3-methyl-1-phenyl-1H-pyrazole-5-O-beta-D-glucoside as well as preparation method, pharmaceutical composition and application of 3-methyl-1-phenyl-1H-pyrazole-5-O-beta-D-glucoside

Abstract

The invention belongs to the field of medicines, and relates to 3-methyl-1-phenyl-1H-pyrazole-5-O-beta-D-glucoside as well as a preparation method, a pharmaceutical composition and application thereof. Specifically, the 3-methyl-1-phenyl-1H-pyrazole-5-O-beta-D-glucoside disclosed by the invention is shown as a formula I, and the preparation method of the 3-methyl-1-phenyl-1H-pyrazole-5-O-beta-D-glucoside comprises the steps of hydroxyl activation, substitution, deprotection, resolution (also can be carried out before the deprotection step) and the like which are carried out in sequence. Through comparative study, the two epimers of the 3-methyl-1-phenyl-1H-pyrazole-5-O-D-glucoside have significant differences in solubility, in-vitro activity and the like, and the effect of the beta-isomer is better, so that the 3-methyl-1-phenyl-1H-pyrazole-5-O-D-glucoside is suitable for being further developed into medicines. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the medical field and relates to 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside, a preparation method thereof, a pharmaceutical composition containing the same, and application thereof in the medical field. Background Art

[0002] Cerebrovascular diseases are known for their high incidence, high disability, high mortality and high recurrence rates, among which stroke (also known as cerebral infarction or cerebral infarction) is the most important clinical cerebrovascular disease. The pathogenesis of stroke is that the sudden rupture of cerebral blood vessels or blood vessel blockage prevents blood from flowing into the brain, causing brain tissue damage, including ischemic stroke and hemorrhagic stroke.

[0003] Similar to stroke, amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is also a disease characterized by the damage, atrophy and death of nerve cells. Although the pathogenesis of ALS has not yet been determined, biological studies have shown that the disease is at least related to superoxide dismutase (SOD). At present, the scientific community generally believes that the mutation of SOD1 is one of the causes of ALS.

[0004] Mitochondrial encephalomyopathy is a type of disease that causes abnormal brain and skeletal muscle function due to abnormal mitochondrial structure caused by the deletion or mutation of mitochondrial genes or nuclear genes. The disease is clinically similar to epilepsy, cerebral infarction, encephalitis, and brain dysplasia, and is a type of disease that seriously affects the health and life of patients. Since mitochondria are one of the most sensitive organelles to various damages, pathological studies have shown that they are attacked by oxygen free radicals, resulting in abnormal function, which in turn affects the central nervous system, thus forming mitochondrial encephalomyopathy.

[0005] The "Notice on the Announcement of the First List of Rare Diseases" (National Health and Medical Development

[2018] No. 10) issued by my country clearly stipulates that amyotrophic lateral sclerosis and mitochondrial encephalomyopathy are rare diseases in my country (also known as orphan diseases). According to incomplete statistics, there are currently about 100,000 to 200,000 people suffering from ALS in my country, and drugs that can cure the disease have not yet been successfully developed. The incidence of mitochondrial encephalomyopathy in adults is about 1 / 4300, and the incidence in children is as high as 1 / 2000. Therefore, the development of drugs for such rare diseases is a common goal of scientists in the biomedical field in my country and even the world, and one of the major research topics in my country's medical and health fields.

[0006] Chinese patent application CN201810959516.7 discloses a series of substituted pyrazole compounds, and tests on stability, solubility, cytotoxicity, and in vitro and in vivo efficacy of some compounds, wherein Example 33 discloses the D-glucoside of 3-methyl-1-phenyl-1H-pyrazole-5-ol (as shown below) and its preparation method, and Test Examples 1-6 disclose relevant effect data, but the specification of the application does not clearly disclose or imply the absolute configuration of the glycosidic bond in the compound structure. Considering that glycoside compounds of different configurations may exhibit differences in physical and chemical properties (e.g., stability, solubility), or even biological activity, it is necessary to conduct relevant research to provide a basis for subsequent drug development.

[0007] Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In view of the fact that there is currently no single-configuration (or single-configuration-enriched) 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside, the present invention hopes to carry out relevant developments in order to provide products with improved physicochemical properties and / or biological activities, as well as preparation methods and applications thereof.

[0010] Solutions for solving problems

[0011] In the first aspect, the present invention provides a compound as shown in formula I

[0012]

[0013] or a pharmaceutically acceptable salt, ester, solvate, isotope-labeled, active metabolite or prodrug thereof;

[0014] or,

[0015] The compound or its pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug enrichment, wherein the content of the compound or its pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug is above 85wt%.

[0016] In a second aspect, the present invention provides a method for preparing the compound as shown in Formula I, comprising the following steps:

[0017] 1) reacting compound a with compound b to obtain intermediate 1;

[0018]

[0019] 2) reacting intermediate 1 with compound c to obtain intermediate 2;

[0020]

[0021] 3) splitting intermediate 2 to obtain intermediate 2A and intermediate 2B;

[0022]

[0023] 4) Deprotect the intermediate 2B to obtain;

[0024]

[0025] Alternatively, step 3) and step 4) are replaced by step 3') and step 4'): 3') deprotecting intermediate 2 to obtain intermediate 3;

[0026]

[0027] 4') split the intermediate 3 to obtain;

[0028]

[0029] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound as shown in Formula I above or a pharmaceutically acceptable salt, ester, solvate, isotope-labeled substance, active metabolite or prodrug or the enrichment thereof.

[0030] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0031] In a fourth aspect, the present invention provides the use of the above-mentioned compound as shown in Formula I or its pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug or the above-mentioned enrichment or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating cerebral stroke, cerebral embolism, sequelae of stroke, cerebral motor dysfunction, mitochondrial encephalomyopathy and / or amyotrophic lateral sclerosis.

[0032] Effects of the Invention

[0033] For 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside, the present invention has developed a preparation method capable of obtaining two single diastereomers, and conducted a comparative study on them. The results show that although the two single isomers and the mixture of the two exhibit excellent performance in terms of solution stability, it is surprisingly found that the two isomers have significant differences in solubility (the solubility of the β-isomer in different solvents is higher than that of the α-isomer, and is about 3 times that of the α-configuration isomer). In addition, through in vitro activity experiments, it is found that the activities of the two single isomers also have significant differences (the β-isomer can significantly improve the survival rate of cells, while the effect of the α-isomer is relatively weak). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the HPLC spectrum of two isomers under the chromatographic conditions of the present invention.

[0035] Figure 2 Schematic diagram of the in vitro neuroprotective effects of the two isomers and their mixture, where ## indicates p<0.01 compared with Con; * indicates p<0.05 compared with OGD. DETAILED DESCRIPTION

[0036] Definition of terms

[0037] Unless otherwise specified, the term "glucoside" used in the present invention refers to an acetal or ketal compound obtained by glycosidation reaction of non-sugar substances (e.g., alcohols, phenols) with glucose. Further, the term "D-glucoside" refers to an acetal or ketal compound obtained by glycosidation reaction of non-sugar substances with D-glucose.

[0038] Unless otherwise specified, the term "diastereomer" used in the present invention refers to a pair of diastereomers with only one asymmetric atom having different configurations in a molecule containing two or more asymmetric atoms. Further, the term "end group diastereomer" refers to an isomer in which the above-mentioned asymmetric atoms with different configurations are located at the end of the main chain structure, for example, α-D-glucoside and β-D-glucoside are end group diastereomers of each other, and the 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside of the present invention contains both of the above-mentioned end group diastereomers.

[0039] Unless otherwise indicated, the term "resolution" as used herein refers to the process of separating and purifying isomers (eg, anomers) in a mixture.

[0040] Unless otherwise specified, the term "pharmaceutically acceptable salt" used in the present invention refers to a salt compound obtained by a free compound reacting with an acid or a base through a salt-forming reaction, and the salt compound can be used normally physiologically or pharmacologically and will not produce unexpected adverse reactions to the subject. Among them, acids suitable for salt formation include inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids, such as formic acid, acetic acid, tartaric acid and citric acid; bases suitable for salt formation include inorganic bases, such as ammonia water, sodium hydroxide, potassium hydroxide and calcium hydroxide, and organic bases, such as triethylamine and diisopropylethylamine.

[0041] Unless otherwise specified, the term "ester" used in the present invention refers to a derivative compound obtained by esterification reaction between a hydroxyl group (or carboxyl group) in a parent compound and a carboxyl group in a carboxylic acid (or a hydroxyl group in an alcohol). Acids suitable for forming esters include formic acid, acetic acid, trifluoroacetic acid, etc.; alcohols suitable for forming esters include methanol, ethanol, and benzyl alcohol.

[0042] Unless otherwise specified, the term "solvate" used in the present invention refers to a derivative compound obtained by solvation between a parent compound and a solvent molecule. Common solvates include hydrates, methanolates, ethanolates, and the like.

[0043] Unless otherwise specified, the term "isotope-labeled compound" used in the present invention refers to a derivative compound obtained by replacing a specific atom in a parent compound with its isotope atom. Common isotope atoms include D( 2 H), T( 3 H), 11 C. 13 C. 14 C, etc.

[0044] Unless otherwise specified, the term "active metabolite" used in the present invention refers to a derivative compound having the same or similar activity that is obtained after the parent compound is administered to a subject through metabolism in vivo.

[0045] Unless otherwise indicated, the term "prodrug" as used herein refers to a derivative compound having improved properties (eg, pharmacokinetic properties) obtained by modifying the structure of a parent compound.

[0046] Unless otherwise specified, the term "enriched product" used in the present invention refers to a composition in which one component is absolutely dominant and this component inevitably coexists with other components or impurities.

[0047] Unless otherwise specified, the term "pharmaceutically acceptable excipients" used in the present invention refers to materials that are used in conjunction with the active pharmaceutical ingredient (API) and play an auxiliary role in the process of preparing pharmaceutical preparations, and the materials can be used normally physiologically or pharmacologically and will not produce unexpected adverse reactions to the subjects. Common excipients include (but are not limited to) diluents (or fillers), binders, disintegrants, lubricants, glidants, wetting agents, surfactants, colorants, olfactory agents, flavoring agents, pH adjusters, osmotic pressure regulators, preservatives, solvents, cosolvents, solubilizers, etc.

[0048] 3-Methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside

[0049] In one embodiment, the 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside of the present invention may exist in a single configuration, for example, may be a β-isomer.

[0050] In a specific embodiment, the 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside of the present invention can be represented by Formula I:

[0051]

[0052] In one embodiment, the 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside of the present invention may exist in a single configuration-enriched form, for example, may be a β-isomer-enriched form.

[0053] In a specific embodiment, the content of the β-isomer in the enrichment of the present invention may be above 85 wt %, for example, above 85 wt %, 90 wt %, 95 wt % or 99 wt %.

[0054] In a specific embodiment, the content of the β-isomer in the enrichment of the present invention may be above 90 wt%, for example, above 90 wt%, 92 wt%, 95 wt% or 99 wt%.

[0055] In a specific embodiment, the content of β-isomer in the enrichment of the present invention may be above 95 wt%, for example, above 95 wt%, 96 wt%, 97 wt%, 98 wt% or 99 wt%.

[0056] In a specific embodiment, the content of β-isomer in the enrichment of the present invention may be above 98 wt%, for example, 98 wt%, 98.5 wt%, 99 wt% or above 99.5 wt%.

[0057] In a specific embodiment, the content of β-isomer in the enrichment of the present invention may be above 99 wt%, for example, above 99 wt%, 99.3 wt%, 99.6 wt% or 99.9 wt%.

[0058] Preparation method of 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside

[0059] In one embodiment, the 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside (compound of formula I) of the present invention can be prepared by method 1 comprising the following steps:

[0060] 1) reacting compound a with compound b to obtain intermediate 1;

[0061]

[0062] 2) reacting intermediate 1 with compound c to obtain intermediate 2;

[0063]

[0064] 3) splitting intermediate 2 to obtain intermediate 2A and intermediate 2B;

[0065]

[0066] 4) Deprotect the intermediate 2B to obtain;

[0067]

[0068] In one embodiment, the 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside (compound of formula I) of the present invention can be prepared by method 2 comprising the following steps:

[0069] 1) reacting compound a with compound b to obtain intermediate 1;

[0070]

[0071] 2) reacting intermediate 1 with compound c to obtain intermediate 2;

[0072]

[0073] 3) Deprotecting intermediate 2 to obtain intermediate 3;

[0074]

[0075] 4) The intermediate 3 is split to obtain;

[0076]

[0077] In a specific embodiment, in step 1) of method 1 or method 2, the molar ratio of compound a to compound b can be 1:1.5-1:3.0, for example 1:1.5, 1:1.8, 1:2.1, 1:2.4, 1:2.7, 1:3.0, etc.

[0078] In one specific embodiment, in step 1) of method 1 or method 2, the reaction temperature can be room temperature.

[0079] In a specific embodiment, in step 1) of method 1 or method 2, the reaction time may be 2-3 hours.

[0080] In one specific embodiment, in step 1) of method 1 or method 2, the reaction may be carried out in a solvent.

[0081] In a specific embodiment, in step 1) of method 1 or method 2, the solvent may be a halogenated hydrocarbon, such as dichloromethane, chloroform, dichloroethane, and the like.

[0082] In one specific embodiment, in step 1) of method 1 or method 2, the reaction may be carried out in the presence of a base.

[0083] In a specific embodiment, in step 1) of method 1 or method 2, the base can be an inorganic base, such as potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, sodium hydride, etc., or can be an organic base, such as triethylamine, diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc.

[0084] In a specific embodiment, in step 1) of method 1 or method 2, the molar ratio of compound a to the base can be 1:0.05-1:0.5, for example 1:0.05, 1:0.08, 1:0.1, 1:0.2, 1:0.5, etc.

[0085] In a specific embodiment, in step 2) of method 1 or method 2, the molar ratio of intermediate 1 to compound c can be 1:1.0-1:1.5, for example 1:1.0, 1:1.01, 1:1.02, 1:1.05, 1:1.1, 1:1.2, 1:1.5, etc.

[0086] In one specific embodiment, in step 2) of method 1 or method 2, the reaction temperature can be room temperature.

[0087] In a specific embodiment, in step 2) of method 1 or method 2, the reaction time can be 2-3 h, for example, 2, 2.5, 3 h, etc.

[0088] In one specific embodiment, in step 2) of method 1 or method 2, the reaction may be carried out in a solvent.

[0089] In a specific embodiment, in step 2) of method 1 or method 2, the solvent may be a halogenated hydrocarbon, such as dichloromethane, chloroform, dichloroethane, and the like.

[0090] In a specific embodiment, in step 2) of method 1 or method 2, the solvent may be an ether, such as diethyl ether.

[0091] In one specific embodiment, in step 2) of method 1 or method 2, the solvent may be a nitrile, such as acetonitrile.

[0092] In one specific embodiment, in step 2) of method 1 or method 2, the reaction may be carried out in the presence of a catalyst.

[0093] In a specific embodiment, in step 2) of method 1 or method 2, the catalyst may be a Lewis acid, such as boron trifluoride ethyl etherate, trimethylsilyl trifluoromethanesulfonate, and the like.

[0094] In one specific embodiment, in step 2) of method 1 or method 2, the amount of the catalyst used can be a catalytic amount, for example, 5% to 10% mol.

[0095] In one specific embodiment, in step 2) of method 1 or method 2, the reaction may be carried out in the presence of a drying agent.

[0096] In a specific embodiment, in step 2) of method 1 or method 2, the desiccant may be 4A molecular sieve.

[0097] In a specific embodiment, in step 4) of method 1 or step 3) of method 2, the deprotection may be performed by catalytic hydrogenation.

[0098] In one specific embodiment, in step 4) of method 1 or step 3) of method 2, the catalyst for catalytic hydrogenation can be a palladium catalyst, such as palladium carbon (Pd / C), preferably palladium carbon (e.g. 5wt%, 7.5wt%, 10wt%, etc.).

[0099] In one specific embodiment, in step 4) of method 1 or step 3) of method 2, the amount of the catalyst used may be a catalytic amount.

[0100] In one specific embodiment, in step 4) of method 1 or step 3) of method 2, the temperature of catalytic hydrogenation can be 20-50°C, for example, 20, 25, 30, 35, 40, 45, 50°C, etc.

[0101] In one specific embodiment, in step 4) of method 1 or step 3) of method 2, the time of catalytic hydrogenation can be 0.5-3 h, for example 0.5, 1, 1.5, 2, 2.5, 3 h, etc.

[0102] In one specific embodiment, in step 4) of method 1 or step 3) of method 2, the catalytic hydrogenation may be carried out in a solvent.

[0103] In a specific embodiment, in step 4) of method 1 or step 3) of method 2, the solvent may be an ester, such as ethyl acetate (EtOAc), methyl acetate (MeOAc), isopropyl acetate (iPrOAc), and the like.

[0104] In a specific embodiment, in step 4) of method 1 or step 3) of method 2, the solvent may be an alcohol, such as methanol, ethanol, and the like.

[0105] In a specific embodiment, in step 3) of method 1 or step 4) of method 2, the resolution can be performed by chromatography.

[0106] In a specific embodiment, in step 3) of method 1, the resolution can be performed by column chromatography (CC).

[0107] In a specific embodiment, in step 3) of method 1, the stationary phase (or support) of column chromatography can be at least one of alumina, silica gel, polyamide and magnesium silicate.

[0108] In a specific embodiment, in step 3) of method 1, the stationary phase of the column chromatography can be silica gel, for example, silica gel with a mesh size of 100-400 mesh, preferably 100-200 mesh.

[0109] In a specific embodiment, in step 3) of method 1, the amount of the stationary phase of the column chromatography can be adjusted according to the amount of the substance to be separated, for example, 10-50 times, preferably 15-20 times the weight of the substance to be separated.

[0110] In a specific embodiment, in step 3) of method 1, the mobile phase (or eluent) of column chromatography can be at least one of esters (e.g., ethyl acetate), halogenated hydrocarbons (e.g., dichloromethane, chloroform, etc.), ethers (e.g., tetrahydrofuran), alcohols (e.g., methanol), ketones (e.g., acetone), aliphatic hydrocarbons (e.g., petroleum ether, n-hexane, etc.), and aromatic hydrocarbons (e.g., benzene, toluene, xylene, etc.).

[0111] In a specific embodiment, in step 3) of method 1, the mobile phase of the column chromatography may be a mixture of two of esters, halogenated hydrocarbons, ethers, alcohols, ketones, aliphatic hydrocarbons and aromatic hydrocarbons.

[0112] In a specific embodiment, in step 3) of method 1, the mobile phase of the column chromatography may be a mixture of three of esters, halogenated hydrocarbons, ethers, alcohols, ketones, aliphatic hydrocarbons and aromatic hydrocarbons.

[0113] In a specific embodiment, in step 3) of method 1, the mobile phase of the column chromatography may be a mixture of esters and aliphatic hydrocarbons.

[0114] In a specific embodiment, in step 3) of method 1, the mobile phase of the column chromatography can be a mixture of ethyl acetate and petroleum ether, for example, a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:5-1:10.

[0115] In a specific embodiment, in step 4) of method 2, the resolution can be performed by high performance liquid chromatography (HPLC).

[0116] In a specific embodiment, in step 4) of method 2, the separation can be performed by preparative high performance liquid chromatography (Prep-HPLC).

[0117] In a specific embodiment, in step 4) of method 2, the stationary phase of the HPLC (or preparative HPLC) may be octadecylsilane bonded silica gel; in other words, the chromatographic column of the HPLC or preparative HPLC may be C 18 Chromatographic column, preferably C 18 Preparative or semi-preparative columns, such as UltimatePlus C 18 Column. Depending on the specific conditions of the sample to be prepared, you can also choose columns of different specifications, such as UltimatePlus C 18 Semi-preparative columns (5μm, 10*250nm), Ultimate Plus C 18 Preparative chromatographic column (5μm, 21.2*250nm), Ultimate Plus C 18 Preparative chromatographic column (5μm, 30*150nm), Ultimate Plus C 18 Prepare chromatographic columns (5μm, 30*250nm), etc.

[0118] In a specific embodiment, in step 4) of method 2, the mobile phase of the high performance liquid chromatography (or preparative high performance liquid chromatography) may be a binary mobile phase system.

[0119] In a specific embodiment, in step 4) of method 2, the binary mobile phase system may consist of a weakly acidic buffered saline solution as the aqueous phase and an alcohol or nitrile as the organic phase.

[0120] In a specific embodiment, in step 4) of method 2, the weakly acidic buffered saline solution can be an aqueous solution of a phosphate buffer system (e.g., phosphoric acid-sodium / ammonium phosphate, phosphoric acid-disodium / ammonium hydrogen phosphate, sodium / ammonium dihydrogen phosphate-sodium / ammonium phosphate, etc.).

[0121] In a specific embodiment, in step 4) of method 2, the pH value of the weakly acidic buffered saline solution can be 6.0-6.9, for example, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, etc.

[0122] In one specific embodiment, in step 4) of method 2, the alcohol may be C 1-4 Aliphatic alcohols, such as methanol, ethanol, isopropanol, etc., preferably methanol.

[0123] In one specific embodiment, in step 4) of method 2, the nitrile may be C 2-4 Aliphatic nitrile, preferably acetonitrile.

[0124] In a specific embodiment, in step 4) of method 2, the elution mode of the high performance liquid chromatography (or preparative high performance liquid chromatography) can be isocratic elution.

[0125] In a specific embodiment, in step 4) of method 2, the volume ratio of the aqueous phase to the organic phase used in the isocratic elution can be 80:20-95:5, for example 80:20, 85:25, 90:10, 95:5, etc.

[0126] In a specific embodiment, in step 4) of method 2, the detection wavelength of the HPLC (or preparative HPLC) can be 240-250 nm, preferably 245 nm.

[0127] In a specific embodiment, in step 4) of method 2, the flow rate of HPLC (or preparative HPLC) can be 1-20 mL / min, for example 1, 5, 10, 12, 13, 15, 18, 20 mL / min, etc.

[0128] In a specific embodiment, in step 4) of method 2, the column temperature of HPLC (or preparative HPLC) can be 20-45° C., for example, 20, 25, 30, 35, 40, 45° C., etc., preferably 25-35° C. Usually, it can be carried out at room temperature or at the ambient temperature during detection.

[0129] In a specific embodiment, in step 4) of method 2, the injection volume of HPLC (or preparative HPLC) can be adjusted according to the specific conditions of the sample to be prepared, for example, using an injection volume of 1 to 5 mL, preferably 2 mL.

[0130] Pharmaceutical composition containing 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside

[0131] In one embodiment, the pharmaceutical composition of the present invention may comprise 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside.

[0132] In one embodiment, the pharmaceutical composition of the present invention may comprise an enrichment of 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside, wherein the content of the β-isomer may be above 85wt%, for example, 85wt%, 90wt%, 95wt% or 99wt% or more, preferably above 90wt%, for example, 90wt%, 92wt%, 95wt% or 99wt% or more, more preferably above 95wt%, for example, 95wt%, 96wt%, 97wt%, 98wt% or 99wt% or more, further preferably above 98wt%, for example, 98wt%, 98.5wt%, 99wt% or 99.5wt% or more, further preferably above 99wt%, for example, 99wt%, 99.3wt%, 99.6wt% or 99.9wt% or more.

[0133] In a specific embodiment, the pharmaceutical composition of the present invention comprises a preventive and / or therapeutically effective amount of 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside or an enrichment thereof.

[0134] In one embodiment, in addition to 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside or its enrichment, the pharmaceutical composition of the present invention may also contain one or more pharmaceutically acceptable excipients, including but not limited to diluents, binders, disintegrants, lubricants, glidants, wetting agents, surfactants, colorants, olfactory agents, flavoring agents, pH regulators, osmotic pressure regulators, preservatives, solvents, solubilizing agents, solubilizing agents, etc.

[0135] In one embodiment, the pharmaceutical composition of the present invention can be administered orally, for example, the pharmaceutical composition of the present invention can be in the form of tablets, granules, capsules, pills, solutions or syrups.

[0136] In one embodiment, the pharmaceutical composition of the present invention can be administered parenterally, for example, the pharmaceutical composition of the present invention can be in the form of injection, infusion or lyophilized powder injection.

[0137] Medical Uses

[0138] Whether it is the 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside of the present invention, or its enrichment, or the pharmaceutical composition containing it or its enrichment, it can be prepared into a medicine.

[0139] In one embodiment, the medicament of the present invention can be used for preventing and / or treating a disease.

[0140] In a specific embodiment, the drug of the present invention can be used to prevent and / or treat at least one of the following diseases: cerebral stroke, cerebral embolism, sequelae of stroke, cerebral motor dysfunction, mitochondrial encephalomyopathy and amyotrophic lateral sclerosis.

[0141] In a specific embodiment, the medicament of the present invention can be used to prevent and / or treat at least one of the following diseases: stroke, mitochondrial encephalomyopathy and amyotrophic lateral sclerosis.

[0142] In a specific embodiment, the medicament of the present invention can be used for preventing and / or treating stroke.

[0143] In a specific embodiment, the medicament of the present invention can be used for preventing and / or treating mitochondrial encephalomyopathy.

[0144] In a specific embodiment, the medicament of the present invention can be used for preventing and / or treating amyotrophic lateral sclerosis.

[0145] The present invention will be further described below in conjunction with specific embodiments. These embodiments should be understood to be only used to illustrate the present invention, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make corresponding modifications after referring to the contents described in the present invention, and the contents of these equivalent modifications also fall into the scope of protection of the present invention.

[0146] Comparative Example: Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside (mixture of two isomers)

[0147] Referring to the preparation method 2 described in Example 33 in CN201810959516.7, the specific steps are as follows:

[0148] (1) Preparation of 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (Intermediate 1):

[0149]

[0150] 2,3,4,6-O-tetrabenzyl-D-glucose (5 g, 9.26 mmol) and DBU (0.14 g, 0.92 mmol) were dissolved in dichloromethane (60 mL), and trichloroacetonitrile (3.2 g, 22.2 mmol) was added dropwise under stirring. The mixture was reacted at room temperature for 2-3 h. TLC (petroleum ether / ethyl acetate = 5:1, v / v) showed that the reaction was complete. The solvent was removed by concentration under reduced pressure and the mixture was used directly in the next step.

[0151] (2) Preparation of 1-O-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (Intermediate 2):

[0152]

[0153] Edaravone (3-methyl-1-phenyl-1H-pyrazole-5-ol) (1.3 g, 7.47 mmol), 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (5 g, 7.32 mmol) and 4A molecular sieves were added to dichloromethane, TMSOTf (0.45 mmol) was added dropwise under stirring, and stirred at room temperature for 3 h. TLC showed that the reaction was complete. The solvent was removed under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1, v / v) to obtain 2.4 g of a light yellow solid with a yield of 47.1%.

[0154] (3) Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-OD-glucoside (target product):

[0155]

[0156] 1-O-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (1 g, 1.44 mmol) was dissolved in ethyl acetate (100 mL), Pd / C (7.5 wt%) was added, hydrogenated at 45°C for 1 h, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (dichloromethane / methanol=20:1, v / v) gave 250 mg of a white solid, with a yield of 51.6%.

[0157] Example 1: Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside

[0158] This example adopts the preparation method including three steps in the comparative example, the difference being that a column chromatography separation step is added between step (2) and step (3), so as to utilize the intermediate 2 having a single configuration to carry out the deprotection reaction in step (3), thereby obtaining the target product having a single configuration accordingly. The specific steps are as follows:

[0159] (1) Preparation of 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (Intermediate 1):

[0160]

[0161] 2,3,4,6-O-tetrabenzyl-D-glucose (5 g, 9.26 mmol) and DBU (0.14 g, 0.92 mmol) were dissolved in dichloromethane (60 mL), and trichloroacetonitrile (3.2 g, 22.2 mmol) was added dropwise under stirring. The mixture was reacted at room temperature for 2-3 h. TLC (petroleum ether / ethyl acetate = 5:1, v / v) showed that the reaction was complete. The solvent was removed by concentration under reduced pressure and the mixture was used directly in the next step.

[0162] (2) Preparation of 1-O-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (Intermediate 2):

[0163]

[0164] Edaravone (3-methyl-1-phenyl-1H-pyrazole-5-ol) (1.3 g, 7.47 mmol), 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (5 g, 7.32 mmol) and 4A molecular sieves were added to dichloromethane, TMSOTf (0.45 mmol) was added dropwise under stirring, and stirred at room temperature for 3 h. TLC showed that the reaction was complete. The solvent was removed under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1, v / v) to obtain 2.4 g of a light yellow solid with a yield of 47.1%.

[0165] The intermediate 2 was separated using silica gel (100-200 mesh) with a weight 15-20 times that of the sample loading, and the eluent was petroleum ether / ethyl acetate (5:1-10:1, v / v), to obtain intermediate 2 of two single configurations, α-type and β-type, respectively.

[0166] (3) Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-O-α-D-glucoside and 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside:

[0167]

[0168] Two configurations of 1-O-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (1 g, 1.44 mmol) were dissolved in ethyl acetate (100 mL), Pd / C (7.5 wt%) was added, hydrogenation reaction was carried out at 45°C for 1 h, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (dichloromethane / methanol = 20:1, v / v) gave a white solid, 250 mg of the α-isomer, a yield of 51.6%, and a purity of 98.8% by HPLC; 200 mg of the β-isomer, a yield of 41.3%, and a purity of 99.0% by HPLC.

[0169] The NMR data of the two isomers are as follows:

[0170] (1) α-isomer:

[0171] 1 H-NMR (400MHz, DMSO-d6): δ7.784(d,2H,J=8.0),7.428(t,2H,J=8.0),7.253(t,1H,J=8.0),5.848(s,1H),5.426(d,1H,J=2.8,1-H),5.38 7(d,1H,J=4.8),5.087(d,1H,J=4.8),5.031(d,1H,J=5.6),4.538(t, 1H,J=5.6),4.090(q,1H,J=5.6),3.606-3.327(m,5H),2.161(s,3H).

[0172] (2) β-isomer:

[0173] 1 H-NMR (400MHz, DMSO-d6): δ7.734(d,2H,J=7.8),7.428(t,2H,J=7.8),7.25 8(t,1H,J=7.8),5.844(s,1H),5.546(d,1H,J=5.4),5.175(d,1H,J=4.8),5. 103(d,1H,J=5.4),4.947(d,1H,J=7.2,1-H),4.612(t,1H,J=5.4),3.685(m, 1H),3.466(m,1H),3.337(m,1H),3.257(m,2H),3.162(m,1H),2.160(s,3H).

[0174] Example 2: Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside

[0175] This example adopts the preparation method including three steps in the comparative example, the difference being that a preparative liquid chromatography separation step is added after step (3) to obtain a target product having a single configuration. The specific steps are as follows:

[0176] (1) Preparation of 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (Intermediate 1):

[0177]

[0178] 2,3,4,6-O-tetrabenzyl-D-glucose (5 g, 9.26 mmol) and DBU (0.14 g, 0.92 mmol) were dissolved in dichloromethane (60 mL), and trichloroacetonitrile (3.2 g, 22.2 mmol) was added dropwise under stirring. The mixture was reacted at room temperature for 2-3 h. TLC (petroleum ether / ethyl acetate = 5:1, v / v) showed that the reaction was complete. The solvent was removed by concentration under reduced pressure and the mixture was used directly in the next step.

[0179] (2) Preparation of 1-O-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (Intermediate 2):

[0180]

[0181] Edaravone (3-methyl-1-phenyl-1H-pyrazole-5-ol) (1.3 g, 7.47 mmol), 2,3,4,6-O-tetrabenzyl-1-O-trichloroimino-D-glucose (5 g, 7.32 mmol) and 4A molecular sieves were added to dichloromethane, TMSOTf (0.45 mmol) was added dropwise under stirring, and stirred at room temperature for 3 h. TLC showed that the reaction was complete. The solvent was removed under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1, v / v) to obtain 2.4 g of a light yellow solid with a yield of 47.1%.

[0182] (3) Preparation of 3-methyl-1-phenyl-1H-pyrazole-5-O-α-D-glucoside and 3-methyl-1-phenyl-1H-pyrazole-5-O-β-D-glucoside:

[0183]

[0184] 1-O-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-2,3,4,6-O-tetrabenzyl-D-glucose (1 g, 1.44 mmol) was dissolved in ethyl acetate (100 mL), Pd / C (7.5 wt%) was added, and the mixture was hydrogenated at 45°C for 1 h. The mixture was filtered and the solvent was removed under reduced pressure to give 350 mg of a white solid (intermediate 3, mixed configuration) with a yield of 72.3%.

[0185] The obtained sample is prepared into a 1 mg / mL solution with methanol (for example, when manually injected, the injection volume is 2 mL), and the isomers are separated by a preparative liquid chromatography column. When the prepared component begins to emit a signal, start to manually take it from the detector outlet until the signal returns to 0, and the sample preparation is completed in a cycle. Using 30 mg of the mixed configuration sample, 10 mg of a single β-isomer can be prepared, with an HPLC purity of 99.8%; 15 mg of a single α-isomer can be prepared, with an HPLC purity of 99.6%.

[0186] The specific chromatographic conditions are as follows:

[0187] Column: Ultimate Plus C 18 Column (21.2*250mm, 5μm);

[0188] Mobile phase: Mobile phase A is 0.01 mol / L diammonium hydrogen phosphate aqueous solution (adjusted to pH = 6.3 by adding phosphoric acid), and mobile phase B is acetonitrile;

[0189] Elution method: isocratic elution, V 流动相A :V 流动相B =90:10;

[0190] Flow rate: 13 mL / min;

[0191] Column temperature: no control (ambient temperature during testing is acceptable);

[0192] Detection wavelength: 245nm;

[0193] Single injection volume: 2mL.

[0194] Test Example 1: Comparison of solubility of different isomers

[0195] This test example is designed to test the solubility of two isomers in water and buffer solutions of different pH values. The buffer solution is a potassium dihydrogen phosphate system, with phosphoric acid or potassium hydroxide as the acid-base regulator. The specific operation is as follows:

[0196] At room temperature (25-30°C), 5 mg of each sample was weighed and the solvent was gradually added under shaking until the sample was completely dissolved. The volume of solvent used was recorded and converted into the amount of sample dissolved in each mL of solvent as the solubility data of the sample. The results are shown in Table 1.

[0197] Table 1. Solubility results of different isomers in various solvents

[0198] Solubility (mg / mL) α configuration β configuration Hybrid Configuration Purified water 2.4 6.3 3.3 pH 4.0 buffer solution 1.6 4.5 2.5 pH 6.8 buffer solution 1.7 5.2 2.8 pH 7.4 buffer solution 1.9 5.6 2.8

[0199] The experimental results show that the solubility of the β-isomer in different solvents is higher than that of the α-isomer, and surprisingly, it is about 3 times that of the α-configuration isomer. Excellent solubility is a prerequisite for the preparation of injection preparations. Under the same dosage volume, the β-isomer can achieve a dosage of about 3 times that of the α-isomer, which can reduce the frequency of administration; in other words, under the same dosage, compared with the α-isomer, the β-isomer can use only 1 / 3 of the volume of the solvent system. Regardless of which aspect is considered, the use of injection preparations with the β-isomer can greatly improve the subject's experience and improve medication compliance.

[0200] For substances with low solubility in water (e.g., compounds of the present invention that are slightly soluble in water), the difference in solubility has a more important impact on the development of pharmaceutical preparations (especially injections). Too low solubility may limit the specifications of the preparation, and the effective dose cannot be achieved when the administration volume is fixed, thereby leading to the failure of preparation development.

[0201] Test Example 2: Comparison of solution stability of different isomers

[0202] This test example is designed to test the stability of the two isomers in water and buffer solutions of different pH values. The buffer solution is a potassium dihydrogen phosphate system, with phosphoric acid or potassium hydroxide as the acid-base regulator. The specific operation is as follows:

[0203] Two samples to be tested were weighed separately, and solvents were added to prepare sample solutions with a concentration of 1 mg / mL. The stability of the samples in the solution state was detected by HPLC. The results are shown in Table 2.

[0204] Table 2. Stability results of different isomers in various solvents

[0205] Stability (1mg / mL) α configuration β configuration Hybrid Configuration Purified water Sample stability, ≥24 hours Sample stability, ≥24 hours Sample stability, ≥24 hours pH 4.0 buffer solution Sample stability, ≥24 hours Sample stability, ≥24 hours Sample unstable, <6.5 hours pH 6.8 buffer solution Sample stability, ≥24 hours Sample stability, ≥24 hours Sample stability, ≥24 hours pH 7.4 buffer solution Sample stability, ≥24 hours Sample stability, ≥24 hours Sample stability, ≥24 hours

[0206] Note: Sample stability means no new impurities or the total amount of new impurities is less than 0.5%

[0207] The experimental results showed that the mixed configuration sample had low stability in pH 4.0 buffer solution, with detectable new impurities appearing after 6.5 hours. However, the individual α-configuration isomer and β-configuration isomer had good stability in different solutions and had the potential to be developed into injectable preparations.

[0208] Experimental Example 3: Comparison of the protective effects of different isomers on neuronal hypoxia and glucose deprivation injury

[0209] 1. Experimental Materials

[0210] 1) Cells

[0211] Human neuroblastoma cells (SH-SY5Y) were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Culture conditions: 37°C, 5% CO2, RPMI Medium 1640 culture medium containing 15% inactivated fetal bovine serum.

[0212] 2) Drugs and reagents

[0213]

[0214] 3) Instruments

[0215]

[0216] 4) Abbreviations

[0217] Sugar-containing culture medium: RPMI Medium 1640

[0218] Sugar-free medium: RPMI Medium 1640 sugar-free medium

[0219] Complete culture medium: RPMI Medium 1640 containing 15% inactivated fetal bovine serum

[0220] 2. Test methods

[0221] 1) Sample preparation:

[0222] Three samples (sample 1 is a mixture of two isomers with a mixing ratio of 1:1; sample 2 is the α-isomer; sample 3 is the β-isomer) were first dissolved to 1 mM with PBS, then diluted 200 times with sugar-free culture medium (anoxic conditions) or complete culture medium (reoxygenation conditions) to a final concentration of 300 μM, and then diluted with corresponding culture medium to form sample solutions with different concentration gradients for later use.

[0223] 2) Cell culture:

[0224] When human neuroblastoma cells (SH-SY5Y cells) grew to 80%, they were seeded in a 96-well plate at a cell seeding density of 3×10 4 After 24 h of culture, replace with complete medium at 150 μL / well.

[0225] 3) Construction of hypoxia model:

[0226] When SH-SY5Y cells grew to 80%, they were plated and the cell density was about 3 × 10 4 / well, 100μL / well. After culturing for 24h, the upper culture medium was discarded. The model group (OGD) group was replaced with sugar-free culture medium. The sample group was given the above-mentioned samples of different concentrations (prepared with sugar-free culture medium). After adding the drug, the 96-well plate was placed in an anoxic chamber, filled with 100% N2 at a flow rate of 20LPM for 10min, and sealed at 37℃ for 6h; then 150μL of complete culture medium containing the drug was added to the sample group, and an equal volume of complete culture medium without the drug was added to the OGD group, and reoxygenated at 37℃, 5% CO2 for 16h.

[0227] 4) MTT assay:

[0228] After reoxygenation, the 96-well plate was removed, and MTT solution (15 μL / well) was added. After incubation at 37°C for 4 h, the supernatant was carefully aspirated, and DMSO (150 μL / well) was added. The plate was shaken on a shaker for 10 min. The absorbance (OD) value was detected at 490 nm using an enzyme reader, and the cell survival rate was calculated based on the measured OD value.

[0229] Cell survival rate % = (OD drug group - OD blank group) / (OD control group - OD blank group) × 100%

[0230] 3. Experimental results:

[0231] Table 3. Results of neuronal cell survival after different sample treatments

[0232]

[0233] Note: OGD is the model group; Con is the blank group.

[0234] The experimental results showed that after incubation of SH-SY5Y cells with samples of different concentrations for 6 hours of hypoxia and 16 hours of reoxygenation, the cell viability of the model group was significantly reduced compared with the blank group (p<0.01). 300μM sample 1 (isomer mixture) and sample 3 (β-isomer) could significantly increase the cell survival rate (p<0.05) and improve hypoxia-glucose deprivation damage, while the effect of sample 2 (α-isomer) was relatively weak.

[0235] In addition, unexpectedly, the β-isomer and the mixture showed a certain dose dependence within the measured concentration range, while the in vitro neuroprotective effect of the α-isomer showed a negative correlation with the concentration, suggesting that splitting the two isomers for use may help improve the safety and efficacy of medication.

Claims

1. The compound shown in formula I or a pharmaceutically acceptable salt, ester, solvate, isotope-labeled substance, active metabolite or prodrug thereof.

2. An enrichment of a compound as shown in formula I according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug thereof, wherein the content of the compound or its pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug is above 85wt%, preferably above 85wt%, 90wt%, 95wt% or 99wt%.

3. A method for preparing the compound of formula I according to claim 1, comprising the following steps: 1) reacting compound a with compound b to obtain intermediate 1; 2) reacting intermediate 1 with compound c to obtain intermediate 2; 3) splitting intermediate 2 to obtain intermediate 2A and intermediate 2B; 4) Deprotect the intermediate 2B to obtain; Alternatively, step 3) and step 4) are replaced by step 3') and step 4'): 3') deprotecting intermediate 2 to obtain intermediate 3; 4') split the intermediate 3 to obtain; 4. The preparation method according to claim 3, characterized in that: In step 1), The molar ratio of the compound a to the compound b is 1:1.5-1:3.0; and / or The reaction is carried out in a solvent; and / or The reaction is carried out in the presence of a base; preferably, the molar ratio of the compound a to the base is 1:0.05-1:0.

5.

5. The preparation method according to claim 3 or 4, characterized in that: In step 2), The molar ratio of the intermediate 1 to the compound c is 1:1.0-1:1.5; and / or The reaction is carried out in a solvent; and / or The reaction is carried out in the presence of a catalyst; and / or The reaction is carried out in the presence of a desiccant.

6. The preparation method according to any one of claims 3 to 5, characterized in that: In step 4) or step 3'), The deprotection is carried out by catalytic hydrogenation; Preferably, the catalyst for the catalytic hydrogenation is a palladium catalyst; and / or Preferably, the catalytic hydrogenation is carried out in a solvent.

7. The preparation method according to any one of claims 3 to 6, characterized in that: In step 3), the separation is performed by column chromatography; Preferably, the stationary phase of the column chromatography is at least one of alumina, silica gel, polyamide and magnesium silicate, preferably silica gel; and / or Preferably, the mobile phase of the column chromatography is at least one of esters, halogenated hydrocarbons, ethers, alcohols, ketones, aliphatic hydrocarbons and aromatic hydrocarbons, preferably a mixture of two of them, more preferably a mixture of esters and aliphatic hydrocarbons, and further preferably a mixture of esters and aliphatic hydrocarbons in a volume ratio of 1:5-1:

10.

8. The preparation method according to any one of claims 3 to 6, characterized in that: In step 4'), The separation is performed by high performance liquid chromatography, preferably by preparative high performance liquid chromatography; Preferably, the stationary phase of the chromatography is octadecylsilane bonded silica gel; and / or Preferably, the mobile phase of the chromatography method adopts a binary mobile phase system; More preferably, the binary mobile phase system consists of a weakly acidic buffered saline solution as an aqueous phase and an alcohol or nitrile as an organic phase; Further preferably, the weakly acidic buffered saline solution is an aqueous solution of a phosphate buffer system, preferably with a pH value of 6.0-6.9; and / or More preferably, the alcohol is C 1-4 fatty alcohol; and / or Further preferably, the nitrile is C 2-4 Fatty nitriles; and / or Preferably, the elution mode of the chromatography is isocratic elution; More preferably, the volume ratio of the aqueous phase to the organic phase used in the isocratic elution is 80:20-95:5; and / or Preferably, the detection wavelength of the chromatography is 240-250 nm; and / or Preferably, the flow rate of the chromatography is 1-20 mL / min; and / or Preferably, the column temperature of the chromatography method is 20-45°C; and / or Preferably, the injection volume of the chromatography method is 1 to 5 mL.

9. A pharmaceutical composition comprising the compound of formula I according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, isotope-labeled substance, active metabolite or prodrug thereof or the enrichment according to claim 2; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

10. Use of the compound of formula I according to claim 1 or its pharmaceutically acceptable salt, ester, solvate, isotope label, active metabolite or prodrug, or the enrichment according to claim 2, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating cerebral stroke, cerebral embolism, sequelae of stroke, cerebral motor dysfunction, mitochondrial encephalomyopathy and / or amyotrophic lateral sclerosis.

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