Use of compound 78c in the preparation of a medicament for the treatment of ischemic cerebrovascular disease

Abstract

Use of compound 78c in preparation of drugs for treating ischemic cerebrovascular disease, the compound can reduce brain tissue damage and cell death caused by ischemic stroke, reduce the volume of cerebral infarction, improve the nerve function after ischemia. Compound 78c is used for treating ischemic cerebrovascular disease, including cerebral thrombosis, cerebral embolism, lacunar cerebral infarction, transient ischemic attack and cerebral ischemia reperfusion injury. Compound 78c can be used for preparing drugs for treating ischemic cerebrovascular disease, can be combined with various adjuvants, can be combined with various existing drugs for treating ischemic cerebrovascular disease, can be suitable for various dosage forms and various administration routes, so as to realize new, safe and effective drugs and effective treatment methods for reducing ischemic cerebrovascular disease, and have important clinical significance and social value.

Description

Technical Field

[0001] This invention relates to the application of compounds in the field of pharmaceutical technology for the treatment of ischemic cerebrovascular diseases, specifically to the application of compound 78c in the preparation of drugs for the treatment of ischemic cerebrovascular diseases. Background Technology

[0002] Ischemic cerebrovascular disease is a common disease of the central nervous system, with high mortality and disability rates, seriously endangering human health. The main cause of ischemic cerebrovascular disease is the blockage of cerebral blood vessels caused by various factors, leading to mechanical interruption of cerebral blood flow and causing cerebral ischemia. This results in insufficient oxygen and nutrient supply to neurons, and mitochondria lacking available oxygen and glucose, causing mitochondrial dysfunction and cell damage. The pathophysiological mechanism of secondary injury caused by cerebral ischemia is very complex, including a series of harmful cascade reactions in cells, such as excitatory neurotoxicity, intracellular Ca2+ overload, oxidative stress, inflammatory response, mitochondrial energy metabolism disorders, opening of mitochondrial membrane permeability transition pores, and apoptosis. These cascade reactions interact, amplifying the cell death signals activated by cerebral ischemia, ultimately leading to the death of nerve cells [de Miguel R, et al., Toxicol Pathol. 2025,53:657-662; Salaudeen MA, et al., Biomolecules.2024,14:305.]. Over the past few decades, although experimental studies have found that many compounds can protect the brain from ischemic stroke damage, the clinical trial results of almost all ischemic brain protectants have been disappointing [Chamorro Á, et al., J Neurol Neurosurg Psychiatry. 2021, 92: 129-135; Moretti A, et al., Pharmacol Ther. 2015, 146: 23-34.]. To date, apart from thrombolytic therapy and mechanical thrombectomy, there are few other effective treatments for ischemic stroke.

[0003] Nicotinamide adenine dinucleotide (NAD+) is a crucial biochemical substance involved in many important biological processes within cells. As a coenzyme of intracellular redox enzymes, NAD+ plays a central role in cellular metabolism and energy production [Hosseini L, et al., Biogerontology. 2019, 20:381-395; Owens K, et al., Transl Stroke Res. 2013, 4:618-634.]. As a substrate for NAD+-consuming enzymes, NAD+ can be hydrolyzed to produce nicotinamide and fragments containing ADP-ribose, which participate in the regulation of many intracellular biological processes, such as DNA repair, post-translational modifications of proteins, gene expression, cellular metabolism, mitochondrial function, inflammatory responses, and oxidative stress [Verdin E. Science. 2015, 350:1208-1213; Lautrup S, et al., Cell Metab. 2019, 30:630-655.].

[0004] CD38 is a multifunctional transmembrane glycoprotein. It not only acts as a cell surface adhesion receptor, binding to its ligand CD31 to promote the transendothelial migration of inflammatory cells in the blood and participate in the inflammatory response, but also possesses NAD+ enzymatic activity. It can cleave NAD+ to generate intracellular Ca2+ mobilization messenger rings (ADP-ribose), promoting the influx of extracellular Ca2+ and increasing intracellular Ca2+ levels. Excessive activation of CD38 can lead to intracellular NAD+ depletion, subsequently affecting the activity of other NAD+-consuming enzymes, such as the silencing signaling protein 2 family and poly(ADP-ribose) polymerase. Experimental studies have found that CD38 expression is upregulated after ischemic stroke, and CD38 gene knockout can reduce secondary damage caused by ischemic stroke [Verdin E. Science. 2015, 50:1208-1213; Covarrubias AJ, et al., Nat Rev Mol Cell Biol. 2021, 22:119-141; Rahi V,Kaundal RK. Life Sci. 2024, 347:122651; Takasawa S. Int J Mol Sci. 2022,23:4306]. However, there are currently no reports on whether CD38 inhibitors can reduce ischemic stroke damage.

[0005] The small molecule compound 78c (4-[[trans-4-(2-methoxyethoxy)cyclohexyl]amino]-1-methyl-6-(5-thiazolyl)quinoline-2(1H)-one, chemical formula C22H27N3O3S, CAS number 1700637-55-3) is a selective CD38 inhibitor. Studies have found that compound 78c can reverse age-related NAD+ decline, improve the motor ability, endurance and metabolic function of mice, and prolong the lifespan of naturally aging mice [Peclat TR, et al., Aging Cell. 2022, 21:e13589; Tarragó MG, et al., Cell Metab. 2018, 27:1081-1095.e10.]. Compound 78c can also inhibit the expression of CD38 and pro-inflammatory factors in macrophages and monocytes induced by myocardial ischemia-reperfusion, reduce inflammatory response, and improve cardiac function [Boslett J, et al., J Pharmacol Exp Ther. 2019, 369:55-64; Piedra-Quintero et al., Front Immunol. 2020,11:597959; Guerreiro S, et al.,Cells. 2020, 9:471.]. However, there are currently no reports in domestic or international literature regarding whether the CD38 inhibitor 78c also has a therapeutic effect on ischemic stroke.

[0006] In view of the shortcomings of the existing technologies, there is an urgent need to find new, safe, and effective drugs that can alleviate cerebral ischemia-reperfusion injury and apply them clinically. Through continuous research, design, and repeated prototype testing and improvements, the inventors have finally created this invention, which has real practical value. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects of the existing technology and provide a novel compound 78c for use in the preparation of drugs for treating ischemic cerebrovascular diseases. The technical problem to be solved is to enable it to be used in the preparation of drugs for treating ischemic cerebrovascular diseases, thereby realizing a novel, safe and effective drug and treatment method for ischemic cerebrovascular diseases that can reduce cerebral ischemia damage, which has important clinical significance and social value.

[0008] The concept of this invention stems from the discovery that compound 78c, as a specific CD38 inhibitor, can improve neurological deficits after ischemia, reduce neuronal cell death and brain tissue damage caused by ischemia, and decrease the volume of cerebral infarction, thus exhibiting a clear anti-cerebral ischemia effect. The main technical problem addressed by this invention is to provide the application of compound 78c in the preparation of drugs for treating ischemic cerebrovascular diseases.

[0009] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0010] The application of compound 78c proposed in this invention in the preparation of a drug for treating ischemic cerebrovascular disease, wherein the drug comprises compound 78c or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and at least one pharmaceutically acceptable carrier or excipient; the chemical name of compound 78c, which can be used in the treatment of ischemic cerebrovascular disease, is 4-[[trans-4-(2-methoxyethoxy)cyclohexyl]amino]-1-methyl-6-(5-thiazolyl)quinoline-2(1H)-one, and its chemical formula is C 22 H 27 N3O3S, CAS number 1700637-55-3.

[0011] Furthermore, the ischemic cerebrovascular diseases mentioned above include: cerebral thrombosis, cerebral embolism, lacunar infarction, transient ischemic attack, and cerebral ischemia-reperfusion injury.

[0012] Furthermore, compound 78c can be combined with one or more pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include: solid dosage form excipients, liquid dosage form excipients, and other functional excipients; wherein, solid dosage form excipients include: fillers and binders, liquid dosage form excipients include: solvents, antioxidants, preservatives, and pH adjusters, and other functional excipients include dispersants and emulsifiers.

[0014] Furthermore, the dosage forms of the drug include oral dosage forms and injectable dosage forms. Oral dosage forms include: powders, tablets, granules, capsules, solutions, emulsions, suspensions, and oral liquids. Injectable dosage forms include: injection solutions and powder injections.

[0015] Furthermore, the drug administration routes include: intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, subcutaneous injection and intradermal injection, oral administration, and sublingual administration.

[0016] Furthermore, the drug can be formulated into a compound preparation with existing drugs for treating ischemic cerebrovascular diseases to achieve synergistic effects or reduce side effects.

[0017] Furthermore, the existing drugs for treating ischemic cerebrovascular disease include thrombolytic drugs, drugs that improve cerebral blood circulation, drugs that lower plasma fibrinogen, anticoagulants, lipid-lowering drugs, and neuroprotective agents.

[0018] Furthermore, the drug is used in combination with revascularization therapy for ischemic cerebrovascular disease, which includes intravenous thrombolysis and / or mechanical thrombectomy, to reduce reperfusion injury and improve patient prognosis.

[0019] Furthermore, while keeping the core skeleton of compound 78c unchanged, structural modifications were made to compound 78c to obtain derivatives with similar activities.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: 1. First discovery of a new use, filling a technological gap: Compared with existing research on the use of compound 78c for anti-inflammatory, anti-aging and cognitive function improvement, this invention is the first to demonstrate the application of compound 78c in the treatment of ischemic cerebrovascular disease, which has outstanding substantive features and significant progress.

[0021] 2. In vivo efficacy verification with sufficient evidence: This invention directly demonstrated the protective effect of compound 78c on the ischemic brain using two gold indicators, namely neurological function and cerebral infarction volume, in a recognized rat model of ischemic stroke, providing a solid experimental foundation for subsequent technology transfer and clinical research.

[0022] 3. Potential Advantages of Multi-Target Action: In the brain, CD38 expression is found in neurons, astrocytes, microglia, and vascular endothelial cells. It is a multifunctional protein that not only acts as a cell surface adhesion receptor, participating in inflammatory responses, but also possesses NAD+ hydrolase and NAD+ cyclase activities. CD38 can cleave extracellular NAD+, reducing intracellular NAD+ levels and thus affecting the activity of other NAD+-consuming enzymes [such as histone deacetylase silencing signaling regulators 2 family and poly(ADP-ribose) polymerase], but also generate intracellular calcium mobilization messenger cyclic ADP-ribose, increasing intracellular calcium levels. Given its widespread distribution and complex functions in brain tissue, compound 78c, as a CD38 inhibitor, can exert neuroprotective effects by influencing multiple signaling pathways associated with ischemia-reperfusion injury (such as apoptosis, autophagy, inflammatory responses, and oxidative stress). Compared to single-target drugs, 78c has better therapeutic potential. This advantage has not been revealed by existing technologies.

[0023] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0024] Figure 1Compound 78c was shown to reduce neurological deficits in rats with transient ischemic stroke. Rats were subjected to focal cerebral ischemia for 1.5 hours, followed by reperfusion. Intraperitoneal injection of compound 78c at 10 minutes of ischemia reduced neurological deficits in rats at 1 day and 3 days after ischemia (i.e., 1.5 hours of ischemia, 22.5 hours of reperfusion, and 70.5 hours of reperfusion). Data are presented as mean ± SD. The number of animals per group was 8. Compared with the ischemic (solvable) control group (Veh), P < 0.01.

[0025] Figure 2 This study demonstrates the effects of compound 78c on brain swelling and infarct volume in rats with transient ischemic stroke. Rats were subjected to focal cerebral ischemia for 1.5 hours, followed by reperfusion for 70.5 hours. Intraperitoneal injection of compound 78c at 10 minutes of ischemia reduced brain swelling and decreased infarct volume in ischemic rats 3 days after ischemia (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion). Data are presented as mean ± SD. Each group consisted of 9 animals. Compared to the ischemic control group (Veh), P < 0.05 P < 0.0001. Wherein: Figure 2 Image a is a large coronal section HE-stained image of the ischemic (solvent) control group (Veh) and the compound 78c treatment group, showing the cerebral infarction foci.

[0026] Figure 2 b shows brain tissue swelling in rats in the ischemic (solvent) control group (Veh) and the compound 78c treatment group.

[0027] Figure 2 c shows the cerebral infarction volume in rats in the ischemic (solvent) control group (Veh) and the compound 78c treatment group.

[0028] Figure 3 This is an HE-stained image of a coronal section of the brain through the caudate putamen, showing the effect of intraperitoneal injection of compound 78c 10 minutes after ischemia on tissue damage and cell death in different brain regions of rats with ischemic stroke. The rats were subjected to focal cerebral ischemia for 1.5 hours, followed by reperfusion for 70.5 hours. Original magnification was 1000. 10. In the ischemic (solvent) control group (Veh), moderate to severe tissue damage and cell death were observed in the frontoparietal cortex somatosensory area (FrPaSS) and the lateral part of the caudate putamen (Cpu-L), while mild to moderate tissue damage and cell death were observed in the frontoparietal cortex motor area (FrPaM) and the medial part of the caudate putamen (Cpu-M). Intraperitoneal injection of compound 78c after 10 minutes of ischemia reduced the brain tissue damage and cell death in the frontoparietal cortex somatosensory area, frontoparietal cortex motor area, lateral part of the caudate putamen, and medial part of the caudate putamen in ischemic rats after 3 days of ischemic injury (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion). FrPaM: Frontoparietal motor area. FrPaSS: Frontoparietal somatosensory area. Cpu-L: Lateral part of the caudate putamen. Cpu-M: Medial part of the caudate putamen.

[0029] Figure 4 This is a semi-quantitative result on the effects of compound 78c on tissue damage and cell death in different brain regions after ischemic stroke. Rats were subjected to focal cerebral ischemia for 1.5 hours, followed by reperfusion for 70.5 hours. Intraperitoneal injection of compound 78c at 10 minutes of ischemia reduced tissue damage and cell death in the somatosensory and motor cortex, as well as the lateral and medial caudate putamen, 3 days after ischemia (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion). Data are presented as mean ± SD. Each group consisted of 8 animals. Compared with the ischemic (solvable) control group (Vehicle), P < 0.05, P < 0.01. FrPaM: Frontoparietal motor cortex; FrPaSS: Frontoparietal somatosensory cortex; Cpu(L): Lateral part of the caudate putamen; Cpu(M): Medial part of the caudate putamen.

[0030] Figure 5 These are images of cresol purple-stained sections of the brain corona via the caudate putamen and semi-quantitative results of neuronal cell death in the cortex and caudate putamen. The rats underwent focal cerebral ischemia for 1.5 hours followed by reperfusion. Among them: Figure 5 'a' is a schematic diagram of the neuronal death index classification.

[0031] Figure 5 b shows the neuronal death index in the cortex and caudate putamen 3 days after ischemic injury (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion). Intraperitoneal injection of compound 78c at 10 minutes of ischemia reduced neuronal death in the cortex and caudate putamen of rats 3 days after ischemic injury (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion). Data are presented as mean ± SD. Each group consisted of 7 animals. Compared with the ischemic (solvent) control group (Vehicle), P < 0.01. Detailed Implementation

[0032] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and preferred embodiments, details the application, features, and effects of compound 78c proposed according to the present invention in the preparation of drugs for treating ischemic cerebrovascular diseases.

[0033] Compound 78c, in the preparation of drugs for treating ischemic cerebrovascular diseases, includes compound 78c or its pharmaceutically acceptable salts, hydrates, solvates or stereoisomers, and at least one pharmaceutically acceptable carrier or excipient; the chemical name of compound 78c, which can be used in the treatment of ischemic cerebrovascular diseases, is: 4-[[trans-4-(2-methoxyethoxy)cyclohexyl]amino]-1-methyl-6-(5-thiazolyl)quinoline-2(1H)-one, with the chemical formula C 22 H 27 N3O3S, CAS number 1700637-55-3.

[0034] The ischemic cerebrovascular diseases include: cerebral thrombosis, cerebral embolism, lacunar infarction, transient ischemic attack, and cerebral ischemia-reperfusion injury.

[0035] Compound 78c is combined with one or more pharmaceutically acceptable excipients. These excipients include solid dosage form excipients, liquid dosage form excipients, and other active pharmaceutical ingredients.

[0036] Solid dosage form excipients include fillers and binders. Specific fillers (i.e., diluents) include lactose, microcrystalline cellulose, starch, powdered sugar, dextrin, mannitol, and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, and calcium carbonate; hydroxypropyl methylcellulose and povidone are also included. Specific binders include hydroxypropyl methylcellulose and povidone.

[0037] Liquid formulation excipients include solvents, preservatives, and pH adjusters. Specific solvents include water, ethanol, and glycerin.

[0038] Specific antioxidants, such as vitamin C (ascorbic acid) and vitamin E, are used to prevent drug components from being oxidized and deteriorated; specific preservatives, such as sodium benzoate and potassium sorbate, can inhibit the growth of microorganisms and extend the shelf life of drugs; specific pH adjusters, such as citric acid and sodium hydroxide, are used to adjust and maintain the pH value of the solution within a suitable range to maintain drug stability and reduce irritation to body tissues.

[0039] Other excipients include dispersants and emulsifiers, which can increase the solubility of drugs in water, improve the bioavailability of drugs, and make the drugs more effective. Excipients such as oxalates can also increase the bioavailability of oral drugs.

[0040] The dosage forms of the drugs include oral dosage forms and injectable dosage forms. Oral dosage forms include powders, tablets, granules, capsules, solutions, emulsions, suspensions, and oral liquids. Injectable dosage forms include injectable solutions and powder injections.

[0041] The routes of administration of the drug include: intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, subcutaneous injection and intradermal injection, oral administration, and sublingual administration.

[0042] Compound 78c can be formulated into a combination drug with existing drugs for treating ischemic cerebrovascular diseases to achieve synergistic effects or reduce side effects.

[0043] The existing drugs for treating ischemic cerebrovascular disease include: thrombolytic drugs, drugs that improve cerebral blood circulation, drugs that lower plasma fibrinogen, anticoagulants, lipid-lowering drugs, and neuroprotective agents.

[0044] The drug is used in combination with revascularization therapy for ischemic cerebrovascular disease. Specific revascularization therapy includes intravenous thrombolysis and / or mechanical thrombectomy to reduce reperfusion injury and improve patient prognosis.

[0045] In the treatment of ischemic cerebrovascular disease, derivatives with similar activities are obtained by structurally modifying compound 78c while keeping the core skeleton of compound 78c unchanged.

[0046] The most important indicator for evaluating the effectiveness of an ischemic brain protectant is its ability to reduce infarct size and improve neurological function. This invention, for the first time at the whole-animal level using a rat model of ischemic stroke, demonstrates that the CD38 inhibitor compound 78c has a clear anti-cerebral ischemia effect. It can reduce brain tissue damage and cell death in ischemic areas, reduce cerebral infarction foci in ischemic rats, and improve neurological function in ischemic rats. These results indicate that the CD38 inhibitor compound 78c is an effective ischemic brain protectant and can be developed into an effective treatment for ischemic cerebrovascular diseases.

[0047] This invention demonstrates the protective effect of CD38 inhibitor compound 78c against ischemic stroke through the following experiments.

[0048] Example 1 1. Experimental Materials (1) Source of experimental animals: Healthy adult male Sprague-Dawley (SD) rats, weighing 260-300g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the SPF-grade animal room of the Beijing Institute of Neurosurgery, in a 12-hour light-dark alternating environment, with free access to food and water.

[0049] (2) Drug: Compound 78c, purity > 99%, purchased from Selleck Chemicals, catalog number: S8960. Before use, it was prepared into a high-concentration stock solution (10 mg / ml) with dimethyl sulfoxide, and diluted with PEG300 to the required concentration (5 mg / ml) immediately before use.

[0050] (3) Main reagents: isoflurane (anesthetic), etc.

[0051] (4) Main equipment: nylon thread, surgical microscope, small animal anesthesia machine, bipolar electrocoagulator, constant temperature heating pad, etc.

[0052] 2. Experimental Methods Establishment of a rat model of ischemic stroke: All experimental protocols complied with the requirements of the "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Ethics Committee of the Beijing Neurosurgical Institute. A rat model of ischemic stroke was established by endovascular ligation of the middle cerebral artery. The procedure is briefly described below: Anesthesia was induced with 5% isoflurane and maintained with 3% isoflurane. A constant temperature blanket was used to maintain the rectal temperature at 37±0.5℃. Rats were fixed supine on a rat board. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The ECA branches were electrocoagulated and ligated, and the pterygopalatine artery, a branch of the ICA, was ligated. The distal end of the ECA was ligated, and the CCA and ICA were closed with arterial clamps. A suture was threaded through the proximal end of the ECA. A small incision was made in the ECA wall near the ligated end, and a nylon suture was inserted to ligate the proximal end of the ECA. The arterial clamps were then released. A nylon suture is slowly inserted through the ECA and ICA, about 2.2 cm (measured from the ECA incision), until resistance is encountered. The suture is then released and gently springs back to block the middle cerebral artery. After 1.5 hours of ischemia, the nylon suture is withdrawn, and reperfusion is initiated.

[0053] Animal grouping: Ischemia (solvent) control group: The solvent (DMSO + PEG300, DMSO:PEG300 = 1:1, 2ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice a day (6 hours apart) for three consecutive days.

[0054] Compound 78c treatment group: Compound 78c (dissolved in DMSO + PEG300, DMSO:PEG300 = 1:1, 5 mg / ml, 2 ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice daily (6 hours apart) for three consecutive days.

[0055] Evaluation of neurological deficits: The neurological function of rats with ischemic injury was evaluated from the aspects of motor function, sensory function and reflexes at 1 day and 3 days after ischemic injury (i.e., 1.5 hours of ischemia, 22.5 hours and 70.5 hours of reperfusion). A modified neurological deficit scale was used to evaluate the neurological function of rats with ischemic injury at 1 day and 3 days after ischemic injury (i.e. 1.5 hours of ischemia, 22.5 hours and 70.5 hours after reperfusion).

[0056] 3. Experimental Results A rat model of ischemic stroke was successfully established. Before ischemia, all rats had normal neurological function with no significant neurological deficits. The ischemic (solvent) control group rats showed significant neurological deficits at 1 and 3 days after ischemic injury (i.e., 1.5 hours of ischemia, 22.5 hours of reperfusion, and 70.5 hours of reperfusion). Intraperitoneal injection of compound 78c at 10 minutes of ischemia significantly reduced the neurological deficits in ischemic rats at 1 and 3 days after ischemic injury (i.e., 1.5 hours of ischemia, 22.5 hours of reperfusion, and 70.5 hours of reperfusion). Figure 1 ).

[0057] Example 2 1. Experimental Materials (1) Source of experimental animals: Healthy adult male Sprague-Dawley (SD) rats, weighing 260-300g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the SPF-grade animal room of the Beijing Institute of Neurosurgery, in a 12-hour light-dark alternating environment, with free access to food.

[0058] (2) Drug: Compound 78c, purity > 99%, purchased from Selleck Chemicals, catalog number: S8960. Before use, it was prepared into a high-concentration stock solution (10 mg / ml) with dimethyl sulfoxide, and diluted with PEG300 to the required concentration (5 mg / ml) immediately before use.

[0059] (3) Main reagents: isoflurane (anesthetic), paraformaldehyde, hematoxylin staining solution, eosin staining solution, phosphate buffer, etc.

[0060] (4) Main equipment: nylon thread, surgical microscope, small animal anesthesia machine, bipolar electrocoagulator, constant temperature heating pad, electronic balance, paraffin slicer, optical microscope, image analyzer (Beijing Konghai Company), etc.

[0061] 2. Experimental Methods Establishment of a rat model of ischemic stroke: Same as in Example 1.

[0062] Animal grouping: Ischemia (solvent) control group: The solvent (DMSO + PEG300, DMSO:PEG300 = 1:1, 2ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice a day (6 hours apart) for three consecutive days.

[0063] Compound 78c treatment group: Compound 78c (dissolved in DMSO + PEG300, DMSO:PEG300 = 1:1, 5 mg / ml, 2 ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice daily (6 hours apart) for three consecutive days.

[0064] Measurement of brain tissue swelling and infarct volume: Three days after ischemic injury (i.e., 1.5 hours of ischemia followed by 70.5 hours of reperfusion), rats were anesthetized, perfused with physiological saline, and then perfused with 4% paraformaldehyde solution. The rats were decapitated, and the entire brain was removed and fixed in 4% paraformaldehyde solution. Coronal sections of the rat brain were prepared, starting from the scalp, with a thickness of 2 mm per section, for a total of 6 sections. These sections were embedded in paraffin, sectioned, and two sections of 5 mm thickness were prepared from each section. m, routine HE staining, and image analysis methods were used to determine the volume of the left (non-ischemic side) cerebral hemisphere (V1), the volume of normal tissue in the right (ischemic side) cerebral hemisphere (V2), and the volume of diseased tissue in the right (ischemic side) cerebral hemisphere (V3). The calculation formula is as follows: V1= A1 d. Among them, A1 represents the sum of the areas of all slices in the left hemisphere (6 slices in total), and d represents the thickness of the tissue block (2mm).

[0065] V2= A2 d. Among them, A2 represents the sum of the normal tissue areas of all sections in the right hemisphere (6 sections in total), and d represents the thickness of the tissue block (2 mm).

[0066] V3= A3 d. Among them, A3 represents the sum of the infarct areas in the right hemisphere (6 slices in total), and d represents the thickness of the tissue block (2 mm).

[0067] Infarct volume (V) = Volume of the left (non-ischemic) cerebral hemisphere (V1) - Volume of normal tissue in the right (ischemic) cerebral hemisphere (V2), expressing the infarct volume as a percentage of the volume of the contralateral normal cerebral hemisphere. Brain tissue swelling volume = Volume of diseased tissue in the right (ischemic) cerebral hemisphere (V3) - Infarct volume (V).

[0068] 3. Experimental Results Three days after ischemic injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion), obvious cerebral infarction foci were visible in the right cerebral hemisphere cortex and caudate putamen region of the ischemic (solvent) control rats (Figure 2a). Administration of compound 78c 10 minutes after ischemia significantly reduced brain tissue swelling in ischemic rats after 3 days of ischemic injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion). Figure 2 b), reducing the infarct volume in ischemic rats ( Figure 2 c).

[0069] Example 3 1. Experimental Materials (1) Source of experimental animals: Healthy adult male Sprague-Dawley (SD) rats, weighing 260-300g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the SPF-grade animal room of the Beijing Institute of Neurosurgery, in a 12-hour light-dark alternating environment, with free access to food and water.

[0070] (2) Drug: Compound 78c, purity > 99%, purchased from Selleck Chemicals, catalog number: S8960. Before use, it was prepared into a high-concentration stock solution (10 mg / ml) with dimethyl sulfoxide, and diluted with PEG300 to the required concentration (5 mg / ml) immediately before use.

[0071] (3) Main reagents: isoflurane (anesthetic), paraformaldehyde, hematoxylin staining solution, eosin staining solution, phosphate buffer, etc.

[0072] (4) Main equipment: nylon thread, surgical microscope, small animal anesthesia machine, bipolar electrocoagulator, constant temperature heating pad, electronic balance, paraffin microtome, optical microscope, etc.

[0073] 2. Experimental Methods Establishment of a rat model of ischemic stroke: Same as in Example 1.

[0074] Animal grouping: Ischemia (solvent) control group: The solvent (DMSO + PEG300, DMSO:PEG300 = 1:1, 2ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice a day (6 hours apart) for three consecutive days.

[0075] Compound 78c treatment group: Compound 78c (dissolved in DMSO + PEG300, DMSO:PEG300 = 1:1, 5 mg / ml, 2 ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice daily (6 hours apart) for three consecutive days.

[0076] Evaluation of tissue damage and cell death in different brain regions: Three days after ischemic injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion), rats were anesthetized and perfused sequentially with 200 ml of physiological saline and 200 ml of 4% paraformaldehyde via the heart. The rats were then decapitated, and the brain tissue was collected. The tissue was fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. Coronal sections of the brain tissue passing through the rat's caudate putamen were collected, with a section thickness of 5 mm. m. Sections were stained with hematoxylin and eosin (HE) and observed under a light microscope for tissue damage and cell death caused by ischemic stroke. Cell death in the frontoparietal motor cortex (supply area of ​​the anterior cerebral artery), the frontoparietal somatosensory cortex (supply area of ​​the middle cerebral artery), the lateral part of the caudate putamen, and the medial part of the caudate putamen was graded as follows: Grade 0: <5% cell death; Grade 1: 5-25% cell damage; Grade 2: 25-50% cell death; Grade 3: 50-75% cell death; Grade 4: more than 75% cell death.

[0077] Experimental results Three days after cerebral ischemia (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion), severe tissue damage and cell death were observed in the somatosensory cortex of the frontoparietal cortex and the lateral part of the caudate putamen of ischemic rats. Mild to moderate tissue damage and cell death were observed in the motor cortex of the frontoparietal cortex and the medial part of the caudate putamen of rats. Figure 3 The tissue damage and cell death caused by ischemic stroke are mainly manifested as follows: under light microscopy, obvious patchy softening lesions are visible, tissue structure is lost, cells within the lesions show eosinophilic degeneration, cell shrinkage, fan-shaped vacuolation changes around the cells, nuclear pyknosis and deep staining, nucleolus disappearance, shadow cells appear, interstitium is loose, and edema is obvious. Intraperitoneal injection of compound 78c 10 minutes after ischemia can significantly reduce the damage to the frontoparietal motor cortex and somatosensory cortex, as well as the lateral and medial parts of the caudate putamen, brain tissue 3 days after ischemic injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion). Figure 3 and Figure 4 ).

[0078] Example 4 1. Experimental Materials (1) Source of experimental animals: Healthy adult male Sprague-Dawley (SD) rats, weighing 260-300g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the SPF-grade animal room of the Beijing Institute of Neurosurgery, in a 12-hour light-dark alternating environment, with free access to food and water.

[0079] (2) Drug: Compound 78c, purity > 99%, purchased from Selleck Chemicals, catalog number: S8960. Before use, it was prepared into a high-concentration stock solution (10 mg / ml) with dimethyl sulfoxide, and diluted with PEG300 to the required concentration (5 mg / ml) immediately before use.

[0080] (3) Main reagents: isoflurane (anesthetic), paraformaldehyde, cresol purple staining solution, phosphate buffer, etc.

[0081] (4) Main equipment: nylon thread, surgical microscope, small animal anesthesia machine, bipolar electrocoagulator, constant temperature heating pad, electronic balance, paraffin microtome, optical microscope, etc.

[0082] 2. Experimental Methods Establishment of a rat model of ischemic stroke: Same as in Example 1.

[0083] Animal grouping: Ischemia (solvent) control group: The solvent (DMSO + PEG300, DMSO:PEG300 = 1:1, 2ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice a day (6 hours apart) for three consecutive days.

[0084] Compound 78c treatment group: Compound 78c (dissolved in DMSO + PEG300, DMSO:PEG300 = 1:1, 5 mg / ml, 2 ml / kg) was administered intraperitoneally 10 minutes after ischemia, twice daily (6 hours apart) for three consecutive days.

[0085] Evaluation of neuronal cell death: Cresol purple staining is mainly used to display Nissl substance in neuronal cells. A decrease or disappearance of Nissl substance is a sensitive indicator of neuronal damage; therefore, cresol purple staining is commonly used to observe neuronal death. Three days after ischemic injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion), rats were anesthetized and perfused sequentially with 200 ml of physiological saline and 200 ml of 4% paraformaldehyde via the heart. The rats were decapitated, and the brain tissue was collected, fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. Coronal sections of the brain passing through the rat's caudate putamen were collected, with a section thickness of 5 mm. m. Sections were stained with cresol purple and Nissl material in nerve cells were observed under a light microscope to evaluate the cell death index in the ischemic cortex and caudate putamen: Grade 0: <5% cell death; Grade 1: 5-25% cell damage; Grade 2: 25-50% cell death; Grade 3: 50-75% cell death; Grade 4: more than 75% cell death.

[0086] Experimental results Cresol violet staining revealed significant neuronal death in the cortex and caudate nucleus of ischemic rats 3 days after cerebral ischemia (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion). Damaged or dead neurons were characterized by cell shrinkage, fan-shaped vacuolation around the cell periphery, reduced or absent Nissl substance within the cell, deeply stained pyknoid nuclei, disappearance of nucleoli, shadow cells, significant edema, and loss of tissue structure. Figure 5a). Intraperitoneal injection of compound 78c 10 minutes after ischemia significantly reduced the death of neurons in the cortex and caudate putamen 3 days after cerebral ischemia injury (i.e., 1.5 hours of ischemia / 70.5 hours of reperfusion). Figure 5 b).

[0087] Based on the above experimental results, the CD38 inhibitor compound 78c has a clear protective effect against ischemic stroke, and therefore it can be developed into a new ischemic brain protection drug.

[0088] This invention, using a rat model of ischemic stroke (middle cerebral artery occlusion / reperfusion), demonstrates for the first time the neuroprotective effect of compound 78c. Experimental results show that administration of compound 78c significantly improves neurological function in rats with ischemic stroke, reduces brain tissue damage and cell death in the ischemic brain region, and significantly reduces infarct volume. This indicates that compound 78c has a clear therapeutic effect on experimental ischemic stroke, thus providing a new candidate drug for the treatment of ischemic cerebrovascular diseases. This invention solves the technical problem of applying compound 78c in the treatment of ischemic cerebrovascular diseases.

[0089] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the scope of the invention. Any person skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. The application of compound 78c in the preparation of drugs for treating ischemic cerebrovascular diseases, characterized in that, The drug comprises compound 78c or a pharmaceutically acceptable salt, hydrate, solvate, or stereoisomer thereof, and at least one pharmaceutically acceptable carrier or excipient; the chemical name of compound 78c is 4-[[trans-4-(2-methoxyethoxy)cyclohexyl]amino]-1-methyl-6-(5-thiazolyl)quinoline-2(1H)-one, and the chemical formula is C 22 H 27 N3O3S, CAS number 1700637-55-3.

2. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The ischemic cerebrovascular diseases include: cerebral thrombosis, cerebral embolism, lacunar infarction, transient ischemic attack, and cerebral ischemia-reperfusion injury.

3. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, Compound 78c is combined with one or more pharmaceutically acceptable excipients, said excipients including: fillers, binders, solvents, antioxidants, preservatives, pH adjusters, dispersants or emulsifiers.

4. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The dosage forms of the drugs include powders, tablets, granules, capsules, solutions, emulsions, suspensions, oral liquids, injections, and powder injections.

5. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The routes of administration of the drug include intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, subcutaneous injection, intradermal injection, oral administration, and sublingual administration.

6. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The drug is formulated into a compound preparation with existing drugs for treating ischemic cerebrovascular diseases, enabling combined use.

7. The use of compound 78c according to claim 6 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The existing drugs for treating ischemic cerebrovascular disease include thrombolytic drugs, drugs that improve cerebral blood circulation, drugs that lower plasma fibrinogen, anticoagulants, lipid-lowering drugs, and neuroprotective agents.

8. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, The drug is used in combination with revascularization therapy for ischemic cerebrovascular disease, which includes intravenous thrombolysis and / or mechanical thrombectomy, to reduce reperfusion injury and improve patient prognosis.

9. The use of compound 78c according to claim 1 in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that, Derivatives with similar activities were obtained by structurally modifying compound 78c while keeping the core skeleton unchanged.

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