Cannabidiol derivatives, their preparation and use

Abstract

The present invention relates to cannabidiol derivatives, their preparation methods and uses, particularly in the treatment of nervous system disorders (e.g. The present invention discloses the use of the above cannabidiol in the prevention and treatment of chronic kidney disease (such as Parkinson's disease). The derivatives were screened from a series of synthetic derivatives, and the results of animal experiments showed that The above cannabidiol derivatives improved the balance beam scores of Parkinson's disease model animals. It reduces dopamine levels and tyrosine hydroxylase (T hydroxylase) levels in the substantia nigra (SubN) of the brain. H) It can improve the positive rate of cells and treat various diseases such as epilepsy and Parkinson's disease. It is useful in the research and development of therapeutic drugs for diseases, and has high application value.

Description

[Technical field]

[0001] The present invention relates to the technical field of chemical medicine, in particular to cannabidiol derivatives, their preparation Methods and uses, particularly in the prevention and treatment of neurological disorders (e.g., Parkinson's disease) Regarding use. [Background technology]

[0002] Cannabidiol, abbreviated as CBD, is extracted from cannabis flowers and leaves and is used in medicines, cosmetics, and other uses. It is a non-toxic, high-value-added phenolic substance that is useful in foods, health foods, etc. Its chemical name is 2-[(1R,6R)-3-methyl-6-(1-methylvinyl)-2-cyclohexene 1,3-benzenediol, CAS Registry Number 1 3956-29-1, the chemical structure of which is shown below. JPEG2025500813000002.jpg5552

[0003] Modern medical research has shown that CBD has significant anti-epileptic, anti-stress, and anti-inflammatory properties. CBD has been shown to be effective in treating epilepsy and for mood improvement and anti-anxiety. It is approved by the U.S. Food and Drug Administration as a dietary supplement.

[0004] However, cannabinoids are poorly water-soluble and have poor oral bioavailability, making them difficult to administer as medicines. Therefore, it is not suitable for the development or use of CBD. This has led to improved bioavailability and expanded use in the medical field. There is a need to provide new cannabidiol derivatives, methods for their preparation and uses. Summary of the Invention [Problem to be solved by the invention]

[0005] In order to solve the shortcomings of the prior art, the present invention provides a cannabidiol derivative, its preparation method and and uses thereof, in particular in the prevention and treatment of nervous system disorders (e.g., Parkinson's disease). Provide. [Means for solving the problem]

[0006] In a first aspect of the invention, there is provided a compound having the structure: JPEG2025500813000003.jpg6062(I) (where: R8 is selected from -NH2, -H, preferably R8 is -NH2; X1 and X2 are independently a single bond, alkylene (e.g., C 1~15 Alkylene, C1 ~10 Alkylene, C 1~5 alkylene), alkylene substituted with one or more substituents an alkylene separated by one or more -C(=O)O-; Alkylene separated by OC(=O)-, one or more alkylimino Separated alkylene, alkylene separated by one or more -OC(=O)O- and alkylene separated by a combination of the above spacer groups, The substituents are alkyl, hydroxy, halogen, aryl, cycloalkyl, thioalkoxy. is selected from aryl, nitro, cyano, amino, heteroaryl, and heterocycloalkyl; Here, H on the C atom is halogen, -OC 0~10 Alkyl, C 1~10 Linear / branched Al may be substituted with alkyl, nitro, cyano, amino; Further, X1 and X2 are independently C 1~5Alkylene, substituted with one or more substituents The substituents are selected from alkyl, hydroxy, halogen, aryl, alkyl, cycloalkyl, thioalkoxy, nitro, cyano, amino, heteroaryl, cycloalkyl, where H on the C atom is selected from halogen, -OC 0~10 Alkyl, C 1~10 Optionally substituted with straight / branched alkyl, nitro, cyano, amino , Further, X1 and X2 are independently -CH2-, -CH2CH2-, -(CH2)2C H2-, -(CH2)3CH2-, -CH2CH(CH3)2-, -(CH2)4CH2 -, -CH(CH3)CH2CH2-, -CH(OH)CH2-, Preferably, X1 and X2 are both -(CH2)3CH2-; R1 and R2 are independently -H, JPEG2025500813000004.jpg1915, -OH, JPEG2025500813000005.jpg1530, JPEG2025500813000006.jpg2534, JPEG2025500813000007.jpg913, JPEG2025500813000008.jpg1024, JPEG2025500813000009.jpg2344, JPEG2025500813000010.jpg3039, JPEG2025500813000011.jpg3728, JPEG2025500813000012.jpg3530, JPEG2025500813000013.jpg3530, alkoxy, where X3 is selected from N, O, S, preferably R 1 and R2 are independently JPEG2025500813000014.jpg1915, JPEG2025500813000015.jpg3728, JPEG2025500813000016.jpg3530, Selected from JPEG2025500813000017.jpg3530, R3 and R4 each independently represent -H, halogen, alkyl, cycloalkyl, aryl, R3 and R4 are preferably selected from the group consisting of alkyl, heteroaryl, and heterocycloalkyl. are both -H, R5, R6, and R7 are each independently -H, -OH, halogen, -NO2, -C R5 is selected from -H or -OH. and R6 and R7 are both -H.

[0007] Further, R1 and R2 are independently -NH2, -OH, JPEG2025500813000018.jpg1222, JPEG2025500813000019.jpg1438, JPEG2025500813000020.jpg1930, JPEG2025500813000021.jpg913, JPEG2025500813000022.jpg1024, JPEG2025500813000023.jpg1440, JPEG2025500813000024.jpg3627, JPEG2025500813000025.jpg3828, alkoxy, where X3 is selected from N, O, S, preferably X 3 is O, and preferably, R1 and R2 are independently -NH2, JPEG2025500813000026.jpg3620, and more preferably, R1 and R2 are both -NH2.

[0008] In one embodiment of the present invention, R1 and R2 are both -NH2.

[0009] Optionally, general formula (I) may be substituted with one or more D atoms.

[0010] In one embodiment of the invention, the compound has the following structure: JPEG2025500813000027.jpg6187, JPEG2025500813000028.jpg6576

[0011] In a second aspect of the present invention, there is provided a stereoisomer of the compound according to the first aspect having the structure do. JPEG2025500813000029.jpg6062(II) (wherein X1, X2, R1, R2, R3, R4, R5, R6, R7, and R8 are (having the definition as set forth in the first aspect of the specification).

[0012] Further, said stereoisomer has the following structure: JPEG2025500813000030.jpg6062(III)

[0013] In some embodiments of the present invention, the stereoisomer has the following structure: JPEG2025500813000031.jpg5787, JPEG2025500813000032.jpg6576

[0014] In a third aspect of the present invention, there is provided a compound according to the first aspect or a salt of a stereoisomer according to the second aspect. provides a eutectic, particularly a pharma- ceutically acceptable salt or eutectic.

[0015] Further, the salt may be a base addition salt or an acid addition salt, the base addition salt being selected from the group consisting of metal and amines. and compounds thereof, in particular basic metal salts, basic earth metal salts, e.g. sodium salts. , potassium salts, magnesium salts, calcium salts, and especially sodium salts, is formed from inorganic or organic acids and compounds, such as hydrochlorides, nitrates, sulfates, phosphates, etc. , citrate, formate, fumarate, maleate, acetate, succinate, tartrate, The preferred salts of the present invention are toluenesulfonates, p-toluenesulfonates, and in particular methanesulfonates. In a fourth aspect, there is provided a prodrug, solvate or metabolite of the compound according to the first aspect. In a fifth aspect of the invention there is provided a process for the preparation of a compound according to the first aspect.

[0016] Furthermore, the preparation method may include the following reaction steps: JPEG2025500813000033.jpg31139

[0017] Additionally, R', R'' can be an amino protecting group, such as an alkoxycarbonyl (e.g., - Boc), acyls, and alkyls.

[0018] Furthermore, the reaction temperature is 20 to 30° C., for example, room temperature.

[0019] In a sixth aspect of the invention, there is provided a compound according to the first aspect of the invention, or a pharma- ceutically acceptable derivative thereof. Salts, co-crystals, stereoisomers, prodrugs, solvates, metabolites and one or more pharmaceutical and excipients acceptable for use in the manufacture of a pharmaceutical composition comprising the compound.

[0020] In addition, pharma- ceutically acceptable auxiliary materials include fillers, adhesives, lubricants, disintegrants, antioxidants, One or more selected from a buffer, a suspension aid, a solubilizing agent, a thickener, a stabilizer, a preservative, etc. There may be multiple types.

[0021] Furthermore, the pharmaceutical composition can be administered gastrointestinal or by other routes, e.g., intravenous, intramuscular, intradermal and subcutaneous. The drug may be administered parenterally, such as by parenteral administration.

[0022] Furthermore, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, lozenges, syrups, etc. capsules, gels, emulsions, suspensions, controlled release formulations, aerosols, powder mists, fillers ointments, injections, drops, transdermal absorption agents, ointments, lotions, patches, suppositories, nasal drops , pulmonary formulations, and the like.

[0023] Furthermore, various dosage forms of the above pharmaceutical compositions can be prepared by conventional methods in the pharmaceutical field. do.

[0024] In addition, the pharmaceutical composition may contain one or more other active ingredients for the same or a different indication. It may also include minutes.

[0025] In a seventh aspect of the invention, there is provided a method for the preparation of a medicament for preventing and / or treating a disease comprising the steps of: The compound according to one embodiment, or a pharma- ceutically acceptable salt, cocrystal, stereoisomer, prodrug thereof, The present invention provides the use of the pharmaceutical composition according to the sixth aspect,

[0026] In addition, the above diseases include nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver damage, etc. and preferably, the disease is a nervous system disease.

[0027] Furthermore, the nervous system disease is a neurodegenerative disease or epilepsy, and the neurodegenerative disease is , Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, and multiple sclerosis, and preferably, said neurodegenerative disease is Parkinson's disease. .

[0028] In addition, cancers include breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, stomach cancer, and gastroesophageal cancer. Adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer , testicular cancer, prostate cancer, leukemia, glioma, etc.

[0029] In addition, autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and ulcers. These include, but are not limited to, chronic colitis, Crohn's disease, autoimmune hepatitis, and the like.

[0030] In an eighth aspect of the present invention, a compound according to the first aspect of the present invention, or a pharma- ceutically acceptable derivative thereof is provided. or a salt, co-crystal, stereoisomer, prodrug, solvate, metabolite or the like according to the sixth aspect. The method includes administering the pharmaceutical composition to a subject in need thereof. provides a method of treatment.

[0031] In addition, the above diseases include nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver damage, etc. and preferably, the disease is a nervous system disease.

[0032] Furthermore, the nervous system disease is a neurodegenerative disease or epilepsy, and the neurodegenerative disease is , Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, and multiple sclerosis, and preferably, said neurodegenerative disease is Parkinson's disease. .

[0033] In addition, cancers include breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, stomach cancer, and gastroesophageal cancer. Adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer , testicular cancer, prostate cancer, leukemia, glioma, etc.

[0034] In addition, autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and ulcers. These include, but are not limited to, chronic colitis, Crohn's disease, autoimmune hepatitis, and the like.

[0035] Further, the subject may be a mammal, such as a human, an orangutan, a monkey, a dog, a rabbit, a rat, or a mouse. The subject matter may be, in particular, a human.

[0036] The inventors of the present invention have carried out structural modifications based on cannabidiol to produce a series of synthetic derivatives. The compounds of the present invention were obtained by screening against the above-mentioned cannabinoids. The diol derivative reduced the balance beam score in Parkinson's disease model animals and levels and positivity of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain This will improve the rate of relapses and will be useful in the research and development of treatments for various diseases, including Parkinson's disease. It is useful and has high application value. [Brief description of the drawings]

[0037] [Figure 1] 1 shows the behavioral balance beam score results of a Parkinson's disease model. [Diagram 2] 1 shows the results of detection of dopamine content in the striatum in a Parkinson's disease model after treatment. [Diagram 3] The positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain is shown. [Figure 4] Shown is a microscopic image of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Unless otherwise defined, all scientific and technical terms used herein are understood by those of ordinary skill in the art. It has the same meaning as commonly understood.

[0039] The term "alkyl" refers to any group that includes unsaturated bonds that are connected to the rest of the molecule through single bonds. As used herein, alkyl generally refers to a straight or branched chain hydrocarbon group that does not include aryl groups. , 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) charcoal It contains 1 to 6 carbon atoms (i.e., C 1~6 alkyl). Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, and n-butyl. , isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl , n-hexyl, isohexyl, and the like.

[0040] The term "alkylene" refers to a hydrocarbon group in which an alkane molecule has lost two hydrogen atoms (a divalent (alkyl) which is a straight or branched chain that is connected to the rest of the molecule by a single bond. Exemplary alkylene herein is 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably It contains 1 to 6 carbon atoms. Examples of alkylene include methylene (-CH2-), ethylene Examples include ethylene, propylene, and butylene.

[0041] The term "alkoxy" refers to a substituent in which the hydrogen in a hydroxyl group is replaced with an alkyl group. As used herein, alkoxy generally refers to an alkyl group having an alkyl group of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms Contains children (i.e., C 1~6 Examples of the alkoxy include methoxy, These include, but are not limited to, ethoxy, propoxy, butoxy and the like.

[0042] The term "cycloalkyl" includes, for example, 1 to 4 single and / or fused rings, 3 to 18 carbon atoms, preferably 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10 ) carbon atoms, e.g., cyclopropyl, cyclobutyl, For example, cyclopentyl, cyclohexyl, or adamantyl.

[0043] The term "aryl" refers to any functional group or substituent derived from a simple aromatic ring. For example, a monocyclic or condensed ring having 6 to 18 (e.g., 6, 8, 10, aryl groups having 12, 14, 16, 18) carbon ring atoms and / or fused rings; Aryl refers to an aryl group. As used herein, aryl generally refers to a monocyclic group containing one or two rings. Aryl having 6 to 12 carbon ring atoms in a ring or fused ring (i.e., C 6~12 Ants In this case, the H on the carbon atom is replaced by, for example, an alkyl or halogen group. Examples of the aryl include phenyl and p-hydroxyphenyl. These include, but are not limited to:

[0044] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0045] The term "eutectic" refers to a mixture of a compound of the present invention and another physiologically acceptable acid, base, non-ionic surfactant, etc. Hydrogen bonds, van der Waals forces, π-π stacking interactions, halogen compounds It refers to crystals that are bound together by non-covalent bonds such as bonds.

[0046] The term "stereoisomers" refers to the forms of enantiomers, diastereomers, and geometric isomers. This includes those that exist in

[0047] The term "solvate" refers to a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, a solvate can be formed, for example, by dissolving one or more solvent molecules in the crystalline solid state. The solvate may be separated from the solution phase when incorporated into the polymer. Representative solvates include ethanolates, methanolates, and the like.

[0048] The term "metabolite" refers to a compound of the present invention that is chemically broken down under physiological conditions in vivo. This refers to the product obtained by

[0049] The term "prodrug" refers to a drug that is effective in treating a patient without excessive toxicity, irritation, or allergic reactions. Acetal, ester and zwitterionic forms suitable for administration and effective for the intended purpose. Prodrugs refer to forms of the compounds of formula I, including forms thereof. Prodrugs are compounds that undergo hydrolysis, e.g., in blood. is converted in vivo to give the parent compound.

[0050] As used herein, "D" refers to deuterium. "Deuterium substituted" refers to one or more This refers to hydrogen atoms being replaced with the corresponding number of deuterium atoms.

[0051] The term "subject" is used herein whether in vitro or in situ. The term "animal" refers to any animal or cell thereof that is amenable to the methods described above. an animal, such as a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey, an orangutan, or Humans, particularly humans, are included.

[0052] The term "treatment" refers to the prevention, cure or amelioration of one or more clinical symptoms of a disease after onset of the disease. It refers to curing, reversing, mitigating, alleviating, minimizing, suppressing, preventing, and / or stopping.

[0053] The term "prevention" refers to avoiding, minimizing, or preventing the onset or progression of a disease before the onset of the disease. refers to performing a treatment to make something more difficult.

[0054] The disclosures of various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety. The body is incorporated by reference.

[0055] The following describes the technical solutions of the present invention clearly and completely with reference to the embodiments of the present invention. However, the described embodiments are merely a part of the embodiments of the present invention, and are not all of the embodiments. It is obvious that the invention can be obtained by a person skilled in the art without any creative work based on the embodiments of the present invention. All of the above embodiments are within the scope of protection of the present invention. Example 1 Preparation of Compound 3

[0056] 1.Synthetic Scheme JPEG2025500813000034.jpg381542.Composition method

[0057] (1) Preparation of Compound 2 JPEG2025500813000035.jpg40136Compound 1 (CBD, 10 g, 31.85 mmol) and salt were added to a 1000 mL single-neck flask. Methylene chloride (500 mL) was added, and the mixture was stirred to dissolve. Then, Nα,Nε-bis(Boc)- L-Lysine (23.14 g, 66.88 mmol), dicyclohexylcarbodiimide ( 13.78 g, 66.88 mmol), and 4-dimethylaminopyridine (8.16 g, 66.88 mmol) was added and stirred at room temperature overnight. The insoluble matter produced by the reaction was filtered off. The filtrate was washed with saturated saline, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n- Compound 2 (13.5 g) was poured into a mixture of hexane and ethyl acetate (5:1 to 3:1) to obtain a foamy solid. ) was obtained. MS(ESI): 988[M+NH4] + . (2) Preparation of Compound 3

[0058] JPEG2025500813000036.jpg441462A 50 mL single-neck flask was charged with compound 2 (5.7 g, 5.88 mmol) and 1,4-dihydropyridine. Add xanthene (60 mL), stir to dissolve, and add methanesulfonate to the reaction flask under nitrogen protection. After the addition was completed, the mixture was stirred at room temperature overnight. A highly viscous solid was formed in the reaction flask. The supernatant was discarded and the remaining solid was dried in vacuum to obtain a crude product. The crude product C18 was purified by column chromatography (acetonitrile:water = 2:1 The chromatography solution was detected by HPLC and collected. The mixture was collected and lyophilized to give compound 3 (2.5 g) as a white foamy solid with a purity of 92%. MS(ESI):286[(M+2H) / 2] + . Example 2 Studies of the Treatment of Parkinson's Disease

[0059] 1. Experimental animals and some reagents C57 / BL6 mice, SPF grade, body weight 18-20g, age 6-8 weeks, male, Shanghai Sichuan University The animals were purchased from K.K. Laboratory Animal Co., Ltd. The temperature and humidity in the rearing environment were 22±1℃ and 65~70℃, respectively. %, the light / dark cycle was 12 hours, and water was available ad libitum. MPTP was purchased from Sigma, and amantadine was purchased from Sigma, anhydrous. Ethanol was purchased from China Pharmaceutical Group Chemical Reagents Co., Ltd.

[0060] 2. Experimental Grouping The experimental animals were randomly divided into 4 groups of 8 animals each: blank group, model group, positive drug group, and compound group.

[0061] 3. Experimental Method (1) Purchase 32 SPF grade C57 mice aged 6 to 8 weeks and raise them for one week until they become adapted. And started modelling. (2) MPTP (25 mg / kg) was intraperitoneally injected once a day for 7 consecutive days. was not processed. (3) Once modeling was completed, administration was started. The positive drug group was amantadine (40 mg / kg), and the compound group (100 mg / kg) was administered with Compound 3 (prepared in Example 1) at a given dose. The drug was administered intragastrically once daily for 14 consecutive days.

[0062] (4) Behavioral testing first: Balance beam detection score A wooden stick 80 cm long and 2.5 cm wide is fixed horizontally at a height of 10 cm from the table. The animals were allowed to walk on the If you can jump onto a balance beam and walk freely without falling over, 0 points, the animal can jump onto the balance beam, but not when walking on it If the animal has a chance of falling, but the probability of falling is less than 50%, 1 point is given. You can jump onto the beam, but there is a chance you might fall off when walking on the balance beam. If the probability of falling is greater than 50%, the animal is given 2 points and is unable to balance using the normal side of its body. The animal is able to jump onto the beam, but the hind limb on the paralyzed side cannot help move the body forward. If not, 3 points; the animal cannot walk on the balance beam, but on the wooden pole. If you can sit on it, give it 4 points. If you put the animal on a stick, it falls over immediately. will be worth 5 points.

[0063] (5) After scoring was completed, material collection began. After the mice were rapidly killed, the mouse brains were harvested and the mouse cerebral cortex (CP) was isolated. After exposing the mouse brain striatum, the striatum was collected and weighed, and PBS was added at a specified ratio. The mixture was then crushed and homogenized. The supernatant was then separated to determine the dopamine levels in the striatum. The amount of nicotine was detected.

[0064] 4. Detection of dopamine content in the striatum using a kit method 4.1 The kit and some of the equipment are as follows:

[0065] [Table 1] Kit information JPEG2025500813000037.jpg163115 [Table 2] Major equipment and consumables JPEG2025500813000038.jpg163115

[0066] 4.2 Protein quantification (1) Preparation of a standard curve: Prepare an enzyme plate and add reagents according to the table below. [Table 3] Reagents JPEG2025500813000039.jpg163115(2) Depending on the number of samples, mix BCA Reagent A and Reagent B in a volume ratio of 50:1 to prepare an appropriate A volume of BCA working solution was prepared and mixed thoroughly. (3) 160 μl of BCA working solution was added to each well. (4) Place the plate on an oscillator, shake for 30 seconds, and then leave it at 37°C for 30 minutes. The absorbance at 562 nm was measured. The horizontal axis represents absorbance, and the vertical axis represents protein concentration (μg / μl). was used to prepare a standard curve. (5) Add 2 μl of the test protein and 18 μl of PBS (10-fold dilution) to the plate. Add 160 μl of BCA working solution, place the plate on an oscillator, shake for 30 sec, and The mixture was allowed to stand at 7° C. for 30 minutes, after which the absorbance at 562 nm was measured. (6) Based on the absorbance value of the measured sample, the corresponding protein concentration on the standard curve is calculated. (μg / μl) and multiplying this by the sample dilution factor (10) gives the actual concentration of the sample. The concentration (unit: μg / μl) was obtained. (7) For the operation and method of the mouse dopamine detection experiment, please refer to the kit instruction manual. Ta.

[0067] 5. Immunohistochemistry of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain measurement 5.1 Experimental materials

[0068] [Table 4] Main reagents JPEG2025500813000040.jpg163115

[0069] [Table 5] Major equipment and consumables JPEG2025500813000041.jpg163115

[0070] Preparation of solutions (1)PBS solution ddH2O600ml contains NaCl8g, KCl0.2g, Na2HPO41.44g, and Dissolve 0.24 g of KH2PO4 in water and adjust the pH of the solution to 7.4 with HCl. The solution was diluted to volume, filtered through a filter, autoclaved, and stored at room temperature. (2) 0.1 mol / L sodium citrate buffer 0.1mol / L citric acid 3.8ml, 0.1mol / L sodium citrate 16.2ml l. Citric acid C6H8O7·H2O: molecular weight 210.14; 0.1 mol / L solution is 21.0 1g / L. Sodium citrate Na3C6H5O7·2H2O: molecular weight 294.12; 0.1 mol / L solution is 29.41 g / mL.

[0071] 5.2 Experimental Method

[0072] 5.2.1 Sample embedding and fixation (1) Collection and fixation of materials When cutting tissue, a sharp knife or scissors was used. When cutting tissue blocks, The tissue was cut by pulling the knife backward from the base. The thickness of the tissue mass was about 0.2-0.3 cm. The appropriate size is 1.5cm x 1.5cm x 0.3cm. The tissue was fixed in ethyl acetate for 48 hours.

[0073] (2) Washing and dehydration The fixed tissue was rinsed in running water to remove residual fixative and impurities. Using alcohol, dilute it to 50%, 70%, 85% or 100% until you get pure alcohol (absolute ethanol). %, 95%, and 100% ethanol for 2 hours each. Dehydration should be carried out in jars with lids to prevent loss of concentration and incomplete dehydration. Materials that require preservation can be dehydrated to 70% ethanol and left to stand, allowing for long-term storage. If extended storage is required, add an equal amount of glycerin.

[0074] (3) Transparency The tissue block was placed in a mixture of equal parts of pure ethanol and xylene and left for 2 hours. , place in pure xylene, leave for 2 hours, place again in pure xylene, and leave for 2 hours. The material becomes transparent, revealing the effect of the dehydration in the previous step. If the tissue is sufficiently dehydrated, it will be transparent. If the tissue has a white cloudy appearance, it is not sufficiently dehydrated. It indicates that you need to start over, but that if you do, the effect will be poor. When xylene is used for clarification, the xylene evaporates and dissolves in the moisture in the air. It is necessary to avoid absorbing the xylene and keep it anhydrous.

[0075] (4) Paraffin infiltration Paraffin infiltration must be performed in a thermostatic oven. First, the tissue material block is infused with molten paraffin. The specimens were immersed in a mixture of equal parts of xylene for 1-2 hours, then transferred to two liquids of molten paraffin. Each was soaked for about 3 hours.

[0076] (5)Embedding For embedding, the tissue block was thoroughly soaked in paraffin and then wrapped in paraffin. The specific method is as follows: First, prepare a paper box, pour the molten wax into the box, and then use pre-warmed tweezers to Use a tool to quickly lift the tissue block and place it flat, cut-side down, on the bottom of the paper box, then The paraffin on the surface was then slowly lifted off and placed flat in cold water. The paper box was quickly pressed into the water, cooled and rapidly solidified, and removed after 30 minutes.

[0077] (6) Slicing Before slicing, place the paraffin block in a refrigerator at -20°C for at least 30 min. The slicer knife holder is attached to the slicer to make it firm. Fix and place the base of the paraffin block or fix the paraffin block and place the base of the paraffin block Adjust the blade and knife to the appropriate position so that the blade and the surface of the paraffin block form a 5 degree angle. The thickness of the slices on the slicer was adjusted to 4-7 μm before slicing.

[0078] 5.2.2 Immunohistochemical staining

[0079] (1) Baking and dewaxing The slides were placed in a thermostatic oven and baked at 65°C for 30 min. min and then in xylene II for 15 min.

[0080] (2) Hydration Deparaffinize sections in 100% alcohol, 95% alcohol, 85% alcohol, and 75% Each was immersed in alcohol for 5 minutes and then rinsed with tap water for 10 minutes.

[0081] (3) Antigen repair The specimen was then high-pressure restored in 0.01M sodium citrate buffer for 15 min, cooled naturally, and then resuspended in 0.02M The sections were washed with PBS for 3 min three times.

[0082] (4) Blocking Slides were placed in 3% H2O2 and incubated in a humidified box for 10 min to determine endogenous The active peroxidase activity was eliminated by washing with 0.02M PBS for 3 min x 3 times.

[0083] (5) Primary antibody incubation Add primary antibody (1:2000 dilution) and incubate in a humidified box at room temperature for 1 The plate was left to stand for 2 hours (or incubated overnight at 4°C). Washed 3 times with 0.02M PBS for 3 minutes each. Purified.

[0084] (6) Dual Antibody Incubation Add HRP-conjugated broad-spectrum secondary antibody dropwise and incubate in a humidified box at room temperature. The plate was left to stand for 20-30 minutes in 0.02M PBS, and then washed three times for 3 minutes each with 0.02M PBS.

[0085] (7) Color development If discoloration was observed in the sections after DAB staining, the staining solution was immediately washed off with tap water.

[0086] (8) Hematoxylin staining Counterstain with hematoxylin for 3 minutes, differentiate with 1% hydrochloric acid alcohol, and observe under a microscope. The degree of dyeing was controlled. Rinse with tap water for 10 minutes and place in a 65℃ oven. The moisture was dried.

[0087] (9) Clearing and mounting The slides were cleared in xylene for 3 min twice, mounted in neutral resin, and incubated at 65°C. It was left in the oven for 15 minutes.

[0088] 5.2.3 Image collection and analysis Photographs were taken under a microscope, relevant parts of the samples were collected and analyzed, and the positive areas were calculated.

[0089] 6. Experimental Results (1) The results of the behavioral balance beam score of the Parkinson's disease model are shown in the following table and Figure 1. .

[0090] [Table 6] Behavioral balance beam score results for Parkinson's disease model JPEG2025500813000042.jpg163115

[0091] A Parkinson's disease model was created in mice by intraperitoneal injection of MPTP. Behavioral evaluation was performed using the balance beam score. Seven days after intraperitoneal injection of MPTP, Modeling was not performed for the blank group, and balance beam scores were The score for the remaining groups was 5 to 6 points. Compared with the blank group, P ≤ 0.05 was statistically significant, proving the success of the model. Ta.

[0092] After that, the positive drug group and the compound group were treated, and then the balance beam score of the model group was The balance beam scores for positive drugs were 2 to 3 points. The balance beam score was 3 to 4 points. The positive drug group and the compound group had a higher score than the model group. There was a significant downward trend compared with control, with P < 0.05 and statistical significance.

[0093] The scores of each group after treatment were as follows: blank group < positive drug group < compound group < model group.

[0094] (2) The dopamine content in the striatum of the Parkinson's disease model after treatment is shown in the following table and figure. Shown in 2.

[0095] Table 7. Striatal dopamine content in Parkinson's disease models after treatment JPEG2025500813000043.jpg163115

[0096] Dopamine, as a neurotransmitter, regulates various physiological functions of the central nervous system. The researchers detected dopamine levels in the striatum of mice treated with Parkinson's disease. By doing so, the effects of drug treatment on the Parkinson's disease model were confirmed.

[0097] The dopamine content of each group was compared with that of the blank group to determine the dopamine content in the model. As a result of judging the degree of dopamine reduction, the dopamine content of the compound group was The decrease was statistically significant, with P≦0.05.

[0098] Next, the dopamine content of the compound group was compared with that of the model group. The pamin content was increased compared with the model group, with P≦0.01, which was statistically significant. This indicates that the compounds have the effect of increasing dopamine content in the brain. The dopamine content in the striatum was as follows: blank group > positive drug group > compound group > model group.

[0099] (3) Immunohistochemistry of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain The experimental results of the objective measurements are shown in the table below and in FIGS.

[0100] [Table 8] Immunohistochemistry of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain Experimental results of biological measurements JPEG2025500813000044.jpg163115

[0101] Tyrosine hydroxylase (TH) is a monooxygenase that produces the organism's own L-dose. It is the rate-limiting enzyme that catalyzes the first step of a series of reactions in the synthesis of damin (DA) in cells. TH is abundant in neurons, and dopamine-β-hydroxylase is expressed only in the midbrain. Due to the lack of xylase, TH-immunoreactive neurons in the midbrain are DA neurons. It can be used as a marker for dopamine neurons in the brain. L-tyrosine is used to produce levodopa under the catalysis of xylase and aromatic decarboxylase The carboxyl group is removed from levodopa by catalysis, ultimately producing levodopamine. Tyrosine hydroxylase plays an important role in the synthesis of dopamine. Lack of function or insufficient expression of dopamine directly affects the synthesis and secretion of dopamine. Dopaminergic neurons synthesize dopamine, which is an important neurotransmitter. Developmental dysfunction or insufficient secretion can lead to the development of Parkinson's disease.

[0102] As a result of this experiment, a mouse model of Parkinson's disease was created by intraperitoneal injection of MPTP. In the mouse brain, tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) were positive. The rate was decreased, and the P value was ≦0.05, which was statistically significant. Compared with the model group. In the positive drug group, tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain were increased. The positive rate of cells was significantly improved, indicating that the modeling was successful.

[0103] After treating a mouse Parkinson's disease model with the compounds, the compound group showed a higher increase in the number of patients with Parkinson's disease compared to the model group. increased the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain. Among these, the compound group inhibited tyrosine hydroxylase (TH) in the substantia nigra (SubN) of the brain. The positive rate of ) cells was compared with that of the model group, and the results were statistically significant (P≦0.01). Ta.

[0104] Positive tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain in each group after treatment The rates were compared as follows: blank group > positive drug group > compound group > model group.

[0105] 7. Summary of the experiment In this experiment, MPTP (25 mg / kg) was intraperitoneally injected once daily for 7 consecutive days. A mouse Parkinson's model was created. The balance beam score was used to measure mouse Parkinson's disease. We performed screening against the 10G model.

[0106] Next, the positive drug group and the compound group were intragastrically administered to the model mice for 14 consecutive days. The balance beam scores of each group were then analyzed, and striatal dopamine content was assessed by ELISA. Detect the amount and positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain. By immunohistochemically detecting the markers, the differences between the compound group and the blank and model groups were confirmed. Statistical analysis was then performed to analyze the efficacy of compound 3.

[0107] As confirmed by the balance beam score after treatment, the positive drug group was significantly higher than the model group. There was a significant downward trend in the and compound groups, with P ≤ 0.05 and statistical significance.

[0108] Next, the dopamine content of the compound group was compared with that of the model group. The dopamine content in the compound group was increased, which was statistically significant (P≦0.01). .

[0109] After treating the mouse Parkinson's disease model with the compound group and the positive drug, the positive drug group and the compound group were The positive rate of the compound group was compared with that of the model group. The positive rate of tyrosine hydroxylase (TH) cells in the rats was compared with that of the model group. The results were statistically significant, with P≦0.01.

[0110] A holistic analysis of the compound group against the model group and a comparison of the various detection indices revealed that the compounds Product 3 was found to have a better therapeutic effect.

[0111] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principle of the present invention are within the scope of protection of the present invention. The scope of the scope of the

[0112] The above-described embodiments and methods described herein are subject to change based on the ability, experience, and preferences of those skilled in the art. It is possible.

[0113] In the present invention, the steps of the method are merely recited in a particular order, which is The order of the steps is not limited.

Claims

1. A compound having the following structure or a pharma- ceutically acceptable salt, cocrystal, stereoisomer, prodrug thereof: Compounds, solvates, and metabolites. （I） (where: R ８ is -NH ２ , -H; X １ and X ２ are independently a single bond, an alkylene, an alkyl group substituted with one or more substituents, alkylene, alkylene separated by one or more -C(=O)O-, one or more Alkylene, one or more alkyliminos separated by -OC(=O)- alkylene separated by one or more -OC(=O)O-; alkylene, alkylene separated by a combination of the above spacer groups. The substituents are alkyl, hydroxy, halogen, aryl, cycloalkyl, thioa, alkoxy, nitro, cyano, amino, heteroaryl, heterocycloalkyl where H on the C atom is halogen, -OC ０～１０ Alkyl, C １～１０ Straight chain / min may be substituted with alkyl, nitro, cyano, or amino; R １ and R ２ are independently -H, , -OH, 、 、 、 、 、 、 、 、 , alkoxy, where X ３ is selected from N, O, S, preferably R １ and R ２ is, independently, 、 、 、 is selected from R ３ and R ４ are each independently -H, a halogen, an alkyl, a cycloalkyl, or an aryl. is selected from alkyl, heteroaryl, and heterocycloalkyl; R ５ , R ６ , and R ７ are each independently -H, -OH, a halogen, or -NO ２ , -C N, alkyl, cycloalkyl; Or, general formula (I) is optionally substituted with one or more D atoms.

2. 2. The compound of claim 1, wherein the stereoisomer has the following structure: (II)

3. The R ８ Ha-NH ２ and X １ and X ２ is independently １～５ Alkylene, 1 or alkylene substituted with multiple substituents, the substituents being selected from alkyl, hydroxyl, alkoxy, halogen, aryl, cycloalkyl, thioalkoxy, nitro, cyano, amino Heteroaryl, heterocycloalkyl, wherein H on the C atom is selected from halo, heteroaryl, heterocycloalkyl, Rogen, -OC ０～１０ Alkyl, C １～１０ Straight chain / branched alkyl, nitro, cyano, a may be substituted with amino; ３ and R ４ are each -H, and the R ５ is -H or —OH, wherein R ６ and R ７ are each -H, and the R １ and R ２ are independently -NH ２ , -OH, 、 、 、 、 、 、 、 , alkoxy, where X ３ is selected from N, O, S, preferably X ３ The compound according to claim 1, characterized in that

4. The R １ and R ２ are independently -NH ２ , 2. The compound according to claim 1, wherein the compound is selected from the group consisting of:

5. The R １ and R ２ are both -NH ２ The compound according to claim 1, thing.

6. The X １ and X ２ is -CH ２ --, --CH ２ CH ２ -, -(CH ２ ) ２ CH ２ -, -(C H ２ ) ３ CH ２ -、-CH ２ CH(CH ３ ) ２ -、-(CH ２ ) ４ CH ２ -、-CH(C H ３ ) CH ２ CH ２ -, -CH(OH)CH ２ - selected from the group consisting of Item 2. The compound according to item 1.

7. The X １ and X ２ are both -(CH ２ ) ３ CH ２ - characterized in that 1. The compound described in

8. 2. The compound of claim 1 having the structure: or

9. The compound according to any one of claims 1 to 8, or a pharma- ceutically acceptable salt, eutectic, or steric thereof. Isomers, prodrugs, solvates, metabolites and one or more pharma- ceutically acceptable co-additives. and auxiliary materials.

10. A compound according to any one of claims 1 to 7 in the preparation of a medicament for preventing and / or treating a disease. or a pharma- ceutically acceptable salt, co-crystal, stereoisomer, prodrug, solvate thereof, Use of a metabolite, The disease is selected from the group consisting of nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, and liver damage. The method of claim 1, wherein the disease is one or more selected from the group consisting of neurological diseases.

11. The nervous system disease is a neurodegenerative disease or epilepsy, and the neurodegenerative disease is Parkinson's disease. Johns Hopkins' disease, Alzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease, multiple sclerosis Preferably, said neurodegenerative disease is Parkinson's disease; The cancers include breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, gastric cancer, and gastroesophageal cancer. esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, sperm Selected from cancer, prostate cancer, leukemia, and glioma. The autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, and the like.

11. The method according to claim 10, characterized in that the disease is selected from the group consisting of enteritis, Crohn's disease, and autoimmune hepatitis. Use of.

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