Use of 2-aminopyrazine derivatives for the preparation of medicaments for the treatment and / or prophylaxis of stroke and its complications

Abstract

The application discloses application of a 2-amino-pyrazine derivative in preparation of a medicine for treating and / or preventing apoplexy and complications, wherein the 2-amino-pyrazine derivative iMAP4K1 is (R)-4-[5-amino-6-[[1-(1-methyl-4-piperidyl)-4-pyrazolyl]oxy]-2-pyrazinyl]-N-[2-(3-fluoro-1-pyrrolidinyl)ethyl]-2,6-dimethylbenzamide; the apoplexy is acute ischemic stroke; and the 2-amino-pyrazine derivative can be used as an active ingredient in a medicine for treating and / or preventing post-stroke brain injury and complications, effectively reducing post-stroke cerebral infarction volume, improving sensory-motor and balance nerve functions, and preventing and treating post-stroke complications such as cerebral edema and acute lung injury.

Description

Technical Field

[0001] This invention relates to the use of a 2-aminopyrazine derivative in the preparation of medicaments for the treatment and / or prevention of stroke and its complications. Background Technology

[0002] Stroke, also known as apoplexy, is an acute cerebrovascular disease that seriously affects human health. It is a leading cause of death worldwide and the leading cause of disability in adults. The report shows that in 2021, there were 11.9 million new stroke cases globally, with a total of 93.8 million people suffering from the condition; ischemic stroke accounted for 65.3% of all strokes. Analysis indicates that the incidence and prevalence of ischemic stroke worldwide will continue to rise until 2030.

[0003] The clinical treatment of ischemic stroke mainly involves thrombolysis or endovascular mechanical thrombectomy to restore blood supply to the ischemic area as early as possible. However, pathological changes such as cerebral infarction and cerebral edema continue to increase after reperfusion therapy. Cerebral infarction exacerbates neurological function impairment and can even lead to death; the increase in infarct volume in the acute phase is associated with poor 90-day prognosis in surviving patients. Severe cerebral edema is a common complication of severe ischemic stroke and a major cause of death in acute-phase patients, independently associated with poor 90-day neurological outcomes. In addition, the incidence of serious complications such as post-stroke pneumonia and pulmonary edema is also high, further increasing patient mortality. The most commonly used drugs after reperfusion therapy are neuroprotective agents, but their clinical efficacy and safety are still unclear. Brain injury after reperfusion involves not only the death of nerve cells but also abnormalities or damage to vascular endothelial cells, pericytes, and various glial cells, as well as the activation and infiltration of peripheral immune cells. However, there is currently a lack of neuroprotective drugs and drugs for treating post-stroke complications. Therefore, there is an urgent need to develop safe and effective therapeutic drugs to improve the neurological prognosis of stroke patients and to prevent and treat serious post-stroke complications. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the use of a 2-aminopyrazine derivative in the preparation of drugs for the treatment and / or prevention of stroke and its complications.

[0005] Technical solution: This invention discloses the application of a 2-aminopyrazine derivative in the preparation of drugs for the treatment and / or prevention of stroke and its complications.

[0006] The 2-aminopyrazine derivative (iMAP4K1) is (R)-4-[5-amino-6-[[1-(1-methyl-4-piperidinyl)-4-pyrazolyl]oxy]-2-pyrazinyl]-N-[2-(3-fluoro-1-pyrrolidinyl)ethyl]-2,6-dimethylbenzamide.

[0007] The stroke mentioned above is an acute ischemic stroke.

[0008] Among them, the complications following stroke are cerebral edema or acute lung injury.

[0009] The application is that 2-aminopyrazine derivatives reduce the volume of cerebral infarction after stroke, improve sensory, motor and balance nerve function, and prevent and treat post-stroke complications such as cerebral edema and acute lung injury.

[0010] The application is administered via the following procedure: 0.1 mg / kg to 2 mg / kg is administered intravenously 0-24 hours after acute ischemic stroke or reperfusion, followed by injection every 12-24 hours for 2-7 consecutive days.

[0011] The present invention also discloses a pharmaceutical composition comprising the above-described 2-aminopyrazine derivative and a pharmaceutically acceptable carrier.

[0012] The dosage form of the pharmaceutical composition is an injection, tablet, powder, granule, capsule, oral liquid, sublingual administration, or nasal administration.

[0013] The above-mentioned pharmaceutical composition can also be used in the preparation of drugs for the treatment and / or prevention of stroke and its complications.

[0014] Invention Principle: This invention discloses that 2-aminopyrazine derivatives can be used to prepare drugs for treating acute brain injury and complications after stroke. Using a mouse model of acute ischemic stroke with middle cerebral artery occlusion (MCAO), the therapeutic effects of 2-aminopyrazine derivative (iMAP4K1) on cerebral infarction and neurological deficits after cerebral ischemia / reperfusion were evaluated, as well as its preventive and therapeutic effects on common post-stroke complications such as cerebral edema and acute lung injury. This provides experimental evidence for the preparation of drugs for the treatment and / or prevention of ischemic stroke. Experimental results show that iMAP4K1 treatment not only reduces the volume of post-stroke cerebral infarction and improves sensory, motor, and balance functions, but also has preventive and therapeutic effects on common post-stroke complications such as cerebral edema and acute lung injury.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The 2-aminopyrazine derivative of the present invention can alleviate cerebral infarction and cerebral edema after acute ischemic stroke, improve neurological function, and reduce acute lung injury after stroke. Attached Figure Description

[0016] Figure 1 To reduce the volume of cerebral infarction after ischemic stroke with 2-aminopyrazine derivatives, where A represents the histological staining results of the cerebral infarction and B represents the quantitative results of the cerebral infarction volume.

[0017] Figure 2 Results of treatment with 2-aminopyrazine derivatives in reducing neurological deficits after acute ischemic stroke;

[0018] Figure 3 Results of treatment with 2-aminopyrazine derivatives in reducing cerebral edema after ischemic stroke;

[0019] Figure 4 Results of treatment with 2-aminopyrazine derivatives in reducing acute lung injury after acute ischemic stroke, where A is the histological staining result of the lung, B is the histological score result of lung injury, and C is the content of protein in bronchoalveolar lavage fluid. Detailed implementation manners

[0020] The technical solutions of the present invention will be further described below in conjunction with embodiments. The test materials used in the embodiments can be obtained through conventional channels.

[0021] Example 1

[0022] Evaluate the therapeutic effect of 2-aminopyrazine derivative (iMAP4K1) on post-stroke brain injury:

[0023] 1. Animals: Adult male C57BL / 6 mice (SPF grade, 25±3 g), purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd., license number: SCXK(Shanghai)2023-0009.

[0024] 2. Drugs and main reagents:

[0025] 2-aminopyrazine derivative ((R)-4-[5-amino-6-[[1-(1-methyl-4-piperidinyl)-4-pyrazolyl]oxy]-2-pyrazinyl]-N-[2-(3-fluoro-1-pyrrolidinyl)ethyl]-2,6-dimethylbenzamide): purchased from SelleckChemicals (Catalog No. E1297). Dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and diluted with normal saline to a working solution before use.

[0026] DMSO: purchased from Shanghai Sigma-Aldrich Trading Co., Ltd.

[0027] Sodium chloride: purchased from Xilong Science Co., Ltd.

[0028] Isoflurane: purchased from Shenzhen Rewod Life Science Co., Ltd.

[0029] 2,3,5-Triphenyltetrazolium chloride (TTC): purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0030] Paraformaldehyde: purchased from Nanjing Chemical Reagent Co., Ltd.

[0031] 3. Instruments and equipment:

[0032] ABS-type mouse anesthesia machine: Shanghai Yuyan Scientific Instruments Co., Ltd.

[0033] FLPI-2 laser speckle blood flow imaging system: Moor Instruments, UK.

[0034] SMZ745 stereomicroscope: Nikon Corporation, Japan.

[0035] 4. Animal grouping: Mice were randomly divided into a solvent control group, a low-dose iMAP4K1 group (1 mg / kg), and a high-dose iMAP4K1 group (2 mg / kg). The drug was administered via tail vein injection immediately 30 minutes after reperfusion, once daily for a total of 2 doses.

[0036] 5. Experimental methods:

[0037] (1) Establishment of a mouse model of acute ischemic stroke:

[0038] An acute ischemic stroke model of middle cerebral artery occlusion (MCAO) was established in mice. Mice were anesthetized (induced by 3% isoflurane, maintained by 1%–1.5% isoflurane), and the blood flow to the middle cerebral artery was blocked with a silicone suture (purchased from Beijing Jitai Yuancheng Co., Ltd.). After 90 minutes of ischemia, the suture was removed to achieve cerebral blood flow reperfusion. Changes in cerebral blood flow before, during, and after ischemia were monitored using a laser speckle imaging system. During the operation, the body temperature of the mice was maintained at 37.0 ± 0.5℃.

[0039] (2) TTC staining to detect cerebral infarction volume:

[0040] 48 hours after MCAO / reperfusion, mice were euthanized by cervical dislocation and their brains were harvested. The brains were frozen at -20°C for 20 min. A vertical incision was made at the midpoint between the anterior pole and the optic chiasm, followed by incisions every 1 mm thereafter to create 1 mm thick coronal sections. The sections were stained in 2% TTC solution at 37°C for 10 min in the dark, fixed with 4% paraformaldehyde, and photographed. The infarct volume was analyzed using ImageJ image analysis software. Infarct volume (%) = (Volume of normal brain tissue on the contralateral side - Volume of non-infarcted brain tissue on the ischemic side) / (2 × Volume of normal brain tissue on the contralateral side) × 100%.

[0041] (3) Modified neurological severity score (mNSS):

[0042] After 48 h of MCAO / reperfusion, the neurological functions of mice, such as movement, sensation, balance, and reflex, were evaluated using the mNSS. The total score of mNSS was 18 points. Scores of 13 - 18 points indicated severe injury, 7 - 12 points indicated moderate injury, and 1 - 6 points indicated mild injury.

[0043] (4) Data statistical analysis: GraphPad Prism 8.0 was used for data statistical analysis. The data were expressed as mean ± standard deviation, and one-way analysis of variance was used. *P < 0.05 was considered statistically significant.

[0044] 6. Results and conclusions:

[0045] (1) Treatment with 2 - aminopyrazine derivatives reduced the cerebral infarction volume after ischemic stroke; as Figure 1 shown, after 48 h of MCAO / reperfusion, the cerebral infarction volumes of mice in the low - dose group (1 mg / kg, n = 5) and high - dose group (2 mg / kg, n = 5) of iMAP4K1 were significantly reduced. Compared with the solvent control group (n = 5), the difference was statistically significant (***P < 0.001); the therapeutic effect of high - dose iMAP4K1 was better, and the difference compared with the low - dose group was statistically significant (***P < 0.001).

[0046] (2) Treatment with 2 - aminopyrazine derivatives improved neurological function after ischemic stroke; as Figure 2 shown, after 48 h of MCAO / reperfusion, the mNSS scores of mice were significantly increased, indicating severe injury; both the low - dose (1 mg / kg, n = 5) and high - dose (2 mg / kg, n = 5) of iMAP4K1 could reduce the mNSS scores, and the differences compared with the solvent control group were statistically significant (**P < 0.01, ***P < 0.001).

[0047] Example 2

[0048] Evaluate the therapeutic effect of 2 - aminopyrazine derivatives (iMAP4K1) on post - stroke complications:

[0049] 1. Animals: Adult male C57BL / 6 mice (SPF level, 25 ± 3 g), purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd., license number: SCXK(Shanghai)2023 - 0009.

[0050] 2. Drugs and main reagents:

[0051] Folin - Ciocalteu phenol: Purchased from Shanghai Macklin Biochemical Co., Ltd.

[0052] Sodium carbonate: Purchased from Nanjing Chemical Reagent Co., Ltd.

[0053] Sodium hydroxide: Purchased from Xilong Scientific Co., Ltd.

[0054] Sodium potassium tartrate: purchased from Shanghai Sangon Biotech Co., Ltd.

[0055] Copper sulfate: purchased from Shanghai Sangon Biotech Co., Ltd.

[0056] Other reagents are the same as in Example 1.

[0057] 3. Instruments and equipment:

[0058] MF52-N optical microscope: Guangzhou Mingmei Optoelectronic Technology Co., Ltd.

[0059] 5424R High-Speed ​​Refrigerated Centrifuge: Eppendorf, USA.

[0060] PTY-A220 Precision Electronic Balance: Fujian Huazhi Electronic Technology Co., Ltd.

[0061] LDO-101-1 Electric Thermostatic Drying Oven: Shanghai Longyue Instrument Equipment Co., Ltd.

[0062] Other equipment is the same as in Example 1.

[0063] 4. Animal grouping: Same as in Example 1.

[0064] 5. Experimental methods:

[0065] (1) Establishment of a mouse model of acute ischemic stroke: Same as in Example 1.

[0066] (2) Brain water content measurement to assess post-stroke cerebral edema:

[0067] 48 hours after MCAO / reperfusion, mice were euthanized by cervical dislocation and their brains were harvested. The brain tissue was cut along the midline into the contralateral and ischemic hemispheres. Surface moisture was absorbed with filter paper, and the tissue was weighed (wet weight). The tissue was then dried in an oven at 100°C for 24 hours and weighed again (dry weight). Brain water content (%) = (wet weight - dry weight) / wet weight × 100%.

[0068] (3) Determination of protein content in bronchoalveolar lavage fluid:

[0069] Mice were euthanized by cervical dislocation 48 h after MCAO / reperfusion. 1 mL of ice-cold PBS was injected into the lungs via the bronchus, and the bronchoalveolar lavage fluid was collected by aspiration three times. The fluid was centrifuged for 10 min (1500×g, 4℃), and the supernatant was collected. The protein content in the bronchoalveolar lavage fluid was determined by the Lowry method.

[0070] (4) Hematoxylin / eosin (HE) staining and lung histological scoring:

[0071] Mouse lung tissue was fixed in paraformaldehyde (4%), cut into 5 μm thick sections, and stained with hematoxylin and eosin (HE). Lung injury severity was assessed under a light microscope using four indicators: alveolar edema, pulmonary hemorrhage, alveolar septal thickening, and leukocyte infiltration. Each indicator was scored from 0 to 3 (normal, 0; mild, 1; moderate, 2; severe, 3). The total score for all four indicators was calculated. Three different fields of view were randomly selected from each section for evaluation, and the average histological score of the three fields was used as the mouse's lung injury score.

[0072] (5) Statistical analysis of data: GraphPad Prism 8.0 was used for statistical analysis of data. Data are expressed as mean ± standard deviation. Brain water content was analyzed using two-way ANOVA; lung injury scores and bronchoalveolar lavage fluid protein content were determined using independent samples t-tests. *P < 0.05 was considered statistically significant.

[0073] 6. Results and Conclusions:

[0074] (1) Treatment with 2-aminopyrazine derivatives can reduce post-stroke cerebral edema; such as... Figure 3 As shown, 48 h after MCAO / reperfusion, the water content of the ischemic cerebral hemisphere increased, and the difference was statistically significant compared with the water content of the contralateral brain tissue (***P<0.001); after high-dose (2mg / kg, n=5) iMAP4K1 treatment, the water content of the ischemic brain was significantly reduced, and the difference was statistically significant compared with the solvent control group (n=5) (***P<0.001).

[0075] (2) Treatment with 2-aminopyrazine derivatives can alleviate acute lung injury after stroke; such as... Figure 4 As shown in Figures A and B, HE staining revealed pulmonary interstitial capillary dilation and congestion with hemorrhage 48 hours after MCAO / reperfusion, thickening of alveolar septa, and a large number of neutrophil aggregates in the pulmonary interstitium. Figure 4 (A); Solvent control group (n=5) histological score greater than 5 ( Figure 4 In the iMAP4K1 group (2 mg / kg, n=5), pathological damage such as pulmonary interstitial hemorrhage, pulmonary interstitial thickening, and neutrophil infiltration was reduced, and the lung injury score was significantly lower, with statistically significant differences compared with the solvent control group (***P<0.001).

[0076] like Figure 4 As shown in Figure C, the total protein level in the bronchoalveolar lavage fluid of mice increased 48 h after MCAO / reperfusion, reaching approximately 1400 μg / mL in the solvent control group (n=5); iMAP4K1 (2 mg / kg, n=5) treatment significantly reduced the protein level in the bronchoalveolar lavage fluid of mice, and the difference was statistically significant compared with the solvent control group (**P<0.01).

[0077] Therefore, the 2-aminopyrazine derivative iMAP4K1, as an active ingredient, is used in the preparation of drugs for the treatment and / or prevention of post-stroke brain injury and complications. It can effectively reduce the volume of post-stroke cerebral infarction, improve sensory, motor and balance nerve function, and prevent and treat post-stroke complications such as cerebral edema and acute lung injury.

Claims

The use of 1,2-aminopyrazine derivatives in the preparation of drugs for the treatment and / or prevention of acute ischemic stroke and its complications, characterized in that, The 2-aminopyrazine derivative is (R)-4-[5-amino-6-[[1-(1-methyl-4-piperidinyl)-4-pyrazolyl]oxy]-2-pyrazinyl]-N-[2-(3-fluoro-1-pyrrolidinyl)ethyl]-2,6-dimethylbenzamide, and the complication is post-stroke cerebral edema or acute lung injury.

2. The application according to claim 1, characterized in that, The application is that 2-aminopyrazine derivatives reduce the volume of cerebral infarction after stroke, improve sensory, motor and balance nerve function, and prevent and treat post-stroke complications such as cerebral edema and acute lung injury.

3. The application according to claim 1, characterized in that, The administration method is as follows: 0.1 mg / kg to 2 mg / kg is administered intravenously 0-24 h after acute ischemic stroke or reperfusion, followed by injection every 12-24 h for 2-7 consecutive days.

4. The use of a pharmaceutical composition comprising the 2-aminopyrazine derivative of claim 1 in the preparation of a medicament for treating and / or preventing stroke and its complications, characterized in that, The complications mentioned are post-stroke cerebral edema or acute lung injury.

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