Medicinal salt of compound and preparation method thereof

Abstract

An alkali addition salt of a compound represented by formula (I), a salt crystal form, a preparation method and applications thereof, a pharmaceutical composition containing a therapeutically effective amount of the salt or the crystal form, and applications of the pharmaceutical composition in treatment of ischemic brain injury related diseases, spinal cord injury and neurodegenerative diseases. # imgabs0 #

Description

Pharmaceutically acceptable salt of compound and preparation method thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a pharmaceutically acceptable salt, crystal form, and pharmaceutical composition of a compound having anti-cerebral ischemia, anti-post-ischemic inflammation, and anticonvulsant effects, capable of improving neurological dysfunction in patients with ischemic stroke, spinal cord injury, and neurodegenerative diseases, as well as their applications, and methods for preparing the salt and crystal form. Technical Background

[0002] White matter is an important component of the central nervous system and is where nerve fibers gather. White matter lesions (WML) are usually caused by reduced blood flow and insufficient vascular oxygen supply. Impaired (insufficient) blood supply to the brain makes it difficult to meet the metabolic needs of brain tissue, resulting in a series of symptoms. Clinically, it can manifest as dizziness, headache, limb numbness or temporary loss of consciousness. In severe cases, it can cause irreversible damage to brain function or even death. Diseases related to cerebral ischemia include transient ischemic attack (TIA), ischemic stroke (cerebral infarction), moyamoya disease, chronic cerebral ischemia, etc., which are also one of the causes of cognitive decline and vascular dementia in patients.

[0003] Spinal cord injury (SCI) is one of the most disabling diseases in modern society, often accompanied by symptoms such as limb sensory impairment and loss of motor function. As an injury to the central nervous system, which is a non-regenerative tissue, recovery from functional impairment caused by nerve damage is difficult. Traumatic brain injury is the main cause of cognitive impairment in patients with SCI. SCI not only causes severe 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 manifestations such as bradykinesia, resting tremor, and postural instability. The underlying cause of PD is the loss, damage, and death of dopamine neurons in the patient's midbrain. The current standard treatment is dopamine replacement therapy, but this only treats the symptoms and not the underlying cause, and it carries significant side effects.

[0005] Currently, many drugs are used to treat diseases caused by central nervous system damage, but few are truly effective. The existing drug nimodipine has a preventive effect on cerebral ischemia, but its therapeutic effect is uncertain. Cerebral nerve damage and necrosis can lead to dysfunction of corresponding human systems, significantly reducing the patient's quality of life. There is a need for other compounds with better efficacy, including those with anti-ischemic, anti-post-ischemic, and anticonvulsant effects, that can improve neurological deficits in patients with ischemic stroke, spinal cord injury, and cognitive impairment in neurodegenerative diseases, as well as improve memory and protect nerve cells and the blood-brain barrier.

[0006] Our company has developed a compound of formula (I), (Z)-3-(1-hydroxybutenyl)benzofuran-2-one, for the treatment of ischemic brain injury, spinal cord injury and neurodegenerative diseases. In subsequent research and development, in order to facilitate the handling, filtration and drying of the product, and to seek a suitable product that is easy to store and has long-term stability, the present invention has conducted comprehensive research on the salts of the above-mentioned substance, and is committed to obtaining the most suitable salt form to ensure the quality, safety, efficacy and application of the drug.

[0007] Summary of the Invention

[0008] The present invention provides a pharmaceutically acceptable salt, crystal form and preparation method of a compound having anti-cerebral ischemia, anti-post-ischemic inflammation and anticonvulsant effects, capable of improving neurological deficits in patients with ischemic stroke, neurological impairment in patients with spinal cord injury and cognitive dysfunction in patients with neurodegenerative diseases.

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

[0010] Wherein, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, meglumine salt, tromethamine salt; wherein the pharmaceutically acceptable salt can also be selected from salt monohydrate, dihydrate, trihydrate or hemihydrate; the salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, meglumine salt, tromethamine salt.

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

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

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

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

[0020] The present invention also provides a method for preparing the aforementioned pharmaceutically acceptable salt, comprising the following steps: forming a salt with a base.

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

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

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

[0024] The present invention also provides use of the composition of the present invention in preparing a drug for treating and / or preventing diseases related to ischemic brain injury.

[0025] The ischemic brain injury-related diseases described in the present invention include but are not limited to cerebral ischemia, ischemic stroke, vascular dementia, post-ischemic inflammation, convulsions, ischemic brain neuron damage or necrosis.

[0026] The present invention also provides use of the composition of the present invention in preparing a drug for treating and / or preventing neuromuscular diseases.

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

[0028] The present invention also provides use of the composition of the present invention in preparing a drug for treating and / or preventing diseases related to impaired neurological function.

[0029] The diseases related to neurological impairment described in the present invention include but are not limited to spinal cord injury or necrosis.

[0030] The present invention also provides use of the composition of the present invention in preparing a drug for treating and / or preventing neurodegenerative diseases.

[0031] The neurodegenerative diseases described in the present invention include but are not limited to Parkinson's disease.

[0032] The pharmaceutically acceptable salts of the compounds of the present invention are easy to handle, filter and dry, are stable in long-term storage, and are suitable for industrial development to ensure the quality, safety and effectiveness of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a differential scanning calorimetry (DSC) diagram of sodium salt monohydrate;

[0034] FIG2 is a thermogravimetric analysis (TGA) diagram of the sodium salt monohydrate;

[0035] FIG3 shows the effect of the tromethamine salt of Example 10 on the survival of SOD1 G93A ALS mice;

[0036] FIG4 shows the results of the pole climbing test of the tromethamine salt of Example 10 on the MPTP-induced Parkinson's disease model in mice (Note: the data in the figure are expressed as mean ± standard error (SEM). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P<0.05 indicates that the difference is statistically significant compared with the model control group. );

[0037] FIG5 shows the grip strength test results of the tromethamine salt of Example 10 on the MPTP-induced Parkinson's disease model in mice (Note: the data in the figure are expressed as mean ± standard error (SEM). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. );

[0038] Figure 6 shows the results of striatal DOPA detection (Note: the data in the figure are expressed as mean ± standard error (SEM). &P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P < 0.05 indicates statistically significant differences compared with the model control group);

[0039] Figure 7 shows the statistical results of the content of TH-positive fibers in the striatum (Note: Figure A shows the field of view of TH immunohistochemical staining of the mouse striatum at 2.5×, and positive results are brown; Figure B shows the statistical results of the TH-positive area in the mouse striatum. Three sections were counted for each animal, and the data in the figures are expressed as mean ± standard error (SEM). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P < 0.05 indicates statistically significant differences compared with the model control group);

[0040] Figure 8 shows the statistical results of the number of TH-positive cells in the substantia nigra pars compacta (Note: Figure A is a 10× microscope field of view of TH immunofluorescence staining of the mouse striatum, and Figure B is a statistical result of the TH-positive area of ​​the mouse striatum. Three sections were counted for each animal, and the data in the figure are expressed as mean ± standard error (SEM). The data in the figure are expressed as mean ± standard error (SEM). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P < 0.05 indicates statistically significant differences compared with the model control group);

[0041] FIG9 is a graphic illustration of the effect of the tromethamine salt of the test compound Example 10 on pathological changes (50× and 200×);

[0042] FIG10 is a graphic illustration of the effect of the tromethamine salt of test compound Example 10 on NF200 (400×);

[0043] FIG11 is a GSEA analysis of the tromethamine salt of the test compound Example 10 on the transcriptomics of the injured spinal cord. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with specific examples, based on common technical knowledge and customary means in the art. The following examples are only some preferred embodiments of the present invention and should not be regarded as limiting the present invention. It is obvious to those skilled in the art that several modifications can be made without departing from the scope of the present invention, and these modifications should also be regarded as the scope of protection of the present invention.

[0045] Example 1: Preparation of (Z)-3-(1-hydroxybutenyl)benzofuran-2-one

[0046] 3-Benzofuranone (5.0 g, 37.3 mmol, 1.0 eq.) was dissolved in dichloromethane (50 mL), cooled to 5°C, and potassium tert-butoxide (6 g, 53.5 mmol, 1.4 eq.) was slowly added. After reacting for 0.5 h, n-butyryl chloride (8 g, 75.0 mmol, 2.0 eq.) was slowly added and reacted for 1 h. Water (50 mL) was added for extraction, and the organic phase was washed with 0.1 N hydrochloric acid until acidic (pH <2). The organic phase was dried over anhydrous sodium sulfate (5 g) for 0.5 h, filtered, and concentrated to obtain an oil. The oil was then purified by high pressure preparative chromatography. The purified solution was freeze-dried to obtain the compound of formula (I) ((Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one (2.5 g, 32.9%) as a white solid.

[0047] 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).

[0048] 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).

[0049] Example 2: Preparation of sodium salt

[0050] (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. The mixture was stirred for 1 h. After concentration, acetone (200 mL) was added. The temperature was raised to 50 ° C for dissolution. The temperature was lowered to room temperature and maintained for 2 h. Filtered and dried to obtain 11 g of an off-white solid.

[0051] Example 3: Preparation of sodium salt monohydrate

[0052] The off-white solid (Z)-3-(1-hydroxybutenyl)benzofuran-2-one 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. for dissolution, cooled to 20° C. and maintained for 2 h, filtered, and dried to obtain 3 g of a white solid. The differential scanning calorimetry (DSC) diagram of the solid powder is shown in FIG1 , and the thermogravimetric analysis (TGA) diagram thereof is shown in FIG2 .

[0053] Example 4: Preparation of potassium salt

[0054] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq.) in acetone (10 mL). Add potassium hydroxide (0.27 g, 5 mmol, 1.0 eq.) after dissolution. Stir and react for 0.5 h. Filter and dry to obtain 0.9 g of a white solid.

[0055] Example 5: Preparation of magnesium salt

[0056] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq.) in methyl acetate (10 mL). Add magnesium chloride (0.46 g, 5 mmol, 1.0 eq.) after dissolution. Stir and react for 1.5 h. Filter and dry to obtain 0.75 g of a white solid.

[0057] Example 6: Preparation of calcium salt

[0058] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (1 g, 5 mmol, 1.0 eq.) in acetone (10 mL). Add calcium chloride (0.54 g, 5 mmol, 1.0 eq.) after dissolution. Stir and react for 1 h. Filter and dry to obtain 1.05 g of a white solid.

[0059] Example 7: Preparation of piperazine salt

[0060] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq.) in methyl isobutyl ketone (20 mL). Add piperazine (0.84 g, 10 mmol, 1.0 eq.) after dissolution. Stir and react for 1 h. Filter and dry to obtain 2.20 g of a white solid.

[0061] Example 8: Preparation of ethanolamine salt

[0062] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq.) in ethanol (20 mL). Add ethanolamine (0.60 g, 10 mmol, 1.0 eq.) after dissolution. Stir and react for 3 h. Filter and dry to obtain 1.88 g of a white solid.

[0063] Example 9: Preparation of meglumine salt

[0064] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (2 g, 10 mmol, 1.0 eq.) in acetonitrile (20 mL). Add meglumine (1.90 g, 10 mmol, 1.0 eq.) after dissolution. Stir and react for 2 h. Filter and dry to obtain 1.94 g of a white solid.

[0065] Example 10: Preparation of tromethamine salt

[0066] Dissolve (Z)-3-(1-hydroxybutenyl)benzofuran-2-one (20 g, 0.1 mol, 1.0 eq.) in isopropanol (200 mL). Add tromethamine (11.8 g, 0.1 mol, 1.0 eq.) after dissolution. Stir and react for 2 h. Filter and dry to obtain 23 g of a white solid.

[0067] Experiments have shown that the salts described in Examples 2-10 can all be liberated into compound (Z)-3-(1-hydroxybutenyl)benzofuran-2-one under conventional experimental conditions.

[0068] Example 11: Study on the Solubility and Hygroscopicity of Tromethamine Salt Crystalline Forms

[0069] The solubility of the tromethamine salt of Example 10 was tested at 20° C., 25° C. and 30° C. The test results are shown in Table 1.

[0070] Table 1 Solubility data of tromethamine salt in pure water at different temperatures

[0071] According to the guidelines for drug hygroscopicity test in Chinese Pharmacopoeia 9103:

[0072] 1. Take a dry stoppered glass weighing bottle (outer diameter 50mm, height 15mm) and place it in a suitable 25℃±1℃ constant temperature desiccator (with ammonium chloride or ammonium sulfate saturated solution placed at the bottom) or an artificial climate box (set temperature at 25℃±1℃, relative humidity at 80%±2%) the day before the test, and accurately weigh the weight (m1).

[0073] 2. Take an appropriate amount of the test sample and spread it evenly in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1mm. Accurately weigh the weight (m2).

[0074] 3. Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours.

[0075] 4. Close the weighing bottle lid and accurately weigh the weight (m3).

[0076] 5. Description of hygroscopic characteristics and definition of hygroscopic weight gain

[0077] Deliquescent: Absorbs sufficient water to form a liquid.

[0078] Highly hygroscopic: weight gain upon moisture absorption is not less than 15%.

[0079] Hygroscopic: Weight gain due to moisture absorption is less than 15% but not less than 2%.

[0080] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%.

[0081] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0082] Experimental results: m1=28.7554; m2=29.7773; m3=29.7773.

[0083] Calculation formula:

[0084] Example 10 Sample Tromethamine salt sample appearance did not change, the weight gain percentage was 0.0%, and there was no hygroscopicity.

[0085] Example 12: Stability Study

[0086] The stability of the sodium salt sample of Example 2 was investigated under high temperature, high humidity and light conditions. The sampling period was 10 days. The test results are shown in Table 2.

[0087] Table 2 Sodium salt (Example 2 sample) Influencing factors Stability experiment

[0088] The potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt and meglumine salt samples of Examples 4 to 9 were respectively tested for their stability under high temperature, high humidity and light conditions. The sampling period was 10 days. The test results are shown in Table 3.

[0089] Table 3 Potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, meglumine salt (Example 4~sample) Influencing factors Stability experiment

[0090] The stability of the tromethamine salt sample of Example 10 was investigated under conditions of high temperature (60° C.), light (bare), light (packaged), and high humidity (92.5%, 75%). The sampling period was 10 days. The test results are shown in Table 4.

[0091] Table 4 Tromethamine salt (Example 10 sample) influence factor stability experiment

[0092] Conclusion: The influencing factor test showed that tromethamine salt had good chemical stability under the conditions of high temperature (60℃), light (naked), light (packaged), and high humidity (92.5%, 75%).

[0093] Biological assays

[0094] Test 1: Compound activity determination

[0095] The following assays demonstrate that Compound I of Example 1 of the present invention has the effect of improving ischemic neurological impairment. The assay results also show that Compound I of the present invention has good bioavailability and efficacy after oral administration.

[0096] 1. Pharmacokinetic test in rats

[0097] Male SD rats (weighing 180-260 g) were administered Compound I at a dose of 1.0 mg / kg via tail vein injection and 10.0 mg / kg orally, with three animals per group. The dosing solvent was 5% DMSO + 5% Cremophor EL in saline. The rats were fasted for approximately 12 hours before dosing and allowed to eat freely 4 hours after dosing; water was not withheld. Approximately 0.2 mL of blood was collected from the orbital cavity before dosing and at 5, 15, and 30 minutes, and 1, 2, 4, 6, 8, and 24 hours after dosing. The blood was collected in EDTA-K2-anticoagulated EP tubes on ice. Plasma was separated by low-speed centrifugation at 3500 rpm for 10 minutes at 4°C and stored at -20°C until analysis. Plasma concentrations of Compound I were quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Pharmacokinetic parameters were calculated using WinNonlin software.

[0098] As shown in the data in Table 5, after oral administration, compared with the values ​​reported in the literature on butylphthalide (Wang Ningning, Li Yue, Li Xiaohong, Jiang Mingyan, Determination of 3-n-butylphthalide in rat plasma by RP-HPLC and its pharmacokinetics, Chinese Journal of New Drugs and Clinical Medicine, December 2012, Vol. 31, No. 12, pp. 743-747), Compound I has a longer elimination time in rats and a higher bioavailability (the bioavailability exceeding 100% is presumably due to nonlinear pharmacokinetic factors).

[0099] Table 5. Pharmacokinetic parameters

[0100] *Calculated based on the AUC0-inf literature values ​​listed in the table

[0101] 2. Rat brain tissue distribution test

[0102] Male SD rats (weighing 200-270g) were orally administered with compound I and butylphthalide (NBP) at a dose of 20mg / kg. Plasma and brain tissue samples were collected from the animals at 0.5, 1, 4, and 24h after administration. Plasma collection: 0.2mL of whole blood was collected in an EP tube containing EDTA-K2. After centrifugation at 3500g for 10 minutes, the upper plasma was collected and stored at -20°C. Brain tissue collection: After euthanasia of the animal, an appropriate amount of brain tissue was weighed and homogenized according to the ratio of brain tissue: 80% methanol water (w / v) = 1:4. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantitatively analyze the compound concentrations in plasma and brain tissue samples.

[0103] As can be seen from the data in Table 6, after oral administration, the distribution concentration of Compound I of the present invention in rat plasma and brain tissue is higher.

[0104] Table 6 Concentrations of compounds in rat plasma and brain tissue

[0105] NA: below the limit of quantification

[0106] 3. Pharmacodynamic study of the compound in a rat stroke model: single treatment administration

[0107] A middle cerebral artery occlusion (MCAO) model was established in SD rats using a suture method to evaluate the neuroprotective effects of compounds against cerebral ischemia-reperfusion. SD rats (240-270 g) were anesthetized with 3.0% isoflurane. Surgery was performed to isolate and expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated, and the ICA was temporarily clamped. Threads were then threaded through the proximal and distal ends of the CCA, with the distal end tied tightly and the proximal end tied loosely for later use. A small incision was made between the two threads, and a 4-0 suture was inserted through the CCA incision. The suture was then slowly and gently advanced into the internal carotid artery, pausing at the ICA clamp. The pre-ligated suture was then tightened further. The clamp occluding blood flow to the ICA was then removed, and the suture was immediately advanced into the ICA until it entered the skull. When the suture plug is inserted approximately 18 mm from the common carotid bifurcation and a slight resistance is felt, the tip of the suture plug has entered the anterior cerebral artery (ACA) and the lateral wall of the suture plug has blocked the middle cerebral artery ostium. Insertion is stopped at this point and the time is recorded. The arterial clamp on the CCA is removed, and the incision is closed after observing for active bleeding. The ischemic rats are kept at room temperature to maintain a body temperature of 37°C. After 120 minutes, anesthesia is induced. While maintaining anesthesia, the suture plug is gently pulled back into the external carotid artery, achieving middle cerebral artery reperfusion. Immediately after reperfusion (within 10 minutes), the animals are administered once. Three groups are divided into the model control group, the intravenous administration of compound I (30 mg / kg), and the oral administration of compound I (60 mg / kg). The animals are euthanized 24 hours after ischemia-reperfusion, and the brains are quickly removed, frozen, sectioned, and stained with TTC. Normal tissue stains rose red, while infarcted tissue appears white. The percentage of infarct tissue weight to total brain weight was used as infarct area (Infraction Area %) to evaluate the extent of cerebral ischemia in rats.

[0108] One day after surgery, TTC staining analysis revealed that the cerebral infarction extent of the model control group was 21.63±5.66%. The cerebral infarction extent of the animals in the intravenous and oral administration groups of Compound I was 13.61±3.66% and 14.88±5.11%, respectively. This indicates that Compound I significantly reduced the extent of cerebral infarction in both the intravenous and oral administration groups (P=0.0025 and P=0.0389). Furthermore, the cerebral infarction inhibition rates of the animals in the intravenous and oral administration groups were 37.1% and 31.2%, respectively. These results suggest that Compound I can significantly improve cerebral infarction in rats, as shown in Table 7.

[0109] Table 7. Cerebral infarction range and cerebral infarction inhibition rate of experimental animals

[0110] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis, "—" indicates that there is no data for this item, * indicates P < 0.05 compared with the animals in the model control group, ** indicates P < 0.01 compared with the animals in the model control group.

[0111] 4. Pharmacodynamic study of the compound in a medium- to long-term rat stroke model

[0112] A middle cerebral artery occlusion (MCAO) model was established in SD rats using the suture embolization method. The test drugs were administered for 28 consecutive days. The pharmacodynamics of the test drugs in stroke were evaluated through general observation and neurobehavioral scores.

[0113] Sprague-Dawley rats (240-280 g) were anesthetized with 3.0% isoflurane and then surgically isolated and exposed. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were then placed. The ECA was ligated, the ICA temporarily clamped, and a suture thread was threaded through the proximal and distal ends of the CCA. The distal end was tied tightly, and the proximal end was tied with a loose knot for later use. A small incision was made between the two threads, and a 4-0 suture was inserted through the CCA incision. The suture was then slowly and gently advanced into the internal carotid artery, pausing at the arterial clamp of the ICA. The suture was further tightened, and the arterial clamp blocking blood flow in the ICA was removed. The suture thread was then immediately advanced into the ICA until it entered the skull. When the suture thread reached a depth of approximately 18 mm from the common carotid bifurcation and a slight resistance was felt, the tip of the suture thread had entered the anterior cerebral artery (ACA), and the lateral wall of the suture thread had blocked the ostium of the middle cerebral artery. Insertion was stopped at this point, and the time was recorded. The arterial clamp on the CCA was removed, and the incision was closed after observing for active bleeding. The ischemic rats were kept at room temperature to maintain a body temperature of 37°C. Anesthesia was induced 120 minutes later. While maintaining anesthesia, the suture was gently pulled back into the external carotid artery, achieving middle cerebral artery reperfusion. The animals were administered medication immediately (within 10 minutes) after reperfusion and then once daily for 28 consecutive days, with the day of surgery designated as Day 1 (Day 0). Five groups were set up: a sham operation group, a model control group, a 60 mg / kg, orally administered NBP group, a low-dose oral Compound I group (6 mg / kg, orally administered, daily), and a high-dose oral Compound I group (20 mg / kg, orally administered, daily). During the drug administration period, all animals were tested with a grid test (D7, D14, D21 and D28, a total of 4 times) and a novel object recognition test (D26 novel object recognition adaptation, D27 detection). After the end of drug administration on D28, all surviving animals in the group were euthanized and their brains were quickly sampled for pathological analysis.

[0114] (1) The results of the grid test showed that one week after surgery, the frequency of wrong steps in the model control group was 7.24±3.59 times, and the frequency of wrong steps in the NBP treatment group, low-dose compound I group, and high-dose compound I group were 7.37±3.03 times, 5.33±2.33 times, and 4.23±1.44 times, respectively. The frequency of wrong steps in the high-dose compound I group was significantly lower than that in the model control group (P=0.0172). 2-3 weeks after surgery, the frequency of wrong steps in the model group gradually decreased due to the gradual recovery of motor function. Although there was no significant difference in the frequency of wrong steps in the high-dose compound I group compared with the model control group, there was still a trend of decrease. 4 weeks after modeling, the frequency of wrong steps in the model animals decreased to the level of the sham-operated group. The above results suggest that compound I has the effect of improving walking dysfunction in stroke animals (see Table 8 for details).

[0115] Table 8. Grid test results

[0116] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis; "1W, 2W, 3W, 4W" represent 1, 2, 3, and 4 weeks after surgery, respectively; * represents P < 0.05 compared with the model control group.

[0117] (2) On the last day of treatment, a novel object recognition test was performed, and the novel object recognition index (NRI) of the animals in each group was calculated. The novel object recognition index of the animals in the model control group was 54.81±21.94%, which was not significantly different from the old object recognition index (FRI). However, the NRI of the animals in the NBP treatment group, the low-dose compound I group, and the high-dose compound I group were 70.97±22.57%, 70.98±22.60%, and 71.66±17.06%, respectively. Among them, the new / old object recognition index of the NBP treatment group, the low-dose compound I group, and the high-dose compound I group were significantly different (P=0.0074, P=0.0212, and P=0.0009). At the same time, the novel object recognition index of the low-dose compound I group and the high-dose compound I group was comparable to that of the sham operation group (66.51±10.80%). The above results suggest that compound I significantly improves cognitive impairment in animals with stroke (see Table 9 for details).

[0118] Table 9. Results of novel object recognition test

[0119] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis; * represents P < 0.05 compared with the model control group, ** represents P < 0.01 compared with the model control group, *** represents P < 0.001 compared with the model control group.

[0120] (3) Pathological examination: 1) Repair range: Samples with a repair area of ​​>30% were considered to have good recovery, while samples with a repair area of ​​≤30% were considered to have poor recovery. The proportion of animals with good recovery in the model control group was 17.6%, while the proportions of animals with a repair area of ​​>30% in the NBP treatment 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 had the best recovery, which was significantly different from the model control group (Chi-square, P=0.0080). The above results suggest that compound I has the effect of promoting 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: the number of red blood cell-containing blood vessels in the infarcted repair area was ≤10 and >10. In the sham operation group, the number of filled blood vessels in all animals was greater than 10, accounting for 100%. In the model control group, the number of animals with erythrocyte-containing vessels in the infarct repair area ≤10 and >10 were 10 and 7, respectively, accounting for 41.2% of the animals. In the NBP treatment group, the number of animals with erythrocyte-containing vessels in the infarct repair area ≤10 and >10 were 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). In the low-dose and high-dose compound I groups, the number of animals with erythrocyte-containing vessels in the infarct repair area ≤10 and >10 were 0 / 12 and 1 / 12, respectively, and the number of animals with erythrocyte-containing vessels >10 was significantly higher than that in the model control group (Chi-square, P=0.0012, P=0.0076). Secondly, the proportion of samples containing more than 10 red blood cell vessels in the repair area of ​​animals in the low-dose and high-dose groups of Compound I were 100% and 92.3%, respectively, and all treatment groups were above 90%. The above results suggest that Compound I can significantly improve the vascular filling degree of the cerebral infarction repair area of ​​stroke animals under 28 consecutive days of treatment (see Table 11 for details).

[0121] Table 10. Statistics of sample recovery in each group

[0122] Note: “.” represents the number of animals in each group for statistical analysis; ** represents Chi-square comparison with the model control group P < 0.01.

[0123] Table 11. Statistics of the abundance of filled blood vessels in each group of samples

[0124] Note: “.” represents the number of animals in each group for statistical analysis; ** represents Chi-square comparison with the model control group P < 0.01.

[0125] Examples 2-10 are salts of the compound of Example 1, which can be freed as the compound of Example 1 in vivo and thus have the same functions as the compound of Example 1.

[0126] Experiment 2: Efficacy of the tromethamine salt of Example 10 on stroke

[0127] The following assays demonstrate that the tromethamine salt of Example 10 has the effect of improving ischemic neurological deficits. The assay results also show that the tromethamine salt of Example 10 has good bioavailability and efficacy after oral administration.

[0128] 1. Pharmacodynamic study of the compound in a rat stroke model

[0129] A middle cerebral artery occlusion (MCAO) model was established in SD rats using a suture method to evaluate the neuroprotective effects of compounds against cerebral ischemia-reperfusion. SD rats (240-270 g) were anesthetized with 3.0% isoflurane. Surgery was performed to isolate and expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated, and the ICA was temporarily clamped. Threads were then threaded through the proximal and distal ends of the CCA, with the distal end tied tightly and the proximal end tied loosely for later use. A small incision was made between the two threads, and a 4-0 suture was inserted through the CCA incision. The suture was then slowly and gently advanced into the internal carotid artery, pausing at the ICA clamp. The pre-ligated suture was then tightened further. The clamp occluding blood flow to the ICA was then removed, and the suture was immediately advanced into the ICA until it entered the skull. When the suture plug is inserted to a depth of approximately 18 mm from the common carotid artery bifurcation and a slight resistance is felt, the tip of the suture plug has entered the anterior cerebral artery (ACA) and the side wall of the suture plug has blocked the opening of the middle cerebral artery. At this point, insertion is stopped and the time is recorded. The artery clamp on the CCA is removed, and the incision is closed after observing for active bleeding. The ischemic rats are placed at room temperature to maintain their body temperature at 37°C. After 120 minutes, anesthesia can be induced. While maintaining anesthesia, the suture plug is slowly and gently pulled back to the tip of the external carotid artery, thus achieving middle cerebral artery reperfusion. The animals were dosed once immediately after reperfusion (within 10 minutes). A total of four groups were set up, namely, a model control group, a gavage group of tromethamine salt of Example 10 (20 and 40 mg / kg), and an oral group of butylphthalide (60 mg / kg). The drug was administered once immediately after reperfusion (within 10 minutes), and then once a day for 7 consecutive times (including the first administration). Except for the first administration, the subsequent administration time was from 4:00 to 7:00 pm. The NSS behavioral score was measured once before modeling and 24 hours (1 day), 72 hours (3 days) and 7 days after modeling. The animals were euthanized 7 days after ischemia-reperfusion and the brain was quickly removed, frozen and sectioned, and TTC staining was performed. Normal tissue was stained rose red, and infarcted tissue was stained white. The percentage of the weight of the infarcted tissue to the total brain weight was used as the infarct area (Infraction Area%) to evaluate the degree of cerebral ischemia damage in rats.

[0130] (1) Cerebral infarction: 7 days after surgery, the results are shown in Table 12. There was no cerebral infarction in the sham-operated control group, the range of cerebral infarction in the model control group was 19.951±3.432%, and the range of cerebral infarction in the positive drug group was 14.241±2.912%, which were significantly lower than those in the model control group (P≤0.05). The range of cerebral infarction in the animals treated with 20 and 40 mg / kg of tromethamine salt of Example 10 was 14.717±2.541% and 13.762±2.225%, respectively, which were significantly lower than those in the model control group (P≤0.05). Based on the range of cerebral infarction, the cerebral infarction inhibition rates of the animals in the 40 mg / kg tromethamine salt group of Example 10 and the positive drug group (butylphthalide, 60 mg / kg) were 31.023±11.15% and 28.624±14.597%, respectively. The above results indicate that under the experimental conditions, at doses of 20 mg / kg and 40 mg / kg, the tromethamine salt of Example 10 has a significant effect on improving the range of cerebral infarction in rats with ischemic stroke, wherein the tromethamine salt of Example 10 at a dose of 40 mg / kg has a better effect on improving cerebral infarction than the positive drug at a dose of 60 mg / kg.

[0131] Table 12 Cerebral infarction range and cerebral infarction inhibition rate of experimental animals

[0132] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis, "—" indicates that there is no data for this item, and * indicates P ≤ 0.05 compared with the animals in the model control group.

[0133] (2) Neurobehavioral Scores: The results are shown in Table 13. All groups achieved a Bederson score of 3 after surgery, indicating successful ischemia recovery. One day after surgery, the NSS score of the model control group was 7.21±1.03. The NSS scores of the positive drug- and tromethamine salt-treated groups showed a decreasing trend relative to the model control group, with no statistically significant differences.

[0134] Three days after surgery, the NSS score of the model control group animals was 5.92 ± 0.70, and the NSS score of the animals in the 40 mg / kg dose group of the tromethamine salt of Example 10 was significantly lower than that of the model control group (P ≤ 0.05). The NSS score of the animals in the positive drug group (butylphthalide, 60 mg / kg) was 5.75 ± 0.92, which was not significantly different from the model control group. During the subsequent observation period, the NSS score of the model control animals dropped to 5 points and remained above 5 points. The NSS scores of the animals in each drug-treated group showed a continuous downward trend. After 7 days of modeling, the NSS score of the model control animals was 5.17 ± 1.56, and the NSS scores of the animals in the 40 mg / kg group of the tromethamine salt of Example 10 and the positive drug group (butylphthalide, 60 mg / kg) were 3.42 ± 1.54 and 4.04 ± 1.20, respectively. Among them, the NSS score of the animals in the 40 mg / kg group of the tromethamine salt of Example 10 was significantly lower than that of the model control group (P ≤ 0.05). The above results indicate that under the experimental conditions, at a dose of 40 mg / kg, the tromethamine salt of Example 10 significantly improves the acute neurological impairment in rats with ischemic stroke, and the effect is better than that of the positive drug group.

[0135] Table 13 Animal neurobehavioral scoring results

[0136] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis; *P ≤ 0.05 indicates that the difference is statistically significant compared with the model control group; "D0" represents 1 day before surgery, and "D2", "D4", "D6" and D8 represent 1 day, 3 days, 5 days and 7 days after surgery, respectively.

[0137] (3) Summary: This study shows that the tromethamine salt of Example 10 can reduce cerebral infarction caused by stroke while also reducing neurological damage; while the positive drug only has an improvement effect on cerebral infarction. The lower dose of the tromethamine salt of Example 10 (40 mg / kg) has a better inhibitory effect on cerebral infarction and a better effect on reducing neurological damage than the higher dose of the positive drug (60 mg / kg), indicating that the tromethamine salt of Example 10 is more effective than the positive drug.

[0138] Experiment 3: Efficacy of the tromethamine salt of Example 10 on amyotrophic lateral sclerosis

[0139] The following assay demonstrates that the tromethamine salt of Example 10 has the effect of improving amyotrophic lateral sclerosis (ALS).

[0140] 1. Pharmacodynamic study on SOD1-G93A transgenic amyotrophic lateral sclerosis mice

[0141] Male SOD1-G93A transgenic ALS model mice (5 weeks old) were used to evaluate the therapeutic effects of the compound on ALS disease animals. After one week of adaptive feeding, the mice were randomly divided into group A (solvent), group B (riluzole 25 mg / kg po), group C (tromethamine salt of Example 10 40 mg / kg po), and group D (tromethamine salt of Example 10 60 mg / kg po) according to body weight, muscle tone, and rotarod. The dosing frequency was two consecutive weeks, Monday to Friday, followed by a one-week drug-free period. Behavioral measurements were performed every two weeks, using rotarod tests and muscle tone tests, and the weight and survival rate of the animals were monitored to evaluate the therapeutic effect of the tromethamine salt of Example 10 on ALS disease.

[0142] (1) Rotarod test (Table 14): The rotarod test evaluates the animals' exercise endurance and coordination by measuring the rotarod time. In this experiment, the 40 mg / kg dose of tromethamine salt of Example 10 significantly improved the rotarod time of ALS mice at 10-16 weeks of age, i.e., in the middle stage of the disease (P < 0.05). The 60 mg / kg dose group significantly improved the rotarod time of mice in the late stage of the disease (P < 0.001, P < 0.05). Riluzole showed an upward trend in the rotarod time of mice under the conditions of this experiment, but there was no significant difference. Under the conditions of this experiment, the efficacy of the tromethamine salt of Example 10 was superior to that of riluzole.

[0143] Table 14 Effect of tromethamine salt of Example 10 on rotarod time (s) in SOD1 G93A ALS mice

[0144] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0145] (2) Muscle tension test (Table 15): 60 mg / kg of the tromethamine salt of Example 10 significantly improved the muscle tension of 18-20 week old SOD1 G93A ALS mice, while riluzole and 40 mg / kg of the tromethamine salt of Example 10 showed an upward trend in the muscle tension of mice, but there was no significant difference.

[0146] Table 15 Effect of the tromethamine salt of Example 10 on muscle tension (g) in SOD1 G93A ALS mice

[0147] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0148] (3) Effect on survival rate: As shown in FIG3 , compared with the control group and the positive drug group, the SOD1 G93A ALS mice administered with the tromethamine salt of Example 10 (20, 40, and 60 mg / kg) showed a longer survival period, among which the survival period of the 40 mg / kg group was extended by about 15 days.

[0149] (4) Effect on body weight: Compared with the solvent group, the various doses of tromethamine salt of Example 10 and the administration of riluzole had no significant effect on the body weight of mice.

[0150] (5) Summary

[0151] The 40 mg / kg and 60 mg / kg doses of the tromethamine salt of Example 10 significantly improved ALS-like motor dysfunction caused by the SOD1G93A transgene and prolonged the survival of ALS mice to a certain extent. Under the experimental conditions, the positive drug riluzole had some improvement in the neurobehavioral function of ALS mice, but there was no significant difference compared to the solvent control group, indicating weaker efficacy than the tromethamine salt of Example 10.

[0152] 2. Pharmacodynamic study on TDP-43-A315T transgenic ALS mice

[0153] TDP-43-A315T transgenic ALS model mice (5 weeks old, half male and half female) were used to evaluate the therapeutic effects of compounds on ALS disease animals. After one week of adaptive feeding, the mice were randomly divided into Group A (vehicle), Group B (riluzole 25 mg / kg po), Group C (tromethamine salt of Example 10 40 mg / kg po), and Group D (tromethamine salt of Example 10 60 mg / kg po) based on body weight, muscle tone, and rotarod. The mice were dosed once daily for 7 consecutive days per week, followed by a one-week rest period. Behavioral assessments were performed every two weeks, using rotarod and muscle tone tests, and the weight and survival rates of the animals were monitored to evaluate the therapeutic effects of the tromethamine salt of Example 10 on ALS disease.

[0154] (1) Muscle tension experiments (Tables 16 and 17) showed that at 13-14 weeks of age, compared with the vehicle group, the 60 mg / kg tromethamine salt of Example 10 significantly improved muscle tension in male TDP-43 ALS mice, while riluzole showed an upward trend in muscle tension, but there was no significant difference. At 14-16 weeks of age, the 20 mg / kg and 40 mg / kg tromethamine salts of Example 10 significantly increased muscle tension in female ALS mice, while riluzole had no significant effect on improving muscle tension in female ALS mice.

[0155] Table 16 Effect of the tromethamine salt of Example 10 on muscle tension (g) in male TDP-43 A315T ALS mice

[0156] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0157] Table 17 Effects of the tromethamine salt of Example 10 on muscle tone in female TDP-43 A315T ALS mice

[0158] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0159] (2) The rotarod test (Tables 18 and 19) showed that the 60 mg / kg tromethamine salt of Example 10 significantly increased the rotarod time of male TDP43 ALS mice at 11-13 weeks of age. The riluzole group also showed an upward trend in rotarod time at 11-13 weeks of age, but there was no significant difference compared to the vehicle group. The 20 mg / kg tromethamine salt of Example 10 also had a certain upward effect on the rotarod time of female mice.

[0160] Table 18 Effect of Example 10 Tromethamine Salt on Rotarod Time (s) in Male TDP-43 A315T ALS Mice

[0161] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0162] Table 19 Effect of tromethamine salt of Example 10 on rotarod time (s) in female TDP-43 A315TALS mice

[0163] Note: The data in the table are expressed as mean ± standard deviation (Mean ± SEM); compared with the solvent group, * indicates P < 0.05,

[0164] ** indicates P < 0.01, *** indicates P < 0.001.

[0165] (3) Body weight determination: The administration of riluzole and the tromethamine salt of Example 10 at different doses had no significant effect on the body weight of mice.

[0166] (4) Summary: The tromethamine salt of Example 10 can significantly improve the motor dysfunction of TDP-43 A315T transgenic ALS mice without affecting the body weight of the mice, and its efficacy is better than that of the control drug riluzole.

[0167] Experiment 4: Therapeutic Effect of the Tromethamine Salt of Example 10 on Parkinson's Disease

[0168] The following assay method demonstrates that the tromethamine salt of Example 10 of the present invention has the effect of alleviating Parkinson's cognitive dysfunction.

[0169] 1. Therapeutic effect of the compound on MPTP-induced Parkinson's disease mouse model

[0170] A Parkinson's disease model was established in mice by intraperitoneal injection of 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 tromethamine salt of the test sample Example 10 was evaluated based on the results of behavioral tests, striatal dopamine (DOPA) and histopathological examinations of the animals.

[0171] Fifty C57 mice were randomly divided into five groups based on the results of the pole climbing test before modeling: a normal control group, a model control group, a low-dose group of the tromethamine salt of Example 10, a high-dose group of the tromethamine salt of Example 10, and a positive control group (levodopa, 20 mg / kg). Drug intervention treatment began on the day of the first MPTP injection, with administration once daily for four consecutive weeks. The tromethamine salt of the test sample 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 pole climbing test and gripping strength test were performed within 48 hours after the last MPTP injection.

[0172] 1) Pole climbing test results

[0173] The results of the pole climbing test statistics are summarized in Table 19 and Figure 4. Before modeling, there was no significant difference in the length of time that animals in each test group climbed the pole. At the end of the test, the length of time that animals in the normal control group climbed the pole was 8.35 ± 0.89 s, and the length of time that animals in the model control group climbed the pole was 11.51 ± 2.39 s, which was significantly increased compared to the normal control group (P < 0.05). The length of time that animals in the positive control group climbed the pole was 8.76 ± 1.57 s, which showed a decreasing trend compared to the model control. The length of time that animals in the low-dose group and high-dose group of the tromethamine salt of Example 10 climbed the pole was 9.67 ± 1.48 s and 8.83 ± 1.77 s, respectively. The length of time that animals in the high-dose group of the tromethamine salt of Example 10 climbed the pole was reduced compared to the model control, and the difference was statistically significant (P < 0.05) compared to the model control. The above results suggest that the tromethamine salt of Example 10 has a significant therapeutic effect on the PD model induced by MPTP.

[0174] Table 19 Climbing pole (s) test results

[0175] Note: The data in the table are expressed as mean (Mean) ± standard deviation (SD). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P<0.05 indicates that the difference is statistically significant compared with the model control group.

[0176] 2) Grip test results

[0177] The statistical summary results of the gripping force test are shown in Table 20 and Figure 5. There was no significant difference in the gripping force of the animals in the experimental groups before modeling. At the end of the experiment, the gripping force of the animals in the normal control group was 260.98±14.81g, and the gripping force of the animals in the model control group was 210.92±10.00g, which were significantly lower than those in the normal control group (P<0.05). The gripping force of the animals in the positive control group (levodopa) was 226.26±8.39g, showing an upward trend compared to the model control. The gripping force of the animals in the low-dose and high-dose groups of the tromethamine salt of Example 10 was 219.05±12.83g and 227.16±5.85g, respectively, showing an upward trend compared to the model control.

[0178] Table 20 Grip force (g) test results

[0179] Note: The data in the table are expressed as mean (Mean) ± standard deviation (SD). & P<0.05 indicates that the difference is statistically significant compared with the normal control group.

[0180] 3) Striatal DOPA test results

[0181] The summary results of striatal DOPA testing are shown in Table 21 and Figure 6. The striatal DOPA content of the normal control group was 4931.52±1607.66 ng / g, and the striatal DOPA content of the model control group was 891.92±285.94 ng / g, which were significantly lower than those of the normal control group (P<0.05). The DOPA content of the positive control group was 1156.72±375.72 ng / g, which was not significantly changed compared with the model control group. The DOPA content of the high-dose tromethamine salt group of Example 10 was 2731.07±868.18 ng / g, which was significantly higher than that of the model control group (P<0.05). The DOPA content of the low-dose tromethamine salt group of Example 10 was 837.82±372.19 ng / g, which was not significantly changed compared with the model control group. The above results indicate that the high-dose group (20 mg / kg) of tromethamine salt in Example 10 has a significant protective effect on dopaminergic neurons in the PD model prepared by MPTP.

[0182] Table 21 Striatal DOPA (ng / g) test results

[0183] Note: The data in the table are expressed as mean (Mean) ± standard deviation (SD). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P<0.05 indicates that the difference is statistically significant compared with the model control group.

[0184] 4) Statistical results of TH-positive fiber content in the striatum

[0185] The statistical summary results of striatal TH-positive fiber content are shown in Table 22 and Figure 7. The TH-positive fiber content of the normal control group was 1133191.93±103519.32, and the TH-positive fiber content of the model control group was 470927.76±115639.36, which was significantly lower than that of the normal control group (P<0.05). The TH-positive fiber content of the positive control group (levodopa) was 536968.78±68050.46, which was not significantly changed compared with the model control group. The TH-positive fiber content of the high-dose tromethamine salt group of Example 10 was 709627.56±100462.19, which was significantly increased compared with the model control group (P<0.05). The TH-positive fiber content of the low-dose tromethamine salt group of Example 10 was 526554.39±169680.75, which was not significantly changed compared with the model control group. The above results indicate that, consistent with the above results, the high-dose group (20 mg / kg) of tromethamine salt of Example 10 has a significant protective effect on dopaminergic neurons in the PD model prepared by MPTP.

[0186] Table 22 Statistical results of TH-positive fiber content in striatum

[0187] Note: The data in the table are expressed as mean (Mean) ± standard deviation (SD). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P<0.05 indicates that the difference is statistically significant compared with the model control group.

[0188] 5) Statistical results of the number of TH-positive cells in the substantia nigra pars compacta

[0189] The statistical summary results of the number of TH-positive cells in the substantia nigra pars compacta are shown in Table 23 and Figure 8, and the individual data are shown in Appendix 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 changed compared with the model control group. The number of TH-positive cells in the high-dose group of tromethamine salt 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 group of tromethamine salt of Example 10 was 31.22 ± 5.97, which was not significantly changed compared with the model control group. The results once again demonstrated that the high-dose group of tromethamine salt of Example 10 (20 mg / kg) had a significant protective effect on dopaminergic neurons in the PD model prepared by MPTP.

[0190] Table 23 Statistical results of the number of TH-positive cells in the substantia nigra pars compacta

[0191] Note: The data in the table are expressed as mean (Mean) ± standard deviation (SD). & P<0.05 indicates that the difference is statistically significant compared with the normal control group. # P<0.05 indicates that the difference is statistically significant compared with the model control group.

[0192] Experiment 5: Therapeutic Effect of the Tromethamine Salt of Example 10 on Spinal Cord Injury

[0193] The following assay method demonstrates that the tromethamine salt of Example 10 of the present invention has the effect of alleviating spinal cord injury.

[0194] 1: Study on compound treatment of spinal cord injury (SCI) in rats

[0195] In a rat spinal cord injury model, the therapeutic effect of the tromethamine salt of Compound Example 10 on spinal cord injury was studied and the mechanism of action was preliminarily explored.

[0196] Male SD rats were anesthetized with isoflurane and the skin of the thoracic vertebrae on the back of the rats was prepared. The skin was incised with the spinous process of the T10 vertebra as the center, and the subcutaneous fascia and muscles were cut layer by layer to expose the spinous process and lamina, exposing the spinal cord dura mater. The rats were fixed under a JK052-type impactor and a 10g impactor was used to hit the T10 thoracic spinal cord from a free fall of 5 cm to prepare a spinal cord injury model. 2 hours after modeling, the rats with successful modeling were divided into 4 groups, with 10 rats in each group. A sham operation group was also set up to count 10 rats. The test drug group was gavaged with the tromethamine salt of Example 10 at doses of 5 and 20 mg / kg, respectively, once a day for 28 consecutive days. The positive control group (methylprednisolone sodium succinate for injection) was given methylprednisolone sodium succinate for injection once through the tail vein at a dose of 180 mg / kg. The sham operation group and the model control group were gavaged with an equal volume of 0.5% CMC. Group dosing is shown in Table 24 below.

[0197] Table 24 Compounds for the treatment of spinal cord injury (SCI) in rats: grouping and administration

[0198] 1) Impact on BBB score

[0199] The rats with spinal cord injury showed obvious neurological dysfunction, and BBB scores were performed at different time points. The scoring method is as follows:

[0200] Rat BBB scoring standard

[0201] With the extension of time after modeling, the BBB score recovered slightly. The test compound Example 10, tromethamine salt, significantly improved the BBB score from 7 days to 28 days after administration at 20 mg / kg (P < 0.05-0.01). The positive control (methylprednisolone sodium succinate for injection) also improved the BBB score from 14 days to 28 days after administration (P < 0.05). The results are shown in Table 25.

[0202] Table 25 Effect of the Tromethamine Salt of Compound Example 10 on BBB Score (mean ± SD)

[0203] Note: 1. Compared with the sham operation group, ### P<0.001; compared with the model control group: * P<0.05, ** P<0.01.

[0204] 2) Impact on pathological changes

[0205] In the sham-operated group, the spinal cord tissue structure was dense, the membrane structure was intact, the gray matter and white matter of the spinal cord had clear structures and distinct boundaries; the outlines of neurons and glial cells were clear, the nerve fibers were continuous, and the nerve cells were neatly arranged.

[0206] Compared to the sham-operated group, the model control group showed severe spinal cord damage, with a noticeable defect on one side of the spinal cord, loose and disorganized tissue, accompanied by inflammatory cell infiltration, and significant neuronal vacuolation and degeneration. No significant differences were observed between the low-dose group and the model control group. In the 20 mg / kg dose group of the tromethamine salt of Test Compound Example 10, over half (4 / 6 cases) experienced lesion reduction. In the positive drug group (methylprednisolone sodium succinate for injection), some (3 / 6 cases) experienced lesion reduction. The results are shown in Figure 9.

[0207] 3) Effects on NF200 expression

[0208] NF200 is a marker for nerve fibers. Compared with the sham-operated group, NF200 expression was slightly increased in rats after spinal cord injury (P < 0.01). Compared with the model control group, the test drug Example 10, tromethamine salt at 20 mg / kg, significantly increased NF200 expression (P < 0.01). There was no significant change in NF200 expression after administration of the positive control (methylprednisolone sodium succinate for injection) (P > 0.05). The results are shown in Table 26 and Figure 10.

[0209] Table 26 Effect of tromethamine salt of Example 10 on NF200 expression (mean ± SD, n = 6)

[0210] Note: 1. Compared with the sham operation group, ## P<0.01; compared with the model control group: ** P<0.01.

[0211] 4) Transcriptomic analysis of injured spinal cord

[0212] To further verify the protective or regenerative effect of the tromethamine salt of Example 10 on the damaged spinal cord, tissue was harvested 1 cm upstream and downstream of the damaged site for transcriptomic 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. Pharmaceutically acceptable salts of compounds of formula (I) The pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt, piperazine salt, ethanolamine salt, meglumine salt, tromethamine salt; Alternatively, the pharmaceutically acceptable salt is selected from the monohydrate, dihydrate, trihydrate or hemihydrate of the aforementioned salt.

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

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

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

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

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

7. A pharmaceutically acceptable salt of the compound according to claim 1, which is a (Z)-3-(1-hydroxybutenyl)benzofuran-2-one piperazine salt.

8. A pharmaceutically acceptable salt of the compound according to claim 1, which is (Z)-3-(1-hydroxybutenyl)benzofuran-2-oneethanolamine salt.

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

10. A pharmaceutically acceptable salt of the compound according to claim 1, which is a (Z)-3-(1-hydroxybutenyl)benzofuran-2-one tromethamine salt.

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 following steps: a step of forming a salt of the compound of formula (I) with a base.

12. The method according to claim 11, characterized in that The solvent used in the salt-forming reaction is selected from at least one of methanol, ethanol, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, acetonitrile, isopropyl alcohol, acetone, isopropyl alcohol, tetrahydrofuran, methyl isobutyl ketone, n-heptane, dichloromethane, toluene, isopropyl ether, methyl tert-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.

14. 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 in the preparation of a drug for treating and / or preventing diseases related to ischemic brain injury, preferably, the diseases related to ischemic brain injury include but are not limited to cerebral ischemia, ischemic stroke, vascular dementia, post-ischemic inflammation, convulsions, ischemic brain neuron damage or necrosis.

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 in the preparation of a drug for treating and / or preventing a neuromuscular disease, preferably, the neuromuscular disease is amyotrophic lateral sclerosis 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 in the preparation of a drug for treating and / or preventing diseases related to neurological impairment, preferably, the diseases related to neurological impairment include but are not limited to spinal cord injury or necrosis.

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 preparing a drug for treating and / or preventing a neurodegenerative disease, preferably, the neurodegenerative disease includes but is not limited to Parkinson's disease.

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