A derivative of adamantyl nitrone and a preparation method and application thereof

Abstract

The application discloses a preparation method of adamantane nitroketone derivatives and application of the adamantane nitroketone derivatives in ischemic stroke drugs. The adamantane nitroketone derivatives are combined by 2,3,5,6-tetramethylpyrazine and adamantane compounds through nitroketone. The compound not only has strong free radical scavenging capacity, but also integrates the nerve protection function of the adamantane compound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an adamantane nitrone derivative, its preparation method, and its application. Background Technology

[0002] Cerebral infarction, as one of the leading causes of death and disability worldwide, affects a large number of people, imposing a heavy economic and psychological burden on society and families. The primary cause is cerebral vascular occlusion by a thrombus, leading to local cerebral ischemia and hypoxia, which in turn triggers a series of pathophysiological processes, including excessive release of excitatory amino acids, calcium ion influx, neuroinflammatory responses, and excessive generation of free radicals. These pathological processes interact, further exacerbating brain tissue damage. Free radicals play a crucial role in the pathological damage of ischemic stroke. Therefore, treatment strategies for ischemic stroke typically include thrombolysis, inhibition of platelet aggregation, scavenging of free radicals, or a comprehensive treatment plan combining these effects.

[0003] TBN (a nitrone derivative of 2,3,5,6-tetramethylpyrazine) is a tetramethylpyrazine nitrone derivative formed by combining the active ingredient TMP (2,3,5,6-tetramethylpyrazine) from Ligusticum chuanxiong with a nitrone structure. TBN can more effectively scavenge free radicals, such as superoxide anions (O2). - ), hydroxyl radicals (·OH) and peroxynitroso anions (ONOO) - In various stroke experimental models, TBN exhibits good neuroprotective effects, mainly attributed to its free radical scavenging ability, reduction of oxidative damage, and inhibition of calcium ion overload.

[0004] Amantadine compounds (such as amantadine and memantine hydrochloride) have shown unique potential in the treatment of neurological diseases. Memantine hydrochloride is a voltage-dependent, moderate-affinity, non-competitive N-methyl-D-aspartate receptor (NMDA receptor) antagonist with rapid binding and dissociation. In recent years, studies have found that memantine hydrochloride also has potential application value in the treatment of ischemic stroke. Its neuroprotective mechanisms mainly include: inhibiting neuronal apoptosis by blocking extrasynaptic NMDA receptor-mediated calcium ion influx; rapid dissociation under physiological conditions to avoid affecting normal physiological function; enhancing synaptic plasticity; promoting the expression of brain-derived neurotrophic factor (BDNF), thereby protecting neurons; reducing glial cell proliferation; and preventing neuronal death through other pathways. Furthermore, amantadine, as an antiviral drug, has been expanded into the treatment of traumatic brain injury due to its significant neuroprotective effects, achieving good clinical results.

[0005] Despite the significant potential of TBNs and adamantane compounds in the treatment of stroke, they each have certain limitations. First, TBNs have relatively low bioavailability, which may limit their effectiveness in clinical applications. Second, adamantane compounds may be accompanied by certain side effects, such as increased central nervous system excitability and gastrointestinal discomfort, which may limit their long-term use. Therefore, overcoming these shortcomings and developing a novel drug that can leverage the advantages of both TBNs and adamantane compounds while possessing better pharmacokinetic properties and lower toxicity has become a key focus for researchers. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and provide a novel compound that can simultaneously improve drug bioavailability, reduce toxic side effects, and has free radical scavenging and NMDA receptor antagonistic effects.

[0007] To achieve the above objectives, this invention has successfully developed a novel drug with multiple therapeutic effects by combining TBN (a nitrone derivative of 2,3,5,6-tetramethylpyrazine) with adamantane compounds. The specific technical solution is as follows: An adamantane nitrone derivative, with the following structural formula: In this context, R is selected from H, CH3, and OH, and R1 is selected from H and OH.

[0008] Or the structural formula is .

[0009] In some embodiments, the adamantane nitrone derivative having the structure shown in general formula I is preferred: , or .

[0010] This invention also provides a method for preparing the above-mentioned adamantane nitrone derivative, the specific steps of which are as follows: Ligustrazine aldehyde and an adamantane derivative are refluxed and dehydrated in toluene to generate an intermediate compound. This intermediate compound is then oxidized to an epoxide under 3-chloroperbenzoic acid conditions. Finally, the epoxide is rearranged in acetonitrile to obtain the adamantane nitrone derivative of this invention. The structural formula of the adamantane derivative is as follows: or In this context, R is selected from H, CH3, and OH, and R1 is selected from H and OH.

[0011] The specific synthesis route is as follows: ; or .

[0012] The present invention also provides a pharmaceutical composition comprising the above-mentioned adamantane nitrone derivative, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient. The carrier may be an oral solid dosage form carrier, a nanoparticle carrier, a sustained-release / controlled-release carrier, etc., and the excipient may be a filler, disintegrant, binder, sustained-release or controlled-release material, etc.

[0013] The pharmaceutical composition can be administered in various known ways, such as orally, parenterally, or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs.

[0014] For example, oral compositions can be any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, etc.

[0015] Alternatively, sterile injectable compositions may be formulated using suitable dispersants or wetting agents and suspending agents in accordance with techniques known in the art. Pharmaceutically acceptable carriers and solvents that may be used include water, mannitol, sodium chloride solution, etc.

[0016] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific compound of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, general health condition, and medical history of the patient being treated, and similar factors known in the medical field.

[0017] The present invention also provides the application of the above-mentioned adamantane nitrone derivative in the preparation of drugs with free radical scavenging and oxidative stress elimination effects, and in the preparation of drugs for neuroinflammatory protection.

[0018] In some embodiments, as a preferred embodiment, the above-mentioned adamantane nitrone derivative is also used in the preparation of drugs for treating ischemic stroke.

[0019] The present invention has the following advantages over the prior art: This invention relates to a novel adamantane nitrone derivative, successfully developing a drug with multiple therapeutic effects by combining TBN (a nitrone derivative of 2,3,5,6-tetramethylpyrazine) with an adamantane compound. This compound not only retains the strong free radical (e.g., DPPH·) scavenging ability of TBN but also combines the neuroprotective effects of adamantane compounds. Through optimization of its chemical structure, this compound exhibits significant advantages in the treatment of ischemic stroke. Existing treatments for stroke have limited comprehensive therapeutic effects, while the development of this invention provides a new therapeutic perspective in this field, particularly in improving drug efficacy and reducing toxic side effects, showing broad application prospects.

[0020] Meanwhile, the compounds of this invention maintained cell viability of over 90% even after treatment at doses up to 100 µM, demonstrating their low cytotoxicity. In an OGD / R-induced cell injury model, compound I1 of this invention showed superior viability enhancement to BV2 cells compared to TBN, I2, I3, and II at the same dose (100 µM), indicating its good protective effect against OGD / R-simulated ischemic stroke.

[0021] The scavenging ability of compound I1 of the present invention against DPPH• is concentration-dependent. At high concentrations (640 µM), the compound of the present invention exhibits high scavenging ability, demonstrating its significant effect on free radical scavenging.

[0022] Compound I1 of this invention exhibited significant neuroprotective effects in a mouse model of cerebral ischemia. TTC staining results showed that compound I1 significantly reduced cerebral infarction volume, and its effect was superior to that of the same dose of TBN.

[0023] The successful development of compound I1 in this invention provides a new potential strategy for the treatment of ischemic stroke. Attached Figure Description

[0024] Figure 1 This is a comparison of the neuroprotective effects of different compounds on glial cells in Example 5 of the present invention; Figure 2 This is a comparison of the anti-apoptotic effects of different concentrations of compounds on glial cells in Example 5 of the present invention; Figure 3 This is a comparison of the ROS scavenging ability of different concentrations of compounds in Example 6 of the present invention; Figure 4 The different compounds ONOO in Example 7 of this invention - Comparison of scavenging effects; Figure 5 This is a comparison of the in vivo therapeutic effects of different dosages of the compound in Example 8 of the present invention on cerebral ischemia. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0028] The preparation method of the adamantane nitrone derivative of the present invention is as follows: Compound 1 is refluxed with adamantane in toluene solvent at 110°C to generate compound 2; compound 2 is oxidized to an epoxide under the condition of 3-chloroperbenzoic acid to generate compound 3; and compound 3 is rearranged in acetonitrile solvent to generate compound I.

[0029] The synthesis route is as follows:

[0030] Example 1 Examples of preparation of compound I1(E)-N-(1-adamantyl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide: 1. Preparation of (E)-N-(1-adamantyl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine (3a): Compounds 3,5,6-trimethylpyrazine-2-carboxaldehyde (1,150.18 mg, 1 mmol) and 1-adamantaneamine (2a, 448.27 mg, 2.5 mmol) were added to a Schlenk culture flask and dissolved in 2 mL of toluene. The mixture was refluxed overnight at 110 °C. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was evaporated to dryness to give a colorless liquid 3a in 80% yield.

[0031] 2. Preparation of 2-(1-adamantyl)-3-(3,5,6-trimethylpyrazin-2-yl)-1,2-oxazolazididine (4a): Compound 3a (300 mg, 1 mmol) was dissolved in a reaction flask with a minimum amount of dichloromethane. Then, a solution of 3-chloroperbenzoic acid (207.08 mg, 1.2 mmol) in chloroform was slowly added dropwise to the reaction flask at 0 °C. After the addition was complete, the mixture was brought to room temperature and stirred vigorously for 5 h. After the reaction was complete, the mixture was filtered, and the filtrate was extracted with a saturated aqueous solution of sodium bicarbonate and dichloromethane. The organic layer was evaporated to dryness to give the crude product 4a, which was a liquid.

[0032] 3. Preparation of (E)-N-(1-adamantyl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide (I1): The crude product 4a was dissolved in an appropriate amount of acetonitrile solvent and heated under reflux at 80°C for 2 days. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give compound I1 in 65% yield.

[0033] The spectral data of compound I1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H, CH=N),2.49-2.36 (m, 9H, CH3), 2.17 (d, J = 3.0 Hz, 9H, CH2, CH), 1.73-1.62 (m, 6H, CH2).

[0034] Its structural formula has been determined to be: .

[0035] Example 2 Examples of preparation of compound I2(Z)-N-(3,5-dimethyladamantane-1-yl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide: 1. Preparation of (E)-N-(3,5-dimethyladamantane-1-yl)-1-(3,5,6-trimethylpyrazin-2-yl)methylimine (3b): Compounds 3,5,6-trimethylpyrazine-2-carboxaldehyde (1,150.18 mg, 1 mmol) and 3,5-dimethyladamantaneamine (2b, 448.27 mg, 2.5 mmol) were added to a Schlenk flask and dissolved in 2 mL of toluene. The mixture was refluxed overnight at 110 °C. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was evaporated to dryness to give a colorless liquid 3b in 80% yield.

[0036] 2. Preparation of 2-(3,5-dimethyladamantane-1-yl)-3-(3,5,6-trimethylpyrazin-2-yl)-1,2-oxazatecyclopropane (4b): Compound 3b (300 mg, 1 mmol) was dissolved in a small amount of dichloromethane in a reaction flask. Then, a solution of 3-chloroperbenzoic acid (207.08 mg, 1.2 mmol) in chloroform was slowly added dropwise to the reaction flask at 0 °C. After the addition was complete, the mixture was brought to room temperature and stirred vigorously for 5 h. After the reaction was complete, the mixture was filtered, and the filtrate was extracted with a saturated aqueous solution of sodium bicarbonate and dichloromethane. The organic layer was evaporated to dryness to give the crude product 4b, which was a liquid.

[0037] 3. Preparation of (Z)-N-(3,5-dimethyladamantane-1-yl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide (I2): The crude product 4b was dissolved in an appropriate amount of acetonitrile solvent and heated under reflux at 80°C for 2 days. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound I2 in 65% yield.

[0038] The spectral data of compound I2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H, CH=N), 2.54 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.47 (s, 3H, CH3), 2.35 (p, J = 3.3 Hz, 1H, CH), 2.10 (d, J = 3.3 Hz, 2H, CH2), 1.93-1.85 (m, 4H, CH2), 1.47-1.38 (m,4H, CH2), 1.26 (s, 2H, CH2), 0.96 (s, 6H, CH3).

[0039] Its structural formula has been determined to be: .

[0040] Example 3 Examples of preparation of compound I3(Z)-N-(3-hydroxyadamantane-1-yl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide: 1. Preparation of (E)-3-(((3,5,6-trimethylpyrazin-2-yl)methylene)amino)adamantane-1-ol (3c): Compounds 3,5,6-trimethylpyrazine-2-carboxaldehyde (150.18 mg, 1 mmol) and 3-aminoadamantane-1-ol (2c, 448.27 mg, 2.5 mmol) were added to a Schlenk flask and dissolved in 2 mL of toluene. The mixture was refluxed overnight at 110 °C. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was evaporated to dryness to give a colorless liquid 3c in 80% yield.

[0041] 2. Preparation of 3-(3-(3,5,6-trimethylpyrazin-2-yl)-1,2-oxazacyclopropane-2-yl)adamantane-1-ol (4c): Compound 3c (300 mg, 1 mmol) was dissolved in a small amount of dichloromethane in a reaction flask. Then, a solution of 3-chloroperbenzoic acid (207.08 mg, 1.2 mmol) in chloroform was slowly added dropwise to the reaction flask at 0 °C. After the addition was complete, the mixture was brought to room temperature and stirred vigorously for 5 h. After the reaction was complete, the mixture was filtered, and the filtrate was extracted with a saturated aqueous solution of sodium bicarbonate and dichloromethane. The organic layer was evaporated to dryness to give the crude product 4c, which was a liquid.

[0042] 3. Preparation of (Z)-N-(3-hydroxyadamantane-1-yl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide (I3): The crude product 4c was dissolved in an appropriate amount of acetonitrile solvent and heated under reflux at 80°C for 2 days. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give compound I3 in 65% yield. The spectral data of compound I3 are as follows: 1 H NMR (400 MHz, DMSO) δ 7.81 (s, 1H), 4.73 (s,1H), 2.39 (s, 3H), 2.37 (s, 3H), 2.24 (d, J = 5.4 Hz, 6H), 1.92 (d, J = 12.4Hz, 4H), 1.53 (s, 3H), 1.16 (s, 1H).

[0043] Its structural formula has been determined to be: .

[0044] Another method for preparing the adamantane nitrone derivative of the present invention is as follows: Compound 1 and 1-aminoethyladamantaneamine 5 are refluxed in toluene solvent at 110°C to generate compound 6. Compound 6 is oxidized to an epoxide under the condition of 3-chloroperbenzoic acid to generate compound 7. Compound 7 is rearranged in acetonitrile solvent to generate compound II.

[0045] The synthesis route is as follows: .

[0046] Example 4 Preparation examples of compound II(Z)-N-(1-adamantane-1-ylethyl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide: 1. Preparation of (E)-N-(1-adamantane-1-ylethyl)-1-(3,5,6-trimethylpyrazin-2-yl)methylimine (6) Compounds 3,5,6-trimethylpyrazine-2-carboxaldehyde (150.18 mg, 1 mmol) and 1-aminoethyladamantane (5,448.27 mg, 2.5 mmol) were added to a Schlenk flask and dissolved in 2 mL of toluene. The mixture was refluxed overnight at 110 °C. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was evaporated to dryness to give a colorless liquid 6 in 80% yield.

[0047] 2. Preparation of 2-(1-adamantane-1-ylethyl)-3-(3,5,6-trimethylpyrazine-2-yl)-1,2-oxazatecyclopropane (7): Compound 6 (300 mg, 1 mmol) was dissolved in a small amount of dichloromethane in a reaction flask. Then, a solution of 3-chloroperbenzoic acid (207.08 mg, 1.2 mmol) in chloroform was slowly added dropwise to the reaction flask at 0 °C. After the addition was complete, the mixture was brought to room temperature and stirred vigorously for 5 h. After the reaction was complete, the mixture was filtered, and the filtrate was extracted with a saturated aqueous solution of sodium bicarbonate and dichloromethane. The organic layer was evaporated to dryness to give crude product 7, which was a liquid.

[0048] 3. Preparation of (Z)-N-(1-adamantane-1-ylethyl)-1-(3,5,6-trimethylpyrazine-2-yl)methylimine oxide (II): The crude product 7 was dissolved in an appropriate amount of acetonitrile solvent and heated under reflux at 80°C for 2 days. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give compound II in 65% yield. The spectral data for compound II are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.46 (s, 1H, CH=N), 2.36 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.15 (p, J = 3.3 Hz, 1H, CH), 2.08 (d, J= 3.3 Hz, 2H, CH2), 1.84-1.75 (m, 4H, CH2), 1.52-1.48 (m,4H, CH2), 1.26 (s, 2H, CH2), 0.86 (s, 6H, CH3).

[0049] Its structural formula has been determined to be: .

[0050] Example 5 Tests on the neuroprotective effects of the compounds of this invention on glial cells: First, the cytotoxicity of the compound of this invention was tested using the Cell Proliferation and Toxicity Assay (CCK-8): A bottle of mouse microglia differentiated cell line BV2 (Shanghai Institute of Cell Biology, China) in good exponential growth phase was digested and prepared into 1×10⁻⁶ cells. 4 Cell suspension of cells / mL was seeded into 96-well plates and cultured at 37°C in the dark for 24 hours. Then, compounds I and II of this invention (0, 6.25, 12.5, 25, 50, 100, 250 µM) were added and cultured for another 24 hours. The absorbance value was then measured using the CCK-8 assay and the cell viability was calculated.

[0051] Experimental results show that compounds I1, I2, I3, and II of this invention all exhibited a survival rate of over 80% for BV2 cells at different concentrations (0, 6.25, 12.5, 25, 50, 100, and 200 µM), demonstrating low toxicity to glial cells.

[0052] The therapeutic effect of the compound of this invention was studied by inducing an inflammatory response in BV2 cells using an oxygen deprivation / glucose deprivation (OGD / R) model. Logarithmically grown BV2 cells were used in a 1×10⁻⁶ cell line. 4 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated for 24 h. The medium was then replaced with sugar-free DMEM complete medium and incubated for another 6 h in an anoxic incubator (95% N2 + 5% CO2 mixed gas). After 6 h, the cells were reoxygenated, and the medium was replaced with DMEM complete medium again. Cells were treated with complete medium at different concentrations (0, 50, 100, 200 µM) of the invented compounds I and II for 24 h to initiate and maintain reperfusion. The absorbance value was detected and cell viability was calculated using the CCK-8 assay.

[0053] Cell survival rate formula: Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%, The OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0054] like Figure 1 The figure shows the cell viability of BV2 cells treated with OGD / R for 6 h after incubation and culture for 24 hours with different concentrations (50, 100, 250 µM) of compounds I1, I2, I3, and II of this invention. The study found that cell viability significantly decreased after OGD / R model injury, with cell survival rate reduced by more than half compared to the uninjured Ctrl group. However, the survival rate was significantly improved after incubation with compounds I1, I2, I3, and II of this invention at different concentrations (50, 100, 250 µM), and was superior to the positive control drug TBN. Among them, compounds I1 and I3 of this invention showed the most significant improvement in survival rate after OGD / R injury, demonstrating that the compounds of this invention have a good protective effect against OGD / simulated ischemic stroke.

[0055] Further validation was performed using flow cytometry: An oxygen deprivation (OGD / R) model was used to induce an inflammatory response in BV2 cells, and the anti-inflammatory and anti-apoptotic effects of the compounds of this invention were investigated. Logarithmically grown BV2 cells were used in a 5×10⁻⁶ cell line. 5 Cells were seeded at a density of 1000 μL / well in 6-well plates and incubated for 24 h. The medium was then replaced with sugar-free DMEM complete medium and incubated for another 6 h in an anoxic incubator (95% N2 + 5% CO2 mixed gas). After 6 h, the cells were reoxygenated, and the medium was replaced with DMEM complete medium again. Cells were then treated with complete medium containing different concentrations of the compound of this invention (0, 50, 100, 200 µM) and TBN for 24 h to initiate and maintain reperfusion. Apoptosis was detected in each group using flow cytometry.

[0056] Figure 2 Apoptosis analysis was performed on OGD / R-treated BV2 cells after incubation and culture for 24 hours with different concentrations (0, 50, 100, 200 µM) of compound I1 of this invention and TBN (0, 50, 100 µM). Figure 2 B represents the quantitative analysis of apoptotic cells in each group.

[0057] The neuroprotective effect of the compounds of this invention on OGD / R-treated BV2 cells was demonstrated by Annexin V-FITC / propidium iodide (PI) double staining. OGD / R-treated BV2 cells showed a high apoptosis rate, while treatment with the compounds of this invention significantly reduced the apoptosis rate, showing a significantly better effect than TBN at the same concentration. This proves that the compounds of this invention have a good protective effect against OGD / simulated ischemic stroke.

[0058] Example 6 ROS scavenging ability test of the compounds of this invention: The ROS scavenging ability of the compounds of this invention was evaluated using the DCFH-DA probe. BV2 cells were seeded into confocal dishes and incubated for a period of time. The culture medium was then replaced with fresh complete medium, and the cells were pretreated for 2 h with different concentrations (0, 50, 100 µM) of compound I1 of this invention and TBN (0, 50, 100 µM). Then, t-BHP was added to a final concentration of 150 μM, and the plates were placed in a cell culture incubator for another 1 h. The culture medium was discarded, and the cells were washed with PBS. 10 μM DCFH-DA was added sequentially, and the cells were incubated for 30 min. After washing with PBS, the cell nuclei were stained using Hoechst 33342, and finally, images were taken using a confocal microscope.

[0059] in, Figure 3 A shows the confocal imaging results of different concentrations (0, 50, 100 µM) of compound I1 and TBN (0, 50, 100 µM) of the present invention in BV2 cells using the DCFH-DA / Hoechst 33342 probe. Figure 3 B represents the average fluorescence intensity of the DCFH-DA probe in Figure A.

[0060] The results (Figure 3) show that the compound of the present invention has a high scavenging ability. At the same concentration, the ROS scavenging ability of the compound of the present invention is higher than that of TBN, which proves that the compound of the present invention has a scavenging effect dependent on t-BHP-induced ROS, and proves that the compound of the present invention has a certain scavenging ability for ROS.

[0061] Example 7 ONOO of the compound of the present invention - Clearance capability test: The ONOO of the compounds of this invention was evaluated using the DHR123 probe. - Scavenging ability. BV2 cells were seeded into confocal dishes and incubated for a period of time. The culture medium was then replaced with fresh complete medium. Cells were pretreated for 2 hours with different concentrations (0, 50, 100, µM) of the compound of this invention and TBN (0, 50, 100 µM). Then, t-BHP was added to a final concentration of 150 μM, and the plates were placed in a cell culture incubator for another 1 hour. The culture medium was discarded, and the cells were washed with PBS. 10 μM DHR123 was added and the cells were incubated for 30 minutes, followed by PBS washing. The cell nuclei were then stained using Hoechst 33342, and finally, images were taken using a confocal microscope.

[0062] in, Figure 4 A represents the confocal imaging results of compounds I1, I2, I3, and II of this invention at a concentration (100 µM) in BV2 cells using the DHR123 / Hoechst33342 probe. Figure 4 B represents the average fluorescence intensity of the DHR 123 probe in Figure A.

[0063] The results show that ( Figure 4 The t-BHP group significantly increased BV2 cell fluorescence; however, the addition of compounds I1, I2, I3, and II of this invention significantly reduced fluorescence signals, indicating that the compounds of this invention have high ONOO. - Scavenging ability, and the compounds of the present invention are effective against t-BHP-induced ONOO. - It exhibits a scavenging effect dependence; these results demonstrate that the compounds of the present invention can scavenge ONOO. - It exerts a neuroprotective effect by reducing oxidative stress levels in inflammation.

[0064] Example 8 In vivo therapeutic efficacy test of the compound of this invention for cerebral ischemia: To evaluate the therapeutic effect of the compound of the present invention in vivo, experimental mice were divided into five groups: Sham group, MCAO group, TBN group, and the compound group of the present invention (different dosages: 67 μmol / kg, 136 μmol / kg). One hour after ischemia, the suture embolus was removed, reperfusion was restored, and TBN (136 μmol / kg) and compound I1 of the present invention (67 μmol / kg, 136 μmol / kg) were immediately injected via the tail vein. Twenty-four hours later, the Longa score was performed to assess neurological deficits. Subsequently, the mice were anesthetized, and brain tissue was harvested for TTC staining to determine infarct volume and evaluate the therapeutic effect of the compound of the present invention.

[0065] in, Figure 5 Image A shows TTC-stained coronal sections of the brains of mice in each group. Figure 5 B is a statistical graph of the cerebral infarction volume of mice in each group (mean ± standard deviation, n = 3).

[0066] The results show that ( Figure 5 Compared with the Sham group, the MCAO group mice showed significantly higher neurological function scores and large-area brain infarction. Figure 5 A, 5B). TTC staining results showed that the compounds of this invention significantly reduced the volume of cerebral infarction, and the effect was superior to that of the TBN group ( Figure 5 (A, 5B). The above results demonstrate that this compound has good neuroprotective effects, can significantly reduce the area of ​​cerebral infarction, and improve the pathological structure of cerebral ischemia mice, providing a new potential strategy for the treatment of cerebral ischemia.

[0067] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An adamantane nitrone derivative, characterized in that, The structural formula of the adamantane nitrone derivative is as follows: In this context, R is selected from H, CH3, and OH, and R1 is selected from H and OH.

2. A adamantane nitrone derivative, characterized in that, The structural formula of the adamantane nitrone derivative is as follows: .

3. The adamantane nitrone derivative according to claim 1, characterized in that, The structural formula of the adamantane nitrone derivative is as follows: , or .

4. The method for preparing the adamantane nitrone derivative according to claim 1, characterized in that, The preparation method includes the following steps: ligustrazine aldehyde (1) and adamantane derivative (2) are refluxed and dehydrated in toluene solvent to generate an imine intermediate compound (3), the intermediate compound (3) is oxidized to an epoxide (4) under the condition of 3-chloroperbenzoic acid, and finally the epoxide (4) is rearranged in acetonitrile solvent to obtain the adamantane nitrone derivative I; the reaction route is shown below: ， R is selected from H, CH3, and OH, and R1 is selected from H and OH.

5. The method for preparing the adamantane nitrone derivative according to claim 2, characterized in that, The preparation method includes the following steps: ligustrazine aldehyde (1) and 1-aminoethyl-adamantane (5) are refluxed and dehydrated in toluene solvent to generate an imine compound (6), the imine compound (6) is oxidized to an epoxide (7) under the condition of 3-chloroperbenzoic acid, and finally the epoxide (7) is rearranged in acetonitrile solvent to obtain the adamantane nitrone derivative II. The reaction route is shown below: 。 6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the adamantane nitrone derivative of claim 1 or 2 and a pharmaceutical carrier or excipient.

7. The use of the adamantane nitrone derivative of claim 1 or 2 in the preparation of drugs with free radical scavenging and oxidative stress elimination effects.

8. The use of the adamantane nitrone derivative of claim 1 or 2 in the preparation of a medicament for the protection of neuroinflammatory effects.

9. The use of the adamantane nitrone derivative of claim 1 or 2 in the preparation of a drug for treating ischemic stroke.

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