Application of magnolol or honokiol derivative in preparation of medicine for treating neurodegenerative diseases and medicine composition

Abstract

The invention provides application of magnolol or honokiol derivatives in preparation of drugs for treating neurodegenerative diseases and a pharmaceutical composition, and belongs to the technical field of biological medicines. According to the application of the magnolol or the honokiol derivative in preparation of the medicine for treating the neurodegenerative diseases, the magnolol or the honokiol derivative is a compound shown in a general formula I, or a stereoisomer, a crystalline hydrate, a deuterated compound, a solvate, a prodrug or a pharmaceutically acceptable salt thereof. The medicine for treating the neurodegenerative diseases is prepared from magnolol or honokiol derivatives, the Parkinson's disease is relieved, and the application has important significance in research on the mechanism of neuronal degeneration of the Parkinson's disease and development of treatment medicines. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a use of magnolol or a magnolol derivative in preparing a medicine for neurodegenerative diseases and a pharmaceutical composition. Background Art

[0002] Neurological diseases, such as neurodegenerative diseases, epilepsy, chronic pain, mental disorders, etc., currently have great unmet medical needs. Parkinson's disease (PD) is a neurodegenerative disease, and there is currently no good treatment to change the course of the disease. PD is defined as a movement disorder with typical symptoms such as resting tremor, rigidity, bradykinesia, and postural instability. PD is the second most common age-related neurodegenerative disease. Due to the poor understanding of the disease and targets, the mechanism of PD neuronal degeneration has not been fully elucidated, and the use of drugs for neurological diseases is very challenging.

[0003] PD is associated with a progressive loss of dopaminergic neurons, leading to loss of dopamine and dysregulation of fine motor control located in the basal ganglia. Ultimately, the death of dopaminergic neurons manifests clinically as parkinsonian symptoms, including bradykinesia, myotonia, and resting tremor, and pathologically involves the presence of Lewy body aggregates composed of α-synuclein. At the cellular level, PD is associated with defects in multiple pathways, including mitochondrial and lysosomal dysfunction, protein accumulation, synaptic and axonal dysfunction, endoplasmic reticulum stress, and increased oxidative stress. Increasing evidence suggests that oxidative stress (OS) contributes to the degeneration of these dopaminergic neurons. OS is currently believed to play an important role in the pathogenesis of PD. Nuclear factor E2-related factor 2 (Nrf2) regulates the coordinated induction of detoxification and antioxidant enzymes by binding to AREs within the regulatory regions of target genes. Nrf2 regulates the coordinated expression of cytoprotective genes, such as heme oxygenase-1 (HO-1).

[0004] Magnolol and honokiol are the main active ingredients of the traditional Chinese medicine Magnolia officinalis.

[0005] The applicant disclosed in Chinese patent CN115581690A that magnolia officinalis or magnolia officinalis derivatives as shown in general formula I can be used as anti-myocardial ischemia related drugs, and the magnolia officinalis or magnolia officinalis derivatives can effectively protect the myocardium during myocardial ischemia and myocardial ischemia-reperfusion injury.

[0006]

[0007] The applicant disclosed in Chinese patent CN103113264A that magnolol and / or magnolol derivatives shown in formula I have anti-thrombotic activity and protective effects against cerebral ischemia-reperfusion injury.

[0008]

[0009] Wherein, in Formula I, R1, R2, R3, R4 or R5 is selected from hydrogen, hydroxyl, modified hydroxyl, amino, modified amino or amino hydrochloride; wherein, when R1, R4, R5 are hydrogen, R2 and R3 are not hydroxyl at the same time; when R1, R3, R4 are hydrogen, R2 and R5 are not hydroxyl at the same time.

[0010] However, there are currently no public reports on the use of magnolol or magnolol derivatives in the treatment of neurodegenerative diseases such as Parkinson's disease. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a use of magnolol or magnolol derivatives in the preparation of drugs for neurodegenerative diseases and a pharmaceutical composition, so as to alleviate the pathogenesis of Parkinson's disease and other neurodegenerative diseases.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] In one aspect, a use of magnolol or a magnolol derivative in the preparation of a drug for a neurodegenerative disease is provided, wherein the magnolol or the magnolol derivative is a compound represented by general formula I, a stereoisomer thereof, a crystalline hydrate thereof, a deuterated product thereof, a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof:

[0014]

[0015] In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups are optionally substituted by N, O, or S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen or hydroxyl groups at the same time; R5 is H, a C1-C8 acyl group, the remaining acyl portion of an amino acid after condensation at the carboxyl group, or the remaining acyl portion of a polypeptide after condensation at the carboxyl group, and R6 is a hydroxyl group.

[0016] Furthermore, the neurodegenerative diseases are: Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0017] Furthermore, the neurodegenerative disease is Parkinson's disease.

[0018] Furthermore, the C1-C8 hydrocarbon groups in R1 and R4 are each independently selected from any one of C1-C8 alkyl groups and C1-C8 alkenyl groups.

[0019] Further, the C1-C8 alkenyl in R1 and R4 is selected from any one of vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, but-3-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-5-enyl, hept-1-enyl, hept-2-enyl, hept-3-enyl, hept-4-enyl, hept-5-enyl, hept-6-enyl, oct-1-enyl, oct-2-enyl, oct-3-enyl, oct-4-enyl, oct-5-enyl, oct-6-enyl, and oct-7-enyl.

[0020] Furthermore, the C1-C8 acyl group in R5 is formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, octanoyl or heptanoyl.

[0021] Furthermore, the amino acid in R5 is lysine, methionine, tryptophan, valine, alanine, phenylalanine, leucine, isoleucine, 6-hydroxynorleucine, glycine, histidine, arginine, proline, glutamic acid, aspartic acid, serine threonine, tyrosine, cystine or cysteine.

[0022] Furthermore, the polypeptide in R5 is a peptide formed by multiple amino acids, and its molecular weight is ≤2500Da.

[0023] Further, magnolol or magnolol derivatives are one or more of the following compounds: 3',5-diallyl-3-amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-acetylamino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-3-phenyl-2-amino-1-propionyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(R)-2,6-diamino-1-hexanoyl]amino ]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-6-hydroxy-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-4-methylthio-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-3-methyl-2-amino-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl, and 3',5-dipropyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl.

[0024] Preferably, the magnolol or magnolol derivative is one or more of the following compounds: 3',5-diallyl-3-amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-4-methylthio-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl and 3',5-dipropyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl.

[0025] In a second aspect, the present invention provides a pharmaceutical composition for treating neurodegenerative diseases, comprising magnolol or a magnolol derivative and a pharmaceutically acceptable carrier; wherein magnolol or a magnolol derivative is a compound represented by general formula I:

[0026]

[0027] In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups are optionally substituted by N, O, or S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen and hydroxyl groups at the same time; R5 is H, a C1-C8 acyl group, the remaining acyl portion of an amino acid after condensation at the carboxyl group, or the remaining acyl portion of a polypeptide after condensation at the carboxyl group.

[0028] In a third aspect, the present invention provides a pharmaceutical preparation for treating neurodegenerative diseases, comprising magnolol or magnolol derivatives, stereoisomers thereof, crystalline hydrates thereof, deuterated products thereof, solvates thereof, prodrugs thereof or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers; the magnolol or magnolol derivatives are compounds represented by general formula I:

[0029]

[0030] In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups are optionally substituted by N, O, and S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen and hydroxyl groups at the same time; R5 is H, a C1-C8 acyl group, the remaining acyl portion of an amino acid after condensation at the carboxyl group, or the remaining acyl portion of a polypeptide after condensation at the carboxyl group.

[0031] Furthermore, the pharmaceutical preparation is a tablet, a pill, a cream, an emulsion, an ointment, a suspension, a lyophilized agent, a capsule, a sustained-release agent, a granule, an granule, an injection or a spray.

[0032] The beneficial effects of the present invention are:

[0033] (1) The magnolol or magnolol derivative is combined with a pharmacologically acceptable excipient or carrier to form a brain protective drug for treating neurodegenerative diseases such as Parkinson's disease.

[0034] (2) Magnolol or its derivatives can effectively protect dopaminergic neurons and other brain tissue cells, thereby alleviating the progression of Parkinson's disease and other neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 The results of TUNEL staining analysis in Application Example 1 of the present invention and the effect of compound 1 on cell apoptosis in the 6-OHDA-induced PD cell model are shown. Red fluorescence indicates TUNEL-positive cells, and DAPI-labeled cell nuclei show blue fluorescence. Scale bar: 240 μm, wherein the group below the black horizontal line is treated with 100 μM 6-OHDA.

[0037] Figure 2 The results of Western blot analysis of the antioxidant effect of compound 2 on the 6-OHDA-induced PD cell model in Application Example 2 of the present invention are shown.

[0038] Figure 3 The results of JC-1 staining analysis of the antioxidant effect of compound 2 on the 6-OHDA-induced PD cell model in Application Example 3 of the present invention are shown. Scale bar: 240 μm.

[0039] Figure 4 The results of Western blot analysis of the antioxidant effects of Compound 3 and Compound 4 on the 6-OHDA-induced PD cell model in Application Example 4 of the present invention are shown.

[0040] Figure 5 The results of the test on the ABTS scavenging ability of compound 2 in Application Example 5 of the present invention are shown. DETAILED DESCRIPTION

[0041] Definition of terms

[0042] Unless otherwise indicated, the following terms and phrases as used herein have the following meanings:

[0043] In the present invention, "room temperature" refers to ambient temperature, which is from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature from 20°C to 30°C; in other embodiments, "room temperature" refers to a temperature from 25°C to 30°C; in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0044] In the present invention, "one or more" means one or two or more.

[0045] "Stereoisomers" refer to compounds with the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0046] The compound shown in Formula I of the present invention may have a chiral center, such as a chiral carbon. The compound therefore includes a racemic mixture of all stereoisomers, including enantiomers, diastereomers and atropisomers. In addition, the compound of the present invention includes an optical isomer enriched or split at any or all asymmetric chiral atoms. In other words, the chiral center similar to the description is provided in the form of a chiral isomer or a racemic mixture. A mixture of racemic and diastereoisomers, as well as a separate or synthesized, substantially free of its enantiomer or diastereoisomer partner, is within the scope of the present invention. The racemic mixture is separated into their separate, substantially optically pure isomers by known techniques, such as separation of diastereoisomer salts formed with optically active auxiliary agents (such as acids or bases), which are then converted back to optically active substances. In most cases, starting from the appropriate stereoisomer of the desired raw material, the desired optical isomer is synthesized by stereospecific reactions.

[0047] "Hydrate" refers to an association formed when the solvent molecule is water.

[0048] "Deuterated" means that one or more hydrogen atoms in a compound or group are replaced by deuterium. Deuterated can be monosubstituted, disubstituted, polysubstituted or fully substituted.

[0049] "Solvate" refers to an association formed between one or more solvent molecules and the compounds of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid and aminoethanol.

[0050] "Prodrug" is used in its broadest sense and includes those derivatives that are converted into the compounds of the present invention in vivo. These derivatives are obvious to those skilled in the art. When designing prodrugs, the most common form is to modify the structure of drugs containing carboxyl, hydroxyl, and amino groups to make prodrugs of the types of esters, carboxylates, amino acid esters, amides, and phosphates. In addition, azo-type prodrugs, nitric oxide-type prodrugs, and prodrugs of new structures such as open-loop and closed-loop structures can also be made.

[0051] The compounds of the present invention may be in crystalline form as a favorable compound or as a solvate, both of which are included within the scope of the present invention. Methods of solvation are well known in the art. Suitable solvates are pharmaceutically acceptable solvates.

[0052] "Pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar untoward reactions, such as gastrointestinal discomfort, dizziness, etc. Preferably, the term "pharmaceutically acceptable" as used in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the Chinese Pharmacopoeia, the United States Pharmacopoeia and other generally recognized pharmacopeias for use in animals, more particularly in humans.

[0053] Examples of "pharmaceutically acceptable salts" include salts derived from suitable bases such as alkali metals or alkaline earth metals (e.g., Na + , Li + , K + , Ca +2 and Mg +2 ) and ammonium. Physiologically acceptable salts of nitrogen atoms or amino groups include: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.; (b) salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalene, etc. (c) salts with elemental anions, such as chlorine, bromine and iodine. Physiologically acceptable salts of hydroxy compounds include salts of the anions of the compounds with hydroxy groups, such as sodium + and NR4 + A combination of appropriate cations.

[0054] For therapeutic use, "acceptable" as used herein is physiologically acceptable, i.e., they are salts derived from physiologically acceptable acids or bases. However, salts that are not physiologically acceptable acids or bases may also be used, for example, to prepare or purify physiologically acceptable compounds. All salts, whether or not derived from physiologically acceptable acids or bases, are within the scope of the present invention.

[0055] The compound shown in formula I or its pharmaceutically acceptable salt can exist as different polymorphs or pseudopolymorphs. The crystal polymorphism used in the present invention refers to the ability of a crystalline compound to exist in different crystal structures. Crystal polymorphism can be derived from differences in crystal packing (stacking polymorphism) or packing differences between different conformers of the same molecule (conformational polymorphism). The crystal pseudopolymorphism used in the present invention refers to the ability of a hydrate or solvate of a compound to exist in different crystal structures. The pseudopolymorphs of the present invention can exist due to differences in crystal packing (stacking pseudopolymorphism) or due to packing differences between different conformers of the same molecule (conformational pseudopolymorphism). The present invention includes all polymorphs and pseudopolymorphs of compounds of formula I and their pharmaceutically acceptable salts.

[0056] The compound of formula I or its pharmaceutically acceptable salt can also exist as an amorphous solid. The amorphous solid used in the present invention is such a solid, wherein there is no long-range order in the positions of the atoms in the solid. This definition also applies when the crystal size is 2 nanometers or less. Additives including solvents can be used to establish the amorphous form of the present invention. The present invention includes all amorphous forms of the compound of formula I and their pharmaceutically acceptable salts.

[0057] Compound preparations

[0058] The compound preparation described in the present invention comprises the compound pharmaceutical composition described above and pharmaceutically acceptable excipients. In certain embodiments, the compound pharmaceutical composition described in the present invention is provided in the compound preparation in an effective amount (eg, a therapeutically effective amount).

[0059] The compound preparation of the present invention is prepared with conventional carriers and excipients, which will be selected according to conventional practice. Although the active ingredient can be used alone, it is preferred that they are made into pharmaceutical preparations. The preparation of the present invention, whether for veterinary or human use, all comprises at least one active ingredient as defined above and one or more acceptable carriers for it, and optionally comprises other therapeutic ingredients, especially those other therapeutic ingredients disclosed by the present invention. The carrier must be "acceptable", which means that it is compatible with other components in the preparation and is physiologically harmless to its recipient.

[0060] The compound preparations described herein can be prepared by any method known in pharmacy. In general, these preparation methods include associating the compound pharmaceutical composition (i.e., "active ingredient") with a carrier or excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single dose or multiple dose unit.

[0061] The compound preparation of the present invention can be prepared according to known methods, such as the methods described in the general rules for preparation of the Chinese Pharmacopoeia, the 16th edition of the Japanese Pharmacopoeia, the United States Pharmacopoeia, and the 9th edition of the European Pharmacopoeia. Depending on the dosage form, the compound preparation of the present invention can be appropriately administered to a patient.

[0062] The compounded preparation can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of a compounded preparation containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient administered to the subject and / or a convenient fraction of the dose (e.g., one-half or one-third of the dose).

[0063] The active ingredients and pharmaceutically acceptable excipients in the compound preparations described herein will vary depending on the identity, body type and / or condition of the subject being treated and further on the route of administration of the composition. The compound preparation may contain between 0.1% and 100% (w / w) of the active ingredient.

[0064] The term "treat", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder, or one or more symptoms of such condition or disorder, to which the term applies. The term "treat", as used herein, refers to the act of treating, as "treat" is defined immediately above.

[0065] The "effective amount" described in the present invention refers to an amount sufficient to induce a desired biological response. The effective amount of the compound described in the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition to be treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. Alternatively, in a separate method or use, the present invention can be used as a preventive treatment when indicated and effective. In certain embodiments, the effective amount is the amount of the active ingredient described herein in a single dose. In certain embodiments, the effective amount is the combined amount of the active ingredients described herein in multiple doses.

[0066] A "therapeutically effective amount" as described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount of an active ingredient means an amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a disorder alone or in combination with other therapies. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any disease or disorder described.

[0067] Route of administration

[0068] The compound pharmaceutical composition or compound preparation of the present invention is administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including oral and sublingual) and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), etc. It should be understood that the preferred route may vary with, for example, the condition of the recipient.

[0069] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0070] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0071] Application Example 1

[0072] TUNEL staining analysis of the effects of magnolol or magnolol derivatives on 6-OHDA-induced apoptosis in SH-SY5Y cells

[0073] 1. Test Compounds

[0074] Compound 1 (Compound 1 in Chinese patent CN115581690A): 3',5-diallyl-3-amino-2,4'-dihydroxy-1,1'-biphenyl, with the following structural formula:

[0075]

[0076] 2. Experimental Methods

[0077] Cultured SH-SY5Y cells were treated with 6-OHDA, a hydroxylated derivative of dopamine with neurotoxicity, to simulate the pathological characteristics of PD. The established PD cell model was treated with small molecule drugs, and the degree of cellular oxidative stress damage was evaluated using corresponding technical means, and molecular changes related to oxidative stress were detected to explore the role of drugs in this model.

[0078] SH-SY5Y cells were used to establish a 6-OHDA model (100 μM), and different doses of compound 1 (1000 nM, 100 nM, 10 nM) were administered. 6-OHDA and the compound were co-treated for 24 hours. TUNEL staining was used to analyze the effect of compound 1 on 6-OHDA-induced apoptosis of SH-SY5Y cells. After a one-step staining reaction, apoptotic cells showing red fluorescence were detected by fluorescence microscopy after washing, and cell nuclei showing blue fluorescence were stained with DAPI.

[0079] 1. Cells: SH-SY5Y cells.

[0080] 2. Model: 6-OHDA model, 100 μM.

[0081] 3.Drug dosage: 1000nM, 100nM, 10nM.

[0082] 4. Period: 6-OHDA and compound 1 were co-treated for 24 hours.

[0083] 5. Detection methods: (1) Preparation of staining working solution: The amount of TUNEL detection solution required for each well of a 6-well plate is 200 μl, containing 20 μl TdT enzyme and 180 μl fluorescent labeling solution. (2) Aspirate the culture medium, wash the cells once with PBS, and fix the cells with 1 ml of 4% paraformaldehyde for 30 minutes. (3) Aspirate the fixative, wash the cells once with PBS, add PBS containing 0.3% TritonX-100, and incubate at room temperature for 5 minutes. (4) Aspirate the solution, wash the cells once with PBS, add 100 μl TUNEL detection solution to the sample well, and incubate at 37°C in the dark for 60 minutes. (5) Aspirate the solution, wash the cells three times with PBS, and observe under a fluorescence microscope after sealing with anti-fluorescence quenching sealing solution (containing DAPI). (6) Fluorescence observation and result analysis: The excitation wavelength of Cy3 is 550 nm and the emission wavelength is 570 nm. It shows red fluorescence to mark apoptotic cells, and DAPI is used to stain the cell nucleus to show blue fluorescence.

[0084] 3. Experimental Results

[0085] TUNEL staining can detect early apoptosis. SH-SY5Y cells were treated with 100 μM 6-OHDA and administered for 24 hours. TUNEL staining was performed at the end of the experiment to analyze the effects of different doses of compound 1 on SH-SY5Y cell apoptosis. Figure 1 As shown, the blank control group was not treated with 100μM 6-OHDA and compound 1, and no apoptotic cells marked with red fluorescence were observed; the vast majority of cells in the model control group were marked as apoptotic cells after being treated with 100μM 6-OHDA; after co-treatment with 6-OHDA and different concentrations of compound 1, compound 1 at a dose of 10nM did not reduce cell apoptosis compared with the model control group, and with the increase of the dose, compound 1 at doses of 100nM and 1000nM reduced the number of apoptotic cells; only compound 1 at a dose of 1000nM, without the addition of 100μM 6-OHDA, no apoptotic cells marked with red fluorescence were observed. In summary, the TUNEL staining results show that compound 1 has a good protective effect on SH-SY5Y cell apoptosis caused by 6-OHDA.

[0086] Application Example 2

[0087] Western blot analysis was performed to analyze the antioxidant effect of magnolol or magnolol derivatives on SH-SY5Y cells damaged by 6-OHDA.

[0088] 1. Test Compounds

[0089] Compound 2 (Compound 3 in Chinese Patent CN 115581690 A): 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl dihydrochloride, with the following structural formula:

[0090]

[0091] 2. Experimental Methods

[0092] SH-SY5Y cells express tyrosine hydroxylase, dopamine 2B2 hydroxylase and dopamine transporter, which are unique to catecholaminergic neurons. This cell line is widely used in the study of the pathogenesis of PD. SH-SY5Y cells were untreated as the blank control group; SH-SY5Y cells were used to establish a 6-OHDA (100μM) model as the model control group; SH-SY5Y cells were used to establish a 6-OHDA (100μM) model and treated with different doses of compound 2 (1000nM, 100nM, 10nM, 1nM) as the experimental group. Compound 2 was pretreated for 16 hours, and 6-OHDA and compound 2 were co-treated for 24 hours. At the end of the experiment, cells were collected and total protein was extracted for Western blot analysis of Nrf2 protein level, and β-actin was used as the internal reference protein. Western blot analysis of Nrf2 content changes evaluated the antioxidant protective effect of compound 2 against cell damage caused by 6-OHDA.

[0093] 1. Cells: SH-SY5Y cells.

[0094] 2. Model: 6-OHDA model, 100 μM.

[0095] 3.Drug dosage: 1000nM, 100nM, 10nM, 1nM.

[0096] 4. Cycle: Pretreatment with compound 2 for 16 hours, co-treatment with 6-OHDA and compound 2 for 24 hours to extract total protein.

[0097] 5. Detection method: Western blot analysis of Nrf2 content changes was used to evaluate the antioxidant protective effect of compound 2 against 6-OHDA-induced cell damage.

[0098] 3. Experimental Results

[0099] Compound 2 was pretreated for 16 hours, 6-OHDA was treated for 24 hours, and the drug group was treated with 6-OHDA at the same time as the drug treatment. At the end of the experiment, cells were collected to extract total protein for Western blot analysis of Nrf2 protein levels. Figure 2 As shown in the figure, with β-actin as the internal reference protein, the protein level of Nrf2 increased significantly with the increase of the dose of compound 2. When the compound 2 was 10 nM, the protein level of Nrf2 showed a high level, indicating that compound 2 had a good antioxidant effect on SH-SY5Y cell damage caused by 6-OHDA.

[0100] Application Example 3

[0101] JC-1 staining was used to analyze the antioxidant effect of magnolol or magnolol derivatives on SH-SY5Y cells damaged by 6-OHDA.

[0102] 1. Test Compounds

[0103] Compound 2: 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl dihydrochloride, the structural formula is as follows:

[0104]

[0105] 2. Experimental Methods

[0106] SH-SY5Y cells were used to establish a 6-OHDA model (100 μM), and different doses of compound 2 (1000 nM, 100 nM, 10 nM, 1 nM) were administered. The experimental group was pretreated with compound 2 for 16 hours, and 6-OHDA and compound 2 were co-treated for 24 hours; the model control group was treated with 6-OHDA only; the blank control group was not treated with compound 2 and 6-OHDA. JC-1 staining analysis was used to evaluate the antioxidant protective effect of compound 2 against cell damage caused by 6-OHDA. Mitochondrial membrane potential detection kit (JC-1) is a widely used fluorescent probe for detecting mitochondrial membrane potential. It can detect the mitochondrial membrane potential of cells, tissues or purified mitochondria. The decrease in cell membrane potential can be easily detected by the transition of JC-1 from red fluorescence to green fluorescence, and the transition of JC-1 from red fluorescence to green fluorescence can also be used as a detection indicator for the early stage of cell apoptosis.

[0107] 1. Cells: SH-SY5Y cells.

[0108] 2. Model: 6-OHDA model, 100 μM.

[0109] 3.Drug dosage: 1000nM, 100nM, 10nM, 1nM.

[0110] 4. Cycle: Pretreatment with compound 2 for 16 hours, co-treatment with 6-OHDA and compound 1 for 24 hours to extract total protein.

[0111] 5. Detection methods: (1) Preparation of JC-1 staining working solution: The amount of JC-1 staining working solution required for each well of a 6-well plate is 1 ml. Take an appropriate amount of JC-1 (200X) and dilute JC-1 at a ratio of 1 ml JC-1 staining buffer for every 5 μl JC-1 (200X). Use a pipette to blow repeatedly and mix to obtain the JC-1 staining working solution. (2) Aspirate the culture medium, wash the cells once with PBS, and add 1 ml cell culture medium. Add 1 ml JC-1 staining working solution and mix thoroughly. Incubate at 37°C in a cell culture incubator for 20 minutes. After the incubation at 37°C, aspirate the supernatant and wash twice with JC-1 staining buffer. Add 2 ml cell culture medium. Observe under a fluorescence microscope. (3) Fluorescence observation and result analysis: When detecting JC-1 monomers, refer to the settings for observing other green fluorescence; when detecting JC-1 polymers, observe red fluorescence. The appearance of green fluorescence indicates that the mitochondrial membrane potential has decreased and the cell is likely in the early stages of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the cell state is also relatively normal.

[0112] 3. Experimental Results

[0113] Compound 2 was pretreated for 16 hours, 6-OHDA was treated for 24 hours, and the experimental group was treated with 6-OHDA at the same time as the drug treatment. The JC-1 kit was used to stain the mitochondrial membrane potential, and the fluorescence microscope was used for fluorescence analysis. In healthy mitochondria, JC-1 formed red fluorescently labeled aggregates. As the membrane potential decreased, JC-1 formed monomers and showed green fluorescence. Figure 3 As shown, the red fluorescence of compound 2 treatment increased significantly with increasing doses, indicating that compound 2 has a good mitochondrial protective effect on SH-SY5Y cell damage caused by 6-OHDA.

[0114] Application Example 4

[0115] Western blot analysis was performed to analyze the antioxidant effect of magnolol or magnolol derivatives on SH-SY5Y cells damaged by 6-OHDA.

[0116] 1. Test Compounds

[0117] Compound 3 (Compound 9 in Chinese Patent CN 115581690 A) 3',5-diallyl-3-[(S)-2-amino-4-methylthio-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl hydrochloride, has the following structural formula:

[0118]

[0119] Compound 4 (Compound 12 in Chinese Patent CN 115581690 A): 3',5-dipropyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl dihydrochloride, with the following structural formula:

[0120]

[0121] 2. Experimental Methods

[0122] SH-SY5Y cells express catecholamine-specific tyrosine hydroxylase, dopamine 2B2 hydroxylase and dopamine transporter. This cell line is widely used in the study of PD pathogenesis. The blank control group SH-SY5Y cells were not treated; the model control group used SH-SY5Y cells to establish a 6-OHDA (100μM) model; the experimental group used SH-SY5Y cells to establish a 6-OHDA (100μM) model, and compound 3 and compound 4 were given at a dose of 1000nM, respectively. The drug was pretreated for 16 hours, and 6-OHDA and the drug were co-treated for 24 hours. At the end of the experiment, the cells were collected and the total protein was extracted for Western blot analysis of the protein level of HO-1, and GAPDH was used as the internal reference protein. Western blot analysis of the changes in HO-1 content evaluated the antioxidant protective effects of compound 3 and compound 4 on cell damage caused by 6-OHDA.

[0123] 1. Cells: SH-SY5Y cells.

[0124] 2. Model: 6-OHDA model, 100 μM.

[0125] 3.Drug dosage: 1000nM.

[0126] 4. Cycle: Pre-treatment with drugs for 16 hours, and co-treatment with 6-OHDA and drugs for 24 hours to extract total protein.

[0127] 5. Detection method: Western blot analysis of HO-1 content changes was used to evaluate the antioxidant protective effects of compounds 3 and 4 against cell damage caused by 6-OHDA.

[0128] 3. Experimental Results

[0129] Compound 3 and compound 4 were pretreated for 16 hours, 6-OHDA was treated for 24 hours, and the drug group was treated with 6-OHDA at the same time as the drug treatment. At the end of the experiment, cells were collected to extract total protein for Western blot analysis of HO-1 protein levels. Figure 4As shown, with GAPDH as the internal reference protein, both compound 3 and compound 4 treatment increased the protein level of HO-1, indicating that compound 3 and compound 4 have good antioxidant potential against 6-OHDA-induced SH-SY5Y cell damage.

[0130] Example 5 ABTS scavenging ability test

[0131] Detection of the ABTS scavenging ability of magnolol or magnolol derivatives.

[0132] 1. Test Compounds

[0133] Compound 2: 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl dihydrochloride, the structural formula is as follows:

[0134]

[0135] 2. Experimental Methods

[0136] 1. According to the instructions of the ABTS free radical scavenging ability test kit (Cat. No. BC4770, Solebao, Beijing, China), preheat the spectrophotometer / enzyme reader for more than 30 minutes, adjust the wavelength to 405nm, and zero the spectrophotometer with distilled water. Reagent 1 can be used directly without preparation. The preparation of other solutions is as follows:

[0137] (1) Reagent 2: Add 3 mL of distilled water before use to fully dissolve (unused reagent can be stored in aliquots at -20°C for four weeks to avoid repeated freezing and thawing);

[0138] (2) Preparation of Reagent 3 working solution: The liquid is placed in the EP tube in the reagent bottle. Before use, prepare the reagent 3 working solution according to the sample volume in the ratio of reagent 3 (μL): distilled water (mL) = 1μL:12mL (prepare it as needed, prepare as much as needed, and try to use it up within 4 hours);

[0139] (3) Preparation of Reagent 4 working solution: Reagent 4 can be stored in aliquots at -20°C. Before use, prepare Reagent 4 working solution according to the sample volume at a ratio of Reagent 4: Reagent 1 (V:V) = 1:9 (prepare and use immediately, unused reagent can be stored at -20°C for two weeks).

[0140] (4) Reagent 5 (positive): Contains 5 mg of vitamin C. Add 2.8 mL of extract solution before use and shake thoroughly to dissolve; prepare a 10 mmol / L vitamin C solution for positive control (can be stored at 2-8°C for two weeks).

[0141] (5) Preparation of ABTS working solution: Before use, prepare ABTS working solution according to the sample volume at a ratio of Reagent 1: Reagent 2: Reagent 3 working solution (V:V:V) = 76:5:4 (prepare before use and store at room temperature away from light).

[0142] 2. Operation Table 1: Add the following reagents into the 96-well plate or EP tube:

[0143] The test samples were compound 2 and honokiol.

[0144] Table 1 ABTS removal ability test sample addition table

[0145] Reagent name Blank tube (μL) Assay tube (μL) Control tube (μL) Test product / 10 10 Distilled water 10 / / Reagent 4 working solution 20 20 / ABTS working fluid 170 170 / Reagent 1 / / 190

[0146] Mix thoroughly and leave it at room temperature away from light for 6 minutes. Measure the absorbance at 405 nm. Record them as A blank, A test, and A control respectively.

[0147] 3. Calculation of ABTS free radical scavenging rate

[0148] The free radical scavenging rate of the sample is calculated by the formula:

[0149] ABTS free radical scavenging rate D% = [A blank - (A measurement - A control)] ÷ A blank × 100%.

[0150] 2. Experimental Results

[0151] The results of the drug's ability to clear ABTS are as follows Figure 5 As shown, the scavenging rate of ABTS free radicals by compound 2 at a dose of 1.25 mg / mL reached 95%, and the scavenging rate of ABTS free radicals by honokiol at a dose of 2.12 mg / mL reached 89%, indicating that compound 2 has a stronger scavenging ability for ABTS than honokiol.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of magnolol or a magnolol derivative in the preparation of a medicament for a neurodegenerative disease, characterized in that: The magnolol or magnolol derivative is a compound represented by general formula I, a stereoisomer thereof, a crystalline hydrate thereof, a deuterated product thereof, a solvate thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof: In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups can be substituted by N, O, and S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen or hydroxyl groups at the same time; R5 is H, C1-C8 acyl groups, the remaining acyl part of amino acids after condensation at carboxyl groups, or the remaining acyl part of polypeptides after condensation at carboxyl groups, and R6 is hydroxyl group.

2. The use according to claim 1, characterized in that The neurodegenerative diseases are: Alzheimer's disease, Parkinson's disease and Huntington's disease.

3. The use according to claim 2, characterized in that: The neurodegenerative disease is Parkinson's disease.

4. The use according to claim 1, characterized in that The C1-C8 hydrocarbon groups in R1 and R4 are independently selected from any one of C1-C8 alkyl groups and C1-C8 alkenyl groups.

5. The use according to claim 4, characterized in that The C1-C8 alkenyl in R1 and R4 is selected from any one of vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, but-3-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-5-enyl, hept-1-enyl, hept-2-enyl, hept-3-enyl, hept-4-enyl, hept-5-enyl, hept-6-enyl, oct-1-enyl, oct-2-enyl, oct-3-enyl, oct-4-enyl, oct-5-enyl, oct-6-enyl, oct-7-enyl.

6. The use according to claim 1, characterized in that: The C1-C8 acyl group in R5 is formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, octanoyl or heptanoyl.

7. The use according to claim 1, characterized in that The amino acid in R5 is lysine, methionine, tryptophan, valine, alanine, phenylalanine, leucine, isoleucine, 6-hydroxynorleucine, glycine, histidine, arginine, proline, glutamic acid, aspartic acid, serine threonine, tyrosine, cystine or cysteine.

8. The use according to claim 1, characterized in that The polypeptide in R5 is a peptide formed by multiple amino acids, and its molecular weight is ≤2500Da.

9. The use according to any one of claims 1 to 8, characterized in that The magnolol or magnolol derivative is one or more of the following compounds: 3',5-diallyl-3-amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-acetylamino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-3-phenyl-2-amino-1-propionyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(R)-2,6-diamino-1-hexanoyl]amino amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-6-hydroxy-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-4-methylthio-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-3-methyl-2-amino-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl and 3',5-dipropyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl.

10. The use according to claim 9, characterized in that The magnolol or magnolol derivative is one or more of the following compounds: 3',5-diallyl-3-amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl, 3',5-diallyl-3-[(S)-2-amino-4-methylthio-1-butyryl]amino-2,4'-dihydroxy-1,1'-biphenyl and 3',5-dipropyl-3-[(S)-2,6-diamino-1-hexanoyl]amino-2,4'-dihydroxy-1,1'-biphenyl.

11. A pharmaceutical composition for treating neurodegenerative diseases, characterized in that: Including magnolol or magnolol derivatives, their stereoisomers, their crystalline hydrates, their deuterated substances, their solvates, and their prodrugs; The magnolol or magnolol derivative is a compound represented by the general formula I: In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups are optionally substituted by N, O, or S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen and hydroxyl groups at the same time; R5 is H, a C1-C8 acyl group, the remaining acyl portion of an amino acid after condensation at the carboxyl group, or the remaining acyl portion of a polypeptide after condensation at the carboxyl group, and R6 is a hydroxyl group.

12. A pharmaceutical preparation for treating neurodegenerative diseases, characterized in that: It includes magnolol or magnolol derivatives, their stereoisomers, their crystalline hydrates, their deuterated products, their solvates, their prodrugs or their pharmaceutically acceptable salts and pharmaceutically acceptable carriers; the magnolol or magnolol derivatives are compounds shown in general formula I: In the general formula I, R1 and R4 are independently selected from C1-C8 hydrocarbon groups, and the carbon atoms in the C1-C8 hydrocarbon groups are optionally substituted by N, O, or S; R2 and R3 are independently selected from hydrogen or hydroxyl groups, and R2 and R3 are not hydrogen and hydroxyl groups at the same time; R5 is H, a C1-C8 acyl group, the remaining acyl portion of an amino acid after condensation at the carboxyl group, or the remaining acyl portion of a polypeptide after condensation at the carboxyl group, and R6 is a hydroxyl group.

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