Application of GLX351322 in preparation of medicine for preventing and treating hemorrhagic stroke and medicine thereof

Abstract

According to the application of the GLX351322 in preparation of the medicine for preventing and treating the hemorrhagic stroke, the molecular formula of the GLX351322 is C21H25N3O5S, the CAS number of the GLX351322 is 835598-94-2, and the molecular weight of the GLX351322 is 431.51. The administration amount of the GLX351322 is 1-5 mg / kg / d, and the administration mode comprises oral administration, intravenous injection or intravenous drip. According to the application disclosed by the invention, the GLX351322 is injected into a mouse ICH model for treatment, so that the volume of cerebral hematoma of a mouse can be remarkably reduced, oxidative stress and inflammatory response can be inhibited, and ISR-eIF2alpha-ATF4-NOX4-ROS circulation can be blocked, and the GLX351322 is prompted to have a certain prevention and treatment effect on hemorrhagic cerebral apoplexy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and particularly relates to application of GLX351322 in preparation of a medicine for preventing and treating hemorrhagic stroke and the medicine. BACKGROUND

[0002] Stroke, also known as cerebral apoplexy or cerebral vascular accident, is an acute cerebrovascular disease. This disease is divided into ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral hemorrhage), and the hemorrhagic stroke accounts for 10%-15% of the mortality rate of stroke.

[0003] Cerebral hemorrhage is due to the rupture of cerebral blood vessels, leading to blood forming a hematoma to compress the brain tissue, and first causing primary brain injury. The hematoma formation and expansion cause the space-occupying effect, and further cause mechanical damage of the adjacent tissue and reduction of cerebral perfusion, and then trigger a series of physiological and pathological changes such as oxidative stress and inflammatory response, leading to cell death and tissue necrosis

see Kil, I.S., et al., Glycation-induced inactivation of NADP(+)-dependent isocitrate dehydrogenase: implications for diabetes and aging. Free Radic Biol Med, 2004. 37(11): p. 1765-78.

[0004] On the other hand, GLX351322 is a NADPH oxidase 4 (NOX4) inhibitor, and the molecular formula is: 21 H 25N3O5S, CAS No. 835598-94-2, molecular weight 431.51. GLX351322 inhibits hydrogen peroxide production in NOX4 overexpressed cells and can penetrate the blood-brain barrier

See Lin, Z., et al., NOX4 exacerbates Parkinson's disease pathology by promoting neuronal ferroptosis and neuroinflammation. Neural Regen Res, 2025. 20(7): p. 2038-2052.

See Tao, W., et al., miR-204-3p / Nox4 Mediates Memory Deficits in a Mouse Model of Alzheimer's Disease. Mol Ther, 2021. 29(1): p. 396-408.

See Lin, Z., et al., NOX4 exacerbates Parkinson's disease pathology by promoting neuronal ferroptosis and neuroinflammation. Neural Regen Res, 2025. 20(7): p. 2038-2052.

See Jiang, Y., et al., Furanodienone induces apoptosis via regulating the PRDX1 / MAPKs / p53 / caspases signaling axis through NOX4-derived mitochondrial ROS in colorectal cancer cells. Biochem Pharmacol, 2024. 227: p. 116456.

See Huang, Y.F., et al., Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1 / PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage. Redox Biol, 2023. 67: p. 102867. and see Liao, J., et al., Inhibition of NOX4 with GLX351322 alleviates acute ocular hypertension-induced retinal inflammation and injury by suppressing ROS mediated redox-sensitive factors activation. Biomed Pharmacother, 2023. 165: p. 115052.

[0005] At present, there is no relevant research and report on the prevention and treatment effect of GLX351322 on hemorrhagic stroke. For this reason, the inventors of the present application established a collagenase-induced mouse brain hemorrhage model (ICH) and studied the protective effect of GLX351322 on the in vivo brain hemorrhage model. SUMMARY

[0006] The present application provides the application of GLX351322 in the preparation of a drug for preventing and treating hemorrhagic stroke and the drug, and the purpose is to seek the application of GLX351322 as an effective ingredient in the preparation of a drug for preventing and treating hemorrhagic stroke and the drug.

[0007] To achieve the above purpose, the first technical solution adopted by the present application is: the application of GLX351322 in the preparation of a drug for preventing and treating hemorrhagic stroke, wherein the molecular formula of GLX351322 is C 21 H 25 N3O5S, the CAS number is 835598-94-2, the molecular weight is 431.51, and the chemical structural formula is shown as formula I: Formula I.

[0008] To achieve the above object, the second technical solution adopted by the present application is: a drug for preventing and treating hemorrhagic stroke, which is characterized by comprising GLX351322 and a pharmaceutically acceptable carrier, wherein the molecular formula of the GLX351322 is C 21 H 25 N3O5S, the CAS number is 835598-94-2, the molecular weight is 431.51, and the chemical structural formula is shown as formula I: Formula I.

[0009] The related content in the above first and second technical solutions is explained as follows: 1. In the above two technical solutions, the GLX351322 is an existing substance, which is an NADPH oxidase 4 (NOX4) inhibitor, inhibits hydrogen peroxide production in NOX4 overexpressing cells, and the IC 50 value is 5 μM, and can penetrate the blood-brain barrier. The molecular formula of GLX351322 is C 21 H 25 N3O5S, the CAS number is 835598-94-2, the molecular weight is 431.51, and the chemical structural formula is shown as formula I. The common name is GLX351322, the English name is GLX351322, and the Chinese name is CS-2871. According to existing research, the mechanism of action of GLX351322 is to inhibit the enzyme activity of NOX4, rather than directly reduce the expression level of NOX4 protein. The specific conclusions are as follows: (1) GLX351322 as a NOX4 activity inhibitor GLX351322 reduces the catalytic production of hydrogen peroxide (H2O2) by blocking the activity of NOX4 protein. In NOX4 overexpressing cell models, the IC50 value of the compound for inhibiting H2O2 production is 5 μM.

[0010] (2) Action mode unrelated to protein expression Existing literature does not mention the effect of GLX351322 on the expression amount of NOX4 protein, and all studies focus on the inhibition of enzyme activity or the generation of downstream oxidation products. For example: one is that GLX351322 does not reduce the NOX4 protein level in tetracycline-induced NOX4 overexpressing cells, but directly inhibits its function. Two is that the mechanism of action of other NOX4 inhibitors (such as GKT136901) is also described as selectively inhibiting enzyme activity rather than regulating protein expression.

[0011] In summary, GLX351322 mainly plays a role by inhibiting the enzyme activity of NOX4, and there is currently no evidence that it affects the expression of NOX4 protein, nor is there evidence that it prevents and treats hemorrhagic stroke. The application of GLX351322 in medicine is described in the background art of the present application.

[0012] 2. In the above two technical solutions, the GLX351322 is administered at a dose of 1-5 mg / kg / d (mg / kg / d represents milligrams per kilogram per day).

[0013] 3. In the above two technical solutions, the GLX351322 is administered orally, intravenously, or by intravenous infusion.

[0014] 4. In the above second solution, in addition to the GLX351322 being used alone as a drug for preventing and treating hemorrhagic stroke, GLX351322 can also be combined with at least one of nicotinamide riboside (NR), reduced nicotinamide adenine dinucleotide phosphate (NADPH), PERK kinase inhibitor GSK2606414, PERK kinase inhibitor GSK2656157, eIF2 phosphatase inhibitor Salubrinal, and integrated stress response inhibitor trans-ISRIB as a drug. Specifically, the following combination methods are included: (1) The GLX351322 is combined with nicotinamide riboside (NR), wherein the nicotinamide riboside (NR) has a CAS number of 23111-00-4, a molecular formula of C 11 H 15 ClN2O5, and a molecular weight of 290.7. The GLX351322 is administered at a dose of 1-5 mg / kg / d, and the administration method includes oral administration, intravenous injection, or intravenous infusion. The nicotinamide riboside (NR) is administered at a dose of 50-1200 mg / kg / d, and the administration method includes oral administration, intravenous injection, or intravenous infusion.

[0015] (2) The GLX351322 is combined with reduced nicotinamide adenine dinucleotide phosphate (NADPH), wherein the reduced nicotinamide adenine dinucleotide phosphate (NADPH) has a CAS number of 53-59-8, a molecular formula of C 21 H 28 N7O 17 P3, and a molecular weight of 744.42. The GLX351322 is administered at a dose of 1-5 mg / kg / d, and the administration method includes oral administration, intravenous injection, or intravenous infusion. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is administered at a dose of 0.1-10 mg / kg / d, and the administration method includes oral administration, intravenous injection, or intravenous infusion.

[0016] (3) the GLX351322 is combined with nicotinamide riboside (NR) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), wherein the CAS number of nicotinamide riboside (NR) is 23111-00-4, the molecular formula is C 11 H 15 ClN2O5, and the molecular weight is 290.7. The CAS number of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 53-59-8, the molecular formula is C 21 H 28 N7O 17 P3, and the molecular weight is 744.42. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The nicotinamide riboside (NR) is administered at a dosage of 50-1200 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

[0017] (4) the GLX351322 is combined with PERK kinase inhibitor GSK2606414, wherein the CAS number of PERK kinase inhibitor GSK2606414 is 1337531-36-8, the molecular formula is C 24 H 20 F3N5O, and the molecular weight is 451.44. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2606414 is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

[0018] (5) the GLX351322 is combined with PERK kinase inhibitor GSK2656157, wherein the CAS number of PERK kinase inhibitor GSK2656157 is 1337532-29-2, the molecular formula is C 23 H 21 FN6O, and the molecular weight is 416.5. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2656157 is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

[0019] (6) the GLX351322 is combined with the integrated stress response inhibitor trans-ISRIB, wherein the CAS number of the integrated stress response inhibitor trans-ISRIB is 1597403-47-8, and the molecular formula is C 22 H 24 Cl2N2O4, and the molecular weight is 451.34. The GLX351322 is administered at a dose of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The integrated stress response inhibitor trans-ISRIB is administered at a dose of 0.1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

[0020] (7) the GLX351322 is combined with the PERK kinase inhibitors GSK2606414 and GSK2656157, wherein the CAS number of the PERK kinase inhibitor GSK2606414 is 1337531-36-8, and the molecular formula is C 24 H 20 F3N5O, and the molecular weight is 451.44. The CAS number of the PERK kinase inhibitor GSK2656157 is 1337532-29-2, and the molecular formula is C 23 H 21 FN6O, and the molecular weight is 416.5. The GLX351322 is administered at a dose of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2606414 is administered at a dose of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2656157 is administered at a dose of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

[0021] (8) the GLX351322 is combined with the PERK kinase inhibitors GSK2606414 and GSK2656157, and the integrated stress response inhibitor trans-ISRIB, wherein the CAS number of the PERK kinase inhibitor GSK2606414 is 1337531-36-8, and the molecular formula is C 24 H 20 F3N5O, and the molecular weight is 451.44. The CAS number of the PERK kinase inhibitor GSK2656157 is 1337532-29-2, and the molecular formula is C 23 H 21 FN6O, and the molecular weight is 416.5. The CAS number of the integrated stress response inhibitor trans-ISRIB is 1597403-47-8, and the molecular formula is C 22 H 24Cl2N2O4 has a molecular weight of 451.34. The dosage of GLX351322 is 1-5 mg / kg / day, administered orally, intravenously, or by intravenous infusion. The dosage of the PERK kinase inhibitor GSK2606414 is 0.1-10 mg / kg / day, administered orally, intravenously, or by intravenous infusion. The dosage of the PERK kinase inhibitor GSK2656157 is 0.1-10 mg / kg / day, administered orally, intravenously, or by intravenous infusion. The dosage of the integrated stress response inhibitor trans-ISRIB is 1-5 mg / kg / day, administered orally, intravenously, or by intravenous infusion.

[0022] (9) The combination of GLX351322 and the eIF2 phosphatase inhibitor Salubrinal, wherein the CAS number of the eIF2 phosphatase inhibitor Salubrinal is 405060-95-9, and its molecular formula is C 21 H 17 Cl3N4OS has a molecular weight of 479.81. The dosage of GLX351322 is 1-5 mg / kg / day, administered orally, intravenously, or by intravenous infusion. The dosage of the eIF2 phosphatase inhibitor Salubrinal is 0.1-10 mg / kg / day, administered orally, intravenously, or by intravenous infusion.

[0023] Mechanism of action of the present invention: 1. Mechanisms of cell death caused by cerebral hemorrhage Cerebral hemorrhage is caused by the rupture of a blood vessel in the brain, which leads to the formation of a hematoma that compresses brain tissue. This initially causes primary brain injury. The formation and expansion of the hematoma result in a space-occupying effect, which in turn causes mechanical damage to adjacent tissues and reduced brain perfusion. This then triggers a series of physiological and pathological changes, such as oxidative stress and inflammatory responses, leading to cell death and tissue necrosis.

[0024] 2. Mechanism of action of GLX351322 in preventing and treating cerebral hemorrhage The inventors have shown that oxidative stress is one of the main pathological mechanisms of hemorrhagic brain injury: when blood pressure presses on brain tissue, red blood cell lysate iron ions, hemoglobin, thrombin, etc. can induce oxidative stress, including when inflammatory cells are stimulated to release a large amount of ROS (Reactive Oxygen Species, ROS), when ROS is produced too much and exceeds the cell's own antioxidant capacity, it will cause cell tissue damage by directly oxidizing cell proteins, lipids and DNA or participating in cell death signaling pathways

See Yang, H., et al., Benzene, 1,2,4-Trimethoxy-5-(2-Methyl-1-Propen-1-yl), a New Neuroprotective Agent, Treats Intracerebral Hemorrhage by Inhibiting Apoptosis, Inflammation, and Oxidative Stress. Neuroscience, 2022. 503: p. 69-82.

See Vermot, A., et al., NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology. Antioxidants (Basel), 2021. 10(6).

See Miller, A.A., et al., NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. Circ Res, 2005. 97(10): p. 1055-62.

see Bedard, K. and K.H. Krause, The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 2007. 87(1): p. 245-313.

see Magid-Bernstein, J., et al., Cerebral Hemorrhage: Pathophysiology, Treatment, and Future Directions. Circ Res, 2022. 130(8): p.1204-1229.

see Park, M.W., et al., NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases. Redox Biol, 2021. 41: p. 101947.

See Kleinschnitz, C., et al., Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol, 2010. 8(9).

[0025] In addition, in this new indication of hemorrhagic stroke, the protective mechanism of GLX351322 not only directly targets NOX4 enzyme activity to inhibit oxidative stress and inflammation, but also relies on the synergistic regulation of the integrated stress response (ISR)-eIF2a-ATF4-NOX4-ROS pathway, which is a completely new mechanism that has not been involved in known studies. After the occurrence of hemorrhagic stroke, oxidative stress and inflammation caused by hematoma compression and blood component decomposition are effective inducing factors for ISR. Activation of ISR can promote eIF2a phosphorylation and induce ATF4 expression

See Lu HJ, et al. Mammalian integrated stress responses in stressed organelles and their functions. Acta Pharmacol Sin. 2024; 45(6): 1095-1114.

see Petry A, et al. CrossTalk Between p22phox and ATF4 in the Endothelial Unfolded Protein Response. Antioxid Redox Signal. 2019;30(1):40-55.

[0026] 3. The essential difference between the induction causes of hemorrhagic stroke and known indications and the mechanism of action of GLX351322 The mechanism of action of GLX351322 in known indications is centered on "directly targeting NOX4 enzyme activity to inhibit oxidative stress and inflammation". For example: in Parkinson's disease, the core cause is the aggregation of alpha-synuclein and the degeneration of dopaminergic neurons, GLX351322 directly inhibits NOX4-mediated ferroptosis of dopaminergic neurons, reduces neuronal damage in the substantia nigra of the midbrain, and the core is the toxic effect of oxidative stress downstream of NOX4 on specific neurons; in Alzheimer's disease, amyloid deposition and tau protein hyperphosphorylation trigger neuroinflammation and synaptic loss, GLX351322 reduces amyloid deposition-related oxidative stress by reducing hippocampal NOX4 enzyme activity to protect synaptic function; in osteoarthritis and retinal inflammation, the main cause is mechanical damage or immune complex deposition to activate the TLR4 / NF-κB inflammatory pathway, GLX351322 reduces local tissue ROS production and inflammatory factor release by inhibiting NOX4 enzyme activity; in cancer (such as colon cancer), the main effect of GLX351322 is to inhibit proliferation by reducing oxidative stress in the tumor microenvironment through inhibition of NOX4. Therefore, in known indications, GLX35122 is limited to a single role of directly inhibiting NOX4 enzyme activity, without involving the influence of GLX351322 on the transcriptional regulation and protein expression of NOX4, and even less involving the whole new mechanism of GLX351322 acting on the ISR-eIF2α-ATF4-NOX4-ROS-eIF2α cycle cascade effect. In the hemorrhagic stroke of the present application, GLX351322 not only directly inhibits NOX4 enzyme activity, but also blocks the ISR-eIF2α-ATF4-NOX4-ROS cycle, blocks the abnormal activation of NOX4 mediated by ATF4, and reduces the protein expression of ATF4 and NOX4, forming a dual action mode of "downstream NOX4 enzyme activity inhibition + upstream ATF4 regulation NOX4 transcription blockage" to reduce brain hemorrhage damage. Therefore, this mechanism breaks through the technical inspiration that cannot be obtained from known uses.

[0027] In addition, the pathogenic cause and pathological changes of hemorrhagic stroke are hematoma formation caused by cerebral vascular rupture, not neuronal degeneration, protein deposition or chronic inflammation, which are completely different from the pathogenic cause and pathological changes of known indications.

[0028] In summary, hemorrhagic stroke and known indications differ significantly in pathogenic cause and pathological change; and in hemorrhagic stroke and known indications, there are essential differences in the regulatory pathways of the mechanism of action of GLX351322, which fully prove the creativity of the new indication of the present application and do not belong to the simple extension based on known mechanisms.

[0029] In addition, the drugs for resisting oxidative stress also include nicotinamide riboside (NR) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). Nicotinamide riboside (NR) can increase the levels of NAD and NADPH, and both nicotinamide riboside (NR) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) can inhibit oxidative stress. Therefore, GLX351322 can be combined with nicotinamide riboside (NR) or / and reduced nicotinamide adenine dinucleotide phosphate (NADPH) and has a synergistic effect.

[0030] In addition, the kinase inhibitors in the ISR signaling pathway can also include PERK kinase inhibitors GSK2606414 and GSK2656157, the eIF2 phosphatase inhibitor Salubrinal, and the integrated stress response inhibitor trans-ISRIB (ISR inhibitor ISRIB). Since the PERK kinase inhibitor GSK2606414 has an inhibitory effect on the activation of PERK in cells, the PERK kinase inhibitor GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity, the eIF2 phosphatase inhibitor Salubrinal is a selective eIF2α dephosphorylation inhibitor, can inhibit ER stress-mediated apoptosis, and the integrated stress response inhibitor trans-ISRIB can enhance the activity of eIF2B to inhibit ISR. Therefore, GLX351322 can be combined with the PERK kinase inhibitor GSK2606414, the PERK kinase inhibitor GSK2656157, the eIF2 phosphatase inhibitor Salubrinal, and the integrated stress response inhibitor trans-ISRIB and has a synergistic effect.

[0031] The application of GLX351322 in the treatment of hemorrhagic stroke has the following advantages: 1. GLX351322 can significantly reduce the volume of hemorrhage by 20-30% in animal experiments, can resist oxidative stress, inflammatory response, and block the ISR-eIF2α-ATF4-NOX4-ROS cycle, and has a significant neuroprotective effect.

[0032] 2. GLX351322 is relatively safe as an effective active ingredient in the prevention and treatment of hemorrhagic stroke. The dose of GLX351322 used in the application is small, and it is presumed to have less toxicity and be relatively safe. In addition, relevant literature has shown that when rats are given a dose of 10 mg / kg by gavage, pharmacokinetic determination shows that GLX351322 can quickly enter the bloodstream with a half-life of 3 hours. GLX351322 has almost no toxicity when given a dose of 5 mg / kg intraperitoneally for 2 weeks in mice. Therefore, it can be considered that GLX351322 has less toxicity and is relatively safe in the treatment of hemorrhagic stroke.

[0033] 3. If GLX351322 has neuroprotective effect due to inhibiting oxidative stress and blocking ISR-eIF2α-ATF4-NOX4-ROS cycle, considering the harmful effects of oxidative stress and ISR-eIF2α-ATF4-NOX4-ROS cycle in some neurodegenerative diseases, GLX351322 can also have therapeutic effect on other neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, etc. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 1 is a diagram of GLX51322 reducing hemorrhagic injury after ICH in mice, wherein, Figure 1 -a indicates the schematic diagram of GLX351322 administration process; Figure 1 -b indicates the figure of neurological deficit score; Figure 1 -c indicates the figure of forelimb placing test; Figure 1 -d indicates the figure of mouse brain section; Figure 1 -e indicates the figure of quantitative analysis of hematoma volume.

[0035] Figure 2 Figure 2 is a diagram of GLX351322 in the time window of hemorrhagic injury in ICH model of mice, wherein, Figure 2 -a indicates the schematic diagram of GLX351322 administration process; Figure 2 -b indicates the figure of neurological deficit score; Figure 2 -c indicates the figure of forelimb placing test; Figure 2 -d indicates the figure of mouse brain section; Figure 2 -e indicates the figure of quantitative analysis of hematoma volume.

[0036] Figure 3 Figure 3 is a diagram of the effect of GLX351322 on long-term survival and recovery of neurological function after cerebral hemorrhage in mice, wherein, Figure 3 -a indicates the schematic diagram of GLX351322 administration process; Figure 3 -b indicates the figure of balance beam test; Figure 3 -c indicates the figure of rotarod test.

[0037] Figure 4 Figure 4 is a diagram of GLX351322 reducing hemorrhagic injury by improving antioxidant stress capacity, wherein, Figure 4 -a indicates the schematic diagram of GLX351322 administration process; Figure 4 -b indicates the figure of NOX4 enzyme activity detection; Figure 4 -c indicates the figure of NADPH content detection; Figure 4- d represents NADP + / NADPH detection graph; Figure 4 - e represents hydrogen peroxide detection graph.

[0038] Figure 5 The GLX351322 of the present application inhibits the expression of proteins related to oxidative stress, inflammatory response and ISR-eIF2α-ATF4-NOX4-ROS cycle in cerebral hemorrhage, wherein, Figure 5 - a to Figure 5 - d represents the effect of GLX351322 on 4-HNE, NOX4 and ATF4 graph; Figure 5 - e to Figure 5 - h represents the effect of GLX351322 on NLRP3, C-Caspase-1 / Caspase-1 and CHOP graph. DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with the accompanying drawings and examples: Example 1:

[0040] The application of GLX351322 in the preparation of a drug for preventing and treating hemorrhagic stroke, wherein the molecular formula of GLX351322 is C 21 H 25 N3O5S, CAS No. 835598-94-2, molecular weight 431.51, and the chemical structural formula is shown as formula I: Formula I.

[0041] The dosage of GLX351322 is 1-5 mg / kg / d (mg / kg / d represents milligrams per kilogram per day), and the administration method includes oral administration, intravenous injection or intravenous infusion. The specific method of using GLX351322 in hemorrhagic stroke: oral administration of 1-5 mg / kg / d, or intravenous injection or intravenous infusion of 1-5 mg / kg / d. Example 2:

[0042] A drug for preventing and treating hemorrhagic stroke, comprising GLX351322 and a pharmaceutically acceptable carrier, wherein the molecular formula of GLX351322 is C 21 H 25 N3O5S, CAS No. 835598-94-2, molecular weight 431.51, and the chemical structural formula is shown as formula I: Formula I.

[0043] The amount of GLX351322 administered is 1-5 mg / kg / d (mg / kg / d means milligrams per kilogram per day), and the administration method includes oral administration, intravenous injection or intravenous infusion. The specific method of using GLX351322 in hemorrhagic stroke: oral administration of 1-5 mg / kg / d, or intravenous injection or intravenous infusion of 1-5 mg / kg / d.

[0044] The following provides the relevant conditions of animal experiments and statistical analysis of data: 1. Experimental method (1) Establishment of collagenase-induced mouse brain hemorrhage model: C57 mice (C57BL / 6, inbred strain of experimental mice), male, body weight 21-23 g, clean grade, hereinafter referred to as "mice". The mice were fixed on the brain stereotactic frame to ensure that the mouse head was firmly fixed and horizontal. A small opening was cut on the head of the mouse to expose the skull, and the fontanel was clearly exposed by wiping with hydrogen peroxide. 0.6 μl of collagenase (0.03 U) was taken up with a micro-injection needle. (It means that in a 0.6 microliter volume of collagenase solution, there are 0.03 units of enzyme activity). With the anterior fontanel as the zero point, the micro-injection needle was slowly lowered to a depth of 3.5 mm in the striatum at coordinates x =−1.8 mm, y = 0.8 mm, and collagenase was injected at a rate of 0.1 μl / min. After injection, the injection needle was slowly withdrawn, the scalp was sutured, and the mouse was placed in a 37°C incubator. After the mouse woke up, it was returned to the cage and could freely obtain water and feed. The mice in the sham operation group were given the same volume of 0.9% normal saline, and the other operations were the same.

[0045] In the dose-effect relationship experiment of GLX351322 ( Figure 1 ), the mice were randomly divided into 5 groups: ICH group, ICH+GLX351322×0.625mg / kg, ICH+GLX351322×1.25mg / kg, ICH+GLX351322×2.5mg / kg and ICH+GLX351322×5mg / kg, GLX351322 was administered immediately after ICH modeling, 8 mice per group. GLX351322 was purchased from Suzhou Smof Bioscience Co., Ltd., MCE.

[0046] In the time window experiment of GLX351322 ( Figure 2 ), the mice were randomly divided into 5 groups: ICH group, ICH+GLX351322×5mg / kg 0 h, 1 h, 3 h, 6 h group, GLX351322 was given intraperitoneally at a dose of 1.25 mg / kg at 0 h, 1 h, 3 h and 6 h after ICH operation, 7 mice per group.

[0047] In the long-term experiment of GLX351322 ( Figure 3 ), mice were randomly divided into 4 groups: Sham group, ICH group, Sham+GLX351322 group, ICH+GLX351322 group, GLX351322 dose was 5 mg / kg, continuous administration for one week. Then reduce the administration frequency, once every 3 days, 10 mice in each group.

[0048] In the mechanism experiment of GLX351322 ( Figure 4 , Figure 5 ), mice were randomly divided into 3 groups: Sham group, ICH group, ICH+GLX351322 group. GLX351322 dose was 5 mg / kg at 0 h, 24 h and 48 h after ICH operation, 6-7 mice in each group for NOX4 enzyme activity, NADPH and H2O2 detection; GLX351322 dose was 5 mg / kg at 0 h after ICH operation, 6-7 mice in each group for Western Blot experiment.

[0049] (2) Neurological deficit score: The 24-point neurological deficit score table was used to evaluate the neurological function of mice in each group after ICH. The score table was divided into 5 parts, each part was scored from 0 to 4. 0 represents normal, no neurological impairment, and 4 represents the most severe neurological impairment.

[0050] Table 1: 24-point neurological deficit score table

[0051] (3) Forelimb placing test: Hold the mouse's neck skin and make the body limbs suspended, touch the edge of the table with the mouse's whisker brush, and observe the reaction degree of the whisker ipsilateral forelimb. Normal mice can quickly lift the forelimb to touch the table, while brain injury mice have slow reaction. Each side of the mouse was tested 10 times, with an interval of 30 s, and the number of successful forelimb placement on the edge of the table / number of experiments (10) represented the success rate.

[0052] (4) Eye blood collection: After isoflurane anesthesia, blood was collected and stored at 4°C for at least 1 h, then centrifuged at 3000 rpm for 15 min at 4°C, and the supernatant serum was stored at -80°C.

[0053] (5) Brain hematoma volume determination: After the mice were sacrificed, the whole brain tissue was taken out, the olfactory bulb and cerebellum were removed, and only the brain tissue was left. The brain tissue was cut into 7 uniform pieces with a thickness of about 1 mm in a coronal section manner, the bleeding area was red, and the non-bleeding area was white, and was placed in paraformaldehyde for fixation. The brain slices were scanned into images using a multifunctional scanner, and the brain slice hematoma volume was counted using Image J.

[0054] (6) Balance beam experiment: The balance beam walking experiment was used to evaluate the motor integration and balance coordination ability of mice. The balance beam used was 44 cm high, 115 cm long and 2 cm wide. Before the experiment, the mice needed to receive pre-training and cross the balance beam twice independently. During the formal test, the crawling time of the mice from one end of the balance beam to the other end was recorded, the residence time was not counted, and the fall was paused and continued from the falling place. Each mouse was tested three times, and the average value was taken, and each test was followed by a 1 h rest.

[0055] (7) Rotarod experiment: The rotarod experiment requires mice to maintain balance on a uniformly rotating rod. This requires proprioception, position sense and fine motor skills, so it is an important method for evaluating the motor coordination of mice. The mice were trained one day in advance, and the instrument was set to rotate at a constant speed of 4 r / min for 60 s to ensure that the mice could maintain balance independently. During the formal test, the instrument was initially set to 4 r / min, and then uniformly accelerated to 40 r / min within 300 s. The movement time and fall time of the mice on the rotating rod were recorded. Each mouse was tested three times, and the results were averaged, with a 30 min rest after each test.

[0056] (8) NOX4 enzyme activity detection: The NADPH oxidase (NAO) kit was purchased from Suzhou Gersite Biological Technology Co., Ltd. (G0138W), and the enzyme label instrument was preheated for more than 30 min, with a temperature setting of 37°C. The protein content was determined by BCA method, and the sample and reagent were added according to the instruction steps, and the absorbance at 450 nm was measured.

[0057] The enzyme activity was calculated according to the following formula: NAO (△OD 450 / min / mg prot) = ΔA÷(V1×Cpr)÷0.01÷T = 250×ΔA÷Cpr, ΔA = A measured- A control, V1---sample volume, 0.02 mL, Cpr---sample protein concentration, mg / mL, T---reaction time, 20 min.

[0058] (9) NADP + / NADPH detection: NADP +NADPH assay kit (WST-8 method) was purchased from Suzhou Smarbiotech Co., Ltd, Beyotime (S0179). Standard preparation: 1 mM standard was diluted with NADP + NADPH extract was diluted to 0, 0.25, 0.5, 1, 2, 4, 6, 8 μM to make standard curve.

[0059] Sample preparation: serum samples and NADPH extract were mixed at a ratio of 2:3, and the mixed sample was used to determine the total amount. 200 μL of the mixed sample was taken into another EP tube and heated at 60°C for 30 min, and the sample was used to determine the NADPH single amount. The protein content was determined in advance by BCA method.

[0060] Sample determination: according to the instruction steps, add sample and reagent, and determine the absorbance at 450 nm.

[0061] (10) Hydrogen peroxide detection: Hydrogen peroxide detection kit was purchased from Suzhou Smarbiotech Co., Ltd, Beyotime (S0038).

[0062] Standard preparation: 1 M H2O2 was diluted with double distilled water to 1, 3, 10, 30, 100 μM to make standard curve.

[0063] Sample preparation: serum samples and phosphate buffer were mixed at a ratio of 1:9 in an EP tube.

[0064] Sample determination: according to the instruction steps, add sample and reagent, and determine the absorbance at 240 nm.

[0065] (11) Western Blot (protein blotting or immunoblotting): The tissue sample was placed in a 1.5 mL EP tube, and the lysis solution was added and incubated on ice for 15 min. After ultrasonic crushing of the cells, centrifugation was performed at 12500 rpm for 15 min, and the supernatant was placed on ice. The protein concentration was measured by BCA kit, and the sample was adjusted for SDS-PAGE electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), membrane transfer, 5% skim milk blocking, primary antibody overnight, secondary antibody, and double-color infrared imager scanning membrane to detect the expression level changes of NOX4 and other related proteins. β-actin (β-actin) was used as an internal reference.

[0066] (12) Data statistics and analysis: The experimental data were statistically analyzed and the charts were made by using GraphPad Prism 8 software, all the experimental data were expressed as mean ± standard deviation, that is, the data were expressed as mean ± SD (Mean ± SD), statistical analysis was performed by using One-way-ANOVA (single factor analysis of variance) Neuman Keuls (Neuman-Keuls test) and 2-way-ANOVA (two-factor analysis of variance) paired t test, P<0.05 indicates that the difference has statistical significance.

[0067] 2. Experimental results (1) GLX351322 reduces hemorrhagic injury after ICH in mice: In the ICH mouse model, GLX351322 significantly reduces the neurological deficit score, increases the success rate of forelimb placement and reduces the hematoma volume in mice after ICH at a dose of 2.5 mg / kg and 5 mg / kg, and the hematoma volume is reduced by about 20%-30% (P<0.05, P<0.01, P<0.001, P<0.0001). Figure 1 b- Figure 1 e). It shows that GLX351322 has a certain preventive effect on hemorrhagic stroke.

[0068] Figure 1 To reduce the hemorrhagic injury of GLX51322 in mice after ICH, wherein, Figure 1 -a represents the schematic diagram of GLX351322 administration process. From Figure 1 -a, it can be seen that GLX351322 is administered immediately after the ICH model, and the neurological deficit score and forelimb placement experiment are performed at 24 h, and the brain hematoma volume is detected. Figure 1 -b represents the neurological deficit score chart. From Figure 1 -b, it can be seen that GLX351322 1.25 mg / kg, 2.5 mg / kg and 5 mg / kg significantly reduce the neurological deficit score in mice after ICH. Figure 1 -c represents the forelimb placement experiment chart. From Figure 1 -c, it can be seen that GLX351322 2.5 mg / kg and 5 mg / kg significantly increase the success rate of forelimb placement in mice after ICH. Figure 1 -d represents the mouse brain section chart, and the hematoma is reddish brown on the brain section. Figure 1 -e represents the quantitative analysis chart of brain hematoma volume. From Figure 1 -d and Figure 1 -e, it can be seen that GLX351322 1.25 mg / kg, 2.5 mg / kg and 5 mg / kg significantly reduce the brain hematoma volume in mice after ICH, and the hematoma volume is reduced by about 20%-30%. Mean ± SD, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0069] (2) Time window study of GLX351322 in mouse ICH model: In an ICH mouse model, GLX351322 was administered at 0 h, 1 h, 3 h, and 6 h post-ICH. Administration at 0 h and 1 h significantly reduced neurological deficit scores after ICH, administration at 0 h significantly improved forelimb placement ability, and administration at 0 h, 1 h, and 3 h significantly reduced hematoma volume 24 h post-ICH by approximately 20%-30%. Figure 2 -b to Figure 2 -e). This indicates that the therapeutic time window of GLX351322 for hemorrhagic stroke can be extended to 3 hours after hemorrhage.

[0070] Figure 2 This is a time window study of GLX351322 in a mouse ICH model of hemorrhage injury, where... Figure 2 -a represents a schematic diagram of the GLX351322 drug delivery process. From Figure 2 -a shows that GLX351322 was administered to mice at 0 h, 1 h, 3 h or 6 h after ICH, and neurological deficit scores and forelimb placement experiments were performed 24 h later to detect cerebral hematoma volume. Figure 2 -b indicates a neurological deficit rating scale. From Figure 2 -b shows that GLX351322 significantly reduced neurological deficit scores when administered to mice 0 h or 1 h after ICH. Figure 2 -c indicates an experimental diagram of forelimb placement. From Figure 2 -c shows that GLX351322 significantly improved the success rate of forelimb placement when administered to mice 0 h after ICH. Figure 2 -d indicates a slice of mouse brain, where hematomas appear reddish-brown on the brain slice. Figure 2 -e indicates a quantitative analysis chart of cerebral hematoma volume. From Figure 2 -d and Figure 2 -e showed that GLX351322 significantly reduced cerebral hematoma volume when administered to mice at 0 h, 1 h, or 3 h after ICH. Mean ± SD, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0071] (3) GLX351322 improves long-term neurological function recovery in mice after cerebral hemorrhage: In an ICH mouse model, mice were injected with 5 mg / kg GLX351322 for one week post-surgery. Balance beam and rotarod tests were performed on days 7, 14, 21, and 28 post-surgery. Figure 3 -b toFigure 3 -c), to detect the effect of GLX351322 on neurological function recovery. The data showed that, compared with the control group, the time of ICH mice passing through the balance beam increased significantly, and the time of ICH mice staying on the rotarod decreased significantly at 7 days and 14 days after ICH modeling, while compared with the ICH group, the time of GLX351322 treatment group passing through the balance beam was shortened, and the time of GLX351322 treatment group staying on the rotarod increased significantly. The above results suggest that the limb function of mice after ICH modeling appears motor dysfunction, and GLX351322 can improve the long-term coordination and functional recovery of ICH model mice.

[0072] Figure 3 Figure showing the effect of GLX351322 on long-term survival and neurological function recovery of mice after intracerebral hemorrhage, wherein, Figure 3 -a shows the schematic diagram of GLX351322 administration process. From Figure 3 -a, it can be seen that the mice were subjected to ICH modeling, and 5 mg / kg GLX351322 was continuously injected after the operation. The balance beam test and the rotarod test were performed on the mice at 7 days, 14 days, 21 days and 28 days after ICH modeling. Figure 3 -b shows the balance beam test diagram. From Figure 3 -b, it can be seen that the time of GLX351322 administration mice passing through the balance beam significantly decreased at 7 days and 14 days after ICH operation, and GLX351322 significantly improved the coordination ability of mice. Figure 3 -c shows the rotarod test diagram. From Figure 3 -c, it can be seen that the time of GLX351322 administration mice staying on the rotarod significantly increased at 7 days, 14 days and 21 days after ICH operation, and GLX351322 had a trend to improve the motor ability of mice. Mean ± SD, n=10, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0073] (4) GLX351322 reduces oxidative stress in brain hemorrhage injury: On the ICH mouse model, 5 mg / kg NOX4 inhibitor GLX351322 was given at 0 h, 24 h, 48 h after operation, and the mice were sacrificed at 72 h to take blood and striatum, to detect NOX4 enzyme activity, NADPH content, NADP + / NADPH level and H2O2 content, to evaluate whether inhibition of NOX4 can improve the antioxidant stress ability to play a protective role. The data showed that the NOX4 enzyme activity of mice significantly increased after ICH modeling, and the reduced NADPH decreased, while the oxidized NADP +and H2O2 levels, indicating that the level of oxidative stress was enhanced after ICH in mice. GLX351322 could significantly inhibit the NOX4 enzyme activity after ICH modeling in mice, and increase the reduced NADPH, while reducing the oxidized NADP + and H2O2 levels Figure 4 -b to Figure 4 -e). It is suggested that GLX351322 can reduce oxidative stress in ICH and play a protective role.

[0074] Figure 4 The GLX351322 of the present application reduces hemorrhagic injury by improving the ability to resist oxidative stress, as shown in the figure, wherein, Figure 4 -a represents the schematic diagram of GLX351322 administration process. From Figure 4 -a, it can be seen that the ICH model of mice was established, and 5 mg / kg of GLX351322 was continuously injected at 0 h, 24 h and 48 h after the operation, and the NOX4 enzyme activity, NADP + / NADPH levels and H2O2 content were detected at 72 h. Figure 4 -b represents the detection diagram of NOX4 enzyme activity. From Figure 4 -b, it can be seen that GLX351322 significantly inhibits the NOX4 enzyme activity after ICH modeling in mice. Figure 4 -c represents the detection diagram of NADPH content. From Figure 4 -c, it can be seen that GLX351322 significantly increases the content level of reduced NADPH after ICH modeling in mice. Figure 4 -d represents the detection diagram of NADP + / NADPH. From Figure 4 -d, it can be seen that GLX351322 significantly reduces the NADP + / NADPH level after ICH modeling in mice. Figure 4 -e represents the detection diagram of hydrogen peroxide. From Figure 5 -e, it can be seen that GLX351322 significantly reduces the H2O2 content level after ICH modeling in mice. Mean ± SD, n=6-7, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0075] (5) GLX351322 reduces oxidative stress, inflammatory response and ISR-eIF2α-ATF4-NOX4-ROS cycle related protein expression in ICH: In the ICH mouse model, 5 mg / kg NOX4 inhibitor GLX351322 was administered immediately after surgery, and 24 h later, the protein expression levels of 4-HNE, NOX4, ATF4, NLRP3, C-Caspase-1 / Caspase-1 and CHOP in the striatum were detected by Western Blot. The results showed that GLX351322 can significantly reduce the expression of 4-HNE, NOX4, ATF4, NLRP3, C-Caspase-1 and CHOP 24 h after intracerebral hemorrhage Figure 5 -a to Figure 5 -h), suggesting that GLX351322 can inhibit oxidative stress, inflammatory response and pyroptosis during intracerebral hemorrhage.

[0076] Figure 5 For the GLX351322 related protein expression diagram of the present application in inhibiting oxidative stress, inflammatory response and ISR-eIF2α-ATF4-NOX4-ROS cycle during intracerebral hemorrhage, wherein, Figure 5 -a represents the Western blot representative diagram of GLX351322 on 4-HNE, NOX4 and ATF4. From Figure 5 -b, it can be seen that GLX351322 significantly reduces the expression of 4-HNE after ICH modeling in mice. From Figure 5 -c, it can be seen that GLX351322 significantly reduces the expression of NOX4 after ICH modeling in mice. From Figure 5 -d, it can be seen that GLX351322 significantly reduces the expression of ATF4 after ICH modeling in mice. Figure 5 -e represents the Western blot representative diagram of GLX351322 on NLRP3, C-Caspase-1 / Caspase-1 and CHOP. From Figure 5 -f, it can be seen that GLX351322 significantly reduces the expression of NLRP3 after ICH modeling in mice. From Figure 5 -g, it can be seen that GLX351322 significantly reduces the expression of C-Caspase-1 after ICH modeling in mice. From ​ -h, it can be seen that GLX351322 significantly reduces the expression of CHOP after ICH modeling in mice. Mean ± SD, n=6-7, * P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001.

[0077] Conclusion: Injection of GLX351322 in the ICH model of mice can significantly reduce the brain hematoma volume, inhibit oxidative stress, inflammatory response and block ISR-eIF2α-ATF4-NOX4-ROS cycle, suggesting that GLX351322 has a certain preventive effect on hemorrhagic stroke.

[0078] For the above embodiments, the variations that the present application can produce are described as follows: The above embodiments are to use GLX351322 alone as the pharmaceutically active ingredient for preventing and treating hemorrhagic stroke. In addition to this, GLX351322 can also be combined with at least one of nicotinamide riboside (NR), reduced nicotinamide adenine dinucleotide phosphate (NADPH), PERK kinase inhibitor GSK2606414, PERK kinase inhibitor GSK2656157, eIF2 phosphatase inhibitor Salubrinal, and integrated stress response inhibitor trans-ISRIB as the pharmaceutically active ingredient. Specifically, the following combination modes are included (but the present application is not limited thereto): 1. The GLX351322 is combined with nicotinamide riboside (NR), wherein the CAS number of nicotinamide riboside (NR) is 23111-00-4, the molecular formula is C 11 H 15 IN2O5, and the molecular weight is 290.7. The administration amount of GLX351322 is 1-5 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion. The administration amount of nicotinamide riboside (NR) is 50-1200 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion. Since nicotinamide riboside (NR) can increase the level of NADPH and relieve oxidative stress, GLX351322 can be combined with nicotinamide riboside (NR) and has a synergistic effect. This is understood and known by those skilled in the art.

[0079] 2. The GLX351322 is combined with reduced nicotinamide adenine dinucleotide phosphate (NADPH), wherein the CAS number of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 53-59-8, the molecular formula is C 21 H 28 N7O 17 P3, and the molecular weight is 744.42. The administration amount of GLX351322 is 1-5 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion. The administration amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 0.1-10 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion. Since reduced nicotinamide adenine dinucleotide phosphate (NADPH) can relieve the level of oxidative stress, GLX351322 can be combined with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and has a synergistic effect. This is understood and known by those skilled in the art.

[0080] 3. The GLX351322 is combined with nicotinamide riboside (NR) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), wherein the CAS number of nicotinamide riboside (NR) is 23111-00-4, the molecular formula is C 11 H 15 ClN2O5, the molecular weight is 290.7. The CAS number of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 53-59-8, the molecular formula is C 21 H 28 N7O 17 P3, the molecular weight is 744.42. The administration amount of the GLX351322 is 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection or intravenous drip. The administration amount of the nicotinamide riboside (NR) is 50-1200 mg / kg / d, and the administration methods include oral administration, intravenous injection or intravenous drip. The administration amount of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection or intravenous drip. Since nicotinamide riboside (NR) can increase the level of NADPH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) can alleviate the level of oxidative stress, the GLX351322 can be combined with nicotinamide riboside (NR) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) and has a synergistic effect. This is understood and known by those skilled in the art.

[0081] 4. The GLX351322 is combined with PERK kinase inhibitor GSK2606414, wherein the CAS number of PERK kinase inhibitor GSK2606414 is 1337531-36-8, the molecular formula is C 24 H 20 F3N5O, the molecular weight is 451.44. The administration amount of the GLX351322 is 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection or intravenous drip. The administration amount of the PERK kinase inhibitor GSK2606414 is 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection or intravenous drip. Since the PERK kinase inhibitor GSK2606414 inhibits endoplasmic reticulum stress by inhibiting the activation of PERK in cells, the GLX351322 can be combined with the PERK kinase inhibitor GSK2606414 and has a synergistic effect. This is understood and known by those skilled in the art.

[0082] 5. The GLX351322 is combined with PERK kinase inhibitor GSK2656157, wherein the CAS number of PERK kinase inhibitor GSK2656157 is 1337532-29-2, the molecular formula is C 23H 21 GLX351322, with a molecular weight of 416.5. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2656157 is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. Since the PERK kinase inhibitor GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity, inhibits endoplasmic reticulum stress, the GLX351322 can be combined with the PERK kinase inhibitor GSK2656157 and has a synergistic effect. This is understood and known by those skilled in the art.

[0083] 6. The GLX351322 is combined with the integrated stress response inhibitor trans-ISRIB, wherein the CAS number of the integrated stress response inhibitor trans-ISRIB is 1597403-47-8, and the molecular formula is C 22 H 24 Cl2N2O4, with a molecular weight of 451.34. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The integrated stress response inhibitor trans-ISRIB is administered at a dosage of 0.1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. Since the integrated stress response inhibitor trans-ISRIB can enhance the activity of eIF2B and inhibit integrated stress response, the GLX351322 can be combined with the integrated stress response inhibitor trans-ISRIB and has a synergistic effect. This is understood and known by those skilled in the art.

[0084] 7. The GLX351322 is combined with the PERK kinase inhibitors GSK2606414 and GSK2656157, wherein the CAS number of the PERK kinase inhibitor GSK2606414 is 1337531-36-8, and the molecular formula is C 24 H 20 F3N5O, with a molecular weight of 451.44. The CAS number of the PERK kinase inhibitor GSK2656157 is 1337532-29-2, and the molecular formula is C 23 H 21FN6O, with a molecular weight of 416.5. The GLX351322 is administered in an amount of 1-5 mg / kg / d, and the administration mode includes oral administration, intravenous injection or intravenous infusion. The PERK kinase inhibitor GSK2606414 is administered in an amount of 0.1-10 mg / kg / d, and the administration mode includes oral administration, intravenous injection or intravenous infusion. The PERK kinase inhibitor GSK2656157 is administered in an amount of 0.1-10 mg / kg / d, and the administration mode includes oral administration, intravenous injection or intravenous infusion. Since the PERK kinase inhibitor GSK2606414 has the effect of inhibiting the activation of PERK in cells, and the PERK kinase inhibitor GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity, the GLX351322 can be combined with the PERK kinase inhibitors GSK2606414 and GSK2656157 and has a synergistic effect. This is understood and known by those skilled in the art.

[0085] 8. The GLX351322 is combined with the PERK kinase inhibitors GSK2606414 and GSK2656157, and the integrated stress response inhibitor trans-ISRIB, wherein the CAS number of the PERK kinase inhibitor GSK2606414 is 1337531-36-8, and the molecular formula is C 24 H 20 F3N5O, with a molecular weight of 451.44. The CAS number of the PERK kinase inhibitor GSK2656157 is 1337532-29-2, and the molecular formula is C 23 H 21 FN6O, with a molecular weight of 416.5. The CAS number of the integrated stress response inhibitor trans-ISRIB is 1597403-47-8, and the molecular formula is C 22 H 24Cl2N2O4, molecular weight 451.34. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2606414 is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The PERK kinase inhibitor GSK2656157 is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The integrated stress response inhibitor trans-ISRIB is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. Since the PERK kinase inhibitor GSK2606414 has the effect of inhibiting the activation of PERK in cells, the PERK kinase inhibitor GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity, and the integrated stress response inhibitor trans-ISRIB can enhance the activity of eIF2B to inhibit ISR, therefore the GLX351322 can be combined with the PERK kinase inhibitors GSK2606414 and GSK2656157 and the integrated stress response inhibitor trans-ISRIB and has a synergistic effect. This is understood and known by those skilled in the art.

[0086] 9. The GLX351322 is combined with the eIF2 phosphatase inhibitor Salubrinal, wherein the eIF2 phosphatase inhibitor Salubrinal has a CAS number of 405060-95-9, a molecular formula of C 21 H 17 Cl3N4OS, molecular weight 479.81. The GLX351322 is administered at a dosage of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. The eIF2 phosphatase inhibitor Salubrinal is administered at a dosage of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion. Since the eIF2 phosphatase inhibitor Salubrinal is a selective eIF2α dephosphorylation inhibitor that can inhibit ER stress-mediated apoptosis, therefore the GLX351322 can be combined with the eIF2 phosphatase inhibitor Salubrinal and has a synergistic effect. This is understood and known by those skilled in the art.

[0087] The above examples are only to illustrate the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. Use of GLX351322 in the preparation of a medicament for the prevention and treatment of hemorrhagic stroke, wherein, The molecular formula of GLX351322 is C 21 H 25 N3O5S, CAS No. 835598-94-2, molecular weight 431.51, and chemical structural formula as shown in Formula I: Formula I.

2. Use according to claim 1, characterized in that: The GLX351322 is administered in an amount of 1-5 mg / kg / d.

3. Use according to claim 2, characterized in that: The GLX351322 is administered by oral administration, intravenous injection or intravenous drip.

4. A medicament for preventing and treating hemorrhagic stroke, characterized by comprising the compound of claim 1. comprising GLX351322 having the formula C 21 H 25 N3O5S, CAS No. 835598-94-2, molecular weight 431.51, and a chemical structural formula as shown in Formula I: Formula I.

5. The medicament according to claim 4, characterized in that: The GLX351322 is administered in an amount of 1-5 mg / kg / d, and the administration is by oral administration, intravenous injection or intravenous drip.

6. The medicament according to claim 4, characterized in that: Nicotinamide riboside (NR) having a CAS number of 23111-00-4, a molecular formula of C 11 H 15 ClN2O5, a molecular weight of 290.7; the nicotinamide riboside (NR) is administered at a dose of 50-1200 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion.

7. The medicament according to claim 4, characterized in that: including reduced nicotinamide-adenine dinucleotide phosphate (NADPH) with CAS No. 53-59-8, molecular formula of C 21 H 28 N7O 17 P3, and a molecular weight of 744.42; the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) is administered in an amount of 0.1-10 mg / kg / d, and the administration mode includes oral administration, intravenous injection, or intravenous infusion.

8. The medicament according to claim 4, characterized in that: including PERK kinase inhibitor GSK2606414, which has a CAS number of 1337531-36-8, a molecular formula of C 24 H 20 F3N5O, and a molecular weight of 451.44; the PERK kinase inhibitor GSK2606414 is administered in an amount of 0.1-10 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

9. The medicament according to claim 4, characterized in that: including PERK kinase inhibitor GSK2656157, which has a CAS number of 1337532-29-2, a molecular formula of C 23 H 21 FN60, which has a molecular weight of 416.5; the PERK kinase inhibitor GSK2656157 is administered in an amount of 0.1-10 mg / kg / d, and the administration method includes oral administration, intravenous injection, or intravenous infusion.

10. The medicament according to claim 4, characterized in that: including eIF2 phosphatase inhibitor Salubrinal, CAS No. 405060-95-9, molecular formula C 21 H 17 Cl3N4OS, molecular weight 479.81; the dosages of the eIF2 phosphatase inhibitor Salubrinal are all 0.1-10 mg / kg / d, and the administration modes all include oral administration, intravenous injection or intravenous infusion.

11. The medicament according to claim 4, characterized in that: including the integrated stress response inhibitor trans-ISRIB, which has a CAS number of 1597403-47-8, a molecular formula of C 22 H 24 Cl2N2O4, with a molecular weight of 451.34; the integrated stress response inhibitor trans-ISRIB is administered in an amount of 1-5 mg / kg / d, and the administration methods include oral administration, intravenous injection, or intravenous infusion.

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