Substituted phenyl glycoside compound as well as preparation method and application thereof

Abstract

The invention discloses a substituted phenyl glycoside compound which has a structure as shown in a formula (I), and R1, R2, R3 and R4 are F, CF3 or H; r < 5 > is CN, OH, NHBoc, N (CH3) Boc, N (CH3) 2, NO2, CHO, COCH3, COOH, COOCH2CH3, OCH3 or OOCCH3; r6 is COCH3 or H; r < 7 > is COCH < 3 >, COOCH < 2 > CH < 3 > or H; and n represents an integer of 0-2. The invention also discloses a preparation method of the substituted phenyl glycoside compound. The substituted phenyl glycoside compound disclosed by the invention shows remarkable quasi-nerve growth factor (NGF) and nerve protection activity, and further verifies that the compound 18 plays the quasi-NGF activity by targeting INSR and ACTN4 and activating a PI3K / Akt signal channel. Besides, in a K6001 yeast replicability life experiment, the substituted phenyl glycoside compound shows remarkable anti-aging activity, is suitable for developing treatment medicines and health-care products for resisting the Alzheimer's disease, not only provides a scientific basis for research and development of new medicines for resisting the Alzheimer's disease, but also lays an important foundation for related basic research, and has a wide application prospect. The method has outstanding application value. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a substituted phenyl glycoside compound, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, approximately 50 million people suffer from Alzheimer's disease (AD) and related dementias, and there is no effective treatment. By 2050, the global AD incidence is expected to triple. AD is a neurodegenerative disease characterized by the pathological accumulation of two types of protein deposits in the brain, including amyloid-β (Aβ) and neurofibrillary tangles (NFT). Oxidative stress plays a key role in Aβ toxicity, and the accumulation of reactive oxygen species (ROS) has been observed in dystrophic neurites associated with Aβ plaques in the brains of AD patients. Elevated ROS levels can mediate neuronal damage and synaptic dysfunction, ultimately leading to AD. Neuroprotective therapies aim to prevent or reverse brain damage caused by Aβ and oxidative stress, and are an ideal strategy for effectively alleviating the development of AD.

[0003] Nerve growth factor (NGF) is the first recognized neurotrophic factor, which can enhance neuroprotection, improve learning and memory, and reduce AD-related pathology (Pentz R, et al. The human brain NGF metabolic pathway is impaired in the pre-clinical and clinical continuum of Alzheimer's disease. Mol Psychiatry. 2021, 26(10): 6023-6037. doi: 10.1038 / s41380-020-0797-2.). However, its therapeutic application in AD patients is limited due to poor pharmacokinetics and insufficient blood-brain barrier (BBB) permeability. Therefore, there is an increasing interest in developing small molecules with potential NGF-mimetic activity for treating neurodegenerative diseases. Currently, many small molecules with NGF-mimetic activity have entered the clinical trial stage, and small molecules with NGF-mimetic activity may provide new treatment approaches for treating AD.

[0004] In addition, AD is generally regarded as a disease closely related to aging. Drugs with anti-aging effects such as resveratrol, metformin, and curcumin have been reported to have significant neuroprotective effects in AD models (Broderick TL, et al. Neuroprotective Effects of Chronic Resveratrol Treatment and Exercise Training in the 3xTg-AD Mouse Model of Alzheimer's Disease. Int J Mol Sci. 2020, 21(19):7337. doi:10.3390 / ijms21197337.; Xu Xiaoyan, et al. Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model. Protein Cell. 2021, 12(10):769-787. doi:10.1007 / s13238-021-00858-3.; Chen Ziyu, et al. Curcumin mitigates Aβ 33- 42 fibril-induced neuronal injury in the hippocampus. Mater. Today Phys. 2024, 44:101436. doi:10.1016 / j.mtphys.2024.101436.), for example, reducing inflammatory factors, enhancing antioxidant capacity, supporting neuron growth and repair, etc. At the same time, this also indicates a close link between aging and neurodegenerative diseases. Therefore, small molecules with anti-aging activity provide a potential direction for the treatment of neurodegenerative diseases by promoting neuroprotection.

[0005] Therefore, based on the relationship between neurodegenerative diseases, aging, and NGF, a compound with NGF-mimicking activity and anti-aging effects can be developed. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a substituted phenyl glycoside compound, which has very significant NGF-mimicking and neuroprotective activities on PC12 cells and obvious anti-aging activity on the K6001 yeast model, and can be used for the preparation of anti-aging and neuroprotective drugs.

[0007] A substituted phenyl glycoside compound, the structure of which is shown in formula (I):

[0008]

[0009] Wherein, R 1 , R 2 , R 3 , R 4 is F, CF 3 or H; R 5 is CN, OH, NHBoc, N(CH 3 )Boc, N(CH 3 ) 2 , NO 2 , CHO, COCH 3 , COOH, COOCH 2 CH 3 , OCH 3 or OOCCH 3 ; R 6 is COCH 3 or H; R 7 is COCH 3 , COOCH 2 CH 3 or H; n is an integer from 0 to 2.

[0010] Preferably, the substituted phenyl glycoside compound is selected from any of the following structures:

[0011]

[0012] When F, CF 3 and ethoxycarbonyl (COOCH 2 CH 3 ) are introduced into some molecules of the substituted phenyl glycoside compounds in the present invention, it has a great influence on the activity. Also, the introduction of an amino group at the 4-position of the benzene ring has a great influence on the activity.

[0013] The present invention also provides a preparation method of the above-mentioned substituted phenyl glycoside compound, including the following steps: adding a phenolic compound and bromoacetyl sugar to an aqueous potassium carbonate / dichloromethane two-phase mixed solution containing tetrabutylammonium bromide for glycosylation reaction to obtain an acetyl glycoside compound (A); then reacting the acetyl glycoside compound (A) in an alkaline solution to obtain a glycoside compound (B); under the catalysis of scandium trifluoromethanesulfonate, reacting the glycoside compound (B) with diethyl pyrocarbonate in a mixed solution of anhydrous toluene and ethanol to obtain a 6-position carbonated glycoside compound (C); reacting the 6-position carbonated glycoside compound (C) with acetic anhydride to obtain a 6-position carbonated acetyl glycoside compound (D).

[0014] The specific reaction formula is as follows:

[0015]

[0016] Among them, TBAB is tetrabutylammonium bromide, DEPC is diethyl pyrocarbonate, and Sc(OTf) 3 is scandium trifluoromethanesulfonate.

[0017] Preferably, the phenol compound is one of 4-hydroxybenzyl alcohol, 4-hydroxy phenethyl alcohol, 2-fluoro-4-hydroxybenzyl alcohol, 2,6-difluoro-4-hydroxybenzyl alcohol, 2-chloro-4-hydroxybenzaldehyde, 3-fluoro-4-hydroxybenzaldehyde, 2,6-difluoro-4-hydroxybenzaldehyde, 3,5-difluoro-4-hydroxybenzaldehyde, 3-fluoro-4-hydroxyacetophenone, 3,5-difluoro-4-methoxyphenol, ethyl 2-methoxy-4-hydroxybenzoate, 2-trifluoromethyl-4-nitrophenol, 2,3-difluoro-4-cyanophenol, N-Boc-4-hydroxybenzylamine, N-Boc-2,6-difluoro-4-hydroxybenzylamine, N-Boc-N-methyl-4-hydroxybenzylamine, 4-dimethylaminoethyl phenol, N-Boc-4-hydroxy-3-trifluoromethylaniline, N-Boc-4-hydroxy-3-fluoroaniline.

[0018] Preferably, the bromoacetyl sugar is one of bromotetraacetylglucose, bromotetraacetylgalactose, bromotriacetylxylose, bromotetraacetylmannose.

[0019] In the present invention, the structural formulas of the bromoacetyl sugars are as follows. From left to right, they are bromotetraacetylglucose, bromotetraacetylgalactose, bromotriacetylxylose, bromotetraacetylmannose;

[0020]

[0021] More preferably, the molar ratio of the bromoacetyl sugar, phenol compound, tetrabutylammonium bromide and potassium carbonate is 1-2:1:0.3-1:1-2.

[0022] Preferably, the basic solution is a methanol solution of any one of sodium methoxide, potassium carbonate, sodium bicarbonate or potassium hydroxide.

[0023] Preferably, the volume ratio of anhydrous toluene to ethanol is 1:0.2-4.

[0024] Preferably, the molar ratio of the glycoside compound (B), diethyl pyrocarbonate and scandium trifluoromethanesulfonate is 1:1-1.5:0.05-0.20.

[0025] Preferably, the molar ratio of the 6-carbonate esterified glycoside compound (C) to acetic anhydride is 1:4-10.

[0026] The present invention also provides the use of the above-mentioned substituted phenyl glycoside compounds in the preparation of drugs, health products or foods for preventing and / or treating senescence-related diseases.

[0027] The yeast mutant strain K6001 derived from W303 is characterized in that only mother cells can divide until death on yeast peptone glucose agar plates, thus simplifying the operation of replicative lifespan and being an ideal model for screening anti-aging small molecules. The substituted phenyl glycoside compounds of the present invention have the effect of prolonging the replicative lifespan of K6001 yeast. By adding pharmaceutically acceptable carriers, diluents, etc., they can be used for the preparation of anti-aging drugs.

[0028] The present invention also provides the use of the above-mentioned substituted phenyl glycoside compounds in the preparation of drugs, health products or foods for preventing and / or treating neurodegenerative diseases.

[0029] PC12 cells are derived from a cell line cloned from rat adrenal pheochromocytoma. Since they stop dividing and grow neurites under NGF stimulation and differentiate into cells with the characteristics of sympathetic neurons, they are often used in in vitro studies of nervous system diseases and are widely used for screening small molecules with NGF-mimicking activity, analyzing the molecular mechanisms of neuron differentiation and NGF action, etc. It is an ideal model for studying the functions and mechanisms of nerve cells at the cellular level.

[0030] The substituted phenyl glycoside compounds 1 to 18 in the present invention have significant activity in promoting neurite outgrowth in the in vitro screening model PC12 cells. The substituted phenyl glycoside compounds can be used as active ingredients, and by adding pharmaceutically acceptable carriers, diluents, etc., drugs for preventing and / or treating neurodegenerative diseases can be prepared.

[0031] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0032] In the present invention, the substituted phenyl glycoside compounds can induce neurite outgrowth in PC12 cells and have significant NGF-mimicking activity. At the same time, compound 18 can also increase the survival rate of PC12 cells induced by H 2 O 2 or Aβ, and reduce the levels of ROS and malondialdehyde (MDA) in the cells.

[0033] The pharmaceutically acceptable carrier refers to the medicinal excipients commonly used in the pharmaceutical field, mainly including the following categories: in terms of fillers, sucrose and its derivatives, starch and its modified products, microcrystalline cellulose, and various inorganic salts can be selected; for binders, cellulose derivatives (such as hydroxypropyl methylcellulose), starch paste, polyvinylpyrrolidone (povidone), gelatin, etc. can be selected; wetting agents include, but are not limited to, solvents such as distilled water and ethanol; lubricants can be selected from magnesium stearate, colloidal silicon dioxide, polyethylene glycol series, etc.; to improve the drug absorption effect, absorption promoters such as polysorbate and lecithin, and surfactants such as sorbitan fatty acid esters and poloxamer can be added. According to needs, sweeteners, flavoring agents and other excipients for improving the taste can also be added to the pharmaceutical composition.

[0034] The substituted phenyl glycoside compounds of the present invention can be administered in unit dosage form, and the routes cover enteral and parenteral methods, specifically including, but not limited to, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, transdermal administration, nasal administration and other methods.

[0035] The drugs of the present invention can be made into various forms, covering solid, semi-solid and liquid preparation types, specifically including tablets, pills, powders, dispersible tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft capsules, hard capsules, sterile injections, liniments and suppositories, etc. These preparations can be prepared by conventional processes, such as mixing the active ingredient with one or more carriers and then processing into the required form.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] The substituted phenyl glycoside compounds of the present invention can induce the elongation of neurites in PC12 cells and have significant NGF-mimicking activity. At the same time, the substituted phenyl glycoside compounds can also extend the replicative lifespan of K6001 yeast and have significant anti-aging activity. This series of compounds can also increase the viability of PC12 cells induced by H 2 O 2 or Aβ, and reduce the levels of ROS and malondialdehyde (MDA) in cells, and have significant neuroprotective activity, and can be used for the preparation of anti-aging and neuroprotective drugs. Description of the Drawings

[0038] Figure 1 Microscopic images and quantification diagrams of PC12 cells at the concentration corresponding to the optimal neurite differentiation rate of the substituted phenyl glycoside compounds (compounds 1-18) prepared in Examples 1-10, wherein,

[0039] A is the microscopic image of PC12 cells at the concentration corresponding to the optimal neurite differentiation rate after treatment with Compounds 1-18 for 48 h, all at the same scale. a is 0.5% dimethyl sulfoxide (DMSO), the negative control (hereinafter abbreviated as C); b is 40 ng / mL NGF, the positive control; c is Compound 1, 10 μM; d is Compound 2, 10 μM; e is Compound 3, 3 μM; f is Compound 4, 3 μM; g is Compound 5, 3 μM; h is Compound 6, 10 μM; i is Compound 7, 10 μM; j is Compound 8, 10 μM; k is Compound 9, 10 μM; l is Compound 10, 10 μM; m is Compound 11, 10 μM; n is Compound 12, 1 μM; o is Compound 13, 1 μM; p is Compound 14, 3 μM; q is Compound 15, 10 μM; r is Compound 16, 10 μM; s is Compound 17, 10 μM; t is Compound 18, 10 μM;

[0040] B is the digital result of Figure A. Each experiment was repeated 3 times, and the results are expressed as mean ± standard error (mean ± SEM). *** indicates P < 0.001, showing a significant difference compared with the negative control group.

[0041] Figure 2 Shows the effects of Compounds 3, 4, 10, 13, 15-18 on the replicative lifespan of K6001 yeast at different concentrations. Among them, A-H are the effects of Compounds 3, 4, 10, 13, 15-18 on the replicative lifespan of K6001 yeast at concentrations of 0.1, 1 or 10 μM, respectively. Each experiment was repeated 3 times, and the results are expressed as mean ± SEM. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, all showing significant differences compared with the negative control group.

[0042] Figure 3 Is the analysis diagram of the neuroprotective effect of Compound 18 on PC12 cells. Among them,

[0043] A is the cell viability of PC12 cells treated with Compound 18 (1, 3 or 10 μM) or resveratrol (10 μM) and H 2 O 2 (0.8 mM);

[0044] B and C are the microscopic photograph (scale bar is 100 μm) and DCF fluorescence intensity quantification of DCFH-DA stained PC12 cells, respectively;

[0045] D is the change in MDA content of PC12 cells treated with Compound 18 (1, 3 or 10 μM) or resveratrol (10 μM) and H 2 O 2 (0.8 mM);

[0046] E is the cell viability of PC12 cells treated with compound 18 (1, 3 or 10 μM) and Aβ 25-35 (30 μM). Each experiment was repeated 3 times, and the results were expressed as mean ± SEM. indicates P < 0.001, showing significant difference compared with the control group; compared with the H 2 O 2 or Aβ 25-35 group, * is P < 0.05, ** is P < 0.01, and *** is P < 0.001, all showing significant differences.

[0047] Figure 4 is the verification diagram of compound 18 and the potential target INSR. Among them,

[0048] A is the Western blot analysis and digital results of INSR protein and phosphorylated INSR protein after transfection with INSR siRNA or NC siRNA and treatment with compound 18 (10 μM);

[0049] B is the Western blot analysis and digital results of INSR protein after treatment with compound 18 (10 μM) and heating at different temperatures;

[0050] C is the PC12 cell protein incubated with compound 18 (10 μM) at room temperature for 3 h, digested with different concentrations of pronase E for 25 min, and the content of INSR protein in the cells was detected by Western blot;

[0051] D is the PC12 cell protein incubated with different concentrations of compound 18 at room temperature for 3 h, digested with pronase E (0 or 1:100) for 25 min, and the content of INSR protein in the cells was detected by Western blot. Each experiment was repeated 3 times, and the results were expressed as mean ± SEM. * is P < 0.05, ** is P < 0.01, and *** is P < 0.001, indicating significant differences compared with the control group; ## and indicate significant differences of P < 0.01 and P < 0.001, respectively.

[0052] Figure 5 Verification diagram of compound 18 and the potential target ACTN4. Among them,

[0053] A and B are the Western blot analysis and digital results of ACTN4 protein at different temperatures after treatment with compound 18 (10 μM), respectively;

[0054] C is the PC12 cell protein incubated with compound 18 (10 μM) at room temperature for 3 h, digested with different concentrations of pronase E for 25 min, and the content of ACTN4 protein in the cells was determined by Western blot;

[0055] PC12 cell proteins were incubated with different doses of compound 18 at room temperature for 3 h, digested with pronase E (0 or 1:100) for 25 min, and the content of ACTN4 protein in the cells was detected by Western blotting. Each experiment was repeated 3 times, and the results were expressed as mean±SEM; * indicated P<0.05 and *** indicated P<0.001, showing significant differences compared with the control group; # and indicated significant differences with P<0.05 and P<0.001, respectively.

[0056] Figure 6 For the PI3K / Akt signaling pathway to be involved in the induced NGF-like activity by compound 18, among which,

[0057] A was the Western blotting analysis and digital results of the phosphorylation levels of INSR and Akt after treatment with an INSR inhibitor and compound 18 or NGF;

[0058] B was the Western blotting analysis and digital results of the phosphorylation levels of PI3K and Akt after treatment with a PI3K inhibitor and compound 18 or NGF;

[0059] C was the Western blotting analysis and digital results of ACTN4 protein and phosphorylated PI3K and Akt proteins after transfection with ACTN4 siRNA or NC siRNA and treatment with compound 18. Each experiment was repeated 3 times, and the results were expressed as mean±SEM; * indicated P<0.05, ** indicated P<0.01, and *** indicated P<0.001, showing significant differences compared with the control group; ## and indicated significant differences with P<0.01 and P<0.001, respectively. Detailed implementation mode

[0060] The following is a further detailed description of the present invention in combination with embodiments, but the implementation modes of the present invention are not limited to the following embodiments.

[0061] The raw materials used in the present invention are all commercially available.

[0062] Example 1: Preparation of compounds 1 and 2

[0063] (1) Gastrodin (286 mg, 1.0 mmol) was added to a mixed solution of DEPC (173 μL, 1.2 mmol) and Sc(OTf) 3 (25 mg, 0.05 mmol) with anhydrous toluene:ethanol volume ratio = 4:1, and stirred for 8 h. Then the reaction mixture was concentrated, and the obtained crude product was purified by a silica gel open column (n-hexane:ethyl acetate = 1:10) to obtain white solid compound 1 (232 mg, yield 65%).

[0064] The structure of compound 1 is shown below, HR ESI-MS and1 The \(^1\)H NMR data are as follows: HR ESI-TOF-MS \(m / z\) 381.1193, calcd. for C 16 \(_{18}\)H 22 \(_{23}\)O 9 Na \([M + Na]^+\) + 381.1156. 1 \(^1\)H NMR (500 MHz, CD 3 \(_3\)OD): \(\delta\) = 7.27 (2H, d, \(J\) = 8.6 Hz), 7.05 (2H, d, \(J\) = 8.6 Hz), 4.87 (1H, d, \(J\) = 7.6 Hz), 4.54 (2H, s), 4.45 (1H, m), 4.29 (1H, m), 4.14 (2H, m), 3.64 (1H, m), 3.45 (2H, m), 3.37 (1H, m), 1.26 (3H, t, \(J\) = 7.1 Hz).

[0065]

[0066] (2) Compound 1 (179 mg, 0.5 mmol) was added with acetic anhydride (0.95 mL, 5.0 mmol) in a pyridine (5 mL) solution and stirred at room temperature for 24 h. The reaction end point was detected by TLC. After the reaction was completed, it was extracted with ethyl acetate and 1N HCl solution. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude sample was separated by column chromatography (n-hexane:ethyl acetate = 75:25) to obtain Compound 2 as a white solid (252 mg, yield 96%).

[0067] The structure of Compound 2 is shown below, and the HRESI-MS and 1 \(^1\)H NMR data are as follows: HRESI-TOF-MS \(m / z\) 549.1528, calcd. for C 24 \(_{26}\)H 30 \(_{31}\)O 13 Na \([M + Na]^+\) + 549.1579. 1 \(^1\)H NMR (500 MHz, CDCl 3 \(_3\)): \(\delta\) = 7.29 (2H, d, \(J\) = 8.5 Hz), 6.97 (2H, d, \(J\) = 8.6 Hz), 5.28 (2H, m), 5.13 (1H, m), 5.07 (1H, d, \(J\) = 7.5 Hz), 5.04 (2H, s), 4.32 (1H, m), 4.24–4.15 (3H, m), 3.88 (1H, m), 2.07 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 2.03 (3H, s), 1.30 (3H, t, \(J\) = 7.1 Hz).

[0068]

[0069] Example 2: Preparation of Compound 3

[0070] (1) First, dissolve 2,6-difluoro-4-hydroxybenzaldehyde (158 mg, 1.0 mmol) in anhydrous methanol. Under an ice bath, slowly add sodium borohydride (NaBH 4 )(76 mg, 2 mmol). After stirring for 2 h, when the reaction is completed as detected by TLC, add water to quench the reaction. Evaporate methanol under reduced pressure, adjust the pH to 7 - 8 with 1N HCl solution, extract with dichloromethane. The combined organic layers are dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3,5-difluoro-4-(hydroxymethyl)phenol (157 mg, yield 98%);

[0071] (2) Dissolve the product obtained in step (1) in a mixed solution of dichloromethane (5 mL) and water (5 mL), and add TBAB (293 mg, 0.9 mmol) and K 2 CO 3 (251 mg, 1.8 mmol). Stir at room temperature for 30 min. Subsequently, add bromotetraacetylglucose (538 mg, 1.3 mmol) to the mixture and stir at 50 °C for 6 h. Extract with dichloromethane. The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated. The crude sample is purified by column chromatography (n-hexane:ethyl acetate = 75:25) to obtain the product (269 mg, yield 56%);

[0072] (3) Dissolve the product obtained in step (2) in saturated NaHCO 3 methanol (10 mL), stir at room temperature for 1 h, concentrate the reaction mixture, filter, and then purify by silica gel open column (dichloromethane:methanol = 88:12) to obtain the product (171 mg, yield 97%);

[0073] (4) The same as step (1) in Example 1, and the eluent used for silica gel open column purification is dichloromethane:methanol = 9:1 by volume ratio to obtain Compound 3 as a white solid (85 mg, yield 41%).

[0074] The structure of Compound 3 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 417.0960, calcd. for C 16 H 20 F 2 O 9 Na [M + Na] + 417.0968. 1 H NMR (500 MHz, CD3 OD): δ = 6.71 (2H, m), 4.92 (1H, d, J = 7.3 Hz), 4.60 (2H, s), 4.47 (1H, m), 4.28 (1H, m), 4.14 (2H, m), 3.70 (1H, m), 3.46 (2H, m), 3.35 (1H, m), 1.25 (3H, t, J = 7.1 Hz).

[0075]

[0076] Example 3: Preparation of Compound 4

[0077] The preparation method was the same as that of Compound 3 in Example 2, except that bromotetraacetylglucose was replaced with bromotetraacetylgalactose to obtain Compound 4 (79 mg, yield 20%).

[0078] The structure of Compound 4 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 417.0969, calcd. for C 16 H 20 F 2 O 9 Na [M+Na] + 417.0968. 1 H NMR (500 MHz, CD 3 OD): δ = 6.71 (2H, m), 4.88 (1H, d, J = 7.6 Hz), 4.60 (2H, s), 4.41 (1H, m), 4.27 (1H, m), 4.16 (2H, m), 3.98 (1H, m), 3.88 (1H, m), 3.78 (1H, m), 3.60 (1H, m), 1.27 (3H, t, J = 7.1 Hz).

[0079]

[0080] Example 4: Preparation of Compound 5

[0081] The preparation method was the same as that of Compound 3 in Example 2, except that 3,5-difluoro-4-(hydroxymethyl)phenol was replaced with 2,3-difluoro-4-cyanophenol to obtain Compound 5 (89 mg, yield 23%).

[0082] The structure of Compound 5 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 412.0861, calcd. for C 16 H17 F 2 NO 8 Na[M+Na] + 412.0815. 1 H NMR(500MHz,CD 3 OD):δ=7.50(1H,m),7.21(1H,m),5.14(1H,d,J=7.5Hz),4.45(1H,m),4.28(1H,m),4.12(2H,m),3.72(1H,m),3.51(2H,m),3.39(1H,m),1.25(3H,t,J=7.1Hz).

[0083]

[0084] Example 5: Preparation of Compounds 6 and 7

[0085] (1) Dissolve 4-hydroxybenzylamine (123 mg, 1.0 mmol) in a solution of 1,4-dioxane (3 mL) and water (3 mL), and add (Boc) 2 O (253 μL, 1.1 mmol) and 2M Na 2 CO 3 (50 μL). Stir at 0 °C for 30 min, heat to room temperature for 30 min, add ethyl acetate to terminate the reaction and extract. The combined organic layers are dried over anhydrous sodium sulfate, filtered and concentrated to obtain tert-butyl (2,6-difluoro-4-hydroxybenzyl)carbamate (135 mg, yield 60%);

[0086] (2) Treat the product obtained in step (1) according to step (2) of the preparation method of compound 3 in Example 2 to obtain compound 6 (153 mg, yield 46%).

[0087] The structure of compound 6 is shown below, and the HR ESI-MS and 1 H NMR data: R ESI-TOF-MS m / z 576.2045, calcd. for C 26 H 35 NO 12 Na[M+Na] + 576.2052. 1 H NMR(500MHz,CDCl 3): δ = 7.21 (2H, d, J = 8.5 Hz), 6.95 (2H, d, J = 8.6 Hz), 5.27 (2H, m), 5.16 (1H, m), 5.05 (1H, d, J = 7.5 Hz), 4.29 (1H, m), 4.26 (2H, s), 4.17 (1H, m), 3.84 (1H, m), 2.08 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 2.04 (3H, s), 1.46 (9H, s).

[0088]

[0089] (3) Compound 6 was processed according to step (3) of the preparation method of compound 3 in Example 2 to obtain compound 7 (102 mg, yield 96%).

[0090] The structure of compound 7 is shown below, along with HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 408.1682, calcd. for C 18 H 27 NO 8 Na[M+Na] + 408.1629. 1 H NMR (500 MHz, CD 3 OD): δ = 7.20 (2H, d, J = 8.6 Hz), 7.05 (2H, d, J = 8.6 Hz), 4.88 (1H, d, J = 7.6 Hz), 4.16 (2H, s), 3.89 (1H, m), 3.69 (1H, m), 3.47–3.35 (4H, m), 1.44 (9H, s).

[0091]

[0092] Example 6: Preparation of Compounds 8 and 9

[0093] (1) The preparation method was the same as that of compound 6 in Example 5, except that bromotetraacetylglucose was replaced with bromotetraacetylgalactose to obtain compound 8 (143 mg, yield 26%).

[0094] The structure of compound 8 is shown below, along with HRESI-MS and 1 H NMR data: HRESI-TOF-MS m / z 576.2047, calcd. for C 26 H 35 NO 12 Na[M+Na] + 576.2052.1 H NMR (500 MHz, CDCl 3 ): δ = 7.21 (2H, d, J = 8.5 Hz), 6.96 (2H, d, J = 8.6 Hz), 5.55–5.38 (3H, m), 5.10 (1H, m), 5.01 (1H, d, J = 8.0 Hz), 4.21 (1H, m), 4.16 (1H, m), 4.12 (1H, m), 4.05 (1H, m), 2.18 (3H, s), 2.06 (3H, s), 2.06 (3H, s), 2.01 (3H, s), 1.45 (9H, s).

[0095]

[0096] (2) The preparation method was the same as that of Compound 7 in Example 5, except that Compound 7 was replaced by Compound 8 to obtain Compound 9 (95 mg, yield 95%).

[0097] The structure of Compound 9 is shown below, and the HR ESI-MS and 1 H NMR data: HRESI-TOF-MS m / z 408.1667, calcd. for C 18 H 27 NO 8 Na[M + Na] + 408.1629. 1 H NMR (500 MHz, CD 3 OD): δ = 7.19 (2H, d, J = 8.6 Hz), 7.06 (2H, d, J = 8.6 Hz), 4.84 (1H, d, J = 7.8 Hz), 4.16 (2H, s), 3.91 (1H, m), 3.81–3.75 (3H, m), 3.67 (1H, m), 3.58 (1H, m), 1.44 (9H, s).

[0098]

[0099] Example 7: Preparation of Compound 10

[0100] The preparation method was the same as that of Compound 6 in Example 5, except that 4-hydroxybenzylamine was replaced by 4-amino-2-fluorophenol to obtain Compound 10 (98 mg, yield 18%).

[0101] The structure of Compound 10 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 580.1870, calcd. for C 25H 32 FNO 12 Na[M+Na] + 580.1801. 1 H NMR(500MHz,CDCl 3 ):δ=7.36(1H,m),7.10(1H,m),6.87(1H,m),6.45(1H,s),5.25(2H,m),5.16(1H,m),4.86(1H,d,J=7.7Hz),4.28(1H,m),4.16(1H,m),3.74(1H,m),2.09(3H,s),2.08(3H,s),2.03(3H,s),2.03(3H,s),1.51(9H,s).

[0102]

[0103] Example 8: Preparation of Compounds 11 and 12

[0104] (1) The preparation method was the same as that of Compound 6 in Example 5, except that 4-hydroxybenzylamine was replaced with 4-[(amino)methyl]phenol, and bromotetraacetylglucose was replaced with bromotetraacetylgalactose to obtain Compound 11 (205 mg, yield 36%).

[0105] The structure of Compound 11 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z590.2248, calcd. for C 27 H 37 NO 12 Na[M+Na] + 590.2208. 1 H NMR(500MHz,CDCl 3 ):δ=7.16(2H,d,J=8.2Hz),6.96(2H,d,J=8.5Hz),5.48(2H,m),5.10(1H,m),5.02(1H,d,J=8.0Hz),4.36(2H,s),4.23(1H,m),4.15(1H,m),4.05(1H,m),2.79(3H,s),2.18(3H,s),2.07(3H,s),2.06(3H,s),2.01(3H,s),1.47(9H,s).

[0106]

[0107] (2) The preparation method was the same as that of Compound 7 in Example 5, except that Compound 7 was replaced with Compound 11 to obtain Compound 12 (132 mg, yield 92%).

[0108] The structure of Compound 12 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 422.1802, calcd. for C 19 H 29 NO 8 Na[M+Na] + 422.1785. 1 H NMR (500 MHz, CD 3 OD): δ = 7.17 (2H, d, J = 8.7 Hz), 7.09 (2H, d, J = 8.6 Hz), 4.85 (1H, m), 4.37 (2H, s), 3.91 (1H, m), 3.81–3.76 (3H, m), 3.68 (1H, m), 3.59 (1H, m), 2.79 (3H, s), 1.48 (9H, s).

[0109]

[0110] Example 9: Preparation of Compounds 13 and 14

[0111] (1) The preparation method was the same as that of Compound 6 in Example 5, except that 4-hydroxybenzylamine was replaced with 4-amino-2-(trifluoromethyl)phenol to obtain Compound 13 (290 mg, yield 48%).

[0112] The structure of Compound 13 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 630.1798, calcd. for C 26 H 32 F 3 NO 12 Na[M+Na] + 630.1769. 1 H NMR (500 MHz, CDCl 3): δ = 7.59 (1H, m), 7.49 (1H, m), 7.21 (1H, m), 6.49 (1H, s), 5.35–5.24 (2H, m), 5.17 (1H, m), 5.00 (1H, d, J = 7.5 Hz), 4.27 (1H, m), 4.22 (1H, m), 3.86 (1H, m), 2.10 (3H, s), 2.05 (6H, m), 2.03 (3H, s), 1.51 (9H, s).

[0113]

[0114] (2) The preparation method was the same as that of Compound 7 in Example 5, except that Compound 7 was replaced with Compound 13 to obtain Compound 14 (207 mg, yield 98%).

[0115] The structure of Compound 14 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 462.1372, calcd. for C 18 H 24 F 3 NO 8 Na[M+Na] + 462.1346. 1 H NMR (500 MHz, CD 3 OD): δ = 7.72 (1H, m), 7.52 (1H, m), 7.29 (1H, m), 4.99 (1H, d, J = 7.3 Hz), 3.90 (1H, m), 3.70 (1H, m), 3.52–3.44 (3H, m), 3.40 (1H, m), 1.51 (9H, s).

[0116]

[0117] Example 10: Preparation of Compounds 15 - 18

[0118] (1) First, dissolve 2,6-difluoro-4-hydroxybenzonitrile (1.00 g, 6.45 mmol) in 1M BH 3 ·THF (25.81 mL, 25.81 mmol), and reflux at 70 °C. After stirring for 6 h, quench with 1N HCl solution, adjust the pH to 10 - 11 with NaOH, and extract with ethyl acetate. The obtained organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated to obtain the crude product 4-(aminomethyl)-2-(trifluoromethyl)phenol (708 mg, yield 69%);

[0119] (2) The preparation method was the same as that of Compound 6 in Example 5, except that 4-hydroxybenzylamine was replaced with 4-(aminomethyl)-2-(trifluoromethyl)phenol to obtain Compound 15 (1.20 g, yield 46%).

[0120] The structure of Compound 15 is shown below, and the HR ESI-MS and 1 H NMR data: HRESI-TOF-MS m / z 612.1865, calcd. for C 22 H 33 F 2 NO 12 Na[M+Na] + 612.1863. 1 H NMR (500 MHz, CDCl 3 ): δ = 6.56 (2H, m), 5.26 (2H, m), 5.12 (1H, m), 5.03 (1H, d, J = 7.6 Hz), 4.33 (2H, s), 4.24 (1H, m), 4.19 (1H, m), 3.88 (1H, m), 2.10 (3H, s), 2.06 (3H, s), 2.06 (3H, s), 2.03 (3H, s), 1.43 (9H, s).

[0121]

[0122] (3) The preparation method was the same as that of Compound 7 in Example 5, except that Compound 7 was replaced with Compound 15 to obtain Compound 16 (844 mg, yield 98%).

[0123] The structure of Compound 16 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 444.1437, calcd. for C 18 H 25 F 2 NO 8 Na[M+Na] + 444.1440. 1 H NMR (500 MHz, CD 3 OD): δ = 6.72 (2H, m), 4.90 (1H, d, J = 7.3 Hz), 4.24 (2H, s), 3.89 (1H, m), 3.69 (1H, m), 3.50–3.41 (3H, m), 3.37 (1H, m), 1.42 (9H, s).

[0124]

[0125] (4) The preparation method was the same as that of Compound 1 in Example 1, except that gastrodin was replaced with Compound 16 to obtain Compound 17 (662 mg, yield 67%).

[0126] The structure of Compound 17 is shown below, and the HR ESI-MS and 1 H NMR data: HRESI-TOF-MS m / z 516.1669, calcd. for C 21 H 29 F 2 NO 10 Na[M+Na] + 516.1652. 1 H NMR (500 MHz, CD 3 OD): δ = 6.69 (2H, m), 4.90 (1H, d, J = 7.3 Hz), 4.46 (1H, m), 4.28 (1H, m), 4.24 (2H, s), 4.14 (2H, m), 3.70 (1H, m), 3.45 (2H, m), 3.35 (1H, m), 1.42 (9H, s), 1.26 (3H, t, J = 7.1 Hz).

[0127]

[0128] (5) The preparation method was the same as that of Compound 2 in Example 1, except that Compound 1 was replaced with Compound 17 to obtain Compound 18 (816 mg, yield 98%).

[0129] The structure of Compound 18 is shown below, and the HR ESI-MS and 1 H NMR data: HR ESI-TOF-MS m / z 642.1975, calcd. for C 27 H 35 F 2 NO 13 Na[M+Na] + 642.1969. 1 H NMR (500 MHz, CDCl 3 ): δ = 6.53 (2H, m), 5.29 (1H, m), 5.23 (1H, m), 5.11 (1H, m), 5.03 (1H, d, J = 7.6 Hz), 4.32–4.28 (3H, m), 4.22–4.15 (3H, m), 3.90 (1H, m), 2.07 (3H, s), 2.06 (3H, s), 2.03 (3H, s), 1.43 (9H, s), 1.30 (3H, t, J = 7.1 Hz).

[0130]

[0131] Compound effect analysis

[0132] I. Bioactivity assay of compounds 1 - 18 on PC12 cells

[0133] 1. Medium preparation

[0134] (1) EM basal medium: High - glucose medium (DMEM) containing 1% (v / v) double antibiotics (10000 U / mL penicillin and 10 mg / mL streptomycin), stored in a 4°C refrigerator for later use.

[0135] (2) CM complete medium: DMEM containing 1% double antibiotics (10000 U / mL penicillin and 10 mg / mL streptomycin), 10% horse serum (HS) and 5% fetal bovine serum (FBS), stored in a 4°C refrigerator for later use.

[0136] 2. Steps for the assay of NGF - like activity of PC12 cells

[0137] When the density of PC12 cells reaches 70% - 80% of the culture dish, sub - culture is carried out. The cells are seeded into a 24 - well plate at a density of 50000 cells per well, and 1 mL of CM complete medium is added to each well. Then it is placed in an incubator at 37°C and 5% CO 2 for 24 h. 0.5% DMSO is used as the negative control, and NGF (40 ng / mL) is used as the positive control. The test samples (compounds 1 - 18) are prepared into the test concentrations (1, 3, 10 μM) with DMSO. DMSO, NGF and the test samples are respectively mixed with EM medium to prepare solutions with a total volume of 1 mL, replacing the original CM medium in the 24 - well plate. After continuing to culture for 48 h, the morphological changes of the cells are observed and the neurite differentiation rate of PC12 cells is calculated (neurite differentiation rate of cells = number of cells with neurites longer than one - fold of the longest diameter of the cell body / total number of cells in the selected field of view × 100%). Approximately 100 - 200 cells are observed in each field of view. Three fields of view are randomly selected for counting and the average value is taken. The images at the concentration corresponding to the optimal neurite differentiation rate of each compound are integrated. The experimental data are analyzed by one - way ANOVA using GraphPad Prism 8.0 statistical software, and the results are expressed as mean ± standard error (mean ± SEM), with the significance level set at P < 0.05.

[0138] 3. Analysis of test results

[0139] Figure 1Microscopic images and quantification graphs of PC12 cells at the concentrations corresponding to the optimal neurite differentiation rates of the substituted phenyl glycoside compounds (Compounds 1 - 18) prepared in Examples 1 - 10. Among them, 0.5% DMSO was used as the negative control, and NGF (40 ng / mL) was used as the positive control. A shows the microscopic photographs of PC12 cells at the concentrations corresponding to the optimal neurite differentiation rates of Compounds 1 - 18; B shows the digitalized results of Figure A. ***P < 0.001 indicates compared with the negative control group. As shown in the figure, the optimal neurite differentiation rates of Compounds 1 - 18 after acting on PC12 cells for 48 h were all above 40%, all of which could significantly promote the neurite differentiation rate of PC12 cells, and Compound 18 had the most significant activity.

[0140] II. Evaluation of the replicative lifespan of substituted phenyl glycoside compounds in K6001 yeast (taking Compounds 3, 4, 10, 13, 15 - 18 as examples)

[0141] 1. Preparation of culture media, common reagents and test samples

[0142] (1) Galactose liquid medium: 1% w / v yeast extract, 2% w / v multilevel peptone, 3% w / v galactose. After adding a quantitative amount of biological pure water to dissolve, it was sterilized by moist heat at 121 °C for 20 min.

[0143] (2) Glucose liquid medium: 1% w / v yeast extract, 2% w / v multilevel peptone, 2% w / v glucose. After adding a quantitative amount of biological pure water to dissolve, it was sterilized by moist heat at 121 °C for 20 min.

[0144] (3) Galactose solid medium and glucose solid medium: 2% w / v agar was added to the liquid media described in (1) and (2) respectively.

[0145] (4) Phosphate buffer solution (PBS): 4 g sodium chloride, 0.1 g potassium chloride, 0.575 g disodium hydrogen phosphate, 0.01 g potassium dihydrogen phosphate were dissolved in 500 mL of biological pure water, sterilized by moist heat at 121 °C for 20 min. After cooling to room temperature, it was stored in a 4 °C refrigerator for later use.

[0146] (5) Preparation of test samples: According to the different polarities of the test samples, they were first dissolved with a quantitative amount of 50% aqueous ethanol solution or ethanol to prepare the required concentrations. The positive control was a 10 μM resveratrol solution prepared with the same solvent, and the negative control was an equal volume of blank solvent. Generally, when measuring the replicative lifespan, the total volume of the sample solution or control was 150 μL.

[0147] 2. Method for measuring the replicative lifespan of K6001 yeast

[0148] (1) Take out the cryopreserved K6001 yeast strain from a -30°C refrigerator, add 5 mL of PBS solution, centrifuge (1500 rpm, 3 min), wash three times to remove the glycerol and culture medium therein, then add 5 mL of galactose liquid medium, and culture at 28°C with constant shaking (160 r / min) for 24 h.

[0149] (2) After the yeast grows to the logarithmic growth phase, take it out from the shaker, mix well, take out 1 mL of yeast solution, centrifuge (1500 rpm, 3 min) three times with 5 mL of PBS to remove the galactose liquid medium therein, observe and count under a microscope using a hemocytometer, and then dilute the yeast solution to a certain concentration for standby.

[0150] (3) Add 5 mL of sterilized glucose solid medium to a sterilized glass petri dish. After the medium solidifies, add the positive control, negative control, and a certain concentration of the test sample solution respectively. After the solvent evaporates, add 4000 yeasts, and spread them evenly with a sterilized spreader, and culture at 28°C for 48 h.

[0151] (4) Observe and count under a microscope (eyepiece 10 times, objective 20 times). Randomly select 40 colonies produced by single yeast cells in each petri dish, count the number of daughter cells produced around the mother cells respectively, and record and analyze by graphing.

[0152] 3. Analysis of test results

[0153] Figure 2 The effects of the substituted phenyl glycoside compounds prepared in the examples, including compounds 3, 4, 10, 13, 15 - 18, on the replicative lifespan of K6001 yeast at different concentrations. Among them, ethanol is the negative control, and resveratrol (10 μM) is the positive control. Compared with the negative control group, compounds 3, 4, 10, 13, 15 - 18 can all significantly extend the replicative lifespan of K6001 yeast, and compound 15 has the most significant effect.

[0154] III. Evaluation of the neuroprotective activity of compound 18 in PC12 cells

[0155] 1. Method for determining the neuroprotective activity of PC12 cells

[0156] (1) Determine the cell survival rate in the H 2 O 2 injury model: Inoculate about 5×10 4 PC12 cells in each well of a 24-well plate, and culture in an incubator at 37°C and 5% CO 2 for 24 h. Then, change the culture medium to 1 mL of EM medium containing different test samples. In H 2 O 2In the dose-dependent experiment, cells were treated with 0.5% DMSO for 24 h and then cultured with different concentrations of H 2 O 2 for 1 h. Finally, it was determined that H 2 O 2 (0.8 mM) had the most significant effect and was selected as the dose for subsequent studies. To study the neuroprotective effect of compound 18, PC12 cells were treated with compound 18 (1, 3, or 10 μM) for 24 h and with 0.8 mM H 2 O 2 for 1 h. The medium was changed to 500 μL of EM medium containing 200 μg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the cells were cultured for another 2 h. The medium was completely removed, and 200 μL of DMSO was added to each well to dissolve the formed formazan crystals. The generated formazan was detected at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0157] (2) Determination of cell viability in the Aβ 25-35 injury model: Approximately 5×10 4 PC12 cells were seeded in each well of a 24-well plate and cultured in an incubator at 37 °C and 5% CO 2 for 24 h. Then, the medium was changed to 1 mL of EM medium containing different test samples. In the dose-dependent experiment of Aβ 25-35 , cells were treated with 0.5% DMSO for 0.5 h and then cultured with different concentrations of Aβ 25-35 for 48 h. Finally, it was determined that Aβ 25-35 (30 μM) had the most significant effect and was selected as the dose for subsequent studies. To study the neuroprotective effect of compound 18, PC12 cells were treated with compound 18 (1, 3, or 10 μM) for 0.5 h and with Aβ 25-35 (30 μM) for 48 h. The medium was changed to 500 μL of EM medium containing 200 μg / mL MTT, and the cells were cultured for another 2 h. The medium was completely removed, and 200 μL of DMSO was added to each well to dissolve the formed formazan crystals. The generated formazan was detected at 570 nm using an ELISA reader.

[0158] (3) Determination of ROS in PC12 cells:

[0159] To determine the ROS level in PC12 cells, approximately 5×10 4 PC12 cells were seeded in each well of a 24-well plate. The cells were treated with compound 18 (1, 3, or 10 μM) for 24 h and then with 0.8 mM H 2 O 2Treat for 1 h. Then add 10 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) to each well and incubate for 30 min. Wash the cells with PBS to remove extracellular DCFH-DA, and observe DCF in PC12 cells using a fluorescence microscope.

[0160] (4) Determination of MDA in PC12 cells: To detect the MDA content in PC12 cells, seed 2×10 6 PC12 cells in a 6-cm culture dish containing 5 mL of complete CM medium and culture for 24 h. Then, treat PC12 cells with compound 18 (1, 3, or 10 μM) for 24 h, and then treat with 0.8 mM H 2 O 2 for another 1 h. Lyse the cells with RIPA lysis buffer, centrifuge, centrifuge the cell lysate, and take the supernatant to evaluate the MDA content. Determine the MDA content using an MDA assay kit according to the manufacturer's instructions.

[0161] 2. Analysis of test results

[0162] As Figure 3 shown, compound 18 significantly increased the survival rate of PC12 cells in the presence of H 2 O 2 (0.8 mM) in a dose-dependent manner ( Figure 3 A in); compound 18 significantly reduced the increase in ROS and MDA in H 2 O 2 -induced PC12 cells ( Figure 3 B–D in); compound 18 significantly increased the survival rate of PC12 cells damaged by Aβ 25-35 (30 μM) ( Figure 3 E in). The results indicate that compound 18 can not only inhibit oxidative stress by reducing ROS and MDA, but also protect PC12 cells from Aβ-induced damage, and has significant neuroprotective activity.

[0163] IV. Target identification and mechanism analysis of compound 18 in PC12 cells

[0164] 1. Methods for target identification and mechanism analysis

[0165] (1) Western blot analysis: To extract proteins, cells were lysed in RIPA lysis buffer supplemented with 1% protease and 1% phosphatase inhibitors. Then the samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was transferred to a new tube. Protein concentration was determined using a BCA kit, and all samples were denatured at 100 °C for 10 min. Approximately 20 μg of protein from each sample was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) gel. After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% skim milk for 60 min. Then the membrane was incubated with diluted primary antibody overnight at 4 °C. After washing, the membrane was incubated with secondary antibody for 45 min. Then a chemiluminescence detection kit was used to visualize protein bands, and ImageJ software was used to quantify the blot density.

[0166] (2) Small interfering RNA (siRNA) experiments: The primer sequences for knocking down INSR and ACTN4 and the negative control (NC) siRNA were as follows: For INSR (NM_017071.2), sense strand: 5'-GUGAAGAGCUGGAGAUGGATT-3' (SEQ ID NO.1), antisense strand: 5'-UCCAUCUCCAGCUCUUCACTT-3' (SEQ ID NO.2); For ACTN4 (NM_031675.2), sense strand: 5'-UUGAAGUGGCUGAGAAAUATT-3' (SEQ ID NO.3), antisense strand: 5'-UAUUUCUCAGCCACUUCAATT-3' (SEQ ID NO.4); For NC siRNA, sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.5), antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.6). According to the manufacturer's instructions, PC12 cells were transfected with FAM-labeled siRNA at concentrations of 50, 100, and 150 nM, respectively, to evaluate the transfection efficiency. INSR siRNA (150 nM) or ACTN4 siRNA (150 nM) could achieve a transfection efficiency of over 90% and was selected as the dose for subsequent studies. To evaluate the protein levels of INSR and ACTN4 after transfection with different siRNAs, approximately 2×10 6Cells were cultured for 24 h. 2 mL of DMEM containing 4 μL Lipofectamine RNAiMAX was mixed with 2 mL of DMEM containing INSR siRNA, ACTN4 siRNA or NC siRNA (the final concentration of siRNA was 150 nM), and the original medium was replaced with this mixture. After 8 h of transfection, the medium in each well was replaced with 5 mL of DMEM containing compound 18 at a concentration of 10 μM, and the cells were cultured for another 2 h. Cell proteins were extracted from each culture dish and analyzed by Western blotting.

[0167] (3) Cellular thermal shift assay (CETSA): First, approximately 2×10 6 cells were seeded into each 6-cm cell culture dish and cultured for 24 h. Subsequently, the medium was replaced with 5 mL of EM medium containing compound 18 at a concentration of 10 μM and incubated for 4 h. Then, cell proteins were extracted from each group, and the protein concentration was determined using a BCA kit. The control group and the experimental group were both divided into seven equal aliquots. Each aliquot contained 100 μL of protein (2 μg / μL) and was heated at 46, 50, 54, 58, 62, and 66 °C for 3 min. The supernatant of each sample was collected, centrifuged at 12,000 rpm at 4 °C for 20 min, and then each sample was mixed with 5× SDS-PAGE protein loading buffer, denatured at 100 °C for 10 min, and analyzed by Western blotting.

[0168] (4) Drug affinity responsive target stability (DARTS): PC12 cell proteins were collected, and their concentration was determined using a BCA kit. Subsequently, the samples were diluted to 2 μg / μL. The protein samples were divided into two equal aliquots and incubated with compound 18 at a concentration of 10 μM or 0.5% DMSO at room temperature for 3 h. After incubation, the control group and the compound 18-treated group were further divided into five equal aliquots. Each sample was treated with pronase E at 500:1, 200:1, 100:1, 50:1 (protein:pronase E = w / w) or an equal volume of TNC buffer (50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl 2 ) at 25 °C for 25 min. After treatment, all samples were mixed with 5× SDS-PAGE protein loading buffer, heated at 100 °C for 10 min, and analyzed by Western blotting. In the dose-dependent experiment, the protein samples were divided into seven equal aliquots and incubated with compound 18 at concentrations of 0.1, 1, 5, 10, 25 μM or 0.5% DMSO at room temperature for 3 h. Each sample was treated with protease E (protein:pronase E = 100:1 w / w) or an equal volume of TNC buffer at 25 °C for 25 min, and the remaining sample treatment methods were the same as above.

[0169] 2. Analysis of Test Results

[0170] As Figure 4 shown, in the siRNA experiment, after transfection with INSR siRNA, the total INSR protein level decreased significantly, confirming the successful knockdown of INSR protein. In addition, INSR siRNA significantly reduced the phosphorylation of INSR protein induced by compound 18 ( Figure 4 A in); in the CETSA experiment, compound 18 enhanced the thermal stability of INSR protein in PC12 cells ( Figure 4 B in); in the DARTS experiment, compound 18 (10 μM) significantly inhibited the degradation of INSR protein in the presence of different concentrations of pronase E, especially 1.0% pronase E ( Figure 4 C in). At the same time, compound 18 increased the stability of INSR protein in a dose-dependent manner ( Figure 4 D in). The results indicate that INSR is the target protein of compound 18 in PC12 cells.

[0171] As Figure 5 shown, in the CETSA experiment, the treatment with compound 18 increased the thermal stability of ACTN4 ( Figure 5 A and B in); in the DARTS experiment, compound 18 (10 μM) significantly inhibited the degradation of ACTN4 protein in the presence of different concentrations of pronase E, especially 1.0% pronase E ( Figure 5 C in). At the same time, after treatment with compound 18, the stability of ACTN4 protein increased in a dose-dependent manner ( Figure 5 D in). The results indicate that ACTN4 is a target of compound 18.

[0172] As Figure 6 shown, the INSR-specific inhibitor (HNMPA-(AM) 3 significantly reduced the phosphorylation of INSR and downstream protein Akt induced by compound 18 ( Figure 6 A in); the PI3K-specific inhibitor LY294002 reversed the increase in the phosphorylation of PI3K and Akt induced by compound 18 ( Figure 6 B in). In the siRNA experiment, transfection with ACTN4 siRNA significantly reduced the levels of ACTN4, p-PI3K, and p-Akt induced by compound 18 ( Figure 6 C in). The results indicate that compound 18 promotes neurite outgrowth by activating the PI3K / Akt signaling pathway through INSR and ACTN4.

[0173] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A substituted phenyl glycoside compound, characterized in that: The structure of the substituted phenyl glycoside compound is shown in formula (I): Among them, R1, R2, R3, R4 are F, CF3 or H; R5 is CN, OH, NHBoc, N(CH3)Boc, N(CH3)2, NO2, CHO, COCH3, COOH, COOCH2CH3, OCH3 or OOCCH3; R6 is COCH3 or H; R7 is COCH3, COOCH2CH3 or H; n is an integer from 0 to 2.

2. The substituted phenyl glycoside compound according to claim 1, characterized in that The substituted phenyl glycoside compound is selected from any of the following structures:

3. The method for preparing a substituted phenyl glycoside compound according to claim 1 or 2, characterized in that: The following steps are involved: adding a phenol compound and brominated acetyl sugar to a two-phase mixture of potassium carbonate aqueous solution / dichloromethane containing tetrabutylammonium bromide to carry out a glycosidation reaction to obtain an acetyl glycoside compound; Subsequently, the acetylated glycoside compound is reacted in an alkaline solution to obtain a glycoside compound; under the catalytic action of scandium trifluoromethanesulfonate, the glycoside compound and diethyl pyrocarbonate are placed in a mixed solution of anhydrous toluene and ethanol to obtain a 6-carbonated glycoside compound after the reaction; the 6-carbonated glycoside compound is reacted with acetic anhydride to obtain a 6-carbonated acetylated glycoside compound.

4. The method for preparing a substituted phenyl glycoside compound according to claim 3, characterized in that: The phenol compounds are 4-hydroxybenzyl alcohol, 4-hydroxyphenylethanol, 2-fluoro-4-hydroxybenzyl alcohol, 2,6-difluoro-4-hydroxybenzyl alcohol, 2-chloro-4-hydroxybenzaldehyde, 3-fluoro-4-hydroxybenzaldehyde, 2,6-difluoro-4-hydroxybenzaldehyde, 3,5-difluoro-4-hydroxybenzaldehyde, 3-fluoro-4-hydroxyacetophenone, 3,5-difluoro-4-methoxyphenol, 2-methoxy-4-hydroxybenzaldehyde One of ethyl formate, 2-trifluoromethyl-4-nitrophenol, 2,3-difluoro-4-cyanophenol, N-tert-butoxycarbonyl-4-hydroxybenzylamine, N-tert-butoxycarbonyl-2,6-difluoro-4-hydroxybenzylamine, N-tert-butoxycarbonyl-N-methyl-4-hydroxybenzylamine, 4-dimethylaminoethylphenol, N-tert-butoxycarbonyl-4-hydroxy-3-trifluoromethylaniline, and N-tert-butoxycarbonyl-4-hydroxy-3-fluoroaniline; The brominated acetyl sugar is one of brominated tetraacetyl glucose, brominated tetraacetyl galactose, brominated triacetyl xylose and brominated tetraacetyl mannose.

5. The method for preparing a substituted phenyl glycoside compound according to claim 3, characterized in that: The molar ratio of the brominated acetyl sugar, the phenol compound, tetrabutylammonium bromide and potassium carbonate is 1-2:1:0.3-1:1-2.

6. The method for preparing a substituted phenyl glycoside compound according to claim 3, characterized in that: The alkaline solution is any one of a methanol solution of sodium methoxide, potassium carbonate, sodium bicarbonate or potassium hydroxide.

7. The method for preparing a substituted phenyl glycoside compound according to claim 3, characterized in that: The molar ratio of the glycoside compound, diethyl pyrocarbonate and scandium trifluoromethanesulfonate is 1:1-1.5:0.05-0.

20.

8. The method for preparing a substituted phenyl glycoside compound according to claim 3, characterized in that: The molar ratio of the 6-carbonated glycoside compound to acetic anhydride is 1:4-10.

9. Use of the substituted phenyl glycoside compound according to claim 1 or 2 in the preparation of medicines, health products, foods or cosmetics for preventing and / or treating aging and aging-related diseases.

10. Use of the substituted phenyl glycoside compound according to claim 1 or 2 in the preparation of medicines, health products or foods for preventing and / or treating neurodegenerative diseases.

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