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WO2017211220A1 - Composé destiné à effectuer un marquage fluorescent sur des plaques ss-amyloïdes et/ou des enchevêtrements neurofibrillaires, procédé pour sa préparation et applications correspondantes et composition pharmaceutique - Google Patents

Composé destiné à effectuer un marquage fluorescent sur des plaques ss-amyloïdes et/ou des enchevêtrements neurofibrillaires, procédé pour sa préparation et applications correspondantes et composition pharmaceutique Download PDF

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WO2017211220A1
WO2017211220A1 PCT/CN2017/086834 CN2017086834W WO2017211220A1 WO 2017211220 A1 WO2017211220 A1 WO 2017211220A1 CN 2017086834 W CN2017086834 W CN 2017086834W WO 2017211220 A1 WO2017211220 A1 WO 2017211220A1
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compound
neurofibrillary tangles
imaging
brain
animal
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Chinese (zh)
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程妍
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Sichuan University
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Sichuan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/12—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
    • C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
    • C07D241/40—Benzopyrazines
    • C07D241/42—Benzopyrazines with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring

Definitions

  • the present invention relates to the field of specific molecular recognition reagents, and more particularly to a compound for fluorescently labeling ⁇ -amyloid plaques and/or neurofibrillary tangles, a preparation method and application thereof, and A ⁇ 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.
  • AD Alzheimer’s Disease
  • AD is a progressive developmental fatal neurodegenerative disease.
  • 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.
  • 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.
  • ⁇ -amyloid plaques A ⁇ plaques
  • a ⁇ ⁇ -amyloid
  • a ⁇ ⁇ -amyloid
  • a ⁇ neurofibrillary tangles with hyperphosphorylated Tau protein as the main component
  • Two major neuropathological features Studies have shown that the deposition of A ⁇ plaques or neurofibrillary tangles in the brain is earlier than the clinical stage of AD, so the development of detection agents targeting A ⁇ plaques or neurofibrillary tangles will provide early diagnosis of AD. , disease monitoring and treatment of therapeutic drugs provide important analytical tools.
  • radioactive imaging agents are also limited by some factors.
  • 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.
  • optical imaging has many advantages such as safe and non-radioactive, short data acquisition time and low cost.
  • 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.
  • photoacoustic imaging PAI can realize three-dimensional scanning imaging through the conversion of photoacoustic signals.
  • 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 A ⁇ 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 ⁇ - ⁇ * 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 A ⁇ plaques or nerve fibers.
  • 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 ⁇ -amyloid deposition disease or a neurofibrillary tangles disease.
  • Another object of the invention is to provide a process and use for the preparation of said compounds.
  • the present invention provides a compound for fluorescently labeling ⁇ -amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in the formula (I):
  • n 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 ⁇ -amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in formula (II):
  • n 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 ⁇ -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 ⁇ -amyloid plaque or neurofibrillary tangles in a test or imaging animal or human tissue. application.
  • the drug is an optical imaging technology drug or a photoacoustic imaging drug.
  • the means of imaging is optical imaging or photoacoustic imaging.
  • 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
  • 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.
  • the pharmaceutically acceptable carrier comprises an excipient and a diluent.
  • 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.
  • the method of detecting or imaging comprises administering a detectable amount of the compound to an animal or human tissue.
  • 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 ⁇ -amyloid plaque or neurofibrillary tangles Compounds were tested sexually.
  • 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 ⁇ -amyloid plaque or nerve fiber entanglement, taking a tissue sample, and Detach animals or humans and detect compounds in tissues.
  • 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 ⁇ -amyloid plaque or neurofibrillary tangles.
  • the invention also provides a synchronous detection method for A ⁇ 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.
  • the invention provides a method for fluorescently labeling A ⁇ plaque on a brain slice of a transgenic mouse according to the above technical solution, comprising the following steps:
  • the concentration of the compound solution is 1 ⁇ mol ⁇ L -1 , and the brain slice is 10 ⁇ m;
  • the rinsing was followed by a 4% 40% ethanol rinse, a 2 minute 40% ethanol rinse, and a 30 s pure water rinse.
  • 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:
  • 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 A ⁇ plaque and neurofibrillary tangles based on a push-pull electronic structure, which has a novel imaging agent for a ⁇ -amyloid deposition disease or disease such as Alzheimer's disease. And the enormous potential of diagnostic drugs.
  • in vitro fluorescent staining experiments show that the compound can clearly bind to A ⁇ 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.
  • 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.
  • ThiS sulphur-sulfur S
  • 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 A ⁇ 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 A ⁇ 1-42 aggregates.
  • the present invention provides a compound for fluorescently labeling ⁇ -amyloid plaques and/or neurofibrillary tangles, characterized in that the structural formula of the compound is as shown in formula (I):
  • n 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 ⁇ -amyloid plaques and/or neurofibrillary tangles, characterized in that the compound has the structural formula as shown in formula (II):
  • n 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 A ⁇ 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.
  • a ⁇ 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.
  • the compound has a great potential as a novel imaging agent and a diagnostic drug for a ⁇ -amyloid deposition disease such as Alzheimer's disease or a neurofibrillary tangles disease.
  • the compound is effective for fluorescently labeling A ⁇ plaques and neurofibrillary tangles in transgenic mice and human brain, and the compound binds to A ⁇ 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 ⁇ -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 ⁇ -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 ⁇ -amyloid plaque or neurofibrillary tangles in a test or imaging animal or human tissue. .
  • 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.
  • 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 frontotemporal dementia
  • the drug in the application is preferably an optical imaging technique drug or a photoacoustic imaging drug.
  • 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
  • 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.
  • 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 ⁇ -amyloid plaques or neurofibrillary tangles in animal or human tissues.
  • 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.
  • 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 ⁇ -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.
  • 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 ⁇ -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 ⁇ -amyloid plaque or neurofibrillary tangles, as is well known to those skilled in the art; in the present invention, the compound is rendered with ⁇ - 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.
  • 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 ⁇ -amyloid plaque or neurofibrillary tangles, as is well known to those skilled in the art; In the case of binding the compound to ⁇ -amyloid plaque or neurofibrillary tangles, and compounds and ⁇ -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 A ⁇ 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 A ⁇ 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 ⁇ -amyloid plaques and neurofibrillary tangles by multispectral imaging of the compounds.
  • the present invention provides a method for fluorescently labeling A ⁇ 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 ⁇ mol ⁇ L -1 , the brain slice is 10 ⁇ m; (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 A ⁇ plaque; the rinsing was followed by 4% 40% ethanol rinsing, 2 min 40% ethanol rinsing, and 30 s pure water rinsing.
  • the brain slice of the transgenic mouse is preferably a brain slice of an APP/PS1 transgenic mouse.
  • the fluorescent spot of the compound staining coincides with the position of the fluorescent spot of the adjacent slice, and the A ⁇ plaque can be selectively bound, and the A ⁇ 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 ⁇ mol ⁇ L -1 , the brain slice is 5 ⁇ m; (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.
  • the compounds of the present invention have good fluorescent properties.
  • the specific procedure is as follows: accurately weigh the compound of the present invention, dissolve it in dichloromethane, and dilute to 1 ⁇ mol ⁇ 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 ( ⁇ ex ) and the maximum emission wavelength ( ⁇ em ) of some of the compounds in the examples of the present invention are shown in Table 1.
  • 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.
  • the specific procedure was as follows: a compound of the present invention having a concentration of 1 ⁇ mol ⁇ L -1 was prepared, and a brain slice (10 ⁇ m) 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 A ⁇ plaque and effectively fluorescently label the A ⁇ plaque.
  • the dyeing results of some of the compounds in the examples of the present invention are shown in FIG.
  • 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 ⁇ mol ⁇ L -1 , and the brain slices (5 ⁇ m) 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.
  • AT-8 phosphorylated Tau protein-specific antibody
  • 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 A ⁇ plaque fluorescence, and can clearly and effectively fluorescently label the A ⁇ 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 A ⁇ 1-42 aggregates
  • the compounds of the present invention have fluorescence-enhancing properties upon binding to A[beta] aggregates.
  • the specific procedure is as follows: accurately weigh the compound of the present invention, dissolve it in ethanol, and dilute it to 1 ⁇ mol ⁇ 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.
  • a ⁇ aggregates were cultured in a 37 ° C water bath using A ⁇ 1-42 protein for simulating A ⁇ aggregates in the human brain.
  • the compound ( 1 ⁇ mol ⁇ L -1 ) was mixed with A ⁇ 1-42 aggregate (2.75 ⁇ mol ⁇ 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 A ⁇ 1-42 aggregate is shown in FIG. 4 .
  • the fluorescence intensity of the compound PB-1 mixed with the A ⁇ 1-42 aggregate was significantly enhanced, which was 76 times the fluorescence intensity of the compound itself.
  • 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 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 ⁇ m 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.

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Abstract

La présente invention concerne un composé destiné à effectuer un marquage fluorescent sur des plaques ß-amyloïdes et/ou des enchevêtrements neurofibrillaires, un procédé pour sa préparation et des applications correspondantes, un procédé pour effectuer un marquage fluorescent sur des plaques Aβ sur des tranches cérébrales d'une souris transgénique, un procédé pour effectuer un marquage fluorescent sur des enchevêtrements neurofibrillaires de tranches cérébrales d'un patient atteint de la maladie d'Alzheimer, une composition pharmaceutique et un procédé de détection synchrone de plaques Aβ et d'enchevêtrements neurofibrillaires chez la souris transgénique et un tissu corporel humain. La présente invention concerne un composé fluorescent de petite molécule n'ayant pas de charges. Un groupe électrodonneur et un groupe électroaccepteur dans la structure forment une structure conjuguée d'électrons donneurs et d'électrons accepteurs et un système conjugué de transition π-π* est augmenté au moyen de doubles liaisons carbone-carbone, de telle sorte que la fluorescence générée par les molécules de composé se déplace vers les ondes longues (l'interférence de bioluminescence est réduite) et la structure satisfait totalement à l'affinité des plaques Aβ ou des enchevêtrements neurofibrillaires.
PCT/CN2017/086834 2016-06-07 2017-06-01 Composé destiné à effectuer un marquage fluorescent sur des plaques ss-amyloïdes et/ou des enchevêtrements neurofibrillaires, procédé pour sa préparation et applications correspondantes et composition pharmaceutique Ceased WO2017211220A1 (fr)

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