Boron difluoride curcumin near-infrared fluorescence diagnosis and treatment probe as well as preparation method and application thereof

Abstract

The invention discloses a boron difluoride curcumin skeleton compound as shown in a formula (I), and belongs to the field of medical imaging molecular probes. The boron difluoride curcumin skeleton compound disclosed by the invention has the characteristics of near-infrared emission and large Stokes shift, can be used as a fluorescent probe for marking key pathological proteins such as beta-amyloid protein (Abeta) aggregate of Alzheimer's disease (AD) and the like, and has the characteristics of high sensitivity, high signal-to-noise ratio and high imaging; in addition, in-vivo level A beta imaging can be realized through a blood brain barrier. The boron difluoride curcumin skeleton compound disclosed by the invention also has a treatment effect and can be used for remarkably inhibiting A beta monomers from forming aggregates and depolymerizing A beta aggregates, and meanwhile, the compound shown as the formula (I) is used for remarkably inhibiting A beta-induced microglial cell inflammatory response. The invention also provides a preparation method of the boron difluoride curcumin skeleton compound, an intermediate compound and application of the boron difluoride curcumin skeleton compound. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medical imaging molecular probes, and in particular to an organic small molecule near-infrared fluorescent diagnostic and therapeutic probe with a boron difluoride curcumin structure and a preparation method thereof, as well as an application of the probe as a fluorescent probe in the pathogenesis, diagnosis and treatment of nervous system diseases with Aβ aggregates as key pathological proteins. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that can lead to a gradual loss of cognitive function, accompanied by a series of changes in mental state, and eventually lead to dementia. According to the 2019 World Alzheimer's Report, there are more than 50 million dementia patients worldwide, and by 2050, this number will increase to 152 million. Recent statistics show that there are more than 10 million AD patients in my country, ranking first in the world, and it is expected to exceed 40 million by 2050. The exact pathogenesis of AD has not yet been elucidated, and the Aβ cascade hypothesis is one of the main hypotheses. The latest research has found that compared with Aβ monomers, Aβ aggregates, especially oligomers, are more potent neurotoxic substances, which have been present in the brain for more than 10 years before the onset of disease symptoms, causing damage to neural tissue. Therefore, efficient early diagnosis and treatment of Aβ aggregates in the brain is expected to become an important strategy for the effective prevention and treatment of AD.

[0003] In recent years, several fluorescent probes have been discovered to detect Aβ oligomers. Ran et al. reported a near-infrared fluorescent probe called CRANAD-58 [J.Am.Chem.Soc., 2013, 135, 16397-16409], which showed a significant increase in fluorescence intensity when incubated with Aβ monomers and oligomers, however, CRANAD-58 still showed significant binding affinity to Aβ aggregates. In 2015, Chang et al. demonstrated a probe BD-Oligo [J.Am.Chem.Soc., 2015, 137, 13503-13509] with high binding specificity for Aβ oligomers, but its blood-brain barrier penetration ability was low and it could only label 12-month-old APP / PS1 mice. Other probes such as AN-SP [Chem. Commun., 2016, 52, 8865-8868] and F-SLOH [Chem. Sci., 2017, 8, 8279-8284] have moderate affinity for Aβ oligomers, with Kd values ​​of only 1.7μM and 0.66μM, respectively. Moreover, the emission wavelengths of these three probes are not in the near-infrared region, and are not suitable for in vivo studies due to the high biological background. In 2017, Ran's group developed another near-infrared probe CRANAD-102 [Chem. Sci., 2017, 8, 7710-7717], which exhibited high binding affinity (7.5nM) for Aβ oligomers. In addition, it can distinguish between 5-month-old APP / PS1 mice and WT mice. However, CRANAD-102 exhibited considerable affinity for Aβ monomers. In 2020, Li's group reported a "V-shaped" structure of PTO-29 [Chem. Commun., 2020, 56, 583-586] that can bind well to Aβ oligomers with a moderate binding affinity (248nM).

[0004] In summary, there is an urgent need in this field to develop a near-infrared organic small molecule fluorescent probe that can highly selectively identify and bind to AD pathological protein Aβ aggregates. Summary of the invention

[0005] One object of the present invention is to provide a near-infrared organic small molecule fluorescent probe that can highly selectively recognize and bind to AD pathological protein Aβ aggregates, thereby achieving early diagnosis of AD while also having a therapeutic function.

[0006] In a first aspect of the present invention, a boron difluoride curcumin skeleton compound as shown in formula I is provided.

[0007]

[0008] In a second aspect of the present invention, a method for preparing a boron difluoride curcumin skeleton compound shown in formula I is provided, comprising the following steps:

[0009] Under alkaline conditions, in an organic solvent, the compound of formula 4 reacts with the compound of formula 7 to obtain the compound of formula (I) XCY13,

[0010]

[0011] In another preferred embodiment, the reaction temperature is 25-100° C. In another preferred embodiment, the amount of the base is 0.1-1.5 molar equivalents of the compound of formula 4; preferably 0.1-1.0; more preferably 0.1-0.3.

[0012] In another preferred embodiment, the base is selected from the following group: n-butylamine, triethylamine, piperidine, pyridine, or a combination thereof.

[0013] In another preferred embodiment, the organic solvent is selected from the following group: C2-C6 nitrile solvents, C1-C6 hydrocarbon solvents, C6-C10 aromatic hydrocarbon solvents, sulfone solvents, amide solvents, or a combination thereof; preferably, toluene, acetonitrile, chloroform, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof.

[0014] In another preferred embodiment, the reaction temperature is 40-100°C; preferably 40-80°C.

[0015] In another preferred embodiment, the molar ratio of the compound of formula 4 to the compound of formula 7 is 1:(1±0.5).

[0016] In another preferred embodiment, the preparation method of the boron difluoride curcumin skeleton compound shown in Formula I further comprises the following steps:

[0017]

[0018] (a) reacting a compound of formula 1 with a compound of formula 2, 4-iodoanisole, to obtain a compound of formula 3;

[0019] (b) reacting the compound of formula 3 obtained in step (a) with boron trifluoride etherate to obtain a compound of formula 4;

[0020] (c) subjecting the compound of formula 5, 5-formaldehyde furan-2-boric acid, and the compound of formula 6, 5-bromo-2-dimethylaminopyridine, to Suzuki coupling reaction to obtain a compound of formula 7;

[0021] (d) Under alkaline conditions, condensing and dehydrating the compound of formula 4 obtained in step (b) and the compound of formula 7 obtained in step (c) to obtain compound XCY13 of formula (I).

[0022] In a third aspect of the present invention, a pharmaceutical composition or formulation is provided, comprising (a) a therapeutically effective amount of a compound of formula I; and (b) a pharmaceutically acceptable carrier.

[0023] In another preferred embodiment, component (a) accounts for 0.001-99.99 wt % of the total weight of the pharmaceutical composition; preferably 0.01-99.9 wt %; more preferably 0.05-90 wt %.

[0024] In another preferred embodiment, the dosage form of the pharmaceutical composition or preparation is an injection, tablet, capsule, pill, suspension or emulsion.

[0025] In the fourth aspect of the present invention, there is provided the use of the compound described in the first aspect of the present invention or the pharmaceutical composition or preparation described in the third aspect of the present invention for preparing (a) a drug for inhibiting the polymerization of β-amyloid protein monomers; (b) a drug for disaggregating β-amyloid protein aggregates; (c) a drug for inhibiting the inflammatory response of microglia induced by β-amyloid protein; and / or (d) a drug for preventing and / or treating neurodegenerative diseases.

[0026] In another preferred embodiment, the neurodegenerative disease is a central nervous system disease associated with β-amyloid protein (Aβ) deposition.

[0027] In another preferred embodiment, the neurodegenerative disease is Alzheimer's disease.

[0028] In another preferred embodiment, the β-amyloid protein (Aβ) includes Aβ aggregates and Aβ monomers.

[0029] In a fifth aspect of the present invention, a near-infrared fluorescent diagnostic and therapeutic probe is provided, wherein the probe is a curcumin skeleton compound shown in Formula I;

[0030]

[0031] In the sixth aspect of the present invention, a method for inhibiting β-amyloid protein-induced microglial inflammatory response is provided, wherein an effective amount of a compound of formula I is administered to a subject in need thereof, thereby inhibiting β-amyloid protein-induced microglial inflammatory response.

[0032] In another preferred embodiment, the method is in vitro.

[0033] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0034] In the seventh aspect of the present invention, a method for inhibiting the polymerization of β-amyloid protein monomers is provided, comprising administering an effective amount of a compound of formula I to a subject in need thereof, thereby inhibiting the polymerization of β-amyloid protein monomers.

[0035] In another preferred embodiment, the method is in vitro.

[0036] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0037] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a staining diagram of the fluorescent probe XCY13 (ie, the compound of formula (I)) of the present invention.

[0039] Figure 2 The graph is a graph showing the change in fluorescence spectrum after the fluorescent probe XCY13 (ie, the compound of formula (I)) of the present invention interacts with Aβ aggregates.

[0040] Figure 3 The figures are in vivo imaging images of transgenic mice and wild-type mice using the fluorescent probe XCY13 of the present invention (ie, the compound of formula (I)).

[0041] Figure 4 The ThT fluorescence intensity diagram shows that the fluorescent probe XCY13 (ie, the compound of formula (I)) of the present invention inhibits Aβ aggregation and depolymerizes Aβ.

[0042] Figure 5 The fluorescent probe XCY13 (i.e., the compound of formula (I)) of the present invention inhibits Aβ-induced microglial inflammation.

[0043] Figure 6 The fluorescent probe XCY13 (i.e., the compound of formula (I)) of the present invention significantly improves the accuracy rate of 5×FAD transgenic mice in the Y-maze. DETAILED DESCRIPTION

[0044] After extensive and in-depth research, the inventors have provided for the first time a near-infrared organic small molecule fluorescent probe that can quickly and efficiently identify Aβ aggregates. The fluorescent probe is a boron difluoride curcumin skeleton compound that can achieve in vivo imaging under near-infrared light. At the same time, the compound can significantly inhibit the formation of aggregates of Aβ monomers and disaggregate Aβ aggregates, and significantly inhibit the Aβ-induced inflammatory response of small cell keratinocytes. The near-infrared fluorescent probe of the present invention has high sensitivity and signal-to-noise ratio, and can quickly and efficiently identify Aβ aggregates. Based on this, the inventors have completed the present invention.

[0045] definition

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0049] Amyloid β (Aβ)

[0050] Aβ can be divided into Aβ monomers and Aβ aggregates (including Aβ oligomers and Aβ fibers) according to their aggregation states. Aβ monomers are the earliest Aβ forms. Current literature shows that they do not have significant neurotoxic effects. Due to their small molecular weight and presence in the blood, electrochemical sensors are often used to detect the Aβ monomer content in the blood. Aβ monomers aggregate to form a series of Aβ oligomers, which appear in the brain before the two major pathological features of AD, senile plaques and neurofibrillary tangles, and stimulate complex cascade reactions at the molecular and cellular levels, leading to neuronal loss, and then to cognitive impairment and brain atrophy, suggesting that Aβ oligomers are neurotoxic forms of Aβ. Aβ oligomers further aggregate into Aβ polymers, which then form Aβ fibers and deposit to form Aβ plaques. Aβ plaques deposited in AD patients are called senile plaques. Considering that Aβ aggregates appear later than Aβ monomers and are more toxic than Aβ monomers, targeting Aβ aggregates in the brain is expected to achieve the dual purposes of diagnosis and treatment.

[0051] Preparation method of curcumin skeleton compound shown in formula I

[0052] The preparation method of the curcumin skeleton compound shown in Formula I of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art, and such a combination can be easily performed by a technician in the field to which the present invention belongs.

[0053] Typically, the present invention provides a method for preparing a boron difluoride curcumin skeleton compound shown in formula I, comprising the following steps:

[0054] Under alkaline conditions, in an organic solvent, the compound of formula 4 reacts with the compound of formula 7 to obtain the compound of formula (I) XCY13,

[0055]

[0056] As used herein, the reaction temperature may be any temperature between room temperature and the boiling point of the reaction solvent. Typically, the reaction temperature is 25-100°C; preferably, the reaction temperature is 40-100°C; more preferably, 40-80°C.

[0057] As used herein, the reaction is carried out under alkaline conditions, and the alkaline conditions can be provided by alkaline substances commonly used in the art. Typically, the base includes but is not limited to n-butylamine, triethylamine, piperidine, pyridine, or a combination thereof. The amount of the base is 0.1-1.5 of the molar equivalent of the compound of formula 4; preferably 0.1-1.0; more preferably 0.1-0.3.

[0058] The reaction of the present invention is carried out in the presence of an organic solvent, and the organic solvent can be a commonly used reaction solvent in the art. Typically, the organic solvent includes but is not limited to C2-C6 nitrile solvents, C1-C6 hydrocarbon solvents, C6-C10 aromatic hydrocarbon solvents, sulfone solvents, amide solvents, or a combination thereof; preferably, toluene, acetonitrile, chloroform, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof.

[0059] As used herein, the molar ratio between the reaction raw materials can be conventionally selected according to the reaction type. For example, the molar ratio of the compound of formula 4 to the compound of formula 7 is 1:(1±0.5).

[0060] Specifically, the preparation method of the boron difluoride curcumin skeleton compound shown in Formula I also includes the following steps:

[0061]

[0062] (a) reacting a compound of formula 1 with a compound of formula 2, 4-iodoanisole, to obtain a compound of formula 3;

[0063] (b) reacting the compound of formula 3 obtained in step (a) with boron trifluoride etherate to obtain a compound of formula 4;

[0064] (c) subjecting the compound of formula 5, 5-formaldehyde furan-2-boric acid, and the compound of formula 6, 5-bromo-2-dimethylaminopyridine, to Suzuki coupling reaction to obtain a compound of formula 7;

[0065] (d) Under alkaline conditions, condensing and dehydrating the compound of formula 4 obtained in step (b) and the compound of formula 7 obtained in step (c) to obtain compound XCY13 of formula (I).

[0066] use

[0067] The curcumin skeleton compound of formula I of the present invention can inhibit the formation of toxic Aβ aggregates by Aβ monomers, and can also depolymerize Aβ aggregates, thereby preventing and treating central nervous system diseases induced by Aβ aggregates and inhibiting Aβ-induced microglial inflammatory reactions.

[0068] In addition, the curcumin skeleton compound of formula I of the present invention can also pass through the blood-brain barrier, mark Aβ aggregates in the body, and emit strong near-infrared fluorescence signals. Therefore, it can be used as a near-infrared fluorescent probe to detect and locate Aβ aggregates.

[0069] Pharmaceutical composition

[0070] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0071] The "active ingredient" described in the present invention refers to the compound of formula I described in the present invention or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0072] As used herein, the term "pharmaceutically acceptable salt" refers to non-toxic acid or alkaline earth metal salts of compounds of Formula I. These salts can be prepared in situ during the final isolation and purification of the compounds of Formula I, or by reacting a suitable organic or inorganic acid or base with a basic or acidic functional group, respectively. Representative salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, gluconate heptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthylsulfonate, oxalate, pamoate, pectinate, thiocyanate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, and undecanoate. In addition, nitrogen-containing basic groups can be quaternized by the following reagents: alkyl halides, such as chlorides, bromides and iodides of methyl, ethyl, propyl, butyl; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates; long-chain halides such as chlorides, bromides and iodides of decyl, lauryl, myristyl and stearyl; aralkyl halides such as benzyl and phenethyl bromides, etc. Water-soluble or oil-soluble or dispersible products are thus obtained. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and organic acids such as oxalic acid, maleic acid, methanesulfonic acid, succinic acid, citric acid. Base addition salts can be prepared in situ during the final isolation and purification of the compound of formula I, or by reacting the carboxylic acid portion with a suitable base (such as a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate) or ammonia, or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, salts based on alkali and alkaline earth metal cations, such as sodium, lithium, potassium, calcium, magnesium, aluminum, and the like, and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines for forming base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.

[0073] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0074] Suitable pharmaceutically acceptable carriers or excipients include: such as treating agents and drug delivery modifiers and promoters, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, sodium methylcellulose, carboxymethylcellulose, glucose, hydroxypropyl-B-cyclodextrin, polyvinylpyrrolidone, low melting point wax, ion exchange resins, etc., and any combination of two or more thereof. Liquid and semisolid excipients can be selected from glycerol, propylene glycol, water, ethanol and various oils, including petroleum, animal oil, vegetable oil or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly carriers for injectable solutions, include water, saline, aqueous glucose solutions and ethylene glycol. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

[0075] The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

[0076] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0077] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0078] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0079] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0080] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0081] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0082] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0083] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0084] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as anti-tumor drugs).

[0085] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0086] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0087] Compared with the prior art, the main advantages of the present invention include:

[0088] (a) The boron difluoride curcumin skeleton compound XCY13 shown in formula (I) of the present invention has the characteristics of near-infrared emission and large Stokes shift, and the maximum absorption wavelength is about 544nm, the maximum emission wavelength is about 673nm, and the Stokes shift is about 129nm in a 10% DMSO and 90% PBS buffer solvent system.

[0089] (b) The compound XCY13 of formula (I) of the present invention can penetrate the blood-brain barrier well as a fluorescent probe; it can label Aβ aggregates and show strong fluorescent signals;

[0090] (c) The compounds of the present invention can inhibit the formation of Aβ monomer aggregates and disaggregate Aβ aggregates, and inhibit the Aβ-induced microglial inflammatory response, which is of great significance for the pathogenesis research, diagnosis and treatment of Aβ-related nervous system diseases and the development of related drugs.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0092] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0093] Example 1: Preparation of Compound 3

[0094]

[0095] Raw material 1 (3.08 g, 22 mmol), raw material 2 (4.68 g, 20 mmol), cesium carbonate (26.06 g, 80 mmol), cuprous iodide (0.38 g, 2 mmol) and L-proline (0.92 g, 8 mmol) were dissolved in 40 mL of dimethyl sulfoxide, and the mixture was heated at 90°C under argon protection. After the reaction was completed, the reaction solution was cooled to room temperature, filtered with diatomaceous earth, added with water and extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and separated and purified with a silica gel column to obtain 2.46 g of compound 3 with a yield of 50%.

[0096] Example 2: Preparation of Compound 4

[0097]

[0098] Compound 3 (2.46 g, 10 mmol) was added to a flask, 20 mL of anhydrous dichloromethane was added to dissolve, and boron trifluoride etherate (14.19 g, 100 mmol) was added, and the reaction was stirred at room temperature. After the raw material was completely converted, sodium bicarbonate aqueous solution was added to quench the reaction. The solution was transferred to a separatory funnel, and dichloromethane was added to extract three times. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed on a rotary evaporator. The residue was separated and purified by silica gel column chromatography to obtain 2.58 g of compound 4 with a yield of 88%. 1H NMR (600MHz, CDCl3) δ7.04-6.99(m,2H),6.98-6.94(m,2H),3.86(s,3H),3. 22(p,J=8.6,8.2Hz,1H),2.47-2.36(m,2H),2.07(s,3H),1.96-1.81(m,4H); 13 C NMR (150MHz, CDCl3) δ194.1,190.8,159.9,132.0,124.7,114.8,55.5,39.8,25.9,23.7,17.8; 19 F NMR (376MHz, CDCl3) δ-138.60,-138.67.

[0099] Example 3: Preparation of Compound 7

[0100]

[0101] The raw material 5 (1.54 g, 11 mmol), the raw material 6 (2.01 g, 10 mmol), palladium acetate (0.11 g, 0.5 mmol), triphenylphosphine (0.26 g, 1 mmol), and potassium carbonate (2.76 g, 20 mmol) were dissolved in a mixed solution of acetonitrile / methanol (20 mL / 10 mL) and stirred for reaction at 60 °C. After the reaction was completed, the reaction solution was cooled to room temperature, water was added and extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and separated and purified by silica gel column to obtain 1.83 g of compound 7 with a yield of 85%. 1 H NMR (600MHz, CDCl3) δ9.56 (s, 1H), 8.61 (d, J = 2.4Hz, 1H), 7.88 (dd, J = 9.0, 2.4Hz, 1H ),7.30(d,J=3.7Hz,1H),6.65(d,J=3.7Hz,1H),6.56(d,J=9.0Hz,1H),3.16(s,6H); 13 C NMR (150MHz, CDCl3) δ176.6,159.4,159.3,151.5,146.2,134.1,113.5,105.6,105.2,38.2.

[0102] Example 4: Preparation of Fluorescent Therapeutic Probe Formula (I)

[0103]

[0104] Compounds 4 (294 mg, 1 mmol) and 7 (216 mg, 1 mmol) were added to a flask, 10 mL of acetonitrile was added to dissolve, piperidine (17 mg, 0.2 mmol) was added, and stirred at 45° C. for 8 hours. After the raw materials were completely converted, the solvent was removed under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain 464 mg of the compound of formula (I), with a yield of 94%. 1 H NMR(400MHz, CDCl3) δ8.45(d,J=2.2Hz,1H),7.77(d,J=14.9Hz,1H),7.61(dd, J=9.0,2.4Hz,1H),7.13-7.06(m,2H),7.03-6.96(m,2H),6.87(d,J=3.7Hz,1H) ,6.59(d,J=3.7Hz,1H),6.51(d,J=9.0Hz,1H),6.23(d,J=14.9Hz,1H),3.92(s, 3H),3.24(p,J=8.7Hz,1H),3.15(s,6H),2.52-2.40(m,2H),1.97–1.79(m,4H); 13 C NMR (100MHz, CDCl3) δ191.3,177.9,159.8,159.0,158.5,150.4,145.6,133.7,132.7,132 .5,124.8,123.4,114.6,114.1,113.7,113.6,107.1,105.7,55.5,39.9,38.2,26.1,17.9; 19 F NMR (376MHz, CDCl3) δ-140.62,-140.68.

[0105] Example 5: XCY13 staining of 5×FAD transgenic mouse brain slices

[0106] Paraffin-embedded brain tissues of 5×FAD transgenic mice (6 months) were subjected to in vitro fluorescent staining. Brain sections (4 μm) were dewaxed and incubated with the fluorescent probe XCY13 (100 μM) for 30 min. The in vitro staining of Aβ by XCY13 was observed using a confocal microscope. The results are shown in Figure 1 .

[0107] like Figure 1 , the fluorescent probe XCY13 can label Aβ in the brain of 5×FAD transgenic mice.

[0108] Example 6: Fluorescent probe XCY13 and Aβ 1-42 Fluorescence spectrum detection after aggregation

[0109] The fluorescent probe XCY13 (300 μl, 2 μM, 10% DMSO and 90% PBS) was mixed with Aβ aggregates according to the following table. The mixture was incubated at room temperature for 30 min and measured with a fluorescence spectrophotometer Fluormax-4. The results are shown in Figure 2 .

[0110] Table 1: Preparation of detection system in fluorescence spectroscopy

[0111]

[0112] from Figure 2 It can be seen that the fluorescent probe XCY13 and Aβ 1-42 The fluorescence intensity was significantly enhanced after the aggregates bound, and the increase in fluorescence intensity was positively correlated with the concentration of Aβ aggregates, proving that XCY13 recognized Aβ aggregates at the molecular level.

[0113] Example 7: In vivo imaging experiment

[0114] 5×FAD transgenic mice (n=3, 12 months old, female) and paired wild-type mice were placed under IVIS (PerkinElmer, Ex=560nm, Em=710nm) for observation and recording. Fluorescence signals were recorded at different time points after caudal vein administration (20 mg / kg) of mice. ROIs were drawn in the brain region and the fluorescence intensity was calculated for quantitative analysis. The results are shown in Figure 3 AB.

[0115] like Figure 3 As shown, 5 minutes after the tail vein injection of XCY13 compound, the signal intensity of wild-type mice and 5×FAD transgenic mice was significantly different, and the signal intensity of 5×FAD transgenic mice increased significantly. Therefore, XCY13 can mark Aβ in the brain of transgenic mice.

[0116] Example 8: XCY13 inhibits Aβ aggregation and disaggregates Aβ

[0117] First, dilute the test compound to 100μM with PBS, and dilute the positive drug EGCG to 80μM. Take PBS as the blank control group, and mix 34μL of Aβ monomer and 34μL of PBS 1:1 as the Aβ group. Add 10μL of compound, 34μL of Aβ monomer and 24μL of PBS to the compound group and mix well. Each group needs to set up a compound control, and add 10μL of compound and 58μL of PBS to the compound control group. After mixing the mixed system, incubate it at 37℃ for 24h in the dark.

[0118] Prepare 15 μM ThT for use, add 17 μL of the mixed system to a 96-well plate, then add 83 μL of ThT, repeat the wells, place on a shaker to mix for 5 minutes, and then use an ELISA reader to measure the fluorescence value (Ex = 425 nm, Em = 490 nm). Calculate using the following formula:

[0119] Fluorescence value (au) = (test group - self-control) / (Aβ42 group - blank control group);

[0120] First, dilute the test compound to 100μM with PBS, and dilute the positive drug EGCG to 80μM. Place the Aβ monomer in a 37℃ constant temperature shaker and incubate at 100rpm for 72h to obtain Aβ aggregates. Use PBS as the blank control group, and mix 34μL of Aβ aggregates with 34μL of PBS in a 1:1 ratio as the Aβ group. Add 10μL of compound, 34μL of Aβ aggregates and 24μL of PBS to the compound group and mix well. Each group needs to set up a compound control, and add 10μL of compound and 58μL of PBS to the compound control group. After mixing the mixed system, incubate it at 37℃ for 24h in the dark.

[0121] The determination and calculation methods are the same as above.

[0122] like Figure 4 As shown in A, after XCY13 was co-incubated with Aβ monomers, the ThT fluorescence value was significantly reduced compared with the Aβ group, indicating that XCY13 inhibited Aβ aggregation at the molecular level. Figure 4 As shown in B, after XCY13 was co-incubated with Aβ aggregates, the ThT fluorescence value was significantly reduced compared with the Aβ aggregate group, indicating that XCY13 can depolymerize Aβ.

[0123] Example 9: XCY13 inhibits Aβ-induced inflammation in BV2 microglia

[0124] Prepare the compound and dilute the compound to 100 μM using DMEM high-glucose culture medium. Replace the cell culture medium with fresh DMEM high-glucose culture medium before adding the drug, and return it to the incubator after changing the medium. Remove the cell plate from the incubator, add the compound to the drug group at different concentrations, add the solvent control to the Control group and Aβ / LPS group, and return the cell plate to the incubator for 2 hours. Prepare the Aβ working solution, dilute Aβ to 5 μM and LPS to 1000 ng / mL using DMEM high-glucose culture medium, add 10 μL of Aβ / LPS working solution to the test compound group and Aβ / LPS group, add the solvent control to the Control group, and continue to culture in the incubator for 24 hours. After 24 hours, remove the culture plate, use a spray gun to aspirate 50 μL of supernatant from each well and add it to a clean uncovered 96-well plate, add 50 μL of Greiss reagent, react in the dark for 15 minutes, read the absorbance value at 540 nm, and calibrate it with the Aβ / LPS group as 100%.

[0125] According to the primer synthesis report sheet of Generay, the primers were prepared to 100μM. The primer sequences are shown in Table 2-4. Aliquot the primers, add 198μL DEPC water to 2μL primers, dilute the primers to 10μM, and place on ice for later use. According to the instructions of the RT-PCR kit, spot the plate: add 5μL of TB green Premix Ex Taq, 1μL of PCR Forward Primer, 1μL of PCR Reverse Primer, 1μL of template DNA, and 2μL of dd H2O to each well, centrifuge at 4000×g in a plate centrifuge for 5min, and then take out for PCR. Use the BioRAD CFX 96PCR instrument to perform PCR, denature at 95℃30s, and then perform 39 cycles (95℃5s, 60℃30s, read the plate), and after the cycle, 95℃5s.

[0126] Table 2: Primer sequences

[0127]

[0128] like Figure 5 As shown in A, after Aβ was added to BV2 microglia, the amount of NO in the cells increased significantly. 5μM and 100μM XCY13 could reduce the NO release of BV2 microglia induced by Aβ in a concentration-dependent manner. Figure 5 As shown in BD, after Aβ was added to BV2 microglia, the mRNA levels of intracellular TNF-α, IL-1β, and IL-6 were significantly upregulated. XCY13 can reduce the upregulation of TNF-α, IL-1β, and IL-6 mRNA levels in BV2 microglia induced by Aβ. In summary, XCY13 can antagonize Aβ-induced microglial inflammation.

[0129] Example 10: XCY13 significantly improves the accuracy of 5×FAD transgenic mice in the Y-maze

[0130] Place the Y maze (each arm is 36cm×6cm×15cm, and the angle between the three arms is 120 degrees) in the predetermined position, test the camera, delineate the ABC and central areas on the supermaze software, set the animal groups and the number of animals, and the measurement time is 5 minutes. Put the mice into the A arm, and record the stay time and the number of shuttles of each mouse in each arm within 5 minutes. Count the total number of mice entering the arm and the correct rate within 5 minutes in the Y maze experiment.

[0131] The results are as follows Figure 6 As shown in the figure, there was no statistically significant difference in the total number of arm entries among the four groups of animals, but the performance of the four groups in terms of accuracy was different. Figure 6As shown in B, the average correct rate of the wild-type mouse group (WT) was 67.49%, the average correct rate of the 5×FAD mouse group was 62.17%, the average correct rate of the 5×FAD mouse plus XCY13 (20 mg / kg) group was 71.66%, and the average correct rate of the 5×FAD mouse plus XCY13 (60 mg / kg) group was 73.28%. Through statistical analysis, it can be seen that both low-dose (20 mg / kg) and high-dose (60 mg / kg) administration of XCY13 can significantly improve the correct rate of 5×FAD transgenic mice in the Y-maze behavior, indicating that XCY13 improves the Y-maze behavior of 5×FAD transgenic mice.

[0132] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A boron difluoride curcumin skeleton compound shown in formula I, 2. A method for preparing a boron difluoride curcumin skeleton compound shown in formula I, characterized in that: The steps include: Under alkaline conditions, in an organic solvent, the compound of formula 4 reacts with the compound of formula 7 to obtain the compound of formula (I) XCY13, 3. The preparation method according to claim 2, characterized in that: The reaction temperature is 25-100°C.

4. A pharmaceutical composition or preparation, characterized in that The invention comprises (a) a therapeutically effective amount of a compound of formula I; and (b) a pharmaceutically acceptable carrier.

5. Use of the compound according to claim 1 or the pharmaceutical composition or preparation according to claim 4, characterized in that: Used to prepare (a) a drug for inhibiting the polymerization of β-amyloid protein monomers; (b) a drug for disaggregating β-amyloid protein aggregates; (c) a drug for inhibiting β-amyloid protein-induced microglial inflammatory response; and / or (d) a drug for preventing and / or treating neurodegenerative diseases.

6. The use according to claim 5, characterized in that The neurodegenerative disease is a central nervous system disease associated with the deposition of β-amyloid protein (Aβ).

7. The use according to claim 5 or 6, characterized in that The neurodegenerative disease is Alzheimer's disease.

8. The use according to claim 6, characterized in that The β-amyloid protein (Aβ) includes Aβ aggregates and Aβ monomers.

9. A near-infrared fluorescent diagnostic probe, characterized in that: The probe is a curcumin skeleton compound shown in formula I; 10. A method for inhibiting β-amyloid protein-induced microglial inflammatory response, characterized in that: An effective amount of a compound of formula I is administered to a subject in need thereof, thereby inhibiting the inflammatory response of microglia induced by β-amyloid protein.

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