A cannabidiol derivative, its preparation method and application

Abstract

The present invention discloses a cannabidiol derivative, its preparation method and application, especially the application in the prevention and treatment of nervous system diseases (such as epilepsy, Parkinson's disease). The above-mentioned cannabidiol derivative is screened from a series of synthetic derivatives. The results of animal experiments show that these compounds can effectively shorten the duration of epileptic seizures in experimental animals, improve the symptoms of epileptic seizures in experimental animals, reduce the balance beam score of Parkinson's model animals, increase the dopamine level and the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain, and can be used for the drug development and research of various diseases such as epilepsy and Parkinson's disease, and has better application value.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical medicine, and particularly relates to a cannabidiol derivative, a preparation method thereof and an application, especially an application in the prevention and treatment of nervous system diseases (such as epilepsy, Parkinson's disease). Background Art

[0002] Cannabinoids are a class of secondary metabolites unique to the cannabis plant, containing an alkyl group and a monoterpene group structure. At present, more than a hundred cannabinoids have been isolated from the cannabis plant, mainly including δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBC), cannabigerol (CBG), cannabinol (CBN), etc., among which the contents of THC and CBD are the highest. Research shows that cannabinoids have a wide range of pharmacological effects.

[0003] Cannabidiol (CBD), with the chemical name of 2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol, CAS registration number: 13956-29-1, has the following chemical structure:

[0004]

[0005] It has been found that CBD has pharmacological effects such as antispasmodic, anti-anxiety, anti-inflammatory, antioxidant, etc., and can be used for drug development. However, high-purity CBD is a white or light yellow crystal, with a melting point of 66-67°C, hardly soluble in water or 10% sodium hydroxide solution, and easily soluble in organic solvents such as ethanol, methanol, ether, benzene, chloroform and petroleum ether. The poor water solubility and low bioavailability of CBD greatly limit the application of CBD in the fields of medicine and the like. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides a cannabidiol derivative, a preparation method thereof and an application, especially an application in the prevention and treatment of nervous system diseases (such as epilepsy, Parkinson's disease).

[0007] In the first aspect of the present invention, a compound is provided, which has the following structure:

[0008]

[0009] Wherein,

[0010] X1 and X2 are independently selected from: a single bond, a substituted or unsubstituted alkylene group, an alkylene group separated by one or more -C(=O)O-, an alkylene group separated by one or more -OC(=O)-, an alkylene group separated by one or more -C(=O)NH-;

[0011] X3 is selected from:

[0012] R1 is selected from: H, alkyl, cycloalkyl;

[0013] R2 is selected from: OH, alkoxy;

[0014] n is an integer from 1 to 5 (such as 1, 2, 3, 4, 5);

[0015] R3 is selected from: H,

[0016] wherein, X4 and X5 are independently selected from: single bond, substituted or unsubstituted alkylene, alkylene separated by one or more -C(=O)O-, alkylene separated by one or more -OC(=O)-, alkylene separated by one or more -C(=O)NH-;

[0017] X6 is selected from:

[0018] R4 is selected from: H, alkyl, cycloalkyl;

[0019] R5 is selected from: OH, alkoxy;

[0020] m is an integer from 1 to 5 (such as 1, 2, 3, 4, 5).

[0021] Specifically, X1 is selected from: single bond, C1-6 straight-chain or branched alkylene, especially C1-3 straight-chain alkyl; in one embodiment of the present invention, X1 is methylene.

[0022] In one embodiment of the present invention, X2 has the following structure: wherein R7 is selected from: H, alkyl, alkyl substituted by alkylthio, alkyl substituted by hydroxyl, alkyl substituted by mercapto, alkyl substituted by -C(=O)NH2, alkyl substituted by carboxyl, alkyl substituted by amino, alkyl substituted by substituted alkyl, heterocyclic alkyl, aralkyl.

[0023] Specifically, R7 can be selected from: H, methyl, isopropyl, sec-butyl, isobutyl, In one embodiment of the present invention, R7 is

[0024] In some embodiments of the present invention, X3 is

[0025] In some embodiments of the present invention, n is 1.

[0026] In some embodiments of the present invention, R1 is H.

[0027] Specifically, R2 is selected from: OH, C1-6 alkoxy; in some embodiments of the present invention, R2 is OH, methoxy or ethoxy.

[0028] In one embodiment of the present invention, the above compound has the following structure:

[0029]

[0030] More specifically, the above compound has the following structure:

[0031]

[0032] In one embodiment of the present invention, R3 is H.

[0033] In another embodiment of the present invention, R3 is

[0034] Specifically, X4 is selected from: a single bond, C1-6 straight-chain or branched-chain alkylene, especially C1-3 straight-chain alkyl; in one embodiment of the present invention, X4 is methylene.

[0035] In one embodiment of the present invention, X5 has the following structure: wherein R8 is selected from: H, alkyl, alkyl substituted by alkylthio, alkyl substituted by hydroxy, alkyl substituted by mercapto, alkyl substituted by -C(=O)NH2, alkyl substituted by carboxyl, alkyl substituted by amino, alkyl substituted by substituted alkyl, heterocyclic alkyl, aralkyl.

[0036] Specifically, R8 can be selected from: H, methyl, isopropyl, sec-butyl, isobutyl, In one embodiment of the present invention, R8 is

[0037] In some embodiments of the present invention, X6 is

[0038] In some embodiments of the present invention, m is 1.

[0039] In some embodiments of the present invention, R4 is H.

[0040] Specifically, R5 is selected from: OH, C1-6 alkoxy; in some embodiments of the present invention, R5 is OH, methoxy or ethoxy.

[0041] In one embodiment of the present invention, the above-mentioned compound has the following structure:

[0042]

[0043] In another embodiment of the present invention, the above-mentioned compound has the following structure:

[0044]

[0045] In some embodiments of the present invention, the above-mentioned compound has the following structure:

[0046]

[0047] In the second aspect of the present invention, there is provided a stereoisomer of the compound described in the first aspect, which has the following structure:

[0048]

[0049] Wherein, X1, X2, X3, R1, R2, R3, and n have the corresponding definitions as described in the first aspect of the present invention.

[0050] In some embodiments of the present invention, the above-mentioned stereoisomer has the following structure:

[0051]

[0052] In the third aspect of the present invention, there is provided a salt of the compound described in the first aspect or the stereoisomer described in the second aspect, particularly a pharmaceutically acceptable salt.

[0053] Specifically, the salt is a base addition salt, which can be formed by a metal or an amine and the compound, particularly an alkali metal salt or an alkaline earth metal salt, such as a sodium salt, a potassium salt, a magnesium salt, or a calcium salt.

[0054] In some embodiments of the present invention, the salt is

[0055] In the fourth aspect of the present invention, there is provided an ester, a prodrug, or a solvate of the compound described in the first aspect.

[0056] In the fifth aspect of the present invention, there is provided a method for preparing the compound described in the first aspect.

[0057] In one embodiment of the present invention, when R3 is H, the preparation method may include the following reaction steps:

[0058]

[0059] Wherein, R2' is an alkoxy group, such as a C1-6 alkoxy group, such as a methoxy group.

[0060] In some embodiments of the present invention, in the above reaction steps is

[0061] Specifically, the preparation method may further include the following reaction steps:

[0062]

[0063] Specifically, the base in the above reaction steps is a hydroxide of an alkali metal, such as lithium hydroxide, sodium hydroxide, potassium hydroxide.

[0064] Specifically, the acid in the above reaction steps is an inorganic acid, such as hydrochloric acid.

[0065] Specifically, the preparation method may further include the following reaction steps:

[0066]

[0067] wherein, R9 is an alkyl group, such as a C1-6 alkyl group, such as a methyl group;

[0068] R 10 is a leaving group, such as -F, -Cl, -Br, -I, (-OTs), (-OMs), (-OBs), (-OTf).

[0069] In some embodiments of the present invention, in the above reaction steps is

[0070] Specifically, the base in the above reaction steps is a hydroxide of an alkali metal, such as lithium hydroxide, sodium hydroxide, potassium hydroxide.

[0071] Specifically, the acid in the above reaction steps is an inorganic acid, such as hydrochloric acid.

[0072] In another embodiment of the present invention, R3 is The preparation method may include passing through separately or simultaneously (in this case and represent the same substance) and reacting with ;

[0073] wherein, R2' and R5' are alkoxy groups, such as C1-6 alkoxy groups, such as methoxy groups.

[0074] In one embodiment of the present invention, and represent the same substance, such as

[0075] In the sixth aspect of the present invention, there is provided a pharmaceutical composition comprising the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, and one or more pharmaceutically acceptable excipients.

[0076] Specifically, the pharmaceutically acceptable excipients may be selected from one or more of the following: fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like.

[0077] Specifically, the pharmaceutical composition can be administered enterally or parenterally, such as by intravenous, intramuscular, intradermal, and subcutaneous routes.

[0078] Specifically, the pharmaceutical composition can be formulated into the following dosage forms: tablets, pills, powders, granules, capsules, lozenges, syrups, gels, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous infusions, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, nasal preparations, pulmonary preparations, and the like.

[0079] Specifically, the various dosage forms of the above pharmaceutical composition can be prepared according to the conventional production methods in the pharmaceutical field.

[0080] Specifically, the pharmaceutical composition may further comprise one or more other active ingredients for the same or different indications.

[0081] In the seventh aspect of the present invention, there is provided the use of the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, and the pharmaceutical composition described in the sixth aspect in the preparation of a drug for preventing and / or treating diseases.

[0082] Specifically, the above diseases are selected from one or more of the following: nervous system diseases, cancers, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver injury, especially nervous system diseases.

[0083] Specifically, the nervous system diseases include, but are not limited to, epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, anxiety disorders, depression, and the like.

[0084] Specifically, epilepsy can be acute epilepsy or chronic epilepsy, especially chronic epilepsy.

[0085] Specifically, cancers include, but are not limited to, breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, gastric cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, testicular cancer, prostate cancer, leukemia, glioma, and the like.

[0086] Specifically, autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, autoimmune hepatitis, etc.

[0087] In the eighth aspect of the present invention, a method for preventing and / or treating a disease is provided, which comprises the step of administering to a subject in need thereof a compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, or the pharmaceutical composition described in the sixth aspect.

[0088] Specifically, the above diseases are selected from one or more of: nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver injury, especially nervous system diseases.

[0089] Specifically, nervous system diseases include, but are not limited to, epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, anxiety disorder, depression, etc.

[0090] Specifically, epilepsy can be acute epilepsy or chronic epilepsy, especially chronic epilepsy.

[0091] Specifically, cancer includes, but is not limited to, breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, testicular cancer, prostate cancer, leukemia, glioma, etc.

[0092] Specifically, autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, autoimmune hepatitis, etc.

[0093] Specifically, the subject can be a mammal, such as, human, orangutan, monkey, dog, rabbit, mouse, etc., especially human.

[0094] The inventors of the present invention carried out structural modification on the basis of cannabidiol, and screened the compounds of the present invention from a series of synthetic derivatives. The results of animal experiments showed that these compounds could effectively shorten the seizure duration of experimental animals, improve the seizure symptoms of experimental animals, reduce the balance beam score of Parkinson's model animals, increase the dopamine level and the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain, and can be used for the drug development and research of various diseases such as epilepsy and Parkinson's disease, and have better application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 Shown are the detection results of the seizure duration of PTZ-kindled epileptic rats before and after treatment.

[0096] Figure 2 Shown are the Ono's classification of the behavioral seizure indices of PTZ-kindled epileptic rats before and after treatment.

[0097] Figure 3 Shown are the detection results of the seizure latency of PTZ-kindled epileptic rats before and after treatment.

[0098] Figure 4 Shown is the PAGE gel image of apoptosis-related proteins Bax and Bcl-2 in the hippocampal tissue of rats.

[0099] Figure 5 Shown are the changes of apoptosis-related proteins Bax and Bcl-2 in the hippocampal tissue of rats.

[0100] Figure 6 Shown are the detection results of the content of γ-aminobutyric acid in the homogenate of rat brain tissue.

[0101] Figure 7 Shown are the detection results of the content of glycine in the homogenate of rat brain tissue.

[0102] Figure 8 Shown are the detection results of the content of glutamate in the homogenate of rat brain tissue.

[0103] Figure 9 Shown are the detection results of the content of aspartic acid in the homogenate of rat brain tissue.

[0104] Figure 10 Shown are the results of the balance beam scoring in the behavioral study of the Parkinson model.

[0105] Figure 11 Shown are the detection results of the dopamine content in the striatum after treatment of the Parkinson model.

[0106] Figure 12 Shown is the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain.

[0107] Figure 13 Shown is the microscopic image of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain. Detailed implementation manners

[0108] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains. For example:

[0109] The term "alkyl" refers to a hydrocarbon group formed by removing one hydrogen atom from an alkane molecule, which can be straight-chain or branched-chain and is connected to the rest of the molecule by a single bond. In this document, the alkyl group generally contains 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms (i.e., C1-C6 alkyl). Examples of the alkyl group include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If the alkyl group is substituted by an aryl group, then it is correspondingly "arylalkyl" (e.g., C7-C 18 arylalkyl, such as C7-C 15 arylalkyl, C7-C 12 arylalkyl; for example, (C1-C6 alkylene)-(C6-C 12 aryl), (C1-C3 alkylene)-phenyl), such as benzyl, diphenylmethyl or phenethyl. If the alkyl group is substituted by a heterocyclic group, then it is correspondingly "heterocyclic alkyl".

[0110] The term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by removing two hydrogen atoms from an alkane molecule, which can be straight-chain or branched-chain and is connected to the rest of the molecule by a single bond. In this document, typical alkylene groups contain 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms. Examples of alkylene groups include methylene (-CH2-), ethylene, propylene, butylene, etc.

[0111] The term "alkoxy" refers to a substituent formed by replacing the hydrogen in a hydroxyl group with an alkyl group. The alkoxy groups used in this document generally contain 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms (i.e., C1-C6 alkoxy). Examples of the alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, etc.

[0112] The term "cycloalkyl" refers to an alicyclic hydrocarbon, such as a monocyclic and / or fused-ring containing 1 to 4 rings, containing 3 - 18 carbon atoms, preferably 3 - 10 (such as 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or adamantyl, etc.

[0113] The term "aryl" refers to any functional group or substituent derived from a simple aromatic ring, including monocyclic aryl groups and / or fused-ring aryl groups, such as those containing 1 - 3 rings, monocyclic or fused-ring and having 6 - 18 (such as 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl groups used in this document are generally aryl groups containing 1 - 2 rings, monocyclic or fused-ring and having 6 - 12 carbon ring atoms (i.e., C6-C12 (aryl), wherein the H on the carbon atom may be substituted, for example, by groups such as alkyl, halogen, etc. Examples of the aryl include, but are not limited to, phenyl, p-hydroxyphenyl, etc.

[0114] The term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group that contains 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may contain a fused, spiro, or bridged ring system. The heterocyclic group can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl).

[0115] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0116] The term "stereoisomer" includes enantiomers, diastereomers, and geometric isomers.

[0117] The term "solvate" refers to the physical association of a compound of the present invention with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solution-phase and isolable solvates. Representative solvates include ethanolates, methanolates, etc.

[0118] The term "prodrug" refers to a form of a compound of formula I that is suitable for administration to a patient, has no excessive toxicity, irritation, allergic reaction, etc., and is effective for its intended purpose, including acetal, ester, and zwitterionic forms. The prodrug is converted in vivo, such as by hydrolysis in the blood, to give the parent compound.

[0119] The term "subject" refers to any animal or its cells that are subjected to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, chimpanzees, or humans, especially humans.

[0120] The term "treatment" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting, and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0121] The term "prevention" refers to avoiding, minimizing, or making it difficult for a disease to occur or develop by treatment before the onset of the disease.

[0122] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0123] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0124] Example 1: Preparation of Compound E1

[0125]

[0126] 1. Synthetic route

[0127]

[0128] 2. Synthetic method

[0129] (1) Synthesis of Compound 2

[0130]

[0131] Experimental procedure:

[0132] CBD (20 g, 64 mmol, 1 eq) was dissolved in 400 ml of methyl ethyl ketone, cesium carbonate (31 g, 96 mmol, 1.5 eq) was added, and the mixture was heated to 80 °C and reacted for 2 h. Methyl bromoacetate (12.6 g, 0.082 mol, 1.3 eq) was added, and the reaction was carried out overnight. Methyl bromoacetate (5 g) and cesium carbonate (15 g) were added, and the reaction was carried out for 2 h. The reaction solution was filtered, concentrated, ethyl acetate (100 ml) was added, washed with 2N hydrochloric acid, washed with saturated sodium chloride, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a brown oily crude product (30 g). Column chromatography (n-hexane / dichloromethane 20:1 - 1:1) gave a yellow oily compound 1 (11 g) and compound 5 (10.5 g).

[0133] Compound 1 (11 g, 28.5 mmol, 1 eq) was dissolved in THF (100 ml), lithium hydroxide (3.6 g, 85 mmol, 3 eq) was dissolved in 15 ml of water, and the mixture was stirred overnight.

[0134] The pH was adjusted to 3 - 4 with 2N dilute hydrochloric acid, ethyl acetate (300 ml) was added, the layers were separated, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily compound 2 (9.5 g), which was directly used in the next step.

[0135] (2) Synthesis of Compound 3

[0136]

[0137] Experimental procedure:

[0138] Dissolve compound 2 (9.5 g, 24.6 mmol, 1 eq), methionine methyl ester hydrochloride (6.4 g, 32 mmol, 1.3 eq), and DMAP (4.5 g, 37 mmol, 1.5 eq) in dichloromethane (60 ml), add DCC (7.6 g, 37 mmol, 1.5 eq), and stir at room temperature overnight.

[0139] Filter the reaction solution, wash the filter cake with 50 ml of dichloromethane, wash the filtrate with 2N hydrochloric acid, saturated sodium chloride, dry over anhydrous sodium sulfate, filter, concentrate, and purify the crude product by column chromatography (n - hexane / dichloromethane: 5 / 1 - 1 / 1, n - hexane / dichloromethane / ethyl acetate: 1 / 1 / 0.1) to obtain compound 3 as a yellow oil (6.6 g).

[0140] (3) Synthesis of compound 4 (free acid)

[0141]

[0142] Experimental procedure:

[0143] Dissolve 6.6 g (0.0128 mol, 1 eq) of compound 3 in 80 ml of THF, dissolve 1.6 g (0.0383 mol, 3 eq) of lithium hydroxide in 10 ml of water and add it to the reaction solution, and react at room temperature overnight.

[0144] Concentrate the reaction solution, add 100 ml of ethyl acetate, adjust the pH to 2 with 2N hydrochloric acid, separate the layers, wash the organic layer with saturated sodium chloride, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 5.5 g of a yellow oil crude product. Purify the crude product by column chromatography (dichloromethane / methanol: 150 / 1 - 50 / 1) to obtain compound 4 as a yellow oil (3.4 g).

[0145] (4) Synthesis of compound E1 (sodium salt)

[0146]

[0147] Experimental procedure:

[0148] Dissolve compound 4 (3.4 g, 6.8 mmol, 1 eq) in 20 ml of ethanol, dissolve sodium bicarbonate (0.625 g, 7.4 mmol, 1.1 eq) in 10 ml of water, mix and stir, and heat to 50 °C for 1 h.

[0149] Concentrate the reaction solution, add 30 ml of ethanol and evaporate to dryness, wash the residue with a mixed solvent of 60 ml of n - hexane and 20 ml of dichloromethane and dry to obtain pink solid E1 (2.4 g), HPLC > 98%.

[0150] MS (ESI, positive): 504.1 [M+H]+.

[0151] 1 1H-NMR (400 MHz, CD3OD): δ 6.30 (d, J = 0.8 Hz, 1H), 6.20 (s, 1H), 5.29 (s, 1H), 4.55 - 4.36 (m, 5H), 4.03 - 4.05 (m, 1H), 2.97 - 2.91 (m, 1H), 2.46 (t, J = 7.6 Hz, 2H), 2.42 - 2.36 (m, 2H), 2.26 - 2.16 (m, 2H), 2.05 (s, 3H), 2.04 - 1.91 (m, 2H), 1.80 - 1.74 (m, 2H), 1.63 (s, 3H), 1.561 (s, 3H), 1.61 - 1.53 (m, 2H), 1.37 - 1.32 (m, 4H), 0.90 (t, J = 6.8 Hz, 3H).

[0152] It was detected that the solubility of compound E1 in water reached 2.7 g / 100 ml of water.

[0153] Example 2: Synthesis of compound E2

[0154]

[0155] 1. Synthetic route

[0156]

[0157] 2. Synthetic method

[0158] (1) Synthesis of compound 6

[0159] Dissolve compound 5 (10.5 g, 0.0229 mol, prepared in Example 1) in 100 ml of methanol, add potassium hydroxide (3.8 g, 0.0688 mol), stir, and reflux for 1 h. Concentrate the reaction solution, wash the residue with 100 ml of ethyl acetate, and filter. Add the filter cake to 200 ml of ethyl acetate, adjust the pH to 2 with 2N dilute hydrochloric acid, and separate the layers. Wash the organic phase with saturated sodium chloride, dry over anhydrous sodium sulfate, filter, and concentrate to obtain yellow oily compound 6 (9 g). MS (ESI); 431 [M+H] +

[0160] (2) Synthesis of compound 7

[0161]

[0162] Compound 6 (6.3 g, 0.0145 mol), methionine methyl ester hydrochloride (11.7 g, 0.0586 mol), and DMAP (7.15 g, 0.0586 mol) were dissolved in 200 ml of dichloromethane. DCC (12 g, 0.0586 mmol) was added, and the reaction was carried out overnight at room temperature. The reaction mixture was filtered, and the filter cake was washed with 50 ml of dichloromethane. The filtrate was successively washed with 2N hydrochloric acid, saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After concentration, column chromatography (n - hexane:dichloromethane = 5:1 - 1:1, n - hexane:dichloromethane:ethyl acetate = 1:1:0.1) gave the yellow oily compound 7 (6 g). MS(ESI): 721[M + H] +

[0163] (3) Synthesis of compound 8

[0164]

[0165] Compound 7 (10 g, 0.014 mol, 1 eq) was dissolved in 80 ml of THF. A solution of lithium hydroxide (1.7 g, 0.042 mol, 3 eq) in 8 ml of water was added, and the reaction was carried out overnight at room temperature. The reaction mixture was concentrated, dried once with 100 ml of ethanol, and the residue was washed with n - hexane. After concentration, 100 ml of ethyl acetate was added, and the pH was adjusted to 2 with 2N hydrochloric acid. The layers were separated, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After concentration, the yellow solid compound 8 (10 g) was obtained. MS(ESI): 693[M + H] +

[0166] (4) Synthesis of compound E2

[0167]

[0168] Compound 8 (10 g, 0.0144 mol) was dissolved in 80 ml of ethanol. Sodium bicarbonate (2.5 g, 0.0304 mol, 2.1 mol) was dissolved in 20 ml of water, and the two solutions were mixed and stirred. The reaction was carried out at 50 °C for 1 h. The reaction mixture was concentrated, 100 ml of ethanol was added and evaporated to dryness. The residue was washed with 20 ml of dichloromethane and dried to give the off - white solid E2 (8.1 g). MS(ESI): 693[M + H] + ; 1H-NMR(CD3OD): 6.48(s, 2H), 5.32(s, 1H), 4.62 - 4.55(m, 4H), 4.44 - 4.39(m, 4H), 4.13 - 4.18(m, 1H), 4.15((d, 1H), 3.01 - 2.95(m, 1H), 2.58 - 2.54(t, 2H), 2.42 - 2.38(t, 4H), 2.17 - 2.19(m, 2H), 2.03(s, 6H), 1.99 - 1.95(m, 3H), 1.84 - 1.81(t, 2H), 1.72(s, 3H), 1.66(s, 3H), 1.64 - 1.60(m, 2H), 1.39 - 1.33(m, 4H), 0.94 - 0.91(t, 3H)

[0169] It was detected that the solubility of compound E2 in water reached 20 g / 100 ml of water.

[0170] Example 3: Anti-epileptic activity study

[0171] 1. Experimental animals and some reagents

[0172] Wistar rats, SPF grade, weighing 240 - 260 g, 6 - 8 weeks old, female, purchased from Shanghai Slake Experimental Animal Co., Ltd. The breeding environment temperature was maintained at 22 ± 1 °C, the humidity was maintained at 65 - 70%, and the 12-hour light / dark cycle was adopted, and they could drink water freely.

[0173] PTZ, purchased from Sigma; Sodium valproate, purchased from Sigma; Absolute ethanol, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0174] 2. Experimental grouping

[0175] The experimental animals were randomly divided into 4 groups: model group, positive drug group, compound 1 group, and compound 2 group, with 8 rats in each group.

[0176] 3. Experimental methods

[0177] (1) Intraperitoneal administration for modeling

[0178] After all rats were adaptively fed for 1 week, modeling began. A 1.75% PTZ solution was freshly prepared with physiological saline, and the prepared PTZ solution was intraperitoneally injected at a dose of 35 mg / kg. PTZ was injected once every other day, and the behavioral changes of rats were observed within 30 minutes after PTZ injection, and the seizure grades were recorded.

[0179] After continuously injecting PTZ 11 times during the experiment, the seizure grades were recorded. For all rats that had continuously had 5 records of grade 2 or above, the models of all rats were successful after continuously injecting PZT 11 times.

[0180] (2)Gavage administration for treatment

[0181] After the model was successfully established, the rats were randomly grouped. Treatment began. All Wistar rats were weighed to calculate the gavage dose. The model group was gavaged with an equal volume of normal saline; the positive drug group (sodium valproate was gavaged at 100 mg / kg); the compound 1 group (compound E1 prepared in Example 1 was gavaged at 100 mg / kg); the compound 2 group (compound E2 prepared in Example 2 was gavaged at 100 mg / kg).

[0182] (3)Ono's grading

[0183] Before drug administration, on the 7th and 14th days after drug administration, 1 h after gavage administration, a prepared PTZ solution was intraperitoneally injected at a dose of 35 mg / kg. The behavioral changes of the rats were observed within 30 min after the injection of PTZ.

[0184] Record the behavioral seizure indices before and after drug administration. According to Ono's grading, record the seizure latency, seizure grade, and seizure duration.

[0185] Table 1 Ono's grading

[0186] Ono's grading Seizure manifestation 0 Non-convulsive 1 Nodding or head twitching 2 Generalized muscle clonus 3 Head twitching plus forelimb clonus 4 Clonic seizure plus hindlimb standing 5 Falling 6 Generalized tonic-clonic seizure

[0187] After the experiment, the brains and hippocampi of the experimental animals were collected and stored at -80 °C.

[0188] 4. Detection of the expression of apoptosis-related proteins Bax and Bcl-2 in the hippocampal tissues of rats by WB:

[0189] 4.1 Reagents, antibodies, and instruments

[0190] Some of the reagents, antibodies, and instruments used in the experiment are shown in Tables 2 - 4 respectively.

[0191] Table 2 Reagents

[0192]

[0193]

[0194] Table 3 Antibodies

[0195]

[0196] Table 4 Instruments

[0197]

[0198] 4.2 Sample preparation

[0199] Cut the hippocampal tissue into small pieces, and add lysis buffer (with protease and phosphatase inhibitors added) according to the ratio of 150 - 250 μl of lysis buffer per 20 mg of tissue. Homogenize with a homogenizer until complete lysis. Centrifuge the lysed sample at 12,000 g for 15 minutes at 4°C, take the supernatant, perform protein quantification, and store it in a -80°C refrigerator.

[0200] 4.3 Protein Quantification

[0201] (1) Preparation of the standard curve: Take an ELISA plate and add reagents according to the following table;

[0202] Table 5 Reagents

[0203] Well number 1 2 3 4 5 6 7 8 Protein standard solution (μl) 0 2 4 6 8 12 16 20 Deionized water (μl) 20 18 16 14 12 8 4 0 Protein concentration (μg / μl) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5

[0204] (2) According to the number of samples, prepare an appropriate amount of BCA working solution by mixing BCA reagent A and reagent B at a volume ratio of 50:1, and mix well;

[0205] (3) Add 160 μl of BCA working solution to each well;

[0206] (4) Place the ELISA plate on an oscillator and shake for 30 sec, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm; Use the absorbance value as the abscissa and the protein concentration (μg / μl) as the ordinate to plot the standard curve;

[0207] (5) Add 2 μl of the protein to be measured and 18 μl of PBS (diluted 10 - fold) to the ELISA plate, add 160 μl of BCA working solution, place the ELISA plate on an oscillator and shake for 30 sec, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm;

[0208] (6) According to the absorbance value of the measured sample, the corresponding protein concentration (μg / μl) can be obtained from the standard curve, and multiplying by the sample dilution factor (10) gives the actual sample concentration (unit: μg / μl).

[0209] 4.4 Loading and Electrophoresis

[0210] (1) Loading

[0211] The loading volume per well is approximately 25 μg of protein, and the loading volume can be increased according to experimental requirements. Take the required protein according to the protein quantification result, add an appropriate amount of loading buffer, centrifuge after boiling water bath for 10 min, and take the supernatant for loading. Place the prepared PAGE gel in the electrophoresis tank, add an appropriate amount of electrophoresis buffer, remove the comb, and gently blow the loading wells with a pipette to avoid residual gel in the wells affecting loading. Slowly add the prepared sample to the corresponding wells with a pipette gun, taking care not to overflow the loading wells.

[0212] (2) Electrophoresis

[0213] For the general stacking gel, run at 80 V for 20 minutes, and for the separating gel, run at 120 V for 60 minutes. The voltage and time can be adjusted according to the specific experimental requirements. When the dye reaches the bottom of the gel, cut off the power supply, stop the electrophoresis, and proceed to the next step of membrane transfer.

[0214] Membrane transfer

[0215] Membrane transfer is divided into wet transfer and semi-dry transfer. In this experiment, semi-dry transfer is selected.

[0216] In semi-dry transfer, the sandwich arrangement is: / filter paper / gel / membrane / filter paper. After wetting with the electrotransfer buffer, place it directly between the positive and negative electrodes of the electrotransfer apparatus. The gel is placed at the negative electrode and the membrane at the positive electrode. The semi-dry electrotransfer buffer is different from the wet electrotransfer buffer. It is recommended to be: 48 mM Tris, 39 mM glycine, 0.04% SDS, 20% methanol. Transfer at 25 V for 30 minutes. Before membrane transfer, soak the PVDF membrane in methanol for 1 - 2 minutes (soak the NC membrane in the electrotransfer solution for 10 - 20 minutes), then incubate it in the ice-cold electrotransfer buffer for 5 minutes. The gel also needs to be equilibrated in the ice-cold electrotransfer buffer for 3 - 5 minutes, otherwise band distortion will occur during membrane transfer.

[0217] Detection of proteins on the membrane

[0218] To detect whether the membrane transfer is successful, Ponceau S staining can be used. The working solution of Ponceau S staining: Dilute the 2% Ponceau S stock solution 1:10, that is, add 9 times of ddH2O.

[0219] Staining method: Wash the membrane once with TBST, then place it in the Ponceau S staining working solution, shake and stain at room temperature for 5 minutes. Wash the membrane with a large amount of water until the water becomes clear and colorless and the protein bands are clear. (The membrane can also be washed with TBST or water again before staining). The PVDF membrane needs to be reactivated with methanol and then washed with TBST before blocking.

[0220] 4.5 Blocking of the membrane and antibody incubation

[0221] (1) Blocking: Block with 5% non-fat milk powder (use BSA for detecting phosphorylated proteins) at room temperature for 1 hour or overnight at 4°C.

[0222] (2) Primary antibody: Dilute the antibody according to the instruction manual Bcl-2 1:500 Bax 1:5000 GAPDH 1:30000 Dilute the antibody into the blocking solution to the required concentration, and incubate with the membrane at room temperature for 2 hours or overnight at 4°C.

[0223] (3) Secondary antibody: Wash the membrane incubated with the primary antibody 3 times with TBST, 5 minutes each time. Subsequently, dilute the HRP-labeled secondary antibody 1:1000 according to the amount used, and incubate with the membrane at 37°C for 1 hour. Wash with TBST 3 times, 5 minutes each time.

[0224] 4.6 Color development

[0225] ECL chemiluminescence detection: Prepare the ECL luminescence solution. According to the dosage, take equal amounts of ECL luminescence solution A and B, mix them evenly, add them to the front side of the membrane, and keep it in the darkroom away from light for 5 minutes. Pour out the developing solution, carefully absorb the developing solution with paper, and cover it with a flat transparent paper on it. Put it into the imaging system for scanning.

[0226] 5. Detection of the contents of γ-aminobutyric acid, glutamate, glycine, and aspartic acid in rat brain tissues by kit method

[0227] 5.1 Experimental materials

[0228] The kits and instruments used in the experiment are shown in the following table.

[0229] Table 6 Kit information

[0230]

[0231] Table 7 Main instruments and consumables

[0232]

[0233] 5.2 Experimental methods

[0234] Refer to the kit instruction manual for the experimental operation and methods.

[0235] 6. Experimental results

[0236] (1) The seizure durations of PTZ-kindled epileptic rats before and after treatment are as shown in the following table and Figure 1 as follows.

[0237] Table 8 Seizure durations of PTZ-kindled epileptic rats before and after treatment

[0238] Grouping Duration before treatment / s Duration after 7 days of treatment / s Duration after 14 days of treatment / s Model group 338.33±27.16 327.50±12.01 311.83±29.02 Positive drug group 347.25±44.10 200.29±18.43 178.71±16.96 Compound 1 group 359.83±14.41 290.50±66.93 299.67±39.25 Compound 2 group 358.57±40.33 280.17±65.01 250.33±48.64

[0239] The results show that: for the seizure durations of PTZ-kindled epileptic seizures before and after treatment, by comparing the data, it is found that the durations of each group before treatment did not change, and the continuous seizure duration was about 350 s; as the treatment stage entered, obvious differences appeared in each group. The seizure duration in the positive drug group decreased at 7 days and 14 days of treatment, and it was statistically significant. The seizure duration in other compound groups also decreased significantly compared with that before treatment at 7 days of treatment, and it was statistically significant; among them, the seizure duration of compound 2 decreased compared with that at 7 days of treatment at 14 days of subsequent treatment, and it was statistically significant; the seizure duration of compound 1 did not change compared with that at 7 days of treatment at 14 days of treatment.

[0240] (2) The Ono's classification of the behavioral seizure index of PTZ-kindled epileptic rats before and after treatment is as shown in the following table and Figure 2As shown

[0241] Table 9 Ono's grading of behavioral seizure indicators in PTZ-kindled epileptic rats before and after treatment

[0242] Grouping Ono's grading before treatment Ono's grading after 7 days of treatment Ono's grading after 14 days of treatment Model group 3.67±0.52 3.50±0.55 3.67±0.52 Positive drug group 3.38±0.52 3.43±0.53 3.57±0.79 Compound 1 group 3.17±0.41 3.33±0.52 3.33±0.52 Compound 2 group 3.29±0.49 3.17±0.41 3.50±0.84

[0243] The results showed that the behavioral seizure indicators before and after treatment were recorded according to Ono's grading. After the model was established and during the treatment period, when the PTZ-kindled epilepsy model was induced by injecting PTZ, the Ono's grading of the seizures was between grades 3 and 4, and there was no significant difference among the groups. After 14 days of treatment, there was also no significant inter-group difference

[0244] During the experiment, the highest Ono's grading recorded was the highest grade of epileptic seizures in rats. During the experiment, epileptic seizures were a progressive process, and the highest seizure grade had a duration during epileptic seizures. It is recommended to record the duration of each seizure grade in the follow-up

[0245] During the experiment, it was found that when the Ono's grading was in the range of 5 - 6, if no measures were taken, most rats would die, and if the rats did not die, they would also die under PTZ-kindling

[0246] (3) The seizure latency of PTZ-kindled epileptic rats before and after treatment is as shown in the following table and Figure 3 As shown

[0247] Table 10 Seizure latency of PTZ-kindled epileptic rats before and after treatment

[0248] Grouping Latency before treatment / s Latency after 7 days of treatment / s Latency after 14 days of treatment / s Model group 99.14±15.52 87.67±16.82 90.00±22.73 Positive drug group 97.38±15.86 87.00±13.90 84.43±14.77 Compound 1 group 85.75±21.45 82.83±20.90 87.00±20.96 Compound 2 group 99.00±1914 80.50±17.51 81.17±21.31

[0249] The results showed that for the seizure latency of PTZ-kindled epilepsy before and after treatment, the mean seizure latency of PTZ-kindled epilepsy was 80 s - 100 s during the pre-treatment and treatment periods. The seizure latency data indicated that the time of PTZ-kindled epilepsy was between 60 s and 120 s. There was no obvious correlation among the groups, and there was also no obvious trend of change in the seizure latency within the same group over time

[0250] (4) The results of WB detection of the changes in apoptosis-related proteins Bax and Bcl-2 in the hippocampal tissues of rats are as Figure 4 and 5 As shown

[0251] The experimental results showed that the pro-apoptotic related Bax protein was highly expressed in the hippocampal tissues of rats in the model group, while the pro-apoptotic related Bax protein was down-regulated in the positive drug group compared with the model group, and it was statistically significant. Both compound E1 and compound E2 showed a significant down-regulation of Bax protein expression, which was statistically significant

[0252] Conversely, the anti-apoptotic Bcl-2 protein in the hippocampal tissues of rats in each group was detected. The experimental results showed that the expression of Bcl-2 protein in the model group was significantly lower than that in other groups. The expression of Bcl-2 protein in the positive drug group was higher than that in other groups. In the compound group, the expressions of compound E1 and compound E2 were significantly higher than that in the model group, and the differences were statistically significant.

[0253] (5) The determination results of the contents of γ-aminobutyric acid, glutamate, glycine, and aspartic acid in the homogenate of rat brain tissues are shown in the following table and Figures 6 - 9 as follows.

[0254] Table 11 Determination results of the contents of γ-aminobutyric acid, glutamate, glycine, and aspartic acid in the homogenate of rat brain tissues

[0255]

[0256]

[0257] The contents of excitatory amino acids: glutamate and aspartic acid, and inhibitory neurotransmitters: γ-aminobutyric acid and glycine in the homogenate of brain tissues were detected by an ELISA detection kit.

[0258] The ELISA experimental results showed that the contents of γ-aminobutyric acid and glycine in the homogenate of brain tissues in the model group were low, while the contents of glutamate and aspartic acid were high.

[0259] Compared with the model group, the expression levels of excitatory amino acids in the positive drug group and the compound group decreased, and the differences were statistically significant; the expression levels of the inhibitory neurotransmitters γ-aminobutyric acid and glycine increased compared with the model group, and the differences were statistically significant.

[0260] 7. Experimental summary

[0261] During the treatment of the PTZ-kindled rat epilepsy model, there were significant differences in the treatment effects of the positive drug and each compound on rats in terms of the duration of PTZ-kindled epilepsy; there were trends in the latency period and the degree of epilepsy model, but there were no significant differences.

[0262] Compared with the model group, the seizure durations on the 7th and 14th days of treatment in the two compound groups were significantly reduced, and the differences were statistically significant. Among them, on the 7th day of treatment, the seizure durations were in the order of compound E2 > compound E1; on the 14th day of treatment, the seizure durations were in the order of compound E2 > compound E1.

[0263] By comparing the apoptosis-related proteins Bax and Bcl-2 in the hippocampal tissues of rats on the 14th day of treatment, and the excitatory amino acids: glutamate and aspartate, and the inhibitory neurotransmitters: γ-aminobutyric acid and glycine in the brain tissue homogenate.

[0264] Example 4: Treatment research on Parkinson's disease

[0265] 1. Experimental animals and some reagents

[0266] C57 / BL6 mice, SPF grade, weighing 18 - 20 g, 6 - 8 weeks old, male, purchased from Shanghai Slake Experimental Animal Co., Ltd. The breeding environment temperature is maintained at 22 ± 1 °C, the humidity is maintained at 65 - 70%, and the 12-hour light / dark cycle is adopted, and they can drink water freely.

[0267] MPTP, purchased from Sigma; amantadine, purchased from Sigma; absolute ethanol, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0268] 2. Experimental grouping

[0269] The experimental animals were randomly divided into 5 groups: blank group, model group, positive drug group, compound 1 group, and compound 2 group, with 8 animals in each group.

[0270] 3. Experimental methods

[0271] (1) Purchase 6 - 8-week-old SPF-grade C57 mice, adaptively breed them for one week, and then start modeling.

[0272] (2) Inject MPTP (25 mg / kg) intraperitoneally once a day for 7 consecutive days. The blank group was not treated.

[0273] (3) After the modeling was completed, drug administration began. The positive drug group was intragastrically administered amantadine (40 mg / kg), and the test substance group (100 mg / kg) was intragastrically administered the determined drug dose once a day for 14 consecutive days.

[0274] (4) First, conduct behavioral tests: balance beam test and scoring

[0275] A wooden strip with a length of 80 cm and a width of 2.5 cm is horizontally fixed at a height of 10 cm from the tabletop, and then the animals are allowed to walk on the wooden strip.

[0276] Can jump onto the balance beam, can walk freely without falling: 0 points; Can jump onto the balance beam, the animal will fall when walking on it, but the chance of falling is less than 50%: 1 point; Can jump onto the balance beam, the animal will fall when walking on it, but the chance of falling is greater than 50%: 2 points; The animal can jump onto the balance beam with the help of the normal side of the body; however, the hind limb on the paralyzed side cannot help the body move forward: 3 points; The animal cannot walk on the balance beam, but can sit on the beam: 4 points; Place the animal on the beam, and the animal will fall off quickly: 5 points.

[0277] (5) After the scoring is completed, the sample collection begins

[0278] After quickly sacrificing the mouse, take the mouse brain and isolate the cerebral cortex (CP) of the mouse. After exposing the striatum of the mouse, take the striatum, weigh it, add PBS according to the proportion, and then grind it into a homogenate. Then separate the supernatant to detect the dopamine content in the striatum.

[0279] 4. Detection of dopamine content in the striatum by kit method

[0280] 4.1 The kits and some instruments are shown in the following table.

[0281] Table 12 Kit information

[0282]

[0283] Table 13 Main instruments and consumables

[0284]

[0285] 4.2 Protein quantification

[0286] (1) Drawing of the standard curve: Take an ELISA plate and add reagents according to the following table;

[0287] Table 14 Reagents

[0288] Well number 1 2 3 4 5 6 7 8 Protein standard solution (μl) 0 2 4 6 8 12 16 20 Deionized water (μl) 20 18 16 14 12 8 4 0 Protein concentration (μg / μl) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5

[0289] (2) According to the number of samples, prepare an appropriate amount of BCA working solution by mixing BCA reagent A and reagent B at a volume ratio of 50:1, and mix well;

[0290] (3) Add 160 μl of BCA working solution to each well;

[0291] (4) Place the ELISA plate on an oscillator and shake for 30 sec, incubate at 37 °C for 30 minutes, and then measure the absorbance at 562 nm. Use the absorbance value as the abscissa and the protein concentration (μg / μl) as the ordinate to draw the standard curve;

[0292] (5) Add 2 μl of the protein to be tested and 18 μl of PBS (diluted 10 times) into the ELISA plate, then add 160 μl of BCA working solution. Place the ELISA plate on an oscillator and shake for 30 sec, incubate at 37 °C for 30 minutes, and then measure the absorbance at 562 nm.

[0293] (6) According to the absorbance value of the measured sample, the corresponding protein concentration (μg / μl) can be obtained from the standard curve, and then multiplied by the sample dilution factor (10) to get the actual concentration of the sample (unit: μg / μl).

[0294] (7) The operation and method of the mouse dopamine detection experiment refer to the kit instruction manual.

[0295] 5. Immunohistochemical determination of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain

[0296] 5.1 Experimental materials

[0297] Table 15 Main reagents

[0298]

[0299] Table 16 Main instruments and consumables

[0300]

[0301]

[0302] Solution preparation

[0303] (1) PBS solution

[0304] Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 in 600 ml of ddH2O, adjust the pH of the solution to 7.4 with HCl, make up the volume to 1 L, filter through a filter, autoclave, and store at room temperature.

[0305] (2) 0.1 mol / L sodium citrate buffer solution

[0306] 3.8 ml of 0.1 mol / L citric acid and 16.2 ml of 0.1 mol / L sodium citrate.

[0307] Citric acid C6H8O7·H2O: molecular weight 210.14; 0.1 mol / L solution is 21.01 g / L.

[0308] Sodium citrate Na3C6H5O7·2H2O: molecular weight 294.12; 0.1 mol / L solution is 29.41 g / ml.

[0309] 5.2 Experimental method

[0310] 5.2.1 Sample Embedding and Fixation

[0311] (1) Tissue Sampling and Fixation

[0312] When cutting tissues, sharp knives and scissors should be used. When cutting tissue blocks, start from the root of the knife and pull backward to cut the tissue. The thickness of the tissue block is about 0.2 - 0.3 cm, and the size is preferably 1.5 cm × 1.5 cm × 0.3 cm. The obtained tissue block is placed in 10% formalin for fixation for 48 hours.

[0313] (2) Washing and Dehydration

[0314] The fixed tissue is rinsed with running water to remove residual fixative and impurities. Dehydration is carried out step by step with ethanol of different concentrations, 50%, 70%, 85%, 95% up to absolute alcohol (anhydrous ethanol), 2 hours for each level. Dehydration must be carried out in a covered bottle to prevent high-concentration ethanol from absorbing moisture in the air, resulting in a decrease in concentration and incomplete dehydration. Materials to be preserved can be dehydrated to 70% ethanol and left in it. If long-term preservation is required, an equal amount of glycerol can be added.

[0315] (3) Clearing

[0316] The tissue block is placed in an equal-volume mixture of absolute alcohol and xylene for 2 hours, then into pure xylene for two hours, and again into pure xylene for two hours. After the material is cleared, it will show how the dehydration in the previous step was. If the dehydration is complete, the tissue will appear transparent. If there are white cloudy substances in the tissue, it means the dehydration is not complete and rework is required, but the rework effect is often not good. When using xylene for clearing, its volatilization and absorption of moisture in the air should be avoided, and its anhydrous state should be maintained.

[0317] (4) Impregnation with Wax

[0318] Impregnation with wax must be carried out in an incubator. First, place the tissue material block in an equal-volume mixture of melted paraffin and xylene for 1 - 2 hours, and then transfer it successively into 2 melted paraffin liquids for impregnation for about 3 hours each.

[0319] (5) Embedding

[0320] Embedding is to wrap the tissue block impregnated with wax in paraffin. The specific method is as follows: First, prepare a paper box, pour the melted wax into the box, quickly pick up the tissue block with pre-warmed forceps and place it flat on the bottom of the paper box, with the cut surface facing down. Then gently lift the paper box and place it flat in cold water. After the surface paraffin solidifies, immediately press the paper box into the water to make it cool and solidify quickly, and take it out after 30 minutes.

[0321] (6) Sectioning

[0322] Before sectioning, place the wax block in a -20°C refrigerator for at least 30 minutes to increase its hardness. Install the microtome knife on the knife holder of the microtome and fix it tightly. Fix the wax block base or the wax block, adjust the wax block and the knife to the appropriate positions, and make the cutting edge form a 5-degree angle with the surface of the wax block. Adjust the section thickness on the microtome to 4 - 7 μm, and then section.

[0323] 5.2.2 Immunohistochemical staining

[0324] (1) Baking and dewaxing

[0325] Place the glass slide in a 65°C constant temperature oven for baking for 30 minutes; soak it in xylene I for 15 minutes, and then soak it in xylene II for 15 minutes.

[0326] (2) Hydration

[0327] Soak the dewaxed sections in 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol for 5 minutes respectively, and rinse with tap water for 10 minutes.

[0328] (3) Antigen retrieval

[0329] Perform high-pressure retrieval in 0.01M sodium citrate buffer solution for 15 minutes. After natural cooling, wash with 0.02M PBS for 3 minutes × 3 times.

[0330] (4) Blocking

[0331] Place the glass slide in 3% H2O2, incubate in a humidified box for 10 minutes to eliminate the activity of endogenous peroxidase. Rinse with 0.02M PBS for 3 minutes × 3 times.

[0332] (5) Primary antibody incubation

[0333] Dropwise add the primary antibody (diluted 1:2000), incubate in a humidified box, and place it at room temperature for 1 hour (or incubate overnight at 4°C). Rinse with 0.02M PBS for 3 minutes × 3 times.

[0334] (6) Secondary antibody incubation

[0335] Dropwise add the HRP-labeled broad-spectrum secondary antibody, incubate in a humidified box, and place it at room temperature for 20 - 30 minutes. Rinse with 0.02M PBS for 3 minutes × 3 times.

[0336] (7) Chromogenic reaction

[0337] Perform DAB staining. When color change is observed in the sections, immediately wash off the staining solution with tap water.

[0338] (8) Hematoxylin staining

[0339] Counterstain with hematoxylin for 3 min, differentiate with 1% hydrochloric acid alcohol, observe under the microscope, and control the staining degree. Rinse with tap water for 10 min, and dry the moisture in an oven at 65 °C.

[0340] (9) Clearing and mounting

[0341] Place the slides in xylene for clearing for 3 min × 2 times, mount with neutral balsam, and place in an oven at 65 °C for 15 min.

[0342] 5.2.3 Image acquisition and analysis

[0343] Take pictures through the microscope, collect and analyze the relevant parts of the samples, and calculate the positive area.

[0344] 6. Experimental results

[0345] (1) The results of the balance beam score in the Parkinson's model behavioral test are shown in the following table and Figure 10 as follows.

[0346] Table 17 Results of the balance beam score in the Parkinson's model behavioral test

[0347] Grouping After model establishment After treatment Blank group 0 0 Model group 4.63±0.52*** 4.50±0.53 Positive drug group 4.75±0.46*** 2.13±0.83### Compound 1 group 4.63±0.52*** 3.75±0.89# Compound 2 group 4.13±0.35*** 3.50±0.93##

[0348] The Parkinson's model was established by intraperitoneal injection of MPTP in mice, and the behavior was evaluated by the balance beam score. Seven days after the intraperitoneal injection of MPTP, the balance beam score was performed. Except for the blank group without modeling and the balance beam score of 0, the scores of other groups of mice were all between 5 and 6 points. The comparison of each group with the blank group showed P ≤ 0.05, which was statistically significant, proving that the model was successful.

[0349] Subsequently, after treatment with the positive drug and each compound group, the balance beam score of the model group was 4 - 5 points, and the balance beam score of the positive drug group was between 2 and 3 points. The balance beam scores of other compound treatment groups were 3 - 4 points. The comparison of the positive drug group and each compound group with the model group showed an obvious downward trend, with P ≤ 0.05, which was statistically significant.

[0350] The scores of each group after treatment were blank group < positive drug group < compound 2 group < compound 1 group < model group.

[0351] (2) The striatal dopamine content after treatment in the Parkinson's model is shown in the following table and Figure 11 as follows.

[0352] Table 18 Striatal dopamine content after treatment in the Parkinson's model

[0353] Grouping DA (ng / mg) Blank group 0.71±0.16 Model group 0.30±0.07*** Positive drug group 0.61±0.12### Compound 1 group 0.45±0.09## Compound 2 group 0.38±0.08#

[0354] Dopamine, as a neurotransmitter, regulates various physiological functions of the central nervous system. By detecting the dopamine content in the striatum of a mouse model of Parkinson's disease treated with drugs, the efficacy of the drugs in treating the Parkinson's model was judged.

[0355] By comparing the dopamine content of each group with that of the blank group, the degree of dopamine decline in the model was judged. Among them, except for the positive drug group, the dopamine content of other groups showed a significant decrease, P≤0.05, which was statistically significant.

[0356] Then, by comparing the dopamine content of the model group with that of other compound groups, the dopamine content of each group increased compared with the model group, P≤0.01, which was statistically significant, indicating that each drug group had the effect of increasing the dopamine content in the brain.

[0357] The dopamine content in the striatum was blank group > positive drug group > compound 1 group > compound 2 group > model group.

[0358] (3) The experimental results of immunohistochemical determination of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain are as follows in the table and Figure 12 and 13 as shown.

[0359] Table 19 Experimental results of immunohistochemical determination of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain

[0360]

[0361]

[0362] Tyrosine hydroxylase (TH) is a monooxygenase. It is the rate-limiting enzyme that catalyzes the first reaction in the series of reactions for the organism to synthesize L-Dopamine (DA) by itself and is only expressed in the cytoplasm. TH is abundant in neurons. Because the midbrain lacks dopamine-β-hydroxylase, the TH immunoreactive neurons in the midbrain are DA neurons and can be used as a marker of dopamine neurons in the brain. The body uses L-tyrosine and synthesizes L-DOPA under the catalysis of tyrosine hydroxylase. L-DOPA is finally decarboxylated under the catalysis of aromatic amino acid decarboxylase to generate L-Dopamine. Due to the important position of tyrosine hydroxylase in dopamine synthesis, the absence or insufficient expression of its function directly affects the synthesis and secretion of dopamine. Dopamine is an important neurotransmitter. The inability of dopaminergic neurons to synthesize or secrete insufficient dopamine will lead to Parkinson's disease.

[0363] The results of this experiment showed that in the mouse Parkinson's model induced by intraperitoneal injection of MPTP, the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the mice decreased, with P ≤ 0.05, showing statistical significance. Compared with the model group, the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the positive drug group increased significantly, indicating successful modeling.

[0364] After treating the mouse Parkinson's model with each compound, by comparing the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of each compound group with that of the model group, an increase was observed in all cases. Among them, compared with the model group, the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the compound 1 group had P ≤ 0.01 and showed statistical significance.

[0365] By comparing the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) after treatment in each group, the blank group > the positive drug group > the compound 1 group > the compound 2 group > the model group.

[0366] 7. Experimental summary

[0367] In this experiment, a mouse Parkinson's model was established by intraperitoneal injection of MPTP (25 mg / kg) once a day for 7 consecutive days, and the mouse Parkinson's model was screened by balance beam scoring.

[0368] Subsequently, the model mice were treated by gavage with positive drugs and each compound. After 14 consecutive days of gavage treatment, by analyzing the balance beam scores of each group after treatment, ELISA was used to detect the dopamine content in the striatum, and immunohistochemistry was used to detect the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) and other detection methods to identify the differences between each compound and the blank group and the model group, and then statistical analysis was performed to analyze the effectiveness of each compound.

[0369] Through the identification of the balance beam scores after treatment, compared with the model group, the positive drug group and each compound group showed an obvious downward trend, with P ≤ 0.05, showing statistical significance.

[0370] By comparing the dopamine content of other compound groups with that of the model group, the dopamine content of each group increased compared with the model group, with P ≤ 0.01, showing statistical significance.

[0371] After treating the mouse Parkinson's model with each compound and the positive drug, by comparing the positive rates of the positive drug group and each compound with that of the model group, among them, compared with the model group, the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the compound 1 group had P ≤ 0.01 and showed statistical significance.

[0372] By comprehensively analyzing each compound group and comparing it with the model group, all have a certain therapeutic effect. Comparing each detection index, compound 1 has a better therapeutic effect.

[0373] The above are only the 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 shall be included within the protection scope of the present invention.

[0374] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0375] Listing the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, the compound having the following structure: Wherein, X1 is a C1-6 straight or branched alkylene group; X2 has the following structure: where R7 is selected from: X3 is R1 is H; R2 is selected from: OH, C1-6 alkoxy; n is 1; R3 is selected from: H, Wherein, X4 is a C1-6 straight or branched alkylene group; X5 has the following structure: wherein R8 is selected from: X6 is R4 is H; R5 is selected from: OH, C1-6 alkoxy; m is 1.

2. The compound according to claim 1, characterized in that, X1 is methylene.

3. The compound according to claim 1, wherein X4 is methylene.

4. The compound according to claim 1, wherein R7 is 5. The compound according to claim 1, characterized in that, R8 is 6. The compound according to claim 1, wherein, R2 is OH, methoxy or ethoxy.

7. The compound according to claim 1, characterized in that, R5 is OH, methoxy or ethoxy.

8. The compound according to any one of claims 1-7, characterized in that, The compound has the following structure:

9. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, and one or more pharmaceutically acceptable excipients.

10. Use of the compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating a disease, wherein, The diseases are selected from one or more of: nervous system diseases, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver injury.

11. The application according to claim 10, characterized in that, The nervous system diseases are selected from: epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, anxiety disorder, depression.

12. The application according to claim 10, characterized in that, The autoimmune diseases are selected from: systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, autoimmune hepatitis.

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