Heptanoate compound and use thereof in preparation of drug or functional food for preventing and / or treating neurodegenerative diseases

Abstract

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and use thereof for preparing an anti-neurodegenerative disease drug or functional food. Research shows that the compound acts against aging-related mechanisms of action, demonstrating a good effect in treating and / or preventing AD.

Description

An enanthate compound and its use in preparing medicine or functional food for preventing and / or treating neurodegenerative diseases Technical Field

[0001] The present invention relates to a new heptanoate structure compound SL-ZF-01 and use thereof in preparing medicines or functional foods for preventing and / or treating neurodegenerative diseases, belonging to the field of medicine. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder classified as a neurocognitive disorder in the DSM-5 (Diagnostic and statistical manual of mental disorders, fifth edition). AD presents with memory impairment and other cognitive impairments, manifesting as dementia, confusion, visual-spatial disorientation, language impairment, aphasia, and impairments in calculation and decision-making. Psychiatric symptoms such as anxiety, depression, sleep disorders, delusions, and hallucinations may also occur. It is the most common type of dementia in the elderly, often developing in people over 65 years of age, with a typical survival of 5-10 years.

[0003] In 1906, German neurologist Alois Alzheimer first reported a case of Alzheimer's disease in a 51-year-old woman whose brain showed significant changes at autopsy, but the high prevalence of the disease was not recognized until the latter decades of the 20th century.

[0004] Alzheimer's disease is currently the fourth leading cause of death in the United States. An estimated 2.5 to 4 million people in the United States suffer from the disease, with approximately 360,000 new cases each year. The disease costs the U.S. economy approximately $75 billion to $100 billion annually. The lifetime risk of developing Alzheimer's disease is approximately 9%, with women approximately 1.5 times more likely to be affected than men. The onset of AD is typically insidious, progressively worsening with aging, which leads to impairments in learning and memory. When recalling and repeating new material, AD patients make intrusive errors—inserting irrelevant words or thoughts, resorting to fabricated knowledge, and experiencing decreased direction. Fifty percent of AD patients experience depression, 20% experience delusions, and 70% experience psychotic symptoms such as agitation. Many medications, including many not considered psychoactive, can exacerbate Alzheimer's disease symptoms. Clinical depression can often mask or mimic dementia, and vice versa. Movement disorders are a common side effect of antipsychotic medications and can significantly impact the quality of life of AD patients. The manifestations of the nervous system may be basically normal, but in the late stage of the disease, muscle tremors, bradykinesia, rigidity, and other symptoms similar to Parkinson's disease and epileptic seizures may occur. Death is usually due to the inability of AD patients to perceive their physical health normally, such as urinary tract or lung infections, and eventually they will die of sepsis due to the delay of these infections. The cerebral cortex atrophies, followed by enlargement of the cerebral sulci and ventricles, which may be very obvious in imaging examinations. Histologically, the cortex, hippocampus, and amygdala atrophy. Vacuoles and argyrophilic granules can be seen in neuronal tissue. Neural plaques composed of granular or filamentous argyrophilic clusters have a core of 42 amino acids in the form of Aβ. 42 The peptide is surrounded by dense astrocytes and microglia. There are also tangles in the nerve cells caused by the hyperphosphorylation of microtubule tau protein. The concentration of tau protein in cerebrospinal fluid is increased, and Aβ 42 Concentrations decrease. Almost all patients with Down syndrome over the age of 40 experience cognitive decline, accompanied by typical AD-like pathology. Advanced age, depression, low education level, smoking, and a history of severe intracranial injury are known high-risk factors for AD.

[0005] The World Health Organization (WHO) pointed out in 2021 that the world today has failed to effectively respond to the challenge of AD. Numerous research results and drug research results show that the available drugs for clinical treatment of AD are very limited so far. In 2022, the US Grant Institute summarized all clinical trials and drugs for the treatment of AD. There are 143 drugs under development, and more drugs have been announced to have failed clinical trials. Among these 143 drugs, disease mechanism improvement therapies account for 83.2% of candidate therapies, and biological products account for 50%. 68% of clinical trials are phase 3 clinical trials submitted by biopharmaceutical companies. 63% of phase 3 clinical trials and 46% of phase 2 clinical trials are conducted in the United States. The main mechanisms of action of these clinical trial drugs can be divided into 12 categories, namely (1) Aβ amyloid protein (2) epigenetic (3) immune (4) metabolism / biosynthesis (5) neurogenesis (6) neurotransmitter receptors (7) others (8) oxidative stress (9) protein homeostasis (10) synaptic plasticity (11) Tau protein (12) vascular system. The (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) involved in the present invention has a mechanism of action and efficacy that can be covered in four categories: (1), (4), (10) and (12).

[0006] Alzheimer's Disease International (ADI) released the "World Alzheimer's Disease 2018 Report", which shows that there are at least 50 million dementia patients in the world, and it is expected to rise to 152 million by 2050, of which about 60%-70% are AD patients. Since AD ​​patients have significant cognitive dysfunctions such as memory, thinking, and emotion, it has led to a heavy socioeconomic burden and disease burden. The disease mechanism of AD is not yet fully understood. The traditional hypothesis is that Aβ 40-42 The abnormal aggregation of Aβ and the subsequent formation of Aβ plaques are the initiating factors of AD. Aβ plaque deposition is also closely linked to tau hyperphosphorylation, forming fibrillary tangles, acetylcholine deficiency, neuroinflammation, hypertension, and diabetes, leading to numerous theoretical hypotheses. Of particular note is the close relationship between AD and aging, as the incidence of AD in people aged 65 to 95 increases exponentially with age.

[0007] Based on these new developments, the National Institute of Neurological Disorders and Stroke–Alzheimer Disease and Related Disorders (NINCDS–ADRDA) has developed the more widely used diagnostic criteria for AD: (1) early and significant episodic memory impairment; (2) atrophy of the middle temporal gyrus; (3) abnormal cerebrospinal fluid biomarkers; (4) specific imaging with PET functional neuroimaging; and (5) a clear autosomal dominant mutation associated with AD in a first-degree relative.

[0008] Item (1) plus any other item or items in the above diagnostic criteria can be used to diagnose AD. Item (4) indicates decreased glucose metabolism in the bilateral temporal and parietal lobes.

[0009] In addition to episodic memory impairment, the clinical symptoms of AD patients also include a variety of psychiatric and other cognitive symptoms. Psychiatric symptoms refer to anxiety, depression, and psychosis symptoms that are common in AD patients. Currently, benzodiazepines and other drugs are used clinically to treat anxiety, agitation, and insomnia. Doxepin (a classic tricyclic antidepressant and anxiety drug) and maprotiline (a norepinephrine reuptake transporter inhibitor, an antidepressant) are used to treat depressive symptoms. Low-dose perphenazine and other drugs are used to treat psychotic symptoms.

[0010] The U.S. Food and Drug Administration (FDA) has approved several anti-AD drugs for marketing. Clinical studies have found that there is no significant difference between patients who took these anti-AD drugs and those who did not take them over a five-year period, suggesting that these anti-AD drugs cannot prevent the progression of AD and may only have the effect of temporarily alleviating cognitive dysfunction. These anti-AD drugs include donepezil, rivastigmine, galantamine, memantine, and a combination of donepezil and memantine. In 2019, the China National Medical Products Administration approved the marketing of sodium mannuronate capsules (trade name "Jiujiyi"), and its mechanism of action may be to improve brain-gut axis function. Almost at the same time, the U.S. FDA approved the monoclonal antibody adulan for the treatment of AD. In January 2023, the U.S. FDA approved the monoclonal antibody lencain for the treatment of AD. These two drugs are biological products, and their mechanism of action is to use monoclonal antibodies to target and clear Aβ amyloid protein. The effectiveness of these three newly marketed drugs in treating AD still needs to be tested with large-sample clinical data.

[0011] Aging may be the initial key factor in the development of AD. The aging process involves changes in glucose metabolism and cerebrovascular systems. Currently, AD patients' glucose metabolism issues are primarily addressed with type 2 diabetes medications, which have shown some effectiveness.

[0012] Reviewing the above progress, the present invention utilizes artificial design and artificial chemical synthesis to design and synthesize enanthate ester compounds. Research has found that one of the enanthate esters, SL-ZF-01, and its biotin-SL-ZF-01, have significant efficacy in improving cognitive dysfunction in AD mice. Its mechanism of action may be to improve the expression and function of genes upstream and downstream of the type 1 glucose transporter, including the tricarboxylic acid cycle, during aging, thereby effectively improving glycolysis and oxidative phosphorylation in AD mice, thereby achieving improved cognitive function. Therefore, this invention shows that SL-ZF-01 can be used as a main ingredient or additive in medicines and foods for the prevention and / or treatment of AD during aging, especially cognitive dysfunction in AD patients. It may also be used to prevent and / or treat the natural decline in cognitive function in normal people during aging.

[0013] Summary of the Invention

[0014] The present invention provides a compound represented by Formula I or a pharmaceutically acceptable salt thereof, and its use in preparing a medicament or functional food for preventing and / or treating neurodegenerative diseases. The compound represented by Formula I is named (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diheptanoate (SL-ZF-01), and its structure is shown below:

[0015] According to an embodiment of the present invention, the patient may be a human.

[0016] According to an embodiment of the present invention, the neurodegenerative diseases include diseases caused by Aβ amyloid protein, immune inflammation, glucose metabolism, synaptic transmission, and vascular lesions.

[0017] According to an embodiment of the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, amyotrophic lateral sclerosis, and the like.

[0018] According to an embodiment of the present invention, the onset characteristics of Alzheimer's disease include memory impairment, aphasia, apraxia, agnosia, visual-spatial ability impairment, abstract thinking and calculation impairment, personality and behavior changes, etc. In severe cases, it includes emotional indifference, erratic crying and laughing, loss of speech ability, inability to take care of oneself, etc.

[0019] According to an embodiment of the present invention, the causes of Alzheimer's disease include Aβ amyloid protein, immune inflammation, glucose metabolism, synaptic transmission, and vascular lesions.

[0020] According to an embodiment of the present invention, the cause of Parkinson's disease includes the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which causes a significant decrease in the dopamine content in the striatum and leads to the disease, and involves the characteristic symptoms of resting tremor, bradykinesia, muscle rigidity and posture and gait disorders.

[0021] According to an embodiment of the present invention, the onset of Huntington's disease includes progressive movement disorders manifested as sudden, rapid jumps or twitches of the limbs, face, and trunk. These movements are not known in advance and cannot be controlled. They can also be manifested as uncontrollable slow movements. Physical examination reveals choreiform involuntary movements and decreased muscle tone. Choreiform involuntary movements are the most prominent feature of this disease. Most of them begin with short, uncontrollable grimacing, nodding, and finger flexion and extension movements, similar to painless convulsions, but slower and non-stereotyped. As the disease progresses, the involuntary movements become more severe, with typical eyebrow raising and head flexion. When looking at an object, the head turns with it. The patient becomes unstable when walking, has a leaping gait, and constantly changes the posture of the hands, and the whole body movements are like dancing. In the late stage of the disease, the patient cannot stand or walk due to involuntary movements of the whole body.

[0022] According to an embodiment of the present invention, the pathogenesis of prion diseases includes related features of kuru, Creutzfeldt-Jakob syndrome, Gerstmann-Straussmann syndrome and fatal familial insomnia.

[0023] According to an embodiment of the present invention, the pathogenesis of amyotrophic lateral sclerosis includes limb-onset and bulbar-onset. The symptoms of the limb-onset type are first progressive atrophy and weakness of the limb muscles, and finally respiratory failure, while the bulbar-onset type is manifested by early swallowing and speaking difficulties, which quickly progress to respiratory failure.

[0024] The present invention also provides a use of a pharmaceutical composition containing the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicine or functional food for preventing and / or treating neurodegenerative diseases.

[0025] According to an embodiment of the present invention, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, amyotrophic lateral sclerosis, etc. The compound represented by Formula I of the present invention or a pharmaceutically acceptable salt thereof can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99% of the compound represented by Formula I or a pharmaceutically acceptable salt thereof, and the rest is a pharmaceutically acceptable, non-toxic, non-inert pharmaceutical carrier and / or excipient to humans and animals.

[0026] The pharmaceutical carrier or excipient is one or more solid, semisolid, and liquid diluents, fillers, and pharmaceutical product adjuvants. Pharmaceutical compositions containing the compound of Formula I or a pharmaceutically acceptable salt thereof are formulated using formulations generally recognized in the pharmaceutical and food industries, such as solid preparations such as tablets, capsules, granules, and granules.

[0027] The drug administration routes of the present invention include injection (intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection), oral administration, sublingual administration, mucosal dialysis, etc.

[0028] The effective dose of the compound shown in Formula I or a pharmaceutically acceptable salt thereof is 10-100 mg / kg / mouse / day, for example 10-50 mg / kg / mouse / day, such as 20 mg / kg / mouse / day. According to the U.S. FDA official website on the "Guidelines for Estimating the Maximum Recommended Starting Dose of Drugs for the First Clinical Trial in Healthy Adult Volunteers", the conversion of animal and human equivalent doses is performed according to the body surface area conversion method. The effective dose for humans should be 0.1-10 mg / kg / person / day, for example 0.5-5 mg / kg / person / day, such as 1 mg / kg / person / day, 1.5 mg / kg / person / day, 1.63 mg / kg / person / day, 2 mg / kg / person / day. Acceptable formulations are used to treat and prevent the above-mentioned diseases, disease causes and symptoms.

[0029] The present invention provides a method for preparing a compound represented by formula I, comprising the following steps:

[0030] (1) condensing the compound represented by formula II with glycerol acetone ketal in the presence of a condensing agent to obtain the compound represented by formula III;

[0031] (2) removing the acetone protecting group from the compound represented by formula III in the presence of an acid to obtain the compound represented by formula IV;

[0032] (3) the compound represented by formula IV is condensed with heptanoic acid in the presence of a condensing agent to obtain the compound represented by formula V;

[0033] (4) The compound represented by formula V is subjected to removal of the R protecting group to obtain the compound represented by formula I.

[0034] Wherein, R is selected from one of trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, benzyl, p-methoxybenzyl and methoxymethyl.

[0035] According to an embodiment of the present invention, the condensing agent in step (1) and step (3) is selected from one or more of DCC, EDCI, HATU, HOBT, BOP, PyBOP, DPP-Cl, and DPPA.

[0036] According to an embodiment of the present invention, the acid in step (2) is selected from one or more of trifluoroacetic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, and nitric acid.

[0037] According to an embodiment of the present invention, steps (1) to (4) can be carried out in a solvent selected from one or more of water, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran, acetone, ether, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, chloroform, 1,4-dioxane, etc.

[0038] According to an embodiment of the present invention, after obtaining the corresponding compound in each step of the reaction in the preparation method, it can be processed by conventional methods in the art, such as extraction, drying, concentration, distillation, beating, crystallization or column chromatography purification. Beneficial effects

[0039] The present invention provides a compound represented by Formula I or a pharmaceutically acceptable salt thereof ((E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01), or a pharmaceutical composition containing the same for use in preparing a drug for treating neurodegenerative diseases, particularly a drug and functional food for preventing and / or treating AD. The present invention provides a new approach for preventing and treating AD, and can also help understand the characteristics of AD and other aging-related brain diseases, and can help discover the pathogenesis of AD. Furthermore, the molecular signaling pathways and potential targets intervened by the compound represented by Formula I or a pharmaceutically acceptable salt thereof may also have similar preventive and therapeutic effects on neurodegenerative diseases such as Parkinson's disease, Huntington's disease, prion disease, and amyotrophic lateral sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1: H NMR spectrum of glyceryl ferulate heptanoate.

[0041] Figure 2: C NMR spectrum of glyceryl ferulate heptanoate.

[0042] Figure 3: Mass spectrum of glyceryl ferulate heptanoate. DETAILED DESCRIPTION

[0043] The technical solutions of the present disclosure will be further described in detail below with reference to specific examples. It should be understood that the following examples are merely illustrative of and explain the present disclosure and should not be construed as limiting the scope of protection of the present disclosure. All technologies implemented based on the above content of the present disclosure are encompassed within the scope of protection intended by the present disclosure. The use of the compounds claimed in the present invention by any other method and the use of the compounds of the present invention in AD and related diseases such as Parkinson's disease, Huntington's disease, prion disease, and amyotrophic lateral sclerosis should also be within the scope of protection of this patent.

[0044] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The present invention uses the following abbreviations: eq represents equivalent; SM1 represents (E)-3-(4-((tert-butyldimethylsilyl)oxy)-3-methoxyphenyl) acrylic acid; M1 represents (2,2-dimethyl-1,3-dioxolane-4-yl)methyl (E)-3-(4-((tert-butyldimethylsilyl)oxy)-3-methoxyphenyl) acrylate; M2 represents 2,3-dihydroxypropyl (E)-3-(4-((tert-butyldimethylsilyl)oxy)-3-methoxyphenyl) acrylate; M3 represents (E)-3-((3-(4-((tert-butyldimethylsilyl)oxy)-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate; DCC represents N,N'-dicyclopentyl Hexylcarbodiimide; EDCI stands for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HATU stands for 2-(7-azobenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT stands for 1-hydroxybenzotriazole; BOP stands for benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; PyBOP stands for 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate; DPP-Cl stands for diphenylphosphinyl chloride; DPPA stands for diphenylphosphoryl azide; TFA stands for trifluoroacetic acid; TBAF stands for tetrabutylammonium fluoride; DCM stands for dichloromethane; PE stands for petroleum ether; EA stands for ethyl acetate; THF stands for tetrahydrofuran; and DMAP stands for 4-dimethylaminopyridine.

[0045] The compounds were named according to conventional nomenclature in the art or using ChemOffice software, and commercially available compounds were named according to the supplier's catalog name.

[0046] A glyceryl ferulate heptanoate as shown in formula I, named (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diheptanoate (SL-ZF-01), and pharmaceutically acceptable salts thereof.

[0047] Example 1: Preparation of glyceryl ferulate heptanoate

[0048] Step 1: Preparation of intermediate M1

[0049] Glycerol acetonide (19.00 g, 143.77 mmol, 1.0 eq.) was dissolved in 500 ml of dichloromethane, the atmosphere was replaced with argon, and the temperature was lowered to 0°C. SM1 (44.34 g, 143.77 mmol, 1.0 eq.), DMAP (8.78 g, 71.88 mmol, 0.5 eq.), and DCC (38.56 g, 186.90 mmol, 1.3 eq.) were added portionwise with stirring. The mixture was then brought to room temperature and stirred for 16 h. The next day, the reaction mixture was filtered to remove insoluble matter. 300 ml of water was added to the filtrate, and the mixture was extracted with DCM (200 ml x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. M1 was purified by column chromatography on a 0-10% EA / PE column to afford 52.11 g of M1 as a pale yellow oil (yield: 86%).

[0050] 1 H NMR(500MHz, CDCl3)δ7.64(dt,J=15.6,4.9Hz,1H),7.00(q,J=5.6Hz,2H),6.83(dt,J=13.1,6.4Hz,1H),6.43–6.18(m,1H),4.42 –4.23(m,2H),4.22–4.03(m,2H),3.90–3.70(m,4H),1.48–1.37(m,3H),1.36(d,J=11.8Hz,3H),1.01–0.79(m,9H),-0.01(s,6H).

[0051] Step 2: Preparation of intermediate M2

[0052] Intermediate M1 (1.00 g, 2.37 mmol, 1.0 eq.) was dissolved in 8 mL of dichloromethane and stirred at room temperature. 2 mL each of TFA and H₂O were added, followed by stirring at room temperature for 16 h. The reaction mixture was diluted with 50 mL of water and extracted with EA (40 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. Column chromatography with 0-50% EA / PE afforded 330.10 mg of M2 as a pale yellow oil (yield: 36%).

[0053] 1H NMR(500MHz, CDCl3)δ7.65(d,J=15.8Hz,1H),7.01(q,J=5.5,4.8Hz,2H),6.94–6.75(m,1H),6.42–6.19(m,1H),4.37–4.23(m,2H),4.00(q,J=5.1H z,1H),3.82(t,J=4.0Hz,3H),3.76–3.69(m,1H),3.65(dd,J=11.2,5.7Hz ,1H),2.86(s,1H),2.43(s,1H),0.99(d,J=2.7Hz,9H),0.18–0.10(m,6H).

[0054] Step 3: Preparation of intermediate M3

[0055] Intermediate M2 (14.71 g, 38.45 mmol, 1.0 eq.) was dissolved in 150 mL of dichloromethane, the atmosphere was replaced with argon, and the mixture was cooled to 0°C. Heptanoic acid (15.02 g, 115.36 mmol, 3.0 eq.), DMAP (4.70 g, 38.45 mmol, 1.0 eq.), and DCC (20.63 g, 99.98 mmol, 2.6 eq.) were added portionwise with stirring. The mixture was then brought to room temperature and stirred for 16 h. The next day, the reaction mixture was filtered to remove insoluble matter. 300 mL of water was added to the filtrate, and the mixture was extracted with DCM (200 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. M3 was purified by column chromatography using 0-5% EA / PE to afford 17.60 g of M3 as a light yellow oil in a 75% yield.

[0056] 1 H NMR (500MHz, CDCl3) δ7.62 (dd, J=15.9, 2.4Hz, 1H), 7.02 (d, J=4.1Hz, 2H), 6.84 (dd, J=8. 7,2.6Hz,1H),6.28(dd,J=15.9,2.4Hz,1H),5.35(t,J=5.2Hz,1H),4.44–4.26(m,3H),4.2 1(ddd,J=12.2,6.2,2.3Hz,1H),3.84(d,J=2.5Hz,3H),2.33(q,J=7.0,6.3Hz,4H),1.70– 1.54(m,4H),1.29(dd,J=15.6,7.0Hz,12H),0.99(s,9H),0.91–0.84(m,6H),0.17(s,6H).

[0057] Step 4: (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diheptanoate

[0058] Intermediate M3 (17.62 g, 29.03 mmol, 1.0 eq.) was dissolved in 100 ml of THF and stirred in an ice bath. TBAF (22.77 g, 87.10 mol, 3.0 eq.) was dissolved in 45 ml of THF and slowly added dropwise to the reaction mixture. After complete addition, the mixture was brought to room temperature and stirred for 0.5 h. The reaction mixture was quenched by pouring into ice water and extracted with EA (100 ml x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. 0-10% EA / PE column chromatography afforded 12.31 g of a white powdery solid (86% yield).

[0059] 1 H NMR (600MHz, CDCl3) δ7.62(d,J=15.9Hz,1H),7.07(d,J=8.2Hz,1H),7.03(s,1 H),6.92(d,J=8.2Hz,1H),6.27(d,J=15.9Hz,1H),5.93(d,J=0.7Hz,1H),5.37– 5.32(m,1H),4.40–4.28(m,3H),4.21(dd,J=11.6,5.8Hz,1H),3.93(s,3H),2. 33(q,J=7.4Hz,4H),1.68–1.56(m,4H),1.35–1.22(m,12H),0.91–0.80(m,6H).

[0060] 13 C NMR (151MHz, CDCl3) δ173.36,172.95,166.64,148.23,146.83,145.77,126.79,123.34, 114.76,114.52,109.39,69.00,62.27,55.98,34.18,31.43,28.75,24.86,22.46,13.99.

[0061] HRMS (ESI): m / z: 491.2592 [MH]-

[0062] Example 2

[0063] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared, 1% DMSO was added as usual, Tween-20 was added to promote dissolution, and water for injection was added as usual. The mixture was finely filtered, and the mixture was sterilized by filling to prepare an injection solution.

[0064] Example 3

[0065] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared, added to the mouse feed, mixed evenly, and pressed into mouse feed pellets.

[0066] Example 4

[0067] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared, and the excipient was added at a weight ratio of 9:1 to the excipient to prepare a powder.

[0068] Example 5

[0069] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared, and the excipients were added at a weight ratio of 1:5-1:10 to the excipients to prepare tablets, capsules, granules, or granules.

[0070] Example 6

[0071] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared, and the excipients were added in a weight ratio of 5:1 to the excipients to prepare capsules, granules or granules.

[0072] Example 7

[0073] According to the method of Example 1, (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) was first prepared. 600 g of starch, 200 g of lactose, 5 g of menthol, and 183 g of sodium carboxymethyl starch were added to every 12.4 g to prepare lozenges as a functional food.

[0074] To better understand the essence of the present invention, the pharmacological effects of a pharmaceutical composition consisting of (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) and a pharmaceutical carrier or excipient are used below to illustrate the essence of the present invention, but the content of the present invention is not limited thereto.

[0075] Example 8 Therapeutic Effect of (E)-3-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)propane-1,2-diyl diheptanoate (SL-ZF-01) on APP / PS1AD Mouse Model

[0076] 1. Experimental methods

[0077] This study used APP / PS1 double transgenic mice and littermate negative control mice (B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax; JacksonLab, purchased from the Model Animal Center of Nanjing University). APP / PS1 mice are double transgenic mice expressing a chimeric Swedish mutation of mouse / human amyloid precursor protein (Mo / HuAPP695swe) and a mutant human presenilin protein 1 (PS1-dE9). Two expression plasmids (Mo / HuAPP695swe and PS1-dE9) are controlled by mouse prion protein (PrP) promoter elements and both direct expression to central nervous system neurons. Both gene mutations are associated with early-onset or familial forms of Alzheimer's disease. The Mo / HuAPP695swe transgene expresses a humanized mouse amyloid precursor protein (A4) gene modified by three amino acids to reflect human-specific residues and further modified to contain the K595N / M596L mutations associated with familial Alzheimer's disease. The PS1-dE9 transgene expresses a human presenilin protein 1 carrying an exon 9 deletion mutation (PSEN1dE9).

[0078] Animals were housed in individually ventilated cages (IVCs) at the Experimental Center of the Kunming Institute of Zoology, Chinese Academy of Sciences. Animals were housed in groups of 4–5 per litter, with free access to food and water. The room temperature was maintained at 24 ± 2°C, with a 12-hour day / night rhythm. All animal manipulations complied with the Life Science Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences.

[0079] Female APP / PS1 mice were randomly divided into two groups at the age of 14 months and fed with control diet and 20 mg / kg SL-ZF-01 diet respectively. Morris water maze test was performed after 2 months of feeding.

[0080] The Morris water maze (MWM) assesses spatial learning and memory in mice. The Morris water maze (MWM), first developed by Richard Morris in 1981, primarily exploits rodents' instinctive escape behavior in water, which involves learning to locate an invisible escape platform hidden underwater based on spatial orientation and cues. It is widely used to assess spatial learning and memory in rodents. The experimental system consists of a water maze apparatus (Med Association, USA) and an image acquisition and analysis system (Noldus, USA). The water maze consists of a 120 cm diameter white plastic pool maintained at 21-22°C. The pool is divided into four quadrants (northeast, southeast, northwest, and southwest). Each quadrant is edged with a spatial marker of a different shape and color: a green triangle, a yellow five-pointed star, a black rectangle, or a red circle. An escape platform (10 cm in diameter) is placed in the center of one quadrant, with the platform aligned with the center of the circle and centered between the two markers. The platform is submerged 1 cm below the water surface. The water surface was covered with white non-toxic polyethylene plastic particles to conceal the escape platform. EthoVision 8.0 software was used for video recording and data analysis.

[0081] The MWM test consists of two phases: a spatial learning task (referred to as "training") and a spatial memory test (also known as the probe test). The day before training, the animals are allowed to swim freely in a pool without a platform for one minute as an acclimatization exercise. The spatial learning phase then begins. Mice are placed from four different quadrants of the pool daily, referred to as four training sessions, with a 10-minute interval between each session. This training is repeated for five consecutive days. During each training session, the animals are placed facing the pool wall and asked to search for a hidden escape platform. If the animal finds the escape platform within 60 seconds, it is allowed to remain on the platform for five seconds, then dried with a towel and returned to its home cage. If the animal fails to find the hidden platform within 60 seconds, it is guided to the platform and allowed to remain there for 20 seconds. Tracking software records various metrics, including the animal's average swimming velocity and latency to the platform. Swimming velocity assesses the animal's motor skills, while latency to the platform assesses the animal's spatial learning ability.

[0082] On the sixth day (24 hours after the last training session), a spatial memory test (also known as an exploration test) was conducted. The hidden platform was removed, and the animal was placed in the pool from the quadrant opposite the platform. The animal was allowed to swim for 60 seconds. The time spent in the target quadrant (the quadrant where the platform was located during training), the number of target entries, the time spent in the platform zone, and the latency to reach the platform zone were recorded. These data reflect the mouse's spatial memory ability.

[0083] 2. Experimental results

[0084] 2.1 Oral administration of 20 mg / kg SL-ZF-01 significantly improved the spatial memory ability of APP / PS1 mice

[0085] Table 1 Effects of 20 mg / kg SL-ZF-01 on the swimming speed of 16-month-old APP / PS1 female mice in the water maze

[0086] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05, ### P<0.01, ### P<0.001, (one-way ANOVA followed by multiple comparisons)

[0087] As shown in Table 1 above, the average swimming speeds of the three groups of mice on day 1 of the adaptation phase were not statistically significantly different among the three groups, but it seemed that the swimming speed of the wild-type group tended to be higher than that of the AD model mouse group.

[0088] Table 2 Effects of 20 mg / kg SL-ZF-01 on the water maze learning curve of 16-month-old APP / PS1 female mice

[0089] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05,### P<0.01, ### P < 0.001 (one-way ANOVA followed by multiple comparisons).

[0090] As shown in Table 2 above, the time required for mice to swim to the underwater platform from the first to the sixth day of training is called escape latency. The average latency of the three groups of mice to reach the platform from the four quadrants each day was calculated. There was no significant difference between the three groups of data on the first day. From the second to the sixth day, the latency of APP / PS1 mice increased significantly compared with WT mice. From the second to the fifth day, the average latency of APP / PS1 mice treated with SL-ZF-01 showed a downward trend. Until the sixth day, the SL-ZF-01 group showed a significant decrease in latency.

[0091] Table 3 Effects of 20 mg / kg SL-ZF-01 on the time to first reach the platform in the water maze of 16-month-old APP / PS1 female mice

[0092] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05, ### P<0.01, ### P < 0.001 (one-way ANOVA followed by multiple comparisons).

[0093] Table 3 shows an exploratory memory test conducted 24 hours after the final training session. APP / PS1 mice required significantly longer to initially locate the platform compared to WT mice, indicating impaired spatial memory. The SL-ZF-01-treated group showed a trend toward faster platform location compared to APP / PS1 mice, demonstrating that SL-ZF-01 improves the spatial memory impairment in APP / PS1 mice, with no significant difference compared to wild-type mice.

[0094] Table 4 Effect of 20 mg / kg SL-ZF-01 on the target quadrant residence time in the water maze of 16-month-old APP / PS1 female mice

[0095] *APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01,*P<0.05,**P<0.01,***P<0.001; # P<0.05, ##P<0.01, ### P < 0.001 (one-way ANOVA followed by multiple comparisons).

[0096] Table 4 shows the exploratory test of memory ability performed 24 hours after the last training session. Compared with WT mice, APP / PS1 mice spent significantly less time in the target quadrant, indicating impaired spatial memory. Compared with APP / PS1 mice, the SL-ZF-01-treated group spent significantly more time in the target quadrant, indicating significant improvement in impaired memory ability. A significant difference was observed between the two groups (##).

[0097] Table 5 Effects of 20 mg / kg SL-ZF-01 on the number of platform shuttles in the water maze of 16-month-old APP / PS1 female mice

[0098] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05, ### P<0.01, ### P < 0.001 (one-way ANOVA followed by multiple comparisons).

[0099] As shown in Table 5 above, the memory exploratory test was performed 24 hours after the last training session. Compared with WT mice, APP / PS1 mice showed a significant decrease in the number of platform crossings, indicating impaired spatial memory. Compared with APP / PS1 mice, the SL-ZF-01-treated group showed a significant increase in the number of platform crossings, indicating that the impaired spatial memory ability was significantly improved, with a statistically significant difference between the two groups (#).

[0100] Example 9 Spatial Transcriptome Analysis Study on the Mechanism of Action of SL-ZF-01 in Improving Alzheimer's Disease (AD)

[0101] 1. Experimental Methods

[0102] After the water maze test, these mice continued to receive SL-ZF-01 treatment until 24 months of age. For the spatial transcriptome analysis, one mouse was randomly selected from each of the WT, APP / PS1, and APP / PS1 + SL-ZF-01 groups. In addition, a 4-month-old WT mouse was selected as an age control.

[0103] Sampling: Place the culture dish on ice in advance, place filter paper on the surface of the culture dish, and drop 2ml of ice PBS on the filter paper to wet the filter paper. Four selected mice were anesthetized by intracavitary injection of 50mg / kg sodium pentobarbital and then killed to remove the brain. Place the brain on the filter paper and cut along the midline of the brain from one end of the olfactory bulb to the cerebellum using a surgical blade (at a 45° angle to the horizontal table). Divide the mouse brain into left and right hemispheres. Select the right hemisphere and wash away excess blood with PBS. Gently place laboratory dust-free paper on the water droplets at the edge of the tissue to absorb excess moisture; the left hemisphere is immediately placed in PFA solution for fixation.

[0104] Quick freezing: First, place the centrifuge tube containing isopentane in liquid nitrogen for pre-cooling for more than 15 minutes, then place the right hemisphere of the mouse brain into the pre-cooled isopentane, and then place the isopentane into liquid nitrogen for quick freezing for 15 seconds.

[0105] Embedding: Remove the brain tissue from the isopentane and place it in an embedding cassette pre-chilled on dry ice. After the excess isopentane evaporates, place the brain tissue in an embedding cassette containing pre-chilled OCT at 4°C. Finally, place the embedding cassette on a flat surface of dry ice for quick freezing and embedding. Record the mouse number and embedding direction on the cassette, seal it, and store at -80°C for use in 10X Visium spatial transcriptome experiments.

[0106] Slicing: Before slicing, thoroughly clean the microtome and set the temperature of the cryostat to -16°C to -20°C. Place the chip, embedded tissue, and 50ml enzyme-free centrifuge tube into the cryostat and pre-cool for more than 30 minutes to allow the temperature of the chip and embedded tissue to equilibrate to the temperature of the microtome. The slice direction is coronal, cut from the olfactory bulb end to the cerebellum end. The thickness of all slices is 16μm. The total trimming, slicing, and mounting time does not exceed 2 hours. After cutting, use a brush to smooth the slice without touching the tissue, then align the top of the chip capture area with the OCT on the top of the tissue on the slice so that the slice is adsorbed on the chip. Turn the chip over so that the slice faces upwards, and use your index finger to slowly move from the top corner of the capture area to the other corner of the bottom of the capture area from the back of the chip. Use the warmth of your finger to evenly fit the slice into the capture area of ​​the chip. The entire process is performed in a freezing microtome. After the capture areas of the chip are covered with tissue, the chip is placed in a pre-cooled 50ml enzyme-free centrifuge tube, which is then placed in dry ice and stored at -80°C.

[0107] Prepare Tris-Acetic Acid Buffer by weighing 11g of Tris Base and dissolving it in 100ml of nuclease-free water. Add 100% acetic acid dropwise to adjust the pH to 6.0, and continue adding nuclease-free water to bring the volume up to 200ml. Finally, filter the solution through a 0.2μm filter and store at room temperature. Add 50ml of ultrapure water to a 50ml centrifuge tube, and add 800ml of ultrapure water to beakers 1, 2, and 3, respectively. Place the Thermocycler Adapter in a PCR instrument and incubate at 37°C for at least 30 minutes. Leave the lid of the PCR instrument open. Place the chip face up on the Thermocycler Adapter and bake at 37°C for 1 minute to prevent the tissue from detaching during subsequent experiments. Then, place the chip in pre-cooled methanol at -20°C and incubate in a -20°C refrigerator for 30 minutes to fix the tissue.

[0108] Remove the chip from -20°C and wipe off any excess methanol from the back of the chip. Incubate the tissue with 500 μl / capture area of ​​isopropanol at room temperature for 1 minute. After incubation, discard the isopropanol and air-dry the chip until the tissue becomes transparent. Add 1 μl of RiboLock RNase Inhibitor to 1 ml of hematoxylin solution and evenly coat the tissue with hematoxylin. Incubate at room temperature for 7 minutes. After incubation, discard the hematoxylin dye. Rinse the chip up and down five times in 50 ml of ultrapure water, then 15 times in beaker 1, and 15 times in beaker 2. Wipe dry the back of the chip. Cover the tissue evenly with 1 ml of blueing solution and incubate at room temperature for 2 minutes. After incubation, rinse the chip up and down five times in beaker 2, and then wipe dry the back of the chip.

[0109] Prepare 1 ml of eosin solution at a ratio of 1:9 stock eosin solution to Tris-Acetic Acid Buffer, evenly covering the tissue. Incubate at room temperature for 1 minute. After incubation, rinse the sample up and down 15 times in beaker 3. Air-dry the chip at room temperature until the tissue is transparent. Bake the chip face-up in a Thermocycler Adapter at 37°C for 5 minutes. Use a microscope to simultaneously capture an H&E image of the capture zone of the chip. The final H&E image should clearly show the cell structure and the boundaries of the capture zone.

[0110] Next, permeabilize the tissue on the chip. Prepare a 0.1× SSC solution in a 50ml centrifuge tube: add 250μl of 20× SSC to 49.75ml of nuclease-free water and store at room temperature. Prepare a 0.2× SSC solution in a 50ml centrifuge tube: add 450μl of 20× SSC to 44.55ml of nuclease-free water and store at room temperature. Prepare a 2× SSC / 0.1% SDS solution in a 50ml centrifuge tube: add 4.5ml of 20× SSC and 450μl of 10% SDS to 40.05ml of nuclease-free water and preheat to 50°C. The permeabilization chip has a total of eight capture zones. Set up a positive control (with Universal Mouse Reference RNA) and a negative control (without tissue). After the remaining six capture zones are attached with tissue, set a gradient permeabilization time to find the optimal permeabilization time for the final experiment. Resuspend the permeabilization enzyme in 1.2 ml of 0.1 N HCl to activate pepsin activity. Pre-equilibrate the permeabilization solution to 37°C. Assemble the chip and slide cassette, isolating each capture zone from each other. Set the PCR instrument lid temperature to 37°C. Add 1 μl of Universal Mouse Reference RNA to the positive control well without subsequent addition of the permeabilization reagent. Then, add 70 μl of the permeabilization solution sequentially, starting with the longest and ending permeabilization time, according to the pre-set permeabilization time. Apply sealing film, shake the chip up and down to ensure even coverage of the permeabilization solution across all capture zones. Place it in the Thermocycler Adapter, close the PCR instrument lid, and start the timer. Remove the chip 30 seconds before the next time point and add the permeabilization solution, ensuring that each capture zone has been permeabilized for the pre-set time. After removing the permeabilization enzyme with a pipette, add 100μl of 0.1× SSC to all but the positive control. Prepare the fluorescent reverse transcription system in advance as follows: 110μl of RT Reagent C, 30.8μl of Template Switch Oligo, 8.8μl of Reducing Agent B, 68.6μl of RT Enzyme D, and 221.8μl of Nuclease-free water. Protect from light and place on ice. After removing the 0.1× SSC with a pipette, set the PCR instrument lid temperature to 53°C. Once the PCR instrument temperature reaches 53°C, add 50μl of the fluorescent reverse transcription system to each capture zone and incubate at 53°C in the PCR instrument for 45 minutes. After reverse transcription, the captured mRNA is converted into cDNA with a fluorescent group.After reverse transcription is complete, remove the reverse transcription system with a pipette and add 0.1× SSC to each capture zone. Prepare the tissue removal system in advance as follows: 539μl of Tissue Removal Buffer and 77μl of Tissue Removal Enzyme, which should be stored at room temperature. After removing the 0.1× SSC with a pipette, add 70μl of the tissue removal system to each capture zone. Set the thermal cycler lid temperature to 56°C. Once the thermal cycler temperature reaches 56°C, incubate the cells at 56°C for 60 minutes. Use tissue removal enzyme to digest any remaining tissue on the chip to prevent interference with subsequent fluorescence imaging. After tissue removal, remove the tissue removal system with a pipette, separate the chip from the slide cassette, and then wash the chip 15 times in 2× SSC / 0.1% SDS, 0.2× SSC, and 0.1× SSC, respectively. Wipe the back of the chip clean, transfer it to a new 50ml centrifuge tube, and centrifuge it at 250°C for 30 seconds to remove excess moisture. Store in dark. The eight capture zones of the entire chip were imaged using a fluorescence confocal microscope under the same light intensity. Based on the fluorescence results (the brightest fluorescence intensity corresponds to the best H&E structure), the optimal permeabilization time was determined to be 15 minutes.

[0111] Subsequent 10X Visium spatial transcriptome experiments were performed based on the optimal permeabilization time determined in the permeabilization experiment. The experimental samples included: WT (4 months old), WT (24 months old), AD (24 months old), and SL (24 months old) (SL-ZF-01-treated) mice. Permeabilization was sequentially applied to each of the four tissues. Sealing film was applied to the chip, and the permeabilization solution was shaken up and down to ensure even coverage of the tissues. The cells were then incubated at 37°C for 15 minutes. After removing the permeabilization solution with a pipette, 100 μl of 0.1× SSC was added to each capture zone. The reverse transcription system was prepared as follows: 82.7 μl of RT Reagent, 22.9 μl of Template Switch Oligo, 6.6 μl of Reducing Agent B, 51.5 μl of RT Enzyme D, and 166.3 μl of Nuclease-free water. After removing 0.1× SSC with a pipette, set the PCR instrument lid temperature to 53°C. After the PCR instrument temperature rises to 53°C, add 75 μl of reverse transcription system to each capture zone and incubate at 53°C in the PCR instrument for 45 minutes.

[0112] Dilute 8.0M KOH to 0.08M with nuclease-free water. After reverse transcription is complete, remove the reverse transcription system with a pipette and add 75μl of 0.08M KOH to each capture zone and incubate at room temperature for 5 minutes. Remove the 0.08M KOH with a pipette and add 100μl of EB buffer to each capture zone. Prepare the second-strand synthesis system as follows: 305.8μl of Second Strand Reagent, 16.7μl of Second Strand Primer, and 6.6μl of Second Strand Enzyme. Remove the EB buffer with a pipette and set the thermal cycler lid temperature to 65°C. Once the thermal cycler temperature reaches 65°C, add 75μl of the second-strand synthesis system to each capture zone and incubate at 65°C for 15 minutes. After incubation, remove the second-strand synthesis system with a pipette, add 100 μl of EB buffer to each capture zone, remove the EB buffer with a pipette, add 35 μl of 0.08 M KOH to each capture zone and incubate at room temperature for 10 minutes. After eluting the cDNA from the chip, transfer the KOH solution to 5 μl of 1 M pH 7.0 Tris-HCl in an eight-tube strip with a pipette, with a total volume of approximately 40 μl.

[0113] Prepare the qPCR Mix as follows: 20.4μl of Nuclease-free Water, 27.5μl of KAPA SYBR FAST qPCR Master Mix, and 1.7μl of cDNA Primers. Add 1μl of the sample from the previous step to 9μl of qPCR Mix. For a negative control, add 1μl of Nuclease-free Water to the qPCR Mix, mix thoroughly, centrifuge briefly, and then measure the Cq value for each sample in a qPCR instrument. The Cq value is then used to determine the number of cycles for cDNA amplification. The final number of cDNA amplification cycles was 15 for the WT sample (4 months of age) and 14 for the remaining samples (WT (24 months of age), AD (24 months of age), and SL (24 months of age).

[0114] Prepare the cDNA amplification system: 220 μl of Amp Mix and 66 μl of cDNA Primers. Add 65 μl of cDNA amplification system to the remaining 35 μl of sample from the previous step. Mix thoroughly by pipetting 15 times with a 90 μl pipette, and briefly centrifuge to start cDNA amplification. After cDNA amplification, add 60 μl of SPRIselect Reagent to each sample, mix well, and incubate at room temperature for 5 minutes. After incubation, insert the sample into the upper position of a 10× Magnetic Separator and let it stand at room temperature for 5 minutes until the solution is clear. Remove the supernatant, add 200 μl of 80% ethanol, incubate at room temperature for 30 seconds, remove the ethanol, repeat the 80% ethanol washing step, centrifuge briefly, and insert the sample into the lower position of a 10× Magnetic Separator. Open the lid and evaporate the residual ethanol at room temperature. Add 40.5 μl of EB buffer to each sample, mix well with a pipette, and incubate at room temperature for 2 minutes. After incubation, insert the sample into the lower position of a 10× Magnetic Separator and let it stand at room temperature for 5 minutes until the solution is clear. Transfer 40 μl of supernatant to a new eight-tube strip and store at 4°C overnight.

[0115] Prepare the fragmentation system on ice as follows: 22μl Fragmentation Buffer, 44μl Fragmentation Enzyme. For each sample, add 10μl of the magnetic bead-purified solution to a new eight-tube strip. Add 25μl of EB buffer and 15μl of the fragmentation system, mix thoroughly with a pipette, and store on ice. Set the PCR cycler to the following program: 4°C (long), 32°C (5 minutes), 65°C (30 minutes), and store at 4°C. Set the heated lid to 65°C, start the PCR, and cool the cycler to 4°C. After adding the samples, skip the 4°C cycle and return to 32°C to begin random cDNA fragmentation. After random cDNA fragmentation, add 30μl of SPRIselect Reagent to each sample, mix thoroughly, and incubate at room temperature for 5 minutes. After incubation, insert the sample into the upper position of a 10× magnetic separator and let it stand at room temperature for 5 minutes until the solution clears. Add 75 μl of supernatant to a new eight-tube strip. Then, add 10 μl of SPRIselect Reagent to each sample, mix thoroughly, and incubate at room temperature for 5 minutes. After incubation, insert the samples into the upper position of a 10× magnetic separator and let them stand at room temperature for 5 minutes until the solution is clear. Remove the supernatant and add 125 μl of 80% ethanol to incubate at room temperature for 30 seconds. Remove the ethanol and repeat the 80% ethanol wash step. Centrifuge briefly and insert the samples into the lower position of a 10× magnetic separator. Open the lid and allow the remaining ethanol to evaporate at room temperature. Add 50.5 μl of EB buffer to each sample, mix thoroughly with a pipette, and incubate at room temperature for 2 minutes. After incubation, insert the samples into the lower position of a 10× magnetic separator and let them stand at room temperature for 5 minutes until the solution is clear. Transfer 50 μl of supernatant to a new eight-tube strip. Prepare the Adaptor Ligation Mix as follows: 88 μl of Ligation Buffer, 44 μl of DNA Ligase, and 88 μl of Adaptor Oligos. Add 50 μl of Adaptor Ligation Mix to each of the above samples and incubate at 20°C for 15 minutes. After incubation, add 80 μl of SPRIselect Reagent to each sample, mix thoroughly, and incubate at room temperature for 5 minutes. After incubation, insert the samples into the upper position of a 10× Magnetic Separator and let them stand at room temperature for 5 minutes until the solution is clear. Remove the supernatant, add 200 μl of 80% ethanol, and incubate at room temperature for 30 seconds. Remove the ethanol, repeat the 80% ethanol wash, centrifuge briefly, and insert the samples into the lower position of a 10× Magnetic Separator. Open the lid and evaporate any remaining ethanol at room temperature. Add 30.5 μl of EB buffer to each sample, mix thoroughly with a pipette, and incubate at room temperature for 2 minutes.After incubation, insert the sample into the lower position of a 10× Magnetic Separator and let it stand at room temperature for 5 minutes until the solution becomes clear. Then transfer 30 μl of the supernatant to a new eight-tube strip.

[0116] After cDNA amplification and magnetic bead purification, 1 μl of each sample was taken and the cDNA concentration of each sample was measured using Qubit 4.0. The cDNA concentration was multiplied by 10 μl to obtain the total amount of cDNA input for library amplification, which was used to determine the number of subsequent PCR cycles. 50 μl Amp Mix and 20 μl of numbered Dual Index Plate TT Set A were added, and the number of each sample was recorded. After amplification, 60 μl SPRIselect Reagent was added to each sample, mixed, and incubated at room temperature for 5 minutes. After incubation, the sample was inserted into the high position of a 10×Magnetic Separator and allowed to stand at room temperature for 5 minutes until the solution clarified. 150 μl of supernatant was added to a new eight-tube strip, and then 20 μl SPRIselect Reagent was added to each sample, mixed, and incubated at room temperature for 5 minutes. After incubation, insert the sample into the upper position of a 10× Magnetic Separator and let it stand at room temperature for 5 minutes until the solution is clear. Remove the supernatant, add 200μl of 80% ethanol, incubate at room temperature for 30 seconds, remove the ethanol, repeat the 80% ethanol wash step, centrifuge briefly, and insert the sample into the lower position of a 10× Magnetic Separator. Open the lid and evaporate the residual ethanol at room temperature. Add 35.5μl of EB buffer to each sample, mix with a pipette, and incubate at room temperature for 2 minutes. After incubation, insert the sample into the lower position of a 10× Magnetic Separator and let it stand at room temperature for 5 minutes until the solution is clear. Then, aspirate 35μl of supernatant and transfer it to a new 1.5ml centrifuge tube. Transport on dry ice to Annoroad's Illumina platform for second-generation paired-end sequencing.

[0117] 2. Experimental results

[0118] 2.1. 20 mg / kg SL-ZF-01 significantly upregulated the RNA transcription levels of type 1 and type 3 glucose transporters Slc2a1 and Slc2a3 in APP / PS1 mice

[0119] Table 6 Effects of 20 mg / kg SL-ZF-01 on glucose transporter RNA transcription levels in the dendritic area of ​​the hippocampus of APP / PS1 mice

[0120] * APP / PS1vs.24months WT, #APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0121] Table 6 lists the transcript levels of glucose transporters in the hippocampal neuropil of mice as determined by spatial transcriptomics. The results showed that 11 glucose transporters, types 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, and 13, were detected. Types 1, 3, and 13 were expressed at higher abundances. Furthermore, the transcript levels of types 1, 3, and 13 increased with age. Compared with 24-month-old WT mice, the transcript levels of type 1 and type 3 glucose transporters in APP / PS1 mice were significantly decreased. Following SL-ZF-01 treatment, these transcript levels were partially restored, showing significant differences compared to untreated AD mice. However, the transcript levels of types 2, 4, 5, 6, 8, 9, 10, and 12 were extremely low, and after SL-ZF-01 treatment, there was no significant difference compared to other groups. These results indicate that SL-ZF-01 improves the transcript levels of type 1 and type 3 glucose transporters in the hippocampal neuropil of mice.

[0122] Table 7 Effects of 20 mg / kg SL-ZF-01 on the RNA transcription level of glucose transporter in the somatic area of ​​the hippocampus of APP / PS1 mice

[0123] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01,△ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0124] Table 7 lists the transcript levels of glucose transporters in the mouse hippocampal somata region as determined by spatial transcriptomic analysis. The results showed that 11 glucose transporters, types 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, and 13, were detected. Types 1, 3, and 13 were expressed at higher abundances. Furthermore, the transcript levels of types 1, 3, and 13 increased with age. Compared with 24-month-old WT mice, the transcript levels of type 3 glucose transporters were significantly decreased in age-matched APP / PS1 mice. Treatment with SL-ZF-01 partially restored the transcript levels of type 3 glucose transporters. However, the transcript levels of types 2, 4, 5, 6, 8, 9, 10, and 12 glucose transporters were extremely low and remained unchanged after SL-ZF-01 treatment. These results indicate that SL-ZF-01 improves the transcript levels of type 3 glucose transporters in the mouse hippocampal somata region.

[0125] 2.2. 20mg / kg SL-ZF-01 can significantly upregulate the thyroid hormone (Thyroid hormone) and hypoxia-inducible factor (HIF-1) signaling pathways upstream of Glut1

[0126] Table 8 Effects of 20 mg / kg SL-ZF-01 on the thyroid hormone signaling pathway in the nerve fiber region of APP / PS1 mice

[0127] * APP / PS1vs.24months WT, #APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0128] Table 8 lists the changes in transcriptional levels of genes related to the thyroid hormone signaling pathway in the neurofiber region of the mouse hippocampus measured by spatial transcriptome analysis in the four groups of mice. The thyroid hormone signaling pathway is an upstream signaling pathway of the type 1 glucose transporter (Glut1) and can regulate the expression of Glut1. The Atp1b1, Prkacb, Slc2a1, Rheb, Atp2a2, Hras, ​​Map2k1, Thra, Actg1, Pfkm, and Mapk1 genes in this pathway all have increased transcriptional levels during aging. At the same time, compared with 24-month-old WT mice, the transcriptional levels of APP / PS1 mice of the same age were significantly decreased. After SL-ZF-01 treatment, these upstream signals were partially restored. These results indicate that SL-ZF-01 may enhance the thyroid hormone signaling pathway upstream of Glut1, thereby increasing the expression level of Glut1.

[0129] Table 9 Effects of 20 mg / kg SL-ZF-01 on the hypoxia-inducible factor (HIF-1) signaling pathway in the nerve fiber region of APP / PS1 mice

[0130] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; *Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0131] Table 9 lists the changes in transcriptional levels of the hypoxia-inducible factor (HIF-1) signaling pathway in the mouse hippocampal neuropil region in the four groups of mice as determined by spatial transcriptome analysis. The hypoxia-inducible factor (HIF-1) signaling pathway is also an upstream signaling pathway of the type 1 glucose transporter (Glut1) and can regulate Glut1 expression. In addition to some genes shared with the thyroid pathway, the Eloc, Aldoa, Rbx1, Elob, and Pgk1 genes in this pathway all have increased transcriptional levels during aging. At the same time, compared with 24-month-old WT mice, the transcriptional levels of APP / PS1 mice of the same age were significantly reduced. After treatment with SL-ZF-01, these signals were partially restored. These results indicate that SL-ZF-01 may enhance the hypoxia-inducible factor signaling pathway upstream of Glut1, thereby increasing the expression level of Glut1.

[0132] 2.3. 20mg / kg SL-ZF-01 can significantly upregulate the glycolysis and oxidative phosphorylation signaling pathways downstream of Glut1

[0133] Table 10 Effects of 20 mg / kg SL-ZF-01 on Glut1 downstream glycolysis in the nerve fiber region of APP / PS1 mice

[0134] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05,** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0135] Table 10 lists the changes in transcriptional levels of genes related to the glycolysis pathway in the neuropil region of the mouse hippocampus measured by spatial transcriptome analysis in the four groups of mice. The glycolysis pathway is a downstream signaling pathway of the type 1 glucose transporter (Glut1), and the expression level of Glut1 is upregulated. The Pfkm, Akr1a1, Pgk1, Pgam1, and Aldoa genes in this pathway all have increased transcriptional levels during aging. At the same time, compared with 24-month-old WT mice, the transcriptional levels of APP / PS1 mice of the same age were significantly reduced. After treatment with SL-ZF-01, the expression levels of these signals were partially restored. These results indicate that SL-ZF-01 can enhance the downstream glycolysis signaling pathway by increasing Glut1 expression.

[0136] Table 11 Effects of 20 mg / kg SL-ZF-01 on Glut1 downstream oxidative phosphorylation in the nerve fiber region of APP / PS1 mice

[0137] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01,### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0138] Table 11 lists the changes in transcriptional levels of genes related to the oxidative phosphorylation pathway in the hippocampal neuropil region of mice in the four groups of mice as determined by spatial transcriptomics. The oxidative phosphorylation pathway is a downstream signaling pathway of the type 1 glucose transporter (Glut1), and Glut1 expression levels are upregulated in response. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Atp5e, Ndufb5, Ndufc1, Ndufb6, Atp6v1g1, Uqcrh, Atp6v0b, Ndufs5, Atp5k, Cox6a1, Atp5j2, Ndufa4, Ndufa5, Atp6v1f, Ndufb2, Atp6v0e2, Cycs, Atp6v1e1, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Atp6v1b2, Ndufa13, Atp6v0d1, Cox4i1, Atp5l, Cox5a, and Cox7 Transcriptional levels of genes such as a2, Uqcr11, Ndufa12, Atp5b, Uqcr10, Uqcrq, Atp5h, Atp6v1d, Uqcrb, Cox7c, Ndufs4, Cox6c, Atp6v1c1, Ndufb9, Cyc1, Ndufa6, Atp5g2, Ndufb4, Cox17, Atp6v1a, Atp5j, Atp6v0c, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, and Cox7b increased during aging. Furthermore, transcript levels were significantly decreased in APP / PS1 mice of the same age compared to WT mice at 24 months of age. These signals were partially restored after SL-ZF-01 treatment. These results suggest that SL-ZF-01 can partially restore impaired glucose metabolism by improving Glut1 expression.

[0139] Example 10 Spatial Transcriptome Analysis SL-ZF-01 Improves Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD), Prion Disease (prion disease), Amyotrophic Lateral Sclerosis (ALS), and Neurodegenerative Diseases (neurodegeneration) Mechanism

[0140] 1. Experimental Methods

[0141] The experimental method is as shown in the experimental method in Example 9.

[0142] 2. Experimental Results

[0143] 2.1 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of Alzheimer's disease-related genes in the neurofibrillary region of APP / PS1 mice

[0144] Table 12 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of Alzheimer's disease-related genes in the nerve fiber region of APP / PS1 mice

[0145] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0146] Table 12 lists the changes in the transcriptional levels of genes related to the Alzheimer's disease pathway in the neuropil region of the mouse hippocampus determined by spatial transcriptomics in the four groups of mice. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Atp5e, Ndufb5, Ndufc1, Ndufb6, Uqcrh, Ndufs5, Cox6a1, Atp2a2, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Snca, Ndufa9, Psenen, Cox6b1, Ndufc2, Ndufab1, Hras, ​​Vdac3, Slc25a4, Ndufa13, Cox4i1, Cox5a, Map2k1, Cox7a2, Uqcr11, Ndufa 12. Transcriptional levels of Atp5b, Uqcr10, Uqcrq, Atp5h, Calm1, Klc1, Uqcrb, Cox7c, Ndufs4, Vdac2, Psmb5, Cox6c, Ndufb9, Cyc1, Ndufa6, Atp5g2, Mapk1, Ndufb4, Atp5j, App, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, Araf, and Cox7b genes all increase during aging. Furthermore, transcript levels in APP / PS1 mice were significantly reduced compared to WT mice at the same age. These signaling pathways were partially restored after SL-ZF-01 treatment. These results suggest that SL-ZF-01 may have a series of effects in addition to improving Glut1 and glucose metabolism, which ultimately work together to achieve the effect of preventing and / or treating AD.

[0147] 2.2 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of Parkinson's disease-related genes in the nerve fiber region of APP / PS1 mice

[0148] Table 13 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of Parkinson's disease-related genes in the nerve fiber region of APP / PS1 mice

[0149] *APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0150] Table 13 lists the changes in transcriptional levels of Parkinson's disease pathway-related genes in the mouse hippocampal neuropil region determined by spatial transcriptomics in the four groups of mice. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Gnas, Atp5e, Ndufb5, Ndufc1, Prkacb, Ndufb6, Txn1, Uqcrh, Ndufs5, Gnai1, Uchl1, Cox6a1, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Snca, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Vdac3, Slc25a4, Ndufa13, Uba52, Cox4i1, Cox5a, Cox7a2, Uqcrh, and Ndufs5 are involved. The transcriptional levels of genes involved in Parkinson's disease pathways increased during aging, including cr11, Ndufa12, Atp5b, Uqcr10, Uqcrq, Ubb, Atp5h, Calm1, Klc1, Uqcrb, Cox7c, Ndufs4, Vdac2, Psmb5, Cox6c, Ndufb9, Cyc1, Ndufa6, Atp5g2, Ndufb4, Atp5j, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, and Cox7b. Furthermore, the transcriptional levels of genes involved in Parkinson's disease pathways increased significantly in APP / PS1 mice compared with WT mice at the same age at 24 months. These results suggest that the transcriptional levels of genes involved in Parkinson's disease pathways were partially restored after SL-ZF-01 treatment.

[0151] 2.3 Effect of 20 mg / kg SL-ZF-01 on the transcriptional levels of Huntington's disease-related genes in the neurofiber region of APP / PS1 mice

[0152] Table 14 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of Huntington's disease-related genes in the neurofiber region of APP / PS1 mice

[0153] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001;# Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0154] Table 14 lists the changes in the transcriptional levels of Huntington's disease pathway-related genes in the neuropil region of the mouse hippocampus determined by spatial transcriptomics in the four groups of mice. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Slc1a2, Atp5e, Ndufb5, Ndufc1, Ndufb6, Uqcrh, Ndufs5, Cox6a1, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Dctn1, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Vdac3, Slc25a4, Ndufa13, Cox4i1, Cox5a, Cox7a2, Uqcr11, Ndufa12, Atp5b, Uqcr10, Transcription levels of genes including Uqcrq, Gria1, Dlg4, Atp5h, Klc1, Uqcrb, Cox7c, Ndufs4, Vdac2, Psmb5, Cox6c, Polr2k, Ndufb9, Cyc1, Polr2f, Ndufa6, Atp5g2, Ap2m1, Ndufb4, Atp5j, Sod2, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, and Cox7b increased with aging. Furthermore, their transcript levels were significantly reduced in APP / PS1 mice compared with WT mice at the same age (24 months). These results suggest that transcription levels of genes involved in the Huntington's disease pathway are partially restored after SL-ZF-01 treatment.

[0155] 2.4 Effect of 20 mg / kg SL-ZF-01 on the transcriptional levels of prion-related genes in the neurofibrillary region of APP / PS1 mice

[0156] Table 15 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of prion disease-related genes in the neurofibrillary region of APP / PS1 mice

[0157] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0158] Table 15 lists the changes in the transcriptional levels of genes related to the prion disease pathway in the neurofibrillary region of the mouse hippocampus determined by spatial transcriptomics in the four groups of mice. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Atp5e, Ndufb5, Ndufc1, Prkacb, Ndufb6, Uqcrh, Ndufs5, Cox6a1, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Vdac3, Slc25a4, The transcriptional levels of Ndufa13, Cox4i1, Cox5a, Cox7a2, Uqcr11, Ndufa12, Atp5b, Uqcr10, Uqcrq, Atp5h, Klc1, Ryr2, Uqcrb, Cox7c, Ndufs4, Vdac2, Psmb5, Cox6c, Ndufb9, Cyc1, Ndufa6, Atp5g2, Mapk1, Ndufb4, Atp5j, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, and Cox7b genes all increased during aging. Furthermore, the transcriptional levels of APP / PS1 mice were significantly decreased compared with WT mice at the same age. These results indicate that the transcription levels of genes involved in the prion disease pathway were partially restored after SL-ZF-01 treatment.

[0159] 2.5 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of ALS-related genes in the nerve fiber region of APP / PS1 mice

[0160] Table 16 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of ALS-related genes in the nerve fiber region of APP / PS1 mice

[0161] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; #Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0162] Table 16 lists the changes in the transcriptional levels of the ALS pathway-related genes in the neurofiber region of the mouse hippocampus determined by spatial transcriptomics in the four groups of mice. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Slc1a2, Atp5e, Ndufb5, Ndufc1, Ndufb6, Uqcrh, Ndufs5, Cox6a1, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Hnrnpa2b1, Dctn1, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Ndufa13, Cox4i1, Cox5a, Cox7a2, Uqcr11, Ndufa12, Atp5b, Transcription levels of genes including Uqcr10, Uqcrq, Gria1, Atp5h, Actg1, Klc1, Uqcrb, Cox7c, Ndufs4, Psmb5, Nefl, Cox6c, Ndufb9, Cyc1, Ndufa6, Atp5g2, Ndufb4, Atp5j, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Matr3, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, and Cox7b increased with aging. Furthermore, their transcription levels were significantly reduced in APP / PS1 mice compared with WT mice at the same age at 24 months. These results suggest that the transcription levels of genes involved in the ALS pathway are partially restored after SL-ZF-01 treatment.

[0163] 2.6 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of neurodegenerative disease-related genes in the nerve fiber region of APP / PS1 mice

[0164] Table 17 Effects of 20 mg / kg SL-ZF-01 on the transcriptional levels of neurodegenerative disease-related genes in the nerve fiber region of APP / PS1 mice

[0165] * APP / PS1vs.24months WT, # APP / PS1vs.APP / PS1+SL-ZF-01, △ 24 months WT vs. 4 months WT; * Adjust Pvalue<0.05, ** Adjust Pvalue<0.01, *** Adjusted P value < 0.001; # Adjust Pvalue<0.05, ## Adjust Pvalue<0.01, ### Adjusted P value < 0.001; △ Adjust Pvalue<0.05, △△ Adjust Pvalue<0.01, △△△ Adjust P value < 0.001 (Wilcoxon rank-sum test to determine whether there is a significant difference in specific oversupply between the two groups, and the P value is corrected by False Discovery Rate).

[0166] Table 17 lists the changes in transcriptional levels of genes related to the neurodegenerative disease pathway in the hippocampal nerve fiber region of mice in the four groups of mice determined by spatial transcriptome analysis. In this pathway, Cox5b, Ndufb3, Ndufs1, Ndufs2, Ndufa8, Atp5e, Ndufb5, Ndufc1, Ndufb6, Uqcrh, Ndufs5, Uchl1, Cox6a1, Atp2a2, Sem1, Ndufa4, Ndufa5, Ndufb2, Cycs, Snca, Dctn1, Ndufa9, Cox6b1, Ndufc2, Ndufab1, Hras, ​​Vdac3, Slc25a4, Ndufa13, Uba52, Cox4i1, Cox5a, Map2k1, Cox7a2, Uqcr11, Ndu fa12, Atp5b, Uqcr10, Uqcrq, Gria1, Ubb, Dlg4, Atp5h, Calm1, Klc1, Ryr2, Uqcrb, Cox7c, Ndufs4, Vdac2, Transcription levels of genes including Psmb5, Nefl, Cox6c, Ndufb9, Cyc1, Ndufa6, Atp5g2, Mapk1, Ndufb4, Atp5j, App, Ndufb10, Ndufa7, Ndufa11, Ndufv2, Cox7a2l, Ndufa2, Atp5a1, Ndufs8, Cox8a, Ndufb8, Ndufb11, Araf, and Cox7b increased with aging. Furthermore, transcription levels were significantly decreased in APP / PS1 mice compared with 24-month-old WT mice. These results suggest that SL-ZF-01 treatment partially restores the transcription of genes involved in neurodegenerative disease pathways.

[0167] Example 11 Verification of the effect of SL-ZF-01 on the expression of type 1 glucose transporter Glut1 in APP / PS1 mice at the protein level - Western blotting

[0168] 1. Experimental methods

[0169] After the water maze test, except for the mice undergoing spatial transcriptome analysis, the rest of the mice were starved overnight and then injected intraperitoneally with 50 mg / kg sodium pentobarbital for 5 minutes before checking whether the mice were deeply anesthetized. After successful anesthesia, the mice were perfused with PBS and then killed. The perfusion time was 5 minutes, the perfusion rate was 11-15, and the brain was removed. The culture dish was placed on ice in advance, filter paper was placed on the surface of the culture dish, and 2 ml of ice PBS was dripped on the filter paper to wet the filter paper. Next, the brain was placed on the filter paper and cut along the midline of the brain from one end of the olfactory bulb to the cerebellum using a surgical blade to divide the mouse brain into left and right hemispheres. The hippocampus and cortical tissue of the right hemisphere were peeled off and immediately placed on ice. They were then collected and sorted and frozen at -80°C.

[0170] Accurately weigh mouse brain tissue: Place an EP tube rack in a liquid nitrogen tank. Wait 15 minutes for the temperature to equilibrate and for white smoke to cease. Using an ophthalmic sniffer, immerse the tip in liquid nitrogen to pre-chill. Quickly open the EP tube containing the mouse brain tissue, remove the brain tissue, and place it in the pre-chilled EP tube on a tared fine balance. Read the reading and quickly transfer the tissue back to the original EP tube.

[0171] Once all tissues have been weighed, calculate the volume of tissue lysis buffer. Calculate the volume of tissue lysis buffer: Based on the mass, add 20 μl of lysis buffer per mg of tissue to calculate the actual volume of lysis buffer. Add the lysis buffer for all tissues to calculate the total volume.

[0172] Prepare cell lysis buffer: Use Beyotime RIPA lysis buffer (P0013B), add 100X protease inhibitor (539131-10VLCN, Millipore) and 100X phosphatase inhibitor (524625-1SET, Millipore), prepare it before use, and place it on ice until use.

[0173] According to the above ratio, add the tissue lysate to the mouse brain sample tube. Add an appropriate amount of zirconium oxide grinding beads (BJ62457, Baigen) and balance on a standard balance. Place the EP tube in the tissue homogenizer using the balanced method. The homogenizer metal rack must have been pre-chilled to -20°C. Homogenize for 60 seconds, let it rest for 1 minute, then homogenize for another 60 seconds and let it rest on ice. After the upper layer of foam disappears, remove the solution and centrifuge at 3000 rpm for 15 minutes. Remove the supernatant and place it in a new EP tube.

[0174] Take 75 μl of tissue, add 25 μl of 4× Laemmli sample buffer, mix thoroughly, and centrifuge. Perform polyacrylamide gel electrophoresis: Align the lower ends of a long WB glass plate with a short WB glass plate, ensuring the raised side of the long plate faces the short one. Place them in a clamp and secure them. Then, clamp them vertically onto a gel casting rack and secure them. Place the gel casting rack on a level, stable table.

[0175] According to the instructions for the 10% PAGE Gel Rapid Preparation Kit (PG111, Yazyme), mix the lower layer solution (2X) and lower gel buffer (2X) in a 1:1 ratio. Pipette 5ml of the lower layer solution (2X) and add 5ml of lower gel buffer (2X). Add 200μl of the modified coagulant. Rapidly pour the gel onto the prepared gel plate. Quickly add an appropriate amount of anhydrous ethanol to level the gel surface. After the gel has completely solidified, pour out the anhydrous ethanol. Tilt the gel rack over 90°, remove excess ethanol with filter paper, and return it to its original level surface to allow it to rest. To prepare the upper gel, according to the instructions, mix the upper layer solution (2X) and upper gel buffer (2X) in a 1:1 ratio. Pipette 1ml of the upper layer solution and add 1ml of upper gel buffer. Add 20μl of the modified coagulant. Rapidly pour the gel onto the lower gel. Quickly insert a 1.5mm 10-hole sample comb and allow the gel to completely solidify.

[0176] Loading and electrophoresis: Gently remove the prepared polyacrylamide gel electrophoresis gel from the gel preparation rack, clamp it into the electrophoresis tank rack, clamp the long plate outward and place it in the electrophoresis tank, add electrophoresis buffer to the center of the electrophoresis tank, the solution height must cover the short plate and be almost aligned with the long plate, and add an appropriate amount of electrophoresis buffer to the outer tank. Slowly pull out the sample comb to ensure that there are no bubbles in the channel, add 3μl protein marker (26616, Thermo Fisher) to the holes on both sides of the sample channel, and add 5-8μl sample to the sample well. Connect the red hole according to the red line and buckle the power supply. Use a constant voltage of 60V for concentrated gel electrophoresis. After the bromophenol blue front runs to the separation gel, adjust the voltage to 120V and perform constant voltage electrophoresis. Finally, turn off the power when the bromophenol blue front reaches about 0.5-1 cm from the glass plate.

[0177] Transfer: Remove the polyacrylamide gel and cut away the concentrated gel. Cut a notch in the upper left corner of the gel and store it in electrophoresis buffer. Cut the PVDF membrane into a 5 cm x 8 cm rectangle and cut a notch in the upper left corner. Activate the PVDF membrane in methanol and shake it on a shaker for 1 minute. Equilibrate it in transfer buffer and shake it on a shaker for 5 minutes. Place the filter paper on the blackboard of the plywood as the base. Next, place the balanced PVDF on the electrophoresis gel, aligning the notches. Place a layer of filter paper on top. Use a glass rod to roll out any bubbles. Clamp the transfer plywood and insert it vertically into the transfer tank. Add an appropriate amount of transfer buffer and pre-cooled ice packs to the transfer buffer. After plugging in the power cord, place the entire electrophoresis tank on ice. Connect the power cord and transfer the membrane at a constant current of 250mA for 90 minutes.

[0178] Antibody incubation: Disconnect the power, unplug the transfer plate, remove the PVDF membrane, cut the membrane into strips according to the desired protein molecular weight, and place them in the incubation box. Add 3 ml of rapid blocking solution (P0235, Beyotime), incubate for 1 hour, then add the diluted primary antibody and incubate at 4°C with shaking overnight. The next day, remove the PVDF membrane, warm it to room temperature, add 5 ml of PBST solution and shake for 10 minutes, repeat this wash three times, add the diluted secondary antibody, incubate at room temperature with shaking for 120 minutes, add 5 ml of PBST solution and shake for 10 minutes, repeat this wash three times, and incubate in TMB colorimetric solution (P0209, Beyotime) in the dark for 2 minutes. Place the membrane in an automatic exposure instrument for chemiluminescence capture to obtain the immunoblot image.

[0179] Antibody preparation: Glut1 antibody (ab40084, abcam) at a dilution of 1:250; CD31 / PECAM-1 (AF3628-SP, R&D) at a dilution of 1:200, diluted in Western primary antibody diluent (P0023A, Beyotime). Mouse secondary antibody was goat anti-mouse IgG (SA00001-1, proteintech) at a dilution of 1:1000 in 5% BSA, and rabbit secondary antibody was goat anti-rabbit IgG (SA00001, proteintech) at a dilution of 1:1000 in 5% BSA.

[0180] 2. Experimental results

[0181] 2.1 20 mg / kg SL-ZF-01 significantly upregulated the expression of type 1 glucose transporter protein in APP / PS1 mice—Western blotting

[0182] Table 18 20 mg / kg SL-ZF-01 can significantly upregulate the expression level of type 1 glucose transporter protein in the hippocampus of APP / PS1 mice

[0183] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05, ## P<0.01, ### P<0.001, (Mann Whitney test, two-tailed).

[0184] Table 18 lists the protein expression levels of glucose transporter type 1 (Glut1) in the mouse hippocampus as determined by immunoblotting. The results showed that Glut1 protein expression was significantly decreased in APP / PS1 mice aged 11 to 24 months compared to WT mice. Compared to the APP / PS1 group, Glut1 expression levels were significantly increased in all four age groups of APP / PS1 mice treated with SL-ZF-01, from 9 to 24 months.

[0185] Example 12: Verification of the Effect of SL-ZF-01 on the Expression of Type 1 Glucose Transporter Glut1 in APP / PS1 Mice at the Protein Level—Immunofluorescence Assay

[0186] 1. Experimental methods

[0187] In Example 11 above, the mouse brain tissue used was the right hemisphere. For this experiment, the left hemisphere was sampled, quickly fixed in PFA solution, and stored at 4°C. After 48 hours, the tissue was removed and, after temperature equilibration, vibratome sectioning was performed. The sections were 40 μm thick and sectioned at a speed of 6. The sections were collected, placed in cryopreservative solution, and stored at 4°C.

[0188] Remove sections from the refrigerator and wash three times with PBS for 10 minutes each. Transfer sections to a 5% BSA solution containing 0.5% Triton X-100 for permeabilization and blocking, then incubate on a rocking platform for 1 hour. Transfer sections to the primary antibody solution: Glut1 (ab40084, abcam, mouse) at a dilution of 1:200, Lectin at a dilution of 1:1000, and GFAP (ab4674, abcam, chicken) at a dilution of 1:1000 in 5% BSA. Incubate at 4°C overnight. Remove sections the next day, wash three times with PBS, and transfer sections to a fluorophore-linked secondary antibody solution: mouse secondary antibody at a dilution of 1:1000 (mouse fluorescent labeling at 647 nm), chicken secondary antibody at a dilution of 1:1000 (chicken fluorescent labeling at 594 nm). Incubate at room temperature for 120 minutes. Wash with BPS three times, 10 minutes each time, and mount on a slide. When the slices are just air-dried, add anti-fluorescence quenching sealing solution (containing DAPI: P0131-25, Biyuntian) and cover with a coverslip.

[0189] Confocal microscopy, Multi-point Time-Lapse (MATL) image acquisition: Click the > button in the Live window and select the Map tab. Move the specimen to the desired field of view, adjust imaging parameters, set the objective lens to 1.25×, and adjust the fluorescence intensity and focal plane to ensure a clear and bright image. Acquire an XYZ image and set the corresponding X sequence. Click the second grid button in the REGISTER list and add a 2×1 grid to register the position information and imaging parameters for the current field of view. Select Range mode, adjust the focal plane to a clear level, click Register, select 3-layer overlay, select Stitch, and project the newly acquired image into the MAP. Click Update, capture the image in the MATL interface, and click Start. After the image is captured, set the objective lens to 10×, adjust the fluorescence intensity and focal plane to optimal levels, open the MAP interface, add a 4×5 grid, select Range mode, adjust the focal plane to a clear level, click Register, select a thickness of 100, optimize, select Stitch, click Update, select a save directory, enter a file name, capture the image in the MATL interface, and click Start.

[0190] In the captured interface, open the file, select the maximum brightness projection of the image, and store it in single channel and multi-channel according to different fluorescence wavelengths. The Lectin signal is 488nm, marked in green; the GFAP signal is 594nm, marked in red; the Glut1 signal is 647nm, marked in white, and DAPI is blue.

[0191] Glut1 signals were counted using Image J, and the relative total area / slice and the relatively large particles / slice were counted.

[0192] 2. Experimental results

[0193] 2.1 20 mg / kg SL-ZF-01 can significantly upregulate the expression level of type 1 glucose transporter protein in APP / PS1 mice - immunofluorescence

[0194] Table 19 20 mg / kg SL-ZF-01 can significantly upregulate the expression level of type 1 glucose transporter protein in the hippocampus of 11-month-old APP / PS1 mice

[0195] * APP / PS1vs.WT, # APP / PS1vs.APP / PS1+SL-ZF-01, * P<0.05, ** P<0.01, *** P < 0.001; # P<0.05, ## P<0.01, ###P < 0.001, (ordinary one-way ANOVA, multiple comparisons, comparing the mean of each group with the mean of APP / PS1).

[0196] Table 19 lists the protein expression levels of glucose transporter type 1 (Glut1) in the mouse hippocampus as determined by immunofluorescence. The results showed that Glut1 protein expression was significantly decreased in 11-month-old APP / PS1 mice compared to WT mice. Glut1 expression levels were significantly increased in APP / PS1 mice treated with SL-ZF-01 compared to the APP / PS1 group.

[0197] Studies have shown that neurodegenerative diseases are more or less related to mechanisms such as Aβ amyloid protein, immune inflammation, glucose metabolism, synaptic transmission, and vascular lesions. Those skilled in the art will appreciate that the present invention is also effective in treating neurodegenerative diseases caused by other causes not listed in the examples.

[0198] The above is an exemplary description of the implementation methods of the technical solutions disclosed herein. It should be understood that the scope of protection of the present disclosure is not limited to the above-mentioned implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of this application.

Claims

1. Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament or functional food for preventing and / or treating neurodegenerative diseases. The structure of the compound of Formula I is shown below:

2. The use according to claim 1, characterized in that The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, and amyotrophic lateral sclerosis.

3. The use according to claim 1 or 2, characterized in that The symptoms of Alzheimer's disease include memory impairment, aphasia, apraxia, agnosia, impairment of visual-spatial ability, impairment of abstract thinking and calculation, personality and behavioral changes, and in severe cases, emotional apathy, erratic crying and laughing, loss of speech ability, and inability to take care of oneself. Preferably, the causes of Alzheimer's disease include amyloid protein, immune inflammation, glucose metabolism, synaptic transmission, and vascular lesions.

4. The use according to any one of claims 1 to 3, characterized in that The cause of Parkinson's disease includes the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which causes a significant decrease in dopamine content in the striatum and leads to the disease. The characteristic symptoms of Parkinson's disease include resting tremor, bradykinesia, muscle rigidity and posture and gait disorders.

5. The use according to any one of claims 1 to 4, characterized in that The onset of Huntington's disease includes a progressive movement disorder manifested as sudden, rapid jumps or twitches of the limbs, face, and trunk.

6. The use according to any one of claims 1 to 5, characterized in that The pathogenesis of the prion disease includes the characteristics of kuru disease, Creutzfeldt-Jakob syndrome, Gerstmann-Strauss syndrome and fatal familial insomnia.

7. The use according to any one of claims 1 to 6, characterized in that The pathogenesis of amyotrophic lateral sclerosis includes limb-onset type and medullary-onset type.

8. Use of a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament or functional food for preventing and / or treating neurodegenerative diseases; Preferably, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, amyotrophic lateral sclerosis.

9. A method for preparing the compound of formula I, comprising the following steps: (1) condensing the compound represented by formula II with glycerol acetone ketal in the presence of a condensing agent to obtain the compound represented by formula III; (2) removing the acetone protecting group from the compound represented by formula III in the presence of an acid to obtain the compound represented by formula IV; (3) the compound represented by formula IV is condensed with heptanoic acid in the presence of a condensing agent to obtain the compound represented by formula V; (4) removing the R protecting group from the compound represented by formula V to obtain the compound represented by formula I; Wherein, R is selected from one of trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, benzyl, p-methoxybenzyl and methoxymethyl.

10. The preparation method according to claim 9, characterized in that The condensing agent in step (1) and step (3) is selected from one or more of DCC, EDCI, HATU, HOBT, BOP, PyBOP, DPP-Cl, and DPPA; Preferably, the acid in step (2) is selected from one or more of trifluoroacetic acid, acetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, and nitric acid. Preferably, steps (1) to (4) can be carried out in a solvent selected from one or more of water, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran, acetone, ether, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, chloroform, 1,4-dioxane, etc. After obtaining the corresponding compound in each step of the reaction in the preparation method, it can be processed by conventional methods in the art, such as extraction, drying, concentration, distillation, beating, crystallization or column chromatography purification.

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