WO2021147971A1 - 治疗阿尔茨海默病的化合物 - Google Patents

治疗阿尔茨海默病的化合物 Download PDF

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WO2021147971A1
WO2021147971A1 PCT/CN2021/073164 CN2021073164W WO2021147971A1 WO 2021147971 A1 WO2021147971 A1 WO 2021147971A1 CN 2021073164 W CN2021073164 W CN 2021073164W WO 2021147971 A1 WO2021147971 A1 WO 2021147971A1
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mice
substituted
unsubstituted
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lps
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French (fr)
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王鑫
马磊
付建军
郑秋阳
周立成
邓青芳
时夕蒙
李桂林
王世华
狄安洁
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厦门大学
华东理工大学
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Priority to US17/759,264 priority Critical patent/US20230104617A1/en
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Definitions

  • the present invention relates to the field of medicine; specifically, the present invention relates to small molecule compounds for the treatment of Alzheimer's disease or Down's syndrome and applications thereof.
  • AD Alzheimer's disease
  • NFT neurofibrillary tangles
  • AD Alzheimer's disease
  • AD has become a global public health problem. It is estimated that by 2020, there will be more than 40 million dementia patients in the world, and AD patients account for about 50% to 60% of them. AD brings a great deal to patients and their families. Pain and economic burden, and bring huge social pressure, therefore, more and more attention of medical and scientific research workers.
  • AD amyloid plaques formed by amyloid ( ⁇ -amyloid, A ⁇ ), neurofibrillary tangles formed by aggregation of highly phosphorylated tau protein, and neuronal loss with glue Plasma cell proliferation and so on.
  • a ⁇ amyloid plaques formed by amyloid
  • a ⁇ amyloid
  • neurofibrillary tangles formed by aggregation of highly phosphorylated tau protein and neuronal loss with glue Plasma cell proliferation and so on.
  • AD may be a chronic central nervous system inflammatory response.
  • the activation of astrocytes and microglia is also an important sign of AD neuroinflammation.
  • microglia In the early stage of AD disease, as the most important immune cell in the brain, microglia play a key role in clearing A ⁇ amyloid plaques, limiting the formation and diffusion of A ⁇ oligomers, and spreading pathological tau protein, suggesting microglia Mediated natural immunity plays an important role in the pathological development of AD. Abnormally activated microglia can induce neurotoxicity by releasing inflammatory factors. In addition, abnormally activated microglia can also cause synapse loss and synapse loss through phagocytosis of synapses. Tactile dysfunction indicates that the abnormal function of microglia is a key factor in the development of AD disease.
  • AD Alzheimer's disease
  • PD Parkinson’s disease
  • the main brain area involved in PD is the midbrain, and the most important pathological change is the degeneration and death of dopamine (DA) neurons in the substantia nigra of the midbrain, which causes a significant decrease in the DA content of the striatum and causes disease.
  • DA dopamine
  • the main function of the current PD treatment drug levodopa is to supplement the dopamine content in the brain.
  • Huntington’s disease (HD), also known as chorea or Huntington’s disease is an autosomal dominant neurodegenerative disease. The main cause is the mutation of the HTT gene on the fourth chromosome of the patient. Mutated protein, the abnormal HTT protein in Huntington's disease patients has many repeated glutamines.
  • Abnormal huntingtin protein is easy to aggregate, has cytotoxicity, and can cause nerve cell death. There is currently no treatment.
  • Amyotrophic lateral sclerosis (ALS) is also called gradual freezing. The main pathological manifestation is motor neuron injury and death, which gradually leads to weakness and atrophy of the muscles including the medullary innervation muscles, limbs, trunk, chest and abdomen, and finally leads to The patient died and there is currently no treatment. Therefore, Alzheimer's disease is very different from other common neurodegenerative diseases in internal mechanism and external symptoms.
  • the abnormal accumulation of neurotoxic proteins in the brain of AD patients suggests that the ubiquitin-proteasome system (UPS) dysfunction may be involved in the occurrence and development of neurodegenerative diseases.
  • the ubiquitination modification pathway like the phosphorylation modification pathway, is also a reversible process.
  • the USP25 gene is located on chromosome 21 q11.2, which belongs to the deubiquitinated protease family and contains the active region of protein deubiquitinated enzyme. In situ hybridization showed that USP25 is highly expressed in the brain, and has a high level of expression in neurons, microglia and astrocytes.
  • Down’s syndrome is the most common genetic disease of intellectual disability, and Down’s syndrome patients carry a third complete or partial chromosome 21.
  • Down’s patient cells there is one more copy of the USP25 gene, and the increased expression of USP25 will affect the ubiquitination level of its substrate and protein homeostasis.
  • 100% of Down’s patients over the age of 40 will have pathological features of AD.
  • This provides a theoretical basis for studying the role of USP25 in the pathogenesis of DS and AD.
  • USP25 inhibitors may be used as a therapeutic drug for AD and DS neuroinflammation and cognitive impairment.
  • the purpose of the present invention is to provide a small molecule compound that can effectively treat Alzheimer's disease and its related diseases Down's syndrome, especially Alzheimer's disease and Down's syndrome.
  • the present invention finds and clarifies for the first time that the ubiquitin-specific protease USP25 restores the homeostasis of microglia in the AD brain by inhibiting the neuroinflammatory response of microglia and the process of synapse phagocytosis, thereby reversing the Alzheimer's disease model Synaptic function and cognitive impairment of 5 ⁇ FAD mice; knocking out Usp25 can improve the homeostasis of microglia in 5 ⁇ FAD mice, and reverse the synaptic and cognitive function defects of 5 ⁇ FAD mice. In addition, knocking out Usp25 can also improve the synaptic and cognitive deficits of Dp16 in Down’s mice. This discovery provides a potential drug target for the clinical treatment of Alzheimer's disease and Down syndrome.
  • the present invention provides compounds of formula I, various crystal forms, hydrates, solvates or pharmaceutically acceptable salts thereof for preparing prevention, treatment or amelioration of Alzheimer’s disease or Down’s syndrome. Use in medicines,
  • X is independently selected from O or NH
  • Ring A and Ring B are each independently a benzene ring
  • R 1 is independently selected from: hydrogen, deuterium, halogen, cyano, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 1 -C 6 )alkoxy;
  • n 0, 1, 2, 3, 4 or 5;
  • R 2 is independently selected from: hydrogen, halogen, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 2 -C 6 )alkenyl, substituted or unsubstituted (C 2- C 6 )alkynyl, substituted or unsubstituted (C 1 -C 6 )alkoxy;
  • n 1 or 2;
  • R 3 is a substituted or unsubstituted (C 1 -C 6 ) aldehyde group
  • each R 3a , R 3b , R 3c is independently selected from: hydrogen, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 2 -C 6 )alkenyl, substituted or Unsubstituted (C 2 -C 6 )alkynyl, Where p is an integer from 1 to 3 (preferably, p is 2).
  • the substitution position of R 3 on the benzene ring is the ortho position or the meta position; the ortho position is preferred.
  • R 1 is independently selected from: hydrogen, fluorine, chlorine, bromine or iodine (preferably fluorine), cyano, substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted ⁇ (C 1 -C 4 )alkoxy.
  • each R 1 is independently selected from: fluorine, trifluoromethyl, and trifluoromethoxy.
  • R 2 is independently selected from: hydrogen, fluorine, chlorine, bromine or iodine (preferably bromine), substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted (C 2 -C 4 )alkenyl, substituted or unsubstituted (C 2 -C 4 )alkynyl, substituted or unsubstituted (C 1 -C 4 )alkoxy.
  • R 2 is independently selected from: hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, allyl, propargyl, -OCH 2 CH 2 CH 3 , -OCH 2 CH 3 , -OCH 3 , -CHF 2 , -CF 3 , -OCHF 2 , -OCF 3 .
  • each of R 3a , R 3b and R 3c is independently selected from: hydrogen, substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted (C 2 -C 4 ) Alkenyl, substituted or unsubstituted (C 2 -C 4 )alkynyl.
  • the substituted (C 1 -C 4 )alkyl group is selected from the group consisting of hydroxy (C 1 -C 4 )alkyl, R 4a R 4b amino (C 1 -C 4 )alkyl or mercapto ( C 1 -C 4 )alkyl, said R 4a and R 4b are independently substituted or unsubstituted (C 1 -C 3 )alkyl; preferably hydroxy (C 1 -C 4 )alkyl or R 4a R 4b Amino (C 1 -C 4 )alkyl.
  • substitution is halo, preferably fluoro.
  • R 1 is independently selected from: hydrogen, fluorine, chlorine, bromine, (C 1 -C 3 ) alkyl, halo (C 1 -C 3 ) alkyl, halo (C 1- C 3 )Alkoxy;
  • n 0, 1, 2 or 3;
  • R 2 is independently selected from: hydrogen, halogen
  • n 1 or 2;
  • each of R 3a and R 3b is independently selected from: hydrogen, hydroxyl substituted (C 1 -C 4 )alkyl, Where p is an integer from 1 to 3 (preferably, p is 2).
  • the compound represented by formula I is selected from the following compounds:
  • the compound represented by Formula I is AZ1, AZ2, MZ77, MZ76, MZ1, MZ30, MZ32, MZ67, MZ66, MZ75, MZ31, MZ34, MZ74, MZ68, MZ29, MZ72, MZ38, or MZ24.
  • the prevention, treatment or improvement of Alzheimer's disease or Down's syndrome refers to the prevention, treatment or improvement of cognitive function impairment caused by Alzheimer's disease or Down's syndrome.
  • the present invention provides the compound represented by formula I, various crystal forms, hydrates, solvates or pharmaceutically acceptable salts thereof,
  • Ring A and Ring B are each independently a benzene ring
  • R 1 is independently selected from: hydrogen, deuterium, halogen, cyano, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 1 -C 6 )alkoxy;
  • n 0, 1, 2, 3, 4 or 5;
  • R 2 is independently selected from: hydrogen, halogen, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 2 -C 6 )alkenyl, substituted or unsubstituted (C 2- C 6 )alkynyl, substituted or unsubstituted (C 1 -C 6 )alkoxy;
  • n 1 or 2;
  • R 3 is a substituted or unsubstituted (C 1 -C 6 ) aldehyde group
  • each R 3a , R 3b , R 3c is independently selected from: hydrogen, substituted or unsubstituted (C 1 -C 6 )alkyl, substituted or unsubstituted (C 2 -C 6 )alkenyl, substituted or Unsubstituted (C 2 -C 6 )alkynyl, Wherein p is an integer from 1 to 3 (preferably, p is 2);
  • the compound of formula I does not include
  • the substitution position of R 3 on the benzene ring is the ortho position or the meta position; the ortho position is preferred.
  • R 1 is independently selected from: hydrogen, fluorine, chlorine, bromine or iodine (preferably fluorine), cyano, substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted ⁇ (C 1 -C 4 )alkoxy.
  • each R 1 is independently selected from: fluorine, trifluoromethyl, and trifluoromethoxy.
  • R 2 is independently selected from: hydrogen, fluorine, chlorine, bromine or iodine (preferably bromine), substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted (C 2 -C 4 )alkenyl, substituted or unsubstituted (C 2 -C 4 )alkynyl, substituted or unsubstituted (C 1 -C 4 )alkoxy.
  • R 2 is independently selected from: hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, allyl, propargyl, -OCH 2 CH 2 CH 3 , -OCH 2 CH 3 , -OCH 3 , -CHF 2 , -CF 3 , -OCHF 2 , -OCF 3 .
  • each of R 3a , R 3b and R 3c is independently selected from: hydrogen, substituted or unsubstituted (C 1 -C 4 )alkyl, substituted or unsubstituted (C 2 -C 4 ) Alkenyl, substituted or unsubstituted (C 2 -C 4 )alkynyl.
  • the substituted (C 1 -C 4 )alkyl group is selected from the group consisting of hydroxy (C 1 -C 4 )alkyl, R 4a R 4b amino (C 1 -C 4 )alkyl or mercapto ( C 1 -C 4 )alkyl, said R 4a and R 4b are independently substituted or unsubstituted (C 1 -C 3 )alkyl; preferably hydroxy (C 1 -C 4 )alkyl or R 4a R 4b Amino (C 1 -C 4 )alkyl.
  • substitution is halo, preferably fluoro.
  • the compound represented by formula I is selected from the following compounds:
  • the compound represented by formula I is MZ77, MZ76, MZ1, MZ30, MZ32, MZ67, MZ66, MZ75, MZ31, MZ34, MZ74, MZ68, MZ29, MZ72, MZ38 or MZ24.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of formula I described in the second aspect, its various crystal forms, hydrates or solvates, and optionally pharmaceutically Acceptable excipients.
  • the present invention provides a compound represented by formula I, various crystal forms, hydrates or solvates thereof, for the prevention, treatment or improvement of Alzheimer's disease and Down's syndrome.
  • the present invention provides a method for preventing, treating or ameliorating Alzheimer’s disease and Down’s syndrome, comprising administering a compound represented by formula I, various crystal forms, hydrates or solvates thereof Steps for this required object.
  • the present invention provides a medicine for preventing, treating or improving Alzheimer's disease and Down syndrome comprising the compound represented by formula I, various crystal forms, hydrates or solvates thereof.
  • Figure 1 is a schematic diagram of knocking out Usp25 to enhance the synaptic and cognitive functions of AD mice; among them, (AF) 6-7 months old WT, Usp25 +/– , 5 ⁇ FAD, 5 ⁇ FAD; Usp25 +/– mice Behavioral analysis results.
  • B Latency to target of mice during Morris water maze (MWM) training period.
  • MVM Morris water maze
  • D Schematic diagram of the swimming trajectory of mice in the Morris water maze platform test.
  • FIG. 2 is a schematic diagram of knocking out Usp25 to enhance the synaptic and cognitive functions of DS mice; among them, (AC) 6-month-old WT, Usp25 +/– , Dp16, Dp16; Usp25 +/– mouse behavioral analysis results.
  • B Latency to target of mice during Morris water maze (MWM) training period.
  • MVM Morris water maze
  • (D) Golgi staining representative image of hippocampus tissue in 6-month-old mice and the results of dendritic spine density analysis. Ruler, 5 ⁇ m. Each group n 18-28 dendrites.
  • Figure (A, C) uses one-way ANOVA for statistical analysis
  • Figure (B) uses repeated-measures ANOVA for statistical analysis
  • Figure (F) uses lKruskal-Wallis test for statistical analysis. *P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001.
  • Figure 3 is a schematic diagram of knocking out Usp25 to improve the homeostasis of mouse microglia; among them, (AC) 6-7 months old WT, Usp25 +/– , 5 ⁇ FAD, 5 ⁇ FAD; Usp25 +/– mice, After anesthesia with 5% chloral hydrate, the brain tissue was perfused with phosphate buffer solution, the brain tissue was taken, fixed in 4% paraformaldehyde overnight, dehydrated by 25% and 30% sucrose solution, and the brain tissue was embedded using OCT.
  • Figure (A) is the result of immunohistochemical staining of hippocampus and cerebral cortex Iba1 + microglia.
  • (D) Imaris software 3D reconstruction of hippocampal tissue Iba1 + microglia phagocytic PSD95 + synapse structure representative diagram. Ruler, 10 ⁇ m.
  • (H) Imaris software 3D reconstruction of cerebral cortex Iba1 + microglia phagocytosis PSD95 + synapse structure representative diagram. Ruler, 10 ⁇ m.
  • Figure (I) shows the statistical results of the last 10 minutes of LTP.
  • n(WT+Vehicle) 5 mice/10 brain slices
  • n(5XFAD+Vehicle) 4 mice/7 brain slices
  • n(5XFAD+AZ1) 7 mice/12 brain slices .
  • Figures (B, C) used repeated-measures ANOVA for statistical analysis
  • Figures (D, F, G, I) used one-way ANOVA for statistical analysis. ns, no significant difference, P>0.05;*P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001.
  • Figure 6 is a schematic diagram showing the reversal of cognitive deficits in DS mice by administration of AZ1; among them,
  • A 5-month-old male WT and Dp16 mice were injected intraperitoneally with AZ1 or a control solvent (5% DMSO+95) at a dose of 20 mg/kg. % Corn oil), a behavioral test related to learning and memory was performed after 4 weeks of continuous administration.
  • C The total distance of the mouse in the open field test (OF test).
  • Figure 7 is a schematic diagram of the toxicological effects of AZ1 administration on mice; among them, 7-month-old WT and 5 ⁇ FAD male mice were intraperitoneally injected with AZ1 or a control solvent (5% DMSO + 95% corn oil) at a dose of 20 mg/kg. ), where WT+Vehicle is the control solvent for intraperitoneal injection of littermate control wild-type mice, 5 ⁇ FAD+Vehicle is the control solvent for intraperitoneal injection of 5 ⁇ FAD mice, and 5 ⁇ FAD+AZ1 is that 5 ⁇ FAD mice are intraperitoneally injected with AZ1 test group. After 4 weeks of continuous administration, perform learning and memory-related behavioral tests. After the test, the mice are administered for another week.
  • a control solvent 5% DMSO + 95% corn oil
  • mice are anesthetized with 5% chloral hydrate, and blood is collected by removing the eyeballs, leaving it at 4°C for more than 30 minutes, 3000rpm Centrifuge at 4°C for 5 minutes, and take serum for blood biochemical test.
  • Figure 8 is an experiment of administering AZ1 to inhibit the inflammation in the brain of AD mice; among them, 7-month-old WT and 5 ⁇ FAD male mice were intraperitoneally injected with AZ1 or a control solvent (5% DMSO + 95% corn at a dose of 20 mg/kg). Oil), where WT+Vehicle is the control solvent for intraperitoneal injection of wild-type mice in the same litter, 5 ⁇ FAD+Vehicle is the control solvent for intraperitoneal injection of 5 ⁇ FAD mice, and 5 ⁇ FAD+AZ1 is the intraperitoneal injection of 5 ⁇ FAD mice AZ1 experimental group. After 4 weeks of continuous administration, conduct learning and memory-related behavioral tests. After the test, the mice are administered for 1 week.
  • mice are anesthetized with 5% chloral hydrate, and the mice are perfused with phosphate buffered saline.
  • RNA was extracted by TRIzol, and after reverse transcription, real-time fluorescent quantitative PCR was used to detect the transcription level of inflammation-related genes.
  • Figure (A) shows the transcription level of pro-inflammatory factor Il1b
  • Figure (B) shows the transcription level of pro-inflammatory factor Il6.
  • n(WT+Vehicle) 7
  • n(5 ⁇ FAD+Vehicle) 7
  • n(5 ⁇ FAD+AZ1) 7.
  • the data uses One-way ANOVA for statistical analysis. **P ⁇ 0.01; ****P ⁇ 0.0001.
  • Figure 9 is an experiment of administering AZ1 to inhibit the proliferation and activation of microglia in the brain of AD mice; among them, 7-month-old WT and 5 ⁇ FAD male mice were injected intraperitoneally with AZ1 or a control solvent (5% DMSO) at a dose of 20 mg/kg. +9% corn oil), where WT+Vehicle is the control solvent for intraperitoneal injection of littermate control wild-type mice, 5 ⁇ FAD+Vehicle is the control solvent for intraperitoneal injection of 5 ⁇ FAD mice, and 5 ⁇ FAD+AZ1 is 5 ⁇ FAD
  • WT+Vehicle is the control solvent for intraperitoneal injection of littermate control wild-type mice
  • 5 ⁇ FAD+Vehicle is the control solvent for intraperitoneal injection of 5 ⁇ FAD mice
  • 5 ⁇ FAD+AZ1 is 5 ⁇ FAD
  • the mice were injected intraperitoneally with AZ1 experimental group. After 4 weeks of continuous administration, conduct learning and memory-related behavioral tests
  • mice are then anesthetized with 5% chloral hydrate, and the mice are perfused with phosphate buffer solution.
  • the brain tissue is taken at 4%. Fixed overnight with paraformaldehyde, dehydrated by 25% and 30% sucrose solution, and embedded brain tissue with OCT. After sectioning, immunofluorescence staining was performed to label the microglia marker protein Iba1 and the nuclear dye 4',6-diamididine Di-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI), images were collected by a laser confocal fluorescence microscope.
  • Figure (A) shows the results of immunohistochemical staining of Iba1 + microglia in the cerebral cortex, hippocampal CA1 area and DG area. Ruler, 100 ⁇ m.
  • E Imaris software 3D reconstructed hippocampal tissue Iba1 + microglia representative image. Ruler, 10 ⁇ m.
  • Figures (B, D, F, H) data were statistically analyzed using One-way ANOVA, and Figures (G, I) data were statistically analyzed using Kruskal-Wallis test. *P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001;****P ⁇ 0.0001.
  • Figure 10 is an experiment of AZ1 administration to inhibit the inflammatory response of microglia induced by lipopolysaccharide; among them, the neonatal mouse microglia of C57BL/6 mice were isolated on the 0th day after birth, cultured for 10 days, and passed 50ng/mL lipopolysaccharide respectively (Lipopolysaccharide, LPS) and 10 ⁇ M AZ1 treatment, where Control is the control group, LPS+Vehicle is 50ng/mL LPS treatment experimental group, LPS+AZ1 is 50ng/mL LPS and 10 ⁇ M AZ1 treatment experimental group at the same time. After 6 hours of treatment, immunofluorescence staining and RNA extraction were performed respectively.
  • Figure (A) is a schematic diagram of immunofluorescence staining labeled microglia marker protein Iba1. Ruler, 75 ⁇ m.
  • Figure (B) is the statistical result of the soma size of microglia in Figure (A).
  • n(Control) 79 microglia
  • n(LPS+Vehicle) 80 microglia
  • n(LPS+AZ1) 74 microglia.
  • Figures (C) and (D) show the transcription levels of inflammation-related genes Il1b and Il6 detected by real-time quantitative fluorescent PCR after reverse transcription of the extracted microglia RNA.
  • Figure (B) uses Kruskal-Wallis test for statistical analysis
  • Figure (C, D) uses One-way ANOVA for statistical analysis. **P ⁇ 0.01; ****P ⁇ 0.0001.
  • Figure 11 is an experiment in which AZ2 is administered to inhibit the inflammatory response in the brain of AD mice; among them, 6-month-old WT and 5 ⁇ FAD male mice were intragastrically administered AZ2 or a control solvent (containing 1% Tween- 80 and 0.5% CMC-Na aqueous solution, pH 3-4), where WT+Vehicle is the control solvent for intragastric administration of wild-type mice in the same litter, and 5 ⁇ FAD+Vehicle is the control solvent for intragastric administration of 5 ⁇ FAD mice
  • the control solvent, 5 ⁇ FAD+AZ2 is the experimental group of 5 ⁇ FAD mice given intragastrically AZ2.
  • FIG. 1 shows the transcription level of pro-inflammatory factor Il1b
  • Figure (B) shows the transcription level of pro-inflammatory factor Il6.
  • the data uses One-way ANOVA for statistical analysis. ns, no significant difference, P>0.05; ****P ⁇ 0.0001.
  • Figure 12 is an experiment of AZ2 inhibiting the proliferation and activation of microglia in the brain of AD mice; among them, (A) 6-month-old WT and 5 ⁇ FAD male mice were administered intragastrically with AZ2 or control at a dose of 20 mg/kg, respectively Solvent (containing 1% Tween-80 and 0.5% CMC-Na aqueous solution, pH 3 ⁇ 4), among which, WT+Vehicle is the control solvent for intragastric administration of wild-type mice in the same litter, and 5 ⁇ FAD+Vehicle is 5 ⁇ FAD mice were given control solvent by gavage, 5 ⁇ FAD+AZ2 was the experimental group of 5 ⁇ FAD mice was gavage AZ2.
  • Solvent containing 1% Tween-80 and 0.5% CMC-Na aqueous solution, pH 3 ⁇ 4
  • WT+Vehicle is the control solvent for intragastric administration of wild-type mice in the same litter
  • 5 ⁇ FAD+Vehicle is 5 ⁇ FAD mice
  • mice were anesthetized with 5% chloral hydrate, and then perfused with phosphate buffer solution. The brain tissue was taken, fixed in 4% paraformaldehyde overnight, and dehydrated with 25% and 30% sucrose solution. OCT was used to embed the brain tissue.
  • FIG. 1 shows the results of immunohistochemical staining of Iba1 + microglia in the cerebral cortex, hippocampus CA1 area and DG area. Ruler, 100 ⁇ m.
  • Figure 13 is an experiment of administering AZ2 to inhibit lipopolysaccharide-induced inflammatory response in microglia; among them, neonatal mouse microglia in C57BL/6 mice were isolated on the 0th day after birth, cultured for 10 days, and passed 50ng/mL lipopolysaccharide respectively (Lipopolysaccharide, LPS) and 10 ⁇ M AZ2 treatment, where Control is the control group, LPS+Vehicle is 50ng/mL LPS treatment group, LPS+AZ2 is 50ng/mL LPS and 10 ⁇ M AZ2 treatment group at the same time.
  • LPS lipopolysaccharide-induced inflammatory response in microglia
  • RNA was extracted and reverse transcribed, and the transcription levels of inflammation-related genes Il1b (A) and Il6 (B) were detected by real-time quantitative fluorescent PCR.
  • the data uses One-way ANOVA for statistical analysis. *P ⁇ 0.05; ****P ⁇ 0.0001.
  • Figure 14 is the USP25 inhibitor screening experiment; among them, the His-USP25 aa157-706 recombinant protein expressed in E. coli BL21 (DE3) was purified by Ni-NTA Agarose (Qiagen, Catalog No. 1018244) (USP25 aa157-706 is USP25). Deubiquitinase catalytic domain), based on Ubiquitin-Rhodamine 110 (R&D System, Catalog No.
  • Ctrl is the control solution treatment group, His-USP25 aa157-706 recombinant protein + control solvent + Ubiquitin-Rhodamine 110.
  • n 3.
  • the data was analyzed by One-way ANOVA. ns, no significant difference, P>0.05;*P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001;****P ⁇ 0.001.
  • Figure 15 is an experiment of administering the AZ1 derivative XMU1 to inhibit the inflammatory response of the microglia cell line BV2 induced by lipopolysaccharide; among them, the mouse microglia cell line BV2 was treated with 50ng/mL lipopolysaccharide (LPS) and 10 ⁇ M XMU1, respectively Among them, Control is the control group, XMU1 is 10 ⁇ M XMU1 treatment control group, LPS+Vehicle is 50ng/mL LPS treatment experimental group, LPS+XMU1 is 50ng/mL LPS and 10 ⁇ M XMU1 simultaneously treats the experimental group. After 6 hours of treatment, RNA was extracted, reverse transcription, and the transcription level of inflammation-related gene Il1b was detected by real-time fluorescent quantitative PCR.
  • LPS lipopolysaccharide
  • Figure 16 is an experiment of administering the AZ1 derivative MZ3 to inhibit the inflammatory response of the microglia cell line BV2 induced by lipopolysaccharide; among them, the mouse microglia cell line BV2 was treated with 25ng/mL Lipopolysaccharide (LPS) and 10 ⁇ M MZ3, respectively Among them, Control is the control group, LPS+Vehicle is 25ng/mL LPS treatment experimental group, LPS+MZ3 is 25ng/mL LPS and 10 ⁇ M MZ3 simultaneously treats the experimental group. After 24 hours of treatment, the RNA was extracted and reverse transcribed, and the transcription level of the inflammation-related gene Il1b was detected by real-time quantitative fluorescent PCR.
  • LPS+Vehicle is 25ng/mL LPS treatment experimental group
  • LPS+MZ3 is 25ng/mL LPS
  • 10 ⁇ M MZ3 simultaneously treats the experimental group.
  • the RNA was extracted and reverse transcribed, and the transcription level of the inflammation
  • the data uses One-way ANOVA for statistical analysis. ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • Figure 17 is an experiment of administering AZ1 derivatives MZ23 and MZ34 to inhibit the inflammatory response of the microglia cell line BV2 induced by lipopolysaccharide; in which, mice small glue was treated with 50ng/mL lipopolysaccharide (LPS) and 10 ⁇ M MZ2 or MZ3, respectively Plasma cell line BV2, where Control is the control group, LPS+Vehicle is 50ng/mL LPS treatment experimental group, LPS+MZ23 is 50ng/mL LPS and 10 ⁇ M MZ23 is simultaneously treated experimental group, LPS+MZ34 is 50ng/mL LPS and 10 ⁇ M MZ34 processed the experimental group at the same time.
  • LPS+Vehicle is 50ng/mL LPS treatment experimental group
  • LPS+MZ23 is 50ng/mL LPS
  • 10 ⁇ M MZ23 is simultaneously treated experimental group
  • LPS+MZ34 is 50ng/mL LPS and 10 ⁇ M MZ34 processed the experimental group at
  • RNA was extracted and reverse transcribed, and the transcription level of the inflammation-related gene Il1b was detected by real-time quantitative fluorescent PCR.
  • the data uses One-way ANOVA for statistical analysis. **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • Figure 18 is an experiment of administering AZ1 derivatives MZ24, MZ25, MZ26, MZ28, MZ29, and MZ31 to inhibit lipopolysaccharide-induced inflammatory response in the microglia cell line BV2; among them, 100ng/mL lipopolysaccharide (LPS) and 10 ⁇ M were passed respectively.
  • LPS lipopolysaccharide
  • MZ24, MZ25, MZ26, MZ28, MZ29, and MZ31 treated mouse microglia cell line BV2, where Control is the control group, LPS+Vehicle is 100ng/mL LPS treatment experimental group, LPS+MZ24 is 100ng/mL LPS and 10 ⁇ M MZ24 treats the experimental group at the same time, LPS+MZ25 is 100ng/mL LPS and 10 ⁇ M MZ25 treats the experimental group at the same time, LPS+MZ26 is 100ng/mL LPS and 10 ⁇ M MZ26 treats the experimental group at the same time, LPS+MZ28 is 100ng/mL LPS and 10 ⁇ M MZ28 at the same time Treat the experimental group, LPS+MZ29 is 100ng/mL LPS and 10 ⁇ M MZ29 treat the experimental group at the same time, LPS+MZ31 is 100ng/mL LPS and 10 ⁇ M MZ31 treat the experimental group at the same time.
  • the data uses One-way ANOVA for statistical analysis. ****P ⁇ 0.0001.
  • Figure 19 is a schematic diagram of the administration of AZ1 derivatives MZ75, MZ76 and MZ77 to inhibit lipopolysaccharide-induced inflammatory response in microglia; among them, neonatal mouse microglia in C57BL/6 mice were isolated on day 0 after birth, and cultured for 10 days , Were treated with 50ng/mL lipopolysaccharide (LPS) and 10 ⁇ M AZ1, MZ75, MZ76 and MZ77 respectively.
  • LPS lipopolysaccharide
  • Control is the control group
  • LPS+Vehicle is 50ng/mL LPS treatment experimental group
  • LPS+MZ76 or LPS+MZ77 is 50ng/mL LPS and 10 ⁇ M AZ1 or MZ75 or MZ76 or MZ77 at the same time to treat the experimental group.
  • the RNA was extracted and reverse transcribed, and the transcription level of the inflammation-related gene Il1b was detected by real-time quantitative fluorescent PCR.
  • Each group n 3.
  • the data uses One-way ANOVA for statistical analysis. ****P ⁇ 0.0001.
  • the inventor unexpectedly discovered a series of compounds that can prevent, treat or ameliorate Alzheimer’s disease and Down’s syndrome, in particular, prevent, treat or ameliorate Alzheimer’s disease And Down’s syndrome caused by impaired cognitive function.
  • Most of these compounds discovered in the present invention have brand-new structures, thereby laying a new material foundation for the development of drugs for the prevention, treatment or improvement of Alzheimer's disease and Down syndrome. The present invention has been completed on this basis.
  • each substituent is selected independently of the other. Therefore, each substituent may be the same or different from another (other) substituent.
  • treating means reversing, alleviating, inhibiting the progression of the disorder or condition or one or more symptoms of the disorder or condition or preventing it.
  • treatment refers to a treatment action as “treatment” as defined just above.
  • treatment also includes adjuvant therapy and neoadjuvant therapy treatment of an individual.
  • C 1-n means that the group has 1-n carbon atoms
  • C 1-6 means that the group has 1, 2, 3, 4, 5 or 6 carbon atoms
  • C2-C6 means that the group has 2, 3, 4, 5, or 6 carbon atoms.
  • C 1 -C 6 alkyl specifically refers to independently disclosed methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • alkyl as used herein has the same meaning as commonly understood by those of ordinary skill in the art, and refers to various saturated or unsaturated linear, side chain or cyclic hydrocarbon groups.
  • the alkyl group described herein refers to a lower alkyl group of 1 to 6 carbon atoms; preferably, it refers to a lower alkyl group of 1 to 4 carbon atoms.
  • the alkyl groups described herein include but are not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and many more.
  • C 1-6 alkoxy as used herein has the same meaning as commonly understood by those of ordinary skill in the art, and refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy Group, ethoxy, propoxy, isopropoxy, butoxy, etc.
  • halogen refers to F, Cl, Br, or I.
  • substituted refers to the replacement of one or more hydrogen atoms on a specific group by a specific substituent.
  • the specific substituents may be the substituents correspondingly described in the foregoing, or may be specific substituents appearing in each embodiment. Therefore, in the present invention, the substituents in formula I can be each independently a corresponding group in the specific compound in the embodiment; that is, the present invention includes the combination of the substituents in the above formula I, and also includes the substituents in the formula I. The combination of some substituents and other specific substituents appearing in the examples is shown.
  • “Pharmaceutically acceptable” in the present invention refers to such compounds, raw materials, compositions and/or dosage forms, which are within the scope of reasonable medical judgment and are suitable for contact with patient tissues without excessive toxicity, irritation, Allergies or other problems and complications that are commensurate with a reasonable benefit/risk ratio, and are effectively used for the intended purpose.
  • the "pharmaceutically acceptable salt” used in the present invention refers to the organic and inorganic salts of the compound of the present invention.
  • Pharmaceutically acceptable salts are well known in the art.
  • the salts formed by pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts, such as hydrochloride, hydrobromide, and phosphoric acid. Salt, sulfate, perchlorate, and organic acid salts, such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, or through books These salts can be obtained by other methods described, such as ion exchange methods.
  • non-toxic acid salts including adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate , Butyrate, camphorate, camphor sulfonate, cyclopentyl propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glutamate Heptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate, lauric acid Salt, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectinate, persulfuric acid Salt, 3-pheny
  • Pharmaceutically acceptable salts obtained with appropriate bases include alkali metal salts and alkaline earth metal salts.
  • Alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.
  • the present invention also intends to contemplate any quaternary ammonium salts formed by compounds containing N groups. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization.
  • Pharmaceutically acceptable salts further comprise suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion, such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -C 8 sulfonate and aromatic sulfonate.
  • the present invention provides compounds capable of preventing, treating or ameliorating Alzheimer's disease and Down's syndrome, in particular, preventing, treating or ameliorating cognitive function impairment caused by Alzheimer's disease and Down's syndrome.
  • the results of the experiment confirmed that the compound of the present invention has a significant improvement effect on the learning and memory impairment and synaptic dysfunction of the 5 ⁇ FAD mouse model of Alzheimer’s disease, and can reduce the microglia in the cerebral cortex and hippocampus.
  • the number and activation state of the anti-Alzheimer's disease and related diseases can be inhibited by inhibiting neuroinflammation.
  • the compound of the invention Compared with other drugs used in clinical practice, the compound of the invention has simple structure, clear drug effect, easy synthesis and preparation, and can effectively pass through the blood-brain barrier, and can effectively improve Alzheimer's disease and Down syndrome mouse models
  • the present invention provides a compound represented by formula I, various crystal forms, hydrates, solvates or pharmaceutically acceptable salts thereof,
  • the present invention provides the following compounds:
  • the compound of the present invention is AZ1, AZ2, MZ77, MZ76, MZ1, MZ30, MZ32, MZ67, MZ66, MZ75, MZ31, MZ34, MZ74, MZ68, MZ29, MZ72, MZ38, or MZ24.
  • the compound of the present invention can be used to prevent, treat or ameliorate Alzheimer's disease and Down's syndrome, especially to prevent, treat or ameliorate cognitive function impairment caused by Alzheimer's disease and Down's syndrome. Therefore, on the basis of the compound of the present invention and various crystal forms, hydrates, solvates or pharmaceutically acceptable salts thereof.
  • the present invention also provides a pharmaceutical composition comprising the compound of the present invention, the pharmaceutical composition optionally comprising pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • “Pharmaceutically acceptable excipients or carriers” refer 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 blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as emulsifiers
  • wetting agents such as sodium lauryl sulfate
  • the method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glycty
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents 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-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous 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 dosage form of the compound of the present invention for topical administration includes ointment, powder, patch, propellant and inhalant.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • 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 when administered is the effective dosage considered pharmaceutically.
  • the compounds and pharmaceutical compositions of the present invention can be administered via oral, nasal, skin, lung or gastrointestinal routes. The most preferred is oral administration, one-time administration or divided-time administration. Regardless of the method of administration, the individual's optimal dosage should be determined based on the specific treatment. Usually, start with a small dose and gradually increase the dose until the most suitable dose is found. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the present invention provides a novel structure for the prevention, treatment or improvement of Alzheimer’s disease and Down’s syndrome, especially the prevention, treatment or improvement of cognitive function impairment caused by Alzheimer’s disease and Down’s syndrome Compound
  • the compound of the present invention has simple structure, clear drug effect, and easy synthesis and preparation;
  • the compound of the present invention can effectively pass through the blood-brain barrier
  • the compound of the present invention will not produce other adverse reactions when applied in a larger dose, and has better safety.
  • the chromatography column used in the following examples is a silica gel column.
  • the silica gel is (200-300 mesh).
  • Compounds AZ1 and AZ2 are commercial drugs, which can also be synthesized according to prior art documents, such as Wrigley, et al., ACS Chem. Biol. 2017.
  • Step 1) Under the protection of nitrogen, potassium carbonate (2.93g, 21.22mmol), 4-(bromomethyl)-1-fluoro-2-(trifluoromethyl)benzene (3g, 11.67mmol) and 5-bromo 2-Hydroxybenzaldehyde (2.13g, 10.61mmol) was sequentially added to a 250ml three-necked flask, and then 60ml DMF was added, and magnetically stirred at room temperature 25°C to obtain a suspension. Stir at room temperature overnight (about 15 hours), and TLC the next day Monitor the disappearance of the raw materials and terminate the reaction.
  • reaction solution was poured into 1.2L ice water, there was a white precipitate, the white solid obtained was filtered, washed with 3 ⁇ 500ml water, and dried under vacuum to obtain 5-bromo-2-(4-fluoro-3-(trifluoromethyl)benzyl (Oxy)benzaldehyde (3.5 g, 87.46%) as a white solid.
  • reaction formula 5-bromo-2-(4-fluoro-3-(trifluoromethyl)benzyl (Oxy)benzaldehyde (3.5 g, 87.46%) as a white solid.
  • Step 2 Under the protection of nitrogen, add 5-bromo-2-(4-fluoro-3-(trifluoromethyl)benzyloxy)benzaldehyde (3g, 7.95mmol) into a 250ml mouth flask, and add 1.0M NH 3 /THF (80 mL) solution, and then the solution was stirred for 10 minutes. Then sodium triacetylborohydride (6.74 g, 31.82 mmol) was added in portions, about 30 minutes. After the addition, the resulting suspension was stirred at 20°C for 16 hours. TLC followed the reaction until the raw material disappeared and stopped the reaction.
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 hours, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.37 g, 89.3%).
  • Step 2 Combine 4-difluoromethoxy-3-(3,5-bistrifluoromethylbenzyl)benzaldehyde (0.21g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), acetic acid (0.17mL , 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, sodium triacetoxyborohydride (0.33g, 1.5mol) was added and the reaction continued for 24h, and extracted with ethyl acetate Adjust the pH to weakly alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.20 g, 87.1%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.37g, 89.3%).
  • Step 2 Combine 4-difluoromethoxy-3-(3,5-bistrifluoromethylbenzyl)benzaldehyde (0.21g, 0.5mmol), N-methylpiperazine (0.17mL, 1.5mL) , Acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added to continue the reaction for 24h , Ethyl acetate extraction, saturated sodium bicarbonate solution to adjust pH to weakly alkaline, combined organic phases, silica gel column chromatography purification, to obtain an oily liquid (0.17g, 68.3%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4,6-dimethoxysalicylic aldehyde (0.18g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and purified by silica gel column to obtain a white solid (0.44g, 89.4%).
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyl)-4,6-dimethoxysalicylaldehyde (0.20g, 0.49mmol), 1-(2-hydroxyethyl)piperazine (0.18mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g , 1.5mol) continue the reaction for 24h, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.15g, 58.6%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.37g, 89.3%).
  • Step 2 Combine 4-difluoromethoxy-3-(3,5-bistrifluoromethylbenzyl)benzaldehyde (0.21g, 0.5mmol), 1-(2-hydroxyethyl)piperazine (0.18 mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were added to a 100mL three-necked round-bottomed flask successively, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5 mol) Continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.18 g, 68.0%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.37g, 89.3%).
  • Step 2 Combine 4-difluoromethoxy-3-(3,5-bistrifluoromethylbenzyl)benzaldehyde (0.21g, 0.5mmol), N,N-diethylethylenediamine (0.21mL , 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mol ) Continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.18 g, 70.03%).
  • Step 1) Combine 2,4,5-trifluorobenzyl bromide (0.16mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.3 g, 90.0%).
  • Step 2 Combine 4-difluoromethoxy-3-(2,4,5-trifluorobenzyl)benzaldehyde (0.167g, 0.5mmol), N-methylpiperazine (0.17mL, 1.5mL), Acetic acid (0.17mL, 1.5mmol) and THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added and the reaction continued for 24 hours. Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.15 g, 71.9%).
  • Step 1) Combine 2,4,5-trifluorobenzyl bromide (0.16mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.3g, 90.0%).
  • Step 2 Combine 4-difluoromethoxy-3-(2,4,5-trifluorobenzyl)benzaldehyde (0.167g, 0.5mmol), 1-(2-hydroxyethyl)piperazine (0.18mL , 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mol ) Continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.13 g, 58.2%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.19mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.32g, 85.3%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyl)-5-bromobenzaldehyde (0.19g, 0.5mmol), tryptamine (0.24g, 1.5mmol), acetic acid (0.17mL, 1.5 mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, sodium triacetoxyborohydride (0.33g, 1.5mol) was added to continue the reaction for 24h, extracted with ethyl acetate, saturated Adjust the pH to weakly alkaline with sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.15 g, 57.6%).
  • Step 1) Combine 2,4,5-trifluorobenzyl bromide (0.16mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF ( 10mL) was sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.30g, 87.2%).
  • Step 2 Combine 2-(2,4,5-trifluorobenzyl)-5-bromobenzaldehyde (0.17g, 0.5mmol), N,N-diethylethylenediamine (0.21mL, 1.5mmol), Acetic acid (0.17mL, 1.5mmol) and THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added and the reaction continued for 24 hours. Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.12 g, 54.05%).
  • Step b) Put compound 2 (0.41g, 1.5mmol) in a 100mL round bottom flask, add DCM (10mL) and di-tert-butyl dicarbonate (0.69mL, 0.3mmol) in sequence, and react at room temperature until TLC detects that the reaction is complete. It was quenched with water, extracted with ethyl acetate, and the organic phases were combined and purified by normal phase chromatography on a silica gel column to obtain compound 3 (0.50 g, 89.8%).
  • Step c) Add compound 3 (0.56g, 1.5mmol), reduced iron powder (0.42g, 7.5mmol), NH 4 Cl (0.80g, 15mmol), MeOH (15mL) into a 100 round bottom flask, and reflux at 80°C After the reaction was detected by TLC, the reaction was quenched by adding water, extracted with ethyl acetate, and the organic phases were combined and purified by normal phase chromatography on a silica gel column to obtain compound 4 (0.37 g, 71.6%).
  • Step d) Under N 2 protection, add 3-trifluoromethyl-4-fluorobenzaldehyde (0.096g, 0.5mmol), DCM (10mL) into a 100mL double-necked round bottom flask, and add compound 4 (0.17g, 0.5mmol), acetic acid (0.28mL, 0.25mmol), react at room temperature for 2h, add sodium triacetoxyborohydride (0.32g, 1.5mmol), until TLC detects the reaction is complete, saturated sodium bicarbonate solution quenched, ethyl acetate After ester extraction, the organic phases were combined and purified by normal phase chromatography on a silica gel column to obtain compound 5-1 (0.20 g, 76.4%).
  • Step e Put compound 5-1 (0.182g, 0.35mmol) in a 100mL round bottom flask, add DCM (10mL) and 2mL trifluoroacetic acid successively, react at room temperature for 1h, quench with saturated sodium bicarbonate solution, and extract with ethyl acetate The organic phases were combined and purified by normal phase chromatography on a silica gel column to obtain compound MZ76 (0.13 g, 87.1%).
  • Step 1) 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF ( 10mL) was sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and purified by silica gel column to obtain a white solid (0.40g, 94.1%).
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyloxy)-5-bromobenzaldehyde (0.21g, 0.5mmol), ethanolamine (0.09mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol) ), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, sodium triacetoxyborohydride (0.33g, 1.5mmol) was added to continue the reaction for 24h, extracted with ethyl acetate, saturated carbonic acid Adjust the pH to weak alkaline with sodium hydrogen solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.14 g, 59.45%).
  • Step 1) Combine 2,4,5-trifluorobenzyl bromide (0.16mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 hours, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and purified by silica gel column to obtain a white solid (0.3 g, 90.0%).
  • Step 2 Combine 4-difluoromethoxy-3-(2,4,5-trifluorobenzyloxy)benzaldehyde (0.167g, 0.5mmol), tetrahydroisoquinoline (0.19mL, 1.5mmol), Acetic acid (0.17mL, 1.5mmol) and THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1h, then sodium triacetoxyborohydride (0.33g, 1.5mmol) was added and the reaction continued for 24h. Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.14 g, 60.4%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.19mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.32g, 85.3%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyloxy)-5-bromobenzaldehyde (0.19g, 0.5mmol), 1-(2-hydroxyethyl)piperazine (0.18mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mmol) Continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.13 g, 53.06%).
  • Step 1) Add 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 2-hydroxybenzaldehyde (0.12g, 1mmol), potassium carbonate (0.41g, 3mmol), and DMF (10mL) in sequence
  • DMF 10mL
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyloxy)benzaldehyde (0.17g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) was added to a 100mL three-necked round bottom flask in sequence, protected by nitrogen, after reacting for 1 hour at room temperature, sodium triacetoxyborohydride (0.33g, 1.5mol) was added to continue the reaction for 24 hours, extracted with ethyl acetate, saturated sodium bicarbonate solution Adjust the pH to weakly alkaline, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.11 g, 56.0%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.18mL, 1.2mmol), 2-hydroxybenzaldehyde (0.12g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) in sequence It was added to a 100ml single-necked round-bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.25g, 82.3%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyloxy)benzaldehyde (0.15g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) was sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added to continue the reaction for 24 hours, extracted with ethyl acetate, saturated sodium bicarbonate The pH of the solution was adjusted to weakly alkaline, the organic phases were combined and purified by silica gel column chromatography to obtain a white solid (0.10 g, 58.3%).
  • Step 2 The 2-(3,5-bistrifluoromethylbenzyloxy)-4,6-dimethoxysalicylic aldehyde (0.20g, 0.49mmol), 2-amino-5-bromopyridine (0.26 g, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, reacted at room temperature for 1h, then added sodium triacetoxyborohydride (0.33g, 1.5 mmol) continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weakly alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.16 g, 58.6%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.18mL, 1.2mmol), 3-hydroxy-4-methoxybenzaldehyde (0.15g, 1mmol), potassium carbonate (0.41g, 3mmol) , DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 hours, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.25 g, 79.2%).
  • Step 2 Combine 3-((4-fluoro-3-(trifluoromethyl)benzyl)oxy)-4-methoxybenzaldehyde (0.16g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol) , Acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added to continue the reaction for 24h , Ethyl acetate extraction, saturated sodium bicarbonate solution to adjust the pH to weakly basic, combined organic phases, silica gel column chromatography purification, to obtain an orange solid (0.10g, 53.6%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.19mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.32g, 85.3%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyloxy)-5-bromobenzaldehyde (0.19g, 0.5mmol), N-ethylpiperazine (0.19mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after 1h reaction at room temperature, sodium triacetoxyborohydride (0.33g, 1.5mmol) was added to continue the reaction for 24h, acetic acid Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.11 g, 54.62%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.18mL, 1.2mmol), 2-hydroxy-4,6-dimethoxybenzaldehyde (0.18g, 1mmol), potassium carbonate (0.41g , 3mmol), DMF (10mL) were sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.30g, 83.4%) .
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyloxy)-4,6-dimethoxybenzaldehyde (0.18g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), Acetic acid (0.17mL, 1.5mmol) and THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added and the reaction continued for 24 hours. Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.11 g, 54.5%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4-methoxy-3-hydroxybenzaldehyde (0.15g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 hours, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.30 g, 80.7%).
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyloxy)-4-methoxy-3-hydroxybenzaldehyde (0.19g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), Acetic acid (0.17mL, 1.5mmol) and THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, and reacted at room temperature for 1 hour, then sodium triacetoxyborohydride (0.33g, 1.5mol) was added and the reaction continued for 24 hours. Extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.11 g, 51.2%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 2-hydroxy-4,6-dimethoxybenzaldehyde (0.18g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) were sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.33g, 81.5%).
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyloxy)-4,6-dimethoxybenzaldehyde (0.20g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mol) to continue the reaction for 24h, acetic acid Extract with ethyl ester, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.11 g, 50.8%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.19mL, 1.2mmol), 2-hydroxy-5-bromobenzaldehyde (0.20g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.32g, 85.3%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyloxy)-5-bromobenzaldehyde (0.19g, 0.5mmol), N-ethylpiperazine (0.19mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1 hour, sodium borohydride (0.05g, 1.5mmol) was added to continue the reaction for 24 hours, and then extracted with ethyl acetate. Adjust the pH to weakly alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.065 g, 29.9%). This compound is a by-product of MZ33.
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyl)-5-bromobenzaldehyde (0.19g, 0.5mmol), 4-(2-aminoethyl)-1-benzylpiperidine (0.33g, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g , 1.5mmol) continue the reaction for 24h, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.21g, 44.82%).
  • Step 2 Combine 2-(4-fluoro-3-trifluoromethylbenzyl)-5-bromobenzaldehyde (0.19g, 0.5mmol), 4-(2-aminoethyl)-1-benzylpiperidine (0.33mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g , 1.5mol) continue the reaction for 24h, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain a white solid (0.15g, 51.77%).
  • Step 1) Combine 2,4,5-trifluorobenzyl bromide (0.16mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.13g, 1mol), potassium carbonate (0.41g, 3mmol) ), DMF (10 mL) was sequentially added to a 100 ml single-necked round bottom flask, reacted at room temperature for 6 hours, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and purified by silica gel column to obtain a white solid (0.3 g, 90.0%).
  • Step 2 Combine 4-difluoromethoxy-3-(2,4,5-trifluorobenzyl)benzaldehyde (0.167g, 0.5mmol), N,N-diethylethylenediamine (0.21mL, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mmol) Continue the reaction for 24 hours, extract with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an oily liquid (0.15 g, 69.4%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 4,6-dimethoxysalicylic aldehyde (0.18g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) was added to a 100ml single-necked round-bottom flask in turn, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.44g, 89.4%).
  • Step 2 The 2-(3,5-bistrifluoromethylbenzyl)-4,6-dimethoxysalicylic aldehyde (0.20g, 0.49mmol), N-methylpiperazine (0.17mL, 1.5 mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mmol) to continue After reacting for 24 hours, extracting with ethyl acetate, adjusting the pH to weak alkaline with saturated sodium bicarbonate solution, combining the organic phases, and purifying by silica gel column chromatography to obtain a white solid (0.19 g, 78.6%).
  • Step 1) Combine 4-fluoro-3-trifluoromethyl benzyl bromide (0.18mL, 1.2mmol), 4-difluoromethoxy-3-hydroxybenzaldehyde (0.19g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) were sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.29g, 79.6%).
  • Step 2 Combine 4-difluoromethoxy-3-((4-fluoro-3-(trifluoromethyl)benzyl)oxy)benzaldehyde (0.18g, 0.5mmol), ethanolamine (0.09ml, 1.5 mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after reacting at room temperature for 1h, add sodium triacetoxyborohydride (0.33g, 1.5mol) to continue After reacting for 24 hours, extracting with ethyl acetate, adjusting the pH to weak alkaline with saturated sodium bicarbonate solution, combining the organic phases, and purifying by silica gel column chromatography to obtain an orange solid (0.14 g, 68.4%).
  • Step 1) Combine 3,5-bistrifluoromethyl benzyl bromide (0.22mL, 1.2mmol), 2-hydroxy-4,6-dimethoxybenzaldehyde (0.18g, 1mmol), potassium carbonate (0.41g, 3mmol), DMF (10mL) were sequentially added to a 100ml single-necked round bottom flask, reacted at room temperature for 6h, extracted with ethyl acetate, combined the organic phases, concentrated under reduced pressure, and then purified by silica gel column to obtain a white solid (0.34g, 82.7%).
  • Step 2 Combine 2-(3,5-bistrifluoromethylbenzyloxy)-4,6-dimethoxybenzaldehyde (0.20g, 0.5mmol), ethanolamine (0.09ml, 1.5mmol), acetic acid (0.17mL, 1.5mmol), THF (10mL) were sequentially added to a 100mL three-necked round bottom flask, protected by nitrogen, after 1h reaction at room temperature, sodium triacetoxyborohydride (0.33g, 1.5mmol) was added to continue the reaction for 24h, acetic acid Extraction with ethyl acetate, adjust the pH to weak alkaline with saturated sodium bicarbonate solution, combine the organic phases, and purify by silica gel column chromatography to obtain an orange solid (0.12 g, 55.1%).
  • Y-maze test (Y-maze test) is used to evaluate spontaneous spatial alternation behavior and working memory of mice. Place the mouse in the center of the Y-maze (length 30cm, width 6cm, height 15cm), and then let the mice explore freely in the maze for 5 minutes. All the limbs of the mouse enter the labyrinth arm as the criterion for entering the labyrinth arm, and the mouse enters the different labyrinth arms three times consecutively as a correct autonomous alternate shuttle (Alternation).
  • Morris water maze test (Morris water maze test) is carried out in a circular water tank (diameter 120 cm).
  • the height of the water in the tank should be 1 cm higher than the platform, and the temperature of the water in the tank is set to 22°C.
  • Four icons of different shapes are affixed to the four directions in the labyrinth arm as reference objects for spatial positioning.
  • the platform was 1 cm below the surface of the water, and then the mice were put into the two water entry points of the maze, and the mice were allowed to search the platform for 60 seconds. The experiment was stopped by the mouse staying on the platform for 10 seconds. If the mouse cannot find the platform within 60 seconds, guide it to the location of the platform and allow it to stay on the platform for 10 seconds.
  • Each mouse was tested twice a day, and two different directions were randomly selected to enter the water.
  • the interval between two experiments for each mouse should be at least 1 hour. Record the latency to target of the mouse to find the platform in each experiment. Continuous learning and training for 6 days. On the 7th day, the platform was removed and the platform test was performed. The mouse was placed in the water from the diagonal position of the platform and allowed to search freely in the water maze for 60 seconds. Record the target quadrant of the platform and the other three differences The swimming time of the quadrant (Time in quadrant).
  • mice were placed in a test box and adapted to the environment for 2 minutes. Then they gave the mice a 60 decibel noise stimulus (conditioning stimulus) for 30 seconds, and In the last 2 seconds of the noise stimulation, the mice were given an electric shock stimulation of 0.05 mA (unconditioned stimulation). Repeat three times with an interval of 60 seconds each time. After the final electric shock stimulation, let the mice stay in the experiment box for 90 seconds.
  • a contextual test was performed. The mice were placed in the same test box for 5 minutes, and the percentage of freezing time (Freezing%) of the mice was recorded to determine the contextual memory. In the afternoon of the first day after training, a Cued test was performed.
  • mice were placed in a test box (different walls and floors) that was different from the previous training environment.
  • the mice were first allowed to stay in a normal environment for 3 minutes. , And then perform sound stimulation at 60 decibels for 3 minutes, and record the percentages (Freezing%) of the mice's freezing time under the sound stimulation and in the normal environment.
  • the neuronal dendritic spine density was analyzed by Golgi staining. As shown in Figure 1G, compared with WT mice, the neuronal dendritic spine density in the brain of 5 ⁇ FAD mice was significantly reduced, while the knockout of Usp25 significantly increased by 5 ⁇ FAD; Dendritic spines density of neurons in Usp25 +/– mouse brain.
  • Electrophysiological recordings of brain slices were performed on WT, Usp25 +/– , 5 ⁇ FAD, 5 ⁇ FAD; Usp25 +/– mice aged 6 to 7 months. After the mice were anesthetized, the brain tissues were quickly taken out and placed in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF) to cool, and then transferred to an oscillating microtome for coronal sectioning. The thickness of the brain slice was 400 ⁇ m. The brain slices were incubated in ACSF saturated with oxygen at 32°C for 1 hour, then transferred to room temperature and incubated for 1 hour. The recording electrode was placed in the radiation layer of the CA1 area of the Schaffer collateral-commissural pathway, and the stimulation electrode was placed in the CA3 area.
  • ASF oxygenated artificial cerebrospinal fluid
  • the stimulation intensity is 30% of the maximum value of field excitatory postsynaptic potential (fEPSP).
  • HFS high-frequency stimulation
  • LTP long-term potentiation
  • Example 39 Knockout Usp25 enhances synaptic and cognitive functions in DS mice
  • T-maze test is used to evaluate spontaneous spatial alternation behavior and working memory of mice. Place the mouse in the center of the T-maze (length 30cm, width 6cm, height 15cm), and then let the mice explore freely in the maze for 5 minutes. All the limbs of the mouse enter the labyrinth arm as the criterion for entering the labyrinth arm, and the mouse enters the different labyrinth arms three times consecutively as a correct autonomous alternate shuttle (Alternation).
  • Morris water maze test (Morris water maze test) is carried out in a circular water tank (diameter 120 cm).
  • the height of the water in the tank should be 1 cm higher than the platform, and the temperature of the water in the tank is set to 22°C.
  • Four icons of different shapes are affixed to the four directions in the labyrinth arm as reference objects for spatial positioning.
  • the platform was 1 cm below the surface of the water, and then the mice were put into the two water entry points of the maze, and the mice were allowed to search the platform for 60 seconds. The experiment was stopped by the mouse staying on the platform for 10 seconds. If the mouse cannot find the platform within 60 seconds, guide it to the location of the platform and allow it to stay on the platform for 10 seconds.
  • Each mouse was tested twice a day, and two different directions were randomly selected to enter the water.
  • the interval between two experiments for each mouse should be at least 1 hour. Record the latency to target of the mouse to find the platform in each experiment. Continue learning and training for 5 days. On the 6th day, the platform was removed and the platform test was performed. The mouse was placed in the water from the diagonal position of the platform and allowed to search freely in the water maze for 60 seconds. Record the number of times the mouse shuttled the quadrant of the platform (Target crossings) .
  • the dendritic spine density of neurons was analyzed by Golgi staining. As shown in Figure 2D, compared with WT mice, the dendritic spine density of neurons in the brain of Dp16 mice was significantly reduced, while the deletion of Usp25 significantly increased Dp16; Usp25 + /– Dendritic spines density of neurons in the mouse brain.
  • Electrophysiological recordings of brain slices were performed on 6-month-old WT, Dp16, Dp16; Usp25 +/– mice. After the mice were anesthetized, the brain tissues were quickly taken out and placed in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF) to cool, and then transferred to an oscillating microtome for coronal sectioning. The thickness of the brain slice was 400 ⁇ m. The brain slices were incubated in ACSF saturated with oxygen at 32°C for 1 hour, then transferred to room temperature and incubated for 1 hour. The recording electrode was placed in the radiation layer of the CA1 area of the Schaffer collateral-commissural pathway, and the stimulation electrode was placed in the CA3 area.
  • ASF oxygenated artificial cerebrospinal fluid
  • the stimulation intensity is 30% of the maximum value of field excitatory postsynaptic potential (fEPSP).
  • HFS high-frequency stimulation
  • LTP long-term potentiation
  • Example 40 Knockout Usp25 improves mouse microglia homeostasis
  • the brain tissue was embedded with OCT, and then sliced and restored with sodium citrate buffer, using 0.2% Triton X-100 was blocked with 3% BSA buffer, and then immunofluorescence staining was performed to label the microglia marker protein Iba1 (Wako) and the nuclear dye 4',6-diamidino-2-phenylindole ( 4',6-diamidino-2-phenylindole, DAPI) (Sigma Company), and then image acquisition by laser confocal fluorescence microscope.
  • knocking out Usp25 significantly reduced the phagocytosis of neuronal synapses by microglia in the brain of 5 ⁇ FAD mice.
  • knocking out Usp25 significantly inhibits the proliferation and activation of microglia and microglia-mediated synaptic phagocytosis in the brain of AD mice.
  • Example 41 Knockout Usp25 inhibits the release of inflammatory factors and synaptic phagocytosis in mouse microglia
  • Isolate Usp25 +/+ and Usp25 –/– mouse neonatal mouse microglia on the 0th day after birth culture them in DMEM medium containing 25ng/mL GM-CSF+10% fetal calf serum for 10 days, then shake at 220rpm for 15 minutes to separate microglia culture for one day, were treated 10 ⁇ MAZ1 or DMSO solvent control, while the processing or control solvent 10 ⁇ M oA ⁇ 42 Vehicle and pHrodo-Red labeled synaptosomes (Syn), 24 hours, the Cells were fixed with 4% paraformaldehyde, 0.2% Triton X-100 solution penetrated, 3% BSA solution blocked, and then the microglia marker protein Iba1 was labeled by immunofluorescence, and the fluorescence of pHrodo-Red in Iba1 + microglia was analyzed.
  • oA ⁇ 42 treatment can induce microglia to phagocytose pHrodo-Red labeled synaptosomes, while knocking out Usp25 can reduce oA ⁇ 42- induced phagocytosis of synaptosomes by microglia; Ciwai, AZ1 process may be reduced oA ⁇ 42 induced phagocytosis of microglia synaptosomes body; Ranhou Usp25 - / - microglia Zhong, AZ1 processing is not longer reduces microglial phagocytosis of synaptosomes of Ti , Indicating that the inhibitory effect of AZ1 on the phagocytic synapse of microglia depends on USP25.
  • Example 42 AZ1 penetration test of the blood-brain barrier
  • ICR mice After intragastric administration of ICR mice, brain tissue and blood samples were collected at different time points to study the blood-brain barrier properties of AZ1 in ICR mice.
  • Preparation of AZ1 solution for intragastric administration accurately weigh about 4 mg of AZ1 into a glass bottle, add an appropriate volume of 0.5% CMC-Na aqueous solution (1% Tween-80), adjust the pH to 3 to 4 with 1M HCl, and vortex. Then, it was sonicated to dissolve, and a final concentration of 1 mg/mL was obtained.
  • CMC-Na aqueous solution 1% Tween-80
  • mice Nine 27.8 ⁇ 33.3g ICR male mice were divided into three groups (different administration time 0.5, 2, 8 hours), and the experimental animals were administered intragastrically at a dose of 10 mg/kg, and then 0.5, 2 and 8 hours after administration. 2. After 8 hours of anesthesia with carbon dioxide, blood was collected from the heart (about 0.2 mL) and executed. The whole blood is placed in a test tube containing the anticoagulant EDTA-K2, stored on wet ice, and centrifuged (1500 ⁇ 1600g) for 10 minutes within 1 hour to separate the plasma.
  • the brain tissues were taken, washed with ice physiological saline, and weighed after absorbing the water, and 20% methanol-water was added according to the weight-volume ratio of 1:5 (tissue: homogenate). Homogenate.
  • the plasma and brain tissue homogenates are stored in a refrigerator at -40 to -20°C for sample analysis.
  • the LC-MS/MS (API 4000: LC-MS-MS-010) method was used by Suzhou Shengsu New Pharmaceutical Development Co., Ltd. to determine the concentration of AZ1 in the plasma and brain of ICR mice after administration.
  • Table 1 after 0.5, 2, and 8 hours after administration, the level of AZ1 in plasma decreased from 68.8 ⁇ 11.9ng/mL to 24.8 ⁇ 5.0ng/mL and 3.9 ⁇ 1.1ng/mL, and the level of AZ1 in the brain The level gradually decreased from 559.2 ⁇ 138.3ng/g to 388.2 ⁇ 49.0ng/g and 80.8 ⁇ 1.2ng/g.
  • AZ1 can penetrate the blood-brain barrier of mice.
  • mice After 4 weeks of continuous administration, behavioral tests related to learning and memory were performed, including Morris water maze test and conditioned panic test. Touch and pet the mice three days before the start of the experiment, once a day, gently grasp the tail of the mouse and pick up the mouse, let the mouse stay on the hand for 30 seconds; on the day of the experiment, transfer the experimental mouse to the preparation before the experiment In between, let the mice adapt for 30 minutes. Animal behavior experiments are carried out between 9:00a.m.-6:00p.m. every day, and the light intensity in the laboratory is 650lux. Use Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis.
  • Morris water maze test (Morris water maze test) is carried out in a circular water tank (diameter 120 cm), the height of the water in the tank should be 1 cm higher than the platform, and the temperature of the water in the tank is set to 22°C.
  • Four icons of different shapes are affixed to the four directions in the labyrinth arm as reference objects for spatial positioning.
  • the platform was 1 cm below the surface of the water, and then the mice were put into the two water entry points of the maze, and the mice were allowed to search the platform for 60 seconds. The experiment was stopped by the mouse staying on the platform for 10 seconds. If the mouse cannot find the platform within 60 seconds, guide it to the location of the platform and allow it to stay on the platform for 10 seconds.
  • Each mouse was tested twice a day, and two different directions were randomly selected to enter the water. The interval between two experiments for each mouse should be at least 1 hour. Record the latency to target of the mouse to find the platform in each experiment. Continuous learning and training for 6 days. On the 7th day, the platform was removed and the platform test was performed. The mouse was placed in the water from the diagonal position of the platform and allowed to search freely in the water maze for 60 seconds. Record the target quadrant of the platform and the other three differences Quadrant swimming time (Time in quadrant) and latency 1 st entrance to target (Latency 1 st entrance to target) for the mouse to reach the platform area for the first time.
  • Quadrant swimming time Time in quadrant
  • latency 1 st entrance to target Latency 1 st entrance to target
  • mice were placed in a test box and adapted to the environment for 2 minutes. Then they gave the mice a 60 decibel noise stimulus (conditioning stimulus) for 30 seconds, and In the last 2 seconds of the noise stimulation, the mice were given an electric shock stimulation of 0.05 mA (unconditioned stimulation). Repeat three times with an interval of 60 seconds each time. After the final electric shock stimulation, let the mice stay in the experiment box for 90 seconds.
  • a Cued test was performed. The mice were placed in a test box (different walls and floors) that was different from the previous training environment. The mice were first allowed to stay in a normal environment for 3 minutes. Then, 60 decibel sound stimulation was performed for 3 minutes, and the percentages (Freezing%) of the time that the mice appeared rigid under the sound stimulation and in the normal environment were recorded respectively.
  • AZ1 administration can reverse the cognitive deficits of AD mice.
  • 5-month-old WT and 5 ⁇ FAD male mice were injected intraperitoneally with AZ1 or a control solvent (5% DMSO+95% corn oil) at a dose of 20 mg/kg, respectively.
  • WT+Vehicle was intraperitoneal injection of wild-type mice in the same litter.
  • As the control solvent 5 ⁇ FAD+Vehicle is the control solvent for 5 ⁇ FAD mice intraperitoneally injected, and 5 ⁇ FAD+AZ1 is the experimental group of 5 ⁇ FAD mice intraperitoneally injected with AZ1.
  • mice After 4 weeks of continuous administration, the mouse brain slices were recorded electrophysiologically. After the mice were anesthetized, the brain tissues were quickly taken out and placed in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF) to cool, and then transferred to an oscillating microtome for coronal sectioning. The thickness of the brain slice was 400 ⁇ m. Place the brain slices in ACSF saturated with oxygen at 32°C and incubate for 1 hour, then transfer to room temperature and incubate for 1 hour. Place the recording electrode on the radiation layer of the CA1 area of the Schaffer collateral-commissural pathway, and place the stimulation electrode on the CA3 area. Stimulation intensity is 30% of the maximum value of excitatory postsynaptic field potential (fEPSP).
  • fEPSP excitatory postsynaptic field potential
  • HFS high-frequency stimulation
  • LTP long-term potentiation
  • Example 44 Administration of AZ1 reverses cognitive deficits in DS mice
  • WT+Vehicle was a littermate control wild-type mouse for intraperitoneal injection of the control solvent
  • Dp16+Vehicle is the control solvent for intraperitoneal injection of Dp16 mice
  • Dp16+AZ1 is the experimental group of Dp16 mice intraperitoneally injected with AZ1 ( Figure 6A). As shown in Figure 6B, AZ1 administration did not affect the body weight of the mice.
  • mice After 4 weeks of continuous administration, behavioral tests were performed, including open field test, T-maze test, new object recognition and Morris water maze test. Touch and pet the mice three days before the start of the experiment, once a day, gently grasp the tail of the mouse and pick up the mouse, let the mouse stay on the hand for 30 seconds; on the day of the experiment, transfer the experimental mouse to the preparation before the experiment In between, let the mice adapt for 30 minutes. Animal behavior experiments are carried out between 9:00a.m.-6:00p.m. every day, and the light intensity in the laboratory is 650lux. Use Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis.
  • the open field test was used to evaluate the exercise ability and anxiety-like emotions of mice. Place the mouse in the center of the open field, and then allow the mouse to explore freely in the maze for 10 minutes, and record the total distance of the mouse in the open field and the time in the center of the open field (Time in center).
  • the administration of AZ1 does not affect the movement distance of the mice in the open field and the activity time in the central area of the open field, indicating that the administration of AZ1 has no significant toxicological effect on the mice.
  • Novel object recognition test is based on mice's innate tendency to explore new things.
  • the process of exploring new things reflects the process of learning, recognition, and memory in mice, and can evaluate the working memory of mice.
  • Dp16+Vehicle mice have significantly reduced the number of explorations in new objects, and administration of AZ1 can significantly increase the exploration of new objects in Dp16 mice, indicating that administration of AZ1 can Enhance the working memory of Dp16 mice.
  • Morris water maze test (Morris water maze test) is carried out in a circular water tank (diameter 120 cm).
  • the height of the water in the tank should be 1 cm higher than the platform, and the temperature of the water in the tank is set to 22°C.
  • Four icons of different shapes are affixed to the four directions in the labyrinth arm as reference objects for spatial positioning.
  • the platform was 1 cm below the surface of the water, and then the mice were put into the two water entry points of the maze, and the mice were allowed to search the platform for 60 seconds. The experiment was stopped by the mouse staying on the platform for 10 seconds. If the mouse cannot find the platform within 60 seconds, guide it to the location of the platform and allow it to stay on the platform for 10 seconds.
  • Each mouse was tested twice a day, and two different directions were randomly selected to enter the water.
  • the interval between two experiments for each mouse should be at least 1 hour. Record the latency to target of the mouse to find the platform in each experiment. Continue learning and training for 5 days. On the 6th day, the platform was removed and the platform test was performed. The mouse was placed in the water from the diagonal position of the platform, and allowed to search freely in the water maze for 60 seconds. Record the target quadrant where the platform is located and the other three differences The swimming time of the quadrant (Time in quadrant) and the number of times the mouse shuttle platform is located in the quadrant (Target crossings).
  • Dp16+Vehicle mice did not show significant learning ability defects during the training process of the water maze.
  • FIG. 6G in the platform test, compared with WT+Vehicle mice, Dp16+Vehicle mice spent less time in the target quadrant, while AZ1 administration significantly increased the time of Dp16 mice in the target quadrant;
  • Fig. 6H after AZ1 administration, the number of shuttles of Dp16 mice in the quadrant of the platform was significantly increased. In summary, it was suggested that AZ1 administration significantly improved the spatial learning and memory ability of DS mice.
  • WT+Vehicle was intraperitoneal injection of wild-type mice in the same litter.
  • control solvent 5 ⁇ FAD+Vehicle is the control solvent for 5 ⁇ FAD mice intraperitoneally injected
  • 5 ⁇ FAD+AZ1 is the experimental group of 5 ⁇ FAD mice intraperitoneally injected with AZ1.
  • liver function includes ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), TP (total protein), ALB (albumin), kidney function includes Urea (Urea), CREA-S (creatinine), blood lipids including TC (total cholesterol), TG (triglycerides), myocardial enzyme spectrum CK (creatine kinase), blood glucose Glu-G.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • TP total protein
  • ALB albumin
  • kidney function includes Urea (Urea), CREA-S (creatinine), blood lipids including TC (total cholesterol), TG (triglycerides), myocardial enzyme spectrum CK (creatine kinase), blood glucose Glu-G.
  • TC total cholesterol
  • TG total cholesterol
  • TG
  • Example 46 Administration of AZ1 inhibits cerebral neuroinflammatory response in AD mice
  • WT+Vehicle was intraperitoneal injection of wild-type mice in the same litter.
  • control solvent 5 ⁇ FAD+Vehicle is the control solvent for 5 ⁇ FAD mice intraperitoneally injected
  • 5 ⁇ FAD+AZ1 is the experimental group of 5 ⁇ FAD mice intraperitoneally injected with AZ1.
  • mice After 4 weeks of continuous administration, conduct learning and memory-related behavioral tests. After the test, the test is completed for another week.
  • the mice are then anesthetized with 5% chloral hydrate, and then perfused with phosphate buffered saline, and brain tissue is taken. , Quick-freeze in liquid nitrogen, and then store in -80°C refrigerator.
  • RNA was extracted by TRIzol (Thermo Fisher Scientific), and then Reverse Tra Ace qPCR RT Kit (TOYOBO) was used for reverse transcription, and FastStart Universal SYBR Green Master (Roche) was used for real-time fluorescent quantitative PCR to detect the inflammation-related genes Il1b and Il6. Transcription level.
  • the expression levels of pro-inflammatory factors Il1b and Il6 in the brains of 5 ⁇ FAD+Vehicle mice were significantly up-regulated, while after administration of AZ1, the brains of 5 ⁇ FAD mice were significantly reduced.
  • the expression of Il1b and Il6 indicates that AZ1 administration can inhibit the inflammatory response in the brain of AD mice.
  • Example 47 Administration of AZ1 inhibits the proliferation and activation of microglia in the brain of AD mice
  • WT+Vehicle was intraperitoneal injection of wild-type mice in the same litter.
  • control solvent 5 ⁇ FAD+Vehicle is the control solvent for 5 ⁇ FAD mice intraperitoneally injected
  • 5 ⁇ FAD+AZ1 is the experimental group of 5 ⁇ FAD mice intraperitoneally injected with AZ1.
  • mice are then anesthetized with 5% chloral hydrate, and then perfused with phosphate buffered saline, and brain tissue is taken. .
  • the brain tissue was fixed overnight with 4% paraformaldehyde at 4°C and dehydrated sequentially with 25% and 30% sucrose solutions.
  • the brain tissue was embedded with OCT, and then sliced and restored with sodium citrate buffer, using 0.2% Triton X-100's 3% BSA buffer was blocked, and then immunofluorescence staining was performed to label the microglia marker protein Iba1 (Wako) and the nuclear dye 4',6-diamidino-2-phenylindole ( 4',6-diamidino-2-phenylindole, DAPI) (Sigma Company), and then image acquisition by laser confocal fluorescence microscope.
  • Example 48 Administration of AZ1 inhibits lipopolysaccharide-induced inflammatory response in microglia
  • LPS Lipopolysaccharide
  • 10 ⁇ M AZ1 10 ⁇ M AZ1 respectively.
  • Control was the control group
  • LPS+Vehicle was 50ng/mL LPS-treated experimental group
  • LPS+AZ1 was 50ng/mL LPS Treat the experimental group simultaneously with 10 ⁇ M AZ1.
  • the cells were fixed with 4% paraformaldehyde, 0.2% Triton X-100 solution penetrated, 3% BSA solution blocked, and then the microglia marker protein Iba1 was labeled with immunofluorescence to analyze the LPS stress of AZ1 treatment Under the influence of microglia activation.
  • the cell body area of microglia increased significantly after LPS treatment, suggesting the activation of microglia, while AZ1 treatment can reduce the area of microglia, thereby inhibiting LPS from activating microglia.
  • LPS treatment significantly increased the expression of pro-inflammatory factors Il1b and Il6, while AZ1 treatment could reduce the expression of Il1b and Il6 induced by LPS, thereby inhibiting the inflammatory response induced by LPS.
  • AZ1 can inhibit LPS-induced microglia activation and inflammation.
  • Example 49 Administration of AZ2 inhibits neuroinflammatory response in the brain of AD mice
  • WT +Vehicle is the control solvent for intragastric administration of wild-type mice in the same litter
  • 5 ⁇ FAD+Vehicle is the control solvent for intragastric administration of 5 ⁇ FAD mice
  • 5 ⁇ FAD+AZ2 is the control solvent for administration of 5 ⁇ FAD mice.
  • AZ2 experimental group is the control solvent for administration of wild-type mice in the same litter.
  • mice were anesthetized with 5% chloral hydrate, and then perfused with phosphate buffer solution.
  • the brain tissue was taken, quick-frozen in liquid nitrogen, and then stored in a refrigerator at -80°C.
  • RNA was extracted by TRIzol (Thermo Fisher Scientific), and then Reverse Tra Ace qPCR RT Kit (TOYOBO) was used for reverse transcription, and FastStart Universal SYBR Green Master (Roche) was used for real-time fluorescent quantitative PCR to detect the inflammation-related genes Il1b and Il6. Transcription level.
  • Example 50 Administration of AZ2 inhibits the proliferation and activation of microglia in the brain of AD mice
  • WT +Vehicle is the control solvent for intragastric administration of wild-type mice in the same litter
  • 5 ⁇ FAD+Vehicle is the control solvent for intragastric administration of 5 ⁇ FAD mice
  • 5 ⁇ FAD+AZ2 is the control solvent for administration of 5 ⁇ FAD mice.
  • AZ2 experimental group Figure 12A). As shown in Figure 12B, AZ2 administration did not affect the body weight of the mice.
  • mice After 4 weeks of continuous administration, the mice were anesthetized with 5% chloral hydrate, and then perfused with phosphate buffer solution, and brain tissues were taken. The brain tissue was fixed overnight with 4% paraformaldehyde at 4°C and dehydrated sequentially with 25% and 30% sucrose solutions.
  • the brain tissue was embedded with OCT, and then sliced and restored with sodium citrate buffer, using 0.2% Triton X-100's 3% BSA buffer was blocked, and then immunofluorescence staining was performed to label the microglia marker protein Iba1 (Wako) and the nuclear dye 4',6-diamidino-2-phenylindole ( 4',6-diamidino-2-phenylindole, DAPI) (Sigma Company), and then image acquisition by laser confocal fluorescence microscope.
  • Example 51 Administration of AZ2 inhibits lipopolysaccharide-induced inflammatory response in microglia
  • LPS lipopolysaccharide
  • AZ2 was the 10 ⁇ M AZ2 treatment control group
  • LPS+Vehicle was the 50ng/mL LPS treatment experimental group.
  • LPS+AZ2 is 50ng/mL LPS and 10 ⁇ M AZ2 are treated at the same time in the experimental group.
  • LPS treatment significantly increased the expression of pro-inflammatory factors Il1b and Il6, while AZ2 treatment could significantly reduce the expression of Il1b and Il6 induced by LPS, thereby inhibiting the inflammatory response induced by LPS.
  • USP25 aa157-706 is the catalytic domain of USP25 deubiquitinating enzyme expressed by E.coli BL21(DE3) by Ni-NTA Agarose (Qiagen company, catalog number 1018244), based on Ubiquitin-Rhodamine 110 (R&D System, Catalog No.
  • MZ77, MZ76, MZ1, MZ30, AZ2, MZ32, AZ1, MZ67, MZ66, MZ75, MZ31, MZ5, XMU1, MZ71, MZ34, MZ33, MZ74, MZ68, MZ29, MZ72, MZ41, MZ43, MZ38, MZ24, MZ40, MZ25, MZ69, MZ42, MZ35, MZ36, MZ26, MZ37, MZ39, MZ23, MZ3, MZ27, MZ28, MZ4 can significantly inhibit the deubiquitination enzyme activity of USP25.
  • Example 53 Administration of XMU1 inhibits lipopolysaccharide-induced inflammatory response in microglia
  • LPS Lipopolysaccharide
  • XMU1 10 ⁇ M XMU1 treatment control group
  • LPS+Vehicle was 50ng/mL LPS treatment experimental group.
  • LPS+XMU1 is 50ng/mL LPS and 10 ⁇ M XMU1 simultaneously treat the experimental group.
  • LPS treatment significantly increased the expression of the pro-inflammatory factor Il1b
  • XMU1 treatment could significantly reduce the LPS-induced Il1b expression, thus indicating that XMU1 administration can inhibit the LPS-induced inflammatory response.
  • Example 54 Administration of AZ1 derivative MZ3 inhibits lipopolysaccharide-induced inflammatory response in microglia cell line BV2
  • the mouse microglia cell line BV2 was treated with 25ng/mL lipopolysaccharide (LPS) and 10 ⁇ M MZ3 respectively.
  • LPS lipopolysaccharide
  • Control is the control group
  • LPS+Vehicle is 25ng/mL LPS treatment experimental group
  • LPS+MZ3 is 25ng/mL LPS and 10 ⁇ M MZ3 were simultaneously treated in the experimental group.
  • LPS treatment significantly increased the expression of pro-inflammatory factor Il1b
  • MZ3 treatment could significantly reduce the expression of Il1b induced by LPS, thereby inhibiting the inflammatory response induced by LPS.
  • Example 55 Administration of AZ1 derivatives MZ23 and MZ34 inhibits lipopolysaccharide-induced inflammatory response in microglia cell line BV2
  • the mouse microglia cell line BV2 was treated with 50ng/mL lipopolysaccharide (LPS) and 10 ⁇ M MZ2 or MZ3 respectively.
  • LPS lipopolysaccharide
  • Control is the control group
  • LPS+Vehicle is 50ng/mL LPS treatment experimental group
  • LPS+ MZ23 is 50ng/mL LPS and 10 ⁇ M MZ23 treat the experimental group at the same time
  • LPS+MZ34 is 50ng/mL LPS and 10 ⁇ M MZ34 treat the experimental group at the same time.
  • LPS treatment significantly increased the expression of pro-inflammatory factor Il1b, while MZ23 and MZ34 treatments could significantly reduce the expression of Il1b induced by LPS, thereby inhibiting the inflammatory response induced by LPS.
  • Example 56 Administration of AZ1 derivatives MZ24, MZ25, MZ26, MZ28, MZ29 and MZ31 inhibits lipopolysaccharide-induced inflammation of the microglia cell line BV2
  • the mouse microglia cell line BV2 was treated with 100ng/mL lipopolysaccharide (LPS) and 10 ⁇ M MZ24, MZ25, MZ26, MZ28, MZ29 and MZ31 respectively.
  • LPS lipopolysaccharide
  • Control was the control group
  • LPS+Vehicle was 100ng/mL.
  • LPS+MZ24 is 100ng/mL LPS and 10 ⁇ M MZ24 simultaneously treats the experimental group
  • LPS+MZ25 is 100ng/mL LPS and 10 ⁇ M MZ25 simultaneously treats the experimental group
  • LPS+MZ26 is 100ng/mL LPS and 10 ⁇ M MZ26 simultaneously Treat the experimental group
  • LPS+MZ28 is 100ng/mL LPS and 10 ⁇ M MZ28 treat the experimental group simultaneously
  • LPS+MZ29 is 100ng/mL LPS and 10 ⁇ M MZ29 treat the experimental group simultaneously
  • LPS+MZ31 is 100ng/mL LPS and 10 ⁇ M MZ31 simultaneously treat the experiment Group.
  • LPS treatment significantly increased the expression of pro-inflammatory factor Il1b, while MZ24, MZ25, MZ26, MZ28, MZ29, and MZ31 treatments could significantly reduce the expression of Il1b induced by LPS, thereby inhibiting the inflammatory response induced by LPS.
  • Example 57 Administration of AZ1 derivatives MZ75, MZ76 and MZ77 to inhibit lipopolysaccharide-induced inflammatory response in microglia
  • the cells were cultured for 1 day and were treated with 50ng/mL lipopolysaccharide (LPS) and 10 ⁇ M AZ1, MZ75, MZ76 and MZ77 respectively.
  • LPS lipopolysaccharide
  • Control is the control group
  • LPS+Vehicle is 50ng/mL LPS treatment experimental group
  • LPS+ AZ1 or LPS+MZ75 or LPS+MZ76 or LPS+MZ77 is 50ng/mL LPS and 10 ⁇ M AZ1 or MZ75 or MZ76 or MZ77 at the same time to treat the experimental group.
  • LPS treatment significantly increased the expression of pro-inflammatory factor Il1b
  • AZ1, MZ75, MZ76, and MZ77 treatments significantly reduced the expression of Il1b induced by LPS, thereby inhibiting the inflammatory response induced by LPS.

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Abstract

本发明提供式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐和药物组合物。本发明的化合物能够有效治疗阿尔茨海默病或唐氏综合征，特别是阿尔茨海默病或唐氏综合征所致的认知功能损伤。本发明还公开了21号染色体编码表达的泛素特异性蛋白酶USP25作为预防、治疗或改善阿尔茨海默病或唐氏综合征的用途。

Description

治疗阿尔茨海默病的化合物 技术领域
本发明涉及医药领域；具体地说，本发明涉及治疗阿尔茨海默病或唐氏综合征的小分子化合物及其应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是发生于老年人最常见的中枢神经系统退行性疾病之一，临床上以进行性记忆、认知障碍及行为异常为特征，典型的病理学表现为淀粉样斑块(Amyloid plaques)、神经纤维缠结(Neurofibrillary tangle,NFT)。随着社会人口老龄化逐步加剧，阿尔茨海默病患病率呈逐渐上升趋势：在年龄为65-74岁的人群中，AD的发病率约为5％，而在85岁以上的人群中，AD的发病率约为50％。AD已成为全球的公共健康问题，预计到2020年，全球将有四千多万痴呆患者，而AD患者约占到其中的50％至60％，AD给患者本身和家庭带来了很大的痛苦和经济负担，并带来巨大的社会压力，因此，越来越引起医疗和科研工作者的关注。
AD患者脑内比较突出的两大病理特征是由淀粉样蛋白(β-amyloid,Aβ)形成的淀粉样斑块和由高度磷酸化tau蛋白聚集形成的神经纤维缠结，以及神经元丢失伴胶质细胞增生等。但是AD的发病机制仍然不清楚，而且至今没有有效的治疗方法。根据近些年对阿尔茨海默病发病机制的研究，AD患者脑内慢性炎症反应的发生也意味着AD可能是一种慢性的中枢神经系统炎症反应。星形胶质细胞和小胶质细胞的激活也是AD神经炎症的一种重要标志。在AD疾病早期，作为脑内最主要的免疫细胞，小胶质细胞在清除Aβ淀粉样斑、限制Aβ寡聚体形成和扩散、病理tau蛋白传播等过程中发挥关键作用，提示小胶质细胞介导的天然免疫在AD病理发展中发挥着重要作用，异常激活的小胶质细胞通过释放炎症因子诱发神经毒性作用，此外异常激活的小胶质细胞还通过吞噬突触导致突触丢失、突触功能障碍，表明小胶质细胞功能异常是AD疾病发生发展的关键因素。
此外，具体的神经退行性疾病之间在机理上存在很大差异，目前治疗神经退行性疾病的药物非常少，有些神经退行性疾病尚无有效的药物可用。例如AD主要受累脑区是主管学习记忆的皮层和海马，患者脑内比较突出的两大病理特征是由淀粉样蛋白Aβ形成的淀粉样斑块和由高度磷酸化tau蛋白聚集形成的神经纤维缠结，以及神经元丢失伴胶质细胞增生等。帕金森病(Parkinson’s disease,PD)是另外一种较常见的神经退行性疾病，在老年人多见，平均发病年龄为60岁左右。PD主要受累脑区是中脑，最主要的病理改变是中脑黑质多巴胺(Dopamine，DA)能神经元的变性死亡，由此而引起纹状体DA含量显著性减少而致病。目前PD的治疗药物左旋多巴主要功能是补充脑中的多巴胺含量。亨廷顿病(Huntington’s disease，HD)，又称大舞蹈病或亨廷顿舞蹈症，是一种常染色体显性遗传性神经退行性疾病，主要病因是患者第四号染色体上的HTT基因发生变异，产生了变异的蛋白质，亨廷顿病患者的异常HTT蛋白有许多重复的谷氨酰胺，异常亨廷顿蛋白容易聚集，具有细胞毒性，可以导致 神经细胞死亡，目前尚无治疗方法。肌萎缩侧索硬化(Amyotrophic lateral sclerosis，ALS)也叫渐冻症，主要病理表现是运动神经元损伤和死亡，逐渐导致包括延髓支配肌肉、四肢、躯干、胸部腹部的肌肉无力和萎缩，最终导致病人死亡，目前尚无治疗方法。因此，阿尔茨海默病与其它常见的神经退行性疾病在内在机理和外在症状均存在很大差异。
常规应用于其它神经退行性疾病，例如帕金森病等的药物对阿尔茨海默病无效。因此，目前对于阿尔茨海默病非常缺乏具有治疗、预防或缓解作用的药物。
因此，本领域急需能够治疗、预防和改善阿尔茨海默病及其临床症状的技术手段。
AD病人脑内发现有神经毒性蛋白的异常堆积，提示泛素蛋白酶体系统(Ubiquitin-proteasome system,UPS)功能障碍可能参与神经退行性疾病的发生发展。泛素化修饰途径与磷酸化修饰途径一样，也是一个可逆过程。细胞内同时还存在一些特异的去泛素化蛋白酶进行负调节。USP25基因定位于21号染色体q11.2上，属于去泛素化蛋白酶家族成员，含有蛋白去泛素化酶活性区域。原位杂交显示，USP25在脑内高表达，且在神经元、小胶质细胞和星形胶质细胞中均有较高表达量。唐氏综合征(Down’s syndrome,DS)是最为常见的智力障碍遗传疾病，唐氏患者携带有第三条完整的或部分的21号染色体。在唐氏患者细胞中，USP25基因多出一个拷贝，USP25的表达增多会影响其底物的泛素化水平和蛋白稳态。作为早发型AD的最重要的遗传风险因素之一，40岁以上的唐氏患者100％会出现AD的病理特征，这就为研究USP25在DS和AD发病中的作用提供理论依据。除此之外，有文章报道USP25参与自身免疫调节和炎症反应。我们的研究发现USP25参与AD与DS的发生发展过程，USP25抑制剂可能作为AD和DS神经炎症和认知损伤的治疗药物。
发明内容
本发明的目的在于提供一种有效治疗阿尔茨海默病和其相关疾病唐氏综合征，特别是阿尔茨海默病和唐氏综合征所致认知功能损伤的小分子化合物。
在第一方面，本发明首次发现并阐明泛素特异性蛋白酶USP25通过抑制小胶质细胞神经炎症反应和突触吞噬过程，恢复AD脑中小胶质细胞稳态，从而逆转阿尔茨海默病模型小鼠5×FAD的突触功能和认知障碍；敲除Usp25可以改善5×FAD小鼠的小胶质细胞稳态，逆转5×FAD小鼠的突触和认知功能缺陷。此外，敲除Usp25也可以改善唐氏小鼠Dp16的突触和认知功能缺陷。本发现为阿尔茨海默病和唐氏综合征的临床治疗提供了一个潜在的药物靶点。
在第二方面，本发明提供式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐在制备预防、治疗或改善阿尔茨海默病或唐氏综合征的药物中的用途，
Figure PCTCN2021073164-appb-000001
其中：
X独立选自O或NH；
A环和B环各自独立为苯环；
R 1独立地选自：氢、氘、卤素、氰基、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 1-C 6)烷氧基；
m为0、1、2、3、4或5；
R 2独立地选自：氢、卤素、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 2-C 6)烯基、取代或未取代的(C 2-C 6)炔基、取代或未取代的(C 1-C 6)烷氧基；
n为1或2；
R 3为取代或未取代的(C 1-C 6)醛基、
Figure PCTCN2021073164-appb-000002
其中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 2-C 6)烯基、取代或未取代的(C 2-C 6)炔基、
Figure PCTCN2021073164-appb-000003
其中p为1-3的整数(优选地，p为2)。
在具体的实施方式中，所述R 3在苯环上的取代位置是邻位或间位；优选邻位。
在优选的实施方式中，当R 1独立选自：氢、氟、氯、溴或碘(优选氟)、氰基、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 1-C 4)烷氧基。
在优选的实施方式中，各R 1独立地选自：氟、三氟甲基、三氟甲氧基。
在优选的实施方式中，R 2独立地选自：氢、氟、氯、溴或碘(优选溴)、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 2-C 4)烯基、取代或未取代的(C 2-C 4)炔基、取代或未取代的(C 1-C 4)烷氧基。
在优选的实施方式中，R 2独立地选自：氢、氟、氯、溴、甲基、乙基、正丙基、烯丙基、炔丙基、-OCH 2CH 2CH 3、-OCH 2CH 3、-OCH 3、-CHF 2、-CF 3、-OCHF 2、-OCF 3。
在优选的实施方式中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 2-C 4)烯基、取代或未取代的(C 2-C 4)炔基。
在优选的实施方式中，所述取代的(C 1-C 4)烷基选自羟基(C 1-C 4)烷基、R 4aR 4b氨基(C 1-C 4)烷基或巯基(C 1-C 4)烷基，所述R 4a和R 4b独立地为取代或未取代的(C 1-C 3)烷基；优选羟基(C 1-C 4)烷基或R 4aR 4b氨基(C 1-C 4)烷基。
在优选的实施方式中，所述“取代”是卤代，优选氟代。
在具体的实施方式中，R 1独立地选自：氢、氟、氯、溴、(C 1-C 3)烷基、卤代(C 1-C 3)烷 基、卤代(C 1-C 3)烷氧基；
m为0、1、2或3；
R 2独立地选自：氢、卤素；
n为1或2；
R 3为
Figure PCTCN2021073164-appb-000004
其中，各R 3a、R 3b独立地选自：氢、羟基取代的(C 1-C 4)烷基、
Figure PCTCN2021073164-appb-000005
其中p为1-3的整数(优选地，p为2)。
在具体的实施方式中，式I所示化合物选自以下化合物：
Figure PCTCN2021073164-appb-000006
Figure PCTCN2021073164-appb-000007
Figure PCTCN2021073164-appb-000008
Figure PCTCN2021073164-appb-000009
Figure PCTCN2021073164-appb-000010
Figure PCTCN2021073164-appb-000011
在具体的实施方式中，式I所示化合物为AZ1、AZ2、MZ77、MZ76、MZ1、MZ30、MZ32、MZ67、MZ66、MZ75、MZ31、MZ34、MZ74、MZ68、MZ29、MZ72、MZ38或MZ24。
在具体的实施方式中，所述预防、治疗或改善阿尔茨海默病或唐氏综合征是指预防、治疗或改善阿尔茨海默病或唐氏综合征所致认知功能损伤。
在第三方面，本发明提供式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐，
Figure PCTCN2021073164-appb-000012
其中：
A环和B环各自独立为苯环；
R 1独立地选自：氢、氘、卤素、氰基、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 1-C 6)烷氧基；
m为0、1、2、3、4或5；
R 2独立地选自：氢、卤素、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 2-C 6)烯基、取代或未取代的(C 2-C 6)炔基、取代或未取代的(C 1-C 6)烷氧基；
n为1或2；
R 3为取代或未取代的(C 1-C 6)醛基、
Figure PCTCN2021073164-appb-000013
其中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 2-C 6)烯基、取代或未取代的(C 2-C 6)炔基、
Figure PCTCN2021073164-appb-000014
其中p为1-3的整数(优选地，p为2)；
其中，式I化合物不包括
Figure PCTCN2021073164-appb-000015
在具体的实施方式中，所述R 3在苯环上的取代位置是邻位或间位；优选邻位。
在优选的实施方式中，当R 1独立选自：氢、氟、氯、溴或碘(优选氟)、氰基、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 1-C 4)烷氧基。
在优选的实施方式中，各R 1独立地选自：氟、三氟甲基、三氟甲氧基。
在优选的实施方式中，R 2独立地选自：氢、氟、氯、溴或碘(优选溴)、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 2-C 4)烯基、取代或未取代的(C 2-C 4)炔基、取代或未取代的(C 1-C 4)烷氧基。
在优选的实施方式中，R 2独立地选自：氢、氟、氯、溴、甲基、乙基、正丙基、烯丙基、炔丙基、-OCH 2CH 2CH 3、-OCH 2CH 3、-OCH 3、-CHF 2、-CF 3、-OCHF 2、-OCF 3。
在优选的实施方式中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 4)烷基、取代或未取代的(C 2-C 4)烯基、取代或未取代的(C 2-C 4)炔基。
在优选的实施方式中，所述取代的(C 1-C 4)烷基选自羟基(C 1-C 4)烷基、R 4aR 4b氨基(C 1-C 4)烷基或巯基(C 1-C 4)烷基，所述R 4a和R 4b独立地为取代或未取代的(C 1-C 3)烷基；优选羟基(C 1-C 4)烷基或R 4aR 4b氨基(C 1-C 4)烷基。
在优选的实施方式中，所述“取代”是卤代，优选氟代。
在具体的实施方式中，式I所示化合物选自以下化合物：
Figure PCTCN2021073164-appb-000016
Figure PCTCN2021073164-appb-000017
Figure PCTCN2021073164-appb-000018
Figure PCTCN2021073164-appb-000019
Figure PCTCN2021073164-appb-000020
Figure PCTCN2021073164-appb-000021
在优选的实施方式中，式I所示化合物为MZ77、MZ76、MZ1、MZ30、MZ32、MZ67、MZ66、MZ75、MZ31、MZ34、MZ74、MZ68、MZ29、MZ72、MZ38或MZ24。
在第四方面，本发明提供一种药物组合物，所述药物组合物包含第二方面所述的式I所示化合物、其各种晶型、水合物或溶剂合物以及任选的药学上可接受的赋形剂。
在优选的实施方式中，本发明提供式I所示化合物、其各种晶型、水合物或溶剂合物，用于预防、治疗或改善阿尔茨海默病和唐氏综合征。
在优选的实施方式中，本发明提供预防、治疗或改善阿尔茨海默病和唐氏综合征的方法，包括将式I所示化合物、其各种晶型、水合物或溶剂合物给予有此需要的对象的步骤。
在优选的实施方式中，本发明提供包含式I所示化合物、其各种晶型、水合物或溶剂合物的用于预防、治疗或改善阿尔茨海默病和唐氏综合征的药物。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是敲除Usp25增强AD小鼠突触和认知功能的示意图；其中，(A-F)6～7月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠行为学分析结果。(A)Y-迷宫测试中自发交替次数百分比分析结果(Alternation％)。每组n＝14～18只小鼠。(B)Morris水迷宫(MWM)训练期小鼠到达平台的潜伏期(Latency to target)。(C)Morris水迷宫平台测试期小鼠在目标象限及其他象限的时间百分比分析结果(Time in quadrant％)。每组n＝14～15只小鼠。(D)Morris水迷宫平台测试中小鼠的游泳轨迹示意图。(E)条件惊恐(FC)场景相关记忆测试中小鼠僵直行为时间百分比分析结果(Freezing％)。(F)条件惊恐线索相关记忆测试中小鼠僵直行为时间百分比分析结果(Freezing％)。每组n＝19～28只小鼠。(G)9月龄小鼠海马组织高尔基染色代表图及树突棘密度分析结果。标尺，10μm。每组n＝4只小鼠，33～46根树突。(H)6～7月龄小鼠海马脑片CA1区长时程增强(Long-term potentiation，LTP)记录结果。(I)LTP记录结果最后10min fEPSP振幅统计分析结果。WT(n＝6只小鼠/11片脑片)，Usp25 +/–(n＝5只小鼠/9片脑片)，5×FAD(n＝5只小鼠/11片脑片)，5×FAD；Usp25 +/–(n＝6只小鼠/12片脑片)。图(A，C，E，F，G，I)数据采用Kruskal-Wallis test进行统计分析，图(B)数据采用repeated-measures ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。
图2是敲除Usp25增强DS小鼠突触和认知功能的示意图；其中，(A-C)6月龄WT、Usp25 +/–、Dp16、Dp16；Usp25 +/–小鼠行为学分析结果。(A)T-迷宫测试中自发交替次数百分比分析结果(Alternation％)。每组n＝9～22只小鼠。(B)Morris水迷宫(MWM)训练期小鼠到达平台的潜伏期(Latency to target)。(C)Morris水迷宫平台测试期小鼠穿梭平台所在象限的次数(Target crossings)。每组n＝9～22只小鼠。(D)6月龄小鼠海马组织高尔基染色代表图及树突棘密度分析结果。标尺，5μm。每组n＝18～28根树突。(E)6月龄小鼠海马脑片CA1区LTP记录结果。(F)LTP记录结果最后10min fEPSP振幅统计分析结果。WT(n＝5只小鼠/10片脑片)，5×FAD(n＝5只小鼠/11片脑片)，5×FAD；Usp25 +/–(n＝5只小鼠/8片脑片)。图(A，C)数据采用one-way ANOVA进行统计分析，图(B)数据采用repeated-measures ANOVA进行统计分析，图(F)数据采用lKruskal-Wallis test进行统计分析。*P<0.05；**P<0.01；***P<0.001。
图3是敲除Usp25改善小鼠小胶质细胞稳态的示意图；其中，(A-C)6～7月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠，5％水合氯醛麻醉后，使用磷酸盐缓冲液进行灌注，取脑组织，于4％多聚甲醛固定过夜，经25％和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，切片后，进行免疫荧光染色，标记小胶质细胞标记蛋白Iba1以及细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)，通过激光共聚焦荧光显微镜采集图像。图(A)是海马组织和大脑皮层Iba1 +小胶质细胞免疫组化染色结果。图(B，C)分别是海马组织(B)和大脑皮层(C)的统计分析结果。标尺，100μm。每组n＝5～7只小鼠。(D)Imaris软件3D重构海马组织Iba1 +小胶质细胞吞噬PSD95 +突触结构代表图。标尺，10μm。(E-G)小胶质细胞胞体大小(E)、分支长度(F)、小胶质细胞吞噬PSD95 +突触结构(G)的统计分析结果。每组n＝3～6只小鼠，24～197个小胶质细胞。(H)Imaris软件3D重构大脑皮层Iba1 +小胶质细胞吞噬PSD95 +突触结构代表图。标尺，10μm。(I-K)小胶质细胞胞体大小(Soma size，I)、分支长度(Total processes，J)、小胶质细胞吞噬PSD95 +突触结构(K)的统计分析结果。每组n＝3～6只小鼠，26～169个小胶质细胞。图(B，C，F，K)数据采用one-way ANOVA进行统计分析，图(E，G，I，J)数据采用Kruskal-Wallis test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。
图4是敲除Usp25抑制小鼠小胶质细胞炎症因子释放和突触吞噬的示意图；其中，(A，B)10月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠海马组织，通过TRIzol提取RNA，经逆转录后，采用实时荧光定量PCR进行炎症相关基因Il6和Tnf的转录水平检测。n＝6。(C，D)Usp25 +/+、Usp25 –/–小鼠小胶质细胞，分别处理10μMAZ1或对照溶剂DMSO，与此同时处理10μM oAβ 42或对照溶剂Vehicle和pHrodo-Red标记的突触小体(Syn)，24小时后分析Iba1 +小胶质细胞内pHrodo-Red的荧光强度。标尺，25μm。n＝140～328。图(A，B)数据采用one-way ANOVA进行统计分析，图(D)数据采用Kruskal-Wallis test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。
图5是给药AZ1逆转AD小鼠突触和认知功能缺陷的示意图；其中，(A)7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，连续给药4周后进行学习记忆相关行为学测试。(B)给药AZ1前后小鼠体重的变化。n (WT+Vehicle)＝16、n(5×FAD+Vehicle)＝8、n(5×FAD+AZ1)＝9。(C)给药1个月后进行Morris水迷宫隐藏平台训练(6天)中小鼠寻找到隐藏平台的潜伏时间(Latency to target)。(D)Morris水迷宫平台测试(第7天)中小鼠在平台所在象限以及其他象限的时间(Time in quadrant％)。(E)Morris水迷宫平台测试中小鼠的游泳轨迹示意图。(F)Morris水迷宫平台测试中小鼠第一次到达平台所在区域的潜伏时间(Latency 1 st entrance)。(G)条件惊恐线索相关记忆测试中小鼠的僵直时间百分比(Freezing％)。n(WT+Vehicle)＝17、n(5×FAD+Vehicle)＝8、n(5×FAD+AZ1)＝9。(H，I)5月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组，连续给药4周后进行小鼠脑片电生理记录。图(H)是小鼠海马脑片CA1区LTP记录结果。图(I)是LTP最后10分钟的统计结果。n(WT+Vehicle)＝5只小鼠/10片脑片，n(5XFAD+Vehicle)＝4只小鼠/7片脑片，n(5XFAD+AZ1)＝7只小鼠/12片脑片。图(B，C)数据采用repeated-measures ANOVA进行统计分析，图(D，F，G，I)数据采用one-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。
图6是给药AZ1逆转DS小鼠认知功能缺陷的示意图；其中，(A)5月龄WT和Dp16雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，连续给药4周后进行学习记忆相关行为学测试。(B)给药AZ1前后小鼠体重的变化。每组n＝20～35只小鼠。(C)旷场测试(Open field test,OF test)中小鼠的运动距离(Total distance)。(D)旷场测试中小鼠在中间区域的运动时间百分比(Time in center％)，每组n＝6～17只小鼠。(E)新物体识别测试(Novel object recognition test,NOR test)中小鼠识别新物体的辨别指数(Recogntion index)。(F)Morris水迷宫隐藏平台训练(5天)中小鼠寻找到隐藏平台的潜伏时间(Latency to target)。(G)Morris水迷宫平台测试(第6天)中小鼠在平台所在象限以及其他象限的时间(Time in quadrant％)。(H)Morris水迷宫平台测试期小鼠穿梭平台所在象限的次数(Target crossings)。每组n＝20～35只小鼠。图(B，F)数据采用repeated-measures ANOVA进行统计分析，图(C，D，G，H)数据采用Kruskal-Wallis test进行统计分析，图(E)数据采用one-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。
图7是给药AZ1对小鼠毒理作用的示意图；其中，7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。连续给药4周后，进行学习记忆相关行为学测试，测试结束后再给药1周，然后采用5％水合氯醛麻醉小鼠，通过摘眼球取血，4℃静置30分钟以上，3000rpm，4℃离心5分钟，取血清进行血液生化检测，其中，肝功能包括(A)丙氨酸氨基转移酶、(B)天门冬氨酸氨基转移酶、(C)天门冬氨酸氨基转移酶/丙氨酸氨基转移酶比值、(D)总蛋白、(E)白蛋白、(F)碱性磷酸酶；肾功能包括(G)尿素、(H)肌酐；血脂包括(I)总胆固醇、(J)甘油三酯；心肌酶谱(K)肌酸激酶；血糖(L)葡萄糖。n(WT+Vehicle)＝10，n(5×FAD+Vehicle)＝8，n(5×FAD+AZ1)＝8。数据采用Kruskal-Wallis test进行统计分 析。ns，无显著性差异，P>0.05。
图8是给药AZ1抑制AD小鼠脑中炎症反应实验；其中，7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。连续给药4周后，进行学习记忆相关行为学测试，测试结束后再给药1周，然后采用5％水合氯醛麻醉小鼠，使用磷酸盐缓冲液进行灌注，取脑组织分离海马组织后，通过TRIzol提取RNA，经逆转录后，采用实时荧光定量PCR进行炎症相关基因转录水平检测。图(A)为促炎因子Il1b转录水平，图(B)为促炎因子Il6转录水平。n(WT+Vehicle)＝7，n(5×FAD+Vehicle)＝7，n(5×FAD+AZ1)＝7。数据采用One-way ANOVA进行统计分析。**P<0.01；****P<0.0001。
图9是给药AZ1抑制AD小鼠脑中小胶质细胞增殖和激活实验；其中，7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+9％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。连续给药4周后，进行学习记忆相关行为学测试，测试结束后再给药1周，然后采用5％水合氯醛麻醉小鼠，使用磷酸盐缓冲液进行灌注，取脑组织，于4％多聚甲醛固定过夜，经25％和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，切片后，进行免疫荧光染色，标记小胶质细胞标记蛋白Iba1以及细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)，通过激光共聚焦荧光显微镜采集图像。图(A)是大脑皮层、海马CA1区和DG区Iba1 +小胶质细胞免疫组化染色结果。标尺，100μm。图(B-D)分别是大脑皮层(B)、海马CA1区(C)和DG区(D)的统计分析结果。每组n＝7～10只小鼠。(E)Imaris软件3D重构海马组织Iba1 +小胶质细胞代表图。标尺，10μm。(F-I)小胶质细胞分支长度(Total processes，F，H)和分支数目(Branch number，G，I)的统计分析结果。每组n＝5只小鼠，31～76个小胶质细胞。图(B，D，F，H)数据采用One-way ANOVA进行统计分析，图(G，I)数据采用Kruskal-Wallis test进行统计分析。*P<0.05；**P<0.01；***P<0.001；****P<0.0001。
图10是给药AZ1抑制脂多糖诱导的小胶质细胞炎症反应实验；其中，分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，培养10天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ1处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ1为50ng/mL LPS和10μM AZ1同时处理实验组。处理6小时后，分别进行免疫荧光染色和RNA提取。图(A)是免疫荧光染色标记小胶质细胞标记蛋白Iba1示意图。标尺，75μm。图(B)是对图(A)中小胶质细胞胞体面积(Soma size)的统计结果。n(Control)＝79个小胶质细胞，n(LPS+Vehicle)＝80个小胶质细胞，n(LPS+AZ1)＝74个小胶质细胞。图(C)和(D)是提取的小胶质细胞RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b和Il6的转录水平。n(Control)＝4，n(LPS+Vehicle)＝4，n(LPS+AZ1)＝4。图(B)数据采用Kruskal-Wallis test进行统计分析，图(C，D)数据采用One-way ANOVA进行统计分析。**P<0.01；****P<0.0001。
图11是给药AZ2抑制AD小鼠脑中炎症反应实验；其中，6月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量灌胃给药AZ2或对照溶剂(含1％Tween-80和0.5％CMC-Na水溶液，pH 3～4)，其中，WT+Vehicle为同窝对照野生型小鼠灌胃给药对照溶剂，5×FAD+Vehicle为5×FAD小鼠灌胃给药对照溶剂，5×FAD+AZ2为5×FAD小鼠灌胃给药AZ2实验组。连续给药4周后，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织分离海马组织后，通过TRIzol提取RNA，经逆转录后，通过实时荧光定量PCR进行炎症相关基因转录水平检测。图(A)为促炎因子Il1b转录水平，图(B)为促炎因子Il6转录水平。n(WT+Vehicle)＝6，n(5×FAD+Vehicle)＝6，n(5×FAD+AZ2)＝5。数据采用One-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；****P<0.0001。
图12是给药AZ2抑制AD小鼠脑中小胶质细胞增殖和激活实验；其中，(A)6月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量灌胃给药AZ2或对照溶剂(含1％Tween-80和0.5％CMC-Na水溶液，pH 3～4)，其中，WT+Vehicle为同窝对照野生型小鼠灌胃给药对照溶剂，5×FAD+Vehicle为5×FAD小鼠灌胃给药对照溶剂，5×FAD+AZ2为5×FAD小鼠灌胃给药AZ2实验组。(B)给药AZ2前后小鼠体重的变化。n(WT+Vehicle)＝16，n(5×FAD+Vehicle)＝9，n(5×FAD+AZ2)＝10。连续给药4周后，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织，于4％多聚甲醛固定过夜，经25％和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，切片后，进行免疫荧光染色，标记小胶质细胞标记蛋白Iba1和细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)，通过激光共聚焦荧光显微镜采集图像。图(C)是大脑皮层、海马CA1区和DG区Iba1 +小胶质细胞免疫组化染色结果。标尺，100μm。图(D-F)分别是大脑皮层(D)、海马CA1区(E)和DG区(F)的统计分析结果。每组n＝4只小鼠。(G)Imaris软件3D重构海马组织Iba1 +小胶质细胞代表图。标尺，10μm。(H-K)小胶质细胞分支长度(Total processes，H，J)和分支数目(Branch number，I，K)的统计分析结果。每组n＝4只小鼠，52～123个小胶质细胞。图(B)数据采用repeated-measures ANOVA进行统计分析，图(D-F)数据采用One-way ANOVA进行统计分析，图(H-K)数据采用Kruskal-Wallis test进行统计分析。*P<0.05；**P<0.01；***P<0.001；****P<0.0001。
图13是给药AZ2抑制脂多糖诱导的小胶质细胞炎症反应实验；其中，分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，培养10天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ2处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ2为50ng/mL LPS和10μM AZ2同时处理实验组。处理6小时后，提取RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b(A)和Il6(B)的转录水平。n(Control)＝3，n(LPS+Vehicle)＝3，n(LPS+AZ2)＝3。数据采用One-way ANOVA进行统计分析。*P<0.05；****P<0.0001。
图14是USP25抑制剂筛选实验；其中，通过Ni-NTA Agarose(Qiagen公司，货号1018244)纯化大肠杆菌E.coli BL21(DE3)表达的His-USP25 a.a.157-706重组蛋白(USP25 a.a.157-706为USP25去泛素化酶催化结构域)，基于C-端偶联有荧光染料Rhodamine 110的Ubiquitin-Rhodamine 110(R&D System公司，货号U-555-050)筛选USP25抑制剂；将10μM (终浓度)化合物与33.3nM(终浓度)His-USP25 157-706重组蛋白和133.3nM(终浓度)Ubiquitin-Rhodamine 110进行孵育，最后加25mM(终浓度)柠檬酸终止反应后，于Tecan Spark多功能酶标仪(瑞士Tecan公司)进行Ubiquitin-Rhodamine 110荧光强度检测。其中，Ctrl为对照溶液处理组，His-USP25 a.a.157-706重组蛋白+对照溶剂+Ubiquitin-Rhodamine 110。n＝3。数据采用One-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。
图15是给药AZ1衍生物XMU1抑制脂多糖诱导的小胶质细胞系BV2炎症反应实验；其中，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM XMU1处理小鼠小胶质细胞系BV2，其中，Control为对照组，XMU1为10μM XMU1处理对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+XMU1为50ng/mL LPS和10μM XMU1同时处理实验组。处理6小时后，提取RNA，经逆转录，通过实时荧光定量PCR检测炎症相关基因Il1b的转录水平。
图16是给药AZ1衍生物MZ3抑制脂多糖诱导的小胶质细胞系BV2炎症反应实验；其中，分别通过25ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ3处理小鼠小胶质细胞系BV2，其中，Control为对照组，LPS+Vehicle为25ng/mL LPS处理实验组，LPS+MZ3为25ng/mL LPS和10μM MZ3同时处理实验组。处理24小时后，提取RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b的转录水平。n(Control)＝4，n(LPS+Vehicle)＝4，n(LPS+MZ2)＝4，n(LPS+MZ3)＝4。数据采用One-way ANOVA进行统计分析。***P<0.001；****P<0.0001。
图17是给药AZ1衍生物MZ23和MZ34抑制脂多糖诱导的小胶质细胞系BV2炎症反应实验；其中，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ2或MZ3处理小鼠小胶质细胞系BV2，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+MZ23为50ng/mL LPS和10μM MZ23同时处理实验组，LPS+MZ34为50ng/mL LPS和10μM MZ34同时处理实验组。处理12小时后，提取RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b的转录水平。n(Control)＝4，n(LPS+Vehicle)＝4，n(LPS+MZ23)＝4，n(LPS+MZ34)＝4。数据采用One-way ANOVA进行统计分析。**P<0.01；***P<0.001；****P<0.0001。
图18是给药AZ1衍生物MZ24、MZ25、MZ26、MZ28、MZ29和MZ31抑制脂多糖诱导的小胶质细胞系BV2炎症反应实验；其中，分别通过100ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ24、MZ25、MZ26、MZ28、MZ29和MZ31处理小鼠小胶质细胞系BV2，其中，Control为对照组，LPS+Vehicle为100ng/mL LPS处理实验组，LPS+MZ24为100ng/mL LPS和10μM MZ24同时处理实验组，LPS+MZ25为100ng/mL LPS和10μM MZ25同时处理实验组，LPS+MZ26为100ng/mL LPS和10μM MZ26同时处理实验组，LPS+MZ28为100ng/mL LPS和10μM MZ28同时处理实验组，LPS+MZ29为100ng/mL LPS和10μM MZ29同时处理实验组，LPS+MZ31为100ng/mL LPS和10μM MZ31同时处理实验组。处理12小时后，提取RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b的转录水平。 每组n＝3。数据采用One-way ANOVA进行统计分析。****P<0.0001。
图19是给药AZ1衍生物MZ75、MZ76和MZ77抑制脂多糖诱导的小胶质细胞炎症反应的示意图；其中，分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，培养10天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ1、MZ75、MZ76和MZ77处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ1或LPS+MZ75或LPS+MZ76或LPS+MZ77为50ng/mL LPS和10μM AZ1或MZ75或MZ76或MZ77同时处理实验组。处理6小时后，提取RNA经逆转录后，通过实时定量荧光PCR检测炎症相关基因Il1b的转录水平。每组n＝3。数据采用One-way ANOVA进行统计分析。****P<0.0001。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现了一系列化合物，这些化合物能够预防、治疗或改善阿尔茨海默病和唐氏综合征，特别是预防、治疗或改善阿尔茨海默病和唐氏综合征导致的认知功能受损。本发明发现的这些化合物中大多数具备全新的结构，从而为开发预防、治疗或改善阿尔茨海默病和唐氏综合征的药物奠定新的物质基础。在此基础上完成了本发明。
定义
除非另外说明，本发明所使用的所有科技属于具有与本发明所述领域技术人员的通常理解相同的含义。为清楚起见，对本发明的一些术语作如下定义。
术语“所述”旨在包括“至少一个”或“一个或多个”。
如果取代基被描述为“独立地选自”一组，则各取代基独立于另一者被选择。因此，各取代基可与另一(其他)取代基相同或不同。
除非另外指明，否则如本文中所用“治疗”意指逆转、缓解、抑制这样的术语所应用的病症或病况的进展或所述病症或病况的一种或多种症状或加以预防。除非另外指明，否则本文所用术语“治疗”是指如上文刚刚定义的“治疗”的处理动作。术语“治疗”还包括个体的辅助疗法及新辅助疗法治疗。
在本文中，形如“C 1-n”的表述是指基团具有1-n个碳原子，例如，“C 1-6”的表述指基团具有1、2、3、4、5或6个碳原子；同样地，“C2～C6”是指基团具有2、3、4、5或6个碳原子。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1-C 6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
本文所用的术语“烷基”与本领域普通技术人员通常理解的含义相同，是指各种饱和或不饱和的直链的、带侧链的或环状的碳氢基团。例如，本文所述的烷基是指1-6个碳原子的低级烷基；优选地，是指1-4个碳原子的低级烷基。在具体的实施方式中，本文所述的烷基包 括但不限于：甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、戊基、己基等等。
本文所用的术语“C 1-6烷氧基”与本领域普通技术人员通常理解的含义相同，是指具有1至6个碳原子的直链或支链烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基和丁氧基等。
本文所用的术语“卤素”是指F、Cl、Br或I。
基于本发明的教导以及本领域的公知常识，本领域技术人员可以知晓本发明的化合物以及上述所定义的各种取代基还可以进一步被取代，例如被卤素、硝基、氨基、羟基等取代，只要所预期的取代基的组合是稳定的或化学上可实现的取代基组合。
本文所用的术语“取代”是指特定基团上的一个或多个氢原子被特定的取代基所替代。特定的取代基可以是前文中相应描述的取代基，也可以是各实施例中出现的具体取代基。因此，在本发明中，式I中的取代基可以各自独立地为实施例中具体化合物中的相应基团；即，本发明包括上述式I中各取代基的组合，也包括式I中所示部分取代基与实施例中出现的其它具体取代基的组合。
本发明所述的“药学上可接受的”是指这样一些化合物、原料、组合物和/或剂型，它们在合理医学判断的范围内，适用于与患者组织接触而无过度毒性、刺激性、变态反应或与合理的利益/风险比相对称的其他问题和并发症，并有效用于既定用途。
本发明所使用的“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，药学上可接受的无毒的酸形成的盐包括，但并不限于，无机酸盐，如盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，和有机酸盐，如乙酸盐，草酸盐，马来酸盐，酒石酸盐，柠檬酸盐，琥珀酸盐，丙二酸盐，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的无毒的酸形成的盐，包括己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯磺酸盐，苯甲酸盐，重硫酸盐，硼酸盐，丁酸盐，樟脑酸盐，樟脑磺酸盐，环戊基丙酸盐，二葡萄糖酸盐，十二烷基硫酸盐，乙磺酸盐，甲酸盐，反丁烯二酸盐，葡庚糖酸盐，甘油磷酸盐，葡萄糖酸盐，半硫酸盐，庚酸盐，己酸盐，氢碘酸盐，2-羟基-乙磺酸盐，乳糖醛酸盐，乳酸盐，月桂酸盐，月桂基硫酸盐，苹果酸盐，丙二酸盐，甲磺酸盐，2-萘磺酸盐，烟酸盐，硝酸盐，油酸盐，棕榈酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，苦味酸盐，特戊酸盐，丙酸盐，硬脂酸盐，硫氰酸盐，对甲苯磺酸盐，十一酸盐，戊酸盐，等等。药学上可接受的通过适当的碱得到的盐，包括碱金属盐，碱土金属盐。碱金属盐或碱土金属盐，包括钠，锂，钾，钙，镁，等等。本发明也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C 1-C 8磺酸化物和芳香磺酸化物。
所属领域的专业人员将认识到：本发明所描述的化学反应可以用来合适地制备许多本发明的其他化合物，且用于制备本发明的化合物的其它方法都被认为是在本发明的范围之内。 例如，根据本发明那些非例证的化合物的合成可以成功地被所属领域的技术人员通过修饰方法完成，如适当的保护干扰基团，通过利用其他已知的试剂除了本发明所描述的，或将反应条件做一些常规的修改。另外，本发明所公开的反应或已知的反应条件也公认地适用于本发明其他化合物的制备。
为了方便以及符合常规理解，术语“任意取代的”、“任选取代的”或“取代或未取代的”只适用于能够被取代基所取代的位点，而不包括那些化学上不能实现的取代。
本发明的化合物
本发明提供了能够预防、治疗或改善阿尔茨海默病和唐氏综合征，特别是预防、治疗或改善阿尔茨海默病和唐氏综合征导致的认知功能受损的化合物。通过试验的结果证实，本发明的化合物对阿尔茨海默病5×FAD小鼠模型的学习记忆障碍和突触功能缺陷有明显的改善作用，并可通过降低其大脑皮层和海马中小胶质细胞的数目和激活状态，抑制神经炎症作用来发挥其抗阿尔茨海默病和相关疾病作用。
与其它临床中应用的药物相比，该发明化合物结构简单、药效明确、合成制备容易，且能有效通过血脑屏障，能够有效地发挥改善阿尔茨海默病和唐氏综合征小鼠模型的突触功能和学习记忆功能缺陷的作用，且较大剂量应用时不会产生其它不良反应，具有较好的安全性，因此可以应用于阿尔茨海默病和唐氏综合征的治疗。
在具体的实施方式中，本发明提供式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐，
Figure PCTCN2021073164-appb-000022
式I中的各取代基如上所述。
更具体地，本发明提供以下化合物：
Figure PCTCN2021073164-appb-000023
Figure PCTCN2021073164-appb-000024
Figure PCTCN2021073164-appb-000025
Figure PCTCN2021073164-appb-000026
Figure PCTCN2021073164-appb-000027
Figure PCTCN2021073164-appb-000028
在具体的实施方式中，本发明的化合物为AZ1、AZ2、MZ77、MZ76、MZ1、MZ30、 MZ32、MZ67、MZ66、MZ75、MZ31、MZ34、MZ74、MZ68、MZ29、MZ72、MZ38或MZ24。
本发明的药物组合物以及施用方法
本发明的化合物能够用于预防、治疗或改善阿尔茨海默病和唐氏综合征，特别是预防、治疗或改善阿尔茨海默病和唐氏综合征导致的认知功能受损。因此，在本发明的化合物及其各种晶型、水合物、溶剂合物或药学上可接受的盐的基础上。本发明还提供了包含本发明化合物的药物组合物，所述药物组合物任选包含药学上可接受的赋形剂。
在具体的实施方式中，本发明的药物组合物包含安全有效量范围内的本发明化合物或其药学上可接受的盐以及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。
“药学上可以接受的赋形剂或载体”是指：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2021073164-appb-000029
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲 酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量。本发明的化合物和药物组合物可通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服，一次性服用或分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点在于：
1.本发明提供了结构全新的预防、治疗或改善阿尔茨海默病和唐氏综合征，特别是预防、治疗或改善阿尔茨海默病和唐氏综合征导致的认知功能受损的化合物；
2.本发明的化合物结构简单、药效明确、合成制备容易；
3.本发明的化合物能有效通过血脑屏障；
4.本发明的化合物在较大剂量应用时不会产生其它不良反应，具有较好的安全性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
以下实施例中使用的色谱柱是硅胶柱。硅胶为(200-300目)。
1H NMR使用Bruker 400MHz磁共振谱仪记录。1H NMR谱以CDC1 3为溶剂(以ppm为单位)，用TMS(0ppm)或氯仿(7.26ppm)作为参照标准。当出现多重峰的时候将使用下面的缩写：s(singlet，单峰)，d(doublet，双峰)，t(triplet，三重峰)，q(quartet，四重峰)，m(multiplet， 多重峰)，dd(doublet of doublets，双二重峰)，ddd(doublet of doublet of doublets，双双二重峰)，dt(doublet of triplets，双三重峰)，td(triplet of doublets，三双重峰)，tt(triplet of triplets，三三重峰)。偶合常数J，用赫兹(Hz)表示。
化合物AZ1和AZ2为商品化药品，也可以根据现有技术文献，例如Wrigley,et al.,ACS Chem.Biol.2017合成。
实施例1.化合物XMU1的制备
Figure PCTCN2021073164-appb-000030
步骤1)在氮气保护下，将碳酸钾(2.93g，21.22mmol),4-(溴甲基)-1-氟-2-(三氟甲基)苯(3g，11.67mmol)和5-溴-2-羟基苯甲醛(2.13g，10.61mmol)依次加入250ml三口瓶中，然后加入60ml DMF，在室温25℃下磁力搅拌，得到一悬浮液，室温搅拌过夜(约15小时)，次日TLC监控原料消失，终止反应。将反应液倒入1.2L冰水中，有白色沉淀，过滤得到的白色固体，用3×500ml水洗涤，真空干燥得到5-溴-2-(4-氟-3-(三氟甲基)苄氧基)苯甲醛(3.5g，87.46％)，为白色固体。如下反应式所示：
Figure PCTCN2021073164-appb-000031
步骤2)在氮气保护下，将5-溴-2-(4-氟-3-(三氟甲基)苄氧基)苯甲醛(3g，7.95mmol)加入到250ml口瓶中，加入1.0M的NH 3/THF(80mL)溶液，然后将溶液搅拌10分钟。然后分批加入三乙酰基硼氢化钠(6.74g，31.82mmol)，大约30分钟。加毕，将所得悬浮液在20℃下搅拌16小时。TLC跟踪反应直到原料消失，停止反应。将混合物倒入10％NaHCO 3溶液(200mL)中，用乙酸乙酯(2×200mL)萃取相。合并的有机相。用硫酸钠干燥，过滤，旋蒸掉溶剂，得到黄色胶状物。粗品通过快速硅胶色谱色谱法纯化产物，洗脱梯度为含有0～10％甲醇的二氯甲烷。合并纯馏分并蒸发至干，得到(5-溴-2-((4-氟-3-(三氟甲基)苄基)氧基)苯基)甲胺(2g，66.48％)白色固体，即XMU1。如下反应式所示：
Figure PCTCN2021073164-appb-000032
1H-NMR(DMSO-d6,400MHz)：7.88-7.72(m,2H),7.62-7.48(m,2H),7.42(d,J＝7.6Hz,1H),6.97(d,J＝8.8Hz,1H),5.17(s,2H),3.68(s,2H),1.75(brs,2H)。
实施例2.化合物MZ39的制备
Figure PCTCN2021073164-appb-000033
将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，5-溴水杨醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.28g，74.2％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H)；
MS(ESI):m/z 376.98[M+H] +。
实施例3.化合物(1)的制备
Figure PCTCN2021073164-appb-000034
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.37g，89.3％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.94(s,2H),7.88(s,1H),7.58(d,J＝1.8Hz,1H),7.55(dd,J＝8.1,1.8Hz,1H),7.39(d,J＝8.1Hz,1H),6.65(t,J＝73.2Hz,1H),5.29(s,2H).
MS(ESI):m/z 415.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(3,5-双三氟甲基苄基)苯甲醛(0.21g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.20g,87.1％)。
1H NMR(400MHz,CDCl 3)δ7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H),3.80(s,2H),3.71–3.65(m,2H),2.82–2.79(m,2H).
MS(ESI):m/z 460.11[M+H] +。
实施例4.化合物(2)的制备
Figure PCTCN2021073164-appb-000035
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.37g,89.3％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.94(s,2H),7.88(s,1H),7.58(d,J＝1.8Hz,1H),7.55(dd,J＝8.1,1.8Hz,1H),7.39(d,J＝8.1Hz,1H),6.65(t,J＝73.2Hz,1H),5.29(s,2H).
MS(ESI):m/z 415.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(3,5-双三氟甲基苄基)苯甲醛(0.21g,0.5mmol)，N-甲基哌嗪(0.17mL,1.5mL)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.17g,68.3％)。
1H NMR(400MHz,CDCl 3)δ7.92(s,2H),7.85(s,1H),7.14(d,J＝8.1Hz,1H),7.05(d,J＝1.9Hz,1H),6.93(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.4Hz,1H),5.23(s,2H),3.45(s,2H),2.42(s,8H),2.28(s,3H).
MS(ESI):m/z 499.16[M+H] +。
实施例5.化合物(3)的制备
Figure PCTCN2021073164-appb-000036
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4,6-二甲氧基水杨醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，硅胶柱纯化，得白色固体(0.44g,89.4％)。
1H NMR(400MHz,CDCl 3)δ10.43(s,1H),8.04(s,2H),7.85(s,1H),6.14(d,J＝2.1Hz,1H),6.11(d,J＝2.1Hz,1H),5.23(s,2H),3.91(s,3H),3.87(s,3H).
MS(ESI):m/z 409.08[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基)-4,6-二甲氧基水杨醛(0.20g,0.49mmol)，1-(2-羟乙基) 哌嗪(0.18mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.15g,58.6％)。
1H NMR(400MHz,CDCl 3)δ7.92(d,J＝1.6Hz,2H),7.83(s,1H),6.16(d,J＝2.2Hz,1H),6.14(d,J＝2.2Hz,1H),5.14(s,2H),3.68(s,2H),3.61(t,J＝5.3Hz,2H),2.68–2.52(m,10H).
MS(ESI):m/z 523.20[M+H] +。
实施例6.化合物(4)的制备
Figure PCTCN2021073164-appb-000037
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.37g,89.3％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.94(s,2H),7.88(s,1H),7.58(d,J＝1.8Hz,1H),7.55(dd,J＝8.1,1.8Hz,1H),7.39(d,J＝8.1Hz,1H),6.65(t,J＝73.2Hz,1H),5.29(s,2H).
MS(ESI):m/z 415.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(3,5-双三氟甲基苄基)苯甲醛(0.21g,0.5mmol)，1-(2-羟乙基)哌嗪(0.18mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.18g,68.0％)。
1H NMR(400MHz,CDCl 3)δ7.95–7.92(m,2H),7.84(s,1H),7.16(d,J＝8.1Hz,1H),7.05(d,J＝1.9Hz,1H),6.93(dd,J＝8.2,1.9Hz,1H),6.51(t,J＝74.3Hz,1H),5.20(s,2H),3.67(s,2H),3.60(t,J＝5.3Hz,2H),2.69–2.50(m,10H).
MS(ESI):m/z 529.17[M+H] +。
实施例7.化合物(5)的制备
Figure PCTCN2021073164-appb-000038
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.37g,89.3％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.94(s,2H),7.88(s,1H),7.58(d,J＝1.8Hz,1H),7.55(dd,J＝8.1,1.8Hz,1H),7.39(d,J＝8.1Hz,1H),6.65(t,J＝73.2Hz,1H),5.29(s,2H).
MS(ESI):m/z 415.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(3,5-双三氟甲基苄基)苯甲醛(0.21g,0.5mmol)，N,N-二乙基乙二胺(0.21mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.18g,70.03％)。
1H NMR(400MHz,CDCl 3)δ7.97–7.93(m,2H),7.85(s,1H),7.18(d,J＝8.1Hz,1H),7.06(d,J＝1.9Hz,1H),6.93(dd,J＝8.2,1.9Hz,1H),6.52(t,J＝74.3Hz,1H),5.22(s,2H),3.81(s,2H),2.64(t,J＝6.4Hz,2H),2.58(t,J＝5.9Hz,2H),2.47(q,J＝7.1Hz,4H),0.99(t,J＝7.1Hz,6H).
MS(ESI):m/z 515.19[M+H] +。
实施例8.化合物(6)的制备
Figure PCTCN2021073164-appb-000039
步骤1)将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.3g,90.0％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.57(d,J＝1.8Hz,1H),7.53(dd,J＝8.1,1.8Hz,1H),7.38(d,J＝10.2Hz,1H),7.36–7.32(m,1H),7.00(td,J＝9.6,6.4Hz,1H),6.64(t,J＝73.6Hz,1H),5.18(q,J＝1.0Hz,2H).
MS(ESI):m/z 333.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(2,4,5-三氟苄基)苯甲醛(0.167g,0.5mmol)，N-甲基哌嗪(0.17mL,1.5mL)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.15g,71.9％)。
1H NMR(400MHz,CDCl 3)δ7.36(ddd,J＝10.3,8.7,6.6Hz,1H),7.11(d,J＝8.1Hz,1H),7.04(d,J＝1.9Hz,1H),7.01–6.93(m,1H),6.91(dd,J＝8.1,1.9Hz,1H),6.51(t,J＝74.8Hz,1H),5.11(q,J＝1.0Hz,2H),3.46(s,2H),2.44(s,8H),2.29(s,3H).
MS(ESI):m/z 417.15[M+H] +。
实施例9.化合物(7)的制备
Figure PCTCN2021073164-appb-000040
步骤1)将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.3g,90.0％)。
1H NMR(400MHz,CDCl 3)δ9.95(s,1H),7.57(d,J＝1.8Hz,1H),7.53(dd,J＝8.1,1.8Hz,1H),7.38(d,J＝10.2Hz,1H),7.36–7.32(m,1H),7.00(td,J＝9.6,6.4Hz,1H),6.64(t,J＝73.6Hz,1H),5.18(q,J＝1.0Hz,2H).
MS(ESI):m/z:333.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(2,4,5-三氟苄基)苯甲醛(0.167g,0.5mmol)，1-(2-羟乙基)哌嗪(0.18mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.13g,58.2％)。
1H NMR(400MHz,CDCl 3)δ7.37(ddd,J＝10.4,8.7,6.6Hz,1H),7.12(d,J＝8.1Hz,1H),7.03(d,J＝1.9Hz,1H),6.97(td,J＝9.6,6.4Hz,1H),6.91(dd,J＝8.2,1.9Hz,1H),6.52(t,J＝74.7Hz,1H),5.11(s,2H),3.71–3.65(m,2H),3.48(s,2H),2.75–2.60(m,6H),2.52(s,4H).
MS(ESI):m/z 447.16(100.0％)[M+H] +。
实施例10.化合物(8)的制备
Figure PCTCN2021073164-appb-000041
步骤1)将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5, 4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄基)-5-溴苯甲醛(0.19g,0.5mmol)，色胺(0.24g,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.15g,57.6％)。
1H NMR(500MHz,CDCl 3)δ9.94(d,J＝8.4Hz,1H),7.62–7.59(m,1H),7.59–7.56(m,1H),7.36–7.30(m,3H),7.14(td,J＝7.5,1.5Hz,1H),7.12–7.10(m,2H),7.05(td,J＝7.5,1.6Hz,1H),6.97(dq,J＝7.5,1.2Hz,1H),6.87(d,J＝7.3Hz,1H),5.16(t,J＝1.0Hz,2H),4.13–4.09(m,2H),3.21–3.16(m,2H),2.98–2.79(m,2H).
MS(ESI):m/z 521.08[M+H] +。
实施例11.化合物(9)的制备
Figure PCTCN2021073164-appb-000042
步骤1)将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.30g,87.2％)。
1H NMR(400MHz,CDCl 3)δ10.38(s,1H)8.02(d,J＝1.5Hz,1H),7.46(dd,J＝7.5,1.5Hz,1H),7.12(dtt,J＝8.0,4.9,1.1Hz,1H),7.02(d,J＝7.5Hz,1H),7.01(td,J＝8.1,5.0Hz,1H),5.18(s,2H).
MS(ESI):m/z 344.97[M+H] +；
步骤2)将2-(2,4,5-三氟苄基)-5-溴苯甲醛(0.17g,0.5mmol)，N,N-二乙基乙二胺(0.21mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.12g,54.05％)。
1H NMR(400MHz,CDCl 3)δ7.44(d,J＝2.5Hz,1H),7.33(td,J＝9.3,8.7,2.8Hz,2H),6.97(td,J＝9.6,6.4Hz,1H),6.77(d,J＝8.7Hz,1H),5.05(s,2H),3.80(s,2H),2.66(t,J＝6.4Hz,2H),2.56(t,J＝5.9Hz,2H),2.48(q,J＝7.1Hz,4H),0.97(t,J＝7.1Hz,6H).
MS(ESI):m/z 445.10[M+H] +。
实施例12.化合物(10)的制备
Figure PCTCN2021073164-appb-000043
a)N 2保护下，将化合物1(0.69g,3mmol)，DCM(20mL)加入100mL双口圆底烧瓶中，依次加入乙醇胺(0.54mL,9mmol)，乙酸(0.17mL,1.5mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(1.27g,6mmol),至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物2(0.70g,85.3％)。
1H NMR(400MHz,DMSO-d 6)δ7.98(d,J＝2.2Hz,1H),7.91(d,J＝8.6Hz,1H),7.72(dd,J＝8.6,2.3Hz,1H),4.51(t,J＝5.3Hz,1H),3.97(s,2H),3.44(q,J＝5.4Hz,2H),2.55(t,J＝5.8Hz,2H).
b)将化合物2(0.41g,1.5mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及二碳酸二叔丁酯(0.69mL,0.3mmol)，室温反应，至TLC检测反应完毕，加水淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物3(0.50g,89.8％)。
1H NMR(400MHz,DMSO-d 6)δ8.06–7.95(m,1H),7.77(dd,J＝8.7,2.2Hz,1H),7.52(d,J＝46.6Hz,1H),4.74(t,J＝5.2Hz,2H),4.70(s,1H),3.51(q,J＝5.6Hz,2H),3.28(t,2H),1.50–1.15(m,9H).
c)H 2条件下，将化合物3(0.37g,1mmol)，10％Pd/C(100mg)，MeOH(10mL)加入100圆底烧瓶中，常温反应，至TLC检测反应完毕，过滤，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物4(0.18g,67.5％)。
1H NMR(400MHz,DMSO-d 6)δ6.97(t,J＝7.5Hz,1H),6.92(s,2H),6.61(d,J＝7.9Hz,1H),6.52(d,J＝7.5Hz,1H),4.68(s,1H),4.29(s,2H),3.43(q,J＝5.7Hz,2H),3.16–3.04(m,2H),1.39(d,J＝14.6Hz,9H).
d)N 2保护下，将3-三氟甲氧基苯甲醛(0.11g,0.6mmol)，DCM(10mL)加入100mL双口圆底烧瓶中，依次加入化合物4(0.32g,1.2mmol)，乙酸(0.03ml,0.3mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(0.25g,1.2mmol)，至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物5(0.20g,75.1％)。 1H NMR(400MHz,DMSO-d 6)δ7.44(t,J＝7.9Hz,1H),7.36(d,J＝7.7Hz,1H),7.24(s,1H),7.22–7.18(m,1H),7.07–6.97(m,2H),6.57(t,J＝7.3Hz,1H),6.44(dd,J＝8.3,1.1Hz,1H),6.06(s,1H),4.72(t,J＝5.5Hz,1H),4.41(d,J＝3.6Hz,4H),3.46(q,J＝6.2Hz,2H),3.14(s,2H),1.36(s,9H).
e)将化合物5(0.15g,0.35mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL) 以及三氟乙酸2mL，室温反应1h，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物MZ75(0.1g,84.6％)。
1H NMR(400MHz,DMSO-d 6)δ7.45(t,J＝7.7Hz,1H),7.42–7.39(m,1H),7.32(s,1H),7.19(dd,J＝7.9,5.1Hz,1H),7.03(dd,J＝7.4,1.6Hz,1H),6.99(td,J＝7.7,1.7Hz,1H),6.79(s,1H),6.52(t,J＝7.3Hz,1H),6.43(d,J＝8.0Hz,1H),4.56(s,1H),4.40(s,2H),3.77(s,2H),3.49(d,J＝7.1Hz,2H),2.59(t,J＝5.7Hz,2H).
实施例13.化合物(11)、(12)的制备
Figure PCTCN2021073164-appb-000044
步骤a)N 2保护下，将化合物1(0.69g,3mmol)，DCM(20mL)加入100mL双口圆底烧瓶中，依次加入乙醇胺(0.54mL,9mmol)，乙酸(0.17mL,1.5mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(1.98g,6mmol),至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物2(0.69g,83.5％)。
1H NMR(400MHz,DMSO-d 6)δ7.98(d,J＝2.2Hz,1H),7.91(d,J＝8.6Hz,1H),7.72(dd,J＝8.6,2.3Hz,1H),4.51(t,J＝5.3Hz,1H),3.97(s,2H),3.44(q,J＝5.4Hz,2H),2.55(t,J＝5.8Hz,2H).
步骤b)将化合物2(0.41g,1.5mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及二碳酸二叔丁酯(0.69mL,0.3mmol)，室温反应，至TLC检测反应完毕，加水淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物3(0.50g,89.8％)。
1H NMR(400MHz,DMSO-d 6)δ8.06–7.95(m,1H),7.77(dd,J＝8.7,2.2Hz,1H),7.52(d,J＝46.6Hz,1H),4.74(t,J＝5.2Hz,2H),4.70(s,1H),3.51(q,J＝5.6Hz,2H),3.28(t,2H),1.50–1.15(m,9H).
步骤c)将化合物3(0.56g,1.5mmol)，还原铁粉(0.42g,7.5mmol)，NH 4Cl(0.80g,15mmol)，MeOH(15mL)加入100圆底烧瓶中，80℃下回流反应，至TLC检测反应完毕，加水淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物4(0.37g,71.6％)。
1H NMR(400MHz,DMSO-d 6)δ7.11(dd,J＝8.6,2.3Hz,1H),7.06(s,1H),6.58(d,J＝8.5Hz,1H),5.45–5.05(m,2H),4.71(s,1H),4.26(s,2H),3.45(t,J＝6.4Hz,2H),3.11(s,2H).
步骤d)N 2保护下，将3-三氟甲基-4-氟苯甲醛(0.096g,0.5mmol)，DCM(10mL)加入100mL双口圆底烧瓶中，依次加入化合物4(0.17g,0.5mmol)，乙酸(0.28mL,0.25mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(0.32g,1.5mmol)，至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物5-1(0.20g,76.4％)。
1H NMR(400MHz,DMSO-d 6)δ7.76–7.60(m,2H),7.47(t,J＝9.8Hz,1H),7.17(s,2H),6.41(d,J＝8.7Hz,1H),6.17(s,1H),4.74(t,J＝5.4Hz,1H),4.45–4.35(m,4H),3.47(q,J＝6.1Hz,2H),3.14(s,2H),1.33(s,9H).
N 2保护下，将3-三氟甲氧基苯甲醛(0.095g,0.5mmol)，DCM(10mL)加入100mL双口圆底烧瓶中，依次加入化合物4(0.17g,0.5mmol)，乙酸(0.28mL,0.25mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(0.32g,1.5mmol)，至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物5-2(0.21g,79.2％)。
1H NMR(400MHz,DMSO-d 6)δ7.45(t,J＝7.9Hz,1H),7.34(d,J＝7.7Hz,1H),7.21(d,J＝9.1Hz,2H),7.16(d,J＝8.2Hz,2H),6.39(d,J＝8.7Hz,1H),6.20(s,1H),4.39(d,J＝3.7Hz,4H),3.48(t,J＝6.3Hz,2H),3.15(s,2H),1.34(d,J＝8.9Hz,9H).
步骤e)将化合物5-1(0.182g,0.35mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及三氟乙酸2mL，室温反应1h，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物MZ76(0.13g,87.1％)。
1H NMR(400MHz,DMSO-d 6)δ7.45(t,J＝7.9Hz,1H),7.34(d,J＝7.7Hz,1H),7.21(d,J＝9.1Hz,2H),7.16(d,J＝8.2Hz,2H),6.39(d,J＝8.7Hz,1H),6.20(s,1H),4.39(d,J＝3.7Hz,4H),3.48(t,J＝6.3Hz,2H),3.15(s,2H),1.34(d,J＝8.9Hz,9H).
将化合物5-2(0.181g,0.35mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及三氟乙酸2mL，室温反应1h，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物MZ77(0.125g,85.7％)。
1H NMR(400MHz,DMSO-d 6)δ7.45(t,J＝7.8Hz,1H),7.38(d,J＝7.7Hz,1H),7.31(s,1H),7.21(q,J＝3.4Hz,2H),7.12(dd,J＝8.6,2.5Hz,1H),6.87(d,J＝6.3Hz,1H),6.36(d,J＝8.6Hz,1H),4.57(s,1H),4.40(d,J＝5.4Hz,2H),3.74(s,2H),3.48(q,J＝5.5Hz,2H),2.58(t,J＝5.8Hz,2H).
实施例14.化合物(13)的制备
Figure PCTCN2021073164-appb-000045
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，硅胶柱纯化，得白色固体(0.40g,94.1％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.90(s,2H),7.87(s,1H),7.47(d,J＝2.5Hz,1H),7.38(dd,J＝8.7,2.5Hz,1H),6.77(d,J＝8.7Hz,1H),5.19(s,2H).
MS(ESI):m/z 426.97[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄氧基)-5-溴苯甲醛(0.21g,0.5mmol)，乙醇胺(0.09mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.14g,59.45％)。
1H NMR(400MHz,CDCl 3)δ7.90(s,2H),7.87(s,1H),7.47(d,J＝2.5Hz,1H),7.38(dd,J＝8.7,2.5Hz,1H),6.77(d,J＝8.7Hz,1H),5.19(s,2H),3.90(s,2H),3.74–3.66(m,2H),2.82(dd,J＝6.0,4.3Hz,2H),1.91(s,1H).
MS(ESI):m/z 472.03[M+H] +。
实施例15.化合物(14)的制备
Figure PCTCN2021073164-appb-000046
步骤1)将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，硅胶柱纯化，得白色固体(0.3g,90.0％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.36(ddd,J＝10.3,8.7,6.6Hz,1H),7.11(d,J＝8.1Hz,1H),7.04(d,J＝1.9Hz,1H),7.01–6.93(m,1H),6.91(dd,J＝8.1,1.9Hz,1H),6.51(t,J＝74.8Hz,1H),5.11(q,J＝1.0Hz,2H).
MS(ESI):m/z 333.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(2,4,5-三氟苄氧基)苯甲醛(0.167g,0.5mmol)，四氢异喹啉(0.19mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL 三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.14g,60.4％)。
1H NMR(400MHz,CDCl 3)δ7.37(ddd,J＝10.4,8.8,6.6Hz,1H),7.13(dtd,J＝10.5,6.0,5.6,3.5Hz,5H),6.98(dd,J＝8.2,1.8Hz,2H),6.89(td,J＝9.6,6.4Hz,1H),6.54(t,J＝74.8Hz,1H),5.10(s,2H),3.64(s,2H),3.61(s,2H),2.90(t,J＝5.9Hz,2H),2.73(t,J＝5.9Hz,2H).
实施例16.化合物(15)的制备
Figure PCTCN2021073164-appb-000047
步骤1)将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5,4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄氧基)-5-溴苯甲醛(0.19g,0.5mmol)，1-(2-羟乙基)哌嗪(0.18mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.13g,53.06％)。
1H NMR(400MHz,CDCl 3)δ7.67(dd,J＝6.8,2.2Hz,1H),7.62(ddd,J＝7.4,4.7,2.2Hz,1H),7.50(d,J＝2.6Hz,1H),7.32(dd,J＝8.7,2.6Hz,1H),7.22(d,J＝9.2Hz,1H),6.76(d,J＝8.7Hz,1H),5.05(s,2H),3.61(t,J＝5.4Hz,2H),3.54(s,2H),2.55(d,J＝6.9Hz,10H).
MS(ESI):m/z 491.09[M+H] +。
实施例17.化合物(16)的制备
Figure PCTCN2021073164-appb-000048
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，2-羟基苯甲醛(0.12g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.31g，89.7％)。
1H NMR(400MHz,DMSO-d 6)δ10.41(s,1H),8.28(s,2H),8.11(s,1H),7.76(dd,J＝7.6,1.8Hz,1H),7.70(ddd,J＝8.8,7.3,1.9Hz,1H),7.32(d,J＝8.4Hz,1H),7.15(t,J＝7.5Hz,1H),5.48(s,2H).
MS(ESI):m/z 349.06[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基氧基)苯甲醛(0.17g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.11g,56.0％)。
1H NMR(400MHz,DMSO-d 6)δ8.19(s,2H),8.08(s,1H),7.35(dd,J＝7.4,1.7Hz,1H),7.25(td,J＝7.8,1.8Hz,1H),7.05(d,J＝8.2Hz,1H),6.96(t,J＝7.4Hz,1H),5.34(s,2H),4.71–4.32(m,1H),3.79(s,2H),3.47(t,J＝5.7Hz,2H),2.61(t,J＝5.7Hz,2H).
MS(ESI):m/z 394.12[M+H] +。
实施例18.化合物(17)的制备
Figure PCTCN2021073164-appb-000049
步骤1)将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，2-羟基苯甲醛(0.12g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.25g，82.3％)。
1H NMR(400MHz,DMSO-d 6)δ10.41(d,J＝0.8Hz,1H),7.95(ddd,J＝16.5,7.4,2.2Hz,2H),7.74(dd,J＝7.7,1.8Hz,1H),7.68(ddd,J＝8.9,7.4,1.9Hz,1H),7.57(dd,J＝10.8,8.5Hz,1H),7.32(d,J＝8.4Hz,1H),7.13(t,J＝7.5Hz,1H),5.35(s,2H).
MS(ESI):m/z 299.07[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄基氧基)苯甲醛(0.15g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.10g,58.3％)。
1H NMR(400MHz,DMSO-d 6)δ7.92–7.84(m,2H),7.55(dd,J＝10.9,8.6Hz,1H),7.33(d,J＝7.3Hz,1H),7.23(t,J＝7.8Hz,1H),7.05(d,J＝8.2Hz,1H),6.94(t,J＝7.4Hz,1H),5.20(s,2H),4.50(s,1H),3.75(s,2H),3.46(s,2H),2.58(d,J＝5.6Hz,2H).
MS(ESI):m/z 344.13[M+H] +。
实施例19.化合物(18)的制备
Figure PCTCN2021073164-appb-000050
将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4,6-二甲氧基水杨醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.44g,89.4％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.93(s,2H),7.84(s,1H),6.18(d,J＝2.2Hz,1H),6.15(d,J＝2.3Hz,1H),5.15(s,2H),
MS(ESI):m/z 409.08[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄氧基)-4，6-二甲氧基水杨醛(0.20g,0.49mmol)，2-氨基-5-溴吡啶(0.26g,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.16g，58.6％)。
1H NMR(400MHz,Chloroform-d)δ8.04(d,J＝2.4Hz,1H),7.90(s,2H),7.85(s,1H),7.40(dd,J＝8.9,2.5Hz,1H),6.43(d,J＝8.9Hz,1H),6.19(d,J＝2.2Hz,1H),6.13(d,J＝2.2Hz,1H),5.17(s,2H),4.46(d,J＝5.9Hz,2H),3.85(s,3H),3.79(s,3H).
MS(ESI):m/z 565.05[M+H] +。
实施例20.化合物(19)的制备
Figure PCTCN2021073164-appb-000051
步骤1)将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，3-羟基-4-甲氧基苯甲醛(0.15g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧 瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.25g，79.2％)。
1H NMR(400MHz,DMSO-d 6)δ9.85(s,1H),7.91(dd,J＝7.1,2.2Hz,1H),7.86(ddd,J＝7.7,4.9,2.1Hz,1H),7.61(dd,J＝8.4,2.0Hz,1H),7.58–7.54(m,1H),7.52(d,J＝1.9Hz,1H),7.23(d,J＝8.3Hz,1H),5.24(s,2H),3.89(s,3H).
MS(ESI):m/z 329.08[M+H] +；
步骤2)将3-((4-氟-3-(三氟甲基)苄基)氧基)-4-甲氧基苯甲醛(0.16g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.10g,53.6％)。
1H NMR(400MHz,DMSO-d 6)δ7.88(dd,J＝7.3,2.1Hz,1H),7.82(ddd,J＝7.6,5.0,2.1Hz,1H),7.54(dd,J＝10.9,8.6Hz,1H),7.06(d,J＝1.8Hz,1H),6.92(d,J＝8.2Hz,1H),6.88(dd,J＝8.3,1.8Hz,1H),5.13(s,2H),3.74(s,3H),3.64(s,2H),3.45(t,J＝5.7Hz,2H),2.53(t,J＝5.8Hz,2H).
MS(ESI):m/z 374.14[M+H] +。
实施例21.化合物(20)的制备
Figure PCTCN2021073164-appb-000052
步骤1)将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5,4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄氧基)-5-溴苯甲醛(0.19g,0.5mmol)，N-乙基哌嗪(0.19mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.11g，54.62％)。
1H NMR(400MHz,CDCl 3)δ7.68–7.60(m,2H),7.49(d,J＝2.6Hz,1H),7.30(dd,J＝8.7,2.6Hz,1H),7.25–7.18(m,1H),6.75(d,J＝8.7Hz,1H),5.03(s,2H),3.54(s,2H), 2.70–2.44(m,8H),2.41(q,J＝7.2Hz,2H),1.07(t,J＝7.2Hz,3H).
MS(ESI):m/z 379.00[M+H] +。
实施例22.化合物(21)的制备
Figure PCTCN2021073164-appb-000053
a)N 2保护下，将化合物1(0.69g,3mmol)，DCM(20mL)加入100mL双口圆底烧瓶中，依次加入乙醇胺(0.54mL,9mmol)，乙酸(0.17mL,1.5mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(1.27g,6mmol),至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物2(0.70g,84.9％)。
1H NMR(400MHz,DMSO-d 6)δ7.98(d,J＝2.2Hz,1H),7.91(d,J＝8.6Hz,1H),7.72(dd,J＝8.6,2.3Hz,1H),4.51(t,J＝5.3Hz,1H),3.97(s,2H),3.44(q,J＝5.4Hz,2H),2.55(t,J＝5.8Hz,2H).
b)将化合物2(0.41g,1.5mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及二碳酸二叔丁酯(0.69mL,0.3mmol)，室温反应，至TLC检测反应完毕，加水淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物3(0.50g,87.8％)。
1H NMR(400MHz,DMSO-d 6)δ8.06–7.95(m,1H),7.77(dd,J＝8.7,2.2Hz,1H),7.52(d,J＝46.6Hz,1H),4.74(t,J＝5.2Hz,2H),4.70(s,1H),3.51(q,J＝5.6Hz,2H),3.28(t,2H),1.50–1.15(m,9H).
c)H 2条件下，将化合物3(0.37g,1mmol)，10％Pd/C(100mg)，MeOH(10mL)加入100圆底烧瓶中，常温反应，至TLC检测反应完毕，过滤，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物4(0.17g,62.7％)。
1H NMR(400MHz,DMSO-d 6)δ6.97(t,J＝7.5Hz,1H),6.92(s,2H),6.61(d,J＝7.9Hz,1H),6.52(d,J＝7.5Hz,1H),4.68(s,1H),4.29(s,2H),3.43(q,J＝5.7Hz,2H),3.16–3.04(m,2H),1.39(d,J＝14.6Hz,9H).
d)N 2保护下，将3-三氟甲基-4-氟苯甲醛(0.12g,0.6mmol)，DCM(10mL)加入100mL双口圆底烧瓶中，依次加入化合物4(0.32g,1.2mmol)，乙酸(0.03ml,0.3mmol)，室温反应2h后，加入三乙酰氧基硼氢化钠(0.25g,1.2mmol)，至TLC检测反应完毕，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物5(0.19g,72.6％)。
1H NMR(400MHz,DMSO-d 6)δ7.68(dd,J＝8.8,5.4Hz,2H),7.51–7.41(m,1H),7.03(t,J＝7.6Hz,2H),6.58(t,J＝7.3Hz,1H),6.46(d,J＝8.1Hz,1H),6.03(s,1H),4.71(t,J＝5.4Hz,1H),4.41(d,J＝5.9Hz,5H),3.45(q,J＝6.2Hz,2H),3.12(s,2H),1.33(s,9H).
e)将化合物5(0.15g,0.35mmol)至于100mL圆底烧瓶中，依次加入DCM(10mL)以及三氟乙酸2mL，室温反应1h，饱和碳酸氢钠溶液淬灭，乙酸乙酯萃取，合并有机相，硅胶柱正相层析纯化，得化合物MZ74(0.1g,81.8％)。
1H NMR(400MHz,DMSO-d 6)δ7.69(dd,J＝8.7,5.4Hz,2H),7.52(m,1H),7.05(t,J＝7.6Hz,2H),6.58(t,J＝7.3Hz,1H),6.46(d,J＝8.1Hz,1H),6.03(s,1H),4.71(t,J＝5.4Hz,1H),4.41(d,J＝5.9Hz,5H),3.44(q,J＝6.3Hz,2H),3.14(s,2H).
实施例23.化合物(22)的制备
Figure PCTCN2021073164-appb-000054
步骤1)将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，2-羟基-4,6-二甲氧基苯甲醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.30g，83.4％)。
1H NMR(400MHz,DMSO-d 6)δ10.27(s,1H),8.03(dd,J＝7.1,2.2Hz,1H),7.89(ddd,J＝7.9,5.0,2.1Hz,1H),7.56(dd,J＝10.8,8.6Hz,1H),6.38(d,J＝2.1Hz,1H),6.32(d,J＝2.1Hz,1H),5.27(s,2H),3.88(s,3H),3.85(s,3H).
MS(ESI):m/z 359.09[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄基氧基)-4,6-二甲氧基苯甲醛(0.18g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.11g,54.5％)。
1H NMR(400MHz,DMSO-d 6)δ7.95(dd,J＝12.7,7.7Hz,2H),7.86(d,J＝8.3Hz,1H),7.56(dd,J＝10.9,8.5Hz,1H),6.33(ddd,J＝25.9,23.3,2.1Hz,2H),5.23(s,2H),4.02(s,2H),3.83(s,3H),3.80(s,3H),2.84(t,J＝5.4Hz,2H).
MS(ESI):m/z 404.15[M+H] +。
实施例24.化合物(23)的制备
Figure PCTCN2021073164-appb-000055
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-甲氧基-3-羟基苯甲醛(0.15g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.30g，80.7％)。
1H NMR(400MHz,DMSO-d 6)δ9.85(s,1H),8.19(s,2H),8.11(s,1H),7.63(dd,J＝8.3,1.9Hz,1H),7.54(d,J＝1.8Hz,1H),7.25(d,J＝8.3Hz,1H),5.38(s,2H),3.90(s,3H).
MS(ESI):m/z 379.08[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基氧基)-4-甲氧基-3-羟基苯甲醛(0.19g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.11g,51.2％)。
1H NMR(400MHz,DMSO-d 6)δ8.16(s,2H),8.08(s,1H),7.12(d,J＝1.8Hz,1H),6.96(d,J＝8.2Hz,1H),6.92(dd,J＝8.3,1.7Hz,1H),5.28(s,2H),3.76(s,3H),3.69(s,2H),3.46(t,J＝5.7Hz,2H),2.57(t,J＝5.7Hz,2H).
MS(ESI):m/z 424.13[M+H] +。
实施例25.化合物(24)的制备
Figure PCTCN2021073164-appb-000056
将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4,6-二甲氧基水杨醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.44g,89.4％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.93(s,2H),7.84(s,1H),6.18(d,J＝2.2Hz,1H),6.15(d,J＝2.3Hz,1H),5.15(s,2H),
MS(ESI):m/z 409.08[M+H] +。
实施例26.化合物(25)的制备
Figure PCTCN2021073164-appb-000057
将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，4,6二甲氧基水杨醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.29g，80.9％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H)；
MS(ESI):m/z 359.09[M+H] +。
实施例27.化合物(26)的制备
Figure PCTCN2021073164-appb-000058
将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，5-溴水杨醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.29g,84.0％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.36(ddd,J＝10.3,8.7,6.6Hz,1H),7.11(d,J＝8.1Hz,1H),7.04(d,J＝1.9Hz,1H),7.01–6.93(m,1H),6.91(dd,J＝8.1,1.9Hz,1H),6.51(t,J＝74.8Hz,1H),5.11(q,J＝1.0Hz,2H).
MS(ESI):m/z 344.97[M+H] +。
实施例28.化合物(27)的制备
Figure PCTCN2021073164-appb-000059
将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，4-羟基-3-二氟甲氧基苯甲醛(0.19g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.29g，79.6％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz, 1H),5.23(s,2H)；
MS(ESI):m/z 365.06[M+H] +。
实施例29.化合物(28)的制备
Figure PCTCN2021073164-appb-000060
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，2-羟基-4,6-二甲氧基苯甲醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.33g，81.5％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H)；
MS(ESI):m/z 409.09[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基氧基)-4,6-二甲氧基苯甲醛(0.20g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.11g,50.8％)。
1H NMR(400MHz,DMSO-d 6)δ8.18(s,2H),8.08(s,1H),6.32(d,J＝2.1Hz,1H),6.26(d,J＝2.1Hz,1H),5.32(s,2H),3.77(s,3H),3.75(s,3H),3.69(s,2H),3.41(t,J＝5.6Hz,2H),2.55–2.51(m,3H).
MS(ESI):m/z 454.15[M+H] +。
实施例30.化合物(29)的制备
Figure PCTCN2021073164-appb-000061
将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.37g，89.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H)；
MS(ESI):m/z 415.05[M+H] +。
实施例31.化合物(30)的制备
Figure PCTCN2021073164-appb-000062
步骤1)将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5,4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄氧基)-5-溴苯甲醛(0.19g,0.5mmol)，N-乙基哌嗪(0.19mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入硼氢化钠(0.05g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.065g，29.9％)。该化合物为MZ33副产物。
1H NMR(400MHz,CDCl 3)δ7.66(dd,J＝6.6,2.2Hz,1H),7.60(ddd,J＝7.1,4.6,2.2Hz,1H),7.51(d,J＝2.5Hz,1H),7.37(dd,J＝8.7,2.5Hz,1H),7.23(d,J＝9.2Hz,1H),6.78(d,J＝8.7Hz,1H),5.08(s,2H),4.71(d,J＝6.3Hz,2H).
MS(ESI):m/z 475.11[M+H] +。
实施例32.化合物(31)的制备
Figure PCTCN2021073164-appb-000063
将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)， 碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5,4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄基)-5-溴苯甲醛(0.19g,0.5mmol)，4-(2-氨基乙基)-1-苄基哌啶(0.33g,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.21g，44.82％)。
1H NMR(400MHz,Chloroform-d)δ7.63(dd,J＝6.9,2.2Hz,2H),7.58–7.52(m,4H),7.32(d,J＝4.4Hz,4H),7.26–7.22(m,3H),7.22–7.15(m,2H),6.68(d,J＝8.7Hz,2H),5.01(s,4H),3.60(s,6H),3.01–2.85(m,2H),2.46(t,J＝7.1Hz,2H),2.01(d,J＝5.1Hz,3H).
MS(ESI):m/z 941.14.00[M+H] +。
实施例33.化合物(32)的制备
Figure PCTCN2021073164-appb-000064
将4-氟-3-三氟甲基溴苄(0.19mL,1.2mmol)，2-羟基-5-溴苯甲醛(0.20g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.32g,85.3％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.96(d,J＝2.6Hz,1H),7.65(dtd,J＝13.1,5.5,4.3,2.3Hz,3H),7.28(d,J＝9.5Hz,1H),6.93(d,J＝8.8Hz,1H),5.17(s,2H).
MS(ESI):m/z 376.97[M+H] +；
步骤2)将2-(4-氟-3-三氟甲基苄基)-5-溴苯甲醛(0.19g,0.5mmol)，4-(2-氨基乙基)-1-苄基哌啶(0.33mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.15g，51.77％)。
1H NMR(400MHz,Chloroform-d)δ7.67(dd,J＝6.8,2.2Hz,1H),7.60(ddd,J＝7.5,4.7,2.3Hz,1H),7.45(d,J＝2.5Hz,1H),7.39–7.27(m,6H),7.23(t,J＝9.4Hz,1H),6.76(d,J＝8.7Hz,1H),5.06(s,2H),3.79(s,2H),3.58(s,2H),2.94(d,J＝11.2Hz,2H),2.62(t,J＝7.4Hz,2H),2.01(d,J＝9.2Hz,2H),1.71–1.31(m,7H).
MS(ESI):m/z 579.16[M+H] +。
实施例34.化合物(33)的制备
Figure PCTCN2021073164-appb-000065
步骤1)将2,4,5-三氟苄基溴(0.16mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.13g,1mol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，硅胶柱纯化，得白色固体(0.3g,90.0％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.36(ddd,J＝10.3,8.7,6.6Hz,1H),7.11(d,J＝8.1Hz,1H),7.04(d,J＝1.9Hz,1H),7.01–6.93(m,1H),6.91(dd,J＝8.1,1.9Hz,1H),6.51(t,J＝74.8Hz,1H),5.11(q,J＝1.0Hz,2H).
MS(ESI):m/z 333.05[M+H] +；
步骤2)将4-二氟甲氧基-3-(2,4,5-三氟苄基)苯甲醛(0.167g,0.5mmol)，N,N-二乙基乙二胺(0.21mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得油状液体(0.15g,69.4％)。
1H NMR(400MHz,CDCl 3)δ7.37(ddd,J＝10.4,8.7,6.6Hz,1H),7.12(d,J＝8.1Hz,1H),7.08(d,J＝1.9Hz,1H),7.00–6.93(m,1H),6.93–6.90(m,1H),6.50(t,J＝74.8Hz,1H),5.11(q,J＝1.0Hz,2H),3.77(s,2H),2.69(td,J＝5.8,1.2Hz,2H),2.62(td,J＝5.8,1.2Hz,2H),2.57(q,J＝7.2Hz,4H),1.02(t,J＝7.2Hz,6H).
MS(ESI):m/z 433.18[M+H] +。
实施例35.化合物(34)的制备
Figure PCTCN2021073164-appb-000066
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，4,6-二甲氧基水杨醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.44g,89.4％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.93(s,2H),7.84(s,1H),6.18(d,J＝2.2Hz,1H),6.15(d,J＝2.3Hz,1H),5.15(s,2H),
MS(ESI):m/z 409.08[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基)-4，6-二甲氧基水杨醛(0.20g,0.49mmol)，N-甲基哌嗪(0.17mL,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得白色固体(0.19g，78.6％)。
1H NMR(400MHz,CDCl 3)δ7.93(s,2H),7.84(s,1H),6.18(d,J＝2.2Hz,1H),6.15(d,J＝2.3Hz,1H),5.15(s,2H),3.80(s,6H),3.65(s,2H),2.51(d,J＝53.4Hz,8H),2.26(s,3H).
MS(ESI):m/z 493.18[M+H] +。
实施例36.化合物(35)的制备
Figure PCTCN2021073164-appb-000067
步骤1)将4-氟-3-三氟甲基溴苄(0.18mL,1.2mmol)，4-二氟甲氧基-3-羟基苯甲醛(0.19g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.29g，79.6％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H)；
MS(ESI):m/z 365.06[M+H] +；
步骤2)将4-二氟甲氧基-3-((4-氟-3-(三氟甲基)苄基)氧基)苯甲醛(0.18g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.14g,68.4％)。
1H NMR(400MHz,CDCl 3)δ7.95–7.92(m,2H),7.86(s,1H),7.17(d,J＝8.1Hz,1H),7.07(d,J＝1.9Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.53(t,J＝74.3Hz,1H),5.23(s,2H),3.80(s,2H),3.71–3.65(m,2H),2.82–2.79(m,2H).
MS(ESI):m/z 410.12[M+H] +。
实施例37.化合物(36)的制备
Figure PCTCN2021073164-appb-000068
步骤1)将3,5-双三氟甲基溴苄(0.22mL,1.2mmol)，2-羟基-4,6-二甲氧基苯甲醛(0.18g,1mmol)，碳酸钾(0.41g,3mmol)，DMF(10mL)依次加入100ml单口圆底烧瓶中，室温下反应6h，乙酸乙酯萃取，合并有机相，减压浓缩，然后硅胶柱纯化，得白色固体(0.34g，82.7％)。
1H NMR(400MHz,CDCl 3)δ10.42(s,1H),δ7.93(s,2H),7.84(s,1H),6.18(d,J＝2.2Hz,1H),6.15(d,J＝2.3Hz,1H),5.15(s,2H),
MS(ESI):m/z 409.09[M+H] +；
步骤2)将2-(3,5-双三氟甲基苄基氧基)-4,6-二甲氧基苯甲醛(0.20g,0.5mmol)，乙醇胺(0.09ml,1.5mmol)，乙酸(0.17mL,1.5mmol)，THF(10mL)依次加入100mL三口圆底烧瓶中，氮气保护，室温下反应1h后，加入三乙酰氧基硼氢化钠(0.33g,1.5mmol)继续反应24h,乙酸乙酯萃取，饱和碳酸氢钠溶液调节pH至弱碱性，合并有机相，硅胶柱层析纯化，得橙色固体(0.12g,55.1％)。
1H NMR(400MHz,Chloroform-d)δ7.94–7.92(m,2H),7.85(s,1H),6.18(d,J＝2.2Hz,1H),6.14(d,J＝2.2Hz,1H),5.16(s,2H),3.90(s,2H),3.83(s,3H),3.80(s,3H),3.39(d,J＝2.5Hz,2H),2.11(t,J＝2.4Hz,1H).
MS(ESI):m/z 448.13[M+H] +。
实施例38.敲除Usp25增强AD小鼠突触和认知功能
6～7月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠，进行学习记忆相关行为学测试，包括Y-迷宫、Morris水迷宫测试和条件惊恐测试。在实验开始前三天接触抚摸小鼠，每天一次，轻柔地抓住鼠尾拿起小鼠，让小鼠在手上停留30秒；实验当天，在实验前先将实验的小鼠转移到准备间，让小鼠适应30分钟。动物行为学实验于每天9:00a.m.-6:00p.m.之间进行，实验室内的光强度为650lux。采用Smart Video Tracking Software(Panlab,Harvard Apparatus)进行数据采集及分析处理。
Y-迷宫测试(Y-maze test)用于评价小鼠的自发性空间交替行为和工作记忆。将小鼠放置于Y-迷宫(长30cm，宽6cm，高15cm)中央，然后让小鼠在迷宫中自由探索5min。以小鼠的四肢都进入迷宫臂作为其进入迷宫臂的标准，以小鼠三次连续进入不同迷宫臂为一次正确的自主交替穿梭(Alternation)。
如图1A所示，与WT小鼠相比较，5×FAD小鼠在Y-迷宫中的自主交替穿梭百分比(Alternation％)显著减少，而敲除Usp25后，5×FAD；Usp25 +/–小鼠的自主交替穿梭百分比显著增加，表明敲除Usp25可以逆转AD小鼠的工作记忆。
Morris水迷宫测试(Morris water maze test)在一个圆形的水箱(直径120厘米)中进行，水箱中注水的高度以超过平台1厘米为宜，水箱内水的温度设定为22℃。迷宫臂内四个方向分别贴有四个不同形状的图标作为空间定位参照物。在训练实验中，平台在水面下1厘米，然后将小鼠从迷宫的两个入水点放入，让小鼠搜索平台60秒，以小鼠停留在平台上10秒为实验停止的标准。如果小鼠在60秒内寻找不到平台，将其引导到平台所在位置，并让其在平台上面停留10秒。每只小鼠每天测试2次，分别随机选择两个不同的方位入水，每只小鼠两次实验的间隔时间至少要1小时。记录小鼠每次实验找到平台的潜伏时间(Latency to target)。连续进行6天的学习训练。第7天撤掉平台，进行平台测试，将小鼠从平台对角线的位置鼠放入水中，让其在水迷宫中自由搜索60秒，记录小鼠在平台所在目标象限以及其他三个不同象限的游泳时间(Time in quadrant)。
如图1B所示，与WT、5×FAD；Usp25 +/–小鼠相比较，5×FAD小鼠在水迷宫的训练过程中表现出显著的学习能力缺陷。如图1C和图1D所示，平台测试中，与WT小鼠相比较，5×FAD小鼠在目标象限所待的时间较少，而敲除Usp25后，显著增加5×FAD；Usp25 +/–小鼠在目标象限的时间，综上提示敲除Usp25显著改善AD小鼠的空间学习记忆能力。
条件惊恐测试(Fear conditioning test)，第一天训练过程中，小鼠被放置在试验箱内，适应环境2分钟，随后对小鼠发出60分贝的噪音刺激(条件刺激)，持续30秒，并在噪音刺激的最后2秒给予小鼠0.05mA的电击刺激(非条件刺激)。重复三次，每次间隔60秒，在最后的电击刺激后，让小鼠在实验箱内停留90秒。训练后第1天上午，进行场景记忆测试(Contextual test)将小鼠放置在同一个试验箱内5分钟，记录小鼠僵直时间的百分比(Freezing％)，以测定场景记忆。训练后第1天下午，进行线索记忆测试(Cued test)，将小鼠放置在一个与之前训练环境不同的试验箱内(不同的墙壁、地面)，先让小鼠在正常环境中待3分钟，然后进行60分贝的声音刺激3分钟，分别记录小鼠在声音刺激下与在正常环境下出现僵直时间的百分比(Freezing％)。
如图1E所示，与WT小鼠相比较，5×FAD小鼠表现出严重的场景记忆缺陷,而敲除Usp25后，显著逆转5×FAD小鼠的场景记忆缺陷；如图1F所示，与WT小鼠相比较，5×FAD小鼠表现出严重的线索记忆缺陷，而敲除Usp25后，显著逆转5×FAD小鼠的线索记忆缺陷。
此外，通过高尔基染色分析神经元树突棘密度，如图1G所示，与WT小鼠相比较，5×FAD小鼠脑中神经元树突棘密度显著减少，而敲除Usp25显著增加5×FAD；Usp25 +/–小鼠脑中神经元树突棘密度。
对6～7月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠进行脑片电生理记录。小鼠经麻醉后，快速取出脑组织置于冰冷且通氧的人工脑脊液(ACSF)中冷却，随后转至振荡切片机进行冠状切片，脑片厚度为400μm。将脑片置于32℃氧饱和的ACSF中孵育1小时，之后转移至室温孵育1小时，将记录电极放置在Schaffer collateral-commissural通路的CA1区 辐射层，刺激电极放置在CA3区。刺激强度为兴奋性突触后场电位(field excitatory postsynaptic potential，fEPSP)最大值的30％，fEPSP基线稳定记录20分钟后，高频刺激(HFS)诱导长时程增强(Long-term potentiation，LTP)(2串刺激，每串刺激包含100个刺激脉冲，每串刺激间隔30秒)，持续记录60分钟。如图1I所示，相对于WT小鼠，5×FAD小鼠海马CA3区至CA1区Schaffer collateral-commissural通路的LTP显著受损，而敲除Usp25后显著增强5×FAD；Usp25 +/–小鼠的LTP，由此表明，敲除Usp25可以逆转AD小鼠的的突触功能障碍。
综上，表明敲除Usp25可以逆转AD小鼠的突触功能和认知缺陷。
实施例39.敲除Usp25增强DS小鼠突触和认知功能
6月龄WT、Usp25 +/–、Dp16、Dp16；Usp25 +/–小鼠，包括T-迷宫和Morris水迷宫测试。在实验开始前三天接触抚摸小鼠，每天一次，轻柔地抓住鼠尾拿起小鼠，让小鼠在手上停留30秒；实验当天，在实验前先将实验的小鼠转移到准备间，让小鼠适应30分钟。动物行为学实验于每天9:00a.m.-6:00p.m.之间进行，实验室内的光强度为650lux。采用Smart Video Tracking Software(Panlab,Harvard Apparatus)进行数据采集及分析处理。
T-迷宫测试(T-maze test)用于评价小鼠的自发性空间交替行为和工作记忆。将小鼠放置于T-迷宫(长30cm，宽6cm，高15cm)中央，然后让小鼠在迷宫中自由探索5min。以小鼠的四肢都进入迷宫臂作为其进入迷宫臂的标准，以小鼠三次连续进入不同迷宫臂为一次正确的自主交替穿梭(Alternation)。
如图2A所示，与WT小鼠相比较，Dp16小鼠在T-迷宫中的自主交替穿梭百分比(Alternation％)显著减少，而敲除Usp25后，Dp16；Usp25 +/–小鼠的自主交替穿梭百分比显著增加，表明敲除Usp25可以逆转DS小鼠的工作记忆。
Morris水迷宫测试(Morris water maze test)在一个圆形的水箱(直径120厘米)中进行，水箱中注水的高度以超过平台1厘米为宜，水箱内水的温度设定为22℃。迷宫臂内四个方向分别贴有四个不同形状的图标作为空间定位参照物。在训练实验中，平台在水面下1厘米，然后将小鼠从迷宫的两个入水点放入，让小鼠搜索平台60秒，以小鼠停留在平台上10秒为实验停止的标准。如果小鼠在60秒内寻找不到平台，将其引导到平台所在位置，并让其在平台上面停留10秒。每只小鼠每天测试2次，分别随机选择两个不同的方位入水，每只小鼠两次实验的间隔时间至少要1小时。记录小鼠每次实验找到平台的潜伏时间(Latency to target)。连续进行5天的学习训练。第6天撤掉平台，进行平台测试，将小鼠从平台对角线的位置鼠放入水中，让其在水迷宫中自由搜索60秒，记录小鼠穿梭平台所在象限的次数(Target crossings)。
如图2B所示，与WT小鼠相比较，Dp16小鼠在水迷宫的训练过程中并无表现出显著的学习能力缺陷。如图2C所示，平台测试中，与WT小鼠相比较，Dp16小鼠在目标象限的穿梭次数显著减少，而敲除Usp25后，显著增加Dp16；Usp25 +/–小鼠在目标象限的穿梭次数，综上提示敲除Usp25显著改善DS小鼠的空间学习记忆能力。
此外，通过高尔基染色分析神经元树突棘密度，如图2D所示，与WT小鼠相比较，Dp16小鼠脑中神经元树突棘密度显著减少，而敲除Usp25显著增加Dp16；Usp25 +/–小鼠脑中神经元树突棘密度。
对6月龄WT、Dp16、Dp16；Usp25 +/–小鼠进行脑片电生理记录。小鼠经麻醉后，快速取出脑组织置于冰冷且通氧的人工脑脊液(ACSF)中冷却，随后转至振荡切片机进行冠状切片，脑片厚度为400μm。将脑片置于32℃氧饱和的ACSF中孵育1小时，之后转移至室温孵育1小时，将记录电极放置在Schaffer collateral-commissural通路的CA1区辐射层，刺激电极放置在CA3区。刺激强度为兴奋性突触后场电位(field excitatory postsynaptic potential，fEPSP)最大值的30％，fEPSP基线稳定记录20分钟后，高频刺激(HFS)诱导长时程增强(Long-term potentiation，LTP)(2串刺激，每串刺激包含100个刺激脉冲，每串刺激间隔30秒)，持续记录60分钟。如图2E-F所示，相对于WT小鼠，Dp16小鼠海马CA3区至CA1区Schaffer collateral-commissural通路的LTP显著受损，而敲除Usp25后显著增强Dp16；Usp25 +/–小鼠的LTP，由此表明，敲除Usp25可以逆转DS小鼠的的突触功能障碍。
综上，表明敲除Usp25可以逆转DS小鼠的突触功能和认知缺陷。
实施例40.敲除Usp25改善小鼠小胶质细胞稳态
6～7月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织。脑组织于4℃用4％多聚甲醛固定过夜，并经25％和30％蔗糖溶液顺序脱水，再使用OCT进行脑组织包埋，切片后经柠檬酸钠缓冲液抗原修复，使用含0.2％Triton X-100的3％BSA缓冲液进行封闭，然后进行免疫荧光染色分别标记小胶质细胞标记蛋白Iba1(Wako公司)以及细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)(sigma公司)，然后通过激光共聚焦荧光显微镜进行图像采集。如图3A-C所示，相较于WT小鼠，5×FAD小鼠海马区和大脑皮层小胶质细胞数目显著增加，而敲除Usp25后显著减少5×FAD小鼠脑中小胶质细胞增生。将免疫荧光染色图片经Imaris软件进行三维重构，如图3D-F和图3H-J所示，敲除Usp25后显著减少5×FAD小鼠海马区和大脑皮层的小胶质细胞胞体面积，增加小胶质细胞的分支长度，提示小胶质细胞激活被抑制。此外，如图3D、3G、3H和3K所示，敲除Usp25后显著减少5×FAD小鼠脑中小胶质细胞对神经元突触的吞噬作用。综上，表明敲除Usp25显著抑制AD小鼠脑中小胶质细胞增殖和激活以及小胶质细胞介导的突触吞噬。
实施例41.敲除Usp25抑制小鼠小胶质细胞炎症因子释放和突触吞噬
10月龄WT、Usp25 +/–、5×FAD、5×FAD；Usp25 +/–小鼠，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织，于液氮速冻，然后保存于-80℃冰箱。通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il6和Tnf的转录水平。如图4A和图4B所示，相较于WT小鼠，5×FAD 小鼠脑中促炎因子Il6和Tnf表达水平显著上调，而敲除Usp25后，显著减少5×FAD小鼠脑中Il6和Tnf表达，表明敲除Usp25可以抑制AD小鼠脑中炎症反应。
分离Usp25 +/+、Usp25 –/–小鼠出生后第0天新生鼠小胶质细胞，使用含25ng/mL GM-CSF+10％胎牛血清的DMEM培养基培养10天后，以220rpm转速摇晃15分钟分离小胶质细胞，培养1天后，分别处理10μMAZ1或对照溶剂DMSO，与此同时处理10μM oAβ 42或对照溶剂Vehicle和pHrodo-Red标记的突触小体(Syn)，24小时后，通过4％多聚甲醛固定细胞，0.2％Triton X-100溶液穿透，3％BSA溶液封闭，然后通过免疫荧光标记小胶质细胞标记蛋白Iba1，分析Iba1 +小胶质细胞内pHrodo-Red的荧光强度。如图4C和图4D所示，oAβ 42处理可以诱导小胶质细胞吞噬pHrodo-Red标记的突触小体，而敲除Usp25可以减少oAβ 42诱导小胶质细胞对突触小体的吞噬；此外，AZ1处理也可以减少oAβ 42诱导小胶质细胞对突触小体的吞噬；然后Usp25 –/–小胶质细胞中，AZ1处理并不能再减少小胶质细胞对突触小体的吞噬，表明AZ1对小胶质细胞吞噬突触的抑制作用依赖于USP25。
实施例42.AZ1透过血脑屏障实验
对ICR小鼠灌胃给药后，于不同时间点同时采集脑组织和血液样品，研究AZ1在ICR小鼠体内血脑屏障特性。
灌胃给药AZ1溶液配制：准确称量AZ1约4mg至玻璃瓶中，加入适量体积的0.5％CMC-Na水溶液(1％Tween-80)，采用1M HCl调节pH至3～4，涡旋振荡后超声至溶解，得终浓度为1mg/mL的给药制剂。
取9只27.8～33.3g ICR雄性小鼠，分为三组(不同给药时间0.5、2、8小时)，以10mg/kg剂量经灌胃给药实验动物，然后分别于给药后0.5、2、8小时采用二氧化碳麻醉，心脏采血(约0.2mL)处死。全血置于含抗凝剂EDTA-K2的试管中，存放于湿冰上，并于1小时之内，离心(1500～1600g)10分钟，分离血浆。ICR小鼠处死后，摘取脑组织，用冰生理盐水清洗干净，吸去水分后，称重，按照重量-体积比1:5(组织：匀浆液)的比例加入20％甲醇-水，进行匀浆。血浆和脑组织匀浆液保存在-40～-20℃的冰箱，以供样品分析。
由苏州圣苏新药开发有限公司采用LC-MS/MS(API 4000:LC-MS-MS-010)方法分别对给药后ICR小鼠血浆和脑中AZ1的浓度进行测定。如表1所示，给药后0.5、2、8小时后，血浆中AZ1的水平由68.8±11.9ng/mL依次递减为24.8±5.0ng/mL和3.9±1.1ng/mL，脑中AZ1的水平由559.2±138.3ng/g依次递减至388.2±49.0ng/g和80.8±1.2ng/g。综上表明AZ1可以透过小鼠血脑屏障。
表1.小鼠灌胃给药(10mg/kg)后脑内AZ1的渗透率
Figure PCTCN2021073164-appb-000069
实施例43.给药AZ1逆转AD小鼠突触和认知功能缺陷
7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组(图5A)。如图5B所示，AZ1给药并不影响小鼠的体重。
连续给药4周后，进行学习记忆相关行为学测试，包括Morris水迷宫测试和条件惊恐测试。在实验开始前三天接触抚摸小鼠，每天一次，轻柔地抓住鼠尾拿起小鼠，让小鼠在手上停留30秒；实验当天，在实验前先将实验的小鼠转移到准备间，让小鼠适应30分钟。动物行为学实验于每天9:00a.m.-6:00p.m.之间进行，实验室内的光强度为650lux。采用Smart Video Tracking Software(Panlab,Harvard Apparatus)进行数据采集及分析处理。
Morris水迷宫测试(Morris water maze test)在一个圆形的水箱(直径120厘米)中进行，水箱中注水的高度以超过平台1厘米为宜，水箱内水的温度设定为22℃。迷宫臂内四个方向分别贴有四个不同形状的图标作为空间定位参照物。在训练实验中，平台在水面下1厘米，然后将小鼠从迷宫的两个入水点放入，让小鼠搜索平台60秒，以小鼠停留在平台上10秒为实验停止的标准。如果小鼠在60秒内寻找不到平台，将其引导到平台所在位置，并让其在平台上面停留10秒。每只小鼠每天测试2次，分别随机选择两个不同的方位入水，每只小鼠两次实验的间隔时间至少要1小时。记录小鼠每次实验找到平台的潜伏时间(Latency to target)。连续进行6天的学习训练。第7天撤掉平台，进行平台测试，将小鼠从平台对角线的位置鼠放入水中，让其在水迷宫中自由搜索60秒，记录小鼠在平台所在目标象限以及其他三个不同象限的游泳时间(Time in quadrant)以及小鼠第一次到达平台区域的潜伏时间(Latency 1 st entrance to target)。
如图5C所示，与WT+Vehicle小鼠相比较，5×FAD+Vehicle小鼠在水迷宫的训练过程中并无表现出显著的学习能力缺陷。如图5D和图5E所示，平台测试中，与WT+Vehicle小鼠相比较，5×FAD+Vehicle小鼠在目标象限所待的时间较少，而AZ1给药后显著增加5×FAD小鼠在目标象限的时间；此外，如图5F所示，AZ1给药后显著减少5×FAD小鼠第一次到达平台位置的潜伏时间，综上提示AZ1给药显著改善AD小鼠的空间学习记忆能力。
条件惊恐测试(Fear conditioning test)，第一天训练过程中，小鼠被放置在试验箱内，适应环境2分钟，随后对小鼠发出60分贝的噪音刺激(条件刺激)，持续30秒，并在噪音刺激的最后2秒给予小鼠0.05mA的电击刺激(非条件刺激)。重复三次，每次间隔60秒，在最后的电击刺激后，让小鼠在实验箱内停留90秒。训练后第1天，进行线索记忆测试(Cued test)，将小鼠放置在一个与之前训练环境不同的试验箱内(不同的墙壁、地面)，先让小鼠在正常环境中待3分钟，然后进行60分贝的声音刺激3分钟，分别记录小鼠在声音刺激下与在正常环境下出现僵直时间的百分比(Freezing％)。
如图5G所示，与WT+Vehicle小鼠相比较，5×FAD+Vehicle小鼠表现出严重的线索记忆缺陷，而AZ1给药后可以显著逆转5×FAD小鼠的线索记忆缺陷。
综上，表明AZ1给药可以逆转AD小鼠的认知功能缺陷。
5月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。
连续给药4周后进行小鼠脑片电生理记录。小鼠经麻醉后，快速取出脑组织置于冰冷且通氧的人工脑脊液(ACSF)中冷却，随后转至振荡切片机进行冠状切片，脑片厚度为400μm。将脑片置于32℃氧饱和的ACSF中孵育1小时，之后转移至室温孵育1小时，将记录电极放置在Schaffer collateral-commissural通路的CA1区辐射层，刺激电极放置在CA3区。刺激强度为兴奋性突触后场电位(field excitatory postsynaptic potential，fEPSP)最大值的30％，fEPSP基线稳定记录20分钟后，高频刺激(HFS)诱导长时程增强(Long-term potentiation，LTP)(2串刺激，每串刺激包含100个刺激脉冲，每串刺激间隔30秒)，持续记录60分钟。如图5H和5I所示，相对于WT+Vehicle小鼠，5×FAD+Vehicle小鼠海马CA3区至CA1区Schaffer collateral-commissural通路的LTP显著受损，而AZ1给药后显著增强5×FAD小鼠的LTP，由此表明AZ1给药可以逆转AD小鼠的突触功能障碍。
实施例44.给药AZ1逆转DS小鼠认知功能缺陷
5月龄WT和Dp16雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，Dp16+Vehicle为Dp16小鼠腹腔注射对照溶剂，Dp16+AZ1为Dp16小鼠腹腔注射AZ1实验组(图6A)。如图6B所示，AZ1给药并不影响小鼠的体重。
连续给药4周后，进行行为学测试，包括旷场测试、T-迷宫测试、新物体识别和Morris水迷宫测试。在实验开始前三天接触抚摸小鼠，每天一次，轻柔地抓住鼠尾拿起小鼠，让小鼠在手上停留30秒；实验当天，在实验前先将实验的小鼠转移到准备间，让小鼠适应30分钟。动物行为学实验于每天9:00a.m.-6:00p.m.之间进行，实验室内的光强度为650lux。采用Smart Video Tracking Software(Panlab,Harvard Apparatus)进行数据采集及分析处理。
旷场实验(Open field test)用于评价小鼠的运动能力和焦虑样情绪。将小鼠放置于旷场中央，然后让小鼠在迷宫中自由探索10min，分别记录小鼠在旷场中的运动距离(Total distance)以及在旷场中央区域的活动时间(Time in center)。
如图6C和6D所示，给药AZ1并不影响小鼠在旷场中的运动距离以及在旷场中央区域的活动时间，表明AZ1给药对小鼠无显著毒理作用。
新物体识别(Novel object recognition test)基于小鼠具有探索新事物的先天性趋向，探索新事物的过程反应了小鼠学习、识别、记忆的过程，可评估小鼠的工作记忆。第一天，让小鼠在单独的旷场内自由探索5min以熟悉环境；第二天，在旷场内放置两个物体A和B，让小鼠自由探索10min；第三天，将其中的一个物体A换为新的物体C，让小鼠自由探索10min。记录小鼠探索B和C的次数，Recognition index＝C/(B+C)。
如图6E所示，与WT+Vehicle小鼠相比较，Dp16+Vehicle小鼠在新物体的探索次数显 著减少，而给药AZ1可以显著增加Dp16小鼠对新物体的探索，表明给药AZ1可以增强Dp16小鼠的工作记忆。
Morris水迷宫测试(Morris water maze test)在一个圆形的水箱(直径120厘米)中进行，水箱中注水的高度以超过平台1厘米为宜，水箱内水的温度设定为22℃。迷宫臂内四个方向分别贴有四个不同形状的图标作为空间定位参照物。在训练实验中，平台在水面下1厘米，然后将小鼠从迷宫的两个入水点放入，让小鼠搜索平台60秒，以小鼠停留在平台上10秒为实验停止的标准。如果小鼠在60秒内寻找不到平台，将其引导到平台所在位置，并让其在平台上面停留10秒。每只小鼠每天测试2次，分别随机选择两个不同的方位入水，每只小鼠两次实验的间隔时间至少要1小时。记录小鼠每次实验找到平台的潜伏时间(Latency to target)。连续进行5天的学习训练。第6天撤掉平台，进行平台测试，将小鼠从平台对角线的位置鼠放入水中，让其在水迷宫中自由搜索60秒，记录小鼠在平台所在目标象限以及其他三个不同象限的游泳时间(Time in quadrant)以及小鼠穿梭平台所在象限的次数(Target crossings)。
如图6F所示，与WT+Vehicle小鼠相比较，Dp16+Vehicle小鼠在水迷宫的训练过程中并无表现出显著的学习能力缺陷。如图6G所示，平台测试中，与WT+Vehicle小鼠相比较，Dp16+Vehicle小鼠在目标象限所待的时间较少，而AZ1给药后显著增加Dp16小鼠在目标象限的时间；此外，如图6H所示，AZ1给药后显著增加Dp16小鼠在平台所在象限的穿梭次数，综上提示AZ1给药显著改善DS小鼠的空间学习记忆能力。
实施例45.给药AZ1无显著毒理作用
7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。
连续给药4周后，进行学习相关行为学测试，测试结束后再给药1周，然后采用5％水合氯醛麻醉，摘眼球取血，4℃静置30分钟以上，3000rpm 4℃离心5分钟，取血清，然后通过全自动生化仪(迈瑞公司，BS-240vet)进行血液生化检测。其中，肝功能包括ALT(丙氨酸氨基转移酶)、AST(天门冬氨酸氨基转移酶)、ALP(碱性磷酸酶)、TP(总蛋白)、ALB(白蛋白)，肾功能包括Urea(尿素)、CREA-S(肌酐)，血脂包括TC(总胆固醇)、TG(甘油三酯)，心肌酶谱CK(肌酸激酶)，血糖Glu-G。如图7所示，AZ1给药后，小鼠没有表现出明显的肝功能、肾功能、心肌功能和血糖血脂异常，提示AZ1对于小鼠无显著毒理作用。
实施例46.给药AZ1抑制AD小鼠大脑神经炎症反应
7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射 AZ1实验组。
连续给药4周后，进行学习记忆相关行为学测试，测试结束后测试结束后再给药1周，然后采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织，于液氮速冻，然后保存于-80℃冰箱。通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b和Il6的转录水平。如图8所示，相较于WT+Vehicle小鼠，5×FAD+Vehicle小鼠脑中促炎因子Il1b和Il6表达水平显著上调，而AZ1给药后，显著减少5×FAD小鼠脑中Il1b和Il6表达，表明AZ1给药可以抑制AD小鼠脑中炎症反应。
实施例47.给药AZ1抑制AD小鼠脑中小胶质细胞增殖和激活
7月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量腹腔注射AZ1或对照溶剂(5％DMSO+95％玉米油)，其中，WT+Vehicle为同窝对照野生型小鼠腹腔注射对照溶剂，5×FAD+Vehicle为5×FAD小鼠腹腔注射对照溶剂，5×FAD+AZ1为5×FAD小鼠腹腔注射AZ1实验组。连续给药4周后，进行学习记忆相关行为学测试，测试结束后测试结束后再给药1周，然后采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织。脑组织于4℃用4％多聚甲醛固定过夜，并经25％和30％蔗糖溶液顺序脱水，再使用OCT进行脑组织包埋，切片后经柠檬酸钠缓冲液抗原修复，使用含0.2％Triton X-100的3％BSA缓冲液进行封闭，然后进行免疫荧光染色分别标记小胶质细胞标记蛋白Iba1(Wako公司)以及细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)(sigma公司)，然后通过激光共聚焦荧光显微镜进行图像采集。如图9A-D所示，相较于WT+Vehicle小鼠，5×FAD+Vehicle小鼠大脑皮层和海马CA1、DG区小胶质细胞数目显著增加，而给药AZ1后显著减少5×FAD小鼠脑中小胶质细胞增生。将免疫荧光染色图片经Imaris软件进行三维重构，如图9E-I所示，给药AZ1后显著增加5×FAD小鼠脑中小胶质细胞的分支数目和分支长度，提示小胶质细胞激活被抑制。综上，表明给药AZ1显著抑制AD小鼠脑中小胶质细胞增殖和激活。
实施例48.给药AZ1抑制脂多糖诱导的小胶质细胞炎症反应
分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，使用含25ng/mL GM-CSF+10％胎牛血清的DMEM培养基培养10天后，以220rpm转速摇晃15分钟分离小胶质细胞，培养1天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ1处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ1为50ng/mL LPS和10μM AZ1同时处理实验组。
处理6小时后，通过4％多聚甲醛固定细胞，0.2％Triton X-100溶液穿透，3％BSA溶液封闭，然后通过免疫荧光标记小胶质细胞标记蛋白Iba1，分析AZ1处理对LPS应激下小胶质细胞激活的影响。如图10A和图10B所示，LPS处理后小胶质细胞胞体面积显著增加， 提示小胶质细胞激活，而AZ1处理可以减少小胶质细胞面积，从而抑制LPS激活小胶质细胞。
处理6小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b和Il6的转录水平。如图10C和图10D所示，LPS处理后显著增加促炎因子Il1b和Il6的表达，而AZ1处理后可以减少LPS诱导的Il1b和Il6表达，从而抑制LPS诱导的炎症反应。
综上，表明AZ1可以抑制LPS诱导的小胶质细胞激活和炎症反应。
实施例49.给药AZ2抑制AD小鼠大脑神经炎症反应
6月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量灌胃给药AZ2或对照溶剂(含1％Tween-80和0.5％CMC-Na水溶液，pH 3～4)，其中，WT+Vehicle为同窝对照野生型小鼠灌胃给药对照溶剂，5×FAD+Vehicle为5×FAD小鼠灌胃给药对照溶剂，5×FAD+AZ2为5×FAD小鼠灌胃给药AZ2实验组。
连续给药4周后，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织，于液氮速冻，然后保存于-80℃冰箱。通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b和Il6的转录水平。如图11所示，相较于WT+Vehicle小鼠，5×FAD+Vehicle小鼠脑中促炎因子Il1b和Il6表达水平显著上调，而AZ2给药后，显著减少5×FAD小鼠脑中Il1b表达，但是AZ2给药对于Il6表达没有显著影响。综上，表明AZ2给药可以抑制AD小鼠脑中炎症反应。
实施例50.给药AZ2抑制AD小鼠脑中小胶质细胞增殖和激活
6月龄WT和5×FAD雄性小鼠，分别以20mg/kg剂量灌胃给药AZ2或对照溶剂(含1％Tween-80和0.5％CMC-Na水溶液，pH 3～4)，其中，WT+Vehicle为同窝对照野生型小鼠灌胃给药对照溶剂，5×FAD+Vehicle为5×FAD小鼠灌胃给药对照溶剂，5×FAD+AZ2为5×FAD小鼠灌胃给药AZ2实验组(图12A)。如图12B所示，AZ2给药并不影响小鼠的体重。
连续给药4周后，采用5％水合氯醛麻醉小鼠，然后使用磷酸盐缓冲液进行灌注，取脑组织。脑组织于4℃用4％多聚甲醛固定过夜，并经25％和30％蔗糖溶液顺序脱水，再使用OCT进行脑组织包埋，切片后经柠檬酸钠缓冲液抗原修复，使用含0.2％Triton X-100的3％BSA缓冲液进行封闭，然后进行免疫荧光染色分别标记小胶质细胞标记蛋白Iba1(Wako公司)和细胞核染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)(sigma公司)，然后通过激光共聚焦荧光显微镜进行图像采集。如图12C-F所示，相较于WT+Vehicle小鼠，5×FAD+Vehicle小鼠大脑皮层和海马CA1、DG区小胶质细胞数目显著增加，而给药AZ2后显著减少5×FAD小鼠脑中小胶质细胞增生。将免疫荧光染色图片经Imaris软件进行三维重 构，如图12G-K所示，给药AZ2后显著增加5×FAD小鼠大脑皮层和海马区小胶质细胞的分支数目和分支长度，提示小胶质细胞激活被抑制。综上，表明给药AZ2显著抑制AD小鼠脑中小胶质细胞增殖和激活。
实施例51.给药AZ2抑制脂多糖诱导的小胶质细胞炎症反应
分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，使用含25ng/mL GM-CSF+10％胎牛血清的DMEM培养基培养10天后，以220rpm转速摇晃15分钟分离小胶质细胞，培养1天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ2处理，其中，Control为对照组，AZ2为10μM AZ2处理对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ2为50ng/mL LPS和10μM AZ2同时处理实验组。
处理6小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b和Il6的转录水平。如图13所示，LPS处理后显著增加促炎因子Il1b和Il6的表达，而AZ2处理后可以显著减少LPS诱导的Il1b和Il6表达，从而抑制LPS诱导的炎症反应。
实施例52.USP25抑制剂筛选
通过Ni-NTA Agarose(Qiagen公司，货号1018244)纯化大肠杆菌E.coli BL21(DE3)表达的His-USP25 a.a.157-706重组蛋白(USP25 a.a.157-706为USP25去泛素化酶催化结构域)，基于C-端偶联有荧光染料Rhodamine 110的Ubiquitin-Rhodamine 110(R&D System公司，货号U-555-050)筛选USP25抑制剂，工作缓冲液为50mM HEPES(pH 7.4)，0.5mM EDTA，1mM tris-(2-Carboxyethyl)phosphine(TCEP)，1mg/ml bovine serum albumin(BSA)。于384孔板(Corning公司，货号3570)中，加6.25μl 33.3μM化合物(18.75μl体系中终浓度为10μM，DMSO终浓度为0.1％)与6.25μl 100nM His-USP25 157-706重组蛋白(18.75μl体系中终浓度为33.3nM)，150g短暂离心30秒后，于室温孵育20分钟；然后加入6.25μl 400nM Ubiquitin-Rhodamine 110(18.75μl体系中终浓度为133.3nM)，150g短暂离心30秒后，于室温孵育30分钟；最后加6.25μl 100mM柠檬酸(25μl体系中终浓度为25mM)终止反应后，150g短暂离心30秒后，于Tecan Spark多功能酶标仪(瑞士Tecan公司)进行Ubiquitin-Rhodamine 110荧光强度检测，设置激发波长为485nm，发射波长为520nm。其中，设置(缓冲液+Ubiquitin-Rhodamine 110+柠檬酸)为阴性对照组，Ctrl为对照溶液处理组(His-USP25 a.a.157-706重组蛋白+对照溶剂+Ubiquitin-Rhodamine 110+柠檬酸)，所有实验组和Ctrl组荧光强度值均扣除阴性对照组的荧光强度值。如图14所示，MZ77、MZ76、MZ1、MZ30、AZ2、MZ32、AZ1、MZ67、MZ66、MZ75、MZ31、MZ5、XMU1、MZ71、MZ34、MZ33、MZ74、MZ68、MZ29、MZ72、MZ41、MZ43、MZ38、MZ24、MZ40、MZ25、MZ69、MZ42、MZ35、MZ36、MZ26、MZ37、MZ39、MZ23、MZ3、MZ27、MZ28、MZ4可以显著抑制USP25的去泛素化酶活性。
实施例53.给药XMU1抑制脂多糖诱导的小胶质细胞炎症反应
分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，使用含25ng/mL GM-CSF+10％胎牛血清的DMEM培养基培养10天后，以220rpm转速摇晃15分钟分离小胶质细胞，培养1天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM XMU1处理，其中，Control为对照组，XMU1为10μM XMU1处理对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+XMU1为50ng/mL LPS和10μM XMU1同时处理实验组。
处理6小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b的转录水平。如图15所示，LPS处理后显著增加促炎因子Il1b的表达，而XMU1处理后可以显著减少LPS诱导的Il1b表达，由此表明XMU1给药可以抑制LPS诱导的炎症反应。
实施例54.给药AZ1衍生物MZ3抑制脂多糖诱导的小胶质细胞系BV2炎症反应
小鼠小胶质细胞系BV2中，分别通过25ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ3处理，其中，Control为对照组，LPS+Vehicle为25ng/mL LPS处理实验组，LPS+MZ3为25ng/mL LPS和10μM MZ3同时处理实验组。
处理24小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b的转录水平。如图16所示，LPS处理后显著增加促炎因子Il1b的表达，而MZ3处理后可以显著减少LPS诱导的Il1b表达，从而抑制LPS诱导的炎症反应。
实施例55.给药AZ1衍生物MZ23和MZ34抑制脂多糖诱导的小胶质细胞系BV2炎症反应
小鼠小胶质细胞系BV2中，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ2或MZ3处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+MZ23为50ng/mL LPS和10μM MZ23同时处理实验组，LPS+MZ34为50ng/mL LPS和10μM MZ34同时处理实验组。
处理12小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b的转录水平。如图17所示，LPS处理后显著增加促炎因子Il1b的表达，而MZ23和MZ34处理后可以显著减少LPS诱导的Il1b表达，从而抑制LPS诱导的炎症反应。
实施例56.给药AZ1衍生物MZ24、MZ25、MZ26、MZ28、MZ29和MZ31抑制脂 多糖诱导的小胶质细胞系BV2炎症反应
小鼠小胶质细胞系BV2中，分别通过100ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM MZ24、MZ25、MZ26、MZ28、MZ29和MZ31处理，其中，Control为对照组，LPS+Vehicle为100ng/mL LPS处理实验组，LPS+MZ24为100ng/mL LPS和10μM MZ24同时处理实验组，LPS+MZ25为100ng/mL LPS和10μM MZ25同时处理实验组，LPS+MZ26为100ng/mL LPS和10μM MZ26同时处理实验组，LPS+MZ28为100ng/mL LPS和10μM MZ28同时处理实验组，LPS+MZ29为100ng/mL LPS和10μM MZ29同时处理实验组，LPS+MZ31为100ng/mL LPS和10μM MZ31同时处理实验组。
处理12小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b的转录水平。如图18所示，LPS处理后显著增加促炎因子Il1b的表达，而MZ24、MZ25、MZ26、MZ28、MZ29和MZ31处理后可以显著减少LPS诱导的Il1b表达，从而抑制LPS诱导的炎症反应。
实施例57.给药AZ1衍生物MZ75、MZ76和MZ77抑制脂多糖诱导的小胶质细胞炎症反应
分离C57BL/6小鼠出生后第0天新生鼠小胶质细胞，使用含25ng/mL GM-CSF+10％胎牛血清的DMEM培养基培养10天后，以220rpm转速摇晃15分钟分离小胶质细胞，培养1天后，分别通过50ng/mL脂多糖(Lipopolysaccharide，LPS)和10μM AZ1、MZ75、MZ76和MZ77处理，其中，Control为对照组，LPS+Vehicle为50ng/mL LPS处理实验组，LPS+AZ1或LPS+MZ75或LPS+MZ76或LPS+MZ77为50ng/mL LPS和10μM AZ1或MZ75或MZ76或MZ77同时处理实验组。
处理6小时后，通过TRIzol(Thermo Fisher Scientific公司)提取RNA，然后使用Rever Tra Ace qPCR RT Kit(TOYOBO公司)进行逆转录，使用FastStart Universal SYBR Green Master(Roche公司)进行实时荧光定量PCR检测炎症相关基因Il1b的转录水平。如图19所示，LPS处理后显著增加促炎因子Il1b的表达，而AZ1、MZ75、MZ76和MZ77处理后则可以显著减少LPS诱导的Il1b表达，从而抑制LPS诱导的炎症反应。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐在制备预防、治疗或改善阿尔茨海默病或唐氏综合征的药物中的用途，
    Figure PCTCN2021073164-appb-100001
    其中：
    X独立选自O或NH；
    A环和B环各自独立为苯环；
    R 1独立地选自：氢、氘、卤素、氰基、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 1-C 6)烷氧基；
    m为0、1、2、3、4或5；
    R 2独立地选自：氢、卤素、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C2-C6)烯基、取代或未取代的(C2-C6)炔基、取代或未取代的(C 1-C 6)烷氧基；
    n为1或2；
    R 3为取代或未取代的(C 1-C 6)醛基、
    Figure PCTCN2021073164-appb-100002
    其中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C2-C6)烯基、取代或未取代的(C2-C6)炔基、
    Figure PCTCN2021073164-appb-100003
    其中p为1-3的整数(优选地，p为2)。
  2. 如权利要求1所述的用途，其特征在于，所述R 3在苯环上的取代位置是邻位或间位；优选邻位。
  3. 如权利要求2所述的用途，其特征在于，R 1独立地选自：氢、氟、氯、溴、(C 1-C 3)烷基、卤代(C 1-C 3)烷基、卤代(C 1-C 3)烷氧基；
    m为0、1、2或3；
    R 2独立地选自：氢、卤素；
    n为1或2；
    R 3为
    Figure PCTCN2021073164-appb-100004
    其中，各R 3a、R 3b独立地选自：氢、羟基取代的(C 1-C 4)烷基、
    Figure PCTCN2021073164-appb-100005
    其中p为 1-3的整数(优选地，p为2)。
  4. 如权利要求1-3中任一项所述的用途，其特征在于，式I所示化合物选自以下化合物：
    Figure PCTCN2021073164-appb-100006
    Figure PCTCN2021073164-appb-100007
    Figure PCTCN2021073164-appb-100008
    Figure PCTCN2021073164-appb-100009
    Figure PCTCN2021073164-appb-100010
    Figure PCTCN2021073164-appb-100011
  5. 如权利要求4所述的用途，其特征在于，式I所示化合物为AZ1、AZ2、MZ77、MZ76、MZ1、MZ30、MZ32、MZ67、MZ66、MZ75、MZ31、MZ34、MZ74、MZ68、MZ29、MZ72、MZ38或MZ24。
  6. 如权利要求1-5中任一项所述的用途，其特征在于，所述预防、治疗或改善阿尔茨海默病或唐氏综合征是指预防、治疗或改善阿尔茨海默病或唐氏综合征所致认知功能损伤。
  7. 式I所示化合物、其各种晶型、水合物、溶剂合物或药学上可接受的盐，
    Figure PCTCN2021073164-appb-100012
    其中：
    X独立选自O或NH；
    A环和B环各自独立为苯环；
    R 1独立地选自：氢、氘、卤素、氰基、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C 1-C 6)烷氧基；
    m为0、1、2、3、4或5；
    R 2独立地选自：氢、卤素、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C2-C6)烯基、取代或未取代的(C2-C6)炔基、取代或未取代的(C 1-C 6)烷氧基；
    n为1或2；
    R 3为取代或未取代的(C 1-C 6)醛基、
    Figure PCTCN2021073164-appb-100013
    其中，各R 3a、R 3b、R 3c独立地选自：氢、取代或未取代的(C 1-C 6)烷基、取代或未取代的(C2-C6)烯基、取代或未取代的(C2-C6)炔基、
    Figure PCTCN2021073164-appb-100014
    其中p为1-3的整数(优 选地，p为2)；
    其中，式I化合物不包括
    Figure PCTCN2021073164-appb-100015
  8. 如权利要求7所述的化合物，其特征在于，所述R 3在苯环上的取代位置是邻位或间位；优选邻位。
  9. 如权利要求7或8所述的化合物，其特征在于，式I所示化合物选自以下化合物：
    Figure PCTCN2021073164-appb-100016
    Figure PCTCN2021073164-appb-100017
    Figure PCTCN2021073164-appb-100018
    Figure PCTCN2021073164-appb-100019
    Figure PCTCN2021073164-appb-100020
    Figure PCTCN2021073164-appb-100021
  10. 如权利要求9所述的化合物，其特征在于，式I所示化合物为MZ77、MZ76、MZ1、MZ30、MZ32、MZ67、MZ66、MZ75、MZ31、MZ34、MZ74、MZ68、MZ29、MZ72、MZ38或MZ24。
  11. 一种药物组合物，所述药物组合物包含权利要求6-10中任一项所述的式I所示化合物、其各种晶型、水合物或溶剂合物以及任选的药学上可接受的赋形剂。
  12. 21号染色体编码表达的泛素特异性蛋白酶USP25作为预防、治疗或改善阿尔茨海默病或唐氏综合征的药物靶点，或在筛选预防、治疗或改善阿尔茨海默病或唐氏综合征的药物中的用途。
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