1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, its manufacturing method and use

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 a patent application filed on April 20, 2023, bearing application number CN 202310430869.9 and titled "1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, and its preparation method and use."

[0002] The present invention relates to the technical field of drug research and development, and in particular to 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds and their preparation and use. [Background technology]

[0003] Spreading depolarization (SD) is a wave in the gray matter of the central nervous system (CNS). Its characteristics include neuronal swelling, dendritic spine deformation, large changes in slow potentials, and suppression of cerebral electrical activity (spreading cerebral electrical activity). There is now clinically convincing electrophysiological evidence that spreading depolarization is ubiquitous in individuals with diseases such as migraine aura, ischemic stroke, traumatic brain injury, aneurysmal subarachnoid hemorrhage (aSAH) and delayed cerebral ischemia (DCI) after subarachnoid hemorrhage, spontaneous cerebral hemorrhage, subdural hematoma, spontaneous intracranial hemorrhage or traumatic brain injury, Alzheimer's disease, Parkinson's disease, and epilepsy. Therefore, spreading depolarization is one of the major and most overlooked pathophysiological processes in acute neurology. Spreading depolarization can be experimentally induced by many injurious conditions, including potassium, glutamate, and sodium pump inhibitors, status epilepticus, hypoxia, hypoglycemia, and ischemia, but it can also penetrate healthy, unaffected tissue. The physiological hemodynamic response resulting from resistance vessel effects can cause transient hyperperfusion in healthy tissue or, in progressively damaged tissue, a retrograde hemodynamic response or spreading ischemia, resulting in profound hypoperfusion and accelerating lesion progression. Therefore, therapies targeting spreading depolarization or retrograde hemodynamic responses may be potentially useful for treating these neurological disorders.

[0004] Therefore, it is of great significance to provide 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds and their preparation methods and uses to effectively inhibit spreading depolarization in the above-mentioned multiple nervous system diseases. Summary of the Invention

[0005] The objective of the present invention is to provide 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compounds and their preparation methods and uses, thereby solving the problem that commercially available drugs in existing technology cannot effectively inhibit spreading depolarization in nervous system diseases.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

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

[0008] [ka]

[0009] The present invention provides a method for preparing 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, which comprises the following steps:

[0010] Step (1) Compound 1a is mixed with dichloromethane, and then compound 1b is added thereto to react with the compound 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 mixed with sodium acetate and acetic anhydride, and then reacted to obtain 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound; The compound 1a

[0011] [ka]

[0012] and the compound 1b is

[0013] [ka]

[0014] and the compound 1d is

[0015] [ka]

[0016] is.

[0017] The present invention provides a use of a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of an antiepileptic drug.

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

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

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

[0021] The present invention provides a use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene in the manufacture of a medicament for preventing and / or treating Parkinson's disease.

[0022] The 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound (hereinafter referred to as CTMB) provided by the present invention can be formulated into various dosage forms by mixing with pharmaceutically acceptable excipients, and can be formulated into liquid dosage forms such as emulsion injections and oral emulsions. Those skilled in the art can also use conventional technical means to formulate it into solid dosage forms such as tablets and capsules. The dosage forms can be administered by routes such as intravenous injection, intramuscular injection, and oral administration when used, as long 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) CTMB of the present invention can reduce the severity and incidence of epileptic seizures induced by pentylenetetrazole, and significantly prolong the latency period of epileptic clonic and tonic seizures, with dose-dependent effects. It also significantly reduces the severity of acute epileptic seizures induced by lithium-pilocarpine in a rat epilepsy model and improves the survival rate of rats, with effects superior to those of sodium valproate, a broad-spectrum antiepileptic drug. (2) The drug produced by CTMB can significantly improve the neurobehavioral function of rats with ischemic stroke and hemorrhagic stroke, and restore the sensory and motor functions of rats. (3) CTMB improves the integrity of the blood-brain barrier and promotes the recovery of motor function in rat models; reduces neuroinflammatory responses; counters glutamate excitotoxicity; reduces neuronal apoptosis; improves neurological dysfunction and emotional restlessness in rat models; and has significant neuroprotective effects against traumatic brain injury. (4) CTMB improves the motor function of Parkinson's disease model mice, improving their coordination and balance ability, increasing the number of Nissl bodies in the substantia nigra of the model mice, alleviating neuronal damage in the striatum and substantia nigra of the model mice, reducing MDA concentration in the striatum of the model mice, and improving GSH concentration and SOD activity in the striatum of the model mice, thereby improving oxidative stress in the striatum. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the survival status of SE model rats after acute drug intervention. [Figure 2] 1 shows the blood-brain barrier permeability test results of Test 3 (content of Evans blue dye per gram of brain tissue of rats in different groups). [Figure 3] The following are the results of measuring TNF-α, IL-1, and IL-6 in the rat cerebral cortex in Test 4. [Figure 4] 1 shows the results of measurements of GLT-1, GABA, and GLT-1 / GABA in different administration groups in Study 5. [Figure 5] This is a photograph of HE staining of the substantia nigra (SN) of a mouse midbrain. [Figure 6] This is a photograph of HE-stained neurons in the mouse striatum (STR). The green arrow indicates a necrotic neuron, and the yellow arrow indicates a proliferative glial cell. [Figure 7] Nissl body staining of neurons in the substantia nigra of a mouse midbrain. The arrows in the image indicate Nissl bodies. [Figure 8] This shows the effect of CTMB administration on the expression level of TH protein in the substantia nigra of mice. DETAILED DESCRIPTION OF THE INVENTION

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

[0026] [ka]

[0027] The present invention provides a method for preparing 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound, which comprises the following steps:

[0028] Step (1) Compound 1a is mixed with dichloromethane, and then compound 1b is added thereto to react with the compound 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 mixed with sodium acetate and acetic anhydride, and then reacted to obtain 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound;

[0029] The compound 1a

[0030] [ka]

[0031] and the compound 1b is

[0032] [ka]

[0033] and the compound 1d is

[0034] [ka] is.

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

[0036] In the present invention, compound 1a and dichloromethane are preferably mixed under a nitrogen atmosphere, and compound 1b and dichloromethane are preferably added dropwise.

[0037] In the present invention, in the 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 is terminated when no more bubbles are generated under stirring conditions.

[0038] In the present invention, in the step (2), 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, preferably 23-26 g: 30.5-32.5 g: 27-29 g: 85-105 mL, and more preferably 23.5-25.5 g: 30.7-32.3 g: 27.5-28.5 g: 90-100 mL;

[0039] The compound 1c

[0040] [ka]

[0041] is.

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

[0043] In the present invention, in the 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, and preferably 2.5 hours.

[0044] In the present invention, in the step (3), the mass / volume ratio of compound 1e, the 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. Among these, the concentration of the 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 compound 1e

[0046] [ka]

[0047] is.

[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 the 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 the step (4), 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 compound 1f

[0053] [ka]

[0054] is.

[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 a use of a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of an antiepileptic drug.

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

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

[0061] The present invention provides the use of a 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound in the manufacture of a medicament for treating and / or preventing hemorrhagic stroke.

[0062] The present invention provides a use of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene in the manufacture of a medicament for preventing and / or treating Parkinson's disease.

[0063] The technical solutions provided by the present invention are described in detail below with reference to examples, which should not be construed as limiting the protection scope of the present invention.

[0064] Example 1 25.00g of compound 1a was dissolved in 150mL of dichloromethane under a nitrogen atmosphere and stirred uniformly, and then compound 1b was added dropwise to cause reaction. The amount of compound 1b used was 95.08g, the reaction temperature was 25℃, and the reaction was carried out under stirring conditions. When bubbles no longer generated under stirring conditions, the reaction was completed, and the mixture was concentrated to obtain 25.00g of an orange-yellow liquid, i.e., 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, and the resulting mixture was cooled to 0 °C. 28.12 g of aluminum trichloride was added. The reaction mixture was then heated to 25 °C and reacted for 2.5 h. The reaction mixture was then quenched with 300 mL of water and extracted with ethyl acetate (3 x 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 slurrying in 40 mL of ethanol for 1 hour to obtain 43.70 g of a white solid, i.e., compound 1e, in a 91.9% yield.

[0066] 43.70 g of compound 1e was dissolved in 150 mL of tetrahydrofuran. The resulting mixture was cooled to 0 °C and 20 mL of aqueous sodium borohydride solution was added dropwise, with the concentration of the aqueous sodium borohydride solution being 0.925 g / mL. Then, 10 drops of 10 wt% sodium hydroxide solution were added dropwise, and the reaction system was heated to 60 °C and reacted for 3 hours. After cooling to 37 °C, the pH was adjusted to 7 with 1 M hydrochloric acid solution, concentrated to remove tetrahydrofuran, extracted with ethyl acetate (3 × 200 mL), and the combined organic phases were dried over anhydrous magnesium sulfate and concentrated to obtain 42.59 g of an orange-yellow semi-solid, i.e., compound 1f, in 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 acetic anhydride, quenched by adding 200 mL of water, extracted with ethyl acetate (4 x 200 mL), and the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. Finally, the crude product was purified by recrystallization from 70% ethanol to obtain 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 27.00g of compound 1a was dissolved in 170mL of dichloromethane under a nitrogen atmosphere and stirred uniformly, and then compound 1b was added dropwise to cause reaction. The amount of compound 1b used was 97g, the reaction temperature was 30℃, and the reaction was carried out under stirring conditions. When bubbles no longer generated under stirring conditions, the reaction was completed, and the mixture was concentrated to obtain 26.62g of an orange-yellow liquid, i.e., 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, and the resulting mixture was cooled to 0 °C. 30 g of aluminum trichloride was added. The reaction mixture was then heated to 30 °C and reacted for 2 h. The reaction product was then quenched with 300 mL of water and extracted with ethyl acetate (3 x 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 slurrying in 40 mL of ethanol for 1 hour to obtain 44.05 g of a white solid, i.e., compound 1e, in an 89.7% yield.

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

[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 acetic anhydride, quenched by adding 200 mL of water, extracted with ethyl acetate (4 x 200 mL), and the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. Finally, the crude product was purified by recrystallization from 70% ethanol to obtain 22.59 g of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound as a white solid. The yield was 59.2%.

[0074] Example 3 23.00g of compound 1a was dissolved in 130mL of dichloromethane under a nitrogen atmosphere and stirred uniformly, and then compound 1b was added dropwise to cause reaction. The amount of compound 1b used was 93g, the reaction temperature was 20℃, and the reaction was carried out under stirring conditions. When bubbles no longer generated under stirring conditions, the reaction was completed, and the mixture was concentrated to obtain 22.87g of an orange-yellow liquid, i.e., 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, and the resulting mixture was cooled to 0 °C. 26 g of aluminum trichloride was added. The reaction mixture was then heated to 20 °C and reacted for 3 h. The reaction product was then quenched with 300 mL of water and 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 slurrying in 40 mL of ethanol for 1 hour to obtain 40.22 g of a white solid, i.e., compound 1e, in a 90.1% yield.

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

[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 acetic anhydride, quenched by adding 200 mL of water, extracted with ethyl acetate (4 x 200 mL), and the organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. Finally, the crude product was purified by recrystallization from 70% ethanol to obtain 21.10 g of 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound as a white solid. The yield was 59.3%.

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

[0079] Preparation process for emulsion injection: 10.0 g of CTMB obtained in Example 1 of the present invention and 200.0 g of injectable soybean oil were placed in a beaker, heated to 75°C under nitrogen-filled conditions, and stirred to dissolve. Next, 12.0 g of egg yolk lecithin was weighed out, added, and stirred to dissolve, preparing the oil phase. Separately, 22.5 g of glycerin was weighed out, and approximately 680 mL of water was weighed out. The mixture was heated to 75°C under nitrogen-filled conditions and stirred to dissolve, preparing the aqueous phase. The oil phase was added to the aqueous phase, and high-speed shearing was performed for 10 minutes. Water was then added to the beaker to make a 1000 mL crude emulsion. The crude emulsion was homogenized twice using a high-pressure homogenizer to reduce the average droplet size to 0.4 μm or less and adjust the pH to 8.5. The emulsion was then filled into 5 mL glass ampoules under nitrogen and sterilized in a rotary autoclave at 121°C for 12 minutes to obtain an emulsion injection (name: CTMB emulsion injection). The CTMB concentration was 10 mg / mL. A blank emulsion injection without CTMB was prepared in the same manner.

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

[0081] (1): Protective effect of CTMB on pentylenetetrazole-induced epilepsy in mouse models; Verification of protective effect of CTMB on lithium-pilocarpine-induced epilepsy in rat models.

[0082] Test 1 CTMB protects against pentylenetetrazole-induced epilepsy in mouse models:

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

[0084] (2) Animal grouping: The test mice were randomly divided into three groups: saline group, low-dose CTMB group 50 mg / kg (CTMB-L), medium-dose CTMB group 75 mg / kg (CTMB-M), and high-dose CTMB group 100 mg / kg (CTMB-H). There were 10 mice in each group, and each group was administered intraperitoneally.

[0085] (3) Evaluation method and results After a certain period of administration, mice in each group were intraperitoneally injected with 75 mg / kg of pentylenetetrazole, a drug used to create epilepsy models. The Racine score, the incidence of clonic seizures (Racine score 1-3) and tonic seizures (Racine score 4-5), and the latency period were observed and recorded within 30 minutes. The Racine classification criteria were: Grade 0: normal non-epileptic activity; Grade 1: wet dog-like shivering or scratching behavior; Grade 2: nodding or tail wagging; Grade 3: unilateral forelimb extension or unilateral limb clonic convulsions; Grade 4: multiple limb clonic convulsions or tonic convulsions; Grade 5: falling and generalized tonic-clonic seizures. If no epileptic seizures occurred within 30 minutes, the latency period was recorded as 1800 seconds. The experimental results are shown in Table 1.

[0086] [Table 1]

[0087] Note: * P<0.05, ** P<0.01, *** P<0.001 compared with the saline group

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

[0089] Test 2 The protective effect of CTMB on the lithium-pilocarpine rat epilepsy model:

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

[0091] (2) Establishment of a lithium-pilocarpine rat epilepsy model Methylscopolamine bromide, lithium chloride, and pilocarpine were weighed and dissolved in 0.9% saline to the working 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), followed 18–20 h later by intraperitoneal injection of 1 mg / kg methylscopolamine bromide. Thirty minutes later, an initial dose of 30 mg / kg pilocarpine was intraperitoneally injected, and the rats were observed for the severity of epileptiform seizures. Subsequently, additional doses of 10 mg / kg pilocarpine were administered every 30 min until the rats developed a clear, non-intermittent status epilepticus (SE)-like seizure of class IV or higher, up to a maximum dose of 60 mg / kg.

[0092] Model selection criteria: If the experimental animal experiences a continuous 1-hour seizure of grade IV or higher, the model can be judged to have been successfully created.

[0093] (3) Grouping of animals The rats that successfully generated the model were randomly divided into three groups: the model group, the CTMB group, and the sodium valproate (VPA) group. Administration was performed 1 h after the SE attack. The model group received a blank emulsion via intraperitoneal injection, the treatment group received a 50 mg / kg CTMB emulsion via intraperitoneal injection, and the VPA group received 300 mg / kg sodium valproate via oral or intragastric administration.

[0094] (4) Evaluation method and results Post-ictal behavioral assessment after drug intervention: The rats in each group were observed for epileptic seizure symptoms after drug intervention and the severity of seizures in each group was recorded according to the Racine scale. The Racine classification criteria were as follows: Grade 0: normal non-epileptic activity; Grade 1: wet dog-like shivering or scratching behavior; Grade 2: nodding or tail wagging; Grade 3: unilateral forelimb extension or unilateral limb 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 CTMB group and model group. *** P<0.001, t-test; comparison between the CTMB group and the VPA group. # P<0.05, t-test.

[0097] Table 2 shows that CTMB (50 mg / kg) significantly reduced the severity of epileptic seizures in the acute phase of the SE model, and its effect was significantly superior to that of the positive control drug, sodium valproate (300 mg / kg).

[0098] One-week survival observation 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 log-rank test was used to analyze the differences in survival curves between rats in each group. The results showed that CTMB (50 mg / kg) significantly improved the survival rate of SE model rats (P<0.05), and its effect was superior to that of the positive control drug, sodium valproate (VPA).

[0100] (2): Study on the mechanism of action of CTMB in the treatment of traumatic brain injury Experimental animals and materials: SPF-grade SD rats, all male, weighing 240–260 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, with the qualification certificate number: SCXK(Chuan)2020-030.

[0101] Evans Blue (C11891158, Shanghai Maclin Biochemical Technology); Formamide (20190716, Tianjin Bodi Chemical Industry); IL-1 detection kit (E-EL-H0149c, Wuhan Yi Rui Biological Science and Technology Co., Ltd.); IL-6 detection kit (E-EL-H2518c, Wuhan Illairui Te Biological Science and Technology 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 grouping and administration scheme are as follows:

[0103] Sham-operated group (P group): Normal saline was administered at the same volume as that of the CTMB group; Model group (T group): CTMB group was administered the same volume of blank emulsion; Medium dose group (Group M): 30 mg / kg of CTMB emulsion injection was administered.

[0104] Two hours after TBI model creation, rats in each group were given an intraperitoneal injection of Evans Blue. 24 hours after administration, 4% Evans Blue solution (2.5 mL / kg) was injected into the right tail vein of the rats. One hour later, the rats were deeply anesthetized and perfused with 100 mL of saline via the heart. The brains were rapidly decapitated and immediately separated into left and right hemispheres, cerebellum, and brainstem. After measuring the wet weight of the brain tissue, the rats were immersed in 10 volumes of pure formamide, incubated at 60°C for 48 hours, and centrifuged at 25°C and 10,000 rpm / min for 30 minutes. The supernatant was subjected to UV spectrophotometry at 622 nm for Evans Blue dye detection. A standard curve was constructed and quantified. The final results were expressed as Evans Blue content per gram of brain tissue (µg / g). Results are shown in Figure 2 (n=6). Compared with the sham-operated group (P group), the hemorrhagic hemisphere of the model group (T group) showed increased blood-brain barrier permeability 24 h after surgery, whereas the medium-dose CTMB group (M group) showed significantly reduced Evans blue leakage, improving the integrity of the blood-brain barrier.

[0105] Test 4 IL-1, IL-6 and TNF-α detection The experimental grouping and administration method were the same as in Study 3. Two hours after TBI model creation, rats in different groups were intraperitoneally administered the drug. 24 hours after administration, the rats were deeply anesthetized, decapitated, and their brains were removed. Approximately 60–120 mg of the cortex from the hemisphere on the bleeding side was collected, and 10 volumes of ice-cold biochemical detection buffer were added. The mixture was homogenized in an ice bath for 10 minutes and centrifuged at 4°C for 30 minutes at 14,000 rpm. The supernatant was collected and the contents of the inflammatory cytokines IL-1, IL-6, and TNF-α were measured according to the instructions for the IL-1, IL-6, and TNF-α content detection kits. The results are shown in Figure 3 (n = 5-8). Compared with group P, the contents of IL-1, IL-6, and TNF-α in the hemorrhagic lateral hemisphere of rats in group T were significantly elevated 24 h after surgery. However, group M significantly reduced the contents of the pro-inflammatory cytokine TNF-α, inflammatory cytokines IL-1, and IL-6 in the cerebral cortical tissue of TBI rats, which is beneficial for reducing neuroinflammation and improving the motor function of rats.

[0106] Test 5 Measurement of GLT-1 and GABA contents The experimental grouping and administration method were the same as in Study 3. After 7 consecutive days of treatment, rats were deeply anesthetized, decapitated, and the brains were removed. Approximately 60–120 mg of cortex from the hemisphere adjacent to the bleeding site was collected and added with 10 volumes of ice-cold biochemical detection buffer. The samples were homogenized for 10 min in an ice bath and centrifuged at 14,000 rpm / min at 4°C for 30 min. The supernatants were collected and assayed for GLT-1 and GABA content, respectively, according to the instructions for the GLT-1 and GABA content assay kits. Glutamate transporter 1 (GLT-1) is an important glutamate transporter in the brain, transporting extracellular glutamate into cells. γ-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter. Under pathological conditions, extracellular glutamate levels rise sharply, leading to excessive neuronal excitotoxicity. Figure 4 shows the GLT-1 and GABA contents and their ratios measured in different treatment groups, which can be used to evaluate the excitatory neurotoxicity caused by TBI injury. The results are shown in Figure 4 (n=6). Compared with group P, group T showed significantly elevated GLT-1 content and GLT-1 / GABA ratios in the hemorrhagic lateral hemisphere of rats on postoperative day 7. However, group M administration reduced GLT-1 content, increased GABA content, and significantly reduced the GLT-1 / GABA ratio in the brains of TBI rats, restoring the balance of excitatory amino acids (EAA) and inhibitory amino acids (IAA) in the brain, alleviating excitatory neurotoxicity caused by excess glutamate, and thereby improving motor function in rats.

[0107] (3) Verification of the acute therapeutic effect of CTMB on ischemic stroke in rats using filament embolization.

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

[0109] 3.2 Experimental grouping: Two hours after MCAO, the success of model creation was determined by the Zea Longa score, and rats with successful model creation were randomly assigned to groups for administration.

[0110] The rats were randomly divided into a sham-operated group (Sham group, administered the same volume of blank emulsion as the CTMB high-dose group), a model group (Vehicle group, administered the same volume of blank emulsion as the CTMB high-dose 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 injectable concentrated solution group (EDB group, commercially available product, 3 mg / kg). Each group consisted of 10 rats, and all were administered intraperitoneally. (Note that rats in the experiments were administered intraperitoneally, and the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the dose per unit mass in humans was 0.2 mg - 4.0 mg / kg. This refers to the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the unit mass in humans when administered intravenously, and was calculated from the effective dose in rats. Taking into account differences in drug bioavailability due to differences in species and administration methods, as well as differences in peak drug concentration and peak time, the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the unit mass in humans was ultimately converted and determined to be 0.2 mg - 4.0 mg / kg.)

[0111] 3.3 Creation of an ischemia-reperfusion model using the filament embolization method Before surgery, rats were fasted for 12 hours and anesthetized with 10% chloral hydrate (350 mg / kg, i.p.). The rats were placed in a supine position, and their body temperature was maintained at approximately 37°C. The neck skin was prepared, a midline incision was made, and the muscles and fascia were separated along the medial border of the sternocleidomastoid muscle to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The proximal ends of the CCA and ECA were ligated, and the ICA was temporarily clamped with an arterial clip. A small hole was made with a needle approximately 4 mm from the CCA bifurcation, and an embolic thread was inserted from the CCA to the ICA. The arterial clip on the ICA was loosened, and the embolic thread was slowly advanced until the mark on the embolic thread reached the bifurcation and slight resistance was felt. The thin thread at the ICA was tightly tied, and the wound was irrigated with saline and sutured. The sham operation was performed in the same manner as the experimental group, except that no embolization thread was inserted. After waking up from anesthesia, the animals were kept in the normal condition.

[0112] 3.4 Criteria for selecting cerebral ischemia models Rats were scored 2 hours after surgery after awakening from anesthesia according to the Zea Longa Neurological 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.). Rats were placed into groups with a score of 1 or 2: 0: no neurological deficits, normal activity; 1: inability to fully extend the contralateral forelimb; 2: circling while walking; 3: leaning toward the paralyzed side; 4: inability to walk spontaneously and loss of consciousness.

[0113] 3.5 Short-term neurological deficit score Twenty-four hours after model creation, rats' neurological function was comprehensively assessed using the modified 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' movement, sensation, climbing, and limb symmetry were assessed. The mNSS score ranged from 0 to 18, with higher scores indicating more severe neurological impairment. The Garcia score ranged from 3 to 18, with lower scores indicating more severe neurological impairment. Scoring was performed independently by a blinded observer who was not involved in model creation or administration.

[0114] [Table 3]

[0115] [Table 4]

[0116] Table 5 shows that compared with the sham-operated group, the model group (vehicle group) had significantly increased mNSS scores (P<0.01) and significantly decreased Garcia scores (P<0.01) 24 hours after surgery, indicating significant neurological deficits in the model group rats 24 hours after MCAO. Administration of different doses of CTMB (CTMB-L, CTMB-H groups) and edaravone dexborneol injectable concentrated solution (EDB group) all reduced mNSS scores and increased Garcia scores to different degrees, improving neurological deficits induced by MCAO, with the most significant effect observed in the CTMB-H group (P<0.01). Furthermore, its efficacy was superior to that of edaravone dexborneol, a drug used clinically to treat ischemic stroke. No toxic side effects associated with CTMB administration were observed during the experiment.

[0117] [Table 5]

[0118] Note: Compared with the sham-operated group, ##P<0.01; compared with the model group (vehicle group); ** P<0.01, * P<0.05.

[0119] (4) Long-term therapeutic effects of CTMB on ischemic stroke in rats treated with filament embolization.

[0120] 4.1 Neurological Deficit Score The experimental materials, grouping (n = 10-15), model preparation method, and score criteria were the same as those for acute treatment. Two hours after the filament embolization model was prepared, the drug was administered according to the group administration form immediately, and then continued for 14 days, once daily, with the improved mNSS score evaluated on days 1, 4, 7, and 14.

[0121] Table 6 shows that compared with the sham-operated group, the model group (vehicle group) had a significantly increased mNSS score on postoperative day 14 (P<0.01), and rats in the model group developed significant neurological deficits on postoperative day 14 after MCAO. For days 1 to 7, CTMB administration (CTMB-H group) and edaravone dexborneol injectable concentrate group (EDB group) both reduced mNSS scores and improved MCAO-induced neurological deficits to varying degrees. The CTMB-H group showed the most significant effect, slightly superior to edaravone dexborneol injectable concentrate, a drug used clinically to treat ischemic stroke. Furthermore, by day 14, the CTMB-H group still achieved a significant reduction in mNSS scores. No toxic side effects associated with CTMB administration were observed during the experiment.

[0122] [Table 6]

[0123] Note: Compared with sham surgery (Sham group), ## P<0.01; compared with the model group (vehicle group); ** P<0.01, *P<0.05.

[0124] 4.2 Adhesive tape removal test Prior to model creation, rats underwent a one-week adhesive tape removal test. Rats that could remove the tape within 10 seconds were selected for model creation. Two hours after filament embolization model creation, rats were immediately administered the group-specific dosage form. The administration continued for 14 days, with daily administration. The adhesive tape removal test was conducted on days 1, 4, 7, 10, and 14 to evaluate the rats' sensory and motor functions. The specific procedure was as follows: a circular piece of tape approximately 12 mm in diameter was attached to the sole of the rat's left forepaw. The time it took for the rat to sense and remove the tape was recorded. If the rat was unable to sense or remove the tape within 60 seconds, the time was recorded as 60 seconds.

[0125] Table 7 shows that compared with the sham group, the model group (Vehicle group) had a significantly prolonged tape sensing time on postoperative day 14 (P<0.05), and the left forelimb sensing ability of rats in the model group was reduced on postoperative day 14. Different doses of CTMB and EDB can promote the recovery of sensory ability in rats.

[0126] [Table 7]

[0127] Note: Compared with sham surgery (Sham group), ## P<0.01, # P<0.05 compared with the model group (vehicle group). ** P<0.01, * P<0.05.

[0128] Table 8 shows that compared with the sham-operated group, the model group (Vehicle group) had a significantly increased tape removal time on postoperative day 14 (P<0.01), and the motor ability of the left forelimb of the model group rats was reduced on postoperative day 14. The CTMB-L group significantly reduced the tape removal time of the rats from day 4 (P<0.05), and the CTMB-H group significantly reduced the tape removal time of the rats from day 10 (P<0.05), demonstrating superior efficacy to edaravone and dexborneol, drugs used clinically to treat ischemic stroke. No toxic side effects associated with CTMB administration were observed during the experiment.

[0129] [Table 8]

[0130] Note: Compared with sham surgery (Sham group), ## P<0.01, # P<0.05 compared with the model group (vehicle group). ** P<0.01, * P<0.05.

[0131] From the above, it was found that long-term administration of CTMB significantly promoted the recovery of motor function and significantly improved the somatosensory ability of rats.

[0132] (5): Verification of the acute therapeutic effect of CTMB on hemorrhagic stroke in rats using the caudate nucleus collagenase VII injection method.

[0133] 5.1 Experimental materials: SPF SD rats (male, weighing 220-250g, purchased from Sichuan Dashuo Experimental Animal Co., Ltd., certificate number: SCXK(Sichuan)2020-0030) Type VII collagenase (purchased from Sigma-Aldrich, USA (specification: 1.5KU; lot number: 0000111586)) Edaravone / dexborneol concentrated solution for injection (purchased from Sunsheng Pharmaceutical Co., Ltd. (specifications: 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, and rats with successful model creation were randomly grouped and administered.

[0135] The rats were randomly divided into a sham-operated group (Sham group, administered the same volume of blank emulsion as the CTMB high-dose group), a model group (Vehicle group, administered the same volume of blank emulsion as the CTMB high-dose group), a CTMB low-dose group (CTMB-L group, 10 mg / kg), a CTMB high-dose group (CTMB-H group, 20 mg / kg), and an edaravone-dexborneol injectable concentrated solution group (EDB group, commercially available product, 3 mg / kg). Each group consisted of 10-13 rats, and all were administered intraperitoneally. (Note that rats in the experiments were administered intraperitoneally, and the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the dose per unit mass in humans was 0.2 mg - 4.0 mg / kg. This refers to the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the unit mass in humans when administered intravenously, and was calculated from the effective dose in rats. Taking into account differences in drug bioavailability due to differences in species and administration methods, as well as differences in peak drug concentration and peak time, the ratio of the effective dose of the active ingredient CTMB in the emulsion injection to the unit mass in humans was ultimately converted and determined to be 0.2 mg - 4.0 mg / kg.)

[0136] 5.3 Creation of a hemorrhagic stroke model using the caudate nucleus collagenase VII injection method Before surgery, rats were fasted for 8 hours and deprived of food and water. Anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. Body temperature was maintained at approximately 37°C. The rat was placed in a prone position in a stereotaxic apparatus. The head skin was prepared, and a longitudinal incision was made along the midline of the head. The skin and subcutaneous tissue were then dissected to expose the anterior fontanel and coronal suture. The right caudate nucleus was located using the stereotaxic apparatus, with the anterior fontanel as the origin. The incision was then offset 3.0 mm to the right from the midline. After marking, a hole was drilled with a skull drill, exposing the dura mater and achieving adequate hemostasis. A microsyringe was attached to the stereotaxic apparatus and the needle was advanced vertically along the skull hole to 5.5 mm (i.e., the caudate nucleus area). 1 μL of collagenase VII (0.5 U / μL) was slowly and uniformly injected into the brain. The injection was allowed to continue for 5 minutes, the needle was left in place for 8 minutes, and the syringe was then slowly withdrawn. The skull defect was closed with sterile bone wax, and the scalp incision was sutured. After disinfection with iodophor, the rats were returned to their cages and kept. The sham-operated (Sham) group underwent the same procedure except for the injection of saline. The rats were allowed to recover from anesthesia and then kept as normal.

[0137] 5.4 Selection criteria for cerebral hemorrhage models Two hours after surgery, rats were scored according to the Zea Longa Neurological 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.). Animals were assigned a score of 1 to 2, with the following classification: 0: no neurological deficits, normal activity; 1: inability to fully extend the contralateral forelimb; 2: the animal circling while walking; 3: leaning toward the affected side; 4: inability to walk spontaneously and loss of consciousness.

[0138] 5.5 Short-term neurological deficit score Twenty-four hours after model creation, rats' neurological function was comprehensively assessed using the modified 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' movement, sensation, climbing, and limb symmetry were assessed. The mNSS score ranged from 0 to 18, with higher scores indicating more severe neurological impairment. The Garcia score ranged from 3 to 18, with lower scores indicating more severe neurological impairment. Scoring was performed independently by a blinded observer who was not involved in model creation or administration.

[0139] [Table 9]

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

[0141] Table 9 shows that compared with the sham-operated group, the model group (vehicle group) had significantly increased mNSS scores (P<0.0001) and significantly decreased Garcia scores (P<0.0001) 24 hours after surgery, indicating significant neurological deficits in the model group rats 24 hours after cerebral hemorrhage surgery. Administration of different doses of CTMB (CTMB-L and CTMB-H groups) reduced mNSS scores and increased Garcia scores to different degrees, whereas administration of edaravone dexborneol injectable concentrate (EDB group) only increased Garcia scores. CTMB administration improved neurological deficits caused by hemorrhagic stroke, with the most significant effect observed in the CTMB-L group (P<0.0001). Furthermore, its efficacy was superior to that of edaravone dexborneol, a drug clinically used to treat ischemic stroke. No toxic side effects related to CTMB administration were observed during the course of the experiment.

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

[0143] 6.2 Experimental grouping: Two weeks after 6-OHDA injection, the mice were scored in the pole climbing test, suspension test, rotarod test, and open field test to determine the success of the model creation. The mice that were successfully created were randomly divided into groups and administered the treatment.

[0144] Mice were randomly divided into a sham-operated group (Group S, administered with the same volume of blank emulsion as the treatment group), a model group (Group M, administered with 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), with 10 to 12 mice in each group, all administered by intraperitoneal injection.

[0145] 6.3 Creation of PD model using 6-OHDA method Mice were fasted for 12 hours before surgery. Anesthesia was induced with 4% isoflurane and maintained with 1.5% isoflurane. The mice were then fixed in a stereotaxic apparatus. After incising the scalp, the skull surface was gently wiped with a cotton swab to expose the anterior fontanel (bregma) and posterior fontanel (lambda). The stereotaxic apparatus was adjusted to match the height of the anterior fontanel and posterior fontanel, keeping the skull level. Using the bregma as the origin, the coordinates of the injection site were determined, and a small hole was drilled in the skull above the injection site.

[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 each of the two points at a rate of 0.5 μL / min. After the injection was completed, the needle was left in place for 3 minutes, then withdrawn, and the animal's scalp was sutured and disinfected. A sham-operated group received an injection of the same volume of saline containing 0.02% ascorbic acid, with the remaining procedures remaining the same. After surgery, the mice were placed in cages at the appropriate temperature. After recovery from anesthesia, they were housed normally.

[0147] 6.4 Selection criteria for Parkinson's disease models Two weeks after surgery, the mice were subjected to a pole climbing test and a suspension test to evaluate their motor function, and mice with significant differences from the sham-operated group were placed into a different group.

[0148] Pole climbing test: The pole climbing test primarily measures the motor ability of mice and evaluates bradykinesia symptoms. This test assesses the ability of mice to grasp and descend a pole and return to their home cage. A cork ball is fixed to an iron rod 1 cm in diameter and 65 cm long. The mouse is placed at the top of the pole with its head facing downwards. The mouse will usually naturally climb down the pole and return to its cage. The time it takes for the mouse to reach the bottom is recorded. The results are the average of three consecutive measurements.

[0149] Suspension test: Normal mice have climbing activity, which requires adequate grip strength and motor coordination, so the suspension test can be used to examine motor dysfunction in mice. A 1.5 mm diameter metal rod is placed horizontally in the box, 30 cm above the bottom. During the experiment, the mouse's front paws are hung from the metal rod, and the time it takes to land is recorded. If the mouse does not fall for more than 60 seconds, the time is recorded as 60 seconds. Two measurements are taken, with an interval of at least 1 minute between each measurement, and the results are the average of the two measurements.

[0150] 6.5 Motor function assessment At 1, 2, 3, and 4 weeks after treatment, the mice were comprehensively evaluated for motor function using the pole climbing test and the suspension test. Scoring was performed independently by a blinded observer who was not involved in the model preparation or treatment.

[0151] [Table 10]

[0152] Note: Compared with the sham-operated group (S group), # P<0.05, ## P<0.01, ### P<0.001 compared with the model group (M group). * P<0.05, ** P<0.01. Data are shown as x±SD and were analyzed by multigroup comparison using ANOVA.

[0153] Table 10 shows that compared with the sham-operated group (Group S), the model group (Group M) had a significantly increased pole-climbing time (P<0.001) before administration (2 weeks after surgery), and the mice in the model group developed obvious bradykinesia 2 weeks after model creation. After intraperitoneal administration of the CTMB group (Group D) and the levodopa group (Group L), the pole-climbing time of the model mice was significantly shortened, improving 6-OHDA-induced bradykinesia. In the PD model, the efficacy of Group D was not significantly different from that of the levodopa group (Group L), a drug used clinically to treat Parkinson's disease. Furthermore, there was no significant difference in the initial pole-climbing time of the mice in each group, excluding any differences in behavioral function due to the severity of the model creation.

[0154] [Table 11]

[0155] Note: Compared with the sham-operated group (S group), # P<0.05, ## P<0.01 compared with the model group (group M). * P<0.05, ** P<0.01. Data are shown as x ± SD, and multigroup comparison analysis was performed using the Kruskal-Wallis test.

[0156] Table 11 shows that compared with the sham-operated group (Group S), the model group (Group M) had a significantly shorter hanging time (P<0.01) before administration (2 weeks after surgery), and mice in the model group developed obvious motor dysfunction 2 weeks after model creation. After intraperitoneal administration of CTMB (Group D), the hanging time of the model mice was extended, and 6-OHDA-induced motor dysfunction was improved. In the PD model, the efficacy of Group D was superior to that of the levodopa group (Group L), a drug used clinically to treat Parkinson's disease, but the difference was not significant. Furthermore, there was no significant difference in the initial hanging time of mice in each group, excluding differences in behavioral function due to the severity of the model creation.

[0157] (7): Study on the mechanism of action of CTMB in the anti-Parkinson's disease 7.1 Experimental materials: SPF C57 mice (male, weighing 18-22g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, China; certificate number: SCXK(Sichuan)2020-030) Hematoxylin and eosin staining solution (BP0211, Hubei Baiosin Biotechnology Co., Ltd.), Toluidine blue staining solution (BP0360, Hubei Baiosu Biological Technology Co., Ltd.), Recombinant Anti-Tyrosine Hydroxylase antibody (ab137869, Abcam (Shanghai) Trading Co., Ltd.), Malondialdehyde (MDA) colorimetric test kit (TBA method) (E-BC-K025-M, Wuhan Yalui Te Biotechnology Co., Ltd.), Total superoxide dismutase (T-SOD) colorimetric test kit (WST-1 method) (E-BC-K020-M, Wuhan Illite Biotechnology Co., Ltd.), Reduced Glutathione (GSH) Colorimetric Test Kit (E-BC-K030-M, Wuhan Illairui Te Biotechnology Co., Ltd.).

[0158] 7.2 Experimental Procedures and Results: 7.2.1 Sample preparation Mice were randomly divided into a sham-operated group (Group S, administered with the same volume of blank emulsion as the treatment group), a model group (Group M, administered with 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), with 10 to 12 mice in each group, all administered by intraperitoneal injection.

[0159] After completing the motor function evaluation 4 weeks after administration, the mice were deeply anesthetized and perfused transcardially with 20 mL of saline and 20 mL of 4% paraformaldehyde. They were then rapidly decapitated to remove the brains, which were then fixed in 4% paraformaldehyde for 72 hours, embedded in paraffin, and cut into 4 μm-thick sections for subsequent histopathological studies.

[0160] 7.2.2 Attenuation of neuronal damage in the substantia nigra and striatum of PD mice HE staining can be used to observe pathological changes in brain tissue. The specific procedure for HE staining is as follows:

[0161] a) Deparaffinization of paraffin sections from water to water: Deparaffinize in environmentally friendly deparaffinizing agent (1), environmentally friendly deparaffinizing agent (2), and environmentally friendly deparaffinizing agent (3) for 10 minutes each, then treat with absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and rinse in tap water for 1 minute.

[0162] b) Stain with hematoxylin stain (Harris) for 4 minutes, then wash with tap water for 2 minutes until excess stain is removed from the sections.

[0163] c) Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, wash with tap water, return to blue color 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 tone directly in 95% ethanol for 5 seconds without washing, and dehydrate in absolute ethanol (1) and absolute ethanol (2) for 2 minutes.

[0165] e) Clear with environmentally friendly clearing agent, mount, and examine microscopically.

[0166] Nissl staining is used to identify Nissl bodies in neurons. The specific procedure for Nissl staining is as follows.

[0167] a) Deparaffinization of paraffin sections from water to water: Deparaffinize in environmentally friendly deparaffinizing agent (1), environmentally friendly deparaffinizing agent (2), and environmentally friendly deparaffinizing agent (3) for 10 minutes each, then treat with absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and wash three times with distilled water.

[0168] b) The sections are placed in a preheated 50°C 1% toluidine blue solution, stained in a 56°C water bath for 20 minutes, and washed with distilled water.

[0169] c) Differentiate with 95% alcohol or 0.1% glacial acetic acid, control under a microscope, and use the clear visualization of Nissl bodies as the criterion.

[0170] d) Rapid dehydration in absolute ethanol.

[0171] e) Clear with environmentally friendly clearing agent, mount, and examine microscopically.

[0172] [Table 12]

[0173] Note: Comparison with the model group (M group), *** P<0.001. Data are shown as x±SD and were analyzed by ANOVA for multiple group comparisons.

[0174] The results, shown in Figures 5 and 6, indicate that 6 weeks after 6-OHDA injection, neurons in the right substantia nigra and striatum of mice underwent coagulation necrosis, with unclear nucleoli and diffuse or focal proliferation of glial cells. Glial cells were observed to invade the cell bodies or processes of necrotic neurons, forming neurophagocytes. Figure 7 and Table 12 also show that neurons in the right substantia nigra shrank and became deeply stained, and the number of Nissl bodies decreased. Compared with the model group (group M), the degree of pathological damage in the substantia nigra and striatum of mice was reduced in the CTMB group (group D) and levodopa group (group L). This indicates that CTMB can attenuate 6-OHDA-induced damage to substantia nigra neurons in mice and has a neuroprotective effect.

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

[0176] a) Paraffin sections were deparaffinized in environmentally friendly deparaffinizing agent (1), environmentally friendly deparaffinizing agent (2), and environmentally friendly deparaffinizing agent (3) for 10 minutes each, followed by treatment with absolute ethanol, 95% ethanol, and 75% ethanol for 5 minutes each. Washed in distilled water three times for 3 minutes each, followed by immersion.

[0177] b) Antigen retrieval is performed.

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

[0179] d) Boxing: After drawing a box with a histochemical pen, the section is placed in TBST.

[0180] e) Mounting: Add a drop of 10% serum that matches the origin 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, prepare the primary antibody working solution, and add 50-100 μL (depending on the size of the tissue) of the primary antibody working solution to each section. Incubate at 4°C overnight.

[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 three times with TBST and then immerse three times, each time for 3 minutes. 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 three times with TBST and then immerse three times, each time for 3 minutes.

[0183] h) DAB color development: Discard the TBST and 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 it with a timer. Rinse with tap water to stop the color development reaction and record the time for color development.

[0184] [Table 13]

[0185] Note: Compared with the sham-operated group (S group), ### P<0.001 compared with the model group (M group). *** P<0.001 compared with the levodopa group (group L). && P<0.01. Data are shown as x±SD and were analyzed by multigroup comparison using ANOVA.

[0186] The results are shown in Figure 8 and Table 13. After 6-OHDA injection, the expression of TH protein in the substantia nigra on the injured side (right side) was significantly reduced, i.e., the number of dopaminergic positive neurons was significantly reduced (P<0.001). The CTMB group (Group D) significantly inhibited 6-OHDA-induced dopaminergic neuronal apoptosis and was significantly superior to the levodopa group (Group L), indicating that CTMB can slow the progression of Parkinson's disease in animal models. The levodopa group (Group L) also inhibited 6-OHDA-induced dopaminergic neuronal apoptosis, but there was no statistically significant difference.

[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 nonenzymatic antioxidants, and superoxide dismutase (SOD) is a common indicator of antioxidant enzymes. After 4 weeks of motor function assessment, mice were deeply anesthetized and perfused with 20 mL of saline via the heart. The brains were rapidly decapitated and the striatum from the injured side (right side) was isolated, weighed, and placed in a 1.5 mL EP tube. PBS was added at a tissue weight (g) to homogenization medium volume (mL) ratio of 1:9. The mixture was then homogenized at low temperature. The supernatant was centrifuged at 10,000 × g for 10 minutes at 4°C. The MDA, GSH, and SOD content and SOD activity were measured according to the instructions of the colorimetric assay kits.

[0188] [Table 14]

[0189] Note: Comparison with the model group (M group), * P<0.05, ** P<0.01. Data are shown as x±SD and were analyzed by multigroup comparison using ANOVA.

[0190] As shown in Table 14, MDA levels in the striatum of mice in the model group (Group M) were significantly elevated compared to the sham-operated group (Group S), and GSH and SOD levels were significantly reduced. The CTMB group (Group D) significantly reduced MDA levels in the striatum of model mice and significantly improved GSH levels and SOD activity in the striatum of model mice. These results indicate that CTMB can exert anti-PD effects by ameliorating oxidative stress.

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

Claims

1. The following formula (I): 【Chemistry 1】 It has a structure represented by A 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound characterized in that

2. Step (1) of mixing compound 1a with dichloromethane, and then adding compound 1b to react to obtain compound 1c; Step (2) of sequentially mixing compound 1c, compound 1d, and aluminum trichloride with dichloromethane and then reacting them to obtain compound 1e; Step (3) of sequentially mixing compound 1e, a sodium borohydride solution, and a sodium hydroxide solution with tetrahydrofuran and then reacting them to obtain compound 1f; and step (4) of mixing compound 1f, sodium acetate and acetic anhydride, followed by reaction to obtain 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound; The compound 1a is 【Chemistry 2】 and The compound 1b is 【Transformation 3】 and The compound 1d is 【Chemistry 4】 is The method for producing the 1-(cyclobutylidenemethyl)-2,4,5-trimethoxybenzene compound according to claim 1.

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

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

5. In the step (2), the mass / volume ratio of compound 1c, compound 1d, aluminum trichloride, and dichloromethane is 22 to 27 g: 30 to 33 g: 26 to 30 g: 80 to 120 mL; The compound 1c is 【Transformation 5】 is The method of claim 4.

6. In step (2), the reaction temperature is 20-30°C, and the reaction time is 2-3 hours. The method of claim 2, 3 or 5.

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

8. In the step (3), the reaction temperature is 50-65°C, and the reaction time is 2-4 hours. The method according to claim 5 or 7.

9. In the step (4), the mass / volume ratio of compound 1f, sodium acetate, and acetic anhydride is 38-45 g:7-10 g:200-230 mL; The compound 1f is 【Transformation 7】 and The reaction temperature is 120-140°C, and the reaction time is 2-4 hours. The method of claim 8.

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

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

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

13. 10. Use of the 1-(cyclobutylmethyl)-2,4,5-trimethoxybenzene compound of claim 1 in the manufacture of a medicament for treating and / or preventing hemorrhagic stroke.

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

Images