Chemiluminescent probe, preparation method thereof and application thereof in detection of beta amyloid

Abstract

The application discloses a chemiluminescent probe, a preparation method thereof and application of the chemiluminescent probe in detection of beta amyloid. The structural formula of the chemiluminescent probe is shown as formula (I). The chemiluminescent probe prepared in the application has a long chemiluminescent wavelength, excellent beta amyloid binding capacity and blood-brain barrier crossing capacity, and can be used for detecting the content of beta amyloid and early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to a chemiluminescent probe, its preparation method, and its application, particularly to a chemiluminescent probe, its preparation method, and its application in the detection of β-amyloid protein, belonging to the field of biomedicine. Background Technology

[0002] Alzheimer's disease (AD) is an insidious neurodegenerative disease clinically characterized by comprehensive dementia manifestations, including memory impairment, aphasia, apraxia, agnosia, and personality and behavioral changes, severely impacting human health and quality of life. Unfortunately, there is currently no effective treatment to halt or reverse the progression of AD, making early diagnosis of AD particularly important. The exact cause of AD remains unclear. Among the many hypotheses regarding its etiology, the β-amyloid beta (Aβ) cascade hypothesis is widely accepted: amyloid precursor protein (APP) is enzymatically cleaved to form Aβ monomers, which aggregate to form plaques, interfering with synaptic transmission of neuronal signals and ultimately leading to neuronal cell death. Numerous studies have shown that Aβ protein deposition begins 10-20 years before the onset of symptoms in AD patients; therefore, sensitive detection of Aβ holds promise for early diagnosis of AD.

[0003] Optical probes, with their high spatiotemporal resolution, have been widely used in in vitro and in vivo detection of Aβ. However, most current optical probes are fluorescent probes, which require high-energy excitation light, resulting in a low signal-to-noise ratio in imaging. Chemiluminescence, on the other hand, generates light based on chemical reactions and does not require an external light source, effectively reducing background signals from the excitation light and thus offering higher detection sensitivity. However, most reported chemiluminescent probes for Aβ detection have relatively short emission wavelengths and poor ability to penetrate the blood-brain barrier. Summary of the Invention

[0004] Objectives of the invention: The objective of this invention is to provide a chemiluminescent probe; another objective of this invention is to provide a method for preparing a chemiluminescent probe; yet another objective of this invention is to provide an application of the chemiluminescent probe in the detection of β-amyloid protein.

[0005] Technical solution: A chemiluminescent probe of the present invention has the structural formula shown in (I):

[0006]

[0007] (I)

[0008] Where R is , , or .

[0009] Preferably, the chemiluminescent probe has any of the following chemical structures:

[0010]

[0011] On the other hand, the present invention provides a method for preparing the above-mentioned chemiluminescent probe, comprising the following steps:

[0012]

[0013] (1) Compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate, and 18-crown ether-6 were added to an organic solvent. After the reaction was completed, the mixture was extracted and concentrated to obtain a crude product. The crude product and hydrochloric acid were added to tetrahydrofuran. After the reaction was completed, the mixture was separated and purified to obtain compound 2.

[0014] (2) In the presence of nitrogen as a protective gas, n-butyllithium and 2,2,6,6-tetramethylpiperidine were added to an organic solvent, followed by bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane and compound 2. After the reaction was completed, compound 3 was obtained by separation and purification.

[0015] (3) Compound 3, 5-bromo-2-aminopyrazine and tetratriphenylphosphine palladium were added to 1,4-dioxane containing a saturated sodium carbonate solution. After the reaction was completed, the compound 4 was obtained by separation and purification.

[0016] (4) Compound 4, methylglyoxal-1,1-dimethylacetal, 6M hydrochloric acid, and water were added to an organic solvent. After the reaction was completed, the chemiluminescent probe was obtained by allowing it to stand and precipitate.

[0017] Preferably, in step (1), the molar ratio of compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate and 18-crown ether-6 is 1:3:4:0.01 to 0.05.

[0018] Preferably, in step (1), the organic solvent is anhydrous toluene.

[0019] Preferably, in step (1), the amount of anhydrous toluene used is calculated as 5 mL of anhydrous toluene per millimole of 4-diethylaminobenzaldehyde.

[0020] In step (1), the amount of hydrochloric acid solution used is calculated based on 0.5 mL of hydrochloric acid per millimole of 4-diethylaminobenzaldehyde.

[0021] Preferably, in step (1), the protective gas is nitrogen or argon.

[0022] Preferably, in step (1), the reaction time is 10 to 12 hours.

[0023] Preferably, in step (1), the extraction is performed using dichloromethane.

[0024] Preferably, in step (1), the separation and purification are performed by silica gel column chromatography.

[0025] The silica gel column chromatography is performed in a 100-200 mesh silica gel column; the silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate as the eluent; wherein the volume ratio of petroleum ether to ethyl acetate is 20:1.

[0026] Preferably, in step (2), the molar ratio of compound 2, bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane, n-butyllithium and 2,2,6,6-tetramethylpiperidine is 1:1.5 to 2:2:3.

[0027] Preferably, in step (2), the organic solvent is anhydrous tetrahydrofuran.

[0028] Preferably, in step (2), the amount of anhydrous tetrahydrofuran is calculated as 3 mL of anhydrous tetrahydrofuran per millimole of compound 2.

[0029] Preferably, in step (2), the reaction time is 2 to 6 hours.

[0030] Preferably, in step (2), the extraction is performed using dichloromethane.

[0031] Preferably, in step (2), the separation and purification is performed by silica gel column chromatography.

[0032] Preferably, the silica gel column chromatography is performed in a 100-200 mesh silica gel column; the silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate as the eluent; wherein the volume ratio of petroleum ether to ethyl acetate is 20:1.

[0033] Preferably, in step (3), the molar ratio of compound 3, 5-bromo-2-aminopyrazine and tetra-triphenylphosphine palladium is 1:1.5 to 2: 0.01 to 0.05.

[0034] Preferably, in step (3), the volume ratio of dioxane to saturated sodium carbonate aqueous solution is 5:1.

[0035] Preferably, in step (3), the reaction time is 8 to 12 hours.

[0036] Preferably, in step (3), the extraction is performed using ethyl acetate and saturated brine.

[0037] Preferably, in step (3), the separation and purification are performed by silica gel column chromatography.

[0038] Preferably, the silica gel column chromatography is performed using a 100-200 mesh silica gel column. The silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate as the eluent; wherein the volume ratio of petroleum ether to ethyl acetate is 20:1.

[0039] Preferably, in step (4), the molar ratio of compound 4 and methylglyoxal-1,1-dimethylacetal is 1:1.3 to 1.8.

[0040] Preferably, in step (4), the organic solvent is ethanol.

[0041] Preferably, in step (4), the reaction time is 8 to 12 hours; more preferably, it is 10 hours.

[0042] Preferably, in step (4), the ethanol is anhydrous ethanol.

[0043] Preferably, in step (4), the amount of hydrochloric acid used is calculated as 1 to 1.2 mL of hydrochloric acid per millimole of compound 4; more preferably, 1 mL of hydrochloric acid per millimole of compound 4.

[0044] Preferably, in step (4), the amount of water used is calculated as 1 to 1.2 mL of water per millimole of compound 4; more preferably, 1 mL of pure water is used per millimole of compound 4.

[0045] Preferably, in step (4), the amount of ethanol used is calculated as 10-15 mL of ethanol per millimole of compound 4; more preferably, it is calculated as 12 mL of ethanol per millimole of compound 4.

[0046] Preferably, in step (4), the washing is performed with ethyl acetate; more preferably, the washing is performed with ethyl acetate three or more times.

[0047] On the other hand, the present invention provides an application of the above-mentioned chemiluminescent probe in the detection of β-amyloid protein.

[0048] Preferably, a chemiluminescent probe is added to a test solution containing β-amyloid protein to obtain a test solution, and the β-amyloid protein in the test solution is qualitatively or quantitatively detected.

[0049] Preferably, there is no excitation light source, and the emission wavelength of the chemiluminescent probe is 570-620 nm.

[0050] In this method, without an excitation light source, the chemiluminescent probe, as a substrate, emits chemiluminescence at around 600 nm in the presence of oxygen with β-amyloid protein.

[0051] On the other hand, the present invention provides an application of the above-mentioned chemiluminescent probe in the preparation of a kit for detecting β-amyloid protein.

[0052] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention optimizes the structure of the existing chemiluminescent probe ADLumin-1 and conducts in vitro performance testing and screening. Therefore, the chemiluminescent probe synthesized by the present invention has the characteristics of longer chemical emission wavelength, stronger ability to penetrate the blood-brain barrier, and better response to Aβ amyloid protein. It can be used to sensitively detect Aβ amyloid protein plaques in vivo and to diagnose early Alzheimer's disease.

[0053] (2) The chemiluminescent probe synthesized in this invention has an emission wavelength of 600 nm, which avoids interference from the autoluminescence of biological tissues; and the chemiluminescent probe synthesized in this invention has a LogP of 2.83, which has good blood-brain barrier penetration ability and an affinity constant of 0.34 μM for Aβ amyloid protein aggregates. Therefore, the chemiluminescent probe of this invention can be used to quantify the content of Aβ amyloid protein in the body and monitor its content changes. Attached Figure Description

[0054] Figure 1 This is a high-resolution mass spectrum of the chemiluminescent probe BH-4 of this invention.

[0055] Figure 2 This is a high-resolution mass spectrum of the chemiluminescent probe EYA-4 of this invention.

[0056] Figure 3 This is a high-resolution mass spectrum of the chemiluminescent probe DJ-4 of this invention.

[0057] Figure 4 This is the chemiluminescence spectrum of the chemiluminescence probe of the present invention (the horizontal axis is wavelength (nm), and the vertical axis is chemiluminescence intensity).

[0058] Figure 5 This is a linear relationship graph showing the fluorescence intensity values ​​of the chemiluminescent probe BH-4 of the present invention in response to Aβ amyloid aggregates.

[0059] Figure 6 This is a linear relationship graph showing the fluorescence intensity values ​​of the chemiluminescent probe C-4 of this invention in response to Aβ amyloid aggregates.

[0060] Figure 7 This is a linear relationship graph showing the fluorescence intensity values ​​of the chemiluminescent probe EYA-4 of this invention in response to Aβ amyloid aggregates.

[0061] Figure 8This is a linear relationship graph showing the fluorescence intensity values ​​of the chemiluminescent probe DJ-4 of this invention in response to Aβ amyloid aggregates. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0063] This invention provides a chemiluminescent probe having the structural formula shown in (I):

[0064]

[0065] (I)

[0066] Where R is , , or .

[0067] This invention provides a method for preparing a chemiluminescent probe, comprising the following steps:

[0068]

[0069] (1) Compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate, and 18-crown ether-6 were added to an organic solvent. After the reaction was completed, the mixture was extracted and concentrated to obtain a crude product. The crude product and hydrochloric acid were added to tetrahydrofuran. After the reaction was completed, the mixture was separated and purified to obtain compound 2.

[0070] (2) In the presence of nitrogen as a protective gas, n-butyllithium and 2,2,6,6-tetramethylpiperidine were added to an organic solvent, followed by bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane and compound 2. After the reaction was completed, compound 3 was obtained by separation and purification.

[0071] (3) Compound 3, 5-bromo-2-aminopyrazine and tetratriphenylphosphine palladium were added to 1,4-dioxane containing a saturated sodium carbonate solution. After the reaction was completed, the compound 4 was obtained by separation and purification.

[0072] (4) Compound 4, methylglyoxal-1,1-dimethylacetal, 6M hydrochloric acid, and water were added to an organic solvent. After the reaction was completed, the chemiluminescent probe was obtained by allowing it to stand and precipitate.

[0073] In some embodiments, in step (1), the molar ratio of compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate and 18-crown ether-6 is 1:3:4:0.01 to 0.05.

[0074] In some embodiments, in step (1), the protective gas is nitrogen or argon.

[0075] In some embodiments, the reaction time in step (1) is 10 to 12 hours.

[0076] In some embodiments, in step (2), the molar ratio of compound 2, bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane, n-butyllithium and 2,2,6,6-tetramethylpiperidine is 1:1.5:~2:2:3.

[0077] In some embodiments, the reaction time in step (2) is 2 to 6 hours.

[0078] In some embodiments, in step (3), the molar ratio of compound 3, 5-bromo-2-aminopyrazine and tetra-triphenylphosphine palladium is 1:1.5 to 2: 0.01 to 0.05.

[0079] In some embodiments, the reaction time in step (3) is 8 to 12 hours.

[0080] In some embodiments, in step (4), the molar ratio of compound 4 and methylglyoxal-1,1-dimethylacetal is 1:1.3 to 1.8.

[0081] In some embodiments, the reaction time in step (4) is 8 to 12 hours; preferably 10 hours.

[0082] In some embodiments, in step (4), the amount of hydrochloric acid used is calculated as 1 to 1.2 mL of hydrochloric acid per millimole of compound 4; preferably, 1 mL of hydrochloric acid per millimole of compound 4.

[0083] In some embodiments, the amount of water used is calculated as 1 to 1.2 mL of water per millimole of compound 4; preferably, 1 mL of pure water is used per millimole of compound 4.

[0084] In some embodiments, the amount of ethanol used is calculated as 10 to 15 mL of ethanol per millimole of compound 4; preferably, it is calculated as 12 mL of ethanol per millimole of compound 4.

[0085] In some embodiments, in step (4), the washing is performed with ethyl acetate; preferably, the washing is performed with ethyl acetate three or more times.

[0086] Example 1

[0087] This invention provides a method for preparing a chemiluminescent probe, comprising the following steps:

[0088] (1) Synthetic steps of intermediate compound 2a

[0089]

[0090] First, 1.77 g (1a, 10 mmol) of 4-diethylaminobenzaldehyde, 12.88 g (30 mmol) of ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, 5.53 g (40 mmol) of anhydrous potassium carbonate, and 2.86 mg (0.2 mmol) of 18-crown ether-6 were added to the reaction flask. After assembling the reaction apparatus, nitrogen was introduced, followed by the addition of 10 mL of anhydrous toluene. The mixture was refluxed in an oil bath at 100±5℃ for 10 h. The reaction was confirmed to be complete by thin-layer chromatography, and the reaction was stopped and allowed to stand at room temperature. The anhydrous toluene was evaporated to dryness, and 80 mL of water was added. The mixture was extracted at least three times with 15 mL of dichloromethane (DCM) and concentrated to obtain the crude product. The crude product was dissolved in tetrahydrofuran and added to the reaction flask. Then, 1 mL of concentrated hydrochloric acid was added dropwise, and the mixture was reacted at room temperature for 2 h. The reaction was confirmed to be complete by thin-layer chromatography, and the reaction was stopped. After the reaction was completed, 100 mL of water was added, and the mixture was extracted at least three times with 15 mL of dichloromethane (DCM). Finally, the mixture was purified by column chromatography (silica gel 100-200 mesh; petroleum ether: ethyl acetate = 20:1, v / v) to give 1.63 g of yellow crystals, namely intermediate compound 2a, with a yield of 80%.

[0091] Characterization by proton nuclear magnetic resonance (NMR):

[0092] 1 H NMR (300 MHz, CDCL3) δ 9.58 (d, J = 7.9 Hz, 1H), 7.48 – 7.36 (m,2H), 6.71 – 6.62 (m, 2H), 6.53 (dd, J = 15.6, 7.9 Hz, 1H), 3.42 (q, J = 7.0Hz, 4H), 1.20 (t, J = 7.1 Hz, 7H).

[0093] (2) Synthesis steps of intermediate compound 3a

[0094]

[0095] In a protective nitrogen atmosphere, 2.5 mL of 2,2,6,6-tetramethylpiperidine (24 mmol) dissolved in 3 mL of anhydrous tetrahydrofuran was added to the reaction flask, followed by 6.4 mL of n-butyllithium (16 mmol). After reacting at 0°C for 5 min, 3.33 g of bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane (12 mmol) dissolved in 5 mL of anhydrous tetrahydrofuran was added to the reaction flask. After reacting at 0°C for 15 min, the reaction flask was moved to -78°C, and 1.63 g of (E)-3-(4-(diethylamino)phenyl)propenal (compound 2a, 8 mmol) dissolved in 6 mL of anhydrous tetrahydrofuran was added to the reaction flask. The reaction was carried out at -78±5°C for 4 h. The reaction was confirmed to be complete by thin-layer chromatography, and the reaction was stopped and allowed to stand at room temperature. The solvent was evaporated to dryness, 60 mL of water was added, and the mixture was extracted at least three times with 15 mL of dichloromethane (DCM). Finally, the mixture was purified by column chromatography (silica gel 100-200 mesh; petroleum ether: ethyl acetate = 20:1, v / v) to give 2.25 g of yellow oil, which is intermediate compound 3a, with a yield of 86%.

[0096] Characterization by proton nuclear magnetic resonance (NMR):

[0097] 1H NMR (400 MHz, CDCL3) δ 7.31 – 7.28 (m, 2H), 7.16 (ddd, J = 17.5,8.6, 1.1 Hz, 1H), 6.66 – 6.54 (m, 4H), 5.52 (d, J = 17.5 Hz, 1H), 3.37 (q, J= 7.0 Hz, 4H), 1.28 (s, 12H), 1.16 (t, J= 7.0 Hz, 6H).

[0098] (3) Synthetic steps of intermediate compound 4a

[0099] First, 2 mL of saturated sodium carbonate solution was added to 10 mL of 1,4-dioxane to obtain a mixed solution of 1,4-dioxane containing saturated sodium carbonate solution. 1.96 g of N,N-diethyl-4-((1E,3E,5E)-6-(4,4,5,5-tetramethyl-1,3,2-dioxoborane-2-yl)hexa-1,3,5-trien-1-yl)aniline (compound 3a, 6 mmol), 1.57 g of 5-bromo-2-aminopyrazine (9 mmol), and 69.33 mg of tetrakis(triphenylphosphine)palladium (0.06 mmol) were successively dissolved in the 1,4-dioxane containing saturated sodium carbonate solution and added to a reaction flask. The reaction was carried out under reflux for 12 h in a protective nitrogen atmosphere and an oil bath at 80 °C. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was confirmed to be complete by thin-layer chromatography. The reaction was then stopped and allowed to stand at room temperature. The solvent was evaporated to dryness, 60 mL of saturated saline was added, and the mixture was extracted at least three times with 15 mL of ethyl acetate (EA). Finally, the mixture was purified by column chromatography (silica gel 100-200 mesh; petroleum ether: ethyl acetate = 5:1, v / v) to give 900 mg of yellow powder, which is intermediate compound 4a, with a yield of 51%.

[0100] Characterization by proton nuclear magnetic resonance (NMR):

[0101] 1H NMR (400 MHz, CDCL3) δ 8.05 – 7.90 (m, 2H), 7.48 – 7.35 (m, 2H), 7.32 (d, J = 9.7 Hz, 1H), 7.28 – 7.02 (m, 1H), 6.89 – 6.74 (m, 1H), 6.74 –6.61 (m, 3H), 4.60 (s, 2H), 3.38 (qd, J = 7.1, 3.4 Hz, 4H), 1.17 (td, J =7.1, 3.4 Hz, 6H).

[0102] (4) Synthesis steps of the chemiluminescent probe EYA-4

[0103]

[0104] 70 mg of 5-((1E, 3E)-4-(4-(diethylamino)phenyl)but-1,3-dien-1-yl)pyrazin-2-amine (compound 4a, 0.2 mmol), 35.5 mg of methylglyoxal-1,1-dimethylacetal (0.3 mmol), 0.2 mL of 6M hydrochloric acid, and 0.2 mL of water were added to 3 mL of ethanol. The mixture was refluxed for 10 h in a sealed oil bath at 70±5 °C. After the reaction was completed, the mixture was cooled to room temperature, and 10 mL of ethyl acetate was added. The mixture was allowed to stand at 4 °C to precipitate a solid. The solid was filtered and washed with ethyl acetate to obtain 30 mg of a reddish-brown solid, which is the chemiluminescent probe EYA-4, with a yield of 43%.

[0105] The following modifications were made to the above preparation method: In step (1), the amounts of compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate, and 18-crown ether-6 added were 10 mol, 30 mol, 40 mol, and 0.1 mol, respectively, and the reaction time under reflux in an oil bath at 100±5℃ was 10 h; In step (2), the amounts of compound 2, bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane, n-butyllithium, and 2,2,6,6-tetramethylpiperidine added were 8 mol, 16 mol, 16 mol, and 24 mol, respectively, and the reaction time under -78±5℃ was 2 h; In step (3), the amounts of compound 3, 5-bromo-2-aminopyrazine, and tetratriphenylphosphine palladium added were 6 mol, 12 mol, and 0.3 mol, respectively. The reaction was carried out under reflux conditions at 80℃ for 8 hours. In step (4), the amounts of compound 4 and methylglyoxal-1,1-dimethylacetal added were 0.2 mol and 0.26 mol, respectively, and the reaction was carried out under reflux conditions at 70±5℃ for 8 hours. The remaining steps were the same as in Example 1, and the chemiluminescent probe EYA-4 was finally obtained.

[0106] The following modifications were made to the above preparation method: In step (1), the amounts of compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate, and 18-crown ether-6 added were 10 mol, 30 mol, 40 mol, and 0.5 mol, respectively, and the reaction time under reflux in an oil bath at 100±5℃ was 12 h; In step (2), the amounts of compound 2, bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane, n-butyllithium, and 2,2,6,6-tetramethylpiperidine added were 8 mol, 16 mol, 16 mol, and 24 mol, respectively, and the reaction time under -78±5℃ was 6 h; In step (3), the amounts of compound 3, 5-bromo-2-aminopyrazine, and tetratriphenylphosphine palladium added were 6 mol, 12 mol, and 0.3 mol, respectively. The reaction time under reflux conditions at 80℃ oil bath was 12h; in step (4), the amount of compound 4 and methylglyoxal-1,1-dimethylacetal added were 0.2 mol and 0.36 mol, respectively, and the reaction time under reflux conditions at 70±5℃ oil bath was 12h. The chemiluminescent probe was EYA-4.

[0107] The chemiluminescent probe EYA-4 prepared in Example 1 was characterized by its 1H NMR spectrum:

[0108] 1H NMR (300 MHz, MeOD) δ 8.87 (d, J = 21.7 Hz, 1H), 8.33 (d, J = 38.9Hz, 1H), 7.99 – 7.87 (m, 1H), 7.79 (dd, J = 8.2, 6.1 Hz, 1H), 7.66 (dd, J =19.1, 8.7 Hz, 3H), 7.56 – 7.36 (m, 1H), 7.26 (dd, J = 15.4, 10.8 Hz, 1H), 6.91 (t, J = 15.4 Hz, 1H), 3.70 (t, J = 6.9 Hz, 4H), 2.52 (d, J = 4.5 Hz,3H), 1.16 (dd, J = 7.2, 3.2 Hz, 6H).

[0109] Example 2

[0110] This invention provides a method for preparing a chemiluminescent probe, wherein the compound luminescent probe is C-4. The preparation method is similar to that of Example 1, except that 4-diethylaminobenzaldehyde in step (1) of Example 1 is replaced with 4-((2-hydroxyethyl)(methyl)amino)benzaldehyde to obtain C-4.

[0111] The 1H NMR characterization of the intermediate compound 2b prepared in Example 2: 1H NMR (300 MHz, CDCL3) δ9.47 (d, J = 7.9 Hz, 1H), 7.71 – 7.49 (m, 1H), 7.39 – 7.30 (m, 2H), 6.68 – 6.63 (m, 2H), 6.43 (dd, J = 15.6, 8.0 Hz, 1H), 4.50 (s, 1H), 3.72 (td, J = 6.2, 1.9 Hz, 2H), 3.54 – 3.47 (m, 2H), 3.03 (d, J = 9.1 Hz, 3H).

[0112] The 1H NMR characterization of the intermediate compound 3b prepared in Example 2 was as follows: 1H NMR (400 MHz, CDCL3) δ 7.31 – 7.28 (m, 2H), 7.16 (ddd, J = 17.5, 8.6, 1.1 Hz, 1H), 6.66 – 6.54 (m, 4H), 5.52 (d, J = 17.5 Hz, 1H), 3.72 (td, J = 6.2, 1.9 Hz, 2H), 3.54 – 3.47 (m, 2H), 3.03 (d, J = 9.1 Hz, 3H), 1.28 (s, 12H).

[0113] 1H NMR characterization of the intermediate compound 4b prepared in Example 2: 1H NMR (400 MHz, MeOD) δ 7.93 (dd, J = 9.5, 1.5 Hz, 2H), 7.34 – 7.30 (m, 2H), 7.16 (dd, J = 15.2, 10.7 Hz, 1H), 6.81 (dd, J = 15.4, 10.8 Hz, 1H), 6.74 – 6.71 (m, 2H), 6.62 (d, J = 15.4 Hz, 1H), 6.50 (d, J = 15.3 Hz, 1H), 3.73 (t, J = 6.1 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 3.03 (s, 3H).

[0114] 1H NMR characterization of the chemiluminescent probe C-4 prepared in Example 2:

[0115] 1H NMR (400 MHz, MeOD) δ 8.83 (d, J = 2.6 Hz, 1H), 8.23 ​​(d, J = 4.0Hz, 1H), 7.71 (dd, J = 8.8, 3.0 Hz, 2H), 7.63 – 7.59 (m, 2H), 7.44 (dd, J =9.5, 5.8 Hz, 1H), 7.19 – 7.13 (m, 1H), 6.84 (td, J = 15.8, 5.8 Hz, 2H), 3.67(d, J = 2.1 Hz, 1H), 3.52 (s, 1H), 3.27 (d, J = 1.8 Hz, 4H), 3.26 – 3.24 (m,3H), 2.46 (d, J = 4.4 Hz, 3H).

[0116] Example 3

[0117] This invention provides a method for preparing a chemiluminescent probe, wherein the compound luminescent probe is BH-4. The preparation method is similar to that of Example 1, except that 4-diethylaminobenzaldehyde in step (1) of Example 1 is replaced with 3,5-dimethyl-1-phenyl-1H-pyrazole-4-carboxaldehyde to obtain BH-4.

[0118] Characterization by proton nuclear magnetic resonance (NMR):

[0119] The intermediate compound 2c prepared in Example 3 was characterized by 1H NMR: 1H NMR (400 MHz, CDCL3) δ 9.62 (d, J = 7.7 Hz, 1H), 7.48 – 7.45 (m, 1H), 7.45 – 7.40 (m, 3H), 6.50 (dd, J = 16.1, 7.7 Hz, 1H), 2.45 (d, J = 9.9 Hz, 6H).

[0120] The intermediate compound 3c prepared in Example 3 was characterized by 1H NMR: 1H NMR (300 MHz, CDCL3) δ7.43 – 7.31 (m, 5H), 7.15 (dd, J = 17.6, 9.6 Hz, 1H), 6.68 – 6.42 (m, 2H), 5.54 (d, J = 17.6 Hz, 1H), 2.33 (d, J = 21.0 Hz, 6H), 1.26 (s, 12H).

[0121] The intermediate compound 4c prepared in Example 3 was characterized by 1H NMR (400 MHz, CDCL3) δ7.97 (s, 2H), 7.48 – 7.33 (m, 5H), 6.66 – 6.61 (m, 2H), 6.54 (d, J = 15.3 Hz, 1H), 5.29 (s, 1H), 4.65 (s, 2H), 2.39 (d, J = 28.5 Hz, 6H).

[0122] 1H NMR characterization of the chemiluminescent probe BH-4 prepared in Example 3:

[0123] 1H NMR (400 MHz, MeOD) δ 8.81 (s, 1H), 8.17 (s, 1H), 7.52 – 7.43 (m,4H), 7.43 – 7.38 (m, 3H), 6.73 (d, J = 7.5 Hz, 2H), 3.22 (s, 3H), 2.38 (s,3H), 2.30 (s, 3H), 1.18 (s, 1H).

[0124] Example 4

[0125] This invention provides a method for preparing a chemiluminescent probe, wherein the compound luminescent probe is DJ-4. The preparation method is similar to that of Example 1, except that 4-diethylaminobenzaldehyde in step (1) of Example 1 is replaced with 4-(dibutylamino)benzaldehyde to obtain DJ-4.

[0126] Characterization by proton nuclear magnetic resonance (NMR):

[0127] The intermediate compound 2d prepared in Example 4 was characterized by 1H NMR spectroscopy:

[0128] 1H NMR (300 MHz, CDCl3) δ 9.57 (d, J = 7.9 Hz, 1H), 7.46 – 7.39 (m,2H), 7.35 (d, J = 15.6 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H), 6.51 (dd, J = 15.6,7.9 Hz, 1H), 3.39 – 3.24 (m, 4H), 1.65 – 1.52 (m, 4H), 1.36 (q, J = 7.4 Hz,4H), 0.96 (t, J = 7.3 Hz, 6H).

[0129] 1H NMR characterization of the intermediate compound prepared in Example 4 (3d):

[0130] 1H NMR (300 MHz, CDCl3) δ 7.29 (d, J = 8.3 Hz, 2H), 7.25 – 7.06 (m,1H), 6.64 (d, J = 7.8 Hz, 2H), 6.58 (d, J = 8.4 Hz, 3H), 3.31 – 3.24 (m, 4H), 1.57 (dq, J = 15.3, 7.3 Hz, 4H), 1.41 – 1.31 (m, 4H), 0.95 (t, J = 7.3 Hz, 12H), 0.89 – 0.82 (m, 6H).

[0131] 1H NMR characterization of the intermediate compound prepared in Example 4, 4d:

[0132] 1H NMR (400 MHz, CDCL3) δ 8.05 – 7.90 (m, 2H), 7.48 – 7.35 (m, 2H), 7.32 (d, J = 9.7 Hz, 1H), 7.28 – 7.02 (m, 1H), 6.89 – 6.74 (m, 1H), 6.74 – 6.61 (m, 3H), 4.60 (s, 2H), 3.39 – 3.24 (m, 4H), 1.65 – 1.52 (m, 4H), 1.36 (q, J = 7.4 Hz, 4H), 0.96 (t, J = 7.3 Hz, 6H).

[0133] 1H NMR characterization of the chemiluminescent probe DJ-4 prepared in Example 4:

[0134] 1H NMR (300 MHz, MeOD) δ 8.87 (d, J = 21.7 Hz, 1H), 8.33 (d, J = 38.9Hz, 1H), 7.99 – 7.87 (m, 1H), 7.79 (dd, J = 8.2, 6.1 Hz, 1H), 7.66 (dd, J =19.1, 8.7 Hz, 3H), 7.56 – 7.36 (m, 1H), 7.26 (dd, J = 15.4, 10.8 Hz, 1H), 6.91 (t, J = 15.4 Hz, 1H), 3.31 – 3.24 (m, 4H), 1.57 (dq, J = 15.3, 7.3 Hz,4H), 1.41 – 1.31 (m, 4H), 2.52 (d, J = 4.5 Hz, 3H), 0.96 (t, J = 7.3 Hz, 6H).

[0135] The high-resolution mass spectra of the chemiluminescent probes finally prepared in specific embodiments 1-4 are shown below. Figure 1-3 (HRMS spectrum) shown.

[0136] Example 5

[0137] Chemiluminescence spectrum of chemiluminescent probe:

[0138] The chemiluminescent probes prepared in this invention were compared with the existing chemiluminescent probe compound ADLumin-1, and the chemical emission wavelengths of the chemiluminescent probes were detected. The emission wavelength of the chemiluminescent probe compound ADLumin-1 can be obtained from the reference (J. Yang, W. Yin, R. Van, K. Yin, P. Wang, C. Zheng, et al. Nature Communications 2020 Vol. 11 Issue 1 Pages 4052), where the chemical emission spectra of individual chemiluminescent probes EYA-4, C-4, BH-4, and DJ-4 (25 μM) were measured in DMSO solution using a small animal in vivo imaging system. Figure 4 ).

[0139] The results are shown in Table 1: The chemiluminescent probes EYA-4, C-4, and DJ-4 prepared in this invention have chemiluminescent wavelengths of 580, 606, and 614 nm, respectively, which are longer than those of the chemiluminescent probe ADLumin-1.

[0140] Example 6

[0141] Linear relationship of fluorescence intensity values ​​in response of chemiluminescent probe to Aβ amyloid aggregates:

[0142] The chemiluminescent probes prepared in this invention were dissolved in DMSO solution to prepare solutions of different concentrations (20, 40, 80, 160, 240, 360, 480, 640, 800, 1000 μM). Then, 1 mL of PBS buffer solution (pH=7.4) containing 500 nM Aβ amyloid aggregates was added sequentially. The changes in fluorescence intensity of the probes were detected using a fluorescence spectrophotometer, and the titration curves for each chemiluminescent probe were obtained using Graphpad fitting. Figure 5-8 The affinity constant is obtained after calculation by the software.

[0143] The results are shown in Table 1: the affinity constants of the chemiluminescent probes prepared in this invention are 0.34, 0.51, 0.41 μM and 1.28 μM, respectively, while the affinity constant of the chemiluminescent probe compound ADLumin-1 is 2.1 μM.

[0144]

[0145] Table 1 shows the chemiluminescence wavelengths of the reported compound ADLumin-1 and the chemiluminescent probes EYA-4, C-4, BH-4, and DJ-4, and their affinity constants for Aβ amyloid aggregates.

[0146]

[0147] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A chemiluminescent probe, characterized in that, The chemiluminescent probe has the structural formula shown in (I): Where R is or .

2. The chemiluminescent probe according to claim 1, characterized in that, The chemiluminescent probe has any of the following chemical structures: .

3. A method for preparing a chemiluminescent probe according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Compound 1, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate, and 18-crown ether-6 were added to an organic solvent. After the reaction was completed, the mixture was extracted and concentrated to obtain a crude product. The crude product and hydrochloric acid were added to tetrahydrofuran. After the reaction was completed, the mixture was separated and purified to obtain compound 2. (2) In the presence of nitrogen gas, n-butyllithium and 2,2,6,6-tetramethylpiperidine were added to an organic solvent, followed by bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane and compound 2. After the reaction was completed, the mixture was separated and purified to obtain compound 3. (3) Compound 3, 5-bromo-2-aminopyrazine and tetratriphenylphosphine palladium were added to 1,4-dioxane containing a saturated sodium carbonate solution. After the reaction was completed, the compound 4 was obtained by separation and purification. (4) Compound 4, methylglyoxal-1,1-dimethylacetal, 6M hydrochloric acid, and water were added to an organic solvent. After the reaction was completed, the chemiluminescent probe was obtained by allowing it to stand and precipitate.

4. The method for preparing the chemiluminescent probe according to claim 3, characterized in that, In step (1), the molar ratio of compound 1, ((1,3-dioxane-2-yl)methyl)triphenylphosphine bromide, anhydrous potassium carbonate and 18-crown ether-6 is 1:3:4:0.01 to 0.

05.

5. The method for preparing the chemiluminescent probe according to claim 3, characterized in that, In step (2), the molar ratio of compound 2, bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)methane, n-butyllithium and 2,2,6,6-tetramethylpiperidine is 1:1.5 to 2:2:

3.

6. The method for preparing the chemiluminescent probe according to claim 3, characterized in that, In step (3), the molar ratio of compound 3, 5-bromo-2-aminopyrazine and tetratriphenylphosphine palladium is 1:1.5 to 2: 0.01 to 0.

05.

7. The method for preparing the chemiluminescent probe according to claim 3, characterized in that, In step (4), the molar ratio of compound 4 and methylglyoxal-1,1-dimethylacetal is 1:1.3 to 1.

8.

8. The use of a chemiluminescent probe according to any one of claims 1-2 in the preparation of a formulation for detecting β-amyloid protein.

9. The use of a chemiluminescent probe according to any one of claims 1-2 in the detection of β-amyloid protein, wherein the use is not for the purpose of treating and / or diagnosing a disease.

10. The application according to claim 9, characterized in that, A chemiluminescent probe is added to a test solution containing β-amyloid protein to obtain a test solution, and the β-amyloid protein in the test solution is then qualitatively or quantitatively detected.

11. The application according to claim 10, characterized in that, Without an excitation source, the chemiluminescent probe emits light in the 570-620 nm range.

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