Pharmaceutically acceptable salts of compounds and methods for preparing same

Abstract

The present invention relates to a base addition salt of the compound of formula (I) and crystalline forms of the salt, methods for preparing and using the salt, and pharmaceutical compositions containing a therapeutically effective amount of the salt or crystalline form, and their use for treating diseases associated with ischemic brain injury, spinal cord injury, and neurodegenerative diseases. [Formula 1] TIFF2026503034000035.tif75170

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and more particularly to pharmaceutically acceptable salts, crystalline forms and pharmaceutical compositions of compounds that have anti-cerebral ischemia, anti-post-ischemic inflammation and anticonvulsant effects and can improve neurological dysfunction in patients with ischemic stroke, spinal cord injury and neurodegenerative diseases, as well as methods for preparing the salts and crystalline forms. [Background technology]

[0002] White matter is an important component of the central nervous system and is the site of nerve fiber accumulation. White matter lesions (WMLs) are usually caused by reduced blood flow or insufficient blood oxygen supply. Impaired (insufficient) cerebral blood supply makes it difficult to meet the metabolic demands of brain tissue, resulting in a series of symptoms. Clinically, symptoms include dizziness, headache, numbness in the limbs, or transient loss of consciousness. In severe cases, irreversible damage to brain function can occur, even leading to death. Diseases associated with cerebral ischemia include transient ischemic attack (TIA), ischemic stroke (cerebral infarction), moyamoya disease, and chronic cerebral blood insufficiency, which are also one of the causes of cognitive decline and vascular dementia in patients.

[0003] Spinal cord injuries (SCI) are one of the diseases with the highest rate of physical disability in modern society, and are often accompanied by symptoms such as sensory impairment and loss of motor function in the limbs. Spinal cord injury is damage to the central nervous system, and the nervous system is a tissue that cannot regenerate, making it difficult to recover from functional impairment caused by nerve damage. Traumatic brain injury is the main cause of cognitive impairment in patients with spinal cord injury. Spinal cord injury not only causes serious physical and psychological harm to the patient, but also places a significant economic burden on society as a whole.

[0004] Parkinson's disease (PD) is a common neurodegenerative disorder that has become a serious problem worldwide. Typical clinical features of PD include motor symptoms such as motor retardation, resting tremor, and postural instability. The underlying cause of PD is the loss, damage, and death of midbrain dopaminergic neurons in patients. The current standard treatment is dopamine replacement therapy, but this is a symptomatic treatment, not a fundamental treatment, and has significant side effects.

[0005] At present, there are many drugs for treating diseases caused by central nervous system damage, but not many are truly effective.Traditional drug nimodipine has preventive effect on cerebral ischemia, but its therapeutic effect is not certain.Cranial nerve damage and necrosis cause the dysfunction of corresponding systems in the human body, and greatly reduce the quality of life of patients.Currently, there is still a need to provide other compounds with better therapeutic effect, anti-cerebral ischemia, anti-ischemic post-inflammation and anticonvulsant effects, can improve the neurological dysfunction in ischemic stroke patients, neurological dysfunction in spinal cord injury patients and cognitive dysfunction in neurodegenerative diseases, and can improve memory, protect nerve cells and blood-brain barrier, etc.

[0006] The present inventors developed the compound (Z)-3-(1-hydroxybutenyl)benzofuran-2-one of formula (I) for the treatment of ischemic brain injury, spinal cord injury, and neurodegenerative diseases. In subsequent research and development, they sought a product that was easy to store and stable for a long period of time, in order to facilitate the handling, filtration, and drying of the product. The present invention involves comprehensive research into the salts of the above substance, in order to obtain the optimal salt form to ensure the quality, safety, efficacy, and application of the drug.

[0007] [ka] Summary of the Invention

[0008] The present invention provides pharmaceutically acceptable salts, crystalline forms, and preparation methods of compounds that have anti-cerebral ischemia, anti-post-ischemic inflammation, and anticonvulsant effects and can improve neurological dysfunction in patients with ischemic stroke, neurological dysfunction in patients with spinal cord injury, and cognitive dysfunction in patients with neurodegenerative diseases, etc.

[0009] The present invention provides pharmaceutically acceptable salts of compounds of structural formula (I): [ka]

[0010] In the present invention, the pharmaceutically acceptable salt is selected from the group consisting of sodium salts, potassium salts, magnesium salts, calcium salts, piperazine salts, ethanolamine salts, meglumine salts, and tromethamine salts, among which the pharmaceutically acceptable salt is selected from the monohydrate, dihydrate, trihydrate, and hemihydrate of the salt, and the salt is selected from the group consisting of sodium salts, potassium salts, magnesium salts, calcium salts, piperazine salts, ethanolamine salts, meglumine salts, and tromethamine salts.

[0011] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I), which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one sodium salt.

[0012] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one sodium monohydrate.

[0013] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one potassium salt.

[0014] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one magnesium salt.

[0015] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one calcium salt.

[0016] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one piperazine salt.

[0017] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one ethanolamine salt.

[0018] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one meglumine salt.

[0019] The present invention further provides a pharmaceutically acceptable salt of the compound of formula (I) which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-one tromethamine salt.

[0020] The present invention further provides a process for preparing the above-mentioned pharmaceutically acceptable salt, which comprises the step of forming a salt between a compound of formula (I) and a base.

[0021] The solvent used in the further salt-forming reaction is at least one selected from methanol, ethanol, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, acetonitrile, isopropanol, acetone, isopropanol, tetrahydrofuran, methyl isobutyl ketone, n-heptane, dichloromethane, toluene, isopropyl ether, methyl t-butyl ether, n-butanol, and water.

[0022] The present invention further provides a pharmaceutical composition comprising a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0023] The present invention further provides the use of a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition.

[0024] The present invention further provides a use of the composition according to the present invention in the preparation of a medicament for treating and / or preventing diseases associated with ischemic brain injury.

[0025] Diseases associated with ischemic brain damage according to the present invention include, but are not limited to, cerebral ischemia, ischemic stroke, vascular dementia, post-ischemic inflammation, convulsion, ischemic cranial nerve damage or necrosis, etc.

[0026] The present invention further provides the use of a composition according to the present invention in the preparation of a medicament for the treatment and / or prevention of a neuromuscular disease.

[0027] The neuromuscular disease according to the present invention is amyotrophic lateral sclerosis (ALS).

[0028] The present invention further provides the use of a composition according to the present invention in the preparation of a medicament for the treatment and / or prevention of a disease associated with neurological dysfunction.

[0029] Diseases associated with neurological dysfunction according to the present invention include, but are not limited to, spinal cord injury or necrosis.

[0030] The present invention further provides the use of a composition according to the present invention in the preparation of a medicament for the treatment and / or prevention of a neurodegenerative disease.

[0031] Neurodegenerative diseases according to the present invention include, but are not limited to, Parkinson's disease.

[0032] The pharmaceutically acceptable salts of the compounds described in the present invention are easy to handle, filter and dry, stable for long-term storage, and suitable for industrial development to ensure the quality, safety and efficacy of the drug. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a differential scanning calorimetry (DSC) diagram of the sodium salt monohydrate. [Figure 2] FIG. 1 is a thermogravimetric analysis (TGA) diagram of the sodium salt monohydrate. [Figure 3] 16 shows the effect of the tromethamine salt of Example 10 on survival of SOD1 G93A ALS mice. [Figure 4] 1 shows the results of the bar lift test on the MPTP-induced mouse Parkinson's model using tromethamine salt of Example 10. (Note: In the figure, all data are shown as mean ± standard error of mean (SEM). &P<0.05 indicates that the difference compared to the normal control group is statistically significant, and #P<0.05 indicates that the difference compared to the model control group is statistically significant.) [Figure 5] 1 shows the results of grip strength detection for the tromethamine salt of Example 10 in an MPTP-induced mouse Parkinson's model. (Note: In the figure, all data are shown as mean ± standard error (SEM). P<0.05 indicates that the difference compared to the normal control group is statistically significant.) [Figure 6] These are the results of detecting DOPA in the striatum. (Note: In the figure, all data are shown as mean ± standard deviation (SEM). &P<0.05 indicates that the difference compared to the normal control group is statistically significant, and #P<0.05 indicates that the difference compared to the model control group is statistically significant.) [Figure 7] Statistical results of the TH-positive fiber content in the striatum. (Note: Panel A is a 2.5x microscope field image of TH immunohistochemical staining in the mouse striatum. Positive results appear brown. Panel B is a statistical result of the TH-positive area in the mouse striatum. Three sections were collected for each animal, and all data in the figure are shown as mean ± standard error of the mean (SEM). P<0.05 indicates a statistically significant difference compared to the normal control group. P<0.05 indicates a statistically significant difference compared to the model control group.) [Figure 8]Statistical results for the number of TH-positive cells in the substantia nigra pars compacta. (Note: Panel A is a 10x microscope field image of TH immunofluorescent staining in the mouse striatum. Panel B is a statistical result of the TH-positive area in the mouse striatum. Three sections were collected for each animal, and all data in the figure are shown as mean ± standard error (SEM). &P<0.05 indicates that the difference compared to the normal control group is statistically significant, and #P<0.05 indicates that the difference compared to the model control group is statistically significant.) [Figure 9] 1 shows examples (50x, 200x) of the effect of the tromethamine salt of Example 10, a test compound, on pathological changes. [Figure 10] 1 is a diagram showing the effect of the tromethamine salt of Example 10, a test compound, on NF200 (400×). [Figure 11] GSEA analysis of the test compound tromethamine salt of Example 10 on the injured spinal cord transcriptome. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be further described below with reference to specific examples based on general technical knowledge and conventional means in this field. The following examples are only some of the preferred embodiments of the present invention and should not be construed as limiting the present invention. Those skilled in the art can make some modifications without departing from the scope of the present invention, and these modifications should also be construed as falling within the scope of the present invention.

[0035] Example 1: Preparation of (Z)-3-(1-hydroxybutenyl)benzofuran-2-one [ka]

[0036] 3-Benzofuranon (5.0 g, 37.3 mmol, 1.0 eq) was dissolved in dichloromethane (50 mL) and cooled to 5°C. Potassium tert-butoxide (6 g, 53.5 mmol, 1.4 eq) was slowly added and the mixture was allowed to react for 0.5 hours. After that, n-butyryl chloride (8 g, 75.0 mmol, 2.0 eq) was slowly added and the mixture was allowed to react for 1 hour. The mixture was then extracted with water (50 mL). The organic phase was separated and washed with 0.1 N hydrochloric acid under acidic conditions (pH < 2). The organic phase was dried over anhydrous sodium sulfate (5 g) for 0.5 hours, filtered, and concentrated to obtain an oil. The product was then purified using high-pressure preparative chromatography. The purified solution was lyophilized to obtain the compound of formula (I) ((Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one (2.5 g, 32.9%) as a white solid.

[0037] 1 H-NMR (CDCl3, 400MHz): δ12.02(s,1H),7.35-7.33(d,1H),7.28-7.17(m,3H),2.76-2.73(m,2H),1.90-1.80(m,2H),1.12-1.08(m,3H).

[0038] 1 H-NMR (CDCl3+D2O, 400MHz): δ7.34-7.32(d,1H),7.28-7.16(m,3H),2.76-2.72(m,2H),1.89-1.80(m,2H),1.12-1.08(m,3H).

[0039] Example 2: Preparation of the sodium salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (10 g, 0.05 mol, 1.0 eq) was dissolved in methanol (100 mL). After dissolution, sodium hydroxide (1.95 g, 0.05 mol, 1.0 eq) was added, and the mixture was stirred for 1 hour and then concentrated. Acetone (200 mL) was then added, and the mixture was heated to 50°C to dissolve the solid. The mixture was then cooled to room temperature and held for 2 hours, filtered, and dried to obtain 11 g of an off-white solid.

[0040] Example 3: Preparation of the sodium salt monohydrate The off-white solid (Z)-3-(1-hydroxybutenyl)benzofuran-2-sodium salt (5 g, 0.22 mol, 1.0 eq), the product of Example 1, was dissolved in water (20 mL), heated to 60°C, dissolved, cooled to 20°C and maintained for 2 hours, filtered, and dried to obtain 3 g of a white solid. The differential scanning calorimetry (DSC) of the solid powder is shown in Figure 1, and its thermogravimetric analysis (TGA) is shown in Figure 2.

[0041] Example 4: Preparation of potassium salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq) was dissolved in acetone (10 mL). After dissolution, potassium hydroxide (0.27 g, 5 mmol, 1.0 eq) was added and the mixture was stirred for 0.5 hours. The mixture was then filtered and dried to give 0.9 g of a white solid.

[0042] Example 5: Preparation of magnesium salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq) was dissolved in methyl acetate (10 mL). After dissolution, magnesium chloride (0.46 g, 5 mmol, 1.0 eq) was added and the mixture was stirred for 1.5 hours. The mixture was filtered and dried to give 0.75 g of a white solid.

[0043] Example 6: Preparation of calcium salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq) was dissolved in acetone (10 mL). After dissolution, calcium chloride (0.54 g, 5 mmol, 1.0 eq) was added and the mixture was stirred for 1 hour. The mixture was filtered and dried to give 1.05 g of a white solid.

[0044] Example 7: Preparation of Piperazine Salts (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq) was dissolved in methyl isobutyl ketone (20 mL). After dissolution, piperazine (0.84 g, 10 mmol, 1.0 eq) was added and the mixture was stirred for 1 hour. The mixture was then filtered and dried to give 2.20 g of a white solid.

[0045] Example 8: Preparation of ethanolamine salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq) was dissolved in ethanol (20 mL). After dissolution, ethanolamine (0.60 g, 10 mmol, 1.0 eq) was added and the mixture was stirred for 3 hours. The mixture was filtered and dried to give 1.88 g of a white solid.

[0046] Example 9: Preparation of meglumine salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq) was dissolved in acetonitrile (20 mL). After dissolution, meglumine (1.90 g, 10 mmol, 1.0 eq) was added and the mixture was stirred for 2 hours. The mixture was then filtered and dried to give 1.94 g of a white solid.

[0047] Example 10: Preparation of tromethamine salt (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (20 g, 0.1 mol, 1.0 eq) was dissolved in isopropanol (200 mL). After dissolution, tromethamine (11.8 g, 0.1 mol, 1.0 eq) was added and the mixture was stirred for 2 hours. The mixture was then filtered and dried to give 23 g of a white solid.

[0048] The experimental results demonstrated that all of the salts described in Examples 2 to 10 were capable of liberating the compound (Z)-3-(1-hydroxybutenyl)benzofuran-2-one under conventional experimental conditions.

[0049] Example 11: Solubility and hygroscopicity studies of tromethamine salt crystalline forms As a sample, the tromethamine salt of Example 10 was used, and the solubility of the sample was measured at 20°C, 25°C, and 30°C. The test results are shown in Table 1.

[0050] [Table 1]

[0051] According to the Chinese Pharmacopoeia, Article 9103, Guidelines for Drug Hygroscopicity Test: 1. Take a dry glass stoppered measuring flask (outer diameter 50 mm, height 15 mm) and, on the day before the test, place it in an appropriate thermostatic oven at 25°C ± 1°C (with saturated ammonium chloride or ammonium sulfate solution placed in the bottom) or an artificial climate chamber (temperature set to 25°C ± 1°C, relative humidity 80% ± 2%), and accurately weigh its weight (m1).

[0052] 2. Take an appropriate amount of sample and spread it evenly in the volumetric flask. The thickness of the sample is generally about 1 mm, and the weight (m2) is precisely measured.

[0053] 3. Open the lid of the volumetric flask and leave it with the lid under the above constant temperature and humidity conditions for 24 hours.

[0054] 4. Cap the volumetric flask and accurately measure its weight (m3).

[0055] 5. Description of Hygroscopic Characteristics and Definition of Hygroscopic Weight Gain Deliquescent: Absorbing sufficient water to form a liquid; Highly hygroscopic: Weight increase due to moisture absorption is 15% or more. Hygroscopic: Weight increase due to moisture absorption is less than 15% but not less than 2%. Slightly hygroscopic: Weight increase due to moisture absorption is less than 2% but not less than 0.2%; No or almost no hygroscopicity: Weight increase due to moisture absorption is less than 0.2%. Experimental results: m1=28.7554, m2=29.7773, m3=29.7773.

[0056]

number

[0057] The tromethamine salt sample of Example 10 showed no change in appearance, had a weight increase percentage of 0.0%, and was not hygroscopic.

[0058] Example 12: Stability studies The stability of the sodium salt sample of Example 2 was examined under high temperature, high humidity, and light exposure conditions. The sampling period was 10 days, and the test results are shown in Table 2.

[0059] [Table 2]

[0060] The stability under high temperature, high humidity, and high light conditions was examined for the potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, and meglumine salt samples of Examples 4 to 9. The sampling period was 10 days, and the test results are shown in Table 3.

[0061] [Table 3]

[0062] The stability of the tromethamine salt sample of Example 10 was examined under high temperature (60°C), light (without packaging), light (with and without packaging), and high humidity (92.5% and 75%) conditions. The sampling period was 10 days, and the test results are shown in Table 4.

[0063] [Table 4]

[0064] Conclusion: The influencing factor test showed that tromethamine salt has higher chemical stability under high temperature (60℃), light (without packaging), light (without packaging), and high humidity (92.5%, 75%) conditions.

[0065] Biological measurements: Test 1: Compound activity measurement The following assays demonstrated that Compound I of Example 1 of the present invention has the effect of improving ischemic neuronal dysfunction. The assay results also demonstrated that Compound I of the present invention has good bioavailability and efficacy after oral administration.

[0066] 1. Pharmacokinetic study in rats Male SD rats (180-260 g) were administered Compound I via tail vein at a dose of 1.0 mg / kg and orally at a dose of 10.0 mg / kg, with three animals per group. The administration vehicle was a saline solution containing 5% DMSO and 5% polyoxyethylene castor oil (Cremophor EL). The animals were fasted for approximately 12 hours before administration, allowed free access to food for 4 hours after administration, and had access to water throughout the entire period. Approximately 0.2 mL of blood was collected via the orbit before administration and at 5, 15, and 30 minutes, and at 1, 2, 4, 6, 8, and 24 hours after administration. The blood was placed in an EDTA-K2 anticoagulated EP tube, placed on ice, and centrifuged at 3500 rpm for 10 minutes at 4°C to separate plasma, which was then stored at -20°C until analysis. Quantitative analysis of Compound I plasma concentrations was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Sample analysis results were used to calculate pharmacokinetic parameters using WinNonlin software.

[0067] The data in Table 5 indicate that after oral administration, Compound I had a longer elimination time in rats and higher bioavailability than the values ​​reported in the literature on butylphthalide (Wang Ningning, Li Yue, Li Xiaohong, Jiang Mingyan, Determination of the content of 3-n-butylphthalide in rat plasma and its pharmacokinetics by RP-HPLC method, Chinese Journal of New Drugs and Clinical Practice, December 2012, Vol. 31, No. 12, pp. 743-747). (The bioavailability exceeding 100% was presumed to be due to nonlinear pharmacokinetics.)

[0068] [Table 5]

[0069] 2. Rat brain tissue distribution test Male SD rats (weight 200-270 g) were orally administered compound I and butylphthalide (NBP) at a dose of 20 mg / kg each. Plasma and brain tissue samples were collected at 0.5, 1, 4, and 24 h after administration. Plasma collection: 0.2 mL of whole blood was collected in EP tubes containing EDTA-K2 and centrifuged at 3500 g for 10 minutes. The upper plasma layer was collected and stored at -20°C. Brain tissue collection: After euthanasia, an appropriate amount of brain tissue was weighed and homogenized in a brain tissue:80% methanol / water (w / v) ratio of 1:4. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantitatively analyze the concentrations of the compounds in plasma and brain tissue samples.

[0070] The data in Table 6 show that Compound I of the present invention has higher distribution concentrations in rat plasma and brain tissue after oral administration.

[0071] [Table 6]

[0072] 3. Pharmacodynamic study of the compound in a rat stroke model: Single therapeutic dose To evaluate the neuroprotective effects of compounds against cerebral ischemia-reperfusion in rats, a middle cerebral artery occlusion (MCAO) model was established in SD rats using the suture embolization method. SD rats (240-270 g) were anesthetized with 3.0% isoflurane and then surgically exposed to isolate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated, the ICA was temporarily clamped, and threads were threaded through the proximal and distal ends of the CCA, tightening the distal end and tying an easy-to-release knot at the proximal end. A small incision was made between the two sutures, and a No. 4-0 suture embolization was inserted through the CCA incision and gently pushed into the internal carotid artery, restraining it against the ICA arterial clip. After further tightening the ligature, the arterial clip blocking blood flow in the ICA was removed, and immediately the suture embolization was pushed into the ICA and into the intracranial cavity. The thread plug was inserted approximately 18 mm from the common carotid artery bifurcation until slight resistance was felt, i.e., the tip of the thread plug had already entered the anterior cerebral artery (ACA) and the side wall of the thread plug had already occluded the opening of the middle cerebral artery. The insertion was stopped and the time recorded. The arterial clip on the CCA was removed, and after observing the absence of active bleeding, the incision was closed. The rats were placed at room temperature, their body temperature maintained at 37°C. After 120 minutes of ischemia, anesthesia was induced. While maintaining anesthesia, the thread plug was gently pulled and its tip was returned to the external carotid artery, achieving middle cerebral artery reperfusion. The animals were administered a single dose immediately (within 10 minutes) after reperfusion. Three groups were established: a model control group, an intravenous compound I group (30 mg / kg), and an oral compound I group (60 mg / kg). The animals were euthanized 24 hours after ischemia and reperfusion. Their brains were rapidly removed, frozen, sectioned, and stained with TTC. After staining, normal tissue appeared rose-colored, while infarcted tissue appeared white. To evaluate the extent of cerebral ischemic injury in rats, the percentage of infarcted tissue to the total brain weight was defined as the infarction area (Infarction Area%).

[0073] According to TTC staining analysis one day after surgery, the cerebral infarction area of ​​the model control group was 21.63±5.66%. The cerebral infarction areas of the animals in the intravenous compound I group and the oral compound I group were 13.61±3.66% and 14.88±5.11%, respectively. This indicates that the intravenous and oral compound I groups described in the present invention significantly reduced the cerebral infarction area of ​​the animals (P=0.0025 and P=0.0389). At the same time, the cerebral infarction inhibition rates of the animals in the intravenous compound I group and the oral compound I group were 37.1% and 31.2%, respectively. The above results indicate that compound I has a significant effect on improving cerebral infarction in rats, as shown in Table 7.

[0074] [Table 7]

[0075] 4. Pharmacodynamic study of the compounds in a medium- to long-term stroke model in rats A middle cerebral artery occlusion (MCAO) model was established in SD rats using the embolization method, and the test drugs were administered for 28 consecutive days. The pharmacological effects of the test drugs on stroke were evaluated based on general observations and neurobehavioral scores.

[0076] Sprague-Dawley rats (240–280 g) were anesthetized with 3.0% isoflurane and then the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated and exposed. The ECA was ligated, the ICA was temporarily clamped, and threads were threaded through the proximal and distal ends of the CCA, tightening the distal end and tying an easy-to-release knot at the proximal end. A small incision was made between the threads, and a No. 4-0 suture obturator was inserted through the CCA incision and gently pushed into the internal carotid artery. The ligature stopped when it contacted the ICA arterial clip. After further tightening, the arterial clip, which had been blocking blood flow through the ICA, was removed. The suture obturator was then immediately pushed into the ICA and into the intracranial cavity. The thread plug was inserted approximately 18 mm from the common carotid artery bifurcation until slight resistance was felt, i.e., the tip of the plug had already entered the anterior cerebral artery (ACA) and the side wall of the plug had already occluded the opening of the middle cerebral artery. The insertion was stopped and the time recorded. The arterial clip on the CCA was removed, and after observing the absence of active bleeding, the incision was closed. The rats were placed at room temperature, with their body temperature maintained at 37°C. After 120 minutes of ischemia, anesthesia was induced. While maintaining anesthesia, the tip of the thread plug was gently pulled back into the external carotid artery, achieving middle cerebral artery reperfusion. The animals were administered acetaminophen immediately (within 10 minutes) after reperfusion and then administered once daily for 28 consecutive days. The day of surgery was defined as Day 0 (D1). Five groups were established: a sham-operated group, a model control group, a butylphthalide (NBP)-treated group (60 mg / kg, po, qd), a low-dose oral administration of Compound I (6 mg / kg, po, qd), and a high-dose oral administration of Compound I (20 mg / kg, po, qd). During the administration period, all animals underwent a grid test (four times in total: on D7, D14, D21, and D28) and a novel object recognition test (novel object recognition adaptation on D26 and detection on D27). After the administration ended on D28, all surviving animals in each group were euthanized, and their brains were immediately removed for pathological analysis.

[0077] (1) Results of the grid test showed that 1 week after surgery, the stepping frequency of animals in the model control group was 7.24 ± 3.59, while the stepping frequencies of the NBP-treated group, low-dose compound I group, and high-dose compound I group were 7.37 ± 3.03, 5.33 ± 2.33, and 4.23 ± 1.44, respectively. The stepping frequency of animals in the high-dose compound I group was significantly reduced compared to the model control group (P = 0.0172). Between 2 and 3 weeks after surgery, the stepping frequency of animals in the model group gradually decreased due to the gradual recovery of motor function. Although the stepping frequency of the high-dose compound I group was not significantly different from that of the model control group, it still tended to decrease. Four weeks after model construction, the stepping frequency of model animals decreased to the level of animals in the sham-operated group. These results demonstrate that compound I has an ameliorative effect on behavioral dysfunction in stroke-affected animals (see Table 8 for details).

[0078] [Table 8]

[0079] (2) On the final day of treatment, a novel object recognition test was performed, and the novel object recognition index (NRI) of each group was calculated. The novel object recognition index of the model control group was 54.81 ± 21.94%, which was not significantly different from the old object recognition index (FRI). The NRIs of the NBP-treated group, low-dose Compound I group, and high-dose Compound I group were 70.97 ± 22.57%, 70.98 ± 22.60%, and 71.66 ± 17.06%, respectively. There were significant differences in the novel / old object recognition index between the NBP-treated group, low-dose Compound I group, and high-dose Compound I group (P = 0.0074, P = 0.0212, and P = 0.0009). At the same time, the novel object recognition index of the low-dose and high-dose Compound I group was comparable to that of the sham-operated group (66.51 ± 10.80%). The above results demonstrated that Compound I significantly improved cognitive impairment in stroke-stricken animals (see Table 9 for details).

[0080] [Table 9]

[0081] (3) Pathological examination: 1) Repair area: Samples with a repair area of ​​>30% were considered to have a good recovery, and samples with a repair area of ​​≤30% were considered to have a poor recovery. The proportion of animals with a good recovery in the model control group was 17.6%, while the proportions of animals with a repair area of ​​>30% in the NBP-treated group, the low-dose compound I group, and the high-dose compound I group were 21.4%, 33.3%, and 75.0%, respectively. The high-dose compound I group showed the best recovery, which was significantly different from the model control group (Chi-square, P=0.0080). These results indicated that compound I had a promoting effect on the repair of the infarcted area (see Table 10 for details). 2) Number of filled blood vessels in the infarcted repair area: Two standards were set: ≤10 and >10 blood vessels containing red blood cells in the infarcted repair area. In the sham-operated group, the number of filled blood vessels was greater than 10 in all animals, accounting for 100%. In the model control group, the number of animals with ≤10 and >10 blood vessels containing red blood cells in the infarct repair area was 10 and 7, respectively, accounting for 41.2% of the animals. In the NBP-treated group, the number of animals with ≤10 and >10 blood vessels containing red blood cells in the infarct repair area was 1 and 14, respectively, accounting for 93.3% of the animals, which was statistically significant compared with the model control group (Chi-square, P = 0.0028). The number of animals with erythrocyte-containing vessels in the infarct repair area in the low-dose and high-dose compound I groups was 0 / 12 and 1 / 12, respectively, and the number of animals with erythrocyte-containing vessels >10 was not significantly higher than that in the model control group (Chi-square, P=0.0012, P=0.0076).Next, the percentage of samples with erythrocyte-containing vessels >10 in the repair area in the low-dose and high-dose compound I groups was 100% and 92.3%, respectively, and each treatment group was above 90%.These results showed that compound I significantly increased the vascular fullness in the cerebral infarct repair area of ​​stroke animals after 28 consecutive days of treatment (see Table 11 for details).

[0082] [Table 10]

[0083] [Table 11]

[0084] Examples 2 to 10 are salts of the compound of Example 1, and can be liberated to the compound of Example 1 in the body, and therefore have the same functions as the compound of Example 1.

[0085] Test 2: Beneficial effect of the tromethamine salt of Example 10 on stroke The following assays demonstrated that the tromethamine salt of Example 10 has the effect of improving ischemic neuronal dysfunction. The assay results also showed that the tromethamine salt of Example 10 has good bioavailability and efficacy after oral administration.

[0086] 1. Pharmacological study of the compound on a rat stroke model To evaluate the neuroprotective effects of compounds against cerebral ischemia-reperfusion in rats, a middle cerebral artery occlusion (MCAO) model was established in SD rats using the suture embolization method. SD rats (240-270 g) were anesthetized with 3.0% isoflurane and then surgically exposed to isolate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated, the ICA was temporarily clamped, and threads were threaded through the proximal and distal ends of the CCA, tightening the distal end and tying an easy-to-release knot at the proximal end. A small incision was made between the two sutures, and a No. 4-0 suture embolization was inserted through the CCA incision and gently pushed into the internal carotid artery. It temporarily stopped when it contacted the ICA arterial clip. The ligature was further tightened, and the arterial clip blocking blood flow in the ICA was removed. Immediately afterwards, the suture embolization was pushed into the ICA and into the intracranial cavity. The thread plug was inserted approximately 18 mm from the common carotid artery bifurcation until slight resistance was felt, i.e., the tip of the thread plug had already entered the anterior cerebral artery (ACA) and the side wall of the thread plug had already occluded the opening of the middle cerebral artery. The insertion was stopped and the time recorded. The arterial clip on the CCA was removed, and after observing that there was no active bleeding, the incision was closed. The ischemic rats were placed at room temperature, their body temperature maintained at 37°C. After 120 minutes, anesthesia was induced. While maintaining anesthesia, the thread plug was gently pulled to return its tip to the external carotid artery, achieving middle cerebral artery reperfusion. The animals received a single dose immediately (within 10 minutes) after reperfusion. Four groups were divided: a model control group, groups receiving intragastric administration of tromethamine salt (Example 10) (20 and 40 mg / kg), and a group receiving oral butylphthalide (60 mg / kg). A single dose was administered immediately after reperfusion (within 10 minutes), followed by one daily dose for seven consecutive doses (including the first dose). All subsequent doses were administered between 4:00 PM and 7:00 PM. NSS behavioral scores were measured before and after modeling, 24 hours (Day 1), 72 hours (Day 3), and 7 days after modeling. Animals were euthanized 7 days after ischemia-reperfusion, and their brains were rapidly removed, frozen, sectioned, and stained with TTC. Normal tissue appeared rose-colored after staining, while infarcted tissue appeared white. To assess the extent of cerebral ischemic injury in rats, the infarct area (% infarction) was calculated as the percentage of infarcted tissue relative to the total brain weight.

[0087] (1) Cerebral infarction: Seven days after the operation, the results are shown in Table 12. There was no cerebral infarction in the animals of the sham-operated control group, the cerebral infarction area in the animals of the model control group was 19.951±3.432%, and the cerebral infarction area in the animals of the positive drug group was 14.241±2.912%, which was significantly reduced compared to the model control group (P≦0.05). The cerebral infarction area in the animals of Example 10 at 20 and 40 mg / kg was 4.241±2.912%, which was significantly reduced compared to the model control group (P≦0.05). The cerebral infarction suppression rates for the 40 mg / kg group of tromethamine salt of Example 10 and the positive drug group (butylphthalide, 60 mg / kg) were 31.023 ± 11.15% and 28.624 ± 14.597%, respectively, based on the area of ​​cerebral infarction. These results suggest that the tromethamine salt of Example 10 at doses of 20 mg / kg and 40 mg / kg significantly improved the cerebral infarction area in rats with ischemic stroke under the test conditions, and that the tromethamine salt of Example 10 at 40 mg / kg had a superior effect on improving cerebral infarction compared to the positive drug at 60 mg / kg.

[0088] [Table 12]

[0089] (2) Neurobehavioral score: As shown in Table 13, all animals in all groups had a Bederson score of 3 after surgery, suggesting that ischemia was successful. One day after surgery, the NSS score of the animals in the model control group was 7.21±1.03, and the NSS scores of the animals treated with the positive drug and the tromethamine salt of Example 10 tended to be lower than those of the model control group, but the difference was not statistically significant.

[0090] Three days after surgery, the NSS score of animals in the model control group was 5.92 ± 0.70, and the NSS score of animals in the tromethamine salt 40 mg / kg group of Example 10 was significantly lower than that of the model control group (P < 0.05). Meanwhile, the NSS score of animals in the positive drug group (butylphthalide, 60 mg / kg) was 5.75 ± 0.92, which was not significantly different from that of the model control group. During the subsequent observation period, the NSS score of the model control animals decreased to 5 and remained above 5. The NSS scores of animals in each treatment group showed a sustained decrease. Seven days after modeling, the NSS score of the model control animals was 5.17 ± 1.56, and the NSS scores of the tromethamine salt 40 mg / kg group of Example 10 and the positive drug group (butylphthalide, 60 mg / kg) were 3.42 ± 1.54 and 4.04 ± 1.20, respectively. The NSS scores of the animals in the 40 mg / kg group treated with tromethamine salt of Example 10 were significantly lower than those in the model control group (P≦0.05). These results suggest that tromethamine salt of Example 10 significantly improved neurological dysfunction in the acute phase of ischemic stroke rats at a dose of 40 mg / kg under the test conditions, and that the effect was stronger than that of the positive drug group.

[0091] [Table 13]

[0092] (3) Summary: This study suggests that the tromethamine salt of Example 10 reduces cerebral infarction due to stroke and also reduces neurological dysfunction, while the positive drug only has the effect of improving cerebral infarction. The cerebral infarction-inhibiting effect and neurological dysfunction-reducing effect of a relatively low dose (40 mg / kg) of the tromethamine salt of Example 10 were both superior to those of a high dose (60 mg / kg) of the positive drug, suggesting that the tromethamine salt of Example 10 has a superior pharmacological effect to the positive drug.

[0093] Test 3: Beneficial effect of the tromethamine salt of Example 10 on amyotrophic lateral sclerosis The tromethamine salt of Example 10 was proven to have an effect of improving amyotrophic lateral sclerosis (ALS) by the following measurement method.

[0094] 1. Pharmacodynamic study of SOD1-G93A transgenic amyotrophic lateral sclerosis mice To evaluate the therapeutic effects of the compound on ALS disease animals, male SOD1-G93A transgenic ALS mice (5 weeks old) were used. After a one-week acclimation period, the mice were randomly assigned to the following groups based on body weight, muscle tone, and rod rotation: Group A: vehicle; Group B: 25 mg / kg riluzole (po); Group C: 40 mg / kg tromethamine salt of Example 10 (po); and Group D: 60 mg / kg tromethamine salt of Example 10 (po). The administration frequency was 2 weeks, from Monday to Friday, followed by a one-week break. Behavioral measurements were performed every two weeks, and the animals' body weight and survival rate were monitored using rod rotation and muscle rotation tests. The therapeutic effects of the tromethamine salt of Example 10 on ALS disease were evaluated.

[0095] (1) A rod-spinning test (Table 14) demonstrated that the rod-spinning time of mice was measured to assess the animals' motor endurance and coordination. In this experiment, the tromethamine salt of Example 10 at 40 mg / kg significantly improved the rod-spinning time of ALS mice aged 10-16 weeks, i.e., in the mid-onset stage (P<0.05). The 60 mg / kg group significantly improved the rod-spinning time of mice in the late-onset stage (P<0.001, P<0.05). Riluzole tended to increase the rod-spinning time of mice under these experimental conditions, but the difference was not significant. Under these experimental conditions, the efficacy of the tromethamine salt of Example 10 was superior to that of riluzole.

[0096] [Table 14]

[0097] (2) Muscle tone test (Table 15): 60 mg / kg of the tromethamine salt of Example 10 significantly improved muscle tone in 18-20 week-old SOD1 G93A ALS mice, whereas riluzole and 40 mg / kg of the tromethamine salt of Example 10 tended to upregulate muscle tone in mice, but the difference was not significant.

[0098] [Table 15]

[0099] (3) Effect on survival rate: As shown in Figure 3, SOD1 G93A ALS mice administered with the tromethamine salt of Example 10 (20, 40, 60 mg / kg) showed a longer survival time than both the control group and the positive drug group. Of these, the survival time of the 40 mg / kg group was extended by approximately 15 days.

[0100] (4) Effect on body weight: The administration of each dose of the tromethamine salt of Example 10 and riluzole did not significantly affect the body weight of mice compared to the vehicle group.

[0101] (5) Summary The 40 mg / kg and 60 mg / kg dose groups of tromethamine salt of Example 10 significantly improved the ALS-like motor dysfunction caused by SOD1 G93A transgenic mice and prolonged the survival time of the ALS mice to some extent. Under the experimental conditions, the positive drug riluzole had a certain improving effect on the neurobehavioral function of ALS mice, but there was no significant difference from the vehicle control group, and its efficacy was weaker than that of tromethamine salt of Example 10.

[0102] 2. Pharmacodynamic study on TDP-43-A315T transgenic ALS mice To evaluate the therapeutic effects of the compound on ALS disease animals, TDP-43-A315T transgenic ALS mice (5 weeks old, half male and half female) were used. After a one-week acclimation period, the mice were randomly assigned to group A: vehicle, group B: 25 mg / kg riluzole (po), group C: 40 mg / kg tromethamine salt of Example 10 (po), and group D: 60 mg / kg tromethamine salt of Example 10 (po). The mice were administered once daily, 7 days a week, with a one-week break between each administration. Behavioral measurements were performed every two weeks, and the animals' weights and survival rates were monitored using rod-spinning and myotonicity tests to evaluate the therapeutic effects of the tromethamine salt of Example 10 on ALS disease.

[0103] (1) Muscle tone experiment (Tables 16 and 17): Compared with the vehicle group, tromethamine salt 60 mg / kg of Example 10 significantly improved muscle tone in male TDP-43 ALS mice at 13-14 weeks of age. Meanwhile, riluzole tended to upregulate muscle tone, but the difference was not significant. At 14-16 weeks of age, tromethamine salt 20 mg / kg and 40 mg / kg of Example 10 significantly upregulated muscle tone in female ALS mice, while riluzole did not significantly improve muscle tone in female ALS mice.

[0104] [Table 16]

[0105] [Table 17]

[0106] (2) Rod-spinning experiment (Tables 18 and 19): The tromethamine salt of Example 10 at a dose of 60 mg / kg significantly upregulated the rod-spinning time of male TDP43 ALS mice aged 11 to 13 weeks. On the other hand, the riluzole group tended to upregulate the rod-spinning time of 11 to 13-week-old mice, although there was no significant difference compared to the vehicle group. The tromethamine salt of Example 10 at 20 mg / kg also showed a certain upregulation effect on the rod-spinning time of female mice.

[0107] [Table 18]

[0108] [Table 19]

[0109] (3) Measurement of body weight: The doses of riluzole and the tromethamine salt of Example 10 did not significantly affect the body weight of the mice.

[0110] (4) Summary: The tromethamine salt of Example 10 significantly improved motor dysfunction in TDP-43 A315T transgenic ALS mice without affecting their body weight, demonstrating superior efficacy to the control drug, riluzole.

[0111] Test 4: Therapeutic effect of the tromethamine salt of Example 10 on Parkinson's disease The following assays demonstrated that the tromethamine salt of Example 10 of the present invention has an effect of alleviating Parkinson's cognitive dysfunction.

[0112] 1. Therapeutic effects of compounds on MPTP-induced mouse Parkinson's disease model A mouse model of Parkinson's disease was established by intraperitoneally injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (aromatic amino-acid decarboxylase, MPTP) at a dose of 30 mg / kg twice a week for 4 consecutive weeks. The efficacy of the test product, tromethamine salt of Example 10, was evaluated based on the animal's behavioral detection, striatal dopamine (DOPA) levels, and histopathological examination results.

[0113] Fifty C57 mice were randomly divided into five groups based on the results of the pre-modeling rod-climbing test: a normal control group, a model control group, a low-dose tromethamine salt group (Example 10), a high-dose tromethamine salt group (Example 10), and a positive control group (levodopa, 20 mg / kg). Drug intervention treatment began on the day of the first injection of MPTP and was administered once daily for four consecutive weeks. The tromethamine salt of Example 10 was administered at doses of 5 mg / kg and 20 mg / kg, and the positive control group (levodopa) was administered at a dose of 20 mg / kg. The rod-climbing test and grip test were performed within 48 h of the final MPTP injection.

[0114] 1) Results of the bar lift test The statistical results of the rod lifting test are shown in Table 19 and Figure 4. There was no significant difference in the rod lifting time of animals in each test group before modeling. At the end of the test, the rod lifting time of animals in the normal control group was 8.35 ± 0.89 s, and that of animals in the model control group was 11.51 ± 2.39 s, significantly increased compared with the normal control group (P < 0.05). The rod lifting time of animals in the positive control group was 8.76 ± 1.57 s, tending to be decreased compared with the model control. The rod lifting times of animals in the low-dose and high-dose groups of tromethamine salt of Example 10 were 9.67 ± 1.48 s and 8.83 ± 1.77 s, respectively. Both rod lifting times were decreased compared with the model control, but the difference in the high-dose group of tromethamine salt of Example 10 was statistically significant compared with the model control (P < 0.05). These results suggest that the tromethamine salt of Example 10 has a significant therapeutic effect on the MPTP-induced PD model.

[0115] [Table 20]

[0116] 2) Grip detection results The statistical results of grip detection are shown in Table 20 and Figure 5. There was no significant difference in grip weight between the animals in each test group before modeling. At the end of the test, the grip weight of the normal control animals was 260.98 ± 14.81 g, and that of the model control animals was 210.92 ± 10.00 g, significantly decreased compared to the normal control group (P < 0.05). The grip weight of the positive control (levodopa) animals was 226.26 ± 8.39 g, tending to increase compared to the model control. The grip weights of the animals in the low-dose and high-dose tromethamine salt groups of Example 10 were 219.05 ± 12.83 g and 227.16 ± 5.85 g, respectively, and both tended to increase compared to the model control.

[0117] [Table 21]

[0118] 3) Striatal DOPA detection results The results of striatal DOPA detection are shown in Table 21 and Figure 6. The DOPA content in the striatum of normal control animals was 4931.52 ± 1607.66 ng / g, and that of model control animals was 891.92 ± 285.94 ng / g, significantly reduced compared to the normal control group (P < 0.05). The DOPA content in the positive control animals was 1156.72 ± 375.72 ng / g, showing no significant change compared to the model control group. The DOPA content in the high-dose tromethamine salt group of Example 10 was 2731.07 ± 868.18 ng / g, significantly increased compared to the model control group (P < 0.05). The DOPA content in the low-dose tromethamine salt group of Example 10 was 837.82 ± 372.19 ng / g, showing no significant change compared to the model control group. The above results demonstrate that the high dose group (20 mg / kg) of tromethamine salt of Example 10 has a significant protective effect on dopaminergic neurons in the MPTP-induced PD model.

[0119] [Table 22]

[0120] 4) Statistical results of TH-positive fiber content in the striatum The statistical results of the striatal TH-positive fiber content are shown in Table 22 and Figure 7. The TH-positive fiber content in the striatum of normal control animals was 1,133,191.93 ± 103,519.32, and the TH-positive fiber content in the striatum of model control animals was 470,927.76 ± 115,639.36, significantly decreased compared to the normal control group (P < 0.05). The TH-positive fiber content in the positive control group (levodopa) animals was 536,968.78 ± 68,050.46, showing no significant change compared to the model control group. The TH-positive fiber content in the high-dose tromethamine salt group of Example 10 animals was 709,627.56 ± 100,462.19, significantly increased compared to the model control group (P < 0.05). The TH-positive fiber content of the animals in the low-dose group of tromethamine salt in Example 10 was 526,554.39±169,680.75, which showed no significant change compared with the model control. These results, consistent with the above results, demonstrate that the high-dose group of tromethamine salt in Example 10 (20 mg / kg) has a significant protective effect on dopaminergic neurons in the MPTP-induced PD model.

[0121] [Table 23]

[0122] 5) Statistical results of the number of TH-positive cells in the substantia nigra pars compacta The statistical results of the number of TH-positive cells in the substantia nigra compacta are shown in Table 23 and FIG. 8, and individual data are shown in Table 23. The number of TH-positive cells in the normal control group was 57.47 ± 5.81, and the number of TH-positive cells in the model control group was 30.57 ± 3.29, which was significantly lower than that in the normal control group (P < 0.05). The number of TH-positive cells in the positive control group was 31.50 ± 4.14, which was not significantly different from that in the model control group. The number of TH-positive cells in the high-dose tromethamine salt group of Example 10 was 42.42 ± 4.36, which was significantly higher than that in the model control group (P < 0.05). The TH-positive fiber content in the low-dose tromethamine salt group of Example 10 was 31.22 ± 5.97, which was not significantly different from that in the model control group. The results again demonstrated that the high dose group (20 mg / kg) of tromethamine salt of Example 10 had a significant protective effect on dopaminergic neurons in the MPTP-induced PD model.

[0123] [Table 24]

[0124] Test 5: Therapeutic effect of the tromethamine salt of Example 10 on spinal cord injury The following assays demonstrated that the tromethamine salt of Example 10 of the present invention has an effect of reducing spinal cord injury.

[0125] 1: Study of compound treatment for spinal cord injury (SCI) in rats The therapeutic effect of the tromethamine salt of Compound Example 10 on spinal cord injury was investigated in a rat spinal cord injury model, and the mechanism of action was preliminarily investigated.

[0126] Male SD rats were anesthetized with isoflurane and the skin of the dorsal thoracic spine region of the rat was prepared. The skin was incised around the spinous process of the T10 vertebra, and the subcutaneous fascia and muscle were incised layer by layer to expose the spinous process, intervertebral disc, and spinal dura mater.

[0127] A rat was fixed under a JK052-type percussion instrument and struck by a 10g percussion rod at T10 thoracic spinal cord from a 5cm free fall to create a spinal cord injury model. Two hours after modeling, rats that were successfully modeled were divided into four groups, with 10 rats in each group. A sham-operated group (10 rats in total) was also established. The test drug groups were orally administered the tromethamine salt of Example 10 at doses of 5 and 20 mg / kg, respectively, once daily for 28 consecutive days. The positive control group (sodium methylprednisolosuccinate for injection) was administered a single dose of 180 mg / kg of sodium methylprednisolosuccinate for injection via the tail vein. The sham-operated group and model control group were orally administered an equivalent amount of 0.5% CMC. The grouping and administration are shown in Table 24 below.

[0128] [Table 25]

[0129] 1) Impact on BBB score The rats with spinal cord injury showed obvious neurological dysfunction, and the BBB scores were calculated at different time points. The scoring method was as follows: [Table 26]

[0130] The BBB score recovered slightly with the extension of the time after modeling, and the BBB score was significantly improved 7 to 28 days after administration of 20 mg / kg of the test compound, tromethamine salt of Example 10 (P<0.05-0.01). The positive control (methylprednisolone sodium for injection) also improved the BBB score 14 to 28 days after administration (P<0.05). The results are shown in Table 25.

[0131] [Table 27]

[0132] 2) Effect on pathological changes The spinal cord tissue in the sham-operated group had a dense structure, intact membranes, clear gray and white matter, distinct boundaries, clear outlines of neurons and glial cells, high continuity of nerve fibers, and an orderly arrangement of nerve cells.

[0133] Compared with the sham-operated group, the model control group showed severe destruction of spinal cord tissue, obvious defects on the spinal cord side, sparse and disorganized tissue arrangement, inflammatory cell infiltration, and significant neuronal vacuolization and degeneration. There was no significant difference between the low-dose group and the model control group. More than half (4 / 6 cases) of the 20 mg / kg dose group of the tromethamine salt of test compound Example 10 showed a reduction in lesions. A portion (3 / 6 cases) of the positive drug group (sodium methylprednisolosuccinate for injection) showed a reduction in lesions. The results are shown in Figure 9.

[0134] 3) Effect on NF200 expression NF200 was a marker of nerve fibers. NF200 expression was slightly increased after spinal cord injury in rats compared with the sham-operated group (P<0.01). Compared with the model control group, the test drug tromethamine salt of Example 10 at 20 mg / kg could significantly increase NF200 expression (P<0.01). After administration of the positive control (sodium methylprednisolosuccinate for injection), there was no significant change in NF200 (P>0.05). The results are shown in Table 26 and Figure 10.

[0135] [Table 28]

[0136] 4) Transcriptome analysis of injured spinal cord To further verify the protective or regenerative effects of the aminotriol salt of Example 10 on the injured spinal cord, 1 cm tissue samples were excised from both the upper and lower reaches of the injury site and subjected to transcriptomics analysis. GSEA analysis showed that the tromethamine salt of Example 10 significantly upregulated axon regeneration and synapse-related genes, and significantly downregulated the expression of extracellular matrix and inflammation-related genes (see Figure 11).

Claims

1. A pharmaceutically acceptable salt of a compound of formula (I), 【Chemistry 1】 the pharmaceutically acceptable salt is selected from the sodium salt, potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, meglumine salt, tromethamine salt, or the pharmaceutically acceptable salt is selected from the monohydrate, dihydrate, trihydrate or hemihydrate of the salt; A pharmaceutically acceptable salt of a compound of formula (I).

2. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one sodium salt. A pharmaceutically acceptable salt of the compound of claim 1.

3. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one sodium monohydrate. A pharmaceutically acceptable salt of the compound of claim 1.

4. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one potassium salt. A pharmaceutically acceptable salt of the compound of claim 1.

5. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one magnesium salt. A pharmaceutically acceptable salt of the compound of claim 1.

6. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one calcium salt. A pharmaceutically acceptable salt of the compound of claim 1.

7. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one piperazine salt. A pharmaceutically acceptable salt of the compound of claim 1.

8. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one ethanolamine salt. A pharmaceutically acceptable salt of the compound of claim 1.

9. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one meglumine salt, A pharmaceutically acceptable salt of the compound of claim 1.

10. (Z)-3-(1-hydroxybutenyl)benzofuran-2-one tromethamine salt. A pharmaceutically acceptable salt of the compound of claim 1.

11. A method for preparing a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10, comprising the step of forming a salt of a compound of formula (I) with a base: method.

12. The method according to claim 11, wherein the solvent used in the salt formation reaction is at least one selected from the group consisting of methanol, ethanol, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, acetonitrile, isopropanol, acetone, tetrahydrofuran, methyl isobutyl ketone, n-heptane, dichloromethane, toluene, isopropyl ether, methyl t-butyl ether, n-butanol, and water.

13. A pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10 and one or more pharmaceutically acceptable carriers, diluents or excipients. Pharmaceutical compositions.

14. Use of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 13 for preparing a medicament for the treatment and / or prevention of diseases associated with ischemic brain injury, preferably the diseases associated with ischemic brain injury include, but are not limited to, cerebral ischemia, ischemic stroke, vascular dementia, post-ischemic inflammation, convulsion, ischemic cranial nerve damage or necrosis. use.

15. Use of a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 13 for the preparation of a medicament for the treatment and / or prevention of a neuromuscular disease, preferably wherein said neuromuscular disease is amyotrophic lateral sclerosis. use.

16. Use of a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 13 for the preparation of a medicament for the treatment and / or prevention of diseases associated with neurological dysfunction, preferably wherein said diseases associated with neurological dysfunction include, but are not limited to, spinal cord injury or necrosis. use.

17. Use of a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 13 for the preparation of a medicament for the treatment and / or prevention of neurodegenerative diseases, preferably said neurodegenerative diseases including but not limited to Parkinson's disease. use.

Images