Drug for preventing and / or treating alzheimer's disease

Abstract

A drug for preventing and / or treating Alzheimer's disease (AD). A provided CF3CN derivative has a structural formula as shown in any one of the following formulas 1-4. Four CF3CN derivatives all have TrkB agonist activity, wherein the CF3CN derivative shown in formula 2 is used as an optimal derivative, and in vivo PK research shows that said CF3CN derivative improves the B / P ratio of CF3CN, and overcomes the defect of CF3CN. Zein and lactoferrin are used to encapsulate a CF3CN derivative to prepare nano particles, such that the oral bioavailability and the concentration of a drug entering the brain can be further promoted, and the curative effect of treating AD is finally promoted. A formulation and an administration mode are further improved, and CF3CN derivatives are coated with a liposome for oral administration and nasal administration, thereby solving the problems of low bioavailability of derivatives and low concentration of drugs entering the brain.

Description

Drugs for the prevention and / or treatment of Alzheimer's disease Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to drugs for preventing and / or treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a globally prevalent neurodegenerative disease, commonly known as senile dementia. The hallmark pathological features of AD include the deposition of extracellular β-amyloid (Aβ) and the hyperphosphorylation and cleavage of microtubule-associated protein (Tau) in neurons, leading to neurofibrillary tangles. These pathological features are accompanied by progressive memory and cognitive dysfunction. Currently, there are only five drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of AD, which are divided into acetylcholinesterase inhibitors (4 types) and glutamate receptor antagonists (memantine), but these drugs can usually only temporarily relieve symptoms and cannot cure or delay the progression of the disease. Brain-derived neurotrophic factor (BDNF) is one of the main members of the neurotrophic factor family and is the most abundant neurotrophic factor in the brain. BDNF primarily exerts its biological functions by specifically binding to its high-affinity tyrosine kinase receptor B (TrkB) and activating tyrosine protein kinases, which in turn activate three major downstream signaling pathways: PI3K / Akt, Ras / ERK, and PLCγ1 / PKC. BDNF promotes the survival, growth, and differentiation of developing neurons, regulates synaptic transmission and synaptic plasticity in multiple brain regions of the adult central nervous system, and improves cognitive function impaired by memory retrieval. BDNF mRNA and protein levels are severely reduced in multiple brain regions, including the hippocampus, temporal lobe, and frontal cortex, in patients with AD. Upregulating BDNF through the ERK / CREB signaling pathway can ameliorate Aβ-induced neuronal loss and dendritic atrophy. BDNF stimulation of neurons leads to tau dephosphorylation by activating the TrkB and PI3K signaling pathways. Therefore, the BDNF / TrkB signaling pathway may serve as a potential drug target for the treatment of AD.

[0003] In order to find a small molecule TrkB agonist, the applicant previously discovered through high-throughput screening of the compound library that 7,8-dihydroxyflavone (7,8-DHF) is a specific TrkB receptor agonist that can mimic the physiological function of BDNF in vivo. However, since 7,8-DHF is a flavonoid compound, it is easily metabolized in the body in phase II, resulting in low oral bioavailability (about 4.6%). Therefore, the applicant optimized the lead compound by chemical structure modification and synthesized CF3CN, a derivative of 7,8-DHF (Chen, C.; Ahn, EH; Liu, X.; Wang, Z.-H.; Luo, S.; Liao, J.; Ye, K. Optimized TrkB agonist ameliorates Alzheimer's disease pathologies and improves cognitive functions via inhibiting delta-secretase. ACS Chemical Neuroscience 2021, 12 (13), 2448-2461.). In vitro experiments have confirmed that CF3CN can tightly bind to the LCM / CC2 motif on the extracellular domain of TrkB, thereby activating the TrkB signaling pathway. In vivo drug metabolism experiments in animals have shown that CF3CN exhibits superior oral bioavailability and in vivo half-life. When 5XFAD mice were orally administered CF3CN, the TrkB receptors and downstream signals in the brain were effectively activated, and their AD symptoms could be treated in a dose-dependent manner. In addition, CF3CN can significantly enhance the learning and memory of 3xTg mice. Although CF3CN has brain permeability, the ratio of its drug concentration entering the brain to the drug concentration in plasma (Brain / Plasma Ratio, B / P Ratio) is approximately 1%, which is not ideal.

[0004] Using nanocarriers to deliver compounds is an effective approach to improving the oral bioavailability of poorly absorbed compounds. Nanoparticle packaging of compounds promotes their stability in the gastrointestinal tract and facilitates absorption, thereby enhancing oral bioavailability. Zein, a hydrophobic protein derived from corn seeds, is readily soluble in ethanol and other organic solvents. In addition to being a generally recognized as safe (GRAS) material, zein is also an FDA-approved pharmaceutical excipient ingredient. Zein's hydrophobicity, biodegradability, and biocompatibility have led to its application in a variety of fields, including food coating and packaging, drug coating, as a drug delivery platform, and in tissue engineering. Zein readily self-assembles into nanoparticles with diverse structures under different solvents and processing conditions. Furthermore, because zein molecules have abundant hydroxyl, amino, and carboxyl groups on their side chains, they can be chemically coupled to compounds through the addition of crosslinking agents. Studies have shown that zein can encapsulate several hydrophobic compounds, including curcumin, resveratrol, folic acid, atorvastatin, and daidzein, improving their oral bioavailability. However, when zein-based nanoparticles are exposed to high ionic strength, high temperature, and pH values ​​near the isoelectric point, they are prone to aggregation or precipitation, which limits their application. To address this problem, a common and effective method is to use surfactants or biopolymers to stabilize zein nanoparticles. For example, pectin, chitosan, and caseinate are common biopolymers. Studies have reported that lactoferrin (LF), a mammalian iron-binding glycoprotein with a molecular weight of 80 kDa, also known as lactotransferrin, can be used as a stabilizer for nanoparticles. In addition, transferrin receptors on intestinal epithelial cells can promote the absorption of nanoparticles by intestinal epithelial cells. Therefore, using zein and stabilizers to prepare nanoparticles for compound delivery is also a common way to improve oral bioavailability.

[0005] Liposomes are promising nanodelivery agents that meet most of the requirements of ideal nanocarriers due to their excellent biocompatibility, safety, and biodegradability. Liposomes have a well-defined structure, are easy to prepare, and possess a wide range of physicochemical properties. They can encapsulate nearly all types of molecules, including lipophilic, hydrophilic, and peptide drugs. Liposomes are biocompatible carriers used to enhance the oral bioavailability of drugs. Due to their similar bilayer structure, they adhere better to biological membranes. Studies have also shown that, like other lipid formulations, they can form mixed micelles with bile salts, which can increase the solubility of poorly soluble drugs and facilitate lymphatic absorption. However, liposomes also have limitations as oral drug carriers. A major challenge with oral liposomes is their stability under gastrointestinal conditions, including pH fluctuations and the presence of bile salts and enzymes. Another drawback is their difficulty in diffusing through the mucus layer. Fortunately, research has proposed solutions to address these issues by modifying the liposome surface with various stabilizers, such as PEG, chitosan, and peptides. These surface-modified materials enhance the stability of liposomes for oral administration.

[0006] Nasal drug delivery is not a new method for delivering drugs to the systemic circulation; however, its novelty lies in utilizing this noninvasive method to rapidly deliver drugs directly from the nasal mucosa to the brain and spinal cord, with the goal of minimizing systemic exposure while treating central nervous system diseases. Although the exact mechanism of nasal drug delivery to the central nervous system is not fully understood, increasing evidence suggests that nasal drug absorption into the brain occurs primarily through three pathways: the olfactory nerve pathway, the trigeminal nerve pathway, and the blood circulation pathway. Through the first two pathways, drugs can be transported along the nerves and directly absorbed into the brain. Through the latter pathway, drugs are first absorbed into the blood circulation through the abundant capillaries and lymphatic vessels under the mucosa and then cross the BBB to enter the brain. The main advantages of nasal administration are as follows: it avoids the first-pass effect of oral drugs by not going through the enterohepatic circulation, and also avoids drug damage to the liver; the activity of nasal mucosal hydrolases is lower than that of the gastrointestinal tract, and generally only 1 / 10 to 1 / 15 of the oral drug dose is required, which is particularly suitable for drugs that are easily decomposed and destroyed by the gastrointestinal tract, such as peptides, hormones, vaccines, etc.; it is more suitable for drugs that act on the brain, and can be directly absorbed into the brain without passing through the BBB, thereby increasing the drug concentration in the brain.

[0007] Summary of the Invention

[0008] The BDNF / TrkB pathway plays a crucial role in neuroplasticity and neuronal survival, but is often deficient in the brains of AD patients, making it a popular drug development target for the treatment of AD. The small molecule compound CF3CN previously developed by the applicant can act as a specific TrkB agonist and exert a therapeutic effect in an AD mouse model, but its brain-to-plasma ratio (B / P ratio) is approximately 1%, which is not ideal. To address the deficiencies in the prior art, the present invention aims to provide a drug for the prevention and / or treatment of Alzheimer's disease.

[0009] The applicant's previous research on drug structure-activity relationships has shown that imidazole derivatives of the CF3CN backbone's Ring A retain TrkB agonist activity, and the electron-withdrawing CN group on Ring B promotes its stability in plasma and liver microsomes. Therefore, the applicant modified only the CN group while maintaining the remaining structural integrity. In the present invention, different CF3CN derivatives #1-4 were synthesized by reducing the CN group to alkylamines and amides to increase their B / P ratios. Nanotechnology was further employed to deliver the compounds, enhancing their oral bioavailability.

[0010] The specific technical solutions are as follows:

[0011] The first aspect of the present invention provides a CF3CN derivative, wherein the CF3CN derivative has a structural formula shown in any one of the following formulas 1-4:

[0012] The second aspect of the present invention provides a pharmaceutical composition comprising one or more of the CF3CN derivatives shown in Formulas 1-4 below, and a pharmaceutically acceptable carrier or excipient.

[0013] The third aspect of the present invention provides a nanoparticle, wherein the nanoparticle comprises one or more CF3CN derivatives shown in the following formulas 1-4 and zein, and the nanoparticle is prepared by an antisolvent coprecipitation method.

[0014] Furthermore, the nanoparticles further include lactoferrin or glycosylated lactoferrin, preferably lactoferrin.

[0015] Furthermore, the mass ratio of the CF3CN derivative to zein is 1:1 to 1:20, preferably 1:10.

[0016] Furthermore, the mass ratio of the CF3CN derivative, zein and lactoferrin or glycosylated lactoferrin is (1-2):(1-20):(1-20), preferably 1:10:10.

[0017] Furthermore, the nanoparticles include the CF3CN derivative shown in Formula 2, zein and lactoferrin, and the mass ratio of the CF3CN derivative shown in Formula 2, zein and lactoferrin is (1-2):(1-20):(1-20), preferably 1:10:10.

[0018] When the nanoparticles include one or more CF3CN derivatives shown in Formulas 1-4 and zein, the nanoparticles are prepared by an antisolvent co-precipitation method: one or more CF3CN derivatives shown in Formulas 1-4 and zein are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution, and the mixed solution is added to deionized water while stirring. After the solvent evaporates, the nanoparticles are obtained.

[0019] When the nanoparticles include one or more of the CF3CN derivatives shown in the following formulas 1-4, zein and lactoferrin, the nanoparticles are prepared by an antisolvent co-precipitation method: one or more of the CF3CN derivatives shown in the following formulas 1-4, zein and lactoferrin are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution, and the mixed solution is added to deionized water while stirring. After the solvent evaporates, the nanoparticles are obtained.

[0020] When the nanoparticles include one or more of the CF3CN derivatives shown in Formulas 1-4, zein, and glycosylated lactoferrin, the one or more of the CF3CN derivatives shown in Formulas 1-4 and the zein are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution; the glycosylated lactoferrin is dissolved in the aqueous solution, and the mixed solution is added to the glycosylated lactoferrin aqueous solution while stirring. After the solvent evaporates, the nanoparticles are obtained.

[0021] The fourth aspect of the present invention provides a liposome, wherein the liposome comprises one or more of the CF3CN derivatives shown in the following formulas 1-4 and lecithin, and the liposome is prepared by an ethanol injection method.

[0022] Preferably, the mass ratio of the lecithin to the CF3CN derivative is (1:1) to (50:1), preferably 20:1.

[0023] Furthermore, the liposome further comprises a stabilizer;

[0024] Preferably, the stabilizer is selected from chitosan;

[0025] Preferably, the mass ratio of the lecithin, chitosan and derivative is (1:1:1) to (50:1:1), preferably 20:2:1.

[0026] Preferably, the liposome comprises the CF3CN derivative shown in Formula 2, lecithin, and chitosan.

[0027] Furthermore, the liposomes are prepared by an ethanol injection method: one or more of the CF3CN derivatives shown in Formulas 1-4 and lecithin are dissolved in anhydrous ethanol to obtain a mixed solution, and the mixed solution is added to deionized water while stirring. After the solvent evaporates, a crude liposome solution is obtained;

[0028] Preferably, the method further comprises subjecting the crude liposome solution to ultrasonic treatment in an ice bath for 5-10 minutes; dissolving chitosan in deionized water, and adding the liposome solution to the chitosan aqueous solution while stirring to obtain liposomes loaded with the derivative.

[0029] The fifth aspect of the present invention provides the use of the CF3CN derivative in the preparation of a TrkB agonist, or the use of the CF3CN derivative, the pharmaceutical composition, the nanoparticle or the liposome in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0030] In a sixth aspect, the present invention provides a method for preventing and / or treating Alzheimer's disease, which comprises administering to a subject a therapeutically effective amount of the above-mentioned CF3CN derivative, the above-mentioned pharmaceutical composition, the above-mentioned nanoparticle or the above-mentioned liposome.

[0031] Furthermore, the administration method is oral administration or nasal administration. Preferably, the optimal administration method for the CF3CN derivative shown in Formula 2 is nasal administration of liposomes.

[0032] The beneficial effects of the present invention are:

[0033] (1) The four CF3CN derivatives #1-4 provided by the present invention all have TrkB agonist activity and can be used to prepare drugs for preventing and / or treating Alzheimer's disease.

[0034] (2) The CF3CN derivative #2 provided by the present invention has good stability in liver microsomes and is less bound to plasma proteins. It is the optimal derivative of the present invention. In vivo PK studies have shown that it improves the B / P ratio of CF3CN and solves the defects of CF3CN.

[0035] (3) The present invention uses zein, LF, or DLF to prepare nanoparticles of CF3CN derivatives in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease to improve the bioavailability of the drug. The use of zein / LF nanoparticles to encapsulate compound #2 to prepare #2-NP improves its oral bioavailability and brain drug concentration, enhances the compound's effectiveness in PK / PD and efficacy experiments, and ultimately promotes the therapeutic efficacy of AD.

[0036] (4) Compound #2 was packaged in liposomes to prepare #2-LIP, which promoted its oral bioavailability and brain drug concentration and improved the compound's effect in PK experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1. Cellular EC50 values ​​of CF3CN cyanide-reduced derivatives and their activation of TrkB receptors. (a) Chemical structures of CF3CN and its four reduced derivatives. (b) EC50 values ​​of CF3CN and its derivatives. BR6 and T48 cells were treated with STS (100 nM) and a TrkB agonist. CF3CN and compounds #1-4 were added at doses of 0, 5, 10, 50, 100, 500, and 1000 nM. MTT assays were performed 24 hours later, and EC50 values ​​were calculated using Prism software. (n = 3; data are expressed as mean ± SD.) (c) Cells were lysed 24 hours after treatment, and the cell lysates were immunoblotted with various antibodies.

[0038] Figure 2. Organic synthesis route of compound #1-4. Reagents and conditions: (a) Tetrahydrofuran borane (BH3 / THF), added at 0°C and reacted at room temperature; (b) Dess-Martin periodinane (DMP), sodium bicarbonate (NaHCO3), dichloromethane (DCM), added at 0°C and reacted at room temperature; (c) Pernitric acid (HNO4), sulfuric acid (H2SO4), reacted at 0°C; (d) Concentrated ammonia, dimethyl sulfoxide (DMSO), reacted at 50°C; (e) 1 mol / L hydrochloric acid (1N HCl), acetonitrile (ACN), reaction at 90℃; (f) sodium hydride (NaH), N,N-dimethylformamide (DMF), added at 0℃, then reacted at room temperature; (g) I2, DMSO, reacted at 130℃; (h) trifluoroacetic anhydride (TFAA), pyridine, added at 0℃, then reacted at room temperature; (i) iron (Fe), acetic acid (AcOH), methanol (MeOH), reaction at 50℃; (j) (preparation of R11a) hydrogen (H2), Raney nickel catalyst ( Ra-Ni), DMF, react at 50°C; (k) (for R11b, R11c) trifluoroacetic acid (TFA), DCM, 25°C; (l) methylamine (MeNH2) (preparation of compound 3); dimethylamine (Me2NH) (preparation of compound 4); 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA), DMF; add at 0°C, then react at room temperature.

[0039] Figure 3. Optimization of nanoparticles with different mass ratios between compound #2 and zein. (A) Appearance of the three nanoparticles. (B-C) PDI and particle size of the three nanoparticles. (D) Particle size distribution of the three nanoparticles by intensity, measured by DLS. (E-F) Turbidity and zeta potential of the three nanoparticles. (G) Zeta potential distribution of the three nanoparticles, measured by DLS. (H-I) Encapsulation efficiency and drug loading of the three nanoparticles. (J) HPLC calibration curve of compound #2. (n = 3; data are presented as mean ± SD).

[0040] Figure 4. Optimization of nanoparticles with different mass ratios between zein and LF. (A) Schematic diagram of the formation mechanism of zein / LF nanoparticles. (B) Appearance of the four nanoparticles. (C) PDI and particle size of the four nanoparticles. (E) Intensity-based size distribution of the four nanoparticles measured by DLS. (F) Turbidity and zeta potential of the four nanoparticles. (H) Zeta potential distribution of the four nanoparticles measured by DLS. (n = 3; data are presented as mean ± SD).

[0041] Figure 5. Characterization of Zein / LF nanoparticles containing compound #2. (a) Schematic diagram of the preparation process for #2 / zein, #2 / zein / LF, and #2 / zein / DLF nanoparticles. (b) Particle size, zeta potential, PDI, and turbidity of the three nanoparticles were measured. (fg) Encapsulation efficiency and drug loading of the three nanoparticles were measured. (n = 3; data are shown as mean ± SD). (h) SEM images taken at 40,000–80,000x magnification. (i) TEM images of the three nanoparticles; scale bar = 500 nm.

[0042] Figure 6. In vivo pharmacokinetic study of #2 and #2-NP. (a) Encapsulation efficiency (EE) and drug loading (LC) of #2-NP before and after lyophilization. (n=3, Student's t-test). (b) Plasma concentration after treatment with #2 or #2-NP. ICR mice were divided into 4 groups and given 2 mg / kg (IV), 5 mg / kg (PO) #2, 5 mg / kg and 10 mg / kg #2-NP (PO), respectively. Blood samples were collected from all 3 mice at the corresponding time points and the #2 concentration was quantitatively analyzed by LC-MS / MS. The pharmacokinetic parameters of each group were calculated using DAS 2.0 software as shown in the table. Cmax: peak plasma concentration; Tmax: time to peak; T 1 / 2: terminal phase half-life; AUC: area under the curve; MRT: mean residence time; CL: clearance; F: relative oral bioavailability. (c) Brain concentrations of #2 and #2-NP after treatment. (d) Comparison of plasma and brain tissue concentrations of #2 and #2-NP after oral gavage at the same dose (5 mg / kg). (Data are expressed as mean ± SD; **p ≤ 0.01, ****p ≤ 0.0001).

[0043] Figure 7. In vivo PK study of #2 and #2-NP. (A) Quantification of #2 concentrations in plasma and brain using LC-MS / MS after intravenous injection of 2 mg / kg #2. (B) Quantification of #2 concentrations in plasma and brain using LC-MS / MS after oral gavage of 5 mg / kg #2. (C) Quantification of #2 concentrations in plasma and brain using LC-MS / MS after oral gavage of 5 mg / kg #2-NP. (D-E) Comparison of plasma and brain drug concentrations after IV or PO administration of #2. (n = 3, data expressed as mean ± SD). (F) Comparison of the B / P ratio after oral administration of the same dose (5 mg / kg) of #2 and #2-NP. (n = 3, Student's t-test).

[0044] Figure 8. In vivo PK / PD study of #2-NP. (a) Analysis of AEP enzyme activity in brain lysates from APP / PS1 mice at different time points after oral administration of #2-NP. Quantification of #2 concentrations in plasma and brain tissue by LC-MS / MS; (n = 3, one-way ANOVA). (bc) APP / PS1 mouse brain tissue was lysed and the expression of the TrkB signaling pathway and AEP was analyzed by immunoblotting, and the results were quantified. (n = 3, one-way ANOVA). (d) Analysis of AEP enzyme activity in brain lysates from Tau P301S mice at different time points after oral administration of #2-NP. Quantification of #2 concentrations in plasma and brain tissue by LC-MS / MS; (n = 3, one-way ANOVA). (ef) Tau P301S mouse brain tissue was lysed and the expression of the TrkB signaling pathway and AEP was evaluated by immunoblotting, and the results were quantified. (n = 3, one-way ANOVA). (g) Effects of acute administration of #2-NP in 3xTg mice. 3xTg mice were administered 5 mg / kg or 10 mg / kg #2-NP (PO). Two hours after acute treatment, blood and brain tissue samples were collected from three mice. #2 concentrations were quantified using LC-MS / MS. (h) AEP enzyme activity in brain lysates from 3xTg mice treated with different oral doses of #2-NP. (ij) Brain tissue from 3xTg mice was lysed and immunoblotted to assess TrkB signaling activation, AEP, APP, and Tau protein cleavage, and quantified. (n = 3, one-way ANOVA) (Data are expressed as mean ± SD, *p < 0.05).

[0045] Figure 9. #2-NP improves cognition in 3xTg mice, reduces Aβ deposition, and reduces brain atrophy. (a) Representative movement trajectories of 3xTg mice in a novel object recognition experiment, from the vehicle group, the #2 3 mg / kg group, the #2-NP 3 mg / kg group, and the #2-NP 10 mg / kg group. (b) Statistical analysis of the recognition index for each group. (n = 6, one-way ANOVA). (c) Representative T2-weighted MRI scans of 3xTg mice, with the cortical region of interest (ROI) marked by a red line. (d) Quantified volume of the cerebral cortical ROI. (n = 3, one-way ANOVA). (ef) Aβ immunohistochemical staining of 3xTg mouse brain sections. Scale bar = 50 μm. (n = 6, one-way ANOVA). (g) [18F]AV-45 PET-CT scans of 3xTg mice treated with the vehicle, #2 3 mg / kg, #2-NP 3 mg / kg, and #2-NP 10 mg / kg groups. (h) Comparison of the standardized uptake ratio (SUVR) of [18F]AV-45 PET per gram of cerebral cortex among the mice in each group (n = 3, one-way ANOVA). (ij) Quantitative analysis of Aβ42 and Aβ40 in brain tissue by ELISA (n = 3, one-way ANOVA) (Data are shown as mean ± SD; **p < 0.01, **p < 0.01, ***p < 0.005, ****p ≤ 0.0001).

[0046] Figure 10. #2-NP activates TrkB and inhibits AEP activity in the brains of 3xTg mice. (a) Immunoblotting was used to examine the expression and cleavage of the TrkB signaling pathway, AEP, APP, and Tau in brain tissue. (b) Brain tissue was harvested for AEP activity assay. (n = 6, one-way ANOVA) (ci) Quantitative analysis of immunoblot results. (n = 6, one-way ANOVA) (Data are shown as mean ± SD; ns indicates not significantly different; *p < 0.05, **p < 0.01, ***p < 0.005).

[0047] Figure 11. #2-NP reduces AD pathology in 3xTg mice. (a) Immunofluorescence staining of p-TrkB and MAP2 in 3xTg mouse brain tissue. Scale bar = 50 μm. (b) Immunofluorescence staining of p-AEP and MAP2 in 3xTg mouse brain tissue. Scale bar = 50 μm. (c) Immunofluorescence staining of AEP and APP C586 in 3xTg mouse brain sections. Scale bar = 50 μm. (d) Immunofluorescence staining of AT8 and Tau N368 in 3xTg mouse brain sections. Scale bar = 50 μm. (ej) Quantification of p-TrkB, p-AEP, AEP, APP C586, Tau N368, and AT8 intensities in the hippocampus. (n = 6, one-way ANOVA) (data are mean ± SD, ns indicates not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

[0048] Figure 12. Bioaccessibility and Caco-2 uptake assays of #2-loaded nanoparticles. (A-B) Effect of in vitro digestion time on the particle size and bioaccessibility of #2-loaded nanoparticles. (C) SEM images of #2 / zein, #2 / zein / LF, and #2 / zein / DLF, magnification: 40,000x, scale bar = 2 μm. (D) Caco-2 cell viability was measured using the CCK8 assay after 24 hours of incubation with different concentrations of #2. (E) Caco-2 cell viability was measured after 24 hours of incubation with #2, #2 / zein, #2 / zein / LF, and #2 / zein / DLF. (F) Transendothelial electrical resistance (TEER) values ​​of Caco-2 cells were measured during 21 days of culture. Caco-2 permeability data are summarized in the table. (n = 3; data are presented as mean ± SD).

[0049] Figure 13. In vivo PK study of #2, #2-NP, and #2-LIP following oral and intranasal administration. (a) Quantitative analysis of #2 plasma concentrations using LC-MS / MS following oral gavage administration of 5 mg / kg #2, #2-NP, and #2-LIP, and intranasal administration of #2 and #2-LIP. (n = 3; data are expressed as mean ± SD). (b) Quantitative analysis of #2 brain tissue concentrations using LC-MS / MS following oral gavage administration of 5 mg / kg #2, #2-NP, and #2-LIP, and intranasal administration of #2 and #2-LIP. (n = 3; data are expressed as mean ± SD). DETAILED DESCRIPTION

[0050] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0051] The present invention synthesizes four different derivatives of CF3CN (compounds #1-4) by reducing the CN group of CF3CN to alkylamines and amides. This increases the compound's side chain length, enhancing its lipophilicity and blood-brain barrier (BBB) ​​penetration without affecting its agonist properties, thereby increasing its B / P ratio. Compounds #1-4 were also validated for TrkB activation in vitro, and their effective concentrations (EC50s) were calculated. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of candidate compounds (#2-4) were analyzed. After comprehensive analysis, the optimal compound #2 was selected for further studies. Although compound #2 exhibited an EC50 comparable to CF3CN and possessed higher stability, it exhibited a higher efflux rate in the Caco-2 assay, indicating poor oral bioavailability. Zein / LF nanoparticles (#2 / zein / LF) were used to deliver compound #2 to improve its oral bioavailability, and the pharmacokinetic-pharmacodynamic (PK / PD) and therapeutic effects of #2 / zein / LF were studied in an AD mouse model.

[0052] The following describes it in detail with reference to the embodiments.

[0053] Example 1

[0054] 1. Synthesis of four CF3CN derivatives #1-4

[0055] The structural formula of the compound CF3CN is shown in Figure 1A. Four different derivatives of CF3CN (Compounds #1-4) were synthesized by reducing the CN group of CF3CN to alkylamines and amides. The structural formulas of the four compounds are shown in Figure 1A. The specific synthesis schemes of the four compounds are shown in Figure 2. All chemicals and solvents used in this synthesis were purchased commercially and used directly without further purification. Recording was performed using a Bruker Avance III 400MHz and a Bruker Fourier 300MHz.1 H NMR spectra were recorded using tetramethylsilane (TMS) as an internal standard. LC / MS was recorded using an Agilent LC / MSD1200 series quadrupole mass spectrometer with an ODS 2000 column (150 × 4.6 mm, 5 μm) in ES (+) or (-) ionization mode. The temperature was 30°C, the flow rate was 1.5 mL / min, and the detection wavelengths were 214 nm and 254 nm. HPLC conditions included a Welchrom C18 column (150 × 20 mm), a detection wavelength of 220 nm, and mobile phases A: acetonitrile (MeCN) containing 0.1% (v / v) trifluoroacetic acid (TFA) and B: water containing 0.1% TFA. The flow rate was 25 mL / min, the injection volume was 2 mL, the detection run time was 30 minutes, and the column equilibration time was 5 minutes.

[0056] Preparation R2. Under 0 ° C., 79.2 mL of BH3 / THF solution (1.0 M, 79.2 mmol) was added to 100 mL of a THF solution containing R1 (10.5 g, 39.6 mmol) and the reaction mixture was stirred at room temperature overnight. The mixture was then quenched with MeOH (20 mL), concentrated, and eluted with petroleum ether containing 15% (volume ratio) ethyl acetate to give R2 (10.4 g, 100% yield) as a brown oil. The molecular weight R2 was found to be 252.3.

[0057] Prepare R3c. 10.0g R2 (10.0g, 40.0mmol) is dissolved in 150mL DCM solution, and NaHCO (6.72g, 80.0mmol) and DMP reagent (18.6g, 44.0mmol) are added to the solution under nitrogen blowing and 0 ℃ condition. Then at room temperature, the reaction mixture is stirred for 2h, and then the product is washed with saturated NaHCO aqueous solution (3×50mL) and brine (2×50mL). The organic portion obtained is dried over anhydrous Na2SO4, filtered and concentrated and passed through silica gel column chromatography, using petroleum ether containing 15% (volume ratio) ethyl acetate as eluent to purify to obtain R3c (8.66g, 84% yield) as a yellow oil. Measuring R3c molecular weight is 250.3.

[0058] Preparation of R5. R4 (90 g, 0.58 mol) was dissolved in a concentrated H2SO4 (400 mL) solution, and HNO3 (41 g, 0.64 mmol) was added under nitrogen and 0°C. The reaction mixture was stirred at 0°C for 3 h, and then the reaction mixture was poured into ice water (2000 mL) and extracted with ethyl acetate (3×1000 mL). The combined organic layer was washed with brine (3×500 mL), then dried over anhydrous Na2SO4, filtered and concentrated, and purified by silica gel column chromatography using a petroleum ether solution containing 20% ​​(volume ratio) ethyl acetate as an eluent to obtain R5 (33 g, 28% yield) as a yellow oil. The molecular weight of R5 was measured to be 200.2, and was used 1 Its structure was further identified by H-NMR: 1 H-NMR (400MHz, CDCl3), 13.29 (s, 1H), 7.92 (dd, J=6.0Hz, 9.2Hz, 1H), 6.81 (t, J=8.8Hz, 1H), 2.67 (s, 3H).

[0059] Preparation of R6. R5 (6.5 g, 32.7 mmol) was dissolved in DMSO (150 mL), and 15 mL of NH H O was added to the suspension under nitrogen at 20° C. The reaction mixture was stirred at 50° C. for 15 h, then 10 mL of water was added and filtered. The filtrate was concentrated to dryness to give R6 (6.4 g, 100% yield) in the form of a yellow solid. The molecular weight of R5 was measured to be 196.0.

[0060] Preparation of R7. R6 (6.4 g, 32.8 mmol) was dissolved in ACN solution (60 mL), and 20 mL of 1N HCl was added to the suspension under nitrogen at 20°C. The reaction mixture was then stirred at 90°C for 4 h, and the mixture was finally concentrated to afford R7 (5.8 g, 92.1% yield) as a yellow solid. The molecular weight of R7 was determined to be 197.3.

[0061] Prepare R8a. Under nitrogen and 0 ° C, R7 (1.0 g, 5.1 mmol) was added to a suspension of sodium hydride (714 mg, 17.9 mmol) in anhydrous DMF (30 mL). The reaction mixture was stirred at 0 ° C for 1 h, and then R3a (1.67 g, 12.8 mmol) was added. The reaction mixture was stirred for another 3 h at room temperature. 100 mL of water was added to the reaction mixture and the mixture was extracted with ethyl acetate (2 × 100 mL). The organic layers were combined, washed with brine (3 × 100 mL), dried over anhydrous Na2SO4, filtered and concentrated. Finally, the residue was purified by silica gel column chromatography using CH2Cl2 containing 10% (volume ratio) MeOH as an eluent to obtain R8a (1.5 g, 91% yield) in the form of a yellow solid. The molecular weight of R8a was measured to be 310.3.

[0062] Prepare R9a. R8a (1.5g, 4.85mmol) is dissolved in DMSO (10mL) solution, and I2 (185mg, 0.72mmol) is then added to the solution. The mixture is stirred for 2h at 130°C. After the reaction is complete, the reaction mixture is quenched and filtered with ice water (20mL). The upper filter cake is dried to obtain R9a (1.35g, 90% yield) in the form of a yellow solid. The molecular weight of R9a measured is 307.9.

[0063] Prepare R10a. R9a (1.35 g, 4.4 mmol) was dissolved in pyridine (14 mL) solution, and TFAA (2.77 g, 13.2 mmol) was added to the solution. Under nitrogen and room temperature, the reaction mixture was stirred for 2 h, then quenched with water (20 mL), the precipitate was filtered, and the filtrate was concentrated to give R10a (1.6 g, 90% yield) in the form of a yellow solid. The molecular weight of R10a was measured to be 404.3.

[0064] Preparation of R11a. R10a (1.6 g, 4.0 mmol) was dissolved in a mixture of MeOH / AcOH (20 mL / 10 mL). Iron powder (1.12 g, 20.0 mmol) was added to the solution and stirred at 50°C for 2 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate concentrated. The residue was purified by silica gel column chromatography using petroleum ether containing 50% (volume ratio) ethyl acetate as eluent to obtain R11a (730 mg, 52% yield) as a yellow solid. The molecular weight of R11a was determined to be 356.3.

[0065] Prepare compound #1. R11a (730 mg, 2.2 mmol) was dissolved in DMF (5 mL) solution, and Ra-Ni (200 mg) was added to the solution. The mixture was stirred at 50°C and hydrogen (1 standard atmosphere) for 12 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated. The obtained residue was purified by C18 column (120 g C18 column, flow rate of 50 mL / min, mobile phase of 5%-95% ACN aqueous solution 1800 mL) to obtain #1 as a yellow solid (140 mg, yield 20%). The molecular weight of compound #1 was measured to be 360.0. And used 1 H-NMR (400 MHz, DMSO-d6) analysis of the compound: 8.30-8.22 (m, 5H), 8.05-8.03 (m, 1H), 7.78-7.70 (m, 3H), 7.23 (s, 1H), 4.18 (s, 2H).

[0066] Preparation of R8b. R7 (5.0 g, 25.5 mmol) was dissolved in anhydrous DMF (180 mL). A suspension of sodium hydroxide (3.57 g, 89.3 mmol) was added at 0°C under nitrogen. The reaction mixture was stirred at 0°C for 1 h, and R3b (13.1 g, 63.7 mmol) was added. The reaction mixture was stirred at room temperature for another 3 h. 200 mL of water was added, and the mixture was extracted with ethyl acetate (2×200 mL). The organic layers were mixed, washed with brine (3×100 mL), dried over anhydrous Na2SO4, and then filtered and concentrated. The residue was purified by silica gel column chromatography using CH2Cl2 containing 10% (volume ratio) methanol as eluent to obtain yellow solid R8b (3.0 g, 26% yield). The molecular weight of R8b was measured to be 385.3.

[0067] Preparation of R9b. R8b (3.0 g, 8.0 mmol) was dissolved in DMSO (30 mL) and I2 (300 mg, 1.1 mmol) was added. The mixture was stirred at 130°C for 2 h. The reaction mixture was quenched with ice water (20 mL) and filtered. The upper filter cake was dried to obtain a yellow solid, R9b (3.0 g, 90% yield). The molecular weight of R9b was determined to be 383.3.

[0068] Preparation of R10b. R9b (2.9 g, 7.6 mmol) was dissolved in pyridine (50 mL) solution, TFAA (4.78 g, 22.8 mmol) was added to the solution, and the reaction was carried out at 0°C. The reaction mixture was stirred at room temperature under nitrogen for 2 h. The mixture was then quenched with water (50 mL), the precipitate was filtered, and the filter cake was dried to obtain R10b as a yellow solid (3.46 g, 95% yield). The molecular weight of R10b was measured to be 479.3.

[0069] Preparation of R11b. R10b (3.36 g, 7.0 mmol) was dissolved in a mixture of MeOH / AcOH (50 mL / 10 mL), followed by the addition of Fe (1.97 g, 35.0 mmol). Stir at 50°C for 2 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate concentrated. The residue was purified by silica gel column chromatography using 50% (v / v) ethyl acetate in petroleum ether as the eluent to afford R11b (2.2 g, 72% yield) as a yellow solid. The molecular weight of R11b was determined to be 431.3.

[0070] Preparation of R12. R11b (2.1 g, 4.88 mmol) was dissolved in DCM (25 mL), and then TFA (8 mL) was added. The mixture was stirred at room temperature for 2 h, and the mixture was concentrated to give a yellow solid R12 (1.9 g, 100% yield). The molecular weight of R12 was determined to be 375.3.

[0071] Preparation of compound #3. R12 (240 mg, 0.64 mmol) and CH3NH2 (2.0 M, 0.35 mL, 0.71 mmol) were dissolved in DMF (7 mL), and then DIEA (165 mg, 1.28 mmol) and HATU (293 mg, 0.77 mmol) were added at 0 ° C. and heated under nitrogen. The reaction mixture was stirred at room temperature for 2 h, concentrated and purified using a C18 column (120 g C18 column, flow rate of 50 mL / min, mobile phase of 5%-95% CAN aqueous solution 1800 mL) to obtain compound #3 as a yellow solid (134 mg, yield 56%). The molecular weight of compound #3 was measured to be 388.3. Use 1 H-NMR identification of the structure of #2: 1 H-NMR (400MHz, DMSO-d6), 8.66 (d, J=4.8Hz, 1H), 8.30 (brs, 2H), 8.07-8.01 (m, 3H, 7.78-7.76 (m, 1H), 7.26 (s, 1H), 2.84 (d, J=4.4Hz, 3H).

[0072] Preparation of compound #4. R12 (240 mg, 0.64 mmol) and dimethylamine hydrochloride (58 mg, 0.71 mmol) were dissolved in DMF (7 mL) solution, and then DIEA (165 mg, 1.28 mmol) and HATU (293 mg, 0.77 mmol) were added at 0 ° C under nitrogen. The reaction mixture was stirred at room temperature for 2 h and concentrated and purified using a C18 column (120 g C18 column, flow rate of 50 mL / min, mobile phase of 5%-95% ACN aqueous solution 1800 mL) to obtain compound #4 as a yellow solid (137 mg, yield 57%). The molecular weight of compound #4 was measured to be 402.3, and the reaction mixture was purified using a C18 column (120 g C18 column, flow rate of 50 mL / min, mobile phase of 5%-95% ACN aqueous solution 1800 mL). 1 H-NMR identification of #4 structure: 1 H-NMR (400MHz, DMSO-d6), 8.26 (brs, 2H), 8.04-8.01 (m, 1H), 7.78-7.75 (m, 1H), 7.65 (d, J=8.4Hz, 2H), 7.23 (s, 1H), 3.03 (s, 3H), 2.95 (s, 3H).

[0073] Preparation of R8c. R7 (2.9 g, 14.8 mmol) was dissolved in anhydrous DMF (100 mL) and a suspension of sodium hydride (2.1 g, 51.8 mmol) was added at 0°C under nitrogen. After the reaction mixture was stirred at 0°C for 1 h, R3c (8.1 g, 51.7 mmol) was added. The reaction mixture was stirred at room temperature for another 3 h. 200 mL of water was added and the mixture was extracted with ethyl acetate (2×200 mL). The organic layers were mixed, washed with brine (3×100 mL), then dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography using a CH2Cl2 solution containing 10% (volume ratio) methanol as eluent to obtain a yellow solid R8c (3.9 g, 62% yield). The molecular weight of R8c was measured to be 428.3.

[0074] Preparation of R9c. R8c (2.7 g, 6.3 mmol) was dissolved in DMSO (60 mL) and then I2 (240 mg, 0.95 mmol) was added. The mixture was stirred at 130 ° C for 2 h. After the reaction was completed, the reaction mixture was quenched with ice water (20 mL) and filtered. The filter cake was concentrated to obtain a yellow solid R9c (600 mg, 18% yield). The molecular weight of R9c was measured to be 426.3.

[0075] Preparation of R10c. R9c (500 mg, 1.2 mmol) was dissolved in pyridine (10 mL) and TFAA (740 mg, 3.6 mmol) was added at 0°C. The reaction mixture was stirred under nitrogen at room temperature for 2 h. The mixture was quenched with water (50 mL) and the precipitate was filtered. The filter cake was dried to afford R10c as a yellow solid (735 mg, 95% yield). The molecular weight of R10c was determined to be 522.3.

[0076] Preparation of R11c. R10c (680 mg, 1.3 mmol) was dissolved in a mixture of MeOH / AcOH (10 mL / 2 mL), followed by the addition of iron powder (365 mg, 6.5 mmol). The reaction was stirred at 50°C for 2 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate concentrated. The residue was purified by silica gel column chromatography using petroleum ether containing 50% (volume ratio) ethyl acetate as the eluent to obtain R11c (560 mg, 92% yield) as a yellow solid. The molecular weight of R11c was determined to be 474.3.

[0077] Preparation of Compound #2. R11c (360 mg, 0.76 mmol) was dissolved in DCM (9 mL) and then TFA (3 mL) was added. The mixture was stirred at room temperature for 2 h and concentrated and purified using a C18 column (120 g C18 column, flow rate of 50 mL / min, 5%-95% ACN aqueous solution 1800 mL) to obtain Compound #2 as a yellow solid (130 g, yield 44%). The molecular weight of Compound #2 was determined to be 374.3. 1 H-NMR identification of compound #2 structure: 1 H-NMR (400MHz, DMSO-d6), 9.09 (s, 2H), 8.29-8-27 (m, 2H), 8.02 (d, J=8.4Hz, 1H), 7.78-7.72 (m, 3H), 7.23 (s, 1H), 4.27 (s, 2H), 2.63 (s, 3H).

[0078] 2. Cyanide-reduced derivatives of CF3CN #1-4 and their cellular EC50 values ​​for activation of TrkB receptors

[0079] The pro-survival effects of the four derivatives were quantitatively analyzed in human SH-SY5Y cells and T48 cells stably transfected with TrkB. After induction of apoptosis with staurosperine (STS), the four derivatives all displayed TrkB-dependent anti-apoptotic effects in both BR6 and T48 cells. Compounds #2 and #4 exhibited pro-survival EC50 values ​​comparable to those of CF3CN (Figure 1b). Immunoblotting revealed that these compounds activated p-TrkB and its downstream effectors p-Akt and p-MAPK in a dose-dependent manner. Since PLCγ1 binds to the p-Y816 residue on the TrkB receptor and is subsequently tyrosine phosphorylated by active TrkB, its tyrosine phosphorylation signal was also monitored in this example (Figure 1c).

[0080] 3. In vitro ADMET analysis

[0081] Because the EC50 value of compound #1 was too high, the applicant selected compounds #2-4 for in vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis and compared them with CF3CN. The results, shown in Table 1, showed similarities between the two compounds for most parameters. Compound #2 showed better stability in liver microsomes and less plasma protein binding, indicating that compound #2 was the optimal derivative.

[0082] Table 1 Comparison of in vitro ADMET between CF3CN and compounds #2-4

[0083] Example 2

[0084] 1. Preparation and characterization of compound #2 / zein / LF nanoparticles

[0085] (1) Determination of the mass ratio of compound #2 and zein

[0086] Compound #2 / zein nanoparticles with varying mass ratios of Compound #2 to zein were prepared using an antisolvent coprecipitation method (ASCP). Specifically, Compound #2 and zein were dissolved in an 80% by volume ethanol-water solution to obtain a Compound #2 / zein ethanol-water solution. This ethanol-water solution was then added to deionized water while stirring. After evaporation of the solvent, Compound #2 / zein nanoparticles were obtained.

[0087] Compound #2 / zein nanoparticles were characterized, including nanoparticle appearance, particle size, zeta potential, low polydispersity index (PDI), and turbidity. The encapsulation efficiency (EE) and drug loading capacity (LC) of the nanoparticles for compound #2 were also tested. The results are shown in Figure 3.

[0088] Table 2 Physical properties of nanoparticles with different mass ratios of compound #2 and zein

[0089] The above results determined that the optimal mass ratio of compound #2 to zein was 1:10.

[0090] (2) Determination of the mass ratio of zein and LF

[0091] LF is an iron-binding glycoprotein that serves as a stabilizer for nanoparticles. Zein / LF nanoparticles with varying mass ratios of zein and LF were prepared using the antisolvent coprecipitation method (ASCP). Specifically, zein was dissolved in an 80% ethanol-water solution to obtain a zein-ethanol-water solution. This solution was then added to deionized water while stirring. After evaporation of the solvent, zein / LF nanoparticles were obtained.

[0092] The zein / LF nanoparticles were characterized, including nanoparticle appearance, particle size, zeta potential, polydispersity index (PDI), and turbidity. The results are shown in FIG4 .

[0093] Table 3 Physical properties of nanoparticles with different mass ratios of zein and LF

[0094] The above results determined that the optimal mass ratio of zein and LF was 1:1.

[0095] (3) Characterization of Compound #2 / zein Nanoparticles, Compound #2 / zein / LF Nanoparticles, and Compound #2 / zein / DLF Nanoparticles

[0096] Compound #2 / zein nanoparticles were prepared by antisolvent coprecipitation (ASCP): Compound #2 and zein were dissolved in an 80% ethanol aqueous solution at a mass ratio of 1:10 to obtain a compound #2 / zein ethanol aqueous solution. The compound #2 / zein ethanol aqueous solution was then added to deionized water while stirring. After the solvent evaporated, compound #2 / zein nanoparticles were obtained.

[0097] Compound #2 / zein / LF nanoparticles were prepared by antisolvent coprecipitation (ASCP): Compound #2, zein, and LF were dissolved in an 80% ethanol aqueous solution at a mass ratio of 1:10:10 to obtain a compound #2 / zein / LF ethanol aqueous solution. The compound #2 / zein / LF ethanol aqueous solution was added to deionized water while stirring. After the solvent evaporated, compound #2 / zein / LF nanoparticles were obtained.

[0098] Compound #2 / zein / DLF nanoparticles were prepared using an antisolvent coprecipitation method (ASCP). Zein / LF nanoparticles have poor salt tolerance in the low pH range. To stabilize Compound #2 / zein / LF nanoparticles, the LF protein was glycosylated using the Maillard reaction to obtain Compound #2 / zein / DLF nanoparticles. Specifically, Compound #2 and zein were dissolved in an 80% ethanol-water solution at a mass ratio of 1:10 to obtain a Compound #2 / zein ethanol-water solution. Glycosylated LF was dissolved in deionized water to obtain a glycosylated LF aqueous solution. The Compound #2 / zein ethanol-water solution was added to the glycosylated LF aqueous solution while stirring, with a mass ratio of Compound #2, zein, and glycosylated LF of 1:10:10. After evaporation of the solvent, Compound #2 / zein / DLF nanoparticles were obtained.

[0099] The #2 / zein, #2 / zein / zein / LF, and #2 / zein / DLF nanoparticles were characterized, including particle size, zeta potential, PDI, and turbidity of the nanoparticles, as well as the encapsulation efficiency (EE) and drug loading capacity (LC) of the nanoparticles for #2. The results showed that the EE and LC of #2 / zein / DLF were significantly lower than those of #2 / zein / LF (Figures 5f & g). Scanning electron microscopy and transmission electron microscopy results showed that the particles were uniformly encapsulated into spherical shapes (Figures 5h & i). Figure 12 shows the bioaccessibility and Caco-2 uptake assays of the nanoparticles loaded with #2.

[0100] Table 4 Physical properties of nanoparticles

[0101] Results showed that the average particle size of #2 / zein / LF nanoparticles was approximately 136.7 nm, and they were dispersed in a solution with low turbidity (<0.22), a low polydispersity index (PDI) (<0.20), and a high encapsulation efficiency (>99%). Based on the physicochemical properties of the three nanoparticles, #2 / zein / LF performed best in most parameters and was therefore selected for further in vivo studies.

[0102] 2. In vivo pharmacokinetic study of compound #2 and its nanoparticles (compound #2 / zein / LF, denoted as #2-NP)

[0103] In order to study the in vivo pharmacokinetic characteristics of #2-NP, the freeze-dried #2-NP powder was redissolved. Although the reconstituted nanoparticles appeared transparent, their EE and LC were slightly reduced (Figure 6a). In order to quantitatively compare the effect of the nanoparticle system in delivering #2 into the blood circulation system and brain, an in vivo pharmacokinetic study was performed using 2-month-old ICR mice. The results showed that after oral administration (PO) of 5 mg / kg of #2, the half-life (t 1 / 2 ) was approximately 4.09 hours, while intravenous (IV) injection showed a t of 0.84 hours (2 mg / kg). The area under the curve (AUC) values ​​for intravenous and oral administration were 2315.89 and 405.15 ng / ml*h, respectively. It is worth noting that at the same 5 mg / kg dose, the plasma t of #2-NP was 1 / 2 The clearance rate (L / h / kg) of the unpackaged drug was 9.91 hours, more than double the 4.09 hours of #2. Comparing the administration of the same dose before and after packaging, it was found that the clearance rate (L / h / kg) decreased from 13.36 in the unpackaged drug to 7.06, and its AUC increased from 405.15 to 756.76 ng / ml*h. The maximum plasma concentration C max The value increased from 90.81 to 112.67 ng / nm. More importantly, the oral bioavailability of compound #2 increased from 6.99% before packaging to 13.07% after nanoparticle encapsulation (Figure 6b). max The levels were 164.38 ng / g after intravenous injection and 18.47 ng / g after oral administration, with AUC values ​​of 84.26 and 35.75 ng / ml*h, respectively. 1 / 2 The t of #2 and #2-NP were 1.41 and 3.49 h, respectively. In contrast, after oral administration of 5 mg / kg #2-NP, the AUC in the brain was 74.64, more than double that of oral administration of #2 (AUC was 35.75). 1 / 2 and T maxThe levels were comparable (Figure 6c). Figures 6d & e depict the in vivo PK profiles in plasma and brain at a 5 mg / kg dose. Detailed plasma and brain distribution analysis of #2 kinetics is shown in Figure 7. Although #2 / zein / LF improved the oral bioavailability of #2 and increased its brain exposure, the nanoparticle delivery method did not significantly improve the B / P ratio of #2 compared to #2 alone (Figure 7F). However, compared to the B / P ratio of approximately 1% for CF3CN, the B / P ratio of #2 was approximately 10%, a significant improvement.

[0104] 3. In vivo PK / PD study of #2-NP

[0105] To assess whether #2-NP binds to its target TrkB receptor in the brain and triggers downstream inhibitory phosphorylation of AEP, we analyzed AEP enzyme activity and TrkB signaling in brain tissue lysates from APP / PS1AD mice following oral gavage. Results showed that brain concentrations of #2, as determined by LC / MS, correlated with the level of active AEP bands detected by 3D analysis, inversely related to increasing brain concentrations of #2 (Figure 8a). Immunoblotting revealed increased expression of p-TrkB Y816 in the brain 1 hour after oral administration of #2-NP, followed by a decrease in signal at 4 hours. Its downstream effector, p-PLCγ1 Y783, exhibited similar kinetics. Notably, p-Akt activation persisted until 4 hours, with time-dependent phosphorylation of AEP T322 by p-Akt. Akt negatively coupled to AEP fragments, leading to enzymatic activation of AEP (Figure 8b). Similarly, quantitative immunoblotting analysis (Figure 8c) yielded consistent results with those in Figure 8b. We performed similar kinetic analyses in Tau P301S mice and observed similar results: as brain levels of #2 increased, TrkB was subsequently activated, accompanied by increased p-AEP signaling and decreased enzyme activity (Figures 8d-f). To further investigate the PK / PD relationship, we orally administered 5 or 10 mg / kg of #2-NP to 3xTg mice and analyzed brain #2 concentrations 2 hours later. We found that AEP enzyme activity was dose-dependently inhibited by #2 (Figures 8g&h). Immunoblotting revealed that with increasing doses of #2-NP, p-TrkB signaling increased, as did the downstream p-PLCγ1Y783. Thus, the p-AEP T322 signal is consistent with upstream p-Akt activity and correlates with decreased AEP cleavage. As expected, the downstream AEP substrates APP N585 and Tau N368 fragments steadily decreased with increasing #2-NP doses (Figures 8i&j). Therefore, in vivo PK / PD studies support that #2 targets the TrkB receptor in the brain and inhibits the enzymatic activity of AEP through phosphorylation of AEP, thereby blocking the cleavage of APP and Tau proteins.

[0106] 4. #2-NP improves cognition, reduces Aβ deposition and brain atrophy in 3xTg mice.

[0107] To investigate whether long-term chronic treatment with #2-NP would alleviate cognitive deficits in 3xTg AD model mice, mice were gavaged with 3 mg / kg of #2 and #2-NP and 10 mg / kg of #2-NP. After 3 months of drug treatment, the novel object recognition experiment showed that 3 mg / kg of #2 failed to improve the cognitive function of 3xTg mice, while #2-NP significantly alleviated cognitive impairment. The recognition index was increased in a dose-dependent manner by #2-NP (Figure 9a&b). MRI scans showed that #2-NP significantly increased the volume of the cerebral cortex compared with the solvent control group, while #2 alone showed an upward trend, but without statistical significance (Figure 9c&d). Immunohistochemical staining of brain sections using anti-Aβ showed that both #2 and #2-NP significantly reduced Aβ aggregation in the hippocampus, with #2-NP showing a dose-dependent effect (Figure 9e&f). Furthermore, we quantitatively analyzed Aβ PET signals using its specific ligand [18F]AV-45, and the results showed that 10 mg / kg #2-NP significantly attenuated Aβ PET signals in 3xTg mice (Figures 9g & h). ELISA analysis showed that both #2 and #2-NP significantly reduced Aβ42 content, but not Aβ40 content, in 3xTg mice (Figures 9i & j). In summary, #2-NP reduced Aβ brain deposition, reduced brain atrophy, and alleviated cognitive deficits in 3xTg mice.

[0108] 5. #2-NP activates TrkB and inhibits AEP enzyme activity in the brain of 3xTg mice

[0109] To investigate whether #2 biochemically activates the TrkB signaling pathway and inhibits AEP, immunoblotting of brain tissue lysates from 3xTg mice revealed a dose-dependent activation of the TrkB receptor by #2-NP, while p-PLCγ1 reflects upstream signaling at p-TrkB Y816. Therefore, p-AEP T322 is associated with the upstream TrkB signaling pathway, which is negatively coupled to the active AEP fragment. Both APP N585 and Tau N368 cleavage gradually decreased. Consistent with Tau N368 cleavage, p-Tau AT8 levels also gradually decreased (Figure 10a). AEP enzymatic assay revealed a dose-dependent inhibitory effect of #2-NP. At the same dose, the biochemical activity of #2-NP was significantly higher than that of #2, consistent with the increased brain concentration of #2 achieved by the nanoparticle system (Figures 10b-i).

[0110] 6. #2-NP reduces AD pathology in 3xTg mice

[0111] To further analyze the therapeutic effect of #2-NP on AD pathology, brain sections of drug-treated 3xTg mice were immunofluorescently stained for p-TrkB staining (IF). IF staining showed that p-TrkB 816 in the hippocampus was significantly activated by both #2 and #2-NP (Figures 7A&E). Therefore, the p-AEP IF signal was closely correlated with upstream p-TrkB activity (Figures 11b&f). At the same time, active AEP IF staining was negatively coupled to p-AEP activity, leading to a gradual decrease in APP C585 levels. Consistent with these findings, Tau N368 signals and AT8 IF intensity were closely correlated with each other (Figures 7C&D). Quantification of fluorescence intensity confirmed these observations (Figures 11g-j). In summary, these results strongly support that #2-NP increases #2 brain exposure, increases TrkB activation and inhibits AEP in the brain, thereby improving AD pathology and cognitive dysfunction in 3xTg mice.

[0112] Example 3

[0113] 1. Preparation and characterization of liposomes

[0114] Preparation of Compound #2-LIP: Compound #2 and phosphatidylcholine (CAS No. 8002-43-5) were dissolved in anhydrous ethanol to obtain a mixed solution. The mixed solution was added to deionized water with stirring. After evaporation of the solvent, a crude liposome solution was obtained. The crude liposome solution was sonicated in an ice bath for 5-10 minutes. Chitosan was dissolved in deionized water and the liposome solution was added to the chitosan aqueous solution with stirring to obtain liposomes loaded with Compound #2 (#2-LIP). The mass ratio of phosphatidylcholine:chitosan:derivative was 20:2:1.

[0115] The prepared liposomes were first physically characterized. The results (Table 5) show that the particle size of #2-LIP was 298.77 nm, the PDI was 0.54, and the zeta potential was 23.97 mV, demonstrating the successful preparation of the liposomes in this example. High-performance liquid chromatography (HPLC) analysis revealed an encapsulation efficiency of 51.23%, slightly lower than that of the nanoparticles.

[0116] Table 5 Physical properties of liposomes

[0117] 2. Comparison of PK parameters between nasal administration and liposomes

[0118] To investigate the brain concentration of nasal and liposomal drugs, an in vivo pharmacokinetic study was conducted using 2-month-old ICR mice and compared the PK parameters with those of the previous study of #2-NP.

[0119] Results showed that intranasal administration (IN) of 5 mg / kg of compound #2 resulted in a maximum plasma concentration (Cmax) of 947.291 ng / mL, approximately 10 times that of oral administration (90.809 ng / mL). However, the drug half-life (t1 / 2) was approximately half that of oral administration (1.542 h and 4.092 h), resulting in a decrease in the mean residence time (MRT: 2.114 h) and clearance (CL: 2.637 L / h / kg) compared to oral administration of compound #2. The area under the curve (AUC) for intranasal administration was 1896.266 ng / mL*h, significantly greater than the AUC for oral administration (405.15 ng / mL*h). Therefore, the bioavailability of compound #2 after intranasal administration increased from 6.99% to 34.3% compared to oral administration (Figure 13a). Regarding drug concentrations in the brain, the maximum concentration level for #2 was 18.47 ng / g following oral administration and 27.663 ng / g following nasal administration, with brain half-lives of 3.49 h and 2.395 h, respectively. The areas under the curve for oral and nasal administration were 35.75 and 78.34 ng / mL*h, respectively, more than doubling (Figure 13b). In summary, nasal administration increased bioavailability by more than fourfold compared to oral administration, and brain exposure more than doubled. Therefore, nasal administration is the superior route of administration for #2.

[0120] Similarly, the in vivo metabolism of #2 after oral liposome delivery was examined. Results showed that, at the same oral dose (5 mg / kg), the maximum blood concentration of the compound after oral administration of #2-LIP was 1408.38 ng / mL, the half-life was 11.24 hours, the area under the curve was 2996.664 ng / mL*h, the mean residence time was 17.75 hours, and the clearance rate was 0.93, all demonstrating superior PK parameters compared to oral administration of #2-NP (Figure 13a). Consequently, the bioavailability of #2-NP after oral administration was 51.76%. In terms of drug exposure in the brain, #2-LIP exhibited similar parameters to #2-NP (Figure 13b). This is inconsistent with expectations, and it is speculated that this may be due to the fact that #2-NP enters the bloodstream in the intact form after oral administration, while #2-LIP may enter the bloodstream in a liposome-derived form, which does not facilitate the cross-brain barrier (BB) of #2. Further experimental confirmation is required.

[0121] After nasal administration, #2-LIP demonstrated excellent PK parameters and brain exposure. Results showed that the maximum blood concentration of the compound after nasal administration of #2-LIP was 7469.15 ng / mL, significantly greater than the 947.29 ng / mL after nasal administration of #2. The time it took for the drug to reach its peak was 0.25 hours, shorter than the 0.5 hours after nasal administration of #2. This may be due to the fact that #2-LIP is more easily absorbed through the nasal mucosa. After IN #2-LIP, the compound had a half-life of 1.254 hours, an average residence time of 1.386 hours, and a clearance rate of 0.9, all of which were lower than those of IN #2. The area under the curve was 5609.404 ng / mL*h, and the bioavailability was 96.89% (Figure 13a). Similarly, we tested brain tissue #2 and found that after IN #2-LIP, it showed similar parameters to IV #2 2 mg / kg, with a maximum brain concentration of 159.68 ng / g, a time to maximum concentration of 0.25 h, a half-life of 1.667 h, and an area under the curve of 134.16 ng / mL*h ( Figure 13b ).

[0122] After a comprehensive comparison of the two preparations (nanoparticles and liposomes) and administration methods (oral and nasal), the present invention concluded that for #2, the optimal administration method is nasal administration of liposomes, and we will also conduct subsequent pharmacodynamic studies based on this.

[0123] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A CF3CN derivative, characterized in that The CF3CN derivative has a structural formula as shown in any one of the following formulas 1-4:

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one or more of the CF3CN derivatives shown in the following formulas 1-4, and a pharmaceutically acceptable carrier or excipient.

3. A nanoparticle, characterized in that: The nanoparticles include one or more of the CF3CN derivatives shown in the following formulas 1-4 and zein, and the nanoparticles are prepared by an anti-solvent co-precipitation method.

4. The nanoparticle according to claim 3, characterized in that The nanoparticles further comprise lactoferrin or glycosylated lactoferrin.

5. The nanoparticle according to claim 3, characterized in that The mass ratio of the CF3CN derivative to zein is 1:1 to 1:

20.

6. The nanoparticle according to claim 4, characterized in that The mass ratio of the CF3CN derivative, zein and lactoferrin or glycosylated lactoferrin is (1-2):(1-20):(1-20).

7. The nanoparticle according to claim 3, characterized in that The nanoparticles include the CF3CN derivative shown in Formula 2, zein and lactoferrin, and the mass ratio of the CF3CN derivative shown in Formula 2, zein and lactoferrin is (1-2):(1-20):(1-20).

8. The nanoparticle according to claim 3, characterized in that The nanoparticles are prepared by an anti-solvent co-precipitation method: one or more of the CF3CN derivatives shown in Formulas 1-4 and zein are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution, and the mixed solution is added to deionized water while stirring. After the solvent evaporates, the nanoparticles are obtained.

9. The nanoparticle according to claim 4, characterized in that The nanoparticles are prepared by an anti-solvent co-precipitation method: one or more of the CF3CN derivatives shown in Formulas 1-4, zein and lactoferrin are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution, and the mixed solution is added to deionized water while stirring, and the nanoparticles are obtained after the solvent evaporates; or One or more of the CF3CN derivatives shown in Formulas 1-4 and zein are dissolved in an ethanol aqueous solution with a volume fraction of 70%-80% to obtain a mixed solution; glycosylated lactoferrin is dissolved in the aqueous solution, and the mixed solution is added to the glycosylated lactoferrin aqueous solution while stirring, and nanoparticles are obtained after the solvent evaporates.

10. A liposome, characterized in that: The liposomes include one or more of the CF3CN derivatives shown in the following formulas 1-4 and lecithin, and the liposomes are prepared by an ethanol injection method. Preferably, the mass ratio of the lecithin to the CF3CN derivative is (1:1) to (50:1).

11. The liposome according to claim 10, characterized in that The liposome also includes a stabilizer; Preferably, the stabilizer is selected from chitosan; Preferably, the mass ratio of the lecithin, chitosan and derivative is (1:1:1) to (50:1:1).

12. The liposome according to claim 10, characterized in that The liposomes are prepared by an ethanol injection method: one or more of the CF3CN derivatives shown in Formula 1-4 and lecithin are dissolved in anhydrous ethanol to obtain a mixed solution, and the mixed solution is added to deionized water while stirring. After the solvent evaporates, a crude liposome solution is obtained; Preferably, the method further comprises subjecting the crude liposome solution to ice bath ultrasonic treatment for 5-10 minutes; dissolving chitosan in deionized water, and adding the liposome solution to the chitosan aqueous solution while stirring, to obtain liposomes loaded with the derivative.

13. Use of the CF3CN derivative according to claim 1 in the preparation of a TrkB agonist, or use of the CF3CN derivative according to claim 1, the pharmaceutical composition according to claim 2, the nanoparticles according to any one of claims 3 to 9, or the liposomes according to any one of claims 10 to 12 in the preparation of a drug for preventing and / or treating Alzheimer's disease.

14. A method for preventing and / or treating Alzheimer's disease, characterized in that: The method comprises administering to a subject a therapeutically effective amount of the CF3CN derivative of claim 1, the pharmaceutical composition of claim 2, the nanoparticles of any one of claims 3-9, or the liposomes of any one of claims 10-12.

15. The method according to claim 14, characterized in that The administration method is oral administration or nasal administration.

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