WO2017211220A1 - Compound for performing fluorescent marking on β-amyloid plaques and/or neurofibrillary tangles, preparation method therefor and applications thereof, and pharmaceutical composition - Google Patents

Abstract

Disclosed in the present invention are a compound for performing fluorescent marking on β-amyloid plaques and/or neurofibrillary tangles, a preparation method therefor and applications thereof, a method for performing fluorescent marking on on Aβ plaques on brain slices of a transgenic mouse, a method for performing fluorescent marking on neurofibrillary tangles of brain slices of an AD patient, a pharmaceutical composition, and a synchronous detection method for Aβ plaques and neurofibrillary tangles in the transgenic mouse and a human body tissue. The present invention provides a small-molecular fluorescent compound having no charges. An electron donating group and an electron withdrawing group in the structure form a conjugated structure of donating electrons and withdrawing electrons, and a conjugated system of π-π* transition is increased by means of carbon-carbon double bonds, so that fluorescence generated by molecules of the compound moves to a long-wave direction (bioluminescence interference is reduced), and the structure wholly satisfies the affinity of Aβ plaques or neurofibrillary tangles.

Description

Compound for fluorescently labeling β-amyloid plaque and/or neurofibrillary tangles, preparation method and application, and pharmaceutical composition

This application claims priority to Chinese Patent Application No. 201610396936.X, filed on June 07, 2016, entitled "Pyridazines and Their Uses in Medicine", the entire contents of which are hereby incorporated by reference. The citations are incorporated herein by reference.

Technical field

The present invention relates to the field of specific molecular recognition reagents, and more particularly to a compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, a preparation method and application thereof, and Aβ on a brain slice of a compound fluorescently labeled transgenic mouse Plaque method and method for labeling neurofibrillary tangles on brain slices of AD patients, a pharmaceutical composition, transgenic mice, and simultaneous detection of A[beta] plaques and neurofibrillary tangles in human tissues.

Background technique

Alzheimer’s Disease (AD) is a progressive developmental fatal neurodegenerative disease. At present, China has entered an aging society ahead of schedule. About 6 million people suffer from AD, ranking first in the world, and the number of patients is increasing at an annual rate of more than 300,000. The prevalence rate of people over 65 years old in China is 7.2%, and AD patients are developing towards a younger trend. AD has become the fourth leading cause of death among the elderly after heart disease, cancer and stroke. AD not only seriously affects the quality of life of middle-aged and elderly people, but also brings a heavy financial burden to patients and families. Because the etiology of AD is complicated, the exact pathogenesis is still unclear. It is mainly diagnosed by evaluating the cognitive function damage of patients. The confirmed patients have entered the middle and late stages of the disease and delayed treatment. The lack of effective detection methods has become a major obstacle to the early diagnosis and treatment of AD.

In the brain of AD patients, β-amyloid plaques (Aβ plaques) with β-amyloid (Aβ) as the main component and neurofibrillary tangles with hyperphosphorylated Tau protein as the main component are the most prominent AD. Two major neuropathological features. Studies have shown that the deposition of Aβ plaques or neurofibrillary tangles in the brain is earlier than the clinical stage of AD, so the development of detection agents targeting Aβ plaques or neurofibrillary tangles will provide early diagnosis of AD. , disease monitoring and treatment of therapeutic drugs provide important analytical tools.

Over the past few years, several Aβ plaque tracers for positron emission tomography (PET) have entered the clinical trial phase, with C-11-labeled [ ¹¹ C]PIB (KlunkWE, etal.Ann.Neurol. 2004, 55(3), 306-319) is the most widely used Aβ plaque PET imaging agent in the world; [ ¹⁸ F]AV-45 (WongDF, etal.J.Nucl.Med.2010,51(6) , 913-920), [ ¹⁸ F]GE-067 (KooleM, etal.J.Nucl.Med.2009, 50(5), 818-822) and [ ¹⁸ F]BAY-94-9172 (RoweCC, etal. Lancet. Neurol. 2008, 7(2), 129-135) and other three F-18-labeled Aβ imaging agents have been approved by the US FDA and European EMA for PET imaging of Aβ plaques in human brain (Villemagne VL. Ageing.Res.Rev.2016, pii: S1568-1637 (16) 30005-8). Small molecular probes for PET imaging of neurofibrillary tangles in the brain have also been clinically tested in humans, including: [ ¹⁸ F]AV-1451, [ ¹⁸ F]THK-5117, [ ¹⁸ F]THK-5351 [ ¹¹ C]PBB3 and [ ¹⁸ F]RO 6958948, etc. (Okamura N, et al. Ageing. Res. Rev. 2016, pii: S1568-1637 (15) 30045-3). However, the application of radioactive imaging agents is also limited by some factors. For example, the radiation emitted by the radioactive imaging agent has certain radiation damage to the human body, and the medical institution is required to have a cyclotron for producing radionuclides, and a radioactive imaging agent must be specialized in technology. Personnel mark preparation, long development time, and time-consuming image processing.

In contrast, optical imaging has many advantages such as safe and non-radioactive, short data acquisition time and low cost. In recent years, its application in medical diagnosis has received extensive attention; especially the emerging near-infrared fluorescence imaging technology, in its spectral range. The autofluorescence interference of biological tissues is small, the background background fluorescence signal is very low, and the penetration tissue distance can be up to several centimeters. In addition, photoacoustic imaging (PAI) can realize three-dimensional scanning imaging through the conversion of photoacoustic signals. Therefore, the development of targeted optical probes with high affinity for Aβ plaques and neurofibrillary tangles, and their direct application to the preparation of imaging agents for detecting or imaging β-amyloid deposition diseases or neurofibrillary tangles, will Has very important scientific significance and practical value. However, there are few reports on fluorescent compounds targeting Aβ plaques or neurofibrillary tangles at home and abroad, and they are still in their infancy. Based on the above technical background, the development of new small-molecule fluorescent compounds with excellent performance is in line with the urgent needs of China's social and people's livelihood and the aging society, which has great application prospects and scientific significance.

Summary of the invention

The technical problem to be solved by the present invention is that the existing imaging probes often require an external fluorophore, and the fluorophore has a large molecular structure and has a charge, and thus is not suitable for preparing Aβ in the brain that needs to pass the blood-brain barrier. A plaque or neurofibrillary tangled imaging agent, and non-specifically bound fluorophore luminescence is susceptible to interference signals for imaging.

The present invention provides a class of uncharged small molecule fluorescent compounds in which the electron-inducing group and the electron-withdrawing group form a conjugated structure of push-pull electrons and increase π-π* by a carbon-carbon double bond. The conjugated system of the transition causes the fluorescence generated by the compound molecules to move in the long-wave direction (reducing the biological autofluorescence interference), and the structure as a whole satisfies the affinity for entanglement with Aβ plaques or nerve fibers. When the compound is combined with Aβ plaque or nerve fiber entanglement, the emission wavelength is blue-shifted or red-shifted, and the fluorescence intensity is significantly increased, thereby effectively eliminating the non-specific binding compound. The luminescence of the object may cause interference with imaging, and thus the compound can be directly used as a drug or a medicinal active ingredient for detecting or imaging a β-amyloid deposition disease or a neurofibrillary tangles disease.

It is an object of the present invention to provide a small molecule fluorescent compound having high affinity for A[beta] plaque and neurofibrillary tangles.

Another object of the invention is to provide a process and use for the preparation of said compounds.

In order to achieve the above object, the present invention provides a compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in the formula (I):

Where n is 1, 2 or 3.

The invention provides a preparation method of the compound described in the above technical scheme, and the invention synthesizes the compound by a Knoevenagel condensation reaction; the compound is obtained according to the following preparation steps: taking 2 mmol of 2-methylpyrazine and 1 mmol of the corresponding aldehyde are dissolved in 20 After drying 40 mL of tetrahydrofuran, 4 mmol of potassium t-butoxide was added thereto, and the mixture was heated under reflux at 80 ° C for 2 hours, and the solvent was removed by rotary evaporation. The solid was precipitated by adding 20 mL of water, and the obtained solid was subjected to column chromatography to obtain the formula (I). compound of.

The present invention also provides a compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in formula (II):

Where n is 1, 2 or 3.

The invention provides a preparation method of the compound described in the above technical solution, wherein the compound is synthesized by a Knoevenagel condensation reaction; the compound is obtained according to the following preparation steps: taking 2 mmol of 2-methylquinoxaline and 1 mmol of the corresponding aldehyde are dissolved in 10 -30 mL of sodium hydroxide solution (5 mol/L), added Aliquat 336 (0.1 mmol), heated under reflux at 100 ° C for 8-15 hours, cooled, filtered, and the obtained solid was subjected to column chromatography to obtain a compound of formula (II). .

The present invention provides the use of the compound described in the above technical solution, in particular, the use of the compound in the preparation of a drug for detecting or imaging β-amyloid plaque or neurofibrillary tangles in an animal or human tissue.

The present invention also provides the use of the compound described in the above technical solution, in particular, the compound is in the preparation of a medicinal active ingredient of β-amyloid plaque or neurofibrillary tangles in a test or imaging animal or human tissue. application.

Preferably, the drug is an optical imaging technology drug or a photoacoustic imaging drug.

Preferably, the means of imaging is optical imaging or photoacoustic imaging.

Preferably, the means of imaging is fluorescence microscopy, laser confocal microscopy, multiphoton microscopy, optical projection tomography, light illumination microscopy, fluorescence imaging system, mesoscopic fluorescence tomography Techniques, fluorescence molecular tomography imaging techniques, multimodal imaging systems, photoacoustic imaging systems, or multispectral photoacoustic tomography.

The present invention provides a pharmaceutical composition comprising a compound according to the above technical solution and an adjuvant; the adjuvant is a pharmaceutically acceptable salt, a pharmaceutically acceptable prodrug or a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier comprises an excipient and a diluent.

Preferably, the pharmaceutically acceptable carrier comprises a liquid, which is water, physiological saline, glycerol or ethanol.

The invention also provides the use of a compound as described in the above technical scheme for the detection or visualization of beta-amyloid plaques or neurofibrillary tangles in animal or human tissues.

Preferably, the method of detecting or imaging comprises administering a detectable amount of the compound to an animal or human tissue.

Preferably, the step of detecting or imaging comprises introducing a detectable amount of the compound into an animal or human and non-invasive after a time sufficient to bind the compound to the β-amyloid plaque or neurofibrillary tangles Compounds were tested sexually.

Preferably, the step of detecting or imaging comprises: introducing the compound into an animal or a human body, and taking a sufficient amount of time to bind the compound to the β-amyloid plaque or nerve fiber entanglement, taking a tissue sample, and Detach animals or humans and detect compounds in tissues.

Preferably, the animal or human body takes a tissue sample and introduces the compound into the removed tissue sample, and the compound is detected after a time sufficient to bind the compound to the β-amyloid plaque or neurofibrillary tangles.

The invention also provides a synchronous detection method for Aβ plaques and neurofibrillary tangles in transgenic mice and human tissues, characterized in that the compounds described in the above technical schemes are fluorescently labeled in transgenic mice and human brains. A[beta] plaques and neurofibrillary tangles, the compound emitting a blue shift in the wavelength of binding to the A[beta] plaque, which, when combined with the nerve fiber knot, red-shifts the emission wavelength.

The invention provides a method for fluorescently labeling Aβ plaque on a brain slice of a transgenic mouse according to the above technical solution, comprising the following steps:

(1) using the compound solution to add brain slices covering the transgenic mice; the concentration of the compound solution is ¹ μmol·L ^-1 , and the brain slice is 10 μm;

(2) incubation at room temperature for 10 min;

(3) rinsing, air drying and fluorescence microscopic observation of the brain slice after the room temperature incubation;

The rinsing was followed by a 4% 40% ethanol rinse, a 2 minute 40% ethanol rinse, and a 30 s pure water rinse.

Preferably, the brain slice of the transgenic mouse is preferably a brain slice of a 26 month old APP/PS1 transgenic mouse.

The present invention also provides a method for fluorescently labeling nerve fiber tangles on brain slices of a patient with AD as described in the above technical solution, comprising the following steps:

(a) using the compound solution to drip the brain slice of the AD patient; the concentration of the compound solution is ¹ μmol·L ^-1 , and the brain slice is 5 μm;

(b) incubation at room temperature for 15 min;

(c) rinsing, air drying, and fluorescence microscopic observation of the brain slices after the room temperature incubation;

The rinsing was followed by a 1% 40% ethanol rinse, a 1 minute 40% ethanol rinse, and a 30 s pure water rinse.

The present invention provides a fluorescent compound having high affinity with Aβ plaque and neurofibrillary tangles based on a push-pull electronic structure, which has a novel imaging agent for a β-amyloid deposition disease or disease such as Alzheimer's disease. And the enormous potential of diagnostic drugs. In the present invention, in vitro fluorescent staining experiments show that the compound can clearly bind to Aβ plaques and neurofibrillary tangles of transgenic mouse brain slices and patient brain tissue sections, and the emission wavelength shifts and the fluorescence intensity is significantly enhanced; The results of the distribution experiments in normal mice show that the compounds have good brain-inducing ability and can be quickly cleared from the brain.

Instruction sheet

Figure 1 is a schematic view showing the synthesis process of the compounds of the formula (I) and formula (II) of the present invention;

Figure 2 shows the results of fluorescent staining of PB-1 and QB-1 for brain slices of APP/PS1 transgenic mice, respectively. The right panel shows the staining results of sulphur-sulfur S (ThS) on brain slices of adjacent mice.

Figure 3 shows the results of fluorescent staining of brain slices of patients with AD by PB-1. Figures A, B and C show the staining results of the same position in the purple, green and red bands after PB-1 staining: the Arrow head PB-1 fluorescently labeled neurofibrillary tangles, arrow pointed to PB-1 fluorescently labeled Aβ plaques; Figure D is the same position of immunostaining results;

Figure 4 is a fluorescence emission spectrum before and after mixing of PB-1 and Aβ _1-42 aggregates.

detailed description

The invention will now be further described with reference to the embodiments and the accompanying drawings.

The present invention provides a compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the structural formula of the compound is as shown in formula (I):

Wherein n is 1, 2 or 3; and the compound of the formula (I) is represented by PB-1.

The present invention also provides a compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in formula (II):

Wherein n is 1, 2 or 3; and the compound represented by the formula (II) is represented by QB-1.

The compound provided by the present invention is a kind of fluorescent compound having high affinity with Aβ plaque and neurofibrillary tangles based on push-pull electronic structure; the compound can be clearly combined with transgenic mouse brain slices and patient brain tissue sections. Aβ plaques are combined with neurofibrillary tangles, the emission wavelength shifts and the fluorescence intensity is significantly enhanced, and the compound has good brain-inducing ability and can be rapidly cleared from the brain. Further, the compound has a great potential as a novel imaging agent and a diagnostic drug for a β-amyloid deposition disease such as Alzheimer's disease or a neurofibrillary tangles disease.

In the present invention, the compound is effective for fluorescently labeling Aβ plaques and neurofibrillary tangles in transgenic mice and human brain, and the compound binds to Aβ plaques and emits a blue shift in wavelength and binds to neurofibrillary tangles. Post-launch The wavelength is red-shifted, and simultaneous real-time detection of β-amyloid plaques and neurofibrillary tangles can be achieved by multispectral imaging of individual compounds.

The present invention also provides a process for the preparation of the compound described in the above technical solution.

The present invention synthesizes the compound of the formula (I) by a Knoevenagel condensation reaction; in the present invention, the compound is obtained according to the following preparation steps: taking 2 mmol of 2-methylpyrazine and 1 mmol of the corresponding aldehyde in 20- After 40 mL of dry tetrahydrofuran, 4 mmol of potassium tert-butoxide was added, and the mixture was heated under reflux at 80 ° C for 2 hours, and the solvent was removed by rotary evaporation. The solid was precipitated by adding 20 mL of water, and the obtained solid was subjected to column chromatography to obtain the formula (I). Compound.

The present invention synthesizes the compound of the formula (II) by a Knoevenagel condensation reaction; in the present invention, the compound is obtained according to the following preparation steps: 2 mmol of 2-methylquinoxaline and 1 mmol of the corresponding aldehyde are dissolved in 10 -30 mL of sodium hydroxide solution (5 mol/L), added Aliquat 336 (0.1 mmol), heated under reflux at 100 ° C for 8-15 hours, cooled, filtered, and the obtained solid was subjected to column chromatography to obtain a compound of formula (II). .

The present invention has no special requirements for the manner of the heating reflux and column chromatography separation, and can be carried out by a method well known to those skilled in the art.

The present invention provides the use of the compound described in the above technical solution, in particular, the use of the compound in the preparation of a drug for detecting or imaging β-amyloid plaque or neurofibrillary tangles in an animal or human tissue.

The invention also provides the use of the compound described in the above technical solution, in particular, the application of the compound in preparing a medicinal active ingredient of β-amyloid plaque or neurofibrillary tangles in a test or imaging animal or human tissue. .

In the present invention, the amyloid deposition disease or neurofibrillary tangles diseases include, but are not limited to, Alzheimer's disease, amyloid cerebrovascular disease, amyloid polyneuropathy, systemic senile amyloidosis, starch Protein-like cardiomyopathy, hereditary cerebral hemorrhage with amyloidosis, Creutzfeldt-Jakob disease, type II diabetes islet tumor, frontotemporal dementia, Pick's disease, tangled-type dementia, progressive supranuclear palsy (PSP) , cortical syndrome (CBS), chronic traumatic brain disease (CTE) or ganglion glioma.

In the present invention, the drug in the application is preferably an optical imaging technique drug or a photoacoustic imaging drug. In the present invention, the means for developing is preferably optical imaging or photoacoustic imaging, further preferably fluorescence microscopy, laser confocal microscopy, multiphoton microscopy, optical projection tomography, light Slice illumination microscopy, fluorescence imaging systems, mesoscopic fluorescence tomography, fluorescence molecular tomography imaging, multimodal imaging systems, photoacoustic imaging systems, or multispectral photoacoustic tomography.

The present invention provides a pharmaceutical composition comprising a compound according to the above technical solution and an adjuvant; the adjuvant is a pharmaceutically acceptable salt, a pharmaceutically acceptable prodrug or a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier preferably includes various excipients and diluents; the pharmaceutically acceptable carrier preferably comprises a liquid, preferably water, physiological saline, glycerol or ethanol. The present invention has no special requirement for the source of the excipient, and a commercially available product well known to those skilled in the art may be used.

The invention also provides the use of the compounds described in the above technical solutions, in particular for the use of the compounds for detecting or imaging β-amyloid plaques or neurofibrillary tangles in animal or human tissues.

In the present invention, the method of detecting or imaging comprises administering a detectable amount of the compound to an animal or human tissue. The present invention has no particular requirements for the mode of administration, and a method of administration well known to those skilled in the art may be employed.

In the present invention, the step of detecting or imaging preferably comprises introducing a detectable amount of the compound into an animal or human and after a time sufficient to bind the compound to the β-amyloid plaque or neurofibrillary tangles , non-invasive detection of compounds. The present invention has no particular requirement for the manner in which the compound is introduced into an animal or a human body, and may be introduced into the animal or the human body by a drug well known to those skilled in the art. The present invention has no particular requirements for the non-invasive manner of detection of the compounds, and is well known to those skilled in the art.

Preferably, the step of detecting or imaging further comprises: introducing the compound into an animal or a human body for a sufficient amount of time to bind the compound to the β-amyloid plaque or nerve fiber entanglement, taking out the tissue sample, and detaching Animals or humans, detecting compounds in tissues. The present invention has no particular requirement for the sufficient amount of time to bind the compound to the β-amyloid plaque or neurofibrillary tangles, as is well known to those skilled in the art; in the present invention, the compound is rendered with β - sufficient time for amyloid plaque or neurofibrillary tangles to bind to compounds and beta-amyloid plaques or nerves known to those skilled in the art for beta-amyloid plaque or neurofibrillary tangles detection or imaging The time of fiber entanglement is consistent. The present invention has no special requirement for the manner in which the compound is introduced into an animal or a human body, and can be introduced into the animal or the human body by using a drug well known to those skilled in the art; the present invention has no special requirement for the manner of taking out the tissue sample. The tissue sample extraction method well known to those skilled in the art can be used; the present invention has no special requirement for the detection method of the compound in the tissue, and the detection method well known to those skilled in the art can be used.

Preferably, the step of detecting or imaging further comprises: removing the tissue sample from the animal or human body and introducing the compound into the removed tissue sample at a level sufficient to bind the compound to the beta-amyloid plaque or nerve fiber After the time of the knot, the compound was detected. The present invention has no particular requirement for the time sufficient to bind the compound to the β-amyloid plaque or neurofibrillary tangles, as is well known to those skilled in the art; In the case of binding the compound to β-amyloid plaque or neurofibrillary tangles, and compounds and β-amyloids known in the art for beta-amyloid plaque or neurofibrillary tangles detection or imaging The time of plaque or nerve fiber tangles is consistent. The present invention has no special requirement for the manner of taking out the tissue sample, and adopts a tissue sample extraction method well known to those skilled in the art; the present invention has no special requirement for introducing the extracted tissue sample into the compound, and adopts The manner in which the drug is introduced into the tissue sample is well known to those skilled in the art; the present invention has no special requirement for the manner of detecting the compound in the tissue, and a detection method well known to those skilled in the art can be used.

The invention also provides a synchronous detection method for Aβ plaque and neurofibrillary tangles in transgenic mice and human tissues, characterized in that the compound described in the above technical scheme is used for fluorescent labeling of transgenic mice and Aβ in human brain. The plaque and neurofibrillary tangles, the compound binding to the A[beta] plaque, the emission wavelength is blue-shifted, and the compound binds to the nerve fiber node and the emission wavelength is red-shifted. The present invention achieves simultaneous detection of β-amyloid plaques and neurofibrillary tangles by multispectral imaging of the compounds.

The present invention provides a method for fluorescently labeling Aβ plaque on a brain slice of a transgenic mouse brain of the compound according to the above technical solution, comprising the following steps: (1) using the compound solution to drip a brain slice covering the transgenic mouse; The concentration of the compound solution is ¹ μmol·L ^-1 , the brain slice is 10 μm; (2) incubation at room temperature for 10 min; (3) the brain slice after the room temperature incubation is subjected to rinsing, air drying and fluorescence microscopic observation; The adjacent sections were stained with thioflavin to confirm the position of Aβ plaque; the rinsing was followed by 4% 40% ethanol rinsing, 2 min 40% ethanol rinsing, and 30 s pure water rinsing. In the present invention, the brain slice of the transgenic mouse is preferably a brain slice of an APP/PS1 transgenic mouse. In the present invention, the fluorescent spot of the compound staining coincides with the position of the fluorescent spot of the adjacent slice, and the Aβ plaque can be selectively bound, and the Aβ plaque is effectively fluorescently labeled.

The present invention also provides a method for fluorescently labeling nerve fiber entanglement on brain slices of a patient with AD according to the above technical solution, comprising the following steps: (a) using the compound solution to drip a brain slice covering an AD patient; The concentration of the compound solution is ¹ μmol·L ^-1 , the brain slice is 5 μm; (b) the room temperature is incubated for 15 min; (c) the brain slice after the room temperature incubation is subjected to rinsing, air drying and fluorescence microscopic observation; The rinsing was followed by a 1% 40% ethanol rinse, a 1 minute 40% ethanol rinse, and a 30 s pure water rinse.

Compound for fluorescence-labeled β-amyloid plaque and/or neurofibrillary tangles provided by the present invention, and preparation method and application thereof, and method for fluorescently labeling Aβ plaque on transgenic mouse brain slice And a description of the method of simultaneous detection of neurofibrillary tangles on brain slices of AD patients, a pharmaceutical composition, β-amyloid plaques and neurofibrillary tangles in animal or human tissues, but cannot be understood For Limitation of the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1

The synthesis of the compound of formula (I) was carried out according to the synthetic route shown in Figure 1 to give PB-1:

2-Methylpyrazine (282.3 mg, 3 mmol) and 4-N,N dimethylaminocinnamaldehyde (175.4 mg, 1 mmol) were dissolved in 20 mL of dry tetrahydrofuran, and potassium t-butoxide (448.8 mg, 4 mmol) was added at 80 ° C The mixture was heated under reflux for 2 hours, and the solvent was evaporated to dryness. The solid was precipitated by adding 20 mL of water, and the obtained solid was subjected to column chromatography to give 105.8 mg of PB-1 (yield: 42.1%). The structure is as follows: 1H-NMR (400MHz, CDCl ₃ ): δ 8.50 (s, 1H), 8.47 (s, 1H), 8.30 (s, 1H), 7.51 (dt, J = 36.4, 18.2 Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 7.0 Hz, 1H), 6.81 (d, J = 4.1 Hz, 1H), 6.70 (d, J = 7.7 Hz, 1H), 6.61 ( s, 1H), 6.58 (s, 1H), 3.00 (s, 6H).

Example 2

The synthesis of the compound of formula (II) was carried out according to the synthetic route shown in Figure 1, to give QB-1:

2-methylquinoxaline (288 mg, 2 mmol) and 4-N,N-dimethylaminocinnamaldehyde (175.4 mg, 1 mmol) were dissolved in 15 mL of sodium hydroxide solution (5 mol/L), and Aliquat 336 (43 mg, 0.1 mmol) was added. The mixture was heated under reflux at 100 ° C for 10 hours, cooled, filtered, and the obtained solid was subjected to column chromatography to give 91.0 mg of QB-1 (yield 30.2%). The structure is as follows: 1H-NMR (400MHz, CDCl3): δ8.90 (s, 1H), 8.03-7.99 (m, 1H), 7.73-7.63 (m, 4H), 7.40 (d, 2H, J = 8.8 Hz) , 6.89 (d, 2H, J = 8.0 Hz), 6.81 (d, 1H, J = 15.6 Hz), 6.70 (d, 2H, J = 8.8 Hz), 3.02 (s, 6H).

Example 3: Determination of optical parameters

1. Determination of maximum absorption wavelength and molar absorption coefficient

Prepare a concentration gradient of the compound, such as 1, 5, 10, 20, 25 μM solution, with methylene chloride as the solvent, scan the UV absorption spectrum with an ultraviolet-visible spectrophotometer, and record the maximum absorption wavelength (λabs) and absorbance. And calculate the molar absorption coefficient ε (M ^-1 cm ^-1 ). The maximum absorption wavelength and molar absorption coefficient of some of the compounds in the examples of the present invention are shown in Table 1.

2. Determination of fluorescence excitation wavelength and fluorescence emission wavelength

The compounds of the present invention have good fluorescent properties. In order to examine the fluorescent properties of the compound of the present invention, the specific procedure is as follows: accurately weigh the compound of the present invention, dissolve it in dichloromethane, and dilute to ¹ μmol·L ^-1 with dichloromethane. Fluorescence detection was performed using a fluorescence spectrophotometer. The excitation/emission wavelength is fixed and the emission/excitation wavelength is continuously scanned at 400-750 nm, and a wave line image is drawn. The maximum excitation wavelength (λ _ex ) and the maximum emission wavelength (λ _em ) of some of the compounds in the examples of the present invention are shown in Table 1.

Optical properties of some of the compounds in the examples in Table 1

Example 4: In vitro fluorescent staining experiment

1. Fluorescent staining of mouse brain slices

The compounds of the present invention are capable of clearly staining A[beta] plaques on brain sections of transgenic mice and efficiently fluorescently labeling A[beta] plaques. In order to examine the ability to label Aβ plaques, the specific procedure was as follows: a compound of the present invention having a concentration of ¹ μmol·L ^-1 was prepared, and a brain slice (10 μm) covering APP/PS1 transgenic mice (26 months old) was separately added dropwise. The cells were incubated at room temperature for 10 minutes; the sections were rinsed in the order of 40% ethanol (2 minutes), 40% ethanol (2 minutes), and pure water (30 seconds), and air-dried and observed under a fluorescence microscope. Adjacent sections were stained with thioflavin to confirm A[beta] plaque location. The fluorescent spot of the compound stained in the present invention is identical to the position of the fluorescent spot of the adjacent slice, and can selectively bind the Aβ plaque and effectively fluorescently label the Aβ plaque. Among them, the dyeing results of some of the compounds in the examples of the present invention are shown in FIG.

2. Fluorescent staining of brain slices of AD patients

The compounds of the present invention are capable of clearly and efficiently fluorescently labeling neurofibrillary tangles on brain slices of AD patients. The specific implementation steps are as follows: the compound of the present invention is prepared at a concentration of ¹ μmol·L ^-1 , and the brain slices (5 μm) of the AD patient are separately added and incubated for 15 minutes at room temperature; the sections are 40% ethanol (1 minute), 40% ethanol. The mixture was rinsed in the order of (1 minute) and pure water (30 seconds), and air-dried and observed with a fluorescence microscope. The same section was confirmed to be entangled with nerve fibers by immunostaining with phosphorylated Tau protein-specific antibody (AT-8) and DAB stain. The results of the dyeing of some of the compounds in the examples of the present invention are shown in Fig. 3. The compound PB-1 of the present invention can clearly and efficiently fluorescently label the nerve fiber in the red light band without displaying the Aβ plaque fluorescence, and can clearly and effectively fluorescently label the Aβ plaque in the violet light band without The neurofibrillary tangles are shown to be fluorescent; therefore, simultaneous multi-spectral imaging of PB-1 in different bands enables simultaneous and specific discrimination between neurofibrillary tangles and A[beta] plaques.

Example 5: Fluorescence spectra of compounds after mixing Aβ _1-42 aggregates

The compounds of the present invention have fluorescence-enhancing properties upon binding to A[beta] aggregates. In order to examine the change in the fluorescence spectral behavior of the compound of the present invention after mixing with Aβ, the specific procedure is as follows: accurately weigh the compound of the present invention, dissolve it in ethanol, and dilute it to ¹ μmol·L ^-1 with PBS. Fluorescence detection was performed using a fluorescence spectrophotometer. The excitation/emission wavelength is fixed and the emission/excitation wavelength is continuously scanned at 400-750 nm, and a wave line image is drawn. Aβ aggregates were cultured in a 37 ° C water bath using Aβ _1-42 protein for simulating Aβ aggregates in the human brain. The compound ( ¹ μmol·L ^-1 ) was mixed with Aβ _1-42 aggregate (2.75 μmol·L ^-1 ), and fluorescence detection was carried out using a fluorescence spectrophotometer. The excitation/emission wavelength is fixed and the emission/excitation wavelength is continuously scanned at 400-750 nm, and a wave line image is drawn. The compounds of the present invention have fluorescence-enhancing properties upon binding to A[beta] aggregates. The fluorescence spectrum behavior of the compound in the examples of the present invention before and after mixing with the Aβ _1-42 aggregate is shown in FIG. 4 . The fluorescence intensity of the compound PB-1 mixed with the Aβ _1-42 aggregate was significantly enhanced, which was 76 times the fluorescence intensity of the compound itself.

Example 6: Brain ingestion and clearance experiment in normal mice

The compounds of the present invention are able to cross the blood-brain barrier into the brain quickly and efficiently with good initial uptake and clearance rates. The specific implementation steps are as follows: taking the compound of the present invention (2.0 mg/kg, containing 20% dimethyl sulfoxide and 80% propylene glycol), and injecting into the normal mouse (n=5) through the tail vein, respectively, after injection 2 At 10, 30, and 60 minutes, the brain was decapitated. The brain tissue was weighed and homogenized, and extracted with acetonitrile 1 mL × 3 times, centrifuged at low temperature (10000 r, 4 ° C) for 5 min, and the supernatant was taken, filtered through a 0.22 μm microporous membrane, and injected into the HPLC instrument to analyze and calculate %injecteddosepergram ( %ID/g). The HPLC analysis conditions of some of the compounds in the examples of the present invention are shown in Table 2. The analysis results of some of the compounds in the examples of the present invention are shown in Table 3. The compounds of the present invention are able to rapidly cross the blood-brain barrier into the brain (which has entered the brain 2 minutes after injection) and have good initial brain uptake and clearance rates.

Table 2 HPLC analysis conditions

The above description of the embodiments is merely to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention. Various modifications to these embodiments are obvious to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but the scope of the invention is to be accorded

Claims (21)

A compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in formula (I):

Where n is 1, 2 or 3. The method for producing a compound according to claim 1, wherein the compound is synthesized by a Knoevenagel condensation reaction, which is obtained according to the following preparation steps: taking 2 mmol of 2-methylpyrazine and 1 mmol of the corresponding aldehyde in 20-40 mL After drying the tetrahydrofuran, 4 mmol of potassium t-butoxide was added, and the mixture was heated under reflux at 80 ° C for 2 hours, and the solvent was removed by rotary evaporation. The solid was precipitated by adding 20 mL of water, and the obtained solid was subjected to column chromatography to give a compound of the formula (I). . A compound for fluorescently labeling β-amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula shown in formula (II):

Where n is 1, 2 or 3. The method for producing a compound according to claim 3, wherein the compound is synthesized by a Knoevenagel condensation reaction, which is obtained according to the following preparation steps: 2 mmol of 2-methylquinoxaline and 1 mmol of the corresponding aldehyde are dissolved in 10- 30 mL of a sodium hydroxide solution (5 mol/L) was added to Aliquat 336 (0.1 mmol), and the mixture was heated under reflux at 100 ° C for 8-15 hours, cooled, filtered, and the obtained solid was subjected to column chromatography to give a compound of formula (II). Use of the compound according to claim 1 or 3, characterized in that the compound is used in the preparation of a medicament for detecting or imaging β-amyloid plaque or neurofibrillary tangles in an animal or human tissue. Use of the compound according to claim 1 or 3, characterized in that the compound is used for the preparation of a pharmaceutically active ingredient for detecting β-amyloid plaque or neurofibrillary tangles in an animal or human tissue. The use according to claim 5 or 6, wherein the drug is an optical imaging drug Physical or photoacoustic imaging drugs. The use according to claim 5 or 6, wherein the means of developing is optical imaging or photoacoustic imaging. The application according to claim 8, wherein the means for developing is fluorescence microscopy, laser confocal microscopy, multiphoton microscopy, optical projection tomography, and light illumination microscopy. , fluorescence imaging systems, mesoscopic fluorescence tomography, fluorescence molecular tomography imaging, multimodal imaging systems, photoacoustic imaging systems, or multispectral photoacoustic tomography. A pharmaceutical composition comprising a compound according to claim 1 or 3 and an adjuvant; the adjuvant being a pharmaceutically acceptable salt, a pharmaceutically acceptable prodrug or a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable carrier comprises an excipient and a diluent. The pharmaceutical composition according to claim 10 or 11, wherein the pharmaceutically acceptable carrier comprises a liquid, and the liquid is water, physiological saline, glycerol or ethanol. Use of the compound of claim 1 or 3 for the detection or visualization of beta-amyloid plaques or neurofibrillary tangles in animal or human tissues. The use according to claim 13 wherein the method of detecting or developing comprises administering a detectable amount of the compound to an animal or human tissue. The use according to claim 13 wherein said step of detecting or imaging comprises introducing a detectable amount of said compound into an animal or human and sufficient to cause a compound with a beta-amyloid plaque or nerve After the time of fiber entanglement, the compound is non-invasively tested. The use according to claim 13 wherein said step of detecting or imaging comprises introducing said compound into an animal or human body for a sufficient amount of time to cause the compound to be associated with a beta-amyloid plaque or nerve. The fiber is entangled, the tissue sample is taken, and the animal or human body is removed, and the compound in the tissue is detected. The use according to claim 13 wherein the animal or human body takes a tissue sample and introduces said compound into said tissue sample for removal sufficient to bind said compound to beta-amyloid plaque or nerve fiber. After the entanglement time, the compound is detected. A method for simultaneous detection of β-amyloid plaques and neurofibrillary tangles in transgenic mice or human brains, characterized in that the compound of claim 1 or 3 is used to fluorescently label transgenic mice or β- in human brain Amyloid plaques and neurofibrillary tangles, the compound emitting a blue shift in the wavelength of binding to the A[beta] plaque, which binds to the nerve fiber knot and red-shifts the emission wavelength. A method of fluorescently labeling A[beta] plaque on a brain slice of a transgenic mouse according to claim 1 or 3, comprising the steps of: (1) using the compound solution to add brain slices covering the transgenic mice; the concentration of the compound solution is ¹ μmol·L ^-1 , and the brain slice is 10 μm; (2) incubation at room temperature for 10 min; (3) rinsing, air drying and fluorescence microscopic observation of the brain slice after the room temperature incubation; The rinsing was followed by a 4% 40% ethanol rinse, a 2 minute 40% ethanol rinse, and a 30 s pure water rinse. The method according to claim 19, wherein the brain slice of the transgenic mouse is a brain slice of a 26-month-old APP/PS1 transgenic mouse. A method of fluorescently labeling a neurofibrillary tangles on a brain slice of an AD patient according to claim 1 or 3, comprising the steps of: (a) using the compound solution to drip the brain slice of the AD patient; the concentration of the compound solution is ¹ μmol·L ^-1 , and the brain slice is 5 μm; (b) incubation at room temperature for 15 min; (c) rinsing, air drying, and fluorescence microscopic observation of the brain slices after the room temperature incubation; The rinsing was followed by a 1% 40% ethanol rinse, a 1 minute 40% ethanol rinse, and a 30 s pure water rinse.

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