Application of 2-aminopyrazine derivative in preparation of medicine for treating and / or preventing cerebral apoplexy and complications

Abstract

The invention discloses an application of a 2-aminopyrazine derivative in preparation of a medicine for treating and / or preventing cerebral apoplexy and complications, and the 2-aminopyrazine 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 cerebral apoplexy is acute ischemic apoplexy; the 2-aminopyrazine derivative can be used as an active component, is applied to drugs for treating and / or preventing cerebral injury and complications after stroke, effectively reduces the volume of cerebral infarction after stroke, improves sensory motility, balances neurological functions, and prevents and treats cerebral edema and acute lung injury which are complications after stroke.

Description

Technical Field

[0001] The present invention relates to application of a 2-aminopyrazine derivative in preparing a medicine for treating and / or preventing cerebral stroke and its complications. Background Art

[0002] Stroke, also known as stroke, is an acute cerebrovascular disease that seriously affects human health. It is the 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 cases of stroke worldwide, with the number of patients reaching 93.8 million; among them, ischemic stroke accounted for 65.3% of all strokes. According to analysis, the incidence and prevalence of ischemic stroke will continue to rise worldwide by 2030.

[0003] The clinical treatment of ischemic stroke mainly adopts drug thrombolysis or intravascular mechanical thrombectomy to achieve cerebral blood flow reperfusion as soon as possible to restore blood supply to the ischemic area. However, pathological changes such as cerebral infarction and cerebral edema continue to increase after reperfusion therapy. Brain tissue infarction aggravates the patient's neurological damage and even leads to death. The increase in acute cerebral infarction volume is associated with poor prognosis of surviving patients at 90 days. Severe cerebral edema is a common complication of severe ischemic stroke and the main cause of death in acute patients. It is independently associated with poor neurological function outcomes at 90 days. In addition, the incidence of serious complications such as post-stroke pneumonia and pulmonary edema is also high, further aggravating the mortality rate of patients. The commonly used drugs after reperfusion therapy are mainly neuroprotective agents, but their clinical efficacy and safety are still unclear. Brain injury after reperfusion is not only the death of neurons, but also involves abnormal or damaged functions of vascular endothelial cells, pericytes and various glial cells, as well as pathological links such as activation and infiltration of peripheral immune cells, but there is currently a lack of brain cell protection drugs and drugs for the treatment of 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 prevent and treat serious complications after stroke. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a 2-aminopyrazine derivative for use in the preparation of a drug for treating and / or preventing stroke and its complications.

[0005] Technical solution: The present invention discloses the use of a 2-aminopyrazine derivative in the preparation of a drug for treating and / or preventing stroke and its complications.

[0006] Wherein, 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] Wherein, the stroke is acute ischemic stroke.

[0008] Wherein, the post-stroke complications are cerebral edema or acute lung injury.

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

[0010] The administration of the application adopts the following operation: 0.1 mg / kg-2 mg / kg is administered by intravenous injection 0-24 hours after acute ischemic stroke or reperfusion, and then injected once every 12-24 hours for 2-7 consecutive days.

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

[0012] The pharmaceutical composition is in the form of injection, tablet, powder, granule, capsule, oral solution, sublingual preparation or nasal preparation.

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

[0014] Principle of the invention: The present invention discloses that 2-aminopyrazine derivatives can be used to prepare drugs for preventing acute brain damage and complications after stroke; the acute ischemic stroke model of middle cerebral artery occlusion (MCAO) in mice is used to evaluate the therapeutic effect of 2-aminopyrazine derivatives (iMAP4K1) on cerebral infarction and neurological dysfunction after cerebral ischemia / reperfusion, as well as the preventive and therapeutic effects on common complications of stroke, cerebral edema and acute lung injury, to provide experimental basis for the preparation of ischemic stroke treatment and / or prevention drugs. The experimental results show that iMAP4K1 treatment not only reduces the volume of cerebral infarction after stroke, improves neurological functions such as sensory movement and balance, but also has preventive and therapeutic effects on common complications of stroke, 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 derivatives of the present invention can reduce cerebral infarction and cerebral edema after acute ischemic stroke, improve neurological function, and reduce acute lung injury after stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The 2-aminopyrazine derivatives can reduce the volume of cerebral infarction after ischemic stroke, where A is the histological staining result of cerebral infarction and B is the quantitative result of cerebral infarction volume;

[0017] Figure 2 The results of 2-aminopyrazine derivatives in alleviating neurological deficits after acute ischemic stroke;

[0018] Figure 3 The results of 2-aminopyrazine derivative treatment in reducing brain edema after ischemic stroke;

[0019] Figure 4 These are the results of 2-aminopyrazine derivatives in alleviating acute lung injury after acute ischemic stroke, wherein A is the histological staining result of the lung, B is the histological score result of lung injury, and C is the protein content of bronchoalveolar lavage fluid. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0021] Example 1

[0022] To evaluate the therapeutic effect of 2-aminopyrazine derivatives (iMAP4K1) on brain damage after stroke:

[0023] 1. Animals: Adult male C57BL / 6 mice (SPF grade, 25±3 g), purchased from Shanghai Bikeway 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 Selleck Chemicals (Catalog No. E1297). Prepare a stock solution by dissolving in dimethyl sulfoxide (DMSO) and dilute with saline to a working solution before use.

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

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

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

[0029] 2,3,5-Triphenyltetrazolium chloride (TTC): purchased from Beijing Solebow 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 Instrument Co., Ltd.

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

[0034] SMZ745 stereo microscope: Nikon Corporation, Japan.

[0035] 4. Animal grouping: The 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 drugs were injected into the tail vein immediately after 30 minutes of reperfusion, once a day, for a total of 2 times.

[0036] 5. Experimental methods:

[0037] (1) Establishment of acute ischemic stroke model in mice:

[0038] The acute ischemic stroke model of middle cerebral artery occlusion (MCAO) was used in mice. The mice were anesthetized (3% isoflurane induced, 1%-1.5% isoflurane maintained), and the blood flow of the middle cerebral artery was blocked by silicone sutures (purchased from Beijing Jitaiyuancheng Company). The sutures were removed after 90 minutes of ischemia to achieve cerebral blood reperfusion. The changes of cerebral blood flow in mice before, during and after ischemia were monitored by laser speckle blood flow 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] 48h after MCAO / reperfusion, mice were killed by cervical dislocation and brains were removed. The brain was frozen at -20℃ for 20min, and the first cut was made vertically at the midpoint of the anterior pole and the optic chiasm, and then cut every 1mm to make 1mm thick coronal slices. The slices were stained in 37℃ constant temperature TTC solution (2%) for 10min in the dark, fixed with paraformaldehyde (4%) and photographed. ImageJ image analysis software was used to analyze the cerebral infarction volume. Cerebral infarction volume (%) = (contralateral normal brain tissue volume - ischemic side non-infarcted brain tissue volume) / (2 × contralateral normal brain tissue volume) × 100%.

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

[0042] 48h after MCAO / reperfusion, the mNSS was used to evaluate the neurological functions of the mice, including movement, sensation, balance, and reflexes. The total mNSS score is 18 points, with 13-18 points indicating severe injury, 7-12 points indicating moderate injury, and 1-6 points indicating mild injury.

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

[0044] 6. Results and conclusions:

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

[0046] (2) 2-aminopyrazine derivatives improve neurological function after ischemic stroke; e.g. Figure 2 As shown, the mNSS score of mice increased significantly 48 hours after MCAO / reperfusion, indicating severe injury; both low-dose (1 mg / kg, n=5) and high-dose (2 mg / kg, n=5) iMAP4K1 reduced the mNSS score, and the difference was statistically significant compared with the solvent control group (**P<0.01, ***P<0.001).

[0047] Example 2

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

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

[0050] 2. Drugs and main reagents:

[0051] Folin phenol: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

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

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

[0054] Potassium sodium tartrate: purchased from Shanghai Sangon Biotechnology Co., Ltd.

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

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

[0057] 3. Instruments and equipment:

[0058] MF52-N optical microscope: Guangzhou Mingmei Optoelectronics 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 constant temperature air drying oven: Shanghai Longyue Instrument Equipment Co., Ltd.

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

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

[0064] 5. Experimental methods:

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

[0066] (2) Determination of brain water content to assess post-stroke brain edema:

[0067] 48 hours after MCAO / reperfusion, mice were killed by cervical dislocation and brains were removed. The brain tissue was cut into the contralateral hemisphere and the ischemic hemisphere along the raphe, and the surface moisture was absorbed by filter paper and weighed (wet weight), and then placed 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] 48 hours after MCAO / reperfusion, mice were killed by cervical dislocation. 1 mL of ice-cold PBS was injected into the lungs from the bronchus, and the alveolar lavage fluid was collected by aspiration 3 times and centrifuged for 10 min (1500×g, 4°C). The supernatant was collected and the protein content in the alveolar lavage fluid was determined by the Lowry method.

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

[0071] After fixing the mouse lung tissue with paraformaldehyde (4%), the lung tissue was cut into 5 μm thick sections and stained with HE. Observed under an optical microscope, the severity of lung injury was evaluated by the following four indicators: alveolar edema, pulmonary hemorrhage, alveolar septum thickening and leukocyte infiltration, each indicator was 0-3 points (normal, 0 points; mild, 1 point; moderate, 2 points; severe, 3 points), and the total score of the above four indicators was calculated. Three different fields of view were randomly selected for each section for evaluation, and the average of the histological scores of the three fields of view was used as the lung injury score of the mouse.

[0072] (5) Data statistical analysis: GraphPad Prism 8.0 was used for data statistical analysis. Data were expressed as mean ± standard deviation. Two-way analysis of variance was used for brain water content. Independent sample t-test was used for lung injury score and bronchoalveolar lavage fluid protein content. *P < 0.05 was considered statistically significant.

[0073] 6. Results and conclusions:

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

[0075] (2) 2-aminopyrazine derivatives can reduce acute lung injury after stroke; Figure 4 As shown in A and B, HE staining showed that 48 hours after MCAO / reperfusion, the pulmonary interstitial capillaries were dilated and congested with hemorrhage, the alveolar septa were thickened, and a large number of neutrophils accumulated in the pulmonary interstitium ( Figure 4 A); solvent control group (n = 5) histological score greater than 5 ( Figure 4 (B); Pathological damage such as pulmonary interstitial hemorrhage, pulmonary interstitial thickening and neutrophil infiltration in mice in the iMAP4K1 group (2 mg / kg, n=5) was alleviated, and the lung injury score was significantly reduced, which was statistically significant compared with the solvent control group (***P<0.001).

[0076] like Figure 4 As shown in C, the total protein level in the bronchoalveolar lavage fluid of mice increased 48 hours after MCAO / reperfusion, reaching about 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 is used as an active ingredient in the preparation of therapeutic and / or preventive drugs for post-stroke brain injury and complications, which can effectively reduce the volume of post-stroke cerebral infarction, improve sensory motor and balance nerve functions, and prevent and treat post-stroke complications such as cerebral edema and acute lung injury.

Claims

1. Use of 2-aminopyrazine derivatives in the preparation of drugs for treating and / or preventing stroke and its complications.

2. The use according to claim 1, 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.

3. The use according to claim 1, characterized in that: The stroke is acute ischemic stroke.

4. The use according to claim 1, characterized in that: The complications are cerebral edema or acute lung injury after stroke.

5. The use 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 functions, and prevent and treat post-stroke complications such as cerebral edema and acute lung injury.

6. The use according to claim 1, characterized in that: The administration for the application is to administer 0.1 mg / kg-2 mg / kg by intravenous injection 0-24 hours after acute ischemic stroke or reperfusion, and then inject once every 12-24 hours for 2-7 consecutive days.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the 2-aminopyrazine derivative according to claim 1 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition is injection, tablet, powder, granule, capsule, oral liquid, sublingual preparation or nasal preparation.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of drugs for treating and / or preventing stroke and its complications.

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