Application of a phenolic acid derivative in treating ischemic stroke

Abstract

The present invention provides the use of a phenolic acid derivative for treating ischemic stroke. Specifically, the present invention provides the use of a compound represented by Formula I below, or a pharmaceutically acceptable salt, optical isomer, hydrate, solvate, or prodrug thereof, for preparing a pharmaceutical composition for treating and / or alleviating ischemic stroke. #imgabs0#.

Description

Technical Field

[0001] The present invention belongs to the fields of life science and medicine, and specifically relates to the use of a phenolic acid derivative in treating ischemic stroke. Background Art

[0002] Stroke is a type of disease caused by impaired blood circulation in the brain, leading to loss of brain function. It is divided into ischemic stroke and hemorrhagic stroke, of which ischemic stroke accounts for as high as 80%.

[0003] The Chinese Stroke Prevention and Treatment Guidelines provide a series of guidance and recommendations for the prevention and treatment of ischemic stroke. Currently, thrombolytic therapy or intravascular thrombectomy is the most effective treatment for ischemic stroke. Despite this, there are still many urgent problems in the treatment of ischemic stroke. First, many patients miss the treatment window after onset and do not receive timely treatment. Second, although some patients can receive medical treatment within the short treatment window, most patients will still be left with varying degrees of disability after treatment, and a small number of patients will experience worsening of their condition due to ischemia-reperfusion injury. Third, there is still no effective treatment for the sequelae of ischemic stroke. Therefore, continued in-depth research on the relevant molecular mechanisms of ischemic stroke and the search and development of new anti-ischemic stroke drugs have potential application value and important research significance.

[0004] In summary, there is still a need in this field to develop drugs suitable for treating ischemic stroke. Summary of the Invention

[0005] The purpose of the present invention is to provide a compound for treating ischemic stroke or a preparation containing the compound and its mechanism of action.

[0006] Specifically, the present invention provides a class of phenolic acid derivatives, or pharmaceutically acceptable salts, or solvates, or prodrugs thereof, and their mechanisms of action and applications in treating ischemic stroke. Specifically, these phenolic acid derivatives can inhibit the proliferation of astrocytes and microglia, reduce the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, iNOS, COX2) and HIF-1α, and thereby achieve the therapeutic effect of ischemic stroke by inhibiting the occurrence and progression of inflammation, providing a new approach for the treatment of ischemic stroke.

[0007] In the first aspect of the present invention, there is provided a compound as shown in the following formula I, or a pharmaceutically acceptable salt, optical isomer, hydrate, solvate or prodrug thereof;

[0008]

[0009] wherein X is selected from the group consisting of O and S;

[0010] R1 and R2 are each independently selected from the group consisting of OH, SH, NH2, X2-PO(OH)2, or X2-PS(OH)2;

[0011] X2 is selected from the following group: O or S;

[0012] express

[0013] The invention is characterized in that it is used for preparing a pharmaceutical composition for treating and / or alleviating ischemic stroke.

[0014] In another preferred embodiment, the compound of formula I has a structure selected from the following group:

[0015]

[0016] Wherein, X is defined as described above.

[0017] In another preferred embodiment, the compound of formula I has a structure selected from the following group:

[0018]

[0019] In another preferred embodiment, the compound of formula I has a structure selected from the following group:

[0020]

[0021] Wherein, X is defined as described above.

[0022] In another preferred embodiment, the compound of formula I is selected from the following group:

[0023]

[0024] In another preferred embodiment, the pharmaceutically acceptable salt is selected from the following group: alkali metal salts, alkaline earth metal salts, and ammonium salts.

[0025] In another preferred embodiment, the pharmaceutically acceptable salt of the compound of formula I is the pentaammonium salt of the compound of formula I.

[0026] In another preferred embodiment, the pharmaceutical composition is also used to inhibit the proliferation of astrocytes and microglia.

[0027] In another preferred embodiment, the pharmaceutical composition is used to improve and / or alleviate the inflammatory response caused by ischemic stroke.

[0028] In another preferred embodiment, the pharmaceutical composition is also used to reduce the expression level of pro-inflammatory factors.

[0029] In another preferred embodiment, the pro-inflammatory factor is selected from the group consisting of TNF-α, IL-1β, IL-6, iNOS or COX2.

[0030] In another preferred embodiment, the pharmaceutical composition is also used to reduce the expression level of the oxygen homeostasis regulatory factor HIF-1α.

[0031] In another preferred embodiment, the pharmaceutical composition improves stroke symptoms by downregulating the expression level of the oxygen homeostasis regulatory factor HIF-1α.

[0032] In another aspect of the present invention, a method for treating and / or alleviating ischemic stroke is provided, characterized in that it comprises the steps of administering a safe and effective amount of Compound 2, or a pharmaceutically acceptable salt, optical isomer, hydrate, solvate or prodrug thereof, to a subject in need thereof.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The MCAO model was established in mice. (A) TTC staining of brain tissue from mice after sham surgery and one day after cerebral ischemia-reperfusion. (B) Statistical results of TTC staining of brain tissue. (C) Laser speckle pattern analysis of cerebral blood flow. (D) Statistical results of cerebral blood flow index. (E) Statistical results of Zea-Longa neurobehavioral scores from mice after sham surgery and one day after cerebral ischemia-reperfusion.

[0035] Figure 2 This is the effect of compound 2 on the neurological function score of MCAO model mice.

[0036] Figure 3 Effect of Compound 2 on ischemic infarct volume in MCAO mice. (A) TTC staining of the brains of mice in the model group and the Compound 2 group. (B) Statistics of ischemic infarct area in the mice in the model group and the Compound 2 group.

[0037] Figure 4 Effects of Compound 2 on balance, neurological impairment, body weight, and survival in model mice. (A) Statistical results of the rotarod test. (B) Neurological impairment on a 14-point scale. (C) Statistical results of body weight change in mice. (D) Survival curve of mice after ischemia-reperfusion.

[0038] Figure 5GFAP immunofluorescence results after ischemia-reperfusion. (A) Representative images of GFAP fluorescence staining in brain tissue from the sham-operated (SHAM), model (MCAO), compound 2, and compound 1 groups. (Scale bar = 100 μm). (B) Statistical results of GFAP-positive cell density in brain tissue from the model (MCAO), compound 2, and compound 1 groups.

[0039] Figure 6 Figure 3. GFAP protein expression in each group after ischemia-reperfusion. (A) Representative Western blot (WB) images of GFAP protein expression in brain tissues of the sham-operated group (SHAM), model group (MCAO), compound 2 group, and compound 1 group; (B) Statistical results of grayscale values of GFAP protein in each group.

[0040] Figure 7 Figure 1 shows the immunofluorescence staining of iba1 after ischemia-reperfusion. (A) Representative images of iba1 fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of the density of iba1-positive cells in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group 72 hours after ischemia-reperfusion.

[0041] Figure 8 Figure 2 shows the expression of iba1 protein in each group after ischemia-reperfusion. (A) Representative Western blotting images of iba1 protein expression in brain tissues of the sham-operated (SHAM) group, the model (MCAO) group, the compound 2 group, and the compound 1 group. (B) Statistical results of grayscale values of iba1 protein in each group.

[0042] Figure 9 Figure 1 shows TNF-α immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of TNF-α fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of TNF-α-positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0043] Figure 10 Figure 1 shows IL-1β immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of IL-1β fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of IL-1β-positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0044] Figure 11 Figure 1 shows IL-6 immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of IL-6 fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of IL-6 positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0045] Figure 12 Figure 2 shows iNOS immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of iNOS fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of iNOS-positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0046] Figure 13 COX2 immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of COX2 fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of COX2-positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0047] Figure 14 Figure 2 shows the mRNA expression of proinflammatory factors in each group after ischemia-reperfusion. Figure 2 shows the mRNA expression and statistical results of TNF-α (A), IL-1β (B), IL-6 (C), iNOS (D), and COX2 (E) in brain tissue of the sham operation group (SHAM), model group (MCAO), compound 2 group, and compound 1 group.

[0048] Figure 15 The expression of TNF-α, IL-1β, and IL-6 in brain tissue after ischemia-reperfusion was determined by enzyme-linked immunosorbent assay. The expression levels of TNF-α (A), IL-1β (B), and IL-6 (C) in brain tissue homogenates of the sham operation group (SHAM), model group (MCAO), compound 2 group, and compound 1 group, as well as the statistical results.

[0049] Figure 16 Figure 2 shows the expression of TNF-α protein in each group after ischemia-reperfusion. (A) Representative Western blotting images of TNF-α protein expression in brain tissues of the sham-operated group (SHAM), model group (MCAO), compound 2 group, and compound 1 group; (B) Statistical results of grayscale values of TNF-α protein in each group.

[0050] Figure 17Figure 2 shows HIF-1α immunofluorescence staining results after ischemia-reperfusion. (A) Representative images of HIF-1α fluorescence staining in brain tissue from the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (Scale bar = 100 μm). (B) Statistical results of HIF-1α-positive cell density in brain tissue from the model (MCAO) group, the Compound 2 group, and the Compound 1 group.

[0051] Figure 18 The expression of HIF-1α mRNA after ischemia-reperfusion. The statistical results of HIF-1α mRNA in brain tissue of sham operation group (SHAM), model group (MCAO), compound 2 group and compound 1 group.

[0052] Figure 19 Figure 2 shows HIF-1α protein expression in each group after ischemia-reperfusion. (A) Representative Western blotting images of HIF-1α protein expression in brain tissues of the sham-operated (SHAM) group, the model (MCAO) group, the Compound 2 group, and the Compound 1 group. (B) Statistical results of grayscale values of HIF-1α protein in each group using Western blotting. DETAILED DESCRIPTION

[0053] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that a compound having the structure represented by Formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is an active ingredient that can effectively treat ischemic stroke. Experiments have shown that the compound represented by Formula I can inhibit the proliferation of astrocytes and microglia, reduce the expression levels of proinflammatory cytokines (TNF-α, IL-1β, IL-6, iNOS, COX2) and HIF-1α, and achieve the therapeutic effect of ischemic stroke by inhibiting the occurrence and progression of inflammation. Based on this, the inventors completed the present invention.

[0054] Active ingredients for the treatment of ischemic stroke

[0055] The present invention provides an active ingredient for treating ischemic stroke. The active ingredient is a compound represented by formula (I), or a pharmaceutically acceptable salt, optical isomer, hydrate, solvate or prodrug thereof;

[0056]

[0057] wherein X is selected from the group consisting of O and S;

[0058] R1 and R2 are each independently selected from the group consisting of OH, SH, NH2, X2-PO(OH)2, or X2-PS(OH)2;

[0059] X2 is selected from the following group: O or S;

[0060] express

[0061] Among them, the preferred compound is Compound 1 or Compound 2:

[0062]

[0063] Experiments have shown that the active ingredients of the present invention can inhibit the proliferation of astrocytes and microglia, reduce the expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6, iNOS, COX2) and HIF-1α, and achieve the effect of treating ischemic stroke by inhibiting the occurrence and development of inflammation.

[0064] As used herein, "active ingredient," "active compound of the present invention," and "active ingredient of the present invention" are used interchangeably to refer to the compound of formula I of the present invention and its structural analogs.

[0065] It should be understood that the active ingredients of the present invention include the compounds of formula I of the present invention, or pharmaceutically acceptable salts, enantiomers, diastereomers or racemates thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include various crystalline forms, amorphous compounds, and deuterated compounds of the compounds of formula I of the present invention.

[0066] The "pharmaceutically acceptable salt" is a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of formula (I) with an inorganic base; or a methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of formula (I) with an organic base; or a corresponding inorganic acid salt formed by the compound of formula (I) with lysine, arginine or ornithine followed by esterification with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or a corresponding organic acid salt formed with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid. In the present invention, a preferred class of pharmaceutically acceptable salts is an ammonium salt, and more preferably a pentaammonium salt.

[0067] Pharmaceutical compositions and applications

[0068] The present invention also provides the use of a compound of formula I, or a mixture of one or more of its pharmaceutically acceptable salts, enantiomers, diastereomers or racemates and prodrugs as an active ingredient in the preparation of a drug for treating and / or alleviating ischemic stroke and other related diseases.

[0069] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in an amount of 0.001-99 wt % by weight, preferably 0.1 wt % to 90 wt % of the total weight of the compound of formula I as the active ingredient, with the remainder being a pharmaceutically acceptable carrier, diluent, solution or saline solution.

[0070] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of the present invention, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0071] The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and can be present in suitable solid or liquid carriers or diluents and suitable sterile devices for injection or infusion.

[0072] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the formulation generally contains 0.05-400 mg of the compound of formula (I), preferably 1 mg-500 mg of the compound of formula (I).

[0073] The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered orally, nasally, dermatologically, pulmonary, or through the gastrointestinal tract, with an injectable formulation (e.g., an infusion) being the most preferred route. The most preferred daily dose is 0.01-400 mg / kg body weight taken as a single dose, or 0.01-200 mg / kg body weight taken in divided doses. Regardless of the route of administration, the optimal individual dose will depend on the specific treatment being used. Typically, a low dose is started and gradually increased until the most suitable dose is found.

[0074] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, or mucosal tissues; or can be mixed or encapsulated in other substances and introduced into the body.

[0075] Advantages of the present invention include:

[0076] (1) The compound of formula I of the present invention can significantly improve cerebral infarction caused by ischemia-reperfusion, significantly improve the motor coordination and balance of ischemia-reperfusion mice, significantly improve their neurological damage, and at the same time significantly inhibit the loss of mouse body weight and reduce the mortality rate of mice.

[0077] (2) The experiments of the present invention show that the compound of formula I can inhibit the proliferation of astrocytes and microglia, reduce the expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6, iNOS, COX2) and HIF-1α, and achieve the effect of treating ischemic stroke by inhibiting the occurrence and development of inflammation.

[0078] (3) The compounds of the present invention have low toxicity and side effects and good drugability.

[0079] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0080] experimental animals

[0081] The experiment was conducted on male C57BL / 6 mice for research and brain tissue extraction. The animals used in this experiment were obtained from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The animals were raised under standard conditions: room temperature (20±2°C) and a 12-hour day and night cycle. The experimental animals were supplied with water and standard feed and adapted for 3 days before the experiment. All animal experiments were conducted from 8 am to 6 pm. The animal experiments involved in this study were strictly carried out in accordance with the Lanzhou University Experimental Animal Ethics Code (License No.: JCYXY Gan 2021-0126).

[0082] Compound 1 and compound 2 were prepared according to the method described in Liu Huiyun. Design, synthesis and activity evaluation of new blood oxygen regulators [D]. Lanzhou University, 2019.

[0083] 1. Construction of Ischemic Stroke Model and Evaluation of Compound 2

[0084] Example 1: Establishment and verification of middle cerebral artery occlusion (MCAO) model

[0085] This study used the method of Longa et al. to construct a mouse MCAO model. This model well simulates the occurrence, development and acute treatment of ischemic stroke in clinical practice, and provides stable application value for the study of the prevention and treatment mechanism of ischemic stroke. To evaluate the success of the model, the brain infarct volume of the mice was measured on the first day after surgery. The experimental results are as follows: Figure 1 A and Figure 1 As shown in Figure B, 2,3,5-triphenyte-trazolium chloride (TTC) staining showed that cerebral infarction occurred one day after surgery, with the infarct volume accounting for 45.14±4.48% (P<0.001). Laser speckle flowmetry was used to measure blood flow at different times before, during, and after ischemia and reperfusion. The experimental results are shown in Figure 2. Figure 1 C and Figure 1As shown in Figure D, 30 minutes after middle cerebral artery occlusion, the blood flow index decreased compared to the pre-ischemic period (138.3±5 vs 52.67±2.3, P<0.001). One hour later, after the suture was removed and reperfusion was performed, blood flow was restored, and the blood flow index increased significantly compared to the ischemic period (52.67±2.3 vs 95±5, P<0.001). At the same time, the behavioral scores of mice were evaluated 24 hours after modeling according to the Zea-Longa scoring method. The experimental results are shown in Figure D. Figure 1 As shown in Figure E, mice developed significant stroke symptoms on the first day after surgery, with a pronounced body tilt when crawling, significantly different from sham surgery (P < 0.001). Based on TTC staining results, blood flow patterns, and mouse behavior, the MCAO model established in this study was successful. n = 6, data are presented as mean ± SEM, and were analyzed using one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001, * indicates comparison with SHAM ( Figure 1 B and Figure 1 E), * indicates comparison with Pre-MCAO ( Figure 1 C and Figure 1 D); # P<0.05, ## P<0.01, ### P<0.001, * indicates comparison with MCAO-30 min.

[0086] Example 2: Neurobehavioral effects of compound 2 on MCAO model mice

[0087] Male C57 / BL mice weighing 23±1g were randomly divided into a sham-operated (SHAM) group, a model (MCAO) group, and a treatment group (50mg / kg, 100mg / kg, or 200mg / kg), each containing 6 mice. Mice in the SHAM group underwent vascular isolation without suture placement and received no pre- and post-operative treatment. Mice in the model group underwent an ischemic MCAO model and received no pre- and post-operative treatment. The treatment groups were treated with Compound 2 in addition to the model group. Mice with a post-reperfusion neurological function score of 0 and mice that died before 72 hours post-surgery were excluded. Drugs were administered via tail vein at 0h, 1 day, and 2 days after reperfusion. The SHAM and MCAO groups received equal volumes of normal saline.

[0088] Neurological function scores reflect the state of nerve damage in MCAO model mice. Neurological function scores were performed 24 hours after model establishment (referring to the Zea-Longa scoring method). Mice with scores of 1-3 were considered successful models, and those with scores of 0 and 4 were excluded. The scoring criteria are as follows:

[0089] Zea-Longa scoring screening method

[0090]

[0091] In this experiment, the Zea-Longa scoring method was used to score the sham hand group (SHAM), model group (MCAO), compound 2 (50 mg / kg), compound 2 (100 mg / kg) and compound 2 (200 mg / kg) groups. The main scoring time points were before surgery, 1 day, 2 days and 3 days. Figure 2 The sham-operated group had a score of 0, indicating no neurological impairment. Compared with the sham-operated group, mice in the MCAO and compound 2 groups exhibited pronounced lateral circling at 24 hours after reperfusion, indicating successful model establishment. No significant differences were observed in the compound 2 group compared with the model group at 24 hours after reperfusion. However, after 48 hours, the scores of all three compound 2 concentrations decreased significantly, with the compound 2 (100 mg / kg) group showing the most significant decrease (P < 0.001). After 72 hours of reperfusion, the neurological function score at the compound 2 (100 mg / kg) concentration was significantly decreased compared with the model group (P < 0.001). n = 6, data are presented as mean ± SEM, and were analyzed using two-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001, *Compared with the model group (MCAO).

[0092] The results showed that compound 2 can effectively improve the neurological deficits in model mice and improve the coordination and balance ability of mice after ischemia-reperfusion, and may have a good protective effect on repairing damage after cerebral ischemia-reperfusion.

[0093] Example 3: Effect of Compound 2 on Cerebral Infarction Volume in MCAO Model Mice

[0094] The cerebral infarction volume of the model mice in the SHAM group, the MCAO group, the Compound 2 (50 mg / kg) group, the Compound 2 (100 mg / kg) group, and the Compound 2 (200 mg / kg) group in Example 2 was detected by TTC staining. Figure 3As shown, significant ischemic infarcts were observed in the brain tissue of model mice. The infarct volumes varied among mice treated with different concentrations of compound 2. Compared with the model group, the infarct volume of model mice treated with compound 2 (100 mg / kg) was significantly reduced (27.17 ± 5.18%, P < 0.001). The infarct volume of mice treated with compound 2 (200 mg / kg) was also significantly reduced (16.19 ± 3.56%, P < 0.05). Compared with the 50 mg / kg compound 2 dose, the infarct volume of model mice treated with compound 2 at a concentration of 100 mg / kg was significantly smaller (26.22 ± 4.68%, P < 0.05). n = 6, data are presented as mean ± SEM, and were analyzed using one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the model group (MCAO). # P<0.05, ## P<0.01, ### P<0.001, compared with compound 2 50 mg / kg.

[0095] 2. Study on the mechanism of the compounds of the present invention in treating ischemic stroke models

[0096] The present invention uses Compound 1 and Compound 2 as examples to study the mechanism of the compounds of the present invention in treating ischemic stroke.

[0097] Grouping and Dosing: Male C57 / BL mice weighing 23±1g were randomly divided into a sham-operated (SHAM) group, a model (MCAO) group, a Compound 2 group (100mg / kg), and a Compound 1 group (50mg / kg), 10 mice each. Mice in the SHAM group underwent vascular isolation without suture placement and received no pre- and post-operative treatment. Mice in the model group underwent a MCAO model and received no pre- and post-operative treatment. The treatment groups received Compound 2 or Compound 1, as well as the treatments in the model group. Mice with a post-reperfusion neurological function score of 0 or those that died before 72 hours post-surgery were excluded. For behavioral testing, dosing was performed seven times: at 0 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days after reperfusion. For other experiments, dosing was performed three times: at 0 hours, 1 day, and 2 days after reperfusion. Dosing was via tail vein administration; the sham-operated and model groups received an equal volume of normal saline.

[0098] Example 4: Effects of the compounds of the present invention on behavioral function in MCAO model mice

[0099] After ischemia-reperfusion, neurons are severely damaged, and synaptic structure is also severely disrupted. The cascade of these injuries ultimately leads to severe behavioral deficits. To investigate the effects of Compound 2 and Compound 1 on behavior after ischemia-reperfusion, the motor coordination of mice after ischemia-reperfusion was tested and analyzed using the rotarod test.

[0100] The motor coordination of mice was tested using a balanced rotarod. The experiment was divided into four groups: sham operation group (SHAM), model group (MCAO), compound 2 group and compound 1 group. The effect of compound 2 on the behavioral function recovery of mice after ischemia-reperfusion was comprehensively analyzed. The rotarod test can test the motor coordination ability of mice. The experimental procedure was set to gradually accelerate the speed of the rotarod from 10 rpm to 40 rpm within 300 s. The time the mice stayed on the rotarod was recorded. The mice were trained 3 days in advance. Mice that could stay on the rotarod for about 300 s were selected for the next experiment. The time the mice stayed on the rotarod before the model was constructed was selected as the preoperative test value. The trained mice were subjected to MCA occlusion for 60 minutes and then reperfused. The rotarod test was performed on the mice in the SHAM group, MCAO group, compound 2 group and compound 1 group on days 1, 3, 5 and 7 after surgery, and the time the mice stayed on the rotarod was recorded.

[0101] The experimental results are as follows Figure 4 As shown in Figure A, compared to the sham-operated group, the rotarod duration in the MCAO group was significantly reduced 1, 3, 5, and 7 days after ischemia-reperfusion (P<0.001), indicating that the behavioral impairment of mice after ischemia-reperfusion was very severe. Motor coordination continued to decline from 1 to 3 days after reperfusion, and gradually recovered after 3 days, but the degree of recovery was extremely low. Compared with the model group (MCAO), compound 2 significantly improved the motor coordination of mice at 5 and 7 days (P<0.05), and significantly improved the motor coordination of mice at 7 days after reperfusion (P<0.001). Compared with the MCAO group, the motor coordination of mice in the compound 1 group was also improved. Compared with the compound 1 group, the motor coordination of mice in the compound 2 group was significantly improved (P<0.001).

[0102] Modified mouse neurological deficit score (mNSS) table (14-point system)

[0103]

[0104]

[0105] In addition, the modified mouse neurological deficit score (mNSS) method shown in the table above was used to perform neurological functional scoring (14-point system) on each group of mice. Neurobehavioral scoring was performed on mice in the SHAM group, MCAO group, compound 2 group, and compound 1 group 1, 3, 5, and 7 days after surgery. Mice with scores of 10 to 14 indicated that the degree of damage to the nervous system was severe. Mice with scores of 5 to 9 indicated that the degree of damage to the nervous system was moderate. 1 to 4 points indicated that the degree of damage to the nervous system was mild. The maximum score was 14 points. Analysis of the neurological function injury score results is as follows: Figure 4 As shown in Figure B, compared with the MCAO group, the neurological function of mice in the compound 2 group was significantly improved from day 1 to day 7 of reperfusion. Compared with the compound 1 group, the neurological function of the compound 2 group was significantly improved.

[0106] At the same time, the weight changes and survival rates of mice after ischemia-reperfusion were analyzed. Figure 4 As shown in Figure C, compared with the sham operation group, the body weight of mice after ischemia-reperfusion was significantly reduced. The weight loss was most severe 3 days after reperfusion, and then gradually recovered, but it did not return to the weight before ischemia until 7 days. Compared with the MCAO group, compound 2 inhibited the decrease in body weight of mice after ischemia-reperfusion 7 days after reperfusion. Compared with the compound 2 group, the body weight of mice in the compound 1 group decreased significantly at 7 days after reperfusion (P<0.05). The survival curve results are shown in Figure 4D. Compared with the MCAO group, compound 2 significantly improved the mortality rate of mice after ischemia-reperfusion. n=10, data are expressed as mean±SEM, and the two-way ANOVA analysis method was used for processing and analysis, and Tukey's HSD test was performed. *P<0.05, **P<0.01, ***P<0.001, # P<0.05, ## P<0.01, ### P<0.001; * indicates comparison with the sham-operated group (SHAM). # Indicates comparison with compound 2 group.

[0107] Example 5: Effects of the compounds of the present invention on astrocytes in MCAO model mice

[0108] To investigate whether compound 2 has an effect on astrocyte activation and proliferation during ischemia-reperfusion, this study used immunofluorescence experiments to observe the fluorescence expression of GFAP in brain tissues of the sham operation group, model group, compound 2 group, and compound 1 group, and then used Western blot to detect the expression level of GFAP, a marker of astrocytes, in brain tissues. The results of fluorescence staining and quantitative analysis are shown in Figure 2. Figure 5As shown. After 72 hours of ischemia-reperfusion, the activation and proliferation of GFAP protein in astrocytes in the brain area were analyzed by immunofluorescence staining. Figure 5 As shown in A, a large number of GFAP-positive cells appeared in the MCAO group, while the number of GFAP-positive cells in the compound 2 and compound 1 groups was significantly reduced. Figure 5 As shown in Figure B, the number of GFAP-positive cells in the MCAO model group increased dramatically. Compared with the MCAO group, the number of GFAP-positive cells decreased significantly after three days of compound 2 administration (195±45 vs 481±67 / mm 2 Compared with the MCAO group, the number of GFAP-positive cells also decreased significantly after three days of compound 1 administration (327±50 vs 481±67 / mm2). 2 Compared with compound 1 group, the number of GFAP-positive cells in compound 2 group decreased more significantly (195±45 vs 327±50 / mm2). 2 , P < 0.001). n = 3. Data are expressed as mean ± SEM. One-way ANOVA was used for analysis and Tukey's HSD test was performed. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. * indicates comparison with the MCAO group. # Indicates comparison with compound 2 group.

[0109] After 72 hours of ischemia-reperfusion, the protein expression level of GFAP, a marker of astrocytes, in brain tissue was detected by Western blot. Representative images of Western blot (WB) of GFAP protein expression in brain tissue of sham operation group (SHAM), model group (MCAO), compound 2 group, and compound 1 group are shown in the figure below. Figure 6 As shown in A, the quantitative statistical results are as follows Figure 6As shown in B. Compared with the sham operation group, the GFAP protein expression in the brain tissue of mice in the MCAO group was significantly increased by 91±30% (P<0.001). Compared with the MCAO group, the GFAP protein expression was significantly decreased by 43±12% after three days of administration of compound 2 (P<0.001). Compared with MCAO, the GFAP protein expression was significantly decreased by 25±10% after three days of administration of compound 1 (P<0.01). Compared with the compound 1 group, the GFAP protein expression in the compound 2 group was significantly decreased by 45±10% (P<0.05). β-actin was used as the internal reference protein in this experiment. n=3, data are expressed as mean±SEM, processed and analyzed by one-way ANOVA analysis method, and Tukey's HSD test was performed. *P<0.05, **P<0.01, ***P<0.001; # P<0.05, ## P<0.01, ### P<0.001. * indicates comparison with the SHAM group, # indicates comparison with the compound 2 group.

[0110] Example 6: Effects of the compounds of the present invention on microglial activation and proliferation in MCAO model mice

[0111] Microglia are resident phagocytes in the nervous system and are the primary defense system for stress and damage of central nervous cells. After cerebral ischemia and reperfusion, microglia are activated after external damage stimulation and release pro-inflammatory and anti-inflammatory mediators. Iba1 (ionized calcium binding adapter molecule 1) is specifically expressed in microglia in the central nervous system and is a calcium-binding protein of approximately 17kDa. To explore whether compounds have an effect on microglial activation and proliferation in the ischemia-reperfusion model, this study used immunofluorescence experiments to detect the fluorescence expression of iba1 in brain tissues of the sham operation group (SHAM), model group (MCAO), compound 2 group, and compound 1 group. The expression level of iba1 in microglia in brain tissue was then detected by Western blot. 72 hours after ischemia and reperfusion, the microglial protein iba1 in the brain region was stained by immunofluorescence to analyze its changes. The results of fluorescence staining are shown in the figure. Figure 7 As shown in A, a large number of iba1-positive cell signals appeared in the MCAO group, while compound 2 and compound 1 reduced the iba1-positive cell signals. Figure 7 As shown in Figure B, the statistical results showed that the expression of iba1 positive cells in the MCAO model group increased dramatically. Compared with the MCAO model group, the number of iba1 positive cells decreased significantly after three days of compound 2 administration (148 ± 40 vs 303 ± 23 / mm 2Compared with the MCAO model group, the number of iba1-positive cells also decreased significantly after three days of compound 1 administration (200±12 vs 303±23 / mm). 2 Compared with compound 1 group, compound 2 group had lower number of iba1-positive cells (148±40 vs 200±12 / mm2). 2 , P < 0.001). n = 3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the MCAO group. # Indicates comparison with compound 2 group.

[0112] The expression level of iba1, a specific protein of microglia in brain tissue, was detected by Western blot. (A) Representative WB images of iba1 protein expression in brain tissue of sham operation group (SHAM), model group (MCAO), compound 2 group, and compound 1 group are shown in Figure 2. Figure 8 As shown in A, the quantitative statistical results are as follows Figure 8 As shown in Figure B. The results showed that compared with the sham operation group, the expression of iba1 protein in the brain tissue of mice in the MCAO model group was significantly increased by 110±50% (P<0.001). Compared with the MCAO group, the expression of iba1 protein was significantly decreased by 50±12% (P<0.05) three days after the administration of compound 2. Compared with the MCAO group, the expression of iba1 protein was decreased three days after the administration of compound 1. Compared with the compound 1 group, the expression of iba1 protein in the compound 2 group was decreased. β-actin was used as the internal reference protein in this experiment. n=3, data are expressed as mean±SEM, processed and analyzed by one-way ANOVA analysis method, and Tukey's HSD test was performed. *P<0.05, **P<0.01, ***P<0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0113] The results showed that compared with the model group, the expression levels of microglia and astrocytes in the compound 2 group and the compound 1 group were significantly decreased, but compound 2 treatment could significantly inhibit the excessive activation of microglia and astrocytes in the post-ischemic brain area.

[0114] Example 7: Effects of the compounds of the present invention on the expression levels of inflammatory factors in MCAO model mice

[0115] The exact etiology of ischemic stroke remains unclear, but studies have shown that inflammation plays a crucial role in the pathogenesis of ischemic stroke. Changes in the expression of inflammatory factors can be used as effective indicators for evaluating the therapeutic efficacy of ischemic brain injury. The infiltration of hematogenous leukocytes into the brain parenchyma and the activation of endogenous microglia contribute to the intense inflammatory response following cerebral ischemia. Under ischemic conditions, the number of microglia and infiltrating immune cells in brain tissue increases significantly. Microglia promote neuroinflammation by releasing proinflammatory cytokines (TNF-α, IL-1β, and IL-6, among others) and cytotoxic molecules (IFN-γ, prostaglandins, ROS, and NO, among others). Microglia may cause neuronal damage through the following mechanisms: 1) secretion of proinflammatory cytokines; 2) activation of nitric oxide (NOX), leading to microglial proliferation and neuroinflammation; 3) expression of iNOS; and 4) endocytosis of neurons. Cyclooxygenase (COX) plays a key role in the biosynthesis of prostaglandins. COX2, acting as a dioxygenase and peroxidase, mediates the conversion of arachidonic acid to prostaglandins, playing a key role in the development of inflammation. COX2 expression is induced by specific events, such as in response to physiological stresses such as infection and inflammation, leading to the production of prostaglandins.

[0116] To investigate the effects of the compounds on the expression of inflammatory factors after ischemia-reperfusion, this study used immunofluorescence to observe the expression of TNF-α, IL-1β, IL-6, iNOS, and COX2 proteins in brain tissue. RT-qPCR was used to examine the mRNA expression of the pro-inflammatory factors TNF-α, IL-1β, IL-6, iNOS, and COX2 in the sham, model, compound 2, and compound 1 groups. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of TNF-α, IL-1β, and IL-6 in brain tissue homogenates, and Western blot was used to determine the expression level of TNF-α protein in brain tissue.

[0117] After 72 hours of ischemia-reperfusion, the expression of inflammatory factors such as TNF-α, IL-1β, IL-6, iNOS and COX2 in the brain was analyzed by immunofluorescence staining. Figures 9 to 13As shown. A large number of pro-inflammatory factor-positive cell signals appeared in the MCAO group, while compound 2 and compound 1 reduced the pro-inflammatory factor-positive cell signals. The statistical results showed that the number of pro-inflammatory factor-positive cells in the MCAO group increased sharply. Compared with the MCAO model group, the number of pro-inflammatory factor-positive cells decreased significantly after three days of administration of compound 2 (P<0.001). The statistical results showed that the number of iba1-positive cells in the MCAO model group increased sharply. Compared with the MCAO group, the number of pro-inflammatory factor-positive cells also decreased significantly after three days of administration of compound 1 (P<0.001). Compared with the compound 1 group, the density of pro-inflammatory factor-positive cells in the compound 2 group was significantly reduced (P<0.001). As Figure 9 As shown in Figure 2, the number of TNF-α positive cells in MCAO model mice was significantly decreased after treatment with compound 2 (140±35 vs 361±48 / mm 2 , P < 0.001), the number of TNF-α positive cells in compound 1 group also decreased to a certain extent (244 ± 36 vs 361 ± 48 / mm 2 , P < 0.001). Other proinflammatory cytokines, IL-1β, IL-6, iNOS, and COX2, showed similar trends to TNF-α. n = 3, data are expressed as mean ± SEM, and were analyzed using one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the MCAO group. # Indicates comparison with compound 2 group.

[0118] The results of RT-qPCR experiment and quantitative analysis are as follows Figure 14As shown, 72 hours after ischemia-reperfusion, the mRNA expression levels of TNF-α, IL-1β, IL-6, iNOS, and COX2 in brain tissue were detected and analyzed by RT-qPCR. The results showed that compared with the sham group, the expression levels of TNF-α (P<0.001), IL-1β (P<0.001), IL-6 (P<0.01), iNOS (P<0.001), and COX2 (P<0.001) in the MCAO group increased to varying degrees. Three days after administration of compound 2, the expression levels of TNF-α (P<0.05), IL-1β (P<0.01), IL-6 (P<0.01), iNOS (P<0.05), and COX2 (P<0.001) were significantly decreased compared with the MCAO group. Compound 2 had the most significant inhibitory effect on IL-6 expression, with a decrease of 51±8%. Compared with the MCAO group, three days after administration of compound 1, the expression levels of TNF-α (P < 0.05), iNOS (P < 0.05), and COX2 (P < 0.001) were significantly decreased to varying degrees. n = 3, data are expressed as mean ± SEM, and were analyzed using one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0119] ELISA test results and quantitative analysis results are as follows Figure 15As shown. 72 hours after ischemia-reperfusion, the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in mouse brain tissue homogenates were measured. The results showed that compared with the sham-operated group, the expression levels of TNF-α (P<0.01), IL-1β (P<0.001), and IL-6 (P<0.01) in the MCAO group increased to varying degrees. Compared with the MCAO group, three days after administration of compound 2, the expression levels of TNF-α, IL-1β, and IL-6 were significantly reduced. TNF-α expression decreased by 40±10% (P<0.01), IL-1β expression decreased by 32±9% (P<0.001), and IL-6 expression decreased by 40±10% (P<0.05). Compared with the MCAO group, three days after administration of compound 1, the expression levels of TNF-α, IL-1β (P<0.05), and IL-6 decreased to varying degrees. n = 3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA and Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0120] After 72 hours of ischemia-reperfusion, the expression level of TNF-α protein in brain tissue was detected by Western blot. Figure 16 As shown, compared with the sham-operated group, the protein expression of TNF-α in the brain tissue of mice in the MCAO group was significantly increased by 120±50% (P<0.001). Compared with the MCAO group, the protein expression of TNF-α was significantly decreased by 40±12% after three days of administration of compound 2 (P<0.001). Compared with the MCAO group, the protein expression of TNF-α was decreased by 20±10% after three days of administration of compound 1 (P<0.001). Compared with the compound 1 group, the protein expression of TNF-α in the compound 2 group was decreased by 31±20% (P<0.05). β-actin was used as the internal reference protein in this experiment. n=3, data are expressed as mean±SEM, and processed and analyzed by one-way ANOVA analysis method, and Tukey's HSD test was performed. *P<0.05, **P<0.01, ***P<0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0121] In this study, the concentrations of TNF-α, IL-1β, and IL-6 in brain tissue homogenates of MCAO model mice were significantly higher than those in the sham-operated group. After treatment with compound 2, the levels of TNF-α, IL-1β, and IL-6 in brain tissue decreased significantly. The production of TNF-α, IL-1β, and IL-6 in brain tissue homogenates of the compound 1 group was also lower than that of the model group. This study suggests that compounds may inhibit the expression of inflammatory factors, reduce inflammation after ischemic cerebral infarction, and improve damage caused by cerebral ischemia when treating ischemia-reperfusion injury, and the therapeutic effect of compound 2 is more pronounced than that of compound 1.

[0122] Example 8: Effects of the compounds of the present invention on hypoxia-inducible factor in MCAO model mice

[0123] HIF-1α is a sensitive regulator of oxygen homeostasis, whose expression is rapidly induced following hypoxia-ischemia. It plays a broad role in the pathophysiology of stroke, including neuronal survival, neuroinflammation, angiogenesis, glucose metabolism, and blood-brain barrier regulation. It not only specifically senses hypoxia but also facilitates the maintenance of oxygen homeostasis. In this study, immunofluorescence and Western blot were used to assess HIF-1α expression in brain tissue. RT-qPCR was also used to examine the gene expression of related factors in the sham, model, compound 2, and compound 1 groups.

[0124] Fluorescence staining results and quantitative analysis results are as follows Figure 17 As shown. After 72 hours of ischemia-reperfusion, the expression changes of the inflammatory factor HIF-1α in the brain area were analyzed by immunofluorescence staining. The staining results showed that there were almost no HIF-1α-positive cells in the sham operation group, a large number of HIF-1α-positive cells appeared in the MCAO group, and compound 2 and compound 1 reduced the number of pro-inflammatory factor-positive cells. The statistical results showed that compared with the sham operation group, the number of HIF-1α-positive cells in the MCAO model group increased sharply. Compared with the MCAO group, the number of HIF-1α-positive cells decreased significantly after three days of administration of compound 2 (197±33 vs 568±60 / mm 2 Compared with the MCAO group, the number of HIF-1α positive cells also decreased significantly after three days of compound 1 administration (250±54 vs 568±60 / mm). 2 , P<0.001). Compared with compound 1 group, the number of HIF-1α positive cells in compound 2 group was lower (197±33 vs 250±54 / mm2). 2, P < 0.001). n = 3, data are expressed as mean ± SEM, and were analyzed by one-way ANOVA followed by Tukey's HSD test. *P < 0.05, **P < 0.01, ***P < 0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with MCAO group. # Indicates comparison with compound 2 group.

[0125] After 72 hours of ischemia-reperfusion, the expression of HIF-1α mRNA in brain tissue was detected and analyzed by RT-qPCR. Figure 18 As shown, compared with the sham group, the expression of HIF-1α in the MCAO group was significantly increased by 417±107% (P<0.001). Compared with the MCAO group, the expression level of HIF-1α was significantly reduced by 75±17% after three days of compound 2 administration (P<0.001); the expression level of HIF-1α was also significantly reduced by 41±13% after three days of compound 1 administration (P<0.01). Compared with the compound 1 group, the expression level of HIF-1α in the compound 2 group was reduced by 53±11% (P<0.001). n=3, data are expressed as mean±SEM, and were analyzed by one-way ANOVA and Tukey's HSD test. *P<0.05, **P<0.01, ***P<0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0126] After 72 h of ischemia-reperfusion, the expression level of HIF-1α protein in brain tissue was detected by Western blot. The experimental and quantitative analysis results are shown in Figure 2. Figure 19As shown, compared with the sham operation group, the protein expression of HIF-1α in the brain tissue of mice in the MCAO model group was significantly increased by 50±20% (P<0.001). Compared with the MCAO model group, the protein expression of HIF-1α was significantly decreased by 53±10% after three days of administration of compound 2 (P<0.001). Compared with the MCAO model group, the protein expression of HIF-1α was decreased by 46±10% after three days of administration of compound 1 (P<0.01). Compared with the compound 1 group, the protein expression of HIF-1α in the compound 2 group was significantly decreased. β-actin was used as the internal reference protein in this experiment. n=3, data are expressed as mean±SEM, and the one-way ANOVA analysis method was used for processing and analysis, and Tukey's HSD test was performed. *P<0.05, **P<0.01, ***P<0.001; # P<0.05, ## P<0.01, ### P<0.001. *Indicates comparison with the SHAM group. # Indicates comparison with compound 2 group.

[0127] The results showed that compared with the sham operation group, the expression of HIF-1α protein in the model group was significantly increased; compared with the model group, the expression of HIF-1α protein in the compound 2 group and the compound 1 group was significantly decreased, and the decrease in the compound 2 group was greater than that in the compound 1 group. This study confirmed through immunofluorescence experiments that compound 2 and compound 1 can significantly reduce the expression of HIF-1α caused by ischemia-reperfusion injury. In addition, the expression of HIF-1α at the gene level was explored through RT-qPCR experiments. The results showed that compound 2 and compound 1 significantly reduced the upregulation of HIF-1α caused by ischemia-reperfusion injury. In summary, compound 2 and compound 1 may play a role in treating ischemia-reperfusion injury by regulating the expression of HIF-1α and triggering downstream pathways.

[0128] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof; in, X is O; R1 and R2 are each independently selected from the group consisting of: X2-PO(OH)2; X2 is 0; express It is characterized in that it is used to prepare a pharmaceutical composition for treating and / or alleviating ischemic stroke, and the pharmaceutical composition is used to improve and / or alleviate the inflammatory response caused by ischemic stroke.

2. The use according to claim 1, characterized in that The compound of formula I has the structure shown below:

3. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from the following group: alkali metal salts, alkaline earth metal salts, and ammonium salts.

4. The use according to claim 1, wherein The pharmaceutical composition is also used to inhibit the proliferation of astrocytes and microglial cells.

5. The use according to claim 1, characterized in that The pharmaceutical composition is also used to reduce the expression level of pro-inflammatory factors.

6. The use according to claim 1, wherein The pro-inflammatory factor is selected from the following group: TNF-α, IL-1β, IL-6, iNOS or COX2.

7. The use according to claim 1, characterized in that The pharmaceutical composition is also used to reduce the expression level of the oxygen homeostasis regulatory factor HIF-1α.

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