CN114957296B - Novel Alzheimer disease detection probes and biological application thereof - Google Patents

Abstract

The present invention provides a novel Alzheimer's disease detection probe and its biological application. Specifically, the present invention provides a compound as shown in the following formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, hydrate or solvate thereof. The compound of the present invention can be used to treat or prevent acetylcholinesterase-related nervous system diseases, or used as an imaging probe for diagnosing acetylcholinesterase-related nervous system diseases.

Description

A new type of Alzheimer's disease detection probe and its biological application

Technical Field

The present invention belongs to the field of integrated biomolecular detection, imaging and diagnosis and treatment, and specifically relates to a novel Alzheimer's disease detection probe based on targeting Aβ and AChE and its biological application.

Background Art

Alzheimer's disease (AD) is a complex degenerative disease of the central nervous system, which is clinically manifested as cognitive impairment, disorientation, gradual memory loss, and ultimately loss of thinking ability, motor skills, and inability to take care of oneself. Among the causes of death among the elderly, AD has become the fourth killer after cardiovascular, tumors, and stroke. It is estimated that there are currently more than 35 million people suffering from the disease worldwide and this number is expected to double by 2030 and quadruple by 2050 to 115 million. The treatment and care of AD has also become a major economic burden on society.

The main pathological characteristics of AD patients are the aggregation of β-amyloid protein (Aβ) into senile plaques, abnormal aggregation of intracellular Tau protein to form neurofibrillary tangles (NFTs) and neuronal death. In recent years, the pathogenesis of AD has mainly focused on the Aβ toxicity hypothesis, the cholinergic neuron hypothesis and the Tau protein hypothesis. Among them, the Aβ toxicity hypothesis is dominant, which is manifested as the neurotoxic aggregation of Aβ to form amyloid fibers. In addition, it is associated with some proteins including apolipoprotein E (apoE), acetylcholinesterase (AChE), α1-antichromycin and heparin sulfate proteoglycans. Among them, AChE plays a key role in cholinergic transmission in the mammalian central nervous system and may also participate in non-cholinergic mechanisms. Studies have reported that most of the cortical AChE activity in the brains of AD patients is mainly related to the amyloid core of senile plaques, rather than the neuritic components found around them. AChE directly promotes the aggregation of Aβ peptides to form amyloid fibers and the formation of complexes with small Aβ peptide fragments, which is consistent with the co-localization points of Aβ deposits in the brains of AD patients.

Biomarkers are indicators that can be used to objectively measure and evaluate normal physiological or pathological processes. They are important bases for early diagnosis of AD, disease course monitoring, intervention treatment, and efficacy judgment. With the development of genomics and imaging technology, people have also made great progress in AD diagnostic technology and biomarkers. In the past few decades, people have gradually established a series of body fluid markers (such as cerebrospinal fluid, blood, urine, etc.) and neuroimaging probes (such as brain structure imaging, functional imaging, and molecular imaging, etc.). In particular, the development of neuroimaging technologies such as positron emission tomography (PET) and single photon emission tomography (SPECT) has made it possible to detect changes in brain regions caused by normal aging and early AD in vivo. When used in conjunction with positron emitters such as 18F and 11C, PET can sensitively detect mild early brain changes and disease progression. 18F-fluorodeoxyglucose (FDG-PET) imaging probes have been used to image brain glucose metabolism and to track the course of asymptomatic AD and detect preventive treatment. So far, three Aβ imaging probes have been approved by the FDA for the diagnosis of AD, such as Florbetapir (18F AV-45), 18F-Flutemetamol, and Florbetaben (18F-BAY94-9172). In addition, some Tau protein PET imaging probes have also made great progress, such as 18F-THK523, 18F-THK5117, 18F-THK5105, 18F-THK5351, 18F-AV1451 (T807), and 11C-PBB3, etc. These probes provide valuable information for understanding the role of Tau protein in the early stage of AD. Although these probes clearly reflect the progression of AD to a certain extent, show good diagnostic value and are included in the new AD diagnostic criteria, and begin to be used for the diagnosis of clinical AD patients, they still have their own shortcomings, such as false positives in many non-demented patients. Therefore, it is very necessary to develop novel and more sensitive diagnostic probes or integrated diagnosis and treatment probes for the diagnosis and treatment of AD.

Summary of the invention

Based on the biological characteristics of Aβ peptide and AChE, the present invention has set up a novel class of AD detection probe molecules targeting Aβ and AChE, and seeks dual-target small molecule probes with high efficiency and high selectivity in binding to Aβ and inhibiting cholinesterase, so as to produce synergistic or complementary effects between different targets, exert linkage effects, and realize the integrated diagnosis or treatment of AD.

In a first aspect of the present invention, there is provided a compound as shown in the following formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, hydrate or solvate thereof:

in,

Ar is selected from the group consisting of substituted or unsubstituted phenyl, or substituted or unsubstituted 5-7 membered aromatic heterocycle; wherein the 5-7 membered aromatic heterocycle contains 1-3 heteroatoms selected from oxygen, sulfur and nitrogen;

X may be CH or N; and when X is CH, the connecting bridge may be located on X (in this case, the H on CH is replaced by the connecting bridge);

The linker can be selected from the following structure fragments:

wherein Y is selected from the group consisting of -(CH ₂ ) _n -, -Ph(CH ₂ ) _n -, -O-, -Ph(CH ₂ ) _n -, -NR-, and -S-;

p is selected from the group consisting of 1, 2, 3, 4, 5 or 6;

p2 is selected from the following group: 0, 1, 2 or 3;

R is selected from the group consisting of H, substituted or unsubstituted C ₁ -C ₄ alkyl;

It is a structural fragment formed by coupling a molecule shown in the following formula with a connecting bridge:

in:

m is 0, 1, 2 or 3;

n is selected from the following group: 0, 1, 2 or 3;

X is selected from the group consisting of (CH ₂ ) _p , CO or SO ₂ , wherein p is 0, 1, 2 or 3; preferably (CH ₂ ) _p or CO, wherein p is preferably 1 or 2;

_R1 is selected from the group consisting of halogen, C1-C6 straight or branched hydrocarbon, cyano, nitro, amino, hydroxy, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxy, mercapto, C2-C6 acyl, sulfonyl, aminosulfonyl and C2-C6 sulfonyl;

_R3 is selected from the group consisting of substituted or unsubstituted _C3 - _C10 cycloalkyl, substituted or unsubstituted C3- _C10 cycloalkenyl, substituted or unsubstituted 3-12 membered heterocyclyl, and substituted or unsubstituted _C6 - _C12 aryl; the substituents in _{the R1 are} the same or different 1, 2, 3, 4 or 5 substituents selected from halogen, _C1 - _C6 alkyl, halogen-substituted _{C1 - C6} alkyl, _{C1 - C6} alkoxy, C1 _{- C6 alkoxycarbonyl, halogen-substituted C1-C6 alkoxy} , _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, C3- _C8 cycloalkyl, cyano, nitro, amino, hydroxyl, hydroxymethyl, carboxyl, mercapto, sulfonyl, C6- _C10 aryl and _3-12 membered heterocyclyl; or the substituents in the _C6 -C ₁₂ Two adjacent substituents on the aryl group and the carbon atoms on the adjacent aromatic ring together form a C ₃ -C ₇ cycloalkyl group, a C ₃ -C ₇ cycloalkenyl group or a 3-7 membered heterocyclic group; each heterocyclic group independently contains 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen;

R ₄ is one or more groups selected from the group consisting of hydrogen, halogen, C ₁ -C ₆ alkyl, C 1 -C ₆ alkyl substituted with halogen, C ₁ -C ₆ alkoxy, and C _{1 -C 6} alkoxy substituted with halogen;

R ₅ and R ₆ are each independently selected from the group consisting of hydrogen, carboxyl, C ₁ -C ₄ alkoxycarbonyl and C ₁ -C ₄ alkyl; or R ₅ and R ₆ together form a C ₁ -C ₄ alkylene group;

Wherein, the substitution refers to that one or more (preferably 1 to 5) hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, C ₁ -C ₆ straight chain or branched alkyl, C ₁ -C ₆ straight chain or branched alkenyl, C ₁ -C ₆ straight chain or branched alkynyl, cyano, nitro, NH ₂ , C ₁ -C ₆ amine (including straight chain or branched hydrocarbon mono-substituted or di-substituted amine), hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁ -C ₆ alkoxy, mercapto, C ₂ -C ₆ acyl, sulfonyl, aminosulfonyl and C ₂ -C ₆ sulfonyl, C ₅ -C ₁₀ aromatic group.

In another preferred embodiment, the It has the structure shown below:

R ₁ and R ₂ are selected from the group consisting of halogen, C1-C6 straight or branched hydrocarbon, cyano, nitro, amino, hydroxy, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxy, mercapto, C1-C4 acyl, sulfonyl, aminosulfonyl, and C1-C4 substituted sulfonyl;

m and n are each independently selected from the group consisting of 0, 1, 2 or 3;

The substitution refers to that one or more (preferably 1 to 5) hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, C ₁ -C ₆ straight chain or branched alkyl, C ₁ -C ₆ straight chain or branched alkenyl, C ₁ -C ₆ straight chain or branched alkynyl, cyano, nitro, NH ₂ , C ₁ -C ₆ amine (including straight chain or branched hydrocarbon mono-substituted or di-substituted amine), hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁ -C ₆ alkoxy, mercapto, C ₂ -C ₆ acyl, sulfonyl, aminosulfonyl and C ₂ -C ₆ sulfonyl, C ₅ -C ₁₀ aromatic group.

In another preferred embodiment, the Select from the following group:

In another preferred embodiment, R ₁ and R ₂ are each independently halogen.

In another preferred embodiment, R ₁ and R ₂ are each independently F.

In another preferred embodiment, the Ar is selected from the following group: a substituted or unsubstituted phenyl group, or a substituted or unsubstituted 5-7 membered aromatic heterocycle; wherein the 5-7 membered aromatic heterocycle contains 1-3 heteroatoms selected from oxygen, sulfur and nitrogen, and the substitution refers to that one or more (preferably 1 to 5) hydrogen atoms on the group are replaced by substituents selected from the following group: halogen, C ₁ -C ₄ straight chain or branched alkyl, C ₁ -C ₄ straight chain or branched alkenyl, C ₁ -C ₄ straight chain or branched alkynyl, cyano, nitro, NH ₂ , C ₁ -C ₄ amine (including straight chain or branched hydrocarbon mono-substituted or di-substituted amine), hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁ -C ₄ alkoxy, mercapto, C ₂ -C ₄ acyl, sulfonyl, aminosulfonyl and C ₂ -C ₄ sulfonyl, C ₅ -C ₁₀ aromatic group.

In another preferred embodiment, the connecting bridge is selected from the following group: Wherein, p1 is selected from the following group: 0, 1 or 2.

In another preferred embodiment, the connecting bridge is selected from the following group:

In another preferred embodiment, the compound of formula I is selected from the following group:

The second aspect of the present invention provides a method for preparing the compound of formula I according to the first aspect of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, hydrate or solvate thereof, characterized in that the compound of formula I has a structure shown in formula I-1, and the compound is prepared by the following method:

In an inert solvent, reacting a compound of formula I-a with a compound of formula I-b to obtain a compound of formula I-1;

or

The compound of formula I has the structure shown in formula I-2, and the compound is prepared by the following method:

In an inert solvent, the compound of formula I-c is reacted with the compound of formula I-d to obtain a compound of formula I-2.

The third aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, hydrate or solvate thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and/or diluents.

The fourth aspect of the present invention provides a use of a compound of formula I as described in the first aspect of the present invention, or its enantiomers, diastereomers, racemates or mixtures thereof, for preparing a pharmaceutical composition for treating or preventing a nervous system disease associated with acetylcholinesterase, or for preparing an imaging probe for diagnosing a nervous system disease associated with acetylcholinesterase.

In another preferred embodiment, the acetylcholinesterase-related nervous system disease is selected from the following group: senile dementia, Alzheimer's disease, Parkinson's syndrome, epilepsy, or schizophrenia.

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

Figure 1 shows the effect of Cy series compounds on fluorescence imaging of APP/PS1 transgenic mouse brain slices. A: cortex; B: hippocampus.

DETAILED DESCRIPTION

After long-term and in-depth research, the inventors have developed a novel class of AD detection probe molecules targeting Aβ and AChE, which have both highly selective Aβ binding ability and cholinesterase inhibitory activity, thus enabling integrated diagnosis or treatment of AD. Based on the above findings, the inventors have completed the present invention.

the term

As used herein, the term "alkyl" includes linear or branched alkyl groups. For example, _C1 - _C8 alkyl groups represent linear or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched alkenyl groups. For example, _C2 - _C6 alkenyl refers to straight or branched alkenyl groups having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includes straight or branched alkynyl groups. For example, _C2 - _C6 alkynyl refers to a straight or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C ₃ -C ₈ cycloalkyl" refers to a cycloalkyl group having 3 to 8 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be a bicyclic ring, such as a bridged ring or a spiro ring.

As used herein, the term "C ₁ -C ₆ alkylamino" refers to an amine group substituted by a C ₁ -C ₆ alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino and the like.

As used herein, the term "C ₁ -C ₆ alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "5-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 5-10 atoms, wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a monocyclic or bicyclic form, such as a bridged ring or a spirocyclic form. Specific examples may include oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl and pyrrolidinyl, etc.

As used herein, the term "C ₆ -C ₁₀ aryl group" refers to an aryl group having 6 to 10 carbon atoms, for example, a phenyl group, a naphthyl group, or the like.

As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms, of which 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a monocyclic ring or a condensed ring. Specific examples may include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

Unless otherwise specified as "substituted or unsubstituted", the groups described in the present invention may be substituted by substituents selected from the following groups: halogen, nitrile, nitro, hydroxyl, amino, C ₁ -C ₆ alkyl-amino, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C _{2 -C 6 alkynyl, C 1 -C 6 alkoxy} , halogenated C ₁ -C ₆ alkyl, halogenated C ₂ -C ₆ alkenyl, halogenated C ₂ -C ₆ alkynyl, halogenated C ₁ -C ₆ alkoxy, allyl, benzyl, C ₆ -C ₁₂ aryl, C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy-carbonyl, phenoxycarbonyl, C ₂ -C ₆ alkynyl-carbonyl, C ₂ -C ₆ alkenyl-carbonyl, C ₃ -C ₆ cycloalkyl-carbonyl, C ₁ -C 6 _6- Alkyl-sulfonyl, etc.

As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I.

Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers with different energies can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropic) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via reorganization of some bonding electrons.

As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

The compounds of the present application can be prepared by a variety of synthesis methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining specific embodiments with other chemical synthesis methods, and equivalent substitution methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

The solvents used in this application can be obtained from commercial sources, and the compounds can be artificially or The software names were used, and commercially available compounds were named using the supplier's catalog names.

New Alzheimer's disease detection probe

The novel Alzheimer's disease detection probe of the present invention may be a compound having the following general formula (I) or a salt thereof, wherein the compound includes its enantiomers, diastereomers, racemates or mixtures thereof,

wherein Ar can be phenyl, substituted phenyl, or a 5-7 membered aromatic heterocycle, wherein the substituted phenyl can be 1 to 5 substituents selected from halogen, C1-C6 straight or branched hydrocarbon, cyano, nitro, amino, C1-C6 straight or branched hydrocarbon monosubstituted or disubstituted amino, hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxy, mercapto, C1-C4 acyl, sulfonyl, aminosulfonyl, and C1-C4 alkyl. The 5-7 membered aromatic heterocyclic ring contains 1-3 heteroatoms selected from oxygen, sulfur and nitrogen, and contains at least one substituent selected from halogen, C1-C6 straight or branched hydrocarbon, cyano, nitro, amino, C1-C6 straight or branched hydrocarbon mono- or di-substituted amino, hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxy, mercapto, C1-C4 acyl and C5-C10 aromatic;

X can be C or N;

The linker can be selected from the following structure fragments:

Wherein, Y may be selected from -(CH ₂ )n-, -Ph(CH ₂ )n-, C1-C6 straight or branched chain alkylene, phenylalkylene;

It can be preferably selected from the following structural fragments:

Wherein, R ₁ and R ₂ may be selected from halogen, C1-C6 straight or branched hydrocarbon, cyano, nitro, amino, hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxy, thiol, C1-C4 acyl, sulfonyl, aminosulfonyl and C1-C4 substituted sulfonyl;

The above m and n can be integers of 0-3.

In a more preferred embodiment of the present invention, the compound of the general formula I of the present invention is preferably the following specific compound:

Preparation method of compound of formula I

The present invention provides a method for preparing a compound probe represented by general formula (I), and the preparation method is carried out according to the following scheme.

Solution 1:

Step a: Add 2-bromo-5-formylpyridine, 1-tert-butoxycarbonylpiperazine and K ₂ CO ₃ into anhydrous DMSO, and heat and stir to react overnight to obtain compound F-2; the heating condition may be 60 to 100 degrees.

Step b: Add acetylacetone to anhydrous toluene, then slowly add BF3*Et2O solution to the above solution, react at 40-80°C for 2h to obtain compound F-4.

Step c: Dissolve F-4 in acetonitrile, then add glacial acetic acid, tetrahydroisoquinoline and p-dimethylaminobenzaldehyde into the above system and react at 40-80°C overnight to obtain compound F-5.

Steps d and e: Dissolve F-5 in acetonitrile, add glacial acetic acid, tetrahydroisoquinoline and F-2 into the above system, and react at 40-80°C overnight to obtain compound F-6.

Step f: Dissolve methyl 3-fluoro-4-methylphenylacetate in anhydrous carbon tetrachloride, then add N-bromosuccinimide and azobisisobutyronitrile to the above system, reflux for 2 hours to obtain compound F-8.

Step g: F-9 was reacted in 50% TFA/DCM system at room temperature, and the reaction solution was spin-dried to obtain compound F-10.

Step h: Dissolve F-10 in DMF, then add K ₂ CO ₃ and F-8 into the above system and react for 8 hours to obtain compound F-8.

Step i: Dissolve F-11 in KOH aqueous solution to react to obtain compound F-12.

Step j: Dissolve F-12 in THF, then add F-6 and a condensing agent into the above system and react at room temperature to obtain the target compound.

Option 2:

The intermediate F-2 in Scheme 1 is replaced by F-13 in this scheme, and the other synthesis methods are the same as Scheme 1.

Option 3:

The intermediate F-12 in Scheme 1 is replaced by F-16 in this scheme, and the other synthesis methods are the same as Scheme 1.

Pharmaceutical compositions and methods of administration

Since the compounds of the present invention have excellent acetylcholinesterase inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent and/or treat (stabilize, alleviate or cure) diseases related to acetylcholinesterase.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically 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 1-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

"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 Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

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.

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 may 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.

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.

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

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.

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.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable therapeutic agents. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable therapeutic agents can be used simultaneously, separately or sequentially with the compound of the present invention to prevent and/or treat cytokine and/or interferon-mediated diseases.

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 1 to 500 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.

Detection probe imaging method

As described herein, the detection probe compound of the present invention has good targeting effects on both Aβ and AChE, and thus can be used for detection of asymptomatic AD course tracking and preventive treatment. Therefore, the present invention also provides a good tool for early detection and diagnosis of Alzheimer's disease.

The present invention also provides a method for detecting β-amyloid protein deposits in vitro or in vivo, comprising the following steps:

i) administering an Alzheimer's detection probe as shown in formula I to a detection subject;

ii) allowing the Alzheimer's detection probe as shown in Formula I of the present invention to bind to the β-amyloid protein in the detection object to obtain a sample to be tested;

iii) staining the sample to be tested, and then detecting the fluorescent signal in the sample;

iv) generating an image representing the location and/or amount of said fluorescent signal; and

v) determining the distribution and extent of said β-amyloid deposits in said subject.

The detection probe of the present invention can be administered by conventional methods, preferably parenterally. After the administration step and before the detection step, the detection probe of the present invention is bound to the β-amyloid protein. For example, when the subject is a complete mammal, the detection probe of the present invention will dynamically pass through the mammal's body and contact various tissues therein. Once the detection probe of the present invention contacts the β-amyloid protein, it will bind to the β-amyloid protein.

The detection method of the present invention preferably includes detecting and imaging the fluorescent signal released from the sample by means of, for example, a fluorescence microscope with a photographic function. The detection step can also be understood as acquiring signal data.

The "test subject" of the present invention can be any human or animal subject. Preferably, the test subject of the present invention is a mammal. Most preferably, the test subject is in vivo in an intact mammalian body. In a particularly preferred embodiment, the test subject of the present invention is a human.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

The experimental methods used in the following examples are all conventional methods unless otherwise specified. The reagents and biological materials used in the following examples are all commercially available unless otherwise specified.

Example 1. Preparation of Cy-3 compound

(1) Synthesis of F-2

2-Bromo-5-formylpyridine (50.0 mg, 0.27 mmol), 1-tert-butoxycarbonylpiperazine (75.1 mg, 0.40 mmol) and K ₂ CO ₃ (74.3 mg, 0.54 mmol) were added to 2.0 ml of anhydrous DMSO, and reacted at 80°C overnight. The reaction solution was spin-dried and extracted with ethyl acetate and water system. The organic layer was washed with water and saturated NaCl respectively, dried over anhydrous Na ₂ SO ₄ , and concentrated to obtain 61.4 mg of yellow solid, with a yield of 78.5%. ESI-MS: [M+H] ⁺ = 292.15; [M+Na] ⁺ = 314.15.

(2) Synthesis of F-4

Acetylacetone (1.0 g, 9.98 mmol) was added to 2.5 ml of anhydrous toluene, and then BF ₃ *Et ₂ O solution was slowly added to the above solution, and the reaction was carried out at 60°C for 2 h. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic layer was washed with water and saturated NaCl, dried over anhydrous Na ₂ SO ₄ , and concentrated to obtain 1.0 g of a light yellow solid with a yield of 71%.

(3) Synthesis of F-5

Dissolve F-4 (150.0 mg, 1.0 mmol) in 2.0 ml acetonitrile, then add glacial acetic acid (0.1 ml), tetrahydroisoquinoline (0.02 ml) and p-dimethylaminobenzaldehyde (149.9 mg, 1.00 mmol) to the above system. React at 60°C overnight, evaporate the solvent, and separate the crude product by flash column chromatography (PE:EA=5:1) to obtain 59 mg of a reddish brown solid with a yield of 21%. ESI-MS: [M+Na] ⁺ =302.10; [M+K] ⁺ =318.10.

(4) Synthesis of F-6

Dissolve F-5 (30.0 mg, 0.11 mmol) in 1.0 ml acetonitrile, then add glacial acetic acid (20 ul), tetrahydroisoquinoline (4.0 ul) and F-2 (31.3 mg, 0.11 mmol) to the above system. React at 60°C overnight, evaporate the solvent, and separate the crude product by flash column chromatography (PE:EA:DCM=4:1:1) to obtain 16.8 mg of dark blue solid, with a yield of 21%. ESI-MS: [M+H] ⁺ =553.35; [M+Na] ⁺ =575.40.

(5) Synthesis of F-8

Methyl 3-fluoro-4-methylphenylacetate (100.0 mg, 0.55 mmol) was dissolved in 15 ml of anhydrous carbon tetrachloride, and N-bromosuccinimide (107.0 mg, 0.60 mmol) and azobisisobutyronitrile (2.7 mg, 0.016 mmol) were added to the above system. The reaction was refluxed for 2 h. The crude reaction product was separated by flash column chromatography (PE:EA=40:1, containing 1% acetic acid) to obtain 51 mg of a yellow solid with a yield of 33%.

(6) Synthesis of F-10

F-9 (155 mg, 0.38 mmol) was reacted in 50% TFA/DCM system at room temperature for 30 min, and the reaction solution was spin-dried to obtain 110 mg of light yellow solid with a yield of 94.3%. ESI-MS: [M+H] ⁺ = 307.16.

(7) F-11 synthesis

F-10 (110 mg, 0.38 mmol) was dissolved in 2.0 ml DMF, and K ₂ CO ₃ (265 mg, 1.9 mmol) and F-8 (150 mg, 0.57 mmol) were added to the above system. The reaction was carried out at 30°C for 8 h. After the solvent was evaporated, the mixture was extracted with ethyl acetate and water. The organic layer was washed with water and saturated NaCl, respectively, and dried over anhydrous Na ₂ SO _4. After concentration, 161 mg of yellow solid was obtained with a yield of 87.3%.

(8) Synthesis of F-12

F-11 (161 mg, 0.38 mmol) was dissolved in 1N/KOH and reacted at 45°C for 6 h. The reaction solution was poured into ice water and adjusted to pH 6.5 with 1M HCl. The organic layer was then extracted with DCM. The organic layer was washed with water and saturated NaCl, dried over anhydrous Na ₂ SO ₄ , and concentrated to obtain 147 mg of a light yellow solid with a yield of 81.8%.

(9) Synthesis of Cy-3

F-12 (100.7 mg, 0.21 mmol) was dissolved in 10.0 ml of anhydrous THF, and then F-6 (98.5 mg, 0.18 mmol), HBTU (80.7 mg, 0.21 mmol) and DIPEA (44 ul, 0.26 mmol) were added to the above system. After reacting at room temperature for 2 h, the reaction solution was evaporated to dryness, and the crude product was separated by C ₁₈ preparative liquid chromatography to obtain a dark blue powder Cy-3 of 41.7 mg in total, with a yield of 25.9%. ESI-MS: [M+H] ⁺ = 908; [M+Na] ⁺ = 930. ¹ H-NMR (CDCl ₃ , 400MHz): δ (ppm) 8.25 (s, 1H), 7.96 (d, 1H, J = 14.7Hz), 7.95 (d, 1H, J = 11.0Hz), 7.59 (d, 1H, J = 15.5Hz), 7.49 (d, 2H, J = 8.8Hz), 7.45-7.50 (m, 1H), 7.13 (s, 1H),7.11(d,2H,J＝9.7Hz),6.96(d,1H,J＝9.4Hz),6.84(s,1H),6.67(d,2H,J＝8.8Hz),6.59( d,1H,J＝15.6Hz),6.43(d,1H,J＝15.0Hz),5.95(s,1H),3.94(s,3H),3.87(s,3H),3.71-3.78(m,10H),3.55(d,2H,J＝9.2Hz),3.27-3.33(m,1H),3.07 (s,6H),3.12(m,2H),2.77-2.82(m,2H),2.37-2.47(m,1H),2.07-2.26(m,4H),1.66-1.69(m,1H).

Using a synthetic method similar to the above method, replacing the corresponding substrate, the compounds shown in the following table were obtained:

Example 2 Inhibitory effect of Cy series compounds on acetylcholinesterase in vitro

Cholinesterase activity was determined using the Ellman colorimetric method ^[8] . Mouse cortical homogenate was used as AChE proenzyme. Before the enzyme activity assay, 0.1 mM tetraisopropylpyrophosphamide (iso-OMPA), a selective inhibitor of butyrylcholinesterase, was added to the mouse cortical homogenate to exclude the influence of butyrylcholinesterase. The enzyme activity assay procedure was as follows: 250 μl of the total enzyme reaction mixture contained 50 μl of sodium phosphate buffer (0.1 M, pH 7.4), 30 μl of thioacetylcholine iodide (2 mM, AChE substrate), 109 μl of water, 1 μl of compound, 50 μl of 3% (w/v) 5,5'-dithio-di(2-nitrobenzene) and 10 μl of proenzyme, and incubated at room temperature for 20 minutes. After 20 minutes, 1 ml of 3% (w/v) sodium dodecyl sulfate was added to terminate the reaction, and the colorimetric value of the colored product was measured at 450 nm on a spectrophotometer. The whole enzyme activity was expressed as a percentage of the enzyme activity without the addition of an inhibitor, and the calculation formula was: inhibition rate % = (whole enzyme activity OD value - test compound OD value) / whole enzyme activity OD value × 100%. The results showed that the IC ₅₀ values of Cy-1 and Cy-4 were 0.131 nM and 1.86 nM, respectively, both of which were nanomolar, indicating that Cy-1 and Cy-4 had a good inhibitory effect on mouse cortical acetylcholinesterase.

Example 3 Effects of Cy series compounds on fluorescence imaging of APP/PS1 transgenic mouse brain slices

12-month-old APP/PS1 mice were anesthetized with chloral hydrate, and the whole brain was removed after cardiac perfusion with normal saline. After being fixed in 4% paraformaldehyde for one week, paraffin sections were made, and the sections included two areas, the cortex and the hippocampus. Before staining, the paraffin sections were dewaxed with xylene, rehydrated with alcohol gradients, pre-treated with distilled water and PBS, and then stained with ThS (positive stain) for 20 minutes in the dark. After staining, the excess dye was washed with 50% alcohol and rinsed with PBS three times. Then, the fluorescent compound was stained in the dark for 30 minutes, and the staining was rinsed with PBS three times. Finally, the antiquenching agent was added and the slices were sealed and dried. The next day, the specific recognition of Aβ plaques by the compound was observed using a Leica upright fluorescence microscope. The results showed that Cy-1, Cy-2, Cy-3 and Cy-4 all had good fluorescence signals in the brain slices of APP/PS1 mice, and co-localized with senile plaques (Figure 1).

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 (8)

1. A compound as shown in the following formula I, or a pharmaceutically acceptable salt thereof, in, Ar is selected from the group consisting of: substituted or unsubstituted phenyl; X is N; The connecting bridge is selected from the following group: Is a structural fragment as shown below: in: _R1 and _R2 are each independently selected from the group consisting of halogen, C1-C6 straight or branched hydrocarbon group, cyano group; Wherein, the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, C ₁ -C ₆ straight chain or branched alkyl, C ₁ -C ₆ straight chain or branched alkenyl, C ₁ -C ₆ straight chain or branched alkynyl, cyano, nitro, NH ₂ , C ₁ -C ₆ amine, hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁ -C ₆ alkoxy, thiol, C ₂ -C ₆ acyl, aminosulfonyl and C ₂ -C ₆ sulfonyl. 2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the connecting bridge is selected from the group consisting of: 3. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the connecting bridge is selected from the group consisting of: 4. A compound selected from the following group, or a pharmaceutically acceptable salt thereof, characterized in that the compound of formula I is selected from the following group: 5. A method for preparing a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I has a structure as shown in formula I-1, and the compound is prepared by the following method: In an inert solvent, a compound of formula Ia is reacted with a compound of formula Ib to obtain a compound of formula I-1, wherein Y is CH ₂ and p is 1. 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and/or diluents. 7. The use of the compound of formula I according to claim 1, characterized in that it is used to prepare a pharmaceutical composition for treating or preventing a nervous system disease associated with acetylcholinesterase, or to prepare an imaging probe for diagnosing a nervous system disease associated with acetylcholinesterase. 8. The use according to claim 7, characterized in that the nervous system disease associated with acetylcholinesterase is selected from the group consisting of senile dementia, Alzheimer's disease, Parkinson's syndrome, epilepsy, or schizophrenia.