Abeta plaque-specific near-infrared fluorescent probe and preparation method and application thereof

Abstract

The application discloses an A beta plaque specific response type near-infrared fluorescent probe and a preparation method and application thereof. The near-infrared fluorescent probe has a red-shifted fluorescent emission, has a significant fluorescent signal in a near-infrared two region, and has higher specificity and sensitivity to A beta plaques, so that the Alzheimer disease can be monitored in a living body level in an early stage, high resolution and long time course and in a nondestructive manner.

Description

Technical Field

[0001] This invention relates to a near-infrared fluorescent probe, specifically to an Aβ plaque-specific responsive near-infrared fluorescent probe and its preparation method, as well as its application in the preparation of products for diagnosing Alzheimer's disease, belonging to the field of near-infrared fluorescence diagnostic technology. Background Technology

[0002] Alzheimer's disease (AD) is a type of dementia in the elderly, characterized by insidious onset and progressive decline in memory and cognitive function, as well as daily living abilities. The etiology and pathogenesis of AD are not fully understood, but the β-amyloid-β (Aβ) cascade hypothesis is considered one of the mainstream theories.

[0003] Aβ is produced by the hydrolysis of amyloid precursor protein (APP) by β-secretase and γ-secretase. Under normal circumstances, Aβ is metabolized and cleared by the body. However, in AD patients, due to genetic factors (such as mutations in the APP gene, presenilin 1 gene, and presenilin 2 gene), Aβ metabolism is imbalanced, leading to an increased proportion of hydrophobic and easily aggregated Aβ subtypes, thus forming Aβ plaques. These plaques can activate microglia, triggering inflammation; damage mitochondria, causing oxidative stress; and promote the abnormal phosphorylation cascade of tau protein, ultimately resulting in neuronal damage and cognitive impairment. Therefore, abnormal Aβ aggregation in the brain is a core element of the AD pathological process. Although whether Aβ aggregation is the initiating event of AD remains controversial, Aβ plaques as a core pathological marker of AD have been widely confirmed. Therefore, highly sensitive and specific detection of Aβ plaques in vivo is crucial for the early diagnosis of AD.

[0004] Near-infrared fluorescence imaging has advantages such as high safety, low cost and short detection cycle, making it a research hotspot for in vivo imaging diagnosis of Alzheimer's disease (AD). However, the Aβ plaque-responsive fluorescent probes that have been developed so far still have the following problems: (1) The probe fluorescence emission wavelength is relatively short, which limits the penetration depth of the skull and scalp; (2) There is a large interference from biological autofluorescence, and the light absorption and scattering in biological tissues are strong, which leads to a decrease in detection sensitivity and signal-to-noise ratio; (3) The binding kinetics of the probe to Aβ plaques are slow and the affinity is low, making it difficult to achieve rapid, stable and long-term monitoring; (4) It can usually only identify mature plaques in the late stage of AD, which cannot meet the needs of early diagnosis. Therefore, developing new near-infrared fluorescent probes with longer emission wavelengths and higher specificity and affinity for Aβ plaques to achieve highly sensitive and specific detection of Aβ plaques at the in vivo level is of great significance for the early diagnosis and efficacy monitoring of AD. Summary of the Invention

[0005] The main objective of this invention is to provide a responsive near-infrared fluorescent probe with high specificity and high sensitivity for detecting Aβ plaques, a biomarker of Alzheimer's disease, and its preparation method, in order to overcome the shortcomings of the prior art.

[0006] Another object of the present invention is to provide applications of the near-infrared fluorescent probe.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] This invention provides an Aβ patch-specific responsive near-infrared fluorescent probe, which has the structure shown in formula (I):

[0009] ;

[0010] Equation (I)

[0011] Where n is 1, 2, or 3; R1 includes -H, , , or Any one of them;

[0012] R2 includes -H, , , , , Any one of them;

[0013] R3 includes , , Any one of the following, where m = 2 or 3;

[0014] X¯ includes any one of ClO4¯, BF4¯, CF3COO¯, CH3COO¯, F¯, Cl¯, Br¯, I¯, and NO3¯.

[0015] This invention also provides a method for preparing an Aβ patch-specific responsive near-infrared fluorescent probe, comprising:

[0016] An aldehyde containing a thiophene ring is subjected to a first condensation reaction with a p-aminophenylboronic acid derivative to obtain a first compound;

[0017] The first compound is subjected to a second condensation reaction with a second compound containing oxonium ions to prepare an Aβ patch-specific responsive near-infrared fluorescent probe.

[0018] The first compound has the structure shown in formula (II):

[0019] ;

[0020] Formula (II)

[0021] R3 includes , , Any one of the following, where m = 2 or 3;

[0022] The second compound has the structure shown in formula (Ⅲ):

[0023] ;

[0024] Formula (III)

[0025] n is 1, 2, or 3; R1 includes -H, , , or Any one of them;

[0026] R2 includes -H, , , , , Any one of them.

[0027] This invention also provides an Aβ patch-specific responsive near-infrared fluorescent probe prepared by the aforementioned method.

[0028] This invention also provides the application of the Aβ plaque-specific responsive near-infrared fluorescent probe in the preparation of products with the function of detecting Aβ plaques.

[0029] Accordingly, embodiments of the present invention also provide a product with the function of detecting Aβ plaques, which includes the Aβ plaque-specific responsive near-infrared fluorescent probe.

[0030] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0031] (1) In this invention, a near-infrared fluorescent probe with specific targeting of β-amyloid protein, large Stokes shift, and high sensitivity and specificity for the detection of Aβ plaques is constructed by using the rhodamine derivative structure as a strong electron-withdrawing group, the thiophene ring structure to extend the conjugated chain, and the p-aminophenylboronic acid derivative as the Aβ plaque recognition unit.

[0032] (2) The near-infrared responsive fluorescent probe constructed in this invention has red-shifted fluorescence emission and has a significant fluorescence signal in the near-infrared II region, which can be used for high-sensitivity and high-resolution fluorescence detection of Alzheimer's disease at the in vivo level. Furthermore, the near-infrared responsive fluorescent probe has the characteristics of long emission wavelength, large Stokes shift, and high specificity and sensitivity to β-amyloid protein. After the probe binds to β-amyloid protein in the brain, the fluorescence signal is significantly enhanced, which has important guiding significance for the early and accurate diagnosis of Alzheimer's disease. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram illustrating the mechanism of binding between the Aβ plaque-specific responsive near-infrared fluorescent probe and β-amyloid protein in a typical embodiment of the present invention;

[0035] Figure 2 This is the 1H NMR spectrum of the AOT-1 platform molecule prepared in Example 1 of this invention;

[0036] Figure 3 This is the carbon NMR spectrum of the AOT-1 platform molecule prepared in Example 1 of this invention;

[0037] Figure 4 This is the 1H NMR spectrum of the AOT-2 platform molecule prepared in Example 2 of this invention;

[0038] Figure 5 This is the carbon NMR spectrum of the AOT-2 platform molecule prepared in Example 2 of this invention;

[0039] Figure 6 This is the 1H NMR spectrum of the AOT-4 platform molecule prepared in Example 4 of this invention;

[0040] Figure 7 This is the carbon NMR spectrum of the AOT-4 platform molecule prepared in Example 4 of this invention;

[0041] Figures 8a-8h These are the absorption and emission spectra of the near-infrared II rhodamine-like probe platform obtained in Examples 1-4 of Test Example 1 of this invention in different solvents;

[0042] Figures 9a-9d These are emission spectra of the near-infrared II rhodamine-like probe platform obtained in Examples 1-4 of Test Example 2 of this invention with different viscosity responses;

[0043] Figures 10a-10d This is the fluorescence spectrum of the near-infrared II rhodamine-like probe platform obtained in Examples 1-4 of Test Example 3 of this invention after binding with Aβ fiber;

[0044] Figure 11 This is a fluorescence imaging image of the near-infrared II rhodamine-like probe platform obtained in Example 1 of Test Example 4 of the present invention, used for staining brain sections of AD mice.

[0045] Figure 12 This is a fluorescence imaging image of the near-infrared II rhodamine-like probe platform obtained in Example 1 of Test Example 5 of the present invention before and after in vivo injection in AD mice;

[0046] Figure 13 This is a quantitative fluorescence bar chart showing the specific response of the near-infrared II rhodamine-like probe platform obtained in Example 1 of Test Example 6 of the present invention to different biomarkers. Detailed Implementation

[0047] In view of the shortcomings of the existing technology, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention, which mainly provides a responsive near-infrared fluorescent probe with high specificity and high sensitivity for detecting Alzheimer's disease marker Aβ plaques and its preparation method, for the early and accurate diagnosis of Alzheimer's disease.

[0048] The following will further explain the technical solution, its implementation process, and its principles. However, it should be understood that within the scope of this invention, the above-mentioned technical features of this invention and the technical features specifically described below (in embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0049] As one aspect of the technical solution of this invention, an Aβ patch-specific responsive near-infrared fluorescent probe has the structure shown in formula (Ⅰ):

[0050] ;

[0051] Equation (I)

[0052] Where n is 1, 2, or 3; R1 includes -H, , , or Any one of them;

[0053] R2 includes -H, , , , , Any one of them;

[0054] R3 includes , , Any one of the following, where m = 2 or 3;

[0055] X¯ includes any one of ClO4¯, BF4¯, CF3COO¯, CH3COO¯, F¯, Cl¯, Br¯, I¯, and NO3¯.

[0056] When the Aβ plaque-specific responsive near-infrared fluorescent probe of the present invention does not bind to the Aβ plaque biomarker of Alzheimer's disease, the fluorescent molecules undergo a torsional charge transfer (TICT) process, thereby inhibiting the fluorescence production of the probe and causing fluorescence quenching. When the Aβ plaque-specific responsive near-infrared fluorescent probe specifically recognizes and binds to the Aβ plaque, the probe's TICT process is inhibited, thereby enabling fluorescence activation and recovery.

[0057] In some specific embodiments, the Aβ plaque-specific responsive near-infrared fluorescent probe has the characteristic of specifically targeting β-amyloid protein, and its specific structural formula is shown below:

[0058] .

[0059] This invention utilizes a rhodamine-derived structure as a strong electron-withdrawing group, a thiophene ring structure to extend the conjugated chain, and a phenylamine derivative as an Aβ plaque recognition unit to construct a near-infrared fluorescent probe that specifically targets β-amyloid protein, has a large Stokes shift, and can perform highly sensitive and specific detection of Aβ plaques.

[0060] Furthermore, the near-infrared fluorescent probe of the present invention has redshifted fluorescence emission, exhibiting a significant fluorescence signal in the near-infrared II region. Moreover, the probe demonstrates high specificity and sensitivity for Aβ plaques, enabling early, accurate, high-resolution, and long-term non-destructive monitoring of Alzheimer's disease at the in vivo level.

[0061] As another aspect of the technical solution of the present invention, a method for preparing an Aβ patch-specific responsive near-infrared fluorescent probe includes:

[0062] An aldehyde containing a thiophene ring is subjected to a first condensation reaction with a p-aminophenylboronic acid derivative to obtain a first compound;

[0063] The first compound is further subjected to a second condensation reaction with a second compound containing oxonium ions to obtain an Aβ patch-specific responsive near-infrared fluorescent probe.

[0064] In some embodiments, the first compound (which may be simply referred to as SF) has the structure shown in formula (II):

[0065] ;

[0066] Formula (II)

[0067] Among them, R3 includes , , Any one of the following, where m = 2 or 3.

[0068] In some embodiments, the second compound (which may be simply referred to as AO) containing oxonium ions used in this invention has the structure shown in formula (Ⅲ):

[0069] ;

[0070] Formula (III)

[0071] Where n is 1, 2, or 3; R1 includes -H, , , or Any one of them;

[0072] R2 includes -H, , , , , Any one of them.

[0073] The reaction equation for preparing a class of Aβ patch-specific responsive near-infrared fluorescent probes of the present invention is as follows:

[0074] .

[0075] In some embodiments, the preparation method of the first compound (hereinafter referred to as SF) specifically includes: mixing an aldehyde containing a thiophene ring, a p-aminophenylboronic acid derivative, a catalyst, an alkaline substance, and a first solvent to form a first mixed reaction system, and carrying out a first condensation reaction at a temperature under a protective atmosphere to obtain the first compound.

[0076] Furthermore, the aldehyde containing the thiophene ring has the structure shown in formula (Ⅳ):

[0077] ;

[0078] Equation (Ⅳ)

[0079] The p-aminophenylboronic acid derivative has the structure shown in formula (V):

[0080] ;

[0081] Formula (V)

[0082] R3 includes , , Any one of the following, where m = 2 or 3.

[0083] In some embodiments, the temperature of the first condensation reaction is 25~120°C and the time is 12~72 h.

[0084] In some preferred embodiments, the molar ratio of the p-aminophenylboronic acid derivative, the catalyst, the thiophene ring-containing aldehyde, and the basic substance is 1~1.3:0.1:1:2.7~3.3.

[0085] In some preferred embodiments, the p-aminophenylboronic acid derivative may include one or more combinations of 4-(dimethylamino)phenylboronic acid, 4-(diethylamino)phenylboronic acid, 4-(N-tetrahydropyrrolyl)phenylboronic acid, 4-(piperidin-1-yl)phenylboronic acid, etc., but is not limited thereto.

[0086] Furthermore, the alkaline substance may include one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, etc., but is not limited to these.

[0087] Furthermore, the catalyst may include one or more combinations of tetra(triphenylphosphine)palladium, tetra(triphenylphosphine)palladium dichloride, palladium chloride, etc., preferably tetra(triphenylphosphine)palladium.

[0088] Furthermore, the first solvent may include, but is not limited to, ethylene glycol dimethyl ether.

[0089] Furthermore, the amount of the first solvent used is 10 to 50 mL of the first solvent per millimole of an aldehyde containing a thiophene ring.

[0090] Furthermore, the protective atmosphere includes nitrogen and / or argon atmospheres, etc.

[0091] In some more specific embodiments, the preparation steps of the first compound (hereinafter referred to as SF) specifically include: dissolving a p-aminophenylboronic acid derivative, tetrakis(triphenylphosphine)palladium, 4-bromo-2-carboxaldehydethiophene and a basic substance in ethylene glycol dimethyl ether to obtain a mixture, reacting it under a protective gas atmosphere at 25~120°C, preferably 60~110°C, and after the reaction is completed, evaporating under reduced pressure, separating and purifying to obtain the first compound (i.e., intermediate SF).

[0092] Furthermore, the separation and purification are carried out using silica gel column chromatography.

[0093] Furthermore, the silica gel column chromatography is performed using a 200-300 mesh silica gel column for chromatographic separation.

[0094] Furthermore, the silica gel column chromatography uses a mixed solvent of petroleum ether and dichloromethane as the eluent for gradient elution; wherein the volume ratio of petroleum ether to dichloromethane is 20:1.

[0095] In some embodiments, the preparation method of the second compound containing the oxonium ion (which may be simply referred to as AO) specifically includes:

[0096] A cyclic fatty ketone compound was added to concentrated sulfuric acid and stirred in an ice bath to obtain a first mixed solution;

[0097] The first mixed solution is mixed with a ketone ester compound and reacted at 60-110°C for 1-4 h to obtain a second mixed solution.

[0098] The second mixed solution was added to ice water containing perchlorate ions to prepare a second compound containing oxonium ions.

[0099] Furthermore, the cyclic aliphatic ketone compound has the structure shown in formula (VI):

[0100] ;

[0101] Formula (VI)

[0102] Where n is 1, 2 or 3.

[0103] Furthermore, the ketone ester compound has the structure shown in formula (VII):

[0104] ;

[0105] Formula (VII)

[0106] Among them, R1 includes -H, , , or Any one of them;

[0107] R2 includes -H, , , , , Any one of them.

[0108] In some embodiments, the cyclic aliphatic ketone compound includes cyclopentanone, cyclohexanone, or cycloheptanone, etc.

[0109] In some embodiments, the ketone ester compound may include one or more combinations of 4-(diethylamino)salicylaldehyde, 4-diethylaminoketo acid, salicylaldehyde, 2,4-dihydroxybenzaldehyde, 4-methoxysalicylaldehyde, 4-(dimethylamino)salicylaldehyde, etc., but is not limited thereto.

[0110] In some preferred embodiments, the amount of concentrated sulfuric acid used is 1.5 to 2 mL of concentrated sulfuric acid per millimole of cyclic aliphatic ketone compound.

[0111] In some preferred embodiments, the cyclic fatty ketone compound is added to concentrated sulfuric acid and stirred in an ice bath for 1 to 4 hours.

[0112] In some preferred embodiments, the molar ratio of the ketone ester compound to the cyclic aliphatic ketone compound is 1:1.5~2.

[0113] In some preferred embodiments, the ice water includes a perchlorate compound, which includes any one of HClO4, KClO4, etc.

[0114] Furthermore, the amount of the perchlorate compound used is 1.5 to 5 mL of perchlorate compound per millimole of cyclic aliphatic ketone compound.

[0115] In some more specific embodiments, the preparation steps of the second compound containing oxonium ions (hereinafter referred to as AO) specifically include: adding a cyclic aliphatic ketone compound dropwise to concentrated sulfuric acid and stirring under an ice bath to obtain a mixed solution A; slowly adding a ketone ester compound to mixed solution A and reacting at 60~110℃ for 1~4 h to obtain a mixed solution B; after the reaction is completed, slowly adding mixed solution B dropwise to ice water containing perchloric acid, allowing it to stand, and then filtering and drying to obtain the second compound (i.e., intermediate AO).

[0116] In some embodiments, the preparation method of the Aβ patch-specific responsive near-infrared fluorescent probe specifically includes: mixing the first compound (hereinafter referred to as SF), the second compound containing oxonium ions (hereinafter referred to as AO), and the second solvent, and carrying out a second condensation reaction under a protective atmosphere and heating conditions. After the reaction is completed, the second solvent is removed, and the mixture is separated and purified to obtain the Aβ patch-specific responsive near-infrared fluorescent probe.

[0117] In some preferred embodiments, the molar ratio of the first compound to the second compound is 1:1.5 to 3.

[0118] Furthermore, the second solvent may include at least one of acetic anhydride, toluene / n-butanol, etc., preferably acetic anhydride.

[0119] Furthermore, the protective atmosphere includes a nitrogen and / or argon atmosphere.

[0120] In some preferred embodiments, the temperature of the second condensation reaction is 25~130°C and the time is 2~12 h, preferably 4~8 h.

[0121] In some preferred embodiments, the preparation method may further include: after the reaction is complete, further purifying the obtained Aβ patch-specific responsive near-infrared fluorescent probe by silica gel column chromatography.

[0122] Furthermore, the silica gel column chromatography is performed by chromatographic separation in a 200-300 mesh silica gel column.

[0123] Furthermore, the silica gel column chromatography uses a mixed solvent of dichloromethane and methanol as the eluent for gradient elution; wherein the volume ratio of dichloromethane to methanol is 10~50:1.

[0124] Furthermore, the preparation method further includes: after obtaining the Aβ patch-specific responsive near-infrared fluorescent probe, an ion exchange step is performed, specifically, perchlorate (ClO4¯) is exchanged for BF4¯, CF3COO¯, CH3COO¯, F¯, Cl¯, Br¯, I¯, NO3¯, etc., as shown in the following reaction formula:

[0125] .

[0126] As another aspect of the technical solution of the present invention, it also relates to an Aβ patch-specific responsive near-infrared fluorescent probe prepared by the aforementioned preparation method.

[0127] As another aspect of the technical solution of the present invention, it also relates to the application of the Aβ patch-specific responsive near-infrared fluorescent probe in the preparation of products with the function of detecting Aβ patches.

[0128] Accordingly, another aspect of the technical solution of the present invention also relates to a product with the function of detecting Aβ plaques, which includes the aforementioned Aβ plaque-specific responsive near-infrared fluorescent probe.

[0129] Furthermore, the product has the ability to diagnose neurodegenerative diseases, including Alzheimer's disease.

[0130] Furthermore, the products include fluorescent probes, reagents, or kits.

[0131] The Aβ plaque-specific responsive near-infrared fluorescent probe synthesized in this invention has the characteristics of long emission wavelength, large Stokes shift, and high specificity and sensitivity to β-amyloid protein. After binding with β-amyloid protein in the brain, the fluorescence signal is significantly enhanced, which has important guiding significance for the early and accurate diagnosis of Alzheimer's disease.

[0132] Furthermore, the β-amyloid protein is an Aβ aggregate, preferably an Aβ1-42 aggregate.

[0133] Specifically, the process of performing in vivo injection fluorescence imaging using the Aβ patch-specific responsive near-infrared fluorescent probe of the present invention includes:

[0134] The synthesized Aβ plaque-specific responsive near-infrared fluorescent probe was dissolved in an organic solvent and added to physiological saline with added solubilizer. The probe was then injected via the tail vein, and fluorescence images of the mouse head were collected before and after administration.

[0135] Furthermore, the solubilizing organic solvent is dimethyl sulfoxide (DMSO).

[0136] Furthermore, the solubilizer is BSA or β-cyclodextrin, preferably β-cyclodextrin.

[0137] Furthermore, the dose administered via tail vein injection is 2 mg / kg.

[0138] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, but the embodiments described do not constitute a limitation of the present invention. All modifications that are conceived or derived from the content disclosed in this invention are considered to be within the scope of protection of this invention.

[0139] All raw materials used in the embodiments of this invention were purchased from the market.

[0140] The instruments and equipment used in the following examples are all conventional equipment in the relevant fields, and the performance tests are all conducted in accordance with the requirements of conventional standards.

[0141] Example 1

[0142] 4-Dimethylaminophenylboronic acid (0.12 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 80 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 4-(diethylamino)salicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-1 (0.1 mmol) and AO-1 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe AOT-1.

[0143] .

[0144] The 1H NMR spectrum of the near-infrared fluorescent probe AOT-1 obtained in this embodiment is shown below. Figure 2 As shown, the carbon NMR spectrum of AOT-1 is as follows: Figure 3 As shown.

[0145] Example 2

[0146] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 100 °C for 24 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 4-diethylaminoketo acid was added, and the reaction was carried out at 90 °C for 3 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-2 was obtained. The intermediates SF-1 (0.1 mmol) and AO-2 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 12 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe AOT-2.

[0147] .

[0148] The near-infrared fluorescent probe AOT-2 obtained in this embodiment is shown in the figure below. Figure 4 As shown, the carbon NMR spectrum of AOT-2 is as follows: Figure 5 As shown.

[0149] Example 3

[0150] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 110 °C for 72 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 1 h. Then, 0.2 mmol of 2,4-dihydroxybenzaldehyde was added, and the mixture was reacted at 90 °C for 1 hour. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-3 was obtained. The intermediates SF-1 (0.1 mmol) and AO-3 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 8 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe AOT-3.

[0151] .

[0152] Example 4

[0153] 4-Dimethylaminophenylboronic acid (0.12 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 24 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 h. Then, 4-methoxysalicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 4 hours. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-4 was obtained. The intermediates SF-1 (0.1 mmol) and AO-4 (0.3 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 4 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe AOT-4.

[0154] .

[0155] The proton NMR spectrum of the near-infrared fluorescent probe AOT-4 obtained in this embodiment is as follows: Figure 6 As shown, the carbon NMR spectrum of AOT-2 is as follows: Figure 7 As shown.

[0156] Example 5

[0157] 4-Diethylaminophenylboronic acid (0.13 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.33 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-2. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 4-(diethylamino)salicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-1 was obtained. The intermediates SF-2 (0.1 mmol) and AO-1 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0158] .

[0159] Example 6

[0160] 0.13 mmol of 4-diethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.33 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 100 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-2. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 4-diethylaminoketo acid was added, and the mixture was reacted at 90 °C for 3 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-2 (0.1 mmol) and AO-2 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 10 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0161] .

[0162] Example 7

[0163] 4-Diethylaminophenylboronic acid (0.13 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.33 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-2. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 1 h. Then, 2,4-dihydroxybenzaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 1 hour. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-3 was obtained. The intermediates SF-2 (0.1 mmol) and AO-3 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 8 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0164] .

[0165] Example 8

[0166] 0.13 mmol of 4-diethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.33 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 100 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-2. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 4-methoxysalicylaldehyde was added, and the mixture was reacted at 90 °C for 4 hours. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-4 was obtained. The intermediates SF-2 (0.1 mmol) and AO-4 (0.3 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 4 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain a near-infrared fluorescent probe.

[0167] .

[0168] Example 9

[0169] 4-(N-tetrahydropyrrolyl)phenylboronic acid (0.12 mmol), tetra(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 48 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-3. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 0.2 mmol of 4-(diethylamino)salicylaldehyde was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-1 was obtained. The intermediates SF-3 (0.1 mmol) and AO-1 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain a near-infrared fluorescent probe.

[0170] .

[0171] Example 10

[0172] 4-(N-tetrahydropyrrolyl)phenylboronic acid (0.12 mmol), tetra(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 48 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-3. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 4-diethylaminoketo acid (0.2 mmol) was added, and the mixture was reacted at 90 °C for 3 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-2 was obtained. The intermediates SF-3 (0.1 mmol) and AO-2 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 10 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0173] .

[0174] Example 11

[0175] 4-(N-tetrahydropyrrolyl)phenylboronic acid (0.12 mmol), tetra(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 48 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-3. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 1 h. Then, 0.2 mmol of 2,4-dihydroxybenzaldehyde was added, and the mixture was reacted at 90 °C for 1 hour. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-3 (0.1 mmol) and AO-3 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 8 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0176] .

[0177] Example 12

[0178] 4-(N-tetrahydropyrrolyl)phenylboronic acid (0.12 mmol), tetra(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 48 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-3. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 h. Then, 4-methoxysalicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 4 hours. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-4 was obtained. The intermediates SF-3 (0.1 mmol) and AO-4 (0.3 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 4 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0179] .

[0180] Example 13

[0181] 4-(piperidin-1-yl)phenylboronic acid (0.1 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.27 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 72 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-4. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 0.2 mmol of 4-(diethylamino)salicylaldehyde was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-1 was obtained. The intermediates SF-4 (0.1 mmol) and AO-1 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0182] .

[0183] Example 14

[0184] 4-(piperidin-1-yl)phenylboronic acid (0.1 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.27 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 72 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-4. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 4-diethylaminoketo acid was added, and the mixture was reacted at 90 °C for 3 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-2 was obtained. The intermediates SF-4 (0.1 mmol) and AO-2 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 10 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0185] .

[0186] Example 15

[0187] 4-(piperidin-1-yl)phenylboronic acid (0.1 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.27 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 72 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-4. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, stirred for 1 h in an ice bath, and then 0.2 mmol of 2,4-dihydroxybenzaldehyde was added. The mixture was reacted at 90 °C for 1 hour. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-3 was obtained. The intermediates SF-4 (0.1 mmol) and AO-3 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 8 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0188] .

[0189] Example 16

[0190] 4-(piperidin-1-yl)phenylboronic acid (0.1 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.27 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 25 °C for 72 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-4. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 4-methoxysalicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 60 °C for 4 hours. The mixture was then added dropwise to a mixture of 2 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-4 (0.1 mmol) and AO-4 (0.3 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at 100 °C for 4 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0191] .

[0192] Example 17

[0193] 4-Dimethylaminophenylboronic acid (0.12 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 24 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.8 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 2,4-dihydroxybenzaldehyde was added, and the mixture was reacted at 90 °C for 3 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-5 was obtained. The intermediates SF-1 (0.1 mmol) and AO-5 (0.15 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 8 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0194] .

[0195] Example 18

[0196] 4-Dimethylaminophenylboronic acid (0.12 mmol), tetrakis(triphenylphosphine)palladium (0.01 mmol), 4-bromo-2-carboxythiophene (0.1 mmol), and K₂CO₃ (0.3 mmol) were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and then evacuated under nitrogen. The mixture was then reacted in an oil bath at 100 °C for 24 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclohexanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 4-(dimethylamino)salicylaldehyde (0.2 mmol) was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.6 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. Intermediate AO-6 was obtained. The intermediates SF-1 (0.1 mmol) and AO-6 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0197] .

[0198] Example 19

[0199] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium dichloride, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of Na₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 120 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cycloheptanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of 4-(dimethylamino)salicylaldehyde was added, and the mixture was reacted at 110 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.6 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-1 (0.1 mmol) and AO-7 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and air was removed using a double-row tube. The reaction was carried out at 130 °C for 4 hours, followed by the addition of 100 mL of petroleum ether to precipitate the product. The precipitate was purified using a silica gel column chromatography eluent of dichloromethane and methanol to obtain the near-infrared fluorescent probe.

[0200] .

[0201] Example 20

[0202] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 80 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.4 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 2 hours. Then, 0.2 mmol of [4-(diethylamino)-2-hydroxybenzene]-benzophenone was added, and the mixture was reacted at 90 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-1 (0.1 mmol) and AO-8 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0203] .

[0204] Example 21

[0205] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 80 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 0.2 mmol of [4-(diethylamino)-2-hydroxyphenyl]-(2-methylphenyl)methyl ketone was added, and the mixture was reacted at 110 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-1 (0.1 mmol) and AO-9 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0206] .

[0207] Example 22

[0208] 0.12 mmol of 4-dimethylaminophenylboronic acid, 0.01 mmol of tetrakis(triphenylphosphine)palladium, 0.1 mmol of 4-bromo-2-carboxythiophene, and 0.3 mmol of K₂CO₃ were dissolved in 5 mL of ethylene glycol dimethyl ether, sonicated, and the mixture was then evacuated and purged with nitrogen. The reaction was then carried out in an oil bath at 80 °C for 12 hours. After the reaction, the solution was evaporated to dryness and separated to obtain intermediate SF-1. 0.4 mmol of cyclopentanone was slowly added dropwise to 0.6 mL of concentrated sulfuric acid, and the mixture was stirred in an ice bath for 4 hours. Then, 0.2 mmol of 2-[4-(diethylamino)-2-hydroxybenzoyl]-1,4-phthalic acid was added, and the mixture was reacted at 100 °C for 2 hours. The mixture was then added dropwise to a mixture of 0.8 mL of perchloric acid and 20 mL of ice, and the precipitate was obtained by filtration. The intermediates SF-1 (0.1 mmol) and AO-10 (0.2 mmol) were dissolved in 15 mL of acetic anhydride, and the air was removed using a double-row tube. The reaction was carried out at room temperature for 2 hours, after which 100 mL of petroleum ether was added to the reaction system to precipitate the product. The precipitate was purified using a silica gel column chromatography with dichloromethane and methanol as eluents to obtain the near-infrared fluorescent probe.

[0209] .

[0210] Example 23

[0211] AOT-1 (0.01 mmol) was dissolved in methanol (1 mL), and then KBF4 (0.02 mmol) was added and sonicated to dissolve. The mixture was refluxed for 2 hours, the organic phase was evaporated to dryness, and the mixture was extracted with water and dichloromethane. The extract was dried over anhydrous MgSO4, concentrated, and recrystallized from the methanol-petroleum ether system to obtain the target product AOT-1-BF4.

[0212] .

[0213] Example 24

[0214] AOT-1 (0.01 mmol) was dissolved in methanol (1 mL), and then KBr (0.02 mmol) was added and sonicated to dissolve. The mixture was refluxed for 2 hours, the organic phase was evaporated to dryness, and the mixture was extracted with water and dichloromethane. The extract was dried over anhydrous MgSO4, concentrated, and recrystallized from the methanol-petroleum ether system to obtain the target product AOT-1-Br.

[0215] .

[0216] Example 25

[0217] AOT-1 (0.01 mmol) was dissolved in methanol (1 mL), and then KNO3 (0.02 mmol) was added and sonicated to dissolve. The mixture was refluxed for 2 hours, the organic phase was evaporated to dryness, and the mixture was extracted with water and dichloromethane. The extract was dried over anhydrous MgSO4, concentrated, and recrystallized from the methanol-petroleum ether system to obtain the target product AOT-1-NO3.

[0218] .

[0219] Test Example 1

[0220] The inventors of this case also characterized the absorption and emission spectra of the near-infrared II fluorescent probe platform obtained in Examples 1-4 above in different solvents: the near-infrared II rhodamine-like probes AOT-1, AOT-2, AOT-3 and AOT-4 were dissolved in different organic solvents (acetone, methanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, tetrahydrofuran and water), and the final probe concentration was 50 μM.

[0221] The absorption and fluorescence spectra of the samples were measured at 37 °C. Figure 8a , Figure 8b The absorption and emission spectra of the fluorescent probe platform molecule AOT-1 in different solvents are shown respectively. Figure 8c , Figure 8d The absorption and emission spectra of the rhodamine-like platform molecule AOT-2 in different solvents are shown respectively. Figure 8e , Figure 8f The absorption and emission spectra of the rhodamine-like platform molecule AOT-3 in different solvents are shown respectively. Figure 8g , Figure 8h The absorption and emission spectra of the rhodamine-like platform molecule AOT-4 in different solvents are shown.

[0222] Test Example 2

[0223] The inventors also characterized the emission spectra of the near-infrared II rhodamine-like probe platforms obtained in Examples 1-4 above at different viscosities. The near-infrared II rhodamine-like probes AOT-1, AOT-2, AOT-3, and AOT-4 were dissolved in solvents with different viscosity gradients: ethanol:glycerol = 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%, respectively, with a final probe concentration of 100 μM. The fluorescence spectra of the samples were measured at 25°C, and the results showed... Figure 9a The fluorescence spectra of the rhodamine-like platform molecule AOT-1 at different viscosities are shown; Figure 9b The fluorescence spectra of the rhodamine-like platform molecule AOT-2 at different viscosities are shown; Figure 9cThe fluorescence spectra of the rhodamine-like platform molecule AOT-3 at different viscosities are shown; Figure 9d The fluorescence spectra of the rhodamine-like platform molecule AOT-4 at different viscosities are shown.

[0224] Test Example 3

[0225] (1) Preparation of Aβ aggregates;

[0226] The purchased Aβ1-42 aggregate protein (purchased from Shanghai Maclean Biotechnology Co., Ltd.) was dissolved in hexafluoroisopropanol (400 μL) for 1 hour at room temperature. After drying with nitrogen, the protein was dissolved in 44.3056 μL of dimethyl sulfoxide to obtain 5 mM Aβ monomer. The solution was then diluted to 50 μM with PBS (10 mM containing 1 mM EDTA) and incubated on a shaker at 37°C and 500 rpm for 5 days to obtain 50 μM Aβ1-42 aggregates.

[0227] (2) Measurement of fluorescence spectrum

[0228] The near-infrared fluorescent probes obtained in Examples 1-4 were subjected to fluorescence spectra determination. Aβ1-42 aggregates obtained after incubation in step (1) were added to PBS buffer solution (pH 7.4, 10 mM) containing 5 μM of fluorescent probe, at a molar ratio of Aβ1-42 aggregate protein to fluorescent probe of 2:1. An equal volume of PBS buffer solution (pH 7.4, 10 mM) was added as a control. The emission spectra were recorded using a fluorescence spectrophotometer. The results showed that... Figure 10a The fluorescence spectrum of the rhodamine-like platform molecule AOT-1 after the response is shown; Figure 10b The fluorescence spectrum of the rhodamine-like platform molecule AOT-2 after the response is shown; Figure 10c The fluorescence spectrum of the rhodamine-like platform molecule AOT-3 after the response is shown; Figure 10d The fluorescence spectrum of the rhodamine-like platform molecule AOT-4 after the response is shown.

[0229] Test Example 4

[0230] The inventors of this case also performed staining and imaging of paraffin-treated brain sections using the near-infrared fluorescent probe obtained in Example 1 above: Brain tissue was extracted from transgenic AD model mice (APP / PS1, 8 months old, male) after perfusion, fixed with paraformaldehyde, and then embedded in paraffin and sectioned (4 μm). Before staining, the sections were first soaked in xylene for 10 min, then dewaxed with a gradient of different concentrations (95%, 85%, 75%) of ethanol for 5 min, and finally washed with PBS for 5 min. The sections were incubated with probe AOT-1 (dissolved in DMSO) at room temperature for 20 min, followed by washing with 50% ethanol and PBS buffer. After wiping away residual liquid with lint-free paper, the sections were stained with 1% thiosulfate T for 10 min. Finally, fluorescence images of the sections were acquired using a wide-field fluorescence microscope. Figure 11 The results of Aβ staining in sections by the rhodamine-like platform molecule AOT-1 and thiosulfate T are shown, and the results are co-localized.

[0231] Test Example 5

[0232] The inventors of this case also used the near-infrared fluorescent probe obtained in Example 1 to inject into living organisms and image the mouse head: In order to increase the water solubility of the prepared AOT-1 probe during injection, AOT-1 was dissolved with 1% DMSO and ultrasonically dripped into physiological saline containing 1% β-cyclodextrin. AOT-1 was injected at 2 mg / kg into transgenic AD model mice APP / PS1 (8 months old, male) via the tail vein, and fluorescence imaging of the brain was performed before and after the injection. Figure 12 The images show fluorescence imaging of the brain in vivo before and after injection of the molecule AOT-1.

[0233] Test Example 6

[0234] The inventors of this case also used the near-infrared fluorescent probe obtained in Example 1 to conduct specific response experiments on different biological markers, and performed bar chart analysis based on the ratio of fluorescence intensity before and after the response. Samples 1. CaCl2, 2. CuSO4, 3. Fe(NO3)2, 4. KCl, 5. MgSO4, 6. Na2S, 7. NaCl, 8. NaHCO3, 9. Na2CO3, 10. NaNO2, 11. NaNO3, 12. ZnCl2, 13. Cysteine, 14. Lysine, 15. Tyrosine, 16. Glutathione, 17. Ascorbic acid, 18. Glutamic acid, 19. Leucine, 20. Arginine, 21. Bovine serum albumin, and 22. Aβ cellulose were added to a PBS buffer solution (pH 7.4, 10 mM) containing 5 μM fluorescent probe. Samples 1-12 were incubated at 100 μM, and samples 13-22 at 10 μM. After incubation at 37 °C for 10 min, the fluorescence intensity at 949 nm was collected using an 808 nm laser on an FLS1000 fluorescence spectrometer. The ratio of this intensity to the fluorescence intensity of the blank fluorescent probe was calculated, and the results were statistically analyzed using a bar chart. Figure 13 As shown.

[0235] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0236] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0237] Although this application has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of this application, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of this application to adapt particular situations or materials to the teachings of this application. Therefore, this application is not intended to be limited to the specific embodiments disclosed for carrying out this application, but rather is intended to include all embodiments falling within the scope of the appended claims.

Claims

1. An Aβ patch-specific responsive near-infrared fluorescent probe, characterized in that, The structural formula of the Aβ patch-specific responsive near-infrared fluorescent probe is as follows: 。 2. The method for preparing the Aβ patch-specific responsive near-infrared fluorescent probe as described in claim 1, characterized in that, include: An aldehyde containing a thiophene ring is subjected to a first condensation reaction with a p-aminophenylboronic acid derivative to obtain a first compound; The first compound is subjected to a second condensation reaction with a second compound containing oxonium ions to prepare an Aβ patch-specific responsive near-infrared fluorescent probe. The first compound has the structure shown in formula (II): Formula (II) R3 is ; The second compound has the structure shown in formula (Ⅲ): Formula (III) n is 2, R1 is R2 is -H.

3. The preparation method according to claim 2, characterized in that, include: An aldehyde containing a thiophene ring, a p-aminophenylboronic acid derivative, a catalyst, an alkaline substance, and a first solvent are mixed to form a first mixed reaction system. Under a protective atmosphere, the temperature is raised to carry out a first condensation reaction to obtain a first compound. The aldehydes containing the thiophene ring have the structure shown in formula (Ⅳ): Equation (Ⅳ) The p-aminophenylboronic acid derivative has the structure shown in formula (V): Formula (V).

4. The preparation method according to claim 3, characterized in that: The temperature of the first condensation reaction is 25~120℃, and the time is 12~72h.

5. The preparation method according to claim 3, characterized in that: The molar ratio of the p-aminophenylboronic acid derivative, the catalyst, the aldehyde containing the thiophene ring, and the alkaline substance is 1~1.3:0.1:1:2.7~3.

3.

6. The preparation method according to claim 3, characterized in that: The alkaline substance includes one or more of potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate.

7. The preparation method according to claim 3, characterized in that: The catalyst comprises one or more of tetra(triphenylphosphine)palladium, tetratriphenylphosphine dichloride palladium, and palladium chloride.

8. The preparation method according to claim 3, characterized in that: The first solvent includes ethylene glycol dimethyl ether.

9. The preparation method according to claim 3, characterized in that: The amount of the first solvent used is 10 to 50 mL of the first solvent per millimole of an aldehyde containing a thiophene ring.

10. The preparation method according to claim 3, characterized in that: The protective atmosphere includes a nitrogen and / or argon atmosphere.

11. The preparation method according to claim 2, characterized in that, include: A cyclic fatty ketone compound was added to concentrated sulfuric acid and stirred in an ice bath to obtain a first mixed solution; The first mixed solution is mixed with a ketone ester compound and reacted at 60-110°C for 1-4 h to obtain a second mixed solution. The second mixed solution was added to ice water containing perchlorate ions to prepare a second compound containing oxonium ions; The cyclic fatty ketone compound is cyclohexanone, and the ketone ester compound is 4-(diethylamino)salicylaldehyde.

12. The preparation method according to claim 11, characterized in that: The amount of concentrated sulfuric acid used is 1.5 to 2 mL per millimole of cyclic aliphatic ketone compound.

13. The preparation method according to claim 11, characterized in that: Cyclic fatty ketone compounds were added to concentrated sulfuric acid and stirred in an ice bath for 1–4 h.

14. The preparation method according to claim 11, characterized in that: The molar ratio of the ketone ester compound to the cyclic fatty ketone compound is 1:1.5~2.

15. The preparation method according to claim 11, characterized in that: The ice water contains perchlorate compounds, and the perchlorate compounds include any one of HClO4 and KClO4. The amount of the perchlorate compound used is 1.5 to 5 mL of perchlorate compound per millimole of cyclic aliphatic ketone compound.

16. The preparation method according to claim 2, characterized in that, include: The first compound, the second compound containing oxonium ions, and the second solvent were mixed and subjected to a second condensation reaction under a protective atmosphere and heating conditions to prepare an Aβ patch-specific responsive near-infrared fluorescent probe. The second solvent includes at least one of acetic anhydride, toluene, and n-butanol; The protective atmosphere includes a nitrogen and / or argon atmosphere.

17. The preparation method according to claim 2, characterized in that: The molar ratio of the first compound to the second compound is 1:1.5~3.

18. The preparation method according to claim 2, characterized in that: The second condensation reaction is carried out at a temperature of 25~130℃ for 2~12 h.

19. The application of the Aβ plaque-specific responsive near-infrared fluorescent probe of claim 1 in the preparation of products with the function of detecting Aβ plaques.

20. A product with the function of detecting Aβ plaques, characterized in that, Includes the Aβ patch-specific responsive near-infrared fluorescent probe as described in claim 1; The product has the ability to diagnose neurodegenerative diseases, including Alzheimer's disease; The products include fluorescent probes, reagents, or kits.

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