An aryl imidazole compound, a preparation method thereof, and a pharmaceutical product prepared therefrom

Abstract

This invention belongs to the field of pharmaceutical technology and relates to neuroprotective drugs, specifically an aryl imidazole compound, its preparation method, and the resulting pharmaceutical product. The compound has the structure represented by the general formula (I): #imgabs0#. Pharmacological experiments conducted in this invention demonstrate that the aryl imidazole compound has a protective effect on nerve cells, inhibiting intracellular oxidative stress and inflammatory damage induced by oxygen and glucose deprivation, and enhancing the survival rate of cells afflicted with oxygen and glucose deprivation and ApoE4 gene overexpression. It can be used as a neuroprotectant for neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to neuroprotective drugs, in particular to an aryl imidazole compound and a preparation method thereof and a pharmaceutical product prepared therefrom. Background Art

[0002] Neurological diseases such as stroke and Alzheimer's disease (AD) impose a huge health burden on individuals and society.

[0003] Neuroprotective agents are an important means of treating acute ischemic stroke. They have the functions of protecting nerve cells, improving cerebral blood flow and restoring neurological function. They are also of great significance for the rehabilitation of patients with acute ischemic stroke.

[0004] Alzheimer's disease (AD) is a chronic neurodegenerative disorder associated with aging. The pathogenesis of AD remains incompletely understood. Widely accepted hypotheses include abnormal apolipoprotein E4 (ApoE4) metabolism, excessive accumulation of amyloid-β (Aβ), and neurofibrillary tangles formed by hyperphosphorylation of tau protein. Furthermore, oxidative stress, inflammation, glial dysfunction, and cholinergic neuron damage also contribute to the development and progression of AD. Currently, because the etiology of AD remains incompletely elucidated, treatment primarily focuses on symptomatic treatment, with drugs primarily including cholinesterase inhibitors and glutamate receptor antagonists, as well as some neuroprotective agents. These drugs can only alleviate or delay the symptoms that occur during the course of the disease.

[0005] Neuroprotectants are typically used to prevent or mitigate nerve cell damage and death caused by neurological diseases, trauma, or injury, and to promote brain cell survival and functional recovery. Their mechanisms of action primarily include reducing oxidative stress in nerve cells, inhibiting inflammatory responses, improving energy metabolism, and enhancing cellular self-repair capabilities. By protecting and supporting damaged nerve cells, neuroprotectants can alleviate symptoms and dysfunction of neurological diseases, help maintain normal neurological function, and improve patient outcomes.

[0006] The development of neuroprotective agents can effectively prevent, delay or treat diseases such as Alzheimer's disease, stroke, Parkinson's disease, etc., fill the current treatment gaps and meet the needs of patients and their families.

[0007] This study aims to prepare a new compound and use it for the protection of nerve cells. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides an aryl imidazole compound, a preparation method thereof, and a pharmaceutical product prepared therefrom.

[0009] The technical solution of the present invention is achieved as follows:

[0010] An aryl imidazole compound, whose structural formula is as follows:

[0011] .

[0012] The preparation method of the above-mentioned aryl imidazole compounds has the following technical route:

[0013] .

[0014] The preparation steps are:

[0015] 2-Hydroxybenzaldehyde, 2,5-dimethoxyaniline, dibenzoyl, ammonium acetate, and acetic acid were sequentially added to a reaction flask under N2 protection and stirred at 110°C. After the raw materials reacted completely, heating was stopped and methanol was added. Water was added dropwise with stirring until a precipitate formed. The mixture was cooled to room temperature, filtered, washed, and dried to obtain the crude product. After vacuum rotary evaporation and column chromatography, a white solid powder was obtained with a yield of 49.68% and a melting point of 131.5-132.7°C. HPLC purity was 97.73%.

[0016] In the above step (1), the molar ratio of 2-hydroxybenzaldehyde, 2,5-dimethoxyaniline and dibenzoyl is 3-5:5-7:3-5; based on the addition amount of 2-hydroxybenzaldehyde of 1 mmol, the addition amount of ammonium acetate is 5 mmol and the addition amount of acetic acid is 2.4 mL.

[0017] The temperature of the above-mentioned heating and stirring reaction is 110°C; the inert gas is N2.

[0018] The volume ratio of acetic acid to methanol is 10:3.

[0019] The pharmaceutically acceptable salts of the above-mentioned aryl imidazole compounds are salts formed with the following acids: one or more of phosphoric acid, carbonic acid, hydrochloric acid, sulfuric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, pantothenic acid, succinic acid, tartaric acid or p-toluenesulfonic acid.

[0020] The present invention relates to the use of aryl imidazole compounds and pharmaceutically acceptable salts thereof in the preparation of neuroprotective drugs.

[0021] The above-mentioned neuroprotection refers to:

[0022] ① Improve the survival rate of nerve cells induced by ischemia and hypoxia and reduce cell death.

[0023] ② Reduce the release of lactate dehydrogenase in nerve cells induced by ischemia and hypoxia, and alleviate nerve cell damage.

[0024] ③Inhibit the production of intracellular reactive oxygen species induced by ischemia and hypoxia, and reduce oxidative stress damage.

[0025] ④Anti-ischemia and hypoxia-induced intracellular oxidative stress and inflammatory response.

[0026] ⑤ Improve the vitality of nerve cells overexpressing the ApoE4 gene and increase cell survival rate.

[0027] The pharmaceutical preparation is prepared by compounding the above-mentioned pharmaceutical composition with conventional excipients in the art.

[0028] The pharmaceutical preparations are any one of tablets, capsules, pills, suppositories, soft capsules, oral solutions, suspensions, injections, sustained-release preparations, controlled-release preparations, sustained-release preparations and microsome delivery systems.

[0029] A medicine, food or health product with neuroprotective effects, characterized by comprising any of the following:

[0030] ① the above-mentioned aryl imidazole compounds;

[0031] ② the above-mentioned salts;

[0032] ③ the above-mentioned pharmaceutical composition;

[0033] ④The above-mentioned pharmaceutical preparations.

[0034] The present invention has the following beneficial effects:

[0035] 1. The present application discloses an aryl imidazole compound having the structure shown in the general formula (I): ; The pharmacological experimental results provided by the present invention show that the aryl imidazole compound has a protective effect on nerve cells, inhibits intracellular oxidative stress and inflammatory damage induced by hypoxia and glucose deprivation, and improves the survival rate of oxygen-glucose deprived and ApoE4 gene-overexpressing cells, and can be used as a neuroprotective agent in neurodegenerative diseases.

[0036] 2. Treatment with the aromatic imidazole compounds of the present application can significantly increase the cell survival rate of HT22 cells induced by oxygen-glucose deprivation, improve the neuroprotective effect of cell damage, reduce the production of ROS in the hypoxia-induced HT22 cell model, alleviate oxidative stress damage, and significantly increase the cell survival rate reduced by Na2S2O4 combined with sugar-free Earles solution. It has certain anti-inflammatory and antioxidant effects, and 32 μmol / L of compound B11 can maximize the improvement of cell survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 This is the hydrogen spectrum of aryl imidazole compound B11.

[0039] Figure 2 This is the carbon spectrum of aryl imidazole compound B11.

[0040] Figure 3 This is the effect of compound B11 on the viability of HT22 cells.

[0041] Figure 4 To screen the time of hypoxia culture (94% N2, 1% O2, 5% CO2)-induced OGD modeling in mouse hippocampal neuron HT22 cells; n=5, all data are mean ± SEM, *** p <0.001.

[0042] Figure 5 Effect of compound B11 on OGD-induced HT22 cell viability; all data are expressed as mean ± SEM, n=6, compared with Con, ### p <0.001; compared with OGD,* p <0.05,** p <0.01.

[0043] Figure 6 Effect of compound B11 on LDH release induced by OGD (physical method) in HT22 cells; all data are expressed as mean ± SEM, n=3, compared with Con, ### p <0.001; compared with OGD, ** p <0.01,*** p <0.001.

[0044] Figure 7 Effect of compound B11 on OGD-induced ROS in HT22 cells; all data are expressed as mean ± SEM, n=3, compared with Con, ### p <0.001; compared with OGD,* p <0.05,** p <0.01,*** p <0.001.

[0045] Figure 8The antioxidant effect of compound B11 on OGD-induced HT22 cells; all data are expressed as mean ± SEM, n=4, compared with Con, ### p <0.001, # p <0.05; compared with OGD,* p <0.05,** p <0.01.

[0046] Figure 9 Screening of OGD cell model conditions induced by Na2S2O4 and Earles' solution; n = 5, all data are mean ± SEM, n = 5; * p <0.05,** p <0.01.

[0047] Figure 10 Effects of compound B11 on the viability of HT22 cells induced by Na2S2O4 and Earles' solution; all data are expressed as mean ± SEM, n = 5; compared with Con, # p <0.05; compared with OGD, ** p <0.01,*** p <0.001.

[0048] Figure 11 Effects of compound B11 on MDA and NO in HT22 cells induced by Na2S2O4 / Earles' solution; all data are mean ± SEM, n = 4; compared with Con, ## p <0.01, ### p <0.001; compared with OGD,* p <0.05,** p <0.01.

[0049] Figure 12 To build ApoE Gene overexpression HT22 cells; A: Immunofluorescence detection ApoE Gene expression; B: ApoE protein representative images and protein quantification results. All data are expressed as mean ± SEM, n = 4; compared with the HT22 group, ** p <0.01.

[0050] Figure 13Figure 2 shows the protective effect of compound B11 on ApoE4-overexpressing HT22 cells. A: Effect of B11 on cell viability at high concentrations; B: Effect of B11 on cell viability at low concentrations. All data are mean ± SEM, n = 5. Compared with ApoE3, # p < 0.05 and ## p < 0.01; compared with ApoE4, * p < 0.5 and ** p < 0.01. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0053] Example 1: Synthesis of Aryl Imidazole Compound B11

[0054] The preparation method of the aryl imidazole compound B11 of this embodiment is as follows:

[0055] 2-Hydroxybenzaldehyde (500.00 mg, 4.10 mmol), 2,5-dimethoxyaniline (940.57 mg, 6.15 mmol), dibenzoyl (861.94 mg, 4.10 mmol), ammonium acetate (1.58 g, 20.50 mmol), and acetic acid (10 mL) were placed in a reaction flask in sequence. Under nitrogen protection, the reaction was stirred at 110°C. After 4 h, TLC (developing solvent PE:EA = 2:1) monitored the completion of the reaction. After the reaction of the starting materials was complete, heating was stopped, methanol (3 mL) was added, and water was added dropwise with stirring until a precipitate formed. The mixture was cooled to room temperature, filtered, washed (MeOH:H2O = 1:1), and dried to obtain the crude product. The product was evaporated under reduced pressure and then purified by column chromatography (PE:EA = 5:1) to obtain 912.5 mg of a white solid powder, aryl imidazole compound B11, with a yield of 49.68% and a melting point of 131.5-132.7°C. HPLC purity: 97.73%.

[0056] The H NMR spectrum of the aryl imidazole compound B11 prepared in this example ( Figure 1 ) and carbon spectrum ( Figure 2 ):

[0057] ESI-HRMS: calcd. for C29 H 25 N2O3[M+H] + : 449.1860, found: 449.1873.

[0058] 1 H NMR (300 MHz, Chloroform- d ) δ 13.69 (br, 1H), 7.63 (d, J = 7.7 Hz,2H), 7.36-7.19 (m, 9H), 7.13 (d, J = 8.1 Hz, 1H), 6.96 (dd, J = 9.1, 3.0 Hz, 1H),6.88 (d, J = 9.1 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 3.0 Hz, 1H), 6.57(t, J = 7.6 Hz, 1H), 3.70 (s, 3H), 3.58 (s, 3H).

[0059] 13 C NMR (75 MHz, Chloroform- d ) δ 158.41, 153.55, 149.68, 145.12,134.82, 133.32, 130.95, 130.57, 130.15, 129.86, 128.48, 128.32, 128.27,126.88, 126.84, 126.40, 125.08,118.06, 117.57, 116.25, 115.46, 113.50,113.22, 56.01, 55.92.

[0060] Example 2: Synthesis of Aryl Imidazole Compound B11

[0061] The preparation method of the aryl imidazole compound B11 of this embodiment is as follows:

[0062] 2-Hydroxybenzaldehyde (3 mmol), 2,5-dimethoxyaniline (5 mmol), dibenzoyl (3 mmol), ammonium acetate (15 mmol), and acetic acid (7.2 mL) were placed sequentially into a reaction flask under nitrogen protection and stirred at 110°C. After 4 h, the reaction was monitored by TLC (developing solvent: PE:EA = 2:1). Once the starting materials had reacted completely, heating was discontinued, methanol (3 mL) was added, and water was added dropwise with stirring until a precipitate formed. The mixture was cooled to room temperature, filtered, washed with MeOH:H2O = 1:1, and dried to obtain the crude product. The product was evaporated under reduced pressure and then purified by column chromatography (PE:EA = 5:1) to afford 687.7 mg of a white solid powder, aryl imidazole compound B11, with a yield of 48.87% and a melting point of 131.5-132.7°C. HPLC purity: 97.16%.

[0063] The NMR data of the aryl imidazole compound B11 prepared in this example are as follows:

[0064] ESI-HRMS: calcd. for C 29 H 25 N2O3[M+H] + : 449.1860, found: 449.1873.

[0065] 1 H NMR (300 MHz, Chloroform- d ) δ 13.69 (br, 1H), 7.63 (d, J = 7.7 Hz,2H), 7.36-7.19 (m, 9H), 7.13 (d, J = 8.1 Hz, 1H), 6.96 (dd, J = 9.1, 3.0 Hz, 1H),6.88 (d, J = 9.1 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 3.0 Hz, 1H), 6.57(t, J = 7.6 Hz, 1H), 3.70 (s, 3H), 3.58 (s, 3H).

[0066] 13 C NMR (75 MHz, Chloroform- d) δ 158.41, 153.55, 149.68, 145.12,134.82, 133.32, 130.95, 130.57, 130.15, 129.86, 128.48, 128.32, 128.27,126.88, 126.84, 126.40, 125.08,118.06, 117.57, 116.25, 115.46, 113.50,113.22, 56.01, 55.92.

[0067] Example 3: Synthesis of Aryl Imidazole Compound B11

[0068] The preparation method of the aryl imidazole compound B11 of this embodiment is as follows:

[0069] 2-Hydroxybenzaldehyde (5 mmol), 2,5-dimethoxyaniline (5 mmol), dibenzoyl (5 mmol), ammonium acetate (25 mmol), and acetic acid (12 mL) were placed sequentially into a reaction flask under N2 protection and stirred at 110°C. After 4 h, the reaction was monitored by TLC (developing solvent: PE:EA = 2:1). Once the starting materials had reacted completely, heating was stopped, methanol (3 mL) was added, and water was added dropwise with stirring until a precipitate formed. The mixture was cooled to room temperature, filtered, washed (MeOH:H2O = 1:1), and dried to obtain the crude product. The product was evaporated under reduced pressure and then purified by column chromatography (PE:EA = 5:1) to obtain 841.5 mg of a white solid powder, aryl imidazole compound B11, with a yield of 49.02% and a melting point of 131.5-132.7°C. HPLC purity was 96.63%.

[0070] The NMR data of the aryl imidazole compound B11 prepared in this example are as follows:

[0071] ESI-HRMS: calcd. for C 29 H 25 N2O3[M+H] + : 449.1860, found: 449.1873.

[0072] 1 H NMR (300 MHz, Chloroform- d ) δ 13.69 (br, 1H), 7.63 (d, J = 7.7 Hz,2H), 7.36-7.19 (m, 9H), 7.13 (d, J = 8.1 Hz, 1H), 6.96 (dd, J= 9.1, 3.0 Hz, 1H),6.88 (d, J = 9.1 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 3.0 Hz, 1H), 6.57(t, J = 7.6 Hz, 1H), 3.70 (s, 3H), 3.58 (s, 3H).

[0073] 13 C NMR (75 MHz, Chloroform- d ) δ 158.41, 153.55, 149.68, 145.12,134.82, 133.32, 130.95, 130.57, 130.15, 129.86, 128.48, 128.32, 128.27,126.88, 126.84, 126.40, 125.08,118.06, 117.57, 116.25, 115.46, 113.50,113.22, 56.01, 55.92.

[0074] Implementation effect example:

[0075] The efficacy of B11 prepared in Example 1 was tested:

[0076] First, the effect of B11 on the viability of normal mouse hippocampal neuronal cells HT22 cells was detected by MTT assay to assess the toxicity of B11. The protective effect of B11 on HT22 cells was then tested in oxygen-glucose deprivation and ApoE4-transfected cell models.

[0077] (I) Effect of B11 on the viability of normal HT22 cells

[0078] 1. Experimental methods

[0079] HT22 cells in the logarithmic growth phase were seeded into 96-well plates at 5,000 cells / well. 100 μL of DMEM was added to each well and cultured at 37°C in a 5% CO2 incubator. After 24 hours, the control group was treated with 0.5% DMEM, while the treatment groups were treated with various concentrations of B11 (2.5, 5, 10, 20, 40, 80, 160, and 320 μmol / L). After 24 hours, 10 μL of MTT was added to each well and incubated in a 37°C oven for 1 hour in the dark. OD values ​​were then measured at 570 nm using a microplate reader for data analysis.

[0080] , Experimental results

[0081] The results are as follows Figure 3 The results showed that within the concentration range of 320 μmol / L, B11 had no significant effect on the survival rate of HT22 cells. These results indicate that B11 has low cytotoxicity and high safety.

[0082] (II) Neuroprotective Effects of B11 in Oxygen-Glucose Deprivation Cell Model

[0083] Hypoxic-ischemic injury is the fundamental pathological process of ischemic cerebrovascular disease. In vitro cell culture is a key research method. Oxygen glucose deprivation (OGD) simulates ischemia / hypoxia at the cellular level. By modifying cell culture conditions, including placing cells in a hypoxic chamber and replacing the culture medium with sugar-free medium, the damage caused by ischemia / hypoxia can be simulated, leading to cell necrosis and apoptosis. This is used in pathological and pharmacological studies of ischemic stroke, among other conditions.

[0084] This part of the experiment constructed an oxygen-glucose deprivation cell model to simulate the cell damage of ischemic stroke and test the protective effect of B11 on nerve cells.

[0085] , Neuroprotective effect of B11 in OGD cell model constructed by physical method

[0086] (1) Determination of model conditions

[0087] In this part of the experiment, hypoxia was used to culture mouse hippocampal neuron HT22 cells to construct an oxygen-glucose deprivation (OGD) cell model.

[0088] Experimental Methods: HT22 cells in the logarithmic growth phase were plated in 96-well plates at 5,000 cells / well, with 100 μl per well. The cells were divided into control and model groups and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, cells in the model group were replaced with sugar-free DMEM medium and placed in a hypoxic incubator (5% CO2, 1% O2, 94% N2) for OGD treatment. Cells were removed from the incubator at 2, 4, 6, and 8 hours, and cell viability was assessed using the MTT assay.

[0089] According to the experimental results, Figure 4 As shown in the figure, the modeling time point of 4 h of hypoxia (cell survival rate of about 50%) was finally selected for subsequent experiments.

[0090] (2) Effect of B11 on the survival rate of HT22 cells induced by OGD

[0091] Experimental Methods: HT22 cells in the logarithmic growth phase were seeded into 96-well plates at 5,000 cells / well, with 100 μL per well. The cells were divided into control, model, and drug-treated groups and cultured at 37°C under 5% CO₂ for 24 hours. After 24 hours, the control and model groups were replaced with 0.5% DMEM, while the drug-treated group was treated with drug-containing medium at varying concentrations (6.25, 12.5, 25, 50, and 100 μmol / L) and cultured in an incubator for an additional 24 hours. Following this, the model group was replaced with sugar-free DMEM, while the drug-treated group was treated with sugar-free medium at varying concentrations. The cells were then placed in a hypoxic incubator (5% CO₂, 1% O₂, 94% N₂) for 4 hours under OGD. The effect of compound B11 on cell viability was then assessed using the MMT assay.

[0092] The results show that ( Figure 5 ), pretreatment with B11 at concentrations of 12.5, 25, and 50 μmol / L significantly increased the cell survival rate of HT22 cells induced by oxygen-glucose deprivation.

[0093] (3) Effect of B11 on LDH release in OGD-treated HT22 cells

[0094] Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is widely present in various organisms. It cannot pass through the cell membrane under normal circumstances. When the cell is damaged or dead, it can be released outside the cell. The released LDH is in the culture supernatant. Therefore, the degree of cell damage can be reflected by detecting the release of intracellular LDH.

[0095] Experimental Methods: HT22 cells in the logarithmic growth phase were seeded in 96-well plates at 5,000 cells / well. 100 μl of DMEM was added to each well. Control, model, and drug-treated groups were assigned and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the control and model groups were replaced with 0.5% DMEM, while the drug-treated groups were treated with varying concentrations of compound B11 (12.5, 25, and 50 μmol / l) and incubated for an additional 24 hours. After 24 hours, the model group was replaced with sugar-free DMEM, while the drug-treated groups were treated with varying concentrations of drug-containing sugar-free medium. The cells were then placed in a hypoxic incubator (5% CO2, 1% O2, 94% N2) and subjected to OGD for 4 hours. Following OGD treatment, the cell culture supernatants from each group were collected and LDH levels were measured using an Elabscience LDH assay.

[0096] The experimental results showed that B11 (12.5, 25, 50 μmol / L) pretreatment significantly reduced the LDH content in cells induced by OGD, with significant differences. Figure 6The experimental results show that compound B11 has a neuroprotective effect in improving cell damage.

[0097] (4) Effect of B11 on reactive oxygen species (ROS) levels in HT22 cells treated with OGD

[0098] Experimental method: DCFH-DA fluorescent probe was used to detect the content of ROS in cells. HT22 cells in the logarithmic growth phase were taken and the density of the digested cell suspension was adjusted to 1×10 5 Cells were seeded into 6-well cell culture plates at 2 ml of cell suspension per well, with triplicate wells per group. Each group was treated for 24 hours. After 24 hours, cells were pre-treated. The control and model groups were treated with 0.5% DMEM, while the drug-treated groups were treated with varying concentrations of compound B11 (12.5, 25, and 50 μmol / L) and incubated in an incubator for an additional 24 hours. After 24 hours, the model and drug-treated groups were established. The model group was replaced with sugar-free DMEM, while the drug-treated groups were treated with varying concentrations of drug-containing sugar-free medium. The cells were then placed in a hypoxic incubator (5% CO₂, 1% O₂, and 94% N₂) for 4 hours of OGD treatment. Following OGD treatment, the culture medium was aspirated, the cells were washed three times with serum-free cell culture medium, and DCFH-DA was diluted 1:1000 in serum-free culture medium to a final concentration of 10 mM and added to the 6-well plates. The cells were incubated at 37°C in a 5% CO₂ incubator for 20 minutes. Invert and mix every 3-5 minutes to ensure full contact between the probe and the cells. Wash the cells three times with serum-free cell culture medium to fully remove any DCFH-DA that has not entered the cells. Measure the fluorescence intensity of each group using a microplate reader set to 488 nm excitation and 525 nm emission.

[0099] The experimental results show that ( Figure 7 Compared with the control group, OGD treatment significantly increased ROS levels in HT22 cells, as evidenced by enhanced green fluorescence intensity. However, treatment with different concentrations of B11 (12.5, 25, and 50 μmol / L) significantly decreased green fluorescence intensity and ROS levels, with the 25 μmol / L dose group showing the greatest effect. These results suggest that B11 can reduce ROS production in the hypoxia-induced HT22 cell model and alleviate oxidative stress damage.

[0100] (5) Antioxidant effect of B11 on OGD-treated HT22 cells

[0101] Oxidative stress produces a large number of oxygen free radicals, causing DNA damage, which in turn leads to varying degrees of cytotoxicity and tissue cell damage. Next, we measured the levels of lipid peroxide (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) to assess the antioxidant effects of B11 on HT22 cells.

[0102] Experimental methods:

[0103] MDA detection method: take no less than 3×10 6 cells, discard the cell culture supernatant, scrape the cells with a cell scraper, transfer the cells to a plastic centrifuge tube with a pipette, add 0.5 mL of reagent V extraction solution, mix for 2 minutes, break the cells into a suspension, and operate according to the MDA kit operation table.

[0104] GSH content detection: take 10 6 Homogenize the cells with 300-500 μL of PBS (0.01 M, pH 7.4) or saline (0.9% NaCl). Centrifuge at 10,000 × g for 10 minutes at 4°C. Remove the supernatant and place it on ice for analysis. Follow the procedures described in the GSH assay kit. Vibrate the plate on a microplate reader for 1 minute, let it sit at room temperature for 5 minutes, and measure the OD value at 450 nm using a microplate reader.

[0105] SOD level assay: Aspirate the supernatant from adherent cells, scrape the cells with a cell scraper, and gently pipette with a micropipette to remove all liquid into an EP tube. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and retain the cell pellet. Add 1 ml of PBS, gently pipette, and centrifuge again at 1000 rpm for 10 minutes, discard the supernatant, and retain the cell pellet for later use. Add a certain amount of PBS to the cell pellet, vortex, and grind on ice for 3 minutes before testing.

[0106] The experimental results show that ( Figure 8 Compared with the control group, the OGD group showed increased MDA levels and significantly decreased GSH and SOD levels. However, pretreatment with B11 (12.5, 25, and 50 μmol / L) reversed these trends, with 25 μmol / L B11 showing a significant effect in reducing MDA levels and increasing GSH and SOD. These results suggest that B11 has a significant antioxidant effect.

[0107] Neuroprotective effect of B11 in an OGD cell model constructed by chemical methods

[0108] (1) Determination of model conditions

[0109] HT22 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5,000 cells / well. 100 μL of DMEM was added to each well. Control and model groups were cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the model was established. The control group culture medium was discarded and replaced with fresh 0.5% DMEM. The model group culture medium was discarded and treated with Na₂S₂O₄ solutions diluted with sugar-free Earles' solution at concentrations of 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 4 mmol / L, 8 mmol / L, and 16 mmol / L, respectively. The cells were then incubated in the incubator for an additional 80 minutes. The 96-well plates were then removed from the incubator and, protected from light, 10 μL of MTT was added to each well. The OD values ​​were measured at 570 nm using a microplate reader for data analysis.

[0110] The results are as follows Figure 9 The results showed that compared with the control group, treatment with 4 mmol / L Na2S2O4 could reduce the cell survival rate to about 50%. Therefore, 4 mmol / L Na2S2O4 plus sugar-free Earles solution was finally selected as the chemical OGD model condition for 80 minutes in a 37°C, 5% CO2 incubator.

[0111] (2) B11 improves the survival rate of HT22 cells induced by Na2S2O4 / Earles' solution

[0112] Experimental Methods: HT22 cells in the logarithmic growth phase were seeded in 96-well plates at 5,000 cells / well. 100 μl of DMEM was added to each well. Control, model, and drug-treated groups were assigned and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, cells were pre-treated with drug treatment. The control and model groups were treated with 0.5% DMEM, while the drug-treated groups were treated with varying concentrations of B11 (1, 3, 10, 30, and 100 μmol / l) and cultured in the incubator for an additional 24 hours. After 24 hours, the model and drug-treated groups were treated with modeling. Cell viability was measured using the MTT assay.

[0113] The experimental results showed that pretreatment with different concentrations of B11 could significantly increase the cell survival rate reduced by Na2S2O4 combined with sugar-free Earles solution ( Figure 10 ).

[0114] (3) B11 reduces the MDA and NO levels in HT22 cells induced by Na2S2O4

[0115] Then, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of lipid peroxide (MDA) and nitric oxide (NO) in HT22 cells treated with OGD.

[0116] Experimental Methods: HT22 cells in the logarithmic growth phase were seeded in 96-well plates at 5,000 cells / well. 100 μl of DMEM was added to each well. Control, model, and drug-treated groups were assigned and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, cells were pre-treated with drug treatment. The control and model groups were treated with 0.5% DMEM. The drug-treated groups were treated with varying concentrations of compound B11 (3, 10, and 30 μmol / l) and cultured in the incubator for an additional 24 hours. After 24 hours, the model and drug-treated groups were treated with modeling. After modeling, the cell culture supernatant and cell pellet were collected and processed using the Elabscience MDA kit and the Nanjing Jiancheng NO kit, respectively.

[0117] The results show that ( Figure 11 Compared with the control group, the levels of MDA and NO in HT22 cells induced by Na₂S₂O₄ and Earles' solution significantly increased, while treatment with different concentrations of B₁₁ (3, 10, and 30 μmol / L) significantly decreased these levels. These results suggest that B₁₁₁ has certain anti-inflammatory and antioxidant effects.

[0118] (III) Neuroprotective effect of B11 on HT22 cells transfected with ApoE4 gene

[0119] In this part of the experiment, an ApoE transgenic cell model was constructed to test the protective effect of B11 on HT22 cells overexpressing the ApoE4 gene.

[0120] , Construction of ApoE transgenic cell model

[0121] (1) Packaging lentivirus

[0122] 1) Inoculate HEK-293FT cells in logarithmic growth phase into 6-well plates and wait for the cells to grow to approximately 50% confluence with lentiviral packaging;

[0123] 2) Dilute 10 μL of Lipo2000 and 4 μg of total DNA (including ApoE4 / ApoE3 / NC, pSpax, and PMD2G plasmids, mixed at a ratio of 2:2:1) in 250 μL of serum-free and double antibody medium and let it stand for 5 minutes;

[0124] 3) Add the mixed plasmid solution to the culture medium containing Lip2000 serum-free and anti-double antibody, mix thoroughly, and let it stand for 20 minutes;

[0125] 4) Aspirate the old culture medium from the 6-well plate, add 1.5 mL of serum-free and anti-double antibody DMEM, and then add the previous mixed solution dropwise to the 6-well plate;

[0126] 5) After 6-8 hours, replace the solution by removing the original medium and adding complete medium;

[0127] 6) Collect the first virus after 24 hours: Transfer the culture medium from the 6-well plate to a 15 mL centrifuge tube. Set the centrifugation parameters to 1000 rpm and centrifuge for 5 minutes. Centrifuge and package the virus. Freeze it in a -80°C refrigerator. Add 2 mL of culture medium to the 6-well plate again and collect the virus again after 24 hours.

[0128] (2) Lentiviral transfection of target cells

[0129] 1) Prepare HT22 cells, resurrect and passage them as described in 2.3. After passage, seed the cells at a density of approximately 10 cells per well. 5 cells in a 6-well plate.

[0130] 2) Transfection was performed when cells reached 60-80% confluency. The experiments were divided into control, experimental, and infection groups. The control group consisted of a negative control lentivirus infection group (NC group), and the experimental groups consisted of an APOE3 lentivirus infection group and an APOE4 lentivirus infection group.

[0131] 3) Thaw the lentiviral solution from the -80°C freezer, add 2 μg / mL polybrene co-transfection reagent, pipette to mix thoroughly, and let it stand for 20 minutes.

[0132] 4) Discard the culture medium on the 6-well plate and replace it with 1 mL of complete culture medium. Then, drop the suspension into the wells and place them in the incubator for culture.

[0133] 5) After 24 hours, observe the cells and replace the culture medium with 2 mL of complete medium before continuing. After 48 hours, replace the medium containing μg / mL puromycin with 2 mL of complete medium to kill untransfected cells. Successful transfection should result in green fluorescence under a fluorescence microscope.

[0134] Experimental results: HT22 cells stably expressing ApoE gene were constructed using lentiviral transfection technology. The results were observed under an inverted fluorescence microscope. Figure 12 As shown in Figure A, the transfection group showed strong green fluorescence because the plasmid vector contained a green fluorescent protein (GFP) tag. 48 hours after transfection, cells were collected and the ApoE protein level in cells was detected by Western blot. The results are shown in Figure 4. Figure 12 As shown in Figure B, the ApoE protein in the transfection group was significantly higher than that in the blank control, indicating that the HT22 cell model with stable overexpression of ApoE3 and ApoE4 was successfully constructed and can be used for subsequent experiments.

[0135] , B11 protective effect on ApoE4-HT22 cells

[0136] The MTT assay was used to examine the effect of B11 on the viability of HT22 cells overexpressing the ApoE4 gene.

[0137] Experimental Methods: Cells in the logarithmic growth phase were seeded into 96-well plates at 5,000 cells / well, with 100 μL added to each well. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, drug treatment was administered. The next day, the 96-well plates were removed from the incubator and, protected from light, 10 μL of MTT was added to each well. The cells were then incubated in a 37°C oven for 1 hour. OD values ​​were then measured at 450 nm using a microplate reader for data processing.

[0138] Experimental results: ApoE3 cells were used as the control group, and ApoE4 overexpressed HT22 cells were used as the model group. Figure 13 As shown in Figure A, after adding compound B11 at concentrations of 8, 16, 32, and 64 μmol / L, the survival rate of ApoE4 cells was improved with the increase of drug concentration after adding compound B11 at concentrations of 8, 16, and 32 μmol / L. However, after adding B11 at a concentration of 64 μmol / L, the survival rate of cells was greatly inhibited.

[0139] In order to determine a more appropriate drug dose, we used ApoE3 cells as the control group and ApoE4-overexpressing HT22 cells as the model group, and selected B11 at a concentration below 32 μmol / L to act on the cells. Figure 13 As can be seen from Figure B, overexpression of the ApoE4 gene can significantly reduce the survival rate of cells. After adding B11 at concentrations of 2, 4, 8, 16, and 32 μmol / L, the survival rate of cells can be improved as the drug concentration gradient increases, indicating that B11 has a protective effect on HT22 cells with overexpression of the ApoE4 gene.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An aryl imidazole compound having the following structural formula: 。 2. The method for preparing the aryl imidazole compound according to claim 1, wherein The steps are: (1) 2-hydroxybenzaldehyde, 2,5-dimethoxyaniline, dibenzoyl, ammonium acetate, and acetic acid are sequentially added to a reaction vessel, and heated and stirred under inert gas protection until the reaction is complete to obtain a reaction solution; (2) Stop heating the reaction solution of step (1), then add methanol, and then add water dropwise under stirring until a precipitate is produced. After cooling to room temperature, the crude product is filtered, washed, and dried. After vacuum rotary evaporation, column chromatography is performed to obtain a white solid powder, which is an aryl imidazole compound.

3. The method for preparing an aryl imidazole compound according to claim 2, wherein: In the step (1), the molar ratio of 2-hydroxybenzaldehyde, 2,5-dimethoxyaniline and dibenzoyl is 3-5:5-7:3-5; based on the addition amount of 2-hydroxybenzaldehyde of 1 mmol, the addition amount of ammonium acetate is 5 mmol, and the addition amount of acetic acid is 2.4 mL.

4. The method for preparing an aryl imidazole compound according to claim 3, wherein: The temperature of the heating and stirring reaction is 110° C.; the inert gas is N 2 .

5. The method for preparing an aryl imidazole compound according to claim 3, wherein: The volume ratio of acetic acid to methanol is 10:

3.

6. The pharmaceutically acceptable salt of the aryl imidazole compound according to claim 1, characterized in that: The salt is a salt formed with one or more of the following acids: phosphoric acid, carbonic acid, hydrochloric acid, sulfuric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, pantothenic acid, succinic acid, tartaric acid or p-toluenesulfonic acid.

7. A pharmaceutical composition, characterized in that: The invention comprises one or more of the aryl imidazole compound according to claim 1 or the salt according to claim 6, and a pharmaceutically acceptable carrier or excipient.

8. A pharmaceutical preparation prepared by compounding the pharmaceutical composition according to claim 7 with conventional excipients in the art.

9. The pharmaceutical preparation according to claim 8, characterized in that: The pharmaceutical preparation is any one of tablets, capsules, pills, suppositories, soft capsules, oral solutions, suspensions, injections, sustained-release preparations, controlled-release preparations, sustained-release preparations and microsome delivery systems.

10. A drug having a neuroprotective effect, characterized in that: Products that include any of the following: ① The aryl imidazole compound according to claim 1; ② The salt according to claim 6; ③ The pharmaceutical composition according to claim 7; 4. The pharmaceutical preparation according to claim 8 or 9.

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