Application of novel compound in preparation of medicine for preventing and treating cerebral arterial thrombosis

Abstract

The invention discloses application of a compound with a novel structure in preparation of a medicine for preventing and treating cerebral arterial thrombosis. The novel-structure compound shown in the formula (I) disclosed by the invention can pass through a blood brain barrier, and after cerebral ischemia-reperfusion, the novel-structure compound can obviously improve neurological function scores and behavioral scores, increase cerebral blood flow, reduce cerebral infarction volume and reduce neuron damage. Therefore, the compound with the novel structure has a remarkable protective effect on cerebral ischemia injury. In addition, the compound with the new structure can induce phagocytosis and elimination of apoptotic neurons by microglial cells after cerebral ischemia-reperfusion, and it is prompted that the compound with the new structure can promote phagocytosis and elimination of apoptotic neurons by the microglial cells to be an important mechanism for playing a cerebral ischemia injury protection role. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and relates to the application of a new compound in the preparation of a drug for preventing and treating ischemic stroke. Background Art

[0002] Stroke is a type of disease with focal or global brain dysfunction caused by cerebrovascular lesions. It is the second leading cause of death and disability worldwide. Stroke is divided into two major categories: ischemic and hemorrhagic, of which more than 87% are ischemic stroke. At present, the main clinical treatments for ischemic stroke include intravenous thrombolysis, mechanical thrombectomy, and drug therapy. However, these treatments have problems such as narrow treatment time window, limited treatment indications, and postoperative reperfusion injury. Therefore, the development of new treatment strategies is crucial to improving the prognosis of patients with ischemic stroke. Studies have found that after an ischemic stroke occurs, local brain tissue will experience ischemia and hypoxia, leading to damage and death of neurons and glial cells. At this time, a large number of microglia are activated, changing from a branched state in a resting state to an ameba-like state to migrate and aggregate to the ischemic injury area, and engulf necrotic cells and tissue fragments. In addition, activated microglia release chemokines and cytokines to attract immune cells such as monocytes in peripheral blood into the brain tissue, further promoting the removal of necrotic cells and tissue fragments. If the phagocytic function of phagocytes is impaired, necrotic cells and tissue fragments cannot be cleared in time, which will trigger an excessive inflammatory response, leading to the massive release of inflammatory mediators, further damaging the surrounding normal tissues and aggravating brain damage. Therefore, research on regulating phagocytic function has brought new hope for the treatment of ischemic stroke and is expected to open up a new treatment approach.

[0003] The new compound provided by the present invention has the function of significantly promoting the phagocytosis of microglia. Currently, no literature has reported the function of the compound and its regulatory effect on ischemic stroke. Summary of the invention

[0004] The purpose of the present invention is to provide a new compound for use in preparing a drug for preventing and treating ischemic stroke.

[0005] To achieve the above objectives, the present invention provides the following solutions:

[0006] The compound represented by formula I:

[0007]

[0008] Use of the compound represented by formula I in the preparation of drugs for preventing and treating ischemic stroke.

[0009] As a preferred embodiment of the present invention, the compound represented by formula I can penetrate the blood-brain barrier.

[0010] As a preferred embodiment of the present invention, the compound represented by formula I can significantly improve the neurological function score and behavioral score after ischemic stroke.

[0011] As a preferred embodiment of the present invention, the compound represented by formula I can significantly increase cerebral blood flow after ischemic stroke.

[0012] As a preferred embodiment of the present invention, the compound represented by formula I can significantly reduce the volume of cerebral infarction after ischemic stroke.

[0013] As a preferred embodiment of the present invention, the compound represented by formula I can significantly reduce neuronal damage after ischemic stroke.

[0014] As a preferred embodiment of the present invention, the compound represented by formula I can promote the phagocytosis and clearance of apoptotic neurons by microglia after ischemic stroke.

[0015] Beneficial effects:

[0016] The present invention discloses a new structure compound, and proves that it can pass through the blood-brain barrier, and finds that it can significantly improve the neurological function score and behavioral score of mice after cerebral ischemia, increase cerebral blood flow, reduce cerebral infarction volume, and reduce neuronal damage. It shows that this new structure compound has a significant protective effect on cerebral ischemic injury. In addition, the new structure compound can promote the phagocytosis and clearance of apoptotic neurons by microglia after cerebral ischemia-reperfusion, suggesting that the new structure compound promoting the phagocytosis and clearance of apoptotic neurons by microglia may be an important mechanism for its protective effect on cerebral ischemic injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 This is a diagram showing the metabolism of this type of new structural compound in the brain of normal mice and tMCAO model mice.

[0019] Figure 2 This is a graph showing the results of this type of new structural compounds improving the neurological function scores and behavioral evaluation of tMCAO model mice.

[0020] Figure 3 This is a graph showing the results of this type of new structural compound increasing cerebral blood flow in tMCAO model mice.

[0021] Figure 4This is a graph showing the results of this type of new structural compound reducing the volume of cerebral infarction in tMCAO model mice.

[0022] Figure 5 This is a graph showing the results of this type of new structural compounds alleviating neuronal damage in tMCAO model mice.

[0023] Figure 6 This figure shows the result of this type of new structural compound promoting the phagocytosis and clearance of apoptotic neurons by microglia in the brain of tMCAO model mice. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1: Experimental grouping, tMCAO model preparation and drug administration

[0026] All experimental animals were adapted to the environment for one week before modeling and randomly divided into sham operation group (Sham), model group (MCAO), and drug administration group (MCAO+Compound) according to body weight. The ZeaLonga method was used to prepare the cerebral ischemia model: fasting was performed the night before modeling. During modeling, rats were anesthetized with sodium phenobarbital, fixed in a supine position, and the neck skin was disinfected with iodine and then cut in the middle. The common carotid artery (CCA) and the external carotid artery (ECA) were separated. The distal end of the external carotid artery was ligated and coagulated, and a slipknot was prepared at the proximal end. A slipknot was tied to the common carotid artery to temporarily block the blood flow. The stump of the external carotid artery was gently lifted to the same direction as the internal carotid artery. The proximal end of the external carotid artery was suspended and a line was prepared to control blood flow. A small incision was made in the external carotid artery with ophthalmic scissors, and the embolism line was gently inserted from the small incision. The embolism line passed through the bifurcation and entered the internal carotid artery until slight resistance was encountered near the embolism mark. At this time, the embolism line was inserted into the anterior cerebral artery (ACA) and the middle cerebral artery (Middle cerebral The bifurcation of the middle cerebral artery (MCA) was blocked at the beginning of the main trunk of the middle cerebral artery, and the spare line of the external carotid artery was tied to fix the line plug. After 45 minutes of ischemia, the line plug was pulled out and the stump of the external carotid artery was ligated. The mice were sutured and disinfected and returned to the cage for monitoring. After 2 hours of reperfusion, the compound shown in formula I (2 mg / kg) was intraperitoneally injected, and the model group and the sham operation group were intraperitoneally injected with normal saline, and the same treatment was given every 24 hours.

[0027] Example 2: Brain penetration experiment

[0028] Mice were randomly divided into sham group and tMCAO group. Both groups of mice were intraperitoneally injected with Compound (2 mg / kg) shown in Formula I after modeling and reperfusion. Blood was collected by eyeball extraction in 1.5 mL anticoagulant tubes at 1, 2, 4, 6, 8, and 12 hours after administration. The mixture was allowed to stand at room temperature for 1-2 hours, centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected into a clean 1.5 mL enzyme-free EP tube. The prepared serum was stored at -80 °C. After blood collection, the mice were fixed on a foam board in a supine position, the chest and abdominal skin of the mice were opened longitudinally, the diaphragm was cut and the ribs were cut, the chest wall was folded and fixed with hemostats to fully expose the heart. The outer wall of the left ventricle was punctured and fixed with hemostats, and a small opening was cut in the right atrial appendage to facilitate the discharge of the perfusion fluid. First, about 40 mL of normal saline was perfused, and then 40 mL of pre-cooled PBS was perfused until the liver turned white. The fixation was removed, the whole brain was removed by decapitation, the surface moisture of the brain tissue was blotted with soft absorbent paper and weighed, and then placed in a 4 mL EP tube and quickly frozen in liquid nitrogen, and then stored at -80°C. Acetonitrile was added according to the weight of the brain for homogenization, ultrasonication for 20 minutes, centrifugation at 12000 rpm for 10 minutes, the supernatant was left to stand at 4°C overnight, centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken as the "brain tissue test sample". Quantitative analysis was performed using a high performance liquid chromatograph (Hitachi Chromaster-5430 detector, Hitachi Chromaster-5310 column oven, Hitachi Chromaster-5210 autosampler, Hitachi Chromaster-5110 pump).

[0029] like Figure 1 As shown, the compound represented by Formula I can effectively cross the blood-brain barrier of normal mice and tMCAO mice, and in both cases, the maximum concentration of the compound in the brain tissue is similar.

[0030] Example 3: Neurological Function Scoring

[0031] After the experimental mice were given tMCAO modeling and / or treated with the compound shown in Formula I, the mice were scored by Bederson. The scoring criteria are: 0 points for normal, no behavioral abnormalities; 1 point, flexion of the left forelimb when the tail is lifted; 2 points, turning to the left when walking; 3 points, leaning to the left; 4 points, no spontaneous walking, decreased consciousness; 5 points, death related to ischemia. If there is no infarction, massive intraoperative bleeding, subarachnoid hemorrhage, or abnormal breathing after surgery, the experimental mice will be eliminated and supplemented.

[0032] like Figure 2 As shown in a, the compound represented by formula I can significantly improve the neurological function of tMCAO model mice at 1d and 3d.

[0033] Example 4: Behavioral testing

[0034] 1) Corner Turn Test is frequently used to assess sensorimotor deficits due to unilateral or asymmetric striatal damage, to measure asymmetric sensorimotor deficits and to detect unilateral abnormalities in sensory and motor function in stroke models. The Corner Turn Test apparatus consists of two walls, and mice are placed between two boards measuring 30×20×1 cm3 while in their cages. The edges of the two boards are connected at a 30° angle, and there is a small opening at the seam between the two boards to facilitate access to the corner. There is an opening on one side and a 30° angle on the other side. Place the open edge of the apparatus close to the edge of the table and allow the rodent to advance to the corner. The open side is located at the edge of the table, allowing only exploration of the corner, but not the outside of the apparatus. Once the animal approaches the narrow corner, it stands forward and upward, then turns to face the open end. A total of 10 consecutive trials are performed, with a rest period of approximately 30 seconds between trials. The side to which the animal turns is recorded for each trial. Turning movements that are not part of the upright movement are not scored. Mice are allowed to enter corners at a 30-degree angle. The rat was placed between two angled plates facing the corner and halfway from the corner. When entering deep into the corner, both sides of the whiskers were stimulated. The rat was then raised forward and upward and turned back to face the open end. Non-ischemic mice turned to the left or right, but ischemic mice preferentially turned to the uninjured ipsilateral side (right side). Ten trials of each test were recorded for turns in one direction versus the other. To leave the corner, the rat could turn left or right, which was recorded. The test was repeated 10 times with at least 30 seconds between each trial, and the percentage of right turns was calculated. Turning movements that were not part of the upright movement were not scored. A total of 10 trials were recorded for each animal before surgery and on the designated day.

[0035] 2) The Adhesive Removal Test is an extremely important experimental method, which is mainly used to evaluate sensorimotor dysfunction and motor asymmetry in rodents. Due to its high sensitivity and objectivity in detecting changes in animal neurological function, it is widely used in research topics such as stroke, brain injury, neurodegenerative diseases, and the evaluation of the neuroprotective effect of new therapies. Gently place the animals that have adapted to the environment and completed training into a transparent test box (15cm×25cm), and let the animals move freely in the box for a period of time to adapt to the test environment, generally 2-3 minutes. Use tweezers to carefully stick the cut tape on the animal's front paws, one strip on each front paw. Note that the tape should be stuck in the same position to ensure the consistency of experimental conditions. For experiments studying sensorimotor dysfunction, focus on the removal of the tape on the contralateral front paw. Start timing from the completion of tape sticking and observe the behavior of the animal. Record the time when the animal first touches the tape with its mouth or other paw, that is, time to contact; continue to observe and record the time when the animal completely removes the tape, that is, time to removal. Each animal was tested 3-5 times, and the average value was taken as the experimental data of the animal.

[0036] like Figure 2 As shown in b, the compound represented by Formula I significantly improved the somatosensory and motor functions of tMCAO mice.

[0037] Example 5: Doppler cerebral blood flow detection

[0038] After the mice were anesthetized, the skin was prepared, disinfected, and the parietal bones were exposed to ensure that the laser spot was located 2 mm behind the anterior fontanelle and 6 mm from the midline. A low-frequency probe was used to transmit ultrasound to the intracranial blood vessels through the thinner part of the skull to detect the hemodynamic parameters of the middle cerebral artery, anterior cerebral artery, posterior cerebral artery, vertebral artery, basilar artery and other blood vessels.

[0039] like Figure 3 As shown, compared with the Sham group mice, the blood flow in the right cerebral region of the tMCAO group mice was significantly reduced, while the blood flow in the right cerebral region of the tMCAO mice was significantly increased after treatment with the compound shown in Formula I.

[0040] Example 6: TTC staining

[0041] After anesthesia, mice were decapitated and the brain tissue was quickly removed and placed on ice. The olfactory bulb and cerebellum were removed and frozen at -20°C. When the mouse brain tissue was frozen (it should not be completely frozen, as brain tissue edema causes high water content, and complete freezing easily causes the brain tissue to be chopped up during slicing), 2 mm thick coronal brain slices were cut continuously from the anterior pole to the posterior pole (6 slices can be cut). 1% TTC solution was prepared with PBS buffer, placed in a 37°C water bath, and the brain slices were immersed in 1% TTC solution for staining in the dark for 15 minutes. During this period, the staining was observed every 5 minutes and turned over in time. When the normal tissue was red and the infarcted area was white, the brain slices were removed and fixed in 4% paraformaldehyde solution overnight, and the slices were taken the next day. The volume of cerebral infarction was analyzed using Image pro plus 5.1 image analysis software.

[0042] like Figure 4 As shown, the compound represented by Formula I can significantly reduce the cerebral infarction volume of model mice, whether 1 day or 3 days after tMCAO modeling.

[0043] Example 7: Nissl staining

[0044] After the slices are returned to room temperature, rinse them in distilled water for 3 times, 5 minutes each time. Immerse the slices in tar violet staining solution for 3-5 minutes (observe the degree of staining under a microscope). After staining, wash the slices in distilled water several times, and then differentiate them in differentiation solution until the background is colorless. Dehydrate quickly with anhydrous ethanol, make xylene transparent, and seal the slices with glycerol.

[0045] like Figure 5 As shown, the compound represented by formula I can significantly reduce the neural damage of tMCAO model mice at 3 days. Figure 2-5 The results showed that the compound represented by formula I had a neuroprotective effect on ischemic stroke model mice.

[0046] Example 8: Tissue Immunofluorescence Staining

[0047] After the experimental animals were anesthetized, the mice required for the experiment were perfused with physiological saline and 4% paraformaldehyde through the left ventricle and systemic circulation. The brains were then decapitated and immersed in 4% paraformaldehyde for external fixation. After fixation, they were placed in a dehydrator for gradient dehydration, transparent and wax-impregnated, and then embedded in a paraffin embedding machine. The slices were then sliced ​​using a paraffin slicer, and 3 brain slices were attached to each slide. One was taken every ten, and the brain slices were dried in a 37°C oven overnight. When staining the slices, the wax was dissolved at 65°C for 1 hour, and after dewaxing with xylene and hydrating with gradient ethanol solution, the slices were placed in a citric acid buffer heated to boiling on high heat, and heated in a microwave oven for 15 minutes on medium heat for antigen repair. After cooling naturally at room temperature, the slices were blocked at room temperature for 1 hour using a blocking solution containing 0.1% Tween-20 and 5% goat serum. After absorbing the blocking solution, an equal volume of primary antibody solution was added to each brain slice and incubated at 4°C overnight. The next day, the primary antibody was recovered and washed three times with phosphate buffer solution (PBS), each time for 5 minutes. After washing, the prepared fluorescent secondary antibody was added dropwise, incubated at room temperature for 1 hour in the dark, and washed three times with PBS, each time for 5 minutes. Ready-to-use DAPI nuclear staining solution was added to each brain slice for sealing, and the corresponding protein indicators were observed under a fluorescence microscope and pictures were taken.

[0048] CD68 is used as a heterologous lysosomal marker and is co-labeled with microglia to reflect the phagocytic ability of microglia. Figure 6 As shown in a, 3 days after tMCAO modeling, the CD68 of microglia increased to a certain extent. However, after treatment with the compound shown in formula I, the expression of CD68 in microglia was significantly increased compared with the modeling group. This shows that the compound shown in formula I has the effect of promoting the phagocytosis of microglia after ischemic stroke. Neuron is a marker for neurons, and IBA1 is a marker for microglia. The two are co-localized and then subjected to three-dimensional reconstruction technology. The positional relationship between the two is analyzed (separation: No Contact; contact: Contact; phagocytosis: Engulfment) to observe the ability of microglia to clear neurons. As shown in Figure 6 As shown in b, on the third day of tMCAO modeling and reperfusion, the microglia in the infarct penumbra of the mouse brain tissue increased significantly and were activated. After treatment with the new structure compound, the number of neurons in the phagocytic state increased significantly compared with the tMCAO group. This indicates that the compound shown in Formula I can promote the phagocytosis and clearance of apoptotic neurons by microglia after ischemic stroke.

Claims

1. Use of the compound represented by formula I in the preparation of drugs for preventing and treating ischemic stroke, 2. The use according to claim 1, characterized in that The compound represented by formula I can penetrate the blood-brain barrier.

3. The use according to claim 1, characterized in that The compound represented by formula I can significantly improve the neurological function score and behavioral score after ischemic stroke.

4. The use according to claim 1, characterized in that The compound represented by formula I can significantly increase cerebral blood flow after ischemic stroke.

5. The use according to claim 1, characterized in that The compound represented by formula I can significantly reduce the volume of cerebral infarction after ischemic stroke.

6. The use according to claim 1, characterized in that The compound represented by formula I can significantly reduce neuronal damage after ischemic stroke.

7. The use according to claim 1, characterized in that The compound represented by formula I can promote microglial cells to phagocytize and eliminate apoptotic neurons after ischemic stroke.

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