1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds, their manufacturing methods, and uses

Abstract

The present invention provides 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds and their preparation and use, which belong to the field of drug research and development. 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds can be used to prepare antiepileptic drugs, and drugs for treating and / or preventing traumatic brain injury, ischemic stroke, hemorrhagic stroke, and Parkinson's disease.

Description

[Technical Field]

[0001] Claims 1 to 10 of the present invention claim priority to the patent application filed on April 20, 2023, application number CN 202310430869.9, title of invention "1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, method for producing the same and uses therefor".

[0002] The present invention relates to the field of drug research and development technology, and more particularly to 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds, as well as methods for producing the same and their uses. [Background technology]

[0003] Diffuse depolarization (SD) is a wave-like phenomenon in the gray matter of the central nervous system, characterized by neuronal swelling, deformation of dendritic spines, significant changes in slow potentials, and suppression of cerebral electrical activity (spreading electroencephalography). Currently, there is clinically conclusive electrophysiological evidence that diffuse depolarization is universally present in individuals with conditions such as migraine aura, ischemic stroke, traumatic brain injury, aneurysmal subarachnoid hemorrhage (aSAH) and delayed ischemia after subarachnoid hemorrhage (DCI), spontaneous cerebral hemorrhage, subdural hematoma, idiopathic intracranial hemorrhage or traumatic brain injury, Alzheimer's disease, Parkinson's disease, and epilepsy. Therefore, diffuse depolarization is one of the major and most often overlooked pathophysiological processes in acute neurology. Diffusion depolarization can be experimentally induced, and its triggers include many harmful conditions such as potassium, glutamate, and sodium pump inhibitors, status epilepticus, hypoxia, hypoglycemia, and ischemia, but it can also invade healthy, unaffected tissues. The physiological hemodynamic response resulting from resistance vasoconstriction causes transient hyperperfusion in healthy tissues or retrograde hemodynamic responses or diffusive ischemia in progressively damaged tissues, leading to severe hypoperfusion and accelerating disease progression. Therefore, therapies targeting diffusion depolarization or retrograde hemodynamic responses may be potentially useful in treating these neurological disorders.

[0004] Therefore, providing 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds, as well as their manufacturing methods and uses, and effectively suppressing diffusion depolarization in the aforementioned neurological diseases is of great significance. [Overview of the Initiative]

[0005] The object of the present invention is to provide a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, as well as a method for producing the same and its uses, thereby solving the problem that commercially available drugs in the existing technology cannot effectively suppress diffusion depolarization in neurological diseases.

[0006] To achieve the objectives of the above invention, the present invention provides the following technical solutions.

[0007] The present invention provides a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound having the structure shown by formula (I).

[0008] [ka]

[0009] The present invention provides a method for producing a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, comprising the following steps.

[0010] Step (1) Mix compound 1a with dichloromethane, then add compound 1b and react to obtain compound 1c. Step (2) Compound 1c, compound 1d, and aluminum trichloride are sequentially mixed with dichloromethane and then reacted to obtain compound 1e. Step (3) Compound 1e, sodium borohydride solution, and sodium hydroxide solution are sequentially mixed with tetrahydrofuran and then reacted to obtain compound 1f. Step (4) After mixing compound 1f, sodium acetate and acetic anhydride, react them to obtain 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound. The said compound 1a is

[0011]

Chemical formula

[0012] and the said compound 1b is

[0013]

Chemical formula

[0014] and the said compound 1d is

[0015]

Chemical formula

[0016] as follows.

[0017] The present invention provides the use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of anti-epileptic drugs.

[0018] The present invention provides the use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of drugs for treating and / or preventing traumatic brain injury diseases.

[0019] The present invention provides the use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of drugs for treating and / or preventing ischemic stroke.

[0020] The present invention provides the use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of drugs for treating and / or preventing hemorrhagic stroke.

[0021] The present invention provides uses for 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene in the manufacture of drugs for the prevention and / or treatment of Parkinson's disease.

[0022] The 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound (hereinafter abbreviated as CTMB) provided by the present invention can be compounded into different dosage forms by mixing with pharmaceutically acceptable excipients, and can be compounded into liquid dosage forms such as emulsion injections and oral emulsions. Those skilled in the art can also compound it into solid dosage forms such as tablets and capsules using conventional technical means. The said dosage forms can be administered by routes such as intravenous injection, intramuscular injection, or oral administration at the time of use, and are administerable insofar as the compound having the structure represented by formula (I) of the present invention reaches the site of action and reaches an effective concentration.

[0023] Beneficial effects of the present invention: (1) The CTMB of the present invention can reduce the severity and incidence of pentetrazole-induced epileptic seizures, and can significantly prolong the latency period of clonic and tonic seizures, and its effects are dose-dependent. Furthermore, it significantly reduces the severity of acute epileptic seizures in a rat epilepsy model induced by lithium-pilocarpine, improves rat survival, and its effect is superior to that of sodium valproate, a broad-spectrum antiepileptic drug. (2) Drugs manufactured at CTMB can significantly improve neurobehavioral function in rats with ischemic stroke and hemorrhagic stroke, and can restore sensory and motor function in rats. (3) CTMB improves the integrity of the blood-brain barrier and promotes the recovery of motor function in model rats. It reduces neuroinflammatory responses. It counteracts glutamate excitatory neurotoxicity. It reduces apoptosis of nerve cells. It improves neurological dysfunction and emotional anxiety in model rats and has a significant neuroprotective effect against traumatic brain injury. (4) CTMB improves motor function in Parkinson's disease model mice, enhances their coordination and balance abilities, increases the number of Nissl bodies in the substantia nigra region of the model mice, reduces neuronal damage in the striatum and substantia nigra region, decreases MDA concentration in the striatum of the model mice, and improves GSH concentration and SOD activity in the striatum of the model mice, thereby improving oxidative stress in the striatum. [Brief explanation of the drawing]

[0024] [Figure 1] This chart shows the survival status of SE model rats after acute drug intervention. [Figure 2] This shows the results of the blood-brain barrier permeability test in Experiment 3 (content of Evans Blue dye per gram of brain tissue from different groups of rats). [Figure 3] These are the measurement results for TNF-α, IL-1, and IL-6 in the rat cerebral cortex from Experiment 4. [Figure 4] These are the measurement results for GLT-1, GABA, and GLT-1 / GABA in different dose groups in Study 5. [Figure 5] This is an HE-stained image of the substantia nigra (SN) in the mouse midbrain. [Figure 6] This is an HE-stained image of mouse striatal (STR) neurons. The gray arrows in the image indicate necrotic neurons, and the white arrows indicate proliferative glial cells. [Figure 7] This image shows Nissl body staining of neurons in the substantia nigra area of ​​the mouse midbrain. The arrows in the figure indicate Nissl bodies. [Figure 8] This study demonstrates the effect of CTMB administration on TH protein expression levels in the substantia nigra of the mouse midbrain. [Modes for carrying out the invention]

[0025] The present invention provides a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound having the structure shown by formula (I).

[0026] [ka]

[0027] The present invention provides a method for producing a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, comprising the following steps.

[0028] Step (1) Mix compound 1a with dichloromethane, then add compound 1b and react to obtain compound 1c. Step (2) Compound 1c, compound 1d, and aluminum trichloride are sequentially mixed with dichloromethane and then reacted to obtain compound 1e. Step (3) Compound 1e, sodium borohydride solution, and sodium hydroxide solution are sequentially mixed with tetrahydrofuran and then reacted to obtain compound 1f. Step (4) Compound 1f is reacted with sodium acetate and acetic anhydride to obtain the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound.

[0029] The compound 1a is

[0030] [ka]

[0031] The compound 1b is

[0032] [ka]

[0033] The compound 1d is

[0034] [ka] That is the case.

[0035] In the present invention, in step (1) above, the mass-volume ratio of compound 1a, dichloromethane, and compound 1b is 23-27g:130-170mL:93-97g, preferably 24-26g:140-160mL:94-96g, and more preferably 25g:150mL:94.5-95.5g.

[0036] In the present invention, when mixing compound 1a and dichloromethane, the mixing is preferably carried out under a nitrogen atmosphere, and when mixing compound 1b and dichloromethane, it is preferably added dropwise.

[0037] In the present invention, in step (1), the reaction temperature is 20 to 30°C, preferably 22 to 28°C, and more preferably 25°C. The reaction is carried out under stirring conditions, and the reaction is terminated when no more bubbles are generated under stirring conditions.

[0038] In the present invention, in step (2) above, the mass-volume ratio of compound 1c, compound 1d, aluminum trichloride, and dichloromethane is 22-27g:30-33g:26-30g:80-120mL, preferably 23-26g:30.5-32.5g:27-29g:85-105mL, and more preferably 23.5-25.5g:30.7-32.3g:27.5-28.5g:90-100mL.

[0039] The aforementioned compound 1c is

[0040] [ka]

[0041] That is the case.

[0042] In the present invention, the mixing temperature of aluminum trichloride is -2 to 2°C, preferably 0°C.

[0043] In the present invention, in step (2), the reaction temperature is 20 to 30°C, preferably 22 to 28°C, and more preferably 25°C. The reaction time is 2 to 3 hours, preferably 2.5 hours.

[0044] In the present invention, in step (3) above, the mass-volume ratio of compound 1e, sodium borohydride solution, and tetrahydrofuran is 40-45 g:18-22 mL:140-160 mL, preferably 41-44 g:19-21 mL:145-155 mL, and more preferably 42-43 g:20 mL:150 mL. Of these, the concentration of sodium borohydride solution is 0.8-1.0 g / mL, preferably 0.85-0.95 g / mL, and more preferably 0.87-0.93 g / mL.

[0045] The aforementioned compound 1e is

[0046] [ka]

[0047] That is the case.

[0048] In the present invention, the mixing temperature of the sodium borohydride solution is -2 to 2°C, preferably 0°C.

[0049] In the present invention, the concentration of the sodium hydroxide solution is 8 to 11 wt%, preferably 9 to 10 wt%, and more preferably 10 wt%. The amount of sodium hydroxide solution used is preferably 10 drops.

[0050] In the present invention, in step (3), the reaction temperature is 50 to 65°C, preferably 55 to 65°C, and more preferably 60°C. The reaction time is 2 to 4 hours, preferably 2.5 to 3.5 hours, and more preferably 3 hours.

[0051] In the present invention, in step (4) above, the mass-volume ratio of compound 1f, sodium acetate, and acetic anhydride is 38-45 g:7-10 g:200-230 mL, preferably 39-44 g:7.5-9.5 g:205-225 mL, and more preferably 40-43 g:8-9 g:210-220 mL.

[0052] The aforementioned compound 1f is

[0053] [ka]

[0054] That is the case.

[0055] In the present invention, the reaction temperature is 120 to 140°C, preferably 125 to 140°C, and more preferably 130 to 140°C. The reaction time is 2 to 4 hours, preferably 2.5 to 3.5 hours, and more preferably 3 hours.

[0056] In the present invention, the reaction pathway is as follows (compound 1 is a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound).

[0057] [ka]

[0058] The present invention provides applications for 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds in the manufacture of antiepileptic drugs.

[0059] The present invention provides a use for a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of a drug for treating and / or preventing traumatic brain injury.

[0060] The present invention provides a use for a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of a drug for treating and / or preventing ischemic stroke.

[0061] The present invention provides a use for 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds in the manufacture of drugs for the treatment and / or prevention of hemorrhagic stroke.

[0062] The present invention provides uses for 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene in the manufacture of drugs for the prevention and / or treatment of Parkinson's disease.

[0063] The technical solutions provided by the present invention will be described in detail below with reference to examples, but these should not be construed as limitations on the scope of protection of the present invention.

[0064] Example 1 25.00 g of compound 1a was dissolved in 150 mL of dichloromethane under a nitrogen atmosphere and stirred uniformly. Then, compound 1b was added dropwise and the reaction was carried out. Of the total amount of compound 1b used, 95.08 g was used, the reaction temperature was 25°C, and the reaction was carried out under stirring conditions. The reaction was completed when no more bubbles were generated under stirring conditions, and the mixture was concentrated to obtain 25.00 g of an orange-yellow liquid, which was compound 1c, with a yield of 84.4%.

[0065] 25.00 g of compound 1c and 31.95 g of compound 1d were dissolved in 100 mL of dichloromethane. The resulting mixture was cooled to 0°C, and 28.12 g of aluminum trichloride was added. The reaction system was then heated to 25°C and reacted for 2.5 hours. The reaction product was then quenched with 300 mL of water, extracted with ethyl acetate (3 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by slurring with 40 mL of ethanol for 1 hour to obtain 43.70 g of a white solid, which was compound 1e, with a yield of 91.9%.

[0066] 43.70 g of compound 1e was dissolved in 150 mL of tetrahydrofuran, and the resulting mixture was cooled to 0°C. 20 mL of sodium borohydride aqueous solution was then added dropwise, with a concentration of 0.925 g / mL of sodium borohydride aqueous solution. Subsequently, 10 drops of 10 wt% sodium hydroxide solution were added dropwise, and the reaction system was heated to 60°C for 3 hours. After cooling to 37°C, the pH was adjusted to 7 with 1 M hydrochloric acid solution. The mixture was concentrated to remove the tetrahydrofuran, extracted with ethyl acetate (3 × 200 mL), combined with the organic phase, dried over anhydrous magnesium sulfate, and then concentrated to obtain 42.59 g of an orange-yellow semi-solid, which was compound 1f, with a yield of 96.7%.

[0067] 42.59 g of compound 1f and 8.31 g of anhydrous sodium acetate were dissolved in 210 mL of acetic anhydride, and the reaction system was heated to 140 °C and reacted for 3 hours. The mixture was then concentrated to remove the acetic anhydride, quenched with 200 mL of water, extracted with ethyl acetate (4 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and then concentrated to obtain the crude product. Finally, recrystallization with 70% ethanol yielded 23.47 g of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound as a white solid. The yield was 59.4%.

[0068] 1 H-NMR (400MHz,CDCl3) δ 6.82 (s,1H), 6.51 (s, 1H), 6.34 (t,J=2.4Hz,1H),3.88 (s,3H), 3.82 (s,3H), 3.81 (s,3H),3.13~2.95 (m,2H), 2.89 (d,J = 6.1 Hz,2H), 2.09 (p,J = 7.8 Hz,2H).

[0069] 13 C-NMR (100 MHz, CDCl3) δ 150.7,148.0,143.1,142.5,119.0,114.5,111.4,98.0,56.9,56.6,56.2,32.8,32.6,18.5.

[0070] Example 2 27.00 g of compound 1a was dissolved in 170 mL of dichloromethane under a nitrogen atmosphere and stirred uniformly. Then, compound 1b was added dropwise and the reaction was carried out. Of the total amount of compound 1b used, 97 g was used, the reaction temperature was 30°C, and the reaction was carried out under stirring conditions. The reaction was completed when no more bubbles were generated under stirring conditions, and the mixture was concentrated to obtain 26.62 g of an orange-yellow liquid, which was compound 1c, with a yield of 83.6%.

[0071] 26.62 g of compound 1c and 33 g of compound 1d were dissolved in 120 mL of dichloromethane. The resulting mixture was cooled to 0°C, and 30 g of aluminum trichloride was added. The reaction system was then heated to 30°C and reacted for 2 hours. The reaction product was then quenched with 300 mL of water, extracted with ethyl acetate (3 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by slurring with 40 mL of ethanol for 1 hour to obtain 44.05 g of a white solid, which was compound 1e, with a yield of 89.7%.

[0072] 44.05 g of compound 1e was dissolved in 160 mL of tetrahydrofuran, and the resulting mixture was cooled to 0°C. 22 mL of sodium borohydride aqueous solution was then added dropwise, with a concentration of 1.0 g / mL of sodium borohydride aqueous solution. Subsequently, 10 drops of 10 wt% sodium hydroxide solution were added dropwise, and the reaction system was heated to 65°C for 2 hours. After cooling to 37°C, the pH was adjusted to 8 with 1 M hydrochloric acid solution. The mixture was concentrated to remove the tetrahydrofuran, extracted with ethyl acetate (3 × 200 mL), combined with the organic phase, dried over anhydrous magnesium sulfate, and then concentrated to obtain 40.10 g of an orange-yellow semi-solid, which was compound 1f, with a yield of 90.3%.

[0073] 40.10 g of compound 1f and 10 g of anhydrous sodium acetate were dissolved in 230 mL of acetic anhydride, and the reaction system was heated to 140 °C and reacted for 2 hours. The mixture was then concentrated to remove the acetic anhydride, quenched with 200 mL of water, extracted with ethyl acetate (4 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and then concentrated to obtain the crude product. Finally, recrystallization with 70% ethanol yielded 22.59 g of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound as a white solid. The yield was 59.2%.

[0074] Example 3 23.00 g of compound 1a was dissolved in 130 mL of dichloromethane under a nitrogen atmosphere and stirred uniformly. Then, compound 1b was added dropwise and the reaction was carried out. Of the total amount of compound 1b used, 93 g was used, the reaction temperature was 20°C, and the reaction was carried out under stirring conditions. The reaction was completed when no more bubbles were generated under stirring conditions, and the mixture was concentrated to obtain 22.87 g of an orange-yellow liquid, which was compound 1c, with a yield of 84.0%.

[0075] 22.87 g of compound 1c and 30 g of compound 1d were dissolved in 80 mL of dichloromethane. The resulting mixture was cooled to 0°C, and 26 g of aluminum trichloride was added. The reaction system was then heated to 20°C and reacted for 3 hours. The reaction product was then quenched with 300 mL of water, extracted with ethyl acetate (3 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by slurring with 40 mL of ethanol for 1 hour to obtain 40.22 g of a white solid, which was compound 1e, with a yield of 90.1%.

[0076] 40.22 g of compound 1e was dissolved in 140 mL of tetrahydrofuran, and the resulting mixture was cooled to 0°C. 18 mL of sodium borohydride aqueous solution was then added dropwise, with a concentration of 0.8 g / mL. Subsequently, 10 drops of 10 wt% sodium hydroxide solution were added dropwise, and the reaction system was heated to 50°C for 4 hours. After cooling to 37°C, the pH was adjusted to 7 with 1 M hydrochloric acid solution. The mixture was concentrated to remove the tetrahydrofuran, extracted with ethyl acetate (3 × 200 mL), and the organic phases were combined. The mixture was dried over anhydrous magnesium sulfate and then concentrated to obtain 38.31 g of an orange-yellow semi-solid, which was compound 1f, with a yield of 94.5%.

[0077] 38.31 g of compound 1f and 7 g of anhydrous sodium acetate were dissolved in 200 mL of acetic anhydride, and the reaction system was heated to 120 °C and reacted for 4 hours. The mixture was then concentrated to remove the acetic anhydride, quenched with 200 mL of water, extracted with ethyl acetate (4 × 200 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, and then concentrated to obtain the crude product. Finally, recrystallization with 70% ethanol yielded 21.10 g of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound as a white solid. The yield was 59.3%.

[0078] Performance testing: 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound (Compound 1, 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene, hereinafter abbreviated as CTMB)

[0079] Manufacturing process for emulsion injection: 10.0 g of CTMB obtained in Example 1 of the present invention and 200.0 g of soybean oil for injection are placed in a beaker, heated to 75°C under nitrogen-filled conditions, and stirred to dissolve. Next, 12.0 g of egg yolk lecithin is weighed and added, and stirred to dissolve to prepare the oil phase. Separately, 22.5 g of glycerin is weighed, and approximately 680 mL of water is measured out, heated to 75°C under nitrogen-filled conditions, and stirred to dissolve to prepare the aqueous phase. The above oil phase is added to the aqueous phase, high-speed shearing is performed for 10 minutes, and water is added to make a total volume of 1000 mL to prepare the crude emulsion. The crude emulsion is homogenized twice using a high-pressure homogenizer to reduce the average particle size of the resulting droplets to 0.4 μm or less, and the pH is adjusted to 8.5. The emulsion is then filled into 5 mL glass ampoules under nitrogen-filled conditions and sterilized in a rotary steam sterilizer at 121°C for 12 minutes to obtain an emulsion injection solution (name: CTMB emulsion injection solution). The concentration of CTMB in this solution is 10 mg / mL. A blank emulsion injection solution without CTMB is prepared using the same method.

[0080] During the trial, unless otherwise specified, CTMB will be administered in the form of an emulsion injection.

[0081] (1): Protective effect of CTMB on a pentetrazole-induced epilepsy mouse model; verification of the protective effect of CTMB on a lithium-pilocarpine-induced epilepsy rat model.

[0082] Test 1 Verification of the protective effect of CTMB on pentetrazol-induced epilepsy mouse models:

[0083] (1) Experimental animals: SPF-grade adult male KM mice, body weight 30-35g.

[0084] (2) Grouping of animals: Test mice were randomly divided into three groups: a saline group, a low-dose CTMB group of 50 mg / kg (CTMB-L), a medium-dose CTMB group of 75 mg / kg (CTMB-M), and a high-dose CTMB group of 100 mg / kg (CTMB-H). There were 10 mice in each group, and all were administered by intraperitoneal injection.

[0085] (3) Evaluation methods and results After administering pentetrazol 75 mg / kg, a drug used to create epilepsy models, to mice in each group for a set period, they were intraperitoneally injected. Clonic and tonic seizures were used as evaluation indicators, and the Racine score within 30 minutes, as well as the incidence and latency of clonic seizures (Racine score 1-3) and tonic seizures (Racine score 4-5) in each group of mice, were observed and recorded. Racine classification criteria: Grade 0: Normal non-epileptic activity; Grade 1: Wet, dog-like tremors or scratching; Grade 2: Nodding or tail wagging; Grade 3: Hemiplegia extension or clonic seizures of one limb; Grade 4: Multiple limb clonic or tonic seizures; Grade 5: Falls and generalized tonic-clonic seizures. If no epileptic seizure occurred in a mouse within 30 minutes, the latency period was recorded as 1800 s. The experimental results are shown in Table 1:

[0086] [Table 1]

[0087] Note: * P<0.05, ** P<0.01, *** P<0.001 indicates comparison with the physiological saline group.

[0088] Table 1 shows that CTMB significantly prolongs the latency period of pentetrazole-induced clonic and tonic seizures, reduces the severity and incidence of epileptic seizures, and its antiepileptic effect is dose-dependent.

[0089] Test 2 Verification of the protective effect of CTMB on lithium-pilocarpine in rat epilepsy models:

[0090] (1) Experimental animals: SPF-grade adult male SD rats, weighing 200-250g.

[0091] (2) Preparation of a lithium-pilocarpine rat epilepsy model Methylscopolamine bromide, lithium chloride, and pilocarpine were weighed and dissolved in 0.9% physiological saline at their effective concentrations (100 mg / mL lithium chloride solution, 1 mg / mL methylscopolamine bromide solution, and 20 mg / mL pilocarpine solution). Rats were intraperitoneally injected with lithium chloride (127 mg / kg), and 18-20 hours later, 1 mg / kg of methylscopolamine bromide was intraperitoneally injected. After 30 minutes, an initial dose of 30 mg / kg of pilocarpine was intraperitoneally injected, and the degree of epileptic seizures in the rats was observed. Subsequently, 10 mg / kg of pilocarpine was administered every 30 minutes until clear, uninterrupted status epilepticus (SE)-like seizures of grade IV or higher appeared, with a maximum dose of 60 mg / kg.

[0092] Model selection criteria: If the experimental animal experiences seizures of grade IV or higher for 1 hour consecutively, the model can be considered successfully created.

[0093] (3) Grouping of animals Rats successfully developed as a model were randomly divided into three groups: the model group, the CTMB group, and the sodium valproate (VPA) group. Administration was performed 1 hour after a seizure episode. The model group received a blank emulsion via intraperitoneal injection, the treatment group received 50 mg / kg of CTMB emulsion via intraperitoneal injection, and the VPA group received 300 mg / kg of sodium valproate via oral intragastric administration.

[0094] (4) Evaluation methods and results Behavioral assessment after epileptic seizures following drug intervention: Referring to the Racine evaluation criteria, epileptic seizure symptoms were observed in each group of rats after drug intervention, and the severity of acute seizures in each group of rats was recorded. Racine classification criteria: Grade 0: Normal non-epileptic activity; Grade 1: Wet, dog-like tremors or scratching behavior; Grade 2: Nodding or tail wagging; Grade 3: Hemiplegia extension or hemiplegia clonic convulsions; Grade 4: Multiple limb clonic convulsions or tonic convulsions; Grade 5: Falls and generalized tonic-clonic seizures; Grade 6: Severe seizures resulting in death. The study results are shown in Table 2.

[0095]

Table 2

[0096] Note: Comparison between the CTMB group and the model group, *** P < 0.001, t-test; Comparison between the CTMB group and the VPA group, # P < 0.05, t-test.

[0097] From Table 2, it can be seen that CTMB (50 mg / kg) significantly reduces the severity of epileptic seizures in the acute phase of the SE model, and its effect is significantly superior to that of sodium valproate (300 mg / kg), which is the positive control drug.

[0098] Observation of one-week survival rate The survival status of SE model rats after acute drug intervention was observed for 2 weeks, and the results are shown in Figure 1.

[0099] The difference in the survival curves of rats in each group was analyzed using the log-rank test (Logrank test). The results showed that CTMB (50 mg / kg) significantly improved the survival rate of SE model rats (P < 0.05), and its effect was shown to be superior to that of sodium valproate VPA, which is the positive control drug.

[0100] (II): Study on the mechanism of action of CTMB in the treatment of traumatic brain injury Experimental animals and experimental materials: SPF-grade SD rats, all male rats, weighing 240 - 260 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, certificate number: SCXK (Chuan) 2020 - 030.

[0101] Evans blue (C1e1891158, Shanghai Macklin Biochemical Technology); Formamide (20190716, Tianjin Bodi Chemical Industry); IL-1 detection kit (E-EL-H0149c, Wuhan Eiareet Biotech Co., Ltd.); IL-6 detection kit (E-EL-H2518c, Wuhan Eiareet Biotech Co., Ltd.); TNF-α detection kit (9680019151122, Aibo Taike Biotechnology); GABA detection kit (ZD0342B47674, Wuhan Yi Rui Te Biological Science and Technology Co., Ltd.); GLT-1 detection kit (Apr2023, Quanzhou Ruixin Biological Technology Co., Ltd.).

[0102] Test 3 Blood-brain barrier permeability test The experimental groupings and administration plans are as follows:

[0103] Sham surgery group (P group): Administered the same volume of saline solution as the CTMB group; Model group (T group): Administered the same volume of blank emulsion as the CTMB group; Intermediate dose group (M group): 30 mg / kg of CTMB emulsion injection was administered.

[0104] Two hours after preparing the TBI model, each group of rats was administered the solution by intraperitoneal injection. Twenty-four hours after administration, a 4% Evans blue solution (2.5 mL / kg) was injected into the right caudal vein of the rats. One hour later, the rats were deeply anesthetized, 100 mL of saline perfused through the heart, and the brains were rapidly decapitated and removed. The brains were immediately separated into the left hemisphere, right hemisphere, cerebellum, and brainstem. After measuring the wet weight of the brain tissue, it was immersed in 10 times its volume of pure formamide and incubated at 60°C for 48 hours. It was then centrifuged at 25°C and 10,000 rpm / min for 30 minutes, and the Evans blue dye was detected in the supernatant at 622 nm using ultraviolet spectrophotometry. A standard curve was created and quantified, and the final result was expressed as the Evans blue content per gram of brain tissue (μg / g). The results are shown in Figure 2 (n=6). Compared to the sham surgery group (P group), the model group (T group) showed increased blood-brain barrier permeability in the hemorrhagic cerebral hemisphere 24 hours postoperatively, while the moderate-dose CTMB group (M group) showed a significant reduction in Evans blue leakage and improved blood-brain barrier integrity.

[0105] Test 4 Detection of IL-1, IL-6, and TNF-α The experimental grouping and administration method were the same as in Experiment 3. Two hours after creating the TBI model, intraperitoneal administration was performed to rats in different groups. 24 hours after administration, the rats were deeply anesthetized, decapitated, and their brains removed. Approximately 60-120 mg of the cortex of the hemisphere on the hemorrhagic side was collected, 10 times the volume of ice-cold centrifugation buffer for biochemical detection was added, and the mixture was homogenized in an ice bath for 10 minutes. The mixture was then centrifuged at 4°C and 14000 rpm / min for 30 minutes, and the supernatant was collected. The content of inflammatory cytokines IL-1, IL-6, and TNF-α was measured according to the instructions for use with the IL-1, IL-6, and TNF-α content detection kit. The results are shown in Figure 3 (n=5-8). Compared to the P group, the T group showed a significant increase in the content of IL-1, IL-6, and TNF-α in the hemorrhagic cerebral hemisphere of rats 24 hours postoperatively. However, the M group significantly reduced the content of the pro-inflammatory cytokine TNF-α and the inflammatory cytokines IL-1 and IL-6 in the cerebral cortical tissue of TBI rats, which is beneficial for reducing neuroinflammation and improving motor function in rats.

[0106] Test 5 Measurement of GLT-1 and GABA content The grouping of the experiments and the dosage forms are the same as in Test 3. After continuous administration to each group for 7 days, the rats were deeply anesthetized, decapitated, and the brains were removed. Approximately 60 - 120 mg of the cortex of the hemorrhagic cerebral hemisphere was collected, and 10 volumes of ice-cold centrifugation buffer for biochemical detection were added. It was homogenized in an ice bath for 10 min, centrifuged at 4°C and 14,000 rpm / min for 30 min, and the supernatant was collected. The GLT-1 and GABA contents were measured according to the instructions of the detection kits for GLT-1 and GABA contents respectively. Glutamate transporter 1 (GLT-1) is an important glutamate transporter in the brain and can transport extracellular glutamate into cells. Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter. Under pathological conditions, the extracellular glutamate concentration rises rapidly, causing excessive excitotoxicity of neurons. Figure 4 shows the contents and their ratios of GLT-1 and GABA measured in different administration groups, which can be used to evaluate the excitatory neurotoxic effect caused by TBI injury. The results are shown in Figure 4 (n = 6). Compared with the P group, in the hemorrhagic cerebral hemisphere of the rats in the T group, the GLT-1 content and the GLT-1 / GABA ratio were significantly increased on the 7th day after surgery. However, the administration of the M group decreased the GLT-1 content in the brain of TBI rats, increased the GABA content, and significantly decreased the GLT-1 / GABA ratio, restored the balance between excitatory amino acids (EAA) and inhibitory amino acids (IAA) in the brain, reduced the excitatory neurotoxicity caused by excessive glutamate, and thereby improved the motor function of the rats.

[0107] (III): Verification of the acute treatment effect of rat ischemic stroke by the filament embolization method of CTMB.

[0108] 3.1 Experimental materials: SPF-grade SD rats (male, body weight 200 - 230 g, purchased from Sichuan Dashuo Experimental Animal Co., Ltd., certificate number: SCXK (Sichuan) 2020 - 0030) MCAO embolization thread (purchased from Beijing Xinuo Technology Co., Ltd., model number: 2432 - A5) Edaravone and dexborneol injection concentrated solution (purchased from Simcere Pharmaceutical Co., Ltd. (specification: edaravone 10 mg / 5 mL, dexborneol 2.5 mg / mL; lot number: 180 - 220522)).

[0109] 3.2 Experimental grouping: Two hours after MCAO surgery, the success of model creation was determined by the Zea Longa score. Rats that successfully created the model were randomly divided into groups and administered the treatment.

[0110] Rats were randomly divided into four groups: a sham group (administered with the same volume of blank emulsion as the high-dose CTMB group), a model group (Vehicle group, administered with the same volume of blank emulsion as the high-dose CTMB group), a low-dose CTMB group (CTMB-L group, 10 mg / kg), a high-dose CTMB group (CTMB-H group, 20 mg / kg), and an edaravone / dexborneol injection concentrate group (EDB group, commercially available product, 3 mg / kg). Ten rats were assigned to each group, and all were administered by intraperitoneal injection. (Note that in the experiment, the rats were administered by intraperitoneal injection, and the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the human dose per unit mass was 0.2 mg to 4.0 mg / kg. This refers to the ratio of the effective dose of the active ingredient CTMB in the emulsion injection during intravenous infusion to the human dose per unit mass, and was calculated from the effective dose in rats. Considering the differences in drug bioavailability due to differences in species and administration methods, as well as differences in the peak concentration and peak time of the drug, the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the human dose per unit mass was ultimately converted to 0.2 mg to 4.0 mg / kg.)

[0111] 3.3 Creation of an ischemia-reperfusion model using filament embolization The rats were fasted for 12 hours prior to the procedure and anesthetized with 10% chloral hydrate (350 mg / kg, ip). They were fixed in a supine position, and their body temperature was maintained at approximately 37°C. The skin of the neck was prepared, a midline cervical incision was made, and the muscle and fascia were separated along the medial edge of the sternocleidomastoid muscle to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Sutures were prepared at the mesial end of the CCA, the ICA, and the ECA. The mesial end of the CCA and the ECA were ligated, the ICA was temporarily clamped with an arterial clip, a small hole was made with a needle approximately 4 mm from the bifurcation of the CCA, and the embolic suture was inserted from the CCA into the ICA. The arterial clip on the ICA was loosened, and the embolic suture was slowly advanced. When the mark on the embolic suture reached the bifurcation and slight resistance was felt, the thin suture at the ICA was tightly tied, the wound surface was washed with saline, and then sutured. The sham surgery was the same as the experimental group except that the embolization sutures were not inserted. After waking from anesthesia, the animals were kept in normal conditions.

[0112] 3.4 Selection Criteria for Cerebral Ischemia Models Refer to the Zea-Longa neurological function score (Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan;20(1):84-91. doi: 10.1161 / 01.str.20.1.84.) and score the rats two hours postoperatively after they have recovered from anesthesia. Group rats with a score of 1-2 points: 0 points: No symptoms of neurological dysfunction, normal activity; 1 point: Unable to fully extend the contralateral forelimb; 2 points: The animal turns when walking; 3 points: The body leans to the paralyzed side; 4 points: Unable to walk spontaneously, loss of consciousness.

[0113] 3.5 Short-term neurological function deficit score Rats' neurological function was comprehensively evaluated 24 hours after model creation using the improved mNSS score and Garcia score. The mNSS score criteria are shown in Table 3, and the Garcia score criteria are shown in Table 4. The rats' motor, sensory, climbing, and limb symmetry were evaluated. The mNSS score ranged from 0 to 18 points, with higher scores indicating more severe neurological impairment, and the Garcia score ranged from 3 to 18 points, with lower scores indicating more severe neurological impairment. The scores were independently administered by blind observers who were not involved in model creation or administration.

[0114] [Table 3]

[0115] [Table 4]

[0116] Table 5 shows that, compared to the sham group, the model group (Vehicle group) showed a significant increase in mNSS score at 24 hours postoperatively (P<0.01) and a significant decrease in Garcia score (P<0.01), indicating that the rats in the model group developed clear neurological dysfunction 24 hours after MCAO surgery. Administration of different doses of CTMB (CTMB-L, CTMB-H groups) and edaravone / dexborneol concentrated solution for injection (EDB group) all reduced mNSS scores and increased Garcia scores to varying degrees, improving neurological dysfunction caused by MCAO, with the effect of the CTMB-H group being the most pronounced (P<0.01). Furthermore, its efficacy was superior to that of edaravone / dexborneol, a drug used clinically to improve ischemic stroke. No toxic side effects associated with CTMB administration were observed during the experiment.

[0117] [Table 5]

[0118] Note: Comparison with the sham surgery group. ##P < 0.01; comparison with the model group (Vehicle group), ** P < 0.01, * P < 0.05.

[0119] (IV): Long-term therapeutic effect of CTMB on rat ischemic stroke by the filament embolization method in rats.

[0120] 4.1 Neurological deficit score The experimental materials, grouping (n = 10 - 15), model preparation method, and scoring criteria were the same as those in the acute-phase treatment. Two hours after the model was prepared by the filament embolization method, the rats were immediately grouped and administered according to the administration form. Then, the administration was continued for 14 days, once a day. The modified mNSS score was evaluated on the 1st, 4th, 7th, and 14th days.

[0121] From Table 6, compared with the sham operation group (Sham group), the mNSS score of the model group (Vehicle group) was significantly increased on the 14th day after surgery (P < 0.01), indicating that obvious neurological deficits occurred in the rats of the model group on the 14th day after MCAO. From the 1st to the 7th day, both the CTMB administration group (CTMB-H group) and the concentrated solution group of edaravone dexborneol for injection (EDB group) decreased the mNSS score to varying degrees and improved the neurological deficits caused by MCAO. Among them, the administration effect of the CTMB-H group was the most significant, and its therapeutic effect was slightly better than that of the concentrated solution of edaravone dexborneol for injection, which is a drug used clinically to improve ischemic stroke. On the 14th day, the CTMB-H group could still significantly decrease the mNSS score. During the experimental process, no toxic side effects related to CTMB administration were observed.

[0122]

Table 6

[0123] Note: Comparison with the sham operation (Sham group), ## P < 0.01; comparison with the model group (Vehicle group), ** P < 0.01, *P<0.05.

[0124] 4.2 Adhesive Tape Removal Test Before creating the models, rats were trained for one week in an adhesive tape removal test, and rats that could remove the tape within 10 seconds were selected to create the models. Two hours after creating the filament embolization models, the rats were immediately administered according to the group administration method, and administration continued for 14 days, once daily. An adhesive tape removal test was performed on the 1st, 4th, 7th, 10th, and 14th days, and the sensory and motor nerve functions of the rats were evaluated. The specific procedure was as follows: A circular tape with a diameter of approximately 12 mm was attached to the sole of the left forelimb of the rat, and the time it took for the rat to sense and remove the tape was recorded. If a mouse could not sense or remove the tape within 60 seconds, it was recorded as 60s.

[0125] Table 7 shows that, compared to the sham group, the model group (Vehicle group) showed a significantly longer tape detection time at 14 days post-surgery (P<0.05), and the left forelimb detection ability of rats in the model group decreased at 14 days post-MCAO. Different doses of CTMB and EDB can promote the recovery of detection ability in rats.

[0126] [Table 7]

[0127] Note: Compared to sham surgery (Sham group). ## P<0.01, # P<0.05; comparison with the model group (Vehicle) group. ** P<0.01, * P<0.05.

[0128] From Table 8, compared with the sham operation group (Sham group), the model group (Vehicle group) had a significantly increased tape removal time on the 14th day after surgery (P<0.01), indicating that the motor ability of the left forelimb of the rats in the model group decreased on the 14th day after MCAO surgery. The CTMB-L group could significantly shorten the tape removal time of rats from the 4th day (P<0.05), and the CTMB-H group could significantly shorten the tape removal time of rats from the 10th day (P<0.05). Its drug effect was superior to that of edaravone dexborneol, a drug used clinically to improve ischemic stroke. During the experiment, no toxic side effects related to CTMB administration were observed.

[0129]

Table 8

[0130] Note: Comparison with sham operation (Sham group), ## P<0.01, # P<0.05; comparison with the model group (Vehicle group), ** P<0.01, * P<0.05.

[0131] From the above, long-term administration of CTMB can significantly promote the recovery of the motor function of rats and significantly improve the limb sensory ability of rats.

[0132] (V): Verification of the acute treatment effect of CTMB on rat hemorrhagic stroke by caudate nucleus collagenase VII injection method.

[0133] 5.1 Experimental materials: SPF-grade SD rats (male, body weight 220 - 250 g, purchased from Sichuan Dashuo Experimental Animal Co., Ltd., certificate number: SCXK(Sichuan)2020 - 0030) Type VII collagenase (purchased from Sigma-Aldrich, USA (specification: 1.5 KU; lot number: 0000111586)) Concentrated solution for injection of edaravone dexborneol (purchased from Simcere Pharmaceutical Co., Ltd. (specification: edaravone 10 mg / 5 mL, dexborneol 2.5 mg / mL; lot number: 181 - 230207)).

[0134] 5.2 Experimental grouping: Two hours after hemorrhagic stroke, the success of model creation was determined by the Zea Longa score. Rats that successfully created the model were randomly divided into groups and administered the treatment.

[0135] Rats were randomly divided into four groups: a sham surgery group (administered the same volume of blank emulsion as the high-dose CTMB group), a model group (vehicle group, administered the same volume of blank emulsion as the high-dose CTMB group), a low-dose CTMB group (CTMB-L group, 10 mg / kg), a high-dose CTMB group (CTMB-H group, 20 mg / kg), and an edaravone / dexborneol injection concentrate group (EDB group, commercially available product, 3 mg / kg). 10-13 rats were assigned to each group, and all were administered by intraperitoneal injection. (Note that in the experiment, the rats were administered by intraperitoneal injection, and the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the human dose per unit mass was 0.2 mg to 4.0 mg / kg. This refers to the ratio of the effective dose of the active ingredient CTMB in the emulsion injection during intravenous infusion to the human dose per unit mass, and was calculated from the effective dose in rats. Considering the differences in drug bioavailability due to differences in species and administration methods, as well as differences in the peak concentration and peak time of the drug, the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the human dose per unit mass was ultimately converted to 0.2 mg to 4.0 mg / kg.)

[0136] 5.3 Creation of a hemorrhagic stroke model using the caudate nucleus collagenase VII injection method Prior to the procedure, rats were fasted and dehydrated for 8 hours. Anesthesia was induced with 4% isoflurane, maintained with 2% isoflurane, and the rats' body temperature was maintained at approximately 37°C. The rats were fixed in a prone position in a stereotactic device, the skin of the head was prepared, a longitudinal sagittal incision was made along the midline of the skull, the skin and subcutaneous tissue were dissected, and the anterior fontanelle and coronal suture were exposed. Using the stereotactic device, the right caudate nucleus was positioned with the rat's anterior fontanelle as the origin, shifted 3.0 mm to the right of the midline, marked, and then a hole was drilled with a skull drill to expose the dura mater, and hemostasis was achieved. A micro-syringe was attached to the stereotactic device beforehand, and the needle was advanced vertically along the hole in the skull to 5.5 mm (i.e., the caudate nucleus site). Collagenase VII (1 μL) at a concentration of 0.5 U / μL was slowly and uniformly injected into the brain, injected for 5 minutes, left in place for 8 minutes, and then slowly withdrawn. The skull defect was closed with sterile bone wax, and the scalp incision was sutured. After iodine disinfection, the rats were returned to their cages and reared. The sham group underwent the same procedure except for the injection of saline solution. The rats were allowed to recover from anesthesia and reared normally.

[0137] 5.4 Selection Criteria for Intracerebral Hemorrhage Models Refer to the Zea-Longa neurological function score (Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan;20(1):84-91. doi: 10.1161 / 01.str.20.1.84.) and assign a score of 1-2 points to each group: 0 points: No symptoms of neurological dysfunction, normal activity; 1 point: Unable to fully extend the contralateral forelimb; 2 points: The animal turns when walking; 3 points: The body leans to the paralyzed side; 4 points: Unable to walk spontaneously, loss of consciousness.

[0138] 5.5 Short-term neurological function deficit score Rats' neurological function was comprehensively evaluated 24 hours after model creation using the improved mNSS score and Garcia score. The mNSS score criteria are shown in Table 3, and the Garcia score criteria are shown in Table 4. The rats' motor, sensory, climbing, and limb symmetry were evaluated. The mNSS score ranged from 0 to 18 points, with higher scores indicating more severe neurological impairment, and the Garcia score ranged from 3 to 18 points, with lower scores indicating more severe neurological impairment. The scores were independently administered by blind observers who were not involved in model creation or administration.

[0139] [Table 9]

[0140] Note: Compared to sham surgery (Sham group). #### P<0.0001, ### P<0.001; comparison with the model group (Vehicle) group. **** P<0.0001, * P<0.05.

[0141] Table 9 shows that, compared to the sham group, the model group (Vehicle group) showed a significant increase in mNSS score at 24 hours postoperatively (P<0.0001) and a significant decrease in Garcia score (P<0.0001), indicating that the rats in the model group developed clear neurological dysfunction 24 hours after cerebral hemorrhage surgery. Different doses of CTMB (CTMB-L and CTMB-H groups) each reduced mNSS scores and increased Garcia scores to varying degrees, but administration of edaravone / dexborneol concentrated solution for injection (EDB group) only increased Garcia scores. CTMB administration was able to improve neurological dysfunction caused by hemorrhagic stroke, with the CTMB-L group showing the most significant effect (P<0.0001). Furthermore, its efficacy was superior to that of edaravone / dexborneol, a drug clinically used to improve ischemic stroke. No toxic side effects associated with CTMB administration were observed during the experimental process.

[0142] (6) Therapeutic effects of CTMB on Parkinson's disease (PD) mice 6.1 Experimental materials: SPF-grade C57 mice (male, body weight 18 - 22 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, certificate number: SCXK(Sichuan)2020 - 030), levodopa raw drug (Shanghai Macklin Biochemical Technology Co., Ltd. (specification: 5 g; lot number: C14100283)), benserazide hydrochloride raw drug (Shanghai Macklin Biochemical Technology Co., Ltd. (specification: 1 g; lot number: C15659767)).

[0143] 6.2 Experimental grouping: Two weeks after 6-OHDA injection, scores were given in pole climbing test, hanging test, rotarod test and open field test to judge the success of model establishment. Mice that successfully established the model were randomly grouped and administered.

[0144] Mice were randomly divided into sham operation group (S group, administered the same volume of blank emulsion as the administration group), model group (M group, administered the same volume of blank emulsion as the administration group), CTMB group (20 mg / kg, D group), levodopa group (levodopa: benserazide hydrochloride = 4:1, 12 mg / kg, L group), with 10 - 12 mice in each group, and all were administered by intraperitoneal injection.

[0145] 6.3 Establishment of PD model by 6-OHDA method Before surgery, mice were fasted for 12 hours, anesthetized with 4% isoflurane, and maintained with 1.5% isoflurane. Then, they were fixed in a stereotaxic apparatus. After incising the scalp, the surface of the skull was gently wiped with a cotton swab to expose the bregma and lambda points. The stereotaxic apparatus was adjusted to keep the skull horizontal with the same height of the bregma and lambda points. Taking the bregma point as the origin, the coordinates of the injection site were found, and a small hole was drilled in the skull above the injection site with a drill.

[0146] 6-OHDA (12.5 μg / μL) was injected into the striatum of mice (AP +0.5 mm; ML +2.00 mm; DV -3.00 mm and -2.00 mm), with 1 μL injected at a rate of 0.5 μL / min into two points. After injection, the needle was left in place for 3 minutes, then withdrawn, and the animal's scalp was sutured and disinfected. The sham surgery group was injected with the same volume of 0.02% ascorbic acid-containing saline, and the other procedures were the same. After the surgery, the mice were placed in cages at an appropriate temperature. After awakening from anesthesia, they were kept in normal care.

[0147] 6.4 Selection Criteria for Parkinson's Disease Models Two weeks post-surgery, mice were subjected to pole climbing and suspension tests to evaluate their motor function, and those showing a significant difference compared to the sham surgery group were added to the group.

[0148] Pole Climbing Test: The pole climbing test primarily measures the motor skills of mice and assesses bradykinesia (slowness of movement). This test evaluates the mouse's ability to grasp a pole, descend, and return to its home cage. A cork ball is attached to an iron pole 1 cm in diameter and 65 cm long. The mouse is placed at the top of the pole with its head facing downwards, and it will normally descend the pole and return to its cage naturally. The time it takes for the mouse to reach the bottom is recorded. The result is the average of three consecutive measurements.

[0149] Suspension Test: Normal mice have climbing ability, which requires adequate grip strength and motor coordination. Therefore, the suspension test can be used to examine motor dysfunction in mice. A 1.5 mm diameter metal rod is placed horizontally in a box 30 cm above the bottom of the box. During the experiment, the mouse's forelegs are suspended from the metal rod, and the time until they land is recorded. If the mouse does not fall for 60 seconds or more, the time is recorded as 60 seconds. Two measurements are taken, with an interval of at least 1 minute between measurements, and the result is the average of the two measurements.

[0150] 6.5 Assessment of motor function The motor function of mice was comprehensively evaluated using pole climbing and suspension tests at weeks 1, 2, 3, and 4 after administration. Scores were independently performed by blind observers who were not involved in model creation or administration.

[0151] [Table 10]

[0152] Note: Comparison with the sham surgery group (Group S). # P<0.05, ## P<0.01, ### P<0.001; Comparison with model group (M group), * P<0.05, ** P<0.01. Data are displayed as x±SD, and multi-group comparative analysis was performed using ANOVA.

[0153] Table 10 shows that, compared to the sham surgery group (group S), the model group (group M) showed a significant increase in pole climbing time before administration (2 weeks post-surgery) (P<0.001), and clear bradykinesia occurred in the model group mice 2 weeks after model creation. After intraperitoneal injection of the CTMB group (group D) and the levodopa group (group L), the pole climbing time of the model mice was significantly reduced, and the bradykinesia caused by 6-OHDA was improved. For the PD model, the efficacy of group D was not significantly different from that of the levodopa group (group L), which is a drug used clinically to improve Parkinson's disease. Furthermore, there was no significant difference in the initial pole climbing time of the mice in each group, and differences in behavioral function due to differences in the severity of model creation can be ruled out.

[0154] [Table 11]

[0155] Note: Comparison with the sham surgery group (Group S). # P<0.05, ## P<0.01; Comparison with model group (M group), * P<0.05, ** P<0.01. Data are presented as x±SD, and multigroup comparative analysis was performed using the Kruskal-Wallis test.

[0156] From Table 11, compared with the sham operation group (S group), the hanging time before administration (2 weeks after surgery) in the model group (M group) was significantly shortened (P<0.01), indicating that obvious motor dysfunction occurred in the mice of the model group 2 weeks after model establishment. After intraperitoneal injection in the CTMB group (D group), the hanging time of the model mice was prolonged, and the motor dysfunction caused by 6-OHDA was improved. For the PD model, the drug effect of group D was superior to that of the levodopa group (L group), which is a drug used clinically to improve Parkinson's disease, but there was no significant difference. In addition, there was no significant difference in the initial hanging time of the mice in each group, excluding the difference in behavioral function due to the severity of model establishment.

[0157] (VII): Study on the mechanism of action of CTMB against Parkinson's disease 7.1 Experimental materials: SPF-grade C57 mice (male, body weight 18 - 22 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, certificate number: SCXK(Sichuan)2020 - 030), Hematoxylin and eosin staining solution (BP0211, Hubei Bioscience Co., Ltd.), Toluidine blue staining solution (BP0360, Hubei Bioscience Co., Ltd.), Recombinant Anti-Tyrosine Hydroxylase antibody (ab137869, Abcam (Shanghai) Trading Co., Ltd.), Malondialdehyde (MDA) colorimetric assay kit (TBA method) (E-BC-K025-M, Wuhan Elabscience Biotechnology Co., Ltd.), Total superoxide dismutase (T-SOD) colorimetric assay kit (WST-1 method) (E-BC-K020-M, Wuhan Elabscience Biotechnology Co., Ltd.), Reduced glutathione (GSH) colorimetric assay kit (E-BC-K030-M, Wuhan Elabscience Biotechnology Co., Ltd.).

[0158] 7.2 Experimental procedures and results: 7.2.1 Sample preparation Mice were randomly divided into a sham surgery group (Group S, administered the same volume of blank emulsion as the treatment group), a model group (Group M, administered the same volume of blank emulsion as the treatment group), a CTMB group (20 mg / kg, Group D), and a levodopa group (levodopa:benserazide hydrochloride = 4:1, 12 mg / kg, Group L). 10-12 mice were assigned to each group, and all were administered by intraperitoneal injection.

[0159] After completing the motor function evaluation for 4 weeks following administration, the mice were deeply anesthetized, perfused with 20 mL of physiological saline and 20 mL of 4% paraformaldehyde through the heart, rapidly decapitated, and the brain was removed. The brain was then fixed in 4% paraformaldehyde for 72 hours, then embedded in paraffin, and 4 μm thick sections were prepared for subsequent histopathological studies.

[0160] 7.2.2 Reduction of neuronal damage in the substantia nigra and striatum of the midbrain in PD mice HE staining allows for the observation of pathological changes in brain tissue. The specific procedure for HE staining is as follows:

[0161] a) From deparaffinization of paraffin sections to water: Deparaffinize sequentially in environmentally friendly deparaffinizer (1), environmentally friendly deparaffinizer (2), and environmentally friendly deparaffinizer (3) for 10 minutes each, then treat with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and wash with tap water for 1 minute.

[0162] b) Stain with hematoxylin (Harris) for 4 minutes, then rinse with tap water for 2 minutes, and rinse until all excess staining solution is removed from the section.

[0163] c) Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, wash with tap water, return to blue with lithium carbonate aqueous solution, and then wash with tap water for 2 minutes.

[0164] d) Immerse in eosin staining solution (alcohol-soluble) for 20 seconds, adjust the color directly with 95% ethanol for 5 seconds without washing, and dehydrate with anhydrous ethanol (1) and anhydrous ethanol (2) for 2 minutes.

[0165] e) Clear the contents using an environmentally friendly clearing agent, then encapsulate and examine them under a microscope.

[0166] Nissl staining is used to identify Nissl bodies within nerve cells. The specific procedure for Nissl staining is as follows:

[0167] a) From deparaffinization of paraffin sections to water: Deparaffinize sequentially in environmentally friendly deparaffinizer (1), environmentally friendly deparaffinizer (2), and environmentally friendly deparaffinizer (3) for 10 minutes each, then treat with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and wash three times with distilled water.

[0168] b) Place the sections in a preheated 1% toluidine blue solution at 50°C, stain them in a 56°C bath for 20 minutes, and wash with distilled water.

[0169] c) Differentiation with 95% alcohol or 0.1% glacial acetic acid, controlled under a microscope, with the clear display of Nissl bodies as the criterion.

[0170] d) Quickly dehydrate with anhydrous ethanol.

[0171] e) Clear the contents using an environmentally friendly clearing agent, then encapsulate and examine them under a microscope.

[0172] [Table 12]

[0173] Note: Comparison with the model group (M group), *** P<0.001. Data are displayed as x±SD, and multi-group comparative analysis was performed using ANOVA.

[0174] The results are shown in Figures 5 and 6. Six weeks after 6-OHDA injection, neurons in the right midbrain substantia nigra and striatum of mice underwent coagulation necrosis, with indistinct nucleoli and widespread diffuse or localized proliferation of glial cells. These glial cells entered the cell bodies or processes of necrotic neurons, forming neurophagous phenomena. Figure 7 and Table 12 show that a large number of neurons in the right midbrain substantia nigra were contracted and deeply stained, and the number of Nissl bodies was reduced. Compared to the model group (M group), the degree of pathological damage to the substantia nigra and striatum was reduced in the CTMB group (D group) and the levodopa group (L group). This indicates that CTMB can reduce 6-OHDA-induced substantia nigra neuronal damage in mice and has a neuroprotective effect.

[0175] 7.2.3 Reduction of dopaminergic neuronal damage in the substantia nigra area of ​​the midbrain in PD mice The expression status of tyrosine hydroxylase (TH) protein is measured using immunohistochemistry. The specific procedure is as follows:

[0176] a) Deparaffinization of paraffin sections to water: Deparaffinize sequentially in environmentally friendly deparaffinizer (1), environmentally friendly deparaffinizer (2), and environmentally friendly deparaffinizer (3) for 10 minutes each, then treat with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 minutes each. Wash three times with distilled water for 3 minutes each, then immerse.

[0177] b) Perform antigen retrieval.

[0178] c) Blocking endogenous peroxidase: After washing, the repaired sections are immersed in 3% H2O2, blocked at room temperature in the dark for 30 minutes, and then immersed in distilled water.

[0179] d) Enclosing: After drawing an outline with a histochemistry pen, place the section in TBST.

[0180] e) Incubation: Add 10% serum matching the source of the secondary antibody and incubate at room temperature for 30 minutes.

[0181] f) Primary antibody incubation: Discard the serum, dilute the primary antibody solution with 10% serum to prepare the primary antibody working solution, add 50-100 μL (depending on the size of the tissue) of the primary antibody working solution to each section, and incubate overnight at 4°C.

[0182] g) Secondary antibody incubation: The next day, remove the sections from the refrigerator and leave them at room temperature for 15 minutes (rewarming). Wash them three times with TBST, then immerse them three more times for 3 minutes each time. Dilute the secondary antibody solution with TBST to prepare the secondary antibody working solution. Add 50-100 μL (depending on the size of the tissue) of the secondary antibody working solution to each section and incubate at 37°C for 45 minutes. Wash them three times with TBST, then immerse them three more times for 3 minutes each time.

[0183] h) DAB color development: Discard the TBST, drop 50-100 μL (depending on the size of the tissue) of freshly prepared DAB color development solution onto each section, observe the color development under a microscope while timing with a timer, wash with tap water to stop the color reaction, and record the color development time.

[0184] [Table 13]

[0185] Note: Comparison with the sham surgery group (Group S). ### P<0.001; Comparison with model group (M group), *** P<0.001; comparison with the levodopa group (L group), && P<0.01. Data are displayed as x±SD, and multi-group comparative analysis was performed using ANOVA.

[0186] The results are shown in Figure 8 and Table 13. After 6-OHDA injection, TH protein expression in the substantia nigra on the injured side (right side) decreased significantly, meaning the number of dopaminergic-positive neurons decreased significantly (P<0.001). The CTMB group (Group D) significantly suppressed 6-OHDA-induced apoptosis of dopaminergic neurons and was clearly superior to the levodopa group (Group L), indicating that CTMB can slow the progression of Parkinson's disease in an animal model. The levodopa group (Group L) also suppressed 6-OHDA-induced apoptosis of dopaminergic neurons, but the difference was not statistically significant.

[0187] 7.2.4 Measurement of MDA, GSH content and SOD activity in the striatum Malondialdehyde (MDA) is a common indicator of membrane lipid peroxidation, reduced glutathione (GSH) is one of the most common non-enzymatic antioxidants, and superoxide dismutase (SOD) is a common indicator of antioxidant enzymes. After completing motor function assessments for 4 weeks after administration, mice were deeply anesthetized, perfused with 20 mL of saline through the heart, rapidly decapitated and the brain removed, the striatal region on the injured side (right side) was isolated, weighed, placed in a 1.5 mL EP tube, and PBS was added in a ratio of animal tissue weight (g):homogenate medium volume (mL) = 1:9, and homogenized at low temperature. The mixture was centrifuged at 4°C and 10000 × g for 10 minutes, and the supernatant was collected and the MDA, GSH content and SOD activity were measured according to the instructions for the MDA, GSH, and SOD colorimetric test kits.

[0188] [Table 14]

[0189] Note: Comparison with the model group (M group), * P<0.05, ** P<0.01. Data are displayed as x±SD, and multi-group comparative analysis was performed using ANOVA.

[0190] As shown in Table 14, the MDA levels in the mouse striatum of the model group (group M) were significantly higher than in the sham surgery group (group S), and GSH and SOD levels were significantly lower. The CTMB group (group D) significantly reduced the MDA concentration in the model mouse striatum and significantly increased the GSH concentration and SOD activity in the model mouse striatum. These results indicate that CTMB can exert its anti-PD effect by improving oxidative stress.

[0191] The above are merely preferred embodiments of the present invention, and it should be noted that those skilled in the art can make some improvements and embellishments without departing from the principles of the present invention, and these improvements and embellishments are also considered to be within the scope of protection of the present invention.

Claims

1. The following equation (I): 【Chemistry 1】 The structure is shown by A 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound characterized by the above.

2. Step (1) involves mixing compound 1a with dichloromethane, then adding compound 1b and reacting to obtain compound 1c, Step (2) involves sequentially mixing compound 1c, compound 1d, and aluminum trichloride with dichloromethane and then reacting them to obtain compound 1e. Step (3) involves sequentially mixing compound 1e, sodium borohydride solution, and sodium hydroxide solution with tetrahydrofuran and then reacting them to obtain compound 1f. The process includes step (4), in which compound 1f is reacted with sodium acetate and acetic anhydride to obtain a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound. The aforementioned compound 1a is 【Chemistry 2】 And, The aforementioned compound 1b is 【Transformation 3】 And, The compound 1d is 【Chemistry 4】 That is A method for producing the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound according to claim 1.

3. In step (1) above, the mass-volume ratio of compound 1a, dichloromethane, and compound 1b is 23-27 g:130-170 mL:93-97 g. The manufacturing method according to claim 2.

4. In step (1) above, the reaction temperature is 20 to 30°C, the reaction is carried out under stirring conditions, and the reaction is completed when no more bubbles are generated under stirring conditions. The manufacturing method according to claim 2 or 3.

5. In step (2) above, the mass-volume ratio of compound 1c, compound 1d, aluminum trichloride, and dichloromethane is 22-27 g:30-33 g:26-30 g:80-120 mL. The compound 1c is, 【Transformation 5】 That is The manufacturing method according to claim 2 or 3.

6. In step (2) above, the reaction temperature is 20 to 30°C and the reaction time is 2 to 3 hours. The manufacturing method according to claim 2.

7. In step (3) above, the mass-volume ratio of compound 1e, sodium borohydride solution, and tetrahydrofuran is 40-45 g:18-22 mL:140-160 mL, and the concentration of sodium borohydride solution is 0.8-1.0 g / mL. The aforementioned compound 1e is 【Transformation 6】 That is The manufacturing method according to claim 2.

8. In step (3) above, the reaction temperature is 50 to 65°C and the reaction time is 2 to 4 hours. The manufacturing method according to claim 2.

9. In step (4) above, the mass-volume ratio of compound 1f, sodium acetate, and acetic anhydride is 38-45 g:7-10 g:200-230 mL. The aforementioned compound 1f is 【Transformation 7】 And, The reaction temperature is 120-140°C, and the reaction time is 2-4 hours. The manufacturing method according to claim 2.

10. Use of the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound according to claim 1 in the manufacture of an antiepileptic drug.

11. Use of the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound according to claim 1 in the manufacture of a drug for treating and / or preventing traumatic brain injury.

12. Use of the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound according to claim 1 in the manufacture of a drug for the treatment and / or prevention of ischemic stroke.

13. Use of the 1-(cyclobutylmethyl)-2,4,5-trimethoxybenzene compound according to claim 1 in the manufacture of a drug for the treatment and / or prevention of hemorrhagic stroke.

14. Use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene according to claim 1 in the manufacture of a drug for the prevention and / or treatment of Parkinson's disease.

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