by silica gel column chromatography to obtain probe AD-Aβ-3.

6. According to claim 5 19 A method for synthesizing an F magnetic resonance probe, characterized in that In step 1), the mass ratio of bromobenzyl cyanide, 3,5-bis(trifluoromethyl)phenylboronic acid, and potassium carbonate is 1.1∶1.6∶1; in step 2), the mass ratio of 4-dimethylaminophenylboronic acid and 2-bromo-5-methoxybenzaldehyde is 1∶1; in step 3), the mass ratio of compound 3-1, compound 3-2, and potassium tert-butoxide is 2.9∶2.4∶1; in step 5), the mass ratio of compound 3-4, 1,3-propanesultone, and sodium hydroxide is 2.3∶1.5∶1.

7. Use of the F magnetic resonance probe obtained by the synthesis method according to any one of claims 1-6 19 in the detection of β-amyloid protein.

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