Dihydromyricetin derivatives, preparation method and application thereof

Abstract

This invention relates to the field of chemical synthesis, specifically to dihydromyricetin derivatives, their preparation methods, and applications. This invention discloses the structures of different dihydromyricetin derivatives, addressing the problem of poor stability of dihydromyricetin itself, which prevents it from exerting its effects stably in the human body. The dihydromyricetin derivatives of this invention can be stably stored at 25℃-45℃ and pH≤9, can regulate the body's immune function, have therapeutic effects on degenerative diseases, and can exert their effects stably in the human body, making them suitable for drug development in humans and possessing significant application prospects.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis, specifically to dihydromyricetin derivatives, their preparation methods, and applications. Background Technology

[0002] Dihydromycetin (DHM, DMY), also known as dihydromycetin bark extract, was first isolated in 1940 by Kotake and Kubota from the leaves of A. meliaefolia, a plant in the Vitaceae family. It is found in plants of the Vitaceae, Ericaceae, Burseraceae, Clusiaceae, and Salicaceae families, and is particularly abundant in vine tea.

[0003] Existing research indicates that dihydromyricetin possesses anti-cancer, antioxidant, anti-inflammatory, and antibacterial effects, as well as the ability to lower blood pressure and blood sugar, lower blood lipids, and protect the cardiovascular system. Furthermore, dihydromyricetin also exhibits some protective and alleviating effects in the progression of diseases such as Alzheimer's disease, brain aging, and muscular atrophy. Dihydromyricetin itself possesses a large π-bond conjugated system and strongly coordinated oxygen atoms, and its suitable spatial structure allows it to chelate with metal ions. The resulting dihydromyricetin metal complexes can be added to pharmaceuticals, foods, and cosmetics to exert their antibacterial, antioxidant, and anti-aging effects. Simultaneously, dihydromyricetin contains six hydroxyl groups and exhibits a certain degree of weak acidity. Esterification can improve its solubility, thereby enhancing its antioxidant activity in oils and fats. Dihydromyricetin esters can be applied to cosmetics to improve antioxidant properties, prevent skin aging, and reduce the need for added antibacterial and preservative agents.

[0004] However, dihydromyricetin, due to its o-triphenol hydroxyl structure, is easily oxidized and has poor stability. pH, temperature, and metal ions all affect its stability. Studies have shown that dihydromyricetin is stable under weakly acidic conditions with a pH ≤ 4, but increasing the pH accelerates its oxidation. Heating at temperatures not exceeding 100℃ for no more than 30 minutes maintains the chemical structure of dihydromyricetin, but its stability decreases with increasing temperature, and irreversible oxidation may even occur. Due to its inherent instability, dihydromyricetin decomposes and loses its medicinal properties during administration, limiting its medical applications.

[0005] Therefore, the development of dihydromyricetin derivatives with improved pharmacological and drug-like properties is of great significance for medical treatment. Summary of the Invention

[0006] To address the issue of poor stability of dihydromyricetin, which prevents it from exerting its effects stably in the human body, this invention provides a class of dihydromyricetin derivatives. While maintaining the pharmacological activity of dihydromyricetin, the physicochemical properties of the compounds are improved and adjusted, enhancing efficacy and increasing safety and stability.

[0007] Specifically, the present invention provides dihydromyricetin derivatives having compounds of formula (I) and pharmaceutically acceptable salts thereof.

[0008]

[0009] Among them, R1 is independently selected from C 1-6 Alkyl, C 1-6 Alkyl carbonyl, C 1-6 alkylsulfonyl, C 1-6 alkyl sulfinyl or H;

[0010] L is selected from -(CR3R4)nC(=X)- or -C(=X)-(CR3R4)n-, -(CR3R4)m-, -(CR3R4)nC(=O)-NH-,

[0011] -(CR3R4)n-NH-C(=O)-, -C(=O)-NH-(CR3R4)n-, -NH-C(=O)-(CR3R4)n-, -(CR3R4)nC(=O)-NH-(CR3R4)n-, -(CR3R4)n-NH-C(=O)-(CR3R4)n-, OC(=O)-(CR3R4)n-; where X is selected from O, S, NH; and n and m are each independently selected from integers from 0 to 6;

[0012] R3 and R4 are independently selected from H and C. 1-6 Alkyl, halogen, hydroxyl, -NR5R6 or -CN;

[0013] R2 is selected from 3-12 membered heterocyclic groups; the heterocyclic group is optionally C 1-6 Alkyl, halogen, hydroxyl, -NR5R6, -CN, -SF5, or C 1-6 Alkyl substitution;

[0014] Or R2 is selected from NR5R6;

[0015] R5 and R6 are independently selected from H and C. 1-6 Alkyl group, C(=NH)NH2.

[0016] As one of the preferred implementation schemes, R1 is independently selected from C. 1-6 Alkyl or H; as one preferred embodiment, L is selected from -(CR3R4)m-, wherein R3 and R4 are independently selected from H, C1-6 alkyl.

[0017] As one of the preferred embodiments, R2 is selected from a 5-10 member heterocyclic group; the heterocyclic group is optionally C 1-6 Alkyl, halogen, hydroxyl, -NR5R6, -CN, or C 1-6 Alkyl-substituted.

[0018] As one of the preferred embodiments, R2 is selected from NR5R6; R5 and R6 are independently selected from H and C. 1-6 Alkyl group, C(=NH)NH2.

[0019] As a further preferred option, C 1-6 The alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl.

[0020] As a further preferred option, the heterocyclic group is selected from azirrobutyl, oxacyclobutyl, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, or diazirrobutyl.

[0021] Furthermore, the heterocyclic group is selected from tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, or piperazineyl.

[0022] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0023]

[0024] Another object of the present invention is to provide a method for preparing a compound of formula (I), comprising the following steps:

[0025]

[0026] In this compound, the compound of formula II reacts with the compound of formula III to generate the compound of formula I, wherein X1 is selected from Br, I or OTf.

[0027] The present invention also provides a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. The pharmaceutical composition is formulated for administration via a route selected from: oral, injection, rectal, nasal, pulmonary, topical, oral and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural.

[0028] The pharmaceutical composition is preferably administered orally. The oral dosage form is not particularly limited and can be any oral dosage form well known in the art, preferably including tablets, capsules, suspensions, or oral solutions, etc. When used as an oral dosage form, the standard dosage is, for example, 500-1500 mg / day, preferably 700-1200 mg / day, more preferably 800-1000 mg / day, and most preferably 1000 mg / day.

[0029] The duration of medication of the pharmaceutical composition of the present invention can be determined according to the severity of the condition, preferably at least 1 month, for example, 1, 2, 3, 4, 5 or 6 months, and may be lifelong as required by the condition.

[0030] According to embodiments of the present invention, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients; preferably, the excipients are selected from at least one of the following: fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, humectants, pH adjusters, solubilizers or co-solvents, and osmotic pressure regulators. Those skilled in the art can readily determine how to select the appropriate excipients and their corresponding amounts according to the needs of the specific dosage form.

[0031] According to embodiments of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0032] The present invention also provides at least one of the compounds or pharmaceutically acceptable salts represented by formula (I), and the use of said pharmaceutical composition in the preparation of a medicament. This pharmaceutical therapy can be used to treat insomnia, sleep disorders, anxiety, cognitive decline, memory impairment, and neurodegenerative diseases. The neurodegenerative diseases include neuroinflammation, Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0033] The present invention also provides at least one of the compounds or pharmaceutically acceptable salts represented by formula (I), and the use of said pharmaceutical composition in the preparation of a medicament, the use of which includes treatment of immune system diseases. These immune system diseases include rheumatoid arthritis, muscular dystrophy, systemic lupus erythematosus, chordal syndrome, multiple sclerosis, systemic sclerosis, scleroderma, small cell lung cancer syndrome, renal tuberculosis, lymphoma, hepatitis B, and chronic lymphocytic leukemia, etc.

[0034] The present invention also provides at least one of the compounds or pharmaceutically acceptable salts represented by formula (I), and the use of said pharmaceutical composition in the preparation of medicaments, including for delaying / counteracting aging.

[0035] Beneficial effects

[0036] (1) The dihydromyricetin derivative of the present invention can be stably stored at 25℃-45℃ and pH≤9, and is suitable for drug development for human use.

[0037] (2) The dihydromyricetin derivative of the present invention can regulate the body's immune function, and its effect is significantly stronger than that of dihydromyricetin.

[0038] (3) The dihydromyricetin derivative of the present invention has a therapeutic effect on degenerative diseases and can exert its effects stably in the human body. Attached Figure Description

[0039] Figure 1 Results of Aβ1-42 detection in rat brains;

[0040] Figure 2 Expression of Beclin1 in the rat brain;

[0041] Figure 3 Expression of LC3-II / LC3-I in the rat brain;

[0042] Figure 4 Expression of p62 in the rat brain;

[0043] Figure 5 Expression of LAMP1 in the rat brain;

[0044] Figure 6 Expression of Cathepsin D in the rat brain.

[0045] Terminology Definitions and Explanations

[0046] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0047] Unless otherwise stated, the numerical ranges described in this specification and claims correspond to at least each specific integer value described therein. For example, the numerical range "0-6" corresponds to each integer value in the numerical range "0-6", namely 0, 1, 2, 3, 4, 5, and 6. When the value is 0, for example -(CR3R4)m-, m is 0, indicating a chemical bond.

[0048] The term "halogen" refers to fluorine, chlorine, bromine, and iodine. In other words, F, Cl, Br, and I can be described as "halogens" in this specification.

[0049] The optional substitution covered cases of no substitution and cases of substitution by one or more substituents. For example, "optionally substituted by one, two or more R" means that it can be unsubstituted (no substitution) or substituted by one, two or more R.

[0050] The term "alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturated bonds, having, for example, 1 to 6 carbon atoms connected to the rest of the molecule by single bonds. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl. Alkyl groups can be unsubstituted or substituted with one or more suitable substituents. Alkyl groups can also be isotopic isomers of naturally abundant alkyl groups rich in carbon and / or hydrogen isotopes (i.e., deuterium or tritium).

[0051] The term "C" refers to the use of the term alone or as part of other substituents. 1-6 "Alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, etc., or their isomers.

[0052] The term "3-12 membered heterocyclic group" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 4-, 5-, 6-, or 7-membered monocyclic ring, a 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic (such as fused rings, bridged rings, spirocyclic rings) or tricyclic ring system, and contains at least one, for example, 1, 2, 3, 4, 5, or more heteroatoms selected from O, S, and N, wherein N and S may optionally be oxidized to various oxidation states to form nitrogen oxides, -S(O)-, or -S(O)2- states. Preferably, the heterocyclic group may be selected from "3-10 membered heterocyclic groups". The term "3-10 membered heterocyclic group" means a saturated or unsaturated non-aromatic ring or ring system containing at least one heteroatom selected from O, S, and N. The heterocyclic group can be attached to the rest of the molecule by any one of the carbon atoms or a nitrogen atom (if present). The heterocyclic group may include fused or bridged rings and spirocyclic rings. Specifically, the heterocyclic group may include, but is not limited to: 4-membered rings, such as azirrobutyl or oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolealkyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, or trithiaalkyl; or 7-membered rings, such as diazacycloheptyl. Optionally, the heterocyclic group may be benzofused. The heterocyclic group may be bicyclic, for example, but not limited to, 5,5-membered rings, such as hexahydrocyclopenta[c]pyrrolo-2(1H)-yl rings, or 5,6-membered bicyclic rings, such as hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl rings. The heterocyclic group can be partially unsaturated, meaning it can contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2,5-dihydro-1H-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 1,2,3,5-tetrahydrooxazolyl, or 4H-[1,4]thiazinyl. Alternatively, it can be benzofused, such as, but not limited to, dihydroisoquinolinyl. When the 3-12-membered heterocyclic group is linked to other groups to form the compounds of the present invention, the carbon atom on the 3-12-membered heterocyclic group can be linked to other groups, or the heterocyclic atom on the ring of the 3-12-membered heterocyclic group can be linked to other groups. For example, when the 3-12-membered heterocyclic group is selected from piperazineyl, the nitrogen atom on the piperazineyl group can be linked to other groups. Or when the 3-12-membered heterocyclic group is selected from piperidinyl, the nitrogen atom on the piperidinyl ring and the carbon atom at its para position can be linked to other groups.

[0053] In this application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, to the extent of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0054] References to "an embodiment," "embodiment," etc., in this specification indicate that the described embodiment may include a particular aspect, feature, structure, part, or characteristic, but not every embodiment must include that aspect, feature, structure, part, or characteristic. Furthermore, such phrases may, but do not necessarily, refer to the same embodiment mentioned in other parts of the specification. Additionally, when a particular aspect, feature, structure, element, or characteristic is described in connection with an embodiment, whether explicitly described or not, it will affect or relate that aspect, feature, structure, element, or characteristic to other embodiments, to the knowledge of those skilled in the art.

[0055] As will be understood by those skilled in the art, all numerical values, including those representing the amount of an ingredient, properties such as molecular weight, reaction conditions, etc., are approximate and are to be understood as optionally modified by the term "about" in all cases. These values ​​may vary according to the desired properties sought to be obtained by those skilled in the art using the teachings described herein. It should also be understood that these values ​​inherently contain variability, which is necessarily caused by the standard deviation found in their respective test measurements.

[0056] "Effective amount" refers to the amount that is effective in treating a disease, condition, and / or symptom or produces the said effect. For example, an effective amount could be the amount that effectively reduces the progression or severity of a condition or symptom being treated.

[0057] The determination of an "effective amount" is entirely within the competence of those skilled in the art. This includes the amount of compounds described herein, or the amount of combinations of compounds described herein, for example, for treating or preventing a disease or disorder in the host, or for treating symptoms of a disease or disorder. Therefore, an "effective amount" generally means the amount that provides the desired effect.

[0058] Alternatively, as used herein, "effective dose" or "therapeutic effective dose" refers to a sufficient quantity of a preparation or composition that provides some degree of relief for one or more symptoms of a disease or condition being treated. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired biological systemic changes. For example, an "effective amount" for therapeutic use is the quantity of a composition comprising the compounds disclosed herein required to provide a clinically significant reduction in the symptoms of a disease. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies. Dosage may be administered once or multiple times. However, the precise determination of an effective dose may be based on individual factors for each patient, including but not limited to the patient's age, body size, type or severity of disease, stage of disease, route of administration of the composition, type or extent of adjunctive therapy used, ongoing disease course, and type of treatment required (e.g., aggressive versus conventional therapy).

[0059] The term "treatment" includes (i) preventing the occurrence of a disease, pathology, or medical condition (e.g., prevention); (ii) suppressing or halting the development of a disease, pathology, or medical condition; (iii) alleviating a disease, pathology, or medical condition; and / or (iv) reducing symptoms associated with a disease, pathology, or medical condition. Therefore, the term "treatment" can be extended to prevention and can include prevention, aversion, prevention, reduction, cessation, or reversal of the progression or severity of a condition or symptom being treated. Therefore, the term "treatment" may, where appropriate, include the administration of medicines, therapies, nutrition, and / or preventative measures.

[0060] As used herein, “subject” or “patient” refers to an individual who has symptoms or risk of a disease or other malignancy. A patient can be human or non-human and can include, for example, animal strains or species used as a “model system” for research purposes, such as the mouse model described herein. Similarly, a patient can refer to any living organism that may include adults or adolescents (e.g., children), preferably mammals (e.g., human or non-human) who may benefit from administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and pigs. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment of the methods provided herein, the mammal is a human. As used herein, the terms “provide” and “administer” are introduced interchangeably herein and refer to the placement of the disclosed compound into the subject body by a method or route that results in at least partial localization of the compound to the desired site. The compound can be administered via any appropriate route, thereby delivering it to the desired location within the subject's body.

[0061] The compounds and compositions described herein may be administered together with other compositions to prolong the stability and activity of the compositions, or in combination with other supplements, nutrients, therapeutics or drugs.

[0062] The term "inhibition" refers to slowing down, stopping, or reversing the growth or progression of a disease, infection, condition, or cell population. For example, the inhibition may be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to growth or progression that occurs without treatment or exposure.

[0063] Whenever the term “comprising” is used herein, the option of using the terms “composed of,” “including,” or “substantially composed of” is considered. As used herein, “comprising” is synonymous with “including” or “characterized by” and is inclusive or open-ended, and does not exclude additional, unlisted elements or method steps. As used herein, “by” excludes any element, step, or component not specified in the elements of an aspect. As used herein, “substantially by” does not exclude materials or steps that do not substantially affect the essential and novel features of that aspect. In each instance herein, any one of the terms “comprising,” “substantially by,” and “by” may be replaced by any of the other two terms. The disclosure described illustratively herein may be practiced without any limitation or restriction on one or more elements not specifically disclosed herein. Detailed Implementation

[0064] The following embodiments are intended to illustrate the invention described above and should not be construed as limiting its scope. Those skilled in the art will readily recognize that the embodiments present many other ways in which the invention can be practiced. It should be understood that many variations and modifications can be made while remaining within the scope of the invention.

[0065] Example 1

[0066]

[0067] Under N2 conditions, 200 mg (0.62 mmol) of dihydromyricetin was dissolved in 7 mL of anhydrous DMF solution, 552 mg (2.5 mmol) of anhydrous K2CO3 was added, and 383 mg (2.7 mmol) of iodomethane was slowly added. The reaction was carried out at 40 °C for 24 h. After stopping the reaction, 1 mL of acetic acid was added, the mixture was filtered, evaporated to dryness under reduced pressure, the solvent was recovered, and the solution was purified by silica gel column chromatography (V). 石油醚 V 乙酸乙酯 =6:1), yielding 7,3',4',5'-tetramethoxydihydromyricetin intermediate. Yield 26.2%.

[0068] Example 2

[0069]

[0070] Add the PdCl2(dppf)CH2Cl2 adduct (0.090 g, 0.123 mmol) to a vial containing a degassed (3× vacuum / Ar) mixture of Br-L-R2 (4.11 mmol), bis(pinacol)diboron (1.25 g, 4.94 mmol), and potassium acetate (1.21 g, 12.3 mmol) in dioxane (10 mL). Degassed the reaction mixture, sealed it, and heated it at 110 °C for 16 hours. Dilute the mixture with water and extract it with EtOAc. Concentrate the organic phase and preliminarily purify it by rapid chromatography (EtOAc / hexane) to give intermediates with the structures shown in the table below for later use.

[0071] Table 1 Intermediates L2-1 to L2-8

[0072]

[0073] Example 3

[0074]

[0075] At room temperature, compound II (0.3 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), followed by the addition of compounds III (L2-1 to L2-8, 1.05 mmol), cesium carbonate (1.07 mmol), and Pd(PPH3)4 (0.036 mmol). The reaction mixture was stirred overnight at 95 °C under a nitrogen atmosphere. After the reaction was complete, the mixture was cooled to room temperature, diluted with water (20 mL), extracted with ethyl acetate (3 × 20 mL), and the combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:2) to give yellow oily compounds L3-1 to L3-8, the structures of which are shown in Table 2.

[0076] Table 2 Dihydromyricetin derivatives L3-1 to L3-8

[0077]

[0078]

[0079] Example 4 Stability Experiment

[0080] This embodiment investigates the stability of dihydromyricetin and the derivatives of this invention under different temperatures and light exposures.

[0081] The experimental method is as follows:

[0082] Stability study of dihydromyricetin and its derivatives at different temperatures: 1 mL of a 1 mg / mL solution of dihydromyricetin and its derivatives (dissolved in 60% anhydrous ethanol) was mixed with 9 mL of PB phosphate buffer (0.2 M, pH 8.0). The mixture was placed in water baths at different temperatures (25, 37, 45 °C) and sealed under natural diffused light for 1 h, 2 h, and 4 h, respectively. The retention was determined by HPLC.

[0083] To investigate the effect of different pH values, 1 mL of a 1 mg / mL solution of dihydromyricetin and its derivatives (dissolved in 60% anhydrous ethanol) was mixed with 9 mL of PB phosphate buffer at different pH values. After being placed at room temperature (25°C) for 8 hours, samples were taken and the retention of DMY was determined by HPLC.

[0084] HPLC analysis conditions: The Agilent 1260 HPLC system was equipped with a photodiode array detector, an autosampler, and a symmetrical C18 column (4.6 mm × 250 mm, 5.0 μm; Waters, USA). The mobile phase was 25% acetonitrile and 75% aqueous phase (containing 0.1% glacial acetic acid) with isocratic elution. The flow rate was 1 mL / min, the column temperature was 40 °C, and the detection wavelength was 291 nm. The injection volume for stability studies was 10 μL.

[0085] Table 3 Temperature stability study

[0086]

[0087]

[0088] It can be seen that when stored at room temperature for more than 4 hours and at a body temperature of 37°C or above, dihydromyricetin undergoes auto-oxidation, generating other components. Therefore, if administered to the human body, dihydromyricetin will quickly lose its drug activity. In contrast, the derivative of this invention is less affected by temperature, and its retention amount hardly changes, making it suitable for drug development.

[0089] Table 4 pH stability study

[0090] Element pH3 pH4 pH5 pH6 pH7 pH8 pH9 Dihydromyricetin 100％ 98％ 97％ 90％ 50％ 0％ 0％ L3-1 100％ 100％ 100％ 100％ 98％ 94％ 89％ L3-2 100％ 100％ 100％ 100％ 99％ 95％ 90％ L3-3 100％ 100％ 100％ 100％ 98％ 96％ 92％ L3-4 100％ 100％ 100％ 100％ 97％ 94％ 90％ L3-5 100％ 100％ 100％ 100％ 98％ 96％ 92％ L3-6 100％ 100％ 100％ 100％ 97％ 95％ 92％ L3-7 100％ 100％ 100％ 100％ 96％ 94％ 91％ L3-8 100％ 100％ 100％ 100％ 98％ 95％ 90％

[0091] It can be seen that both dihydromyricetin and the derivative of this invention are stable under acidic conditions. Under neutral conditions, dihydromyricetin undergoes structural changes and becomes unstable, while the derivative of this invention remains relatively stable under neutral conditions, which makes injection, oral administration, and other methods of administration possible. Under alkaline conditions, dihydromyricetin undergoes complete structural changes and can no longer be detected in samples preserved in alkaline solutions, while the derivative of this invention remains relatively stable. Since many parts of the human body are in a slightly alkaline environment, the derivative of this invention provides possibilities for applications in various scenarios. The 8-hour treatment simulates the drug's condition in the human body, and 8 hours is sufficient for the body to complete absorption.

[0092] Example 5: Immune Regulation Related

[0093] a. 110 SPF-grade male mice were selected and grouped according to the table below. Group 1 was the blank group (normal mice). Groups 2-10 were treated with cyclophosphamide 80 mg / kg / day intraperitoneally for 3 consecutive days to induce the model. The treated mice were called model mice. The blank group mice were injected intraperitoneally with an equal volume of physiological saline for 3 consecutive days. After modeling, groups 3-10 were injected with dihydromyricetin and its derivatives 1-8 at 2 mg / kg / day, respectively. Groups 1 and 2 were injected intraperitoneally with an equal volume of physiological saline. Each group was injected once a day for 17 consecutive days. After the intraperitoneal injection ended, the mice were fasted for 24 hours, and their body weight was measured using an electronic balance. Blood was collected by enucleation, and the mice were euthanized by cervical dislocation. The spleen and thymus tissue were collected and stored at -80℃ for later use.

[0094] b. The immune organ index was calculated using a weighing method. After removing connective and adipose tissue from the extracted thymus and spleen, the organs were rinsed with physiological saline, blotted dry with filter paper, and the mass of the mouse thymus and spleen (immune organ mass) was measured using a micro-analytical balance. The thymus index and spleen index were then calculated. Immune organ index = immune organ mass (mg) / body mass (g).

[0095] Table 4 Immune Organ Index

[0096]

[0097] The results are shown in the table above. The results show that, compared with group 2, the spleen index and thymus index of the model mice in groups 3-11 were increased. The increase of dihydromyricetin derivative was significantly higher than that of dihydromyricetin, indicating that both dihydromyricetin and dihydromyricetin derivative can enhance the body's immune function. However, compared with dihydromyricetin, the dihydromyricetin derivative of this invention can effectively antagonize the immunosuppression caused by cyclophosphamide and has a stronger enhancing effect.

[0098] c. After weighing and grinding the spleens of mice in each group, single spleen cells were isolated using lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., Mouse Spleen Lymphocyte Separation Solution Kit). Based on the cell count results, a concentration of 1×10⁻⁶ cells was prepared. 4 Cell suspensions of 1 cell / μL were prepared. 50 μL of each pre-prepared single-cell suspension were added to anti-mouse CD3 inhibitors. + CD4 + CD8 + 1 μL of fluorescent antibody was added. After incubation at 4°C in the dark for 30 min, the cells were washed once with PBS buffer, centrifuged for 5 min at 2000 rpm, the supernatant was discarded, and 500 μL of PBS buffer was added to the pellet. The mixture was gently mixed and the percentage of each cell subpopulation was detected by flow cytometry (Accui C5, BD Biosciences, USA).

[0099] The results are as follows:

[0100] Table 5. Indicators of T lymphocyte subsets

[0101]

[0102] The results above show that the proportions of CD3+, CD4+, and CD8+ T lymphocyte subsets and the CD4+ / CD8+ ratio in the spleen of model mice (groups 2-11) were all higher in group 3-11 than in group 2. This indicates that dihydromyricetin and the dihydromyricetin derivative of this invention have an ameliorative effect on the T lymphocyte subset indicators in the spleen of immunosuppressed model mice, and the dihydromyricetin derivative has a more significant ameliorative effect.

[0103] d. Biomarker detection: On day 17 of the experiment, blood was collected from the eyes of mice in each group. After standing for 2-3 hours, the blood was centrifuged at 3500 r / min for 15 minutes, and the supernatant serum was collected. The levels of TNF-α, IL-6 and IL-8 in the mouse serum were detected by enzyme-linked immunosorbent assay (the detection kit was purchased from Shanghai Enzyme Link Co., Ltd.).

[0104] Table 6. Serum levels of TNF-α, IL-6, and IL-8 in mice.

[0105]

[0106]

[0107] The results are shown in Table 6. It can be seen that compared with group 1, the levels of TNF-α, IL-6, and IL-8 in groups 2-11 were significantly increased, while compared with group 2, the levels of TNF-α, IL-6, and IL-8 in groups 3-10 were significantly decreased. TNF-α, IL-6, and IL-8 are all inflammatory markers. Immunosuppression is an abnormal state characterized by impaired collective immune function and a decreased ability to respond to antigens. The significantly increased levels of TNF-α, IL-6, and IL-8 in the model mice after injection of cyclophosphamide indicate that cyclophosphamide caused damage to the mouse immune system. The addition of the dihydromyricetin derivative of this invention significantly reduced these levels, thus playing a role in regulating the body's immune function.

[0108] Example 5: Degenerative Disease Related

[0109] Alzheimer's disease, commonly known as senile dementia, mostly occurs in people over 60 years of age. Its clinical features are mainly spatial cognition, executive function impairment, and memory decline. It is a common neurodegenerative disease. In this embodiment, AD mice are used to explore the effect of the compound of the present invention on the treatment of neurodegenerative diseases.

[0110] Current research indicates that excessive Aβ deposition is a major cause of AD. This study investigates Aβ... 1-42 A rat AD model was induced by microinjection into the bilateral hippocampus.

[0111] a、Aβ 1-42 Oligomer preparation: Take 1 mg of Aβ 1-42 The monomer powder and hexafluoroisopropanol (HFIP) were pre-cooled on ice, and then added to a container containing 1 mg of Aβ. 1-42 Add 200 μL of HFIP to an EP tube containing the monomer powder, seal, vortex to mix, and incubate at room temperature for 60 minutes until the liquid becomes clear to obtain 1 mmol / L Aβ. 1-42 -HFIP solution. Then add Aβ 1-42 -Incubate the HFIP solution on ice for 5 minutes. Take 4 sterile EP tubes and dispense Aβ. 1-42 55 μL of each HFIP solution was evaporated in a fume hood to obtain a colorless and transparent Aβ solution. 1-42 Peptide membranes were stored at -20°C. Before use, one aliquoted EP tube was placed on an ice pack, and 11 μL of DMSO was added. The tube was then sonicated in a water bath (300 W, 35 Hz) for 10 min. 539 μL of PBS solution was then added, vortexed, and incubated at 4°C for 1 day. After incubation, the EP tube was centrifuged at 1000 rpm for 10 min at 4°C. The supernatant was collected to obtain 100 μmol / L Aβ. 1-42 Oligomer.

[0112] b. One hundred and one hundred healthy male SD rats aged 8-10 weeks (normal rats), weighing (20±20) g, were randomly grouped according to Table 7, including Aβ... 1-42 After anesthetizing the rats, the bilateral hippocampal regions were located according to the rat brain atlas. The anterior fontanelle was used as the zero point, 2.4 mm lateral to the midline and 3.8 mm posterior to the anterior fontanelle, with a needle insertion depth of 3.0 mm. Aβ was injected into both hippocampi of the rats. 1-42 (5μg / μL) 2μL, the treated rats were named AD rats. The + dihydromyricetin derivative group was treated with 120mg / kg dihydromyricetin derivative, while the control group was administered an equal volume of physiological saline by gavage, for 4 weeks.

[0113] c. Morris Water Maze: For the first 5 days after treatment, all rats underwent a navigational test. Rats were placed in the water maze from four different quadrants, and the time required for each group of rats to find and stand on the platform was recorded. If the platform was not found within 60 seconds, the escape latency was recorded as 60 seconds, and the rat was guided to rest on the platform for 10 seconds before proceeding with the next experiment. One day later, the platform was hidden, and a spatial exploration experiment was conducted. Rats from each group entered the water maze from the quadrant opposite to the original platform. The rats' movement trajectory, the number of times they crossed the original platform, and the percentage of time spent in the target quadrant were recorded within 60 seconds.

[0114] Table 7 Effects of dihydromyricetin derivatives 1-8 on memory impairment in mice.

[0115]

[0116] It can be seen that, compared with "normal rats + saline", the escape latency of "AD rats + saline" was significantly prolonged after day 4 of navigation training (P < 0.05), while the number of times the original platform was crossed and the proportion of time spent in the target quadrant were significantly reduced (P < 0.01). Compared with the "AD rats + saline" group, the escape latency of "AD rats + dihydromyricetin derivative" was significantly shortened (P < 0.05), while the number of times the original platform was crossed and the proportion of time spent in the target quadrant were significantly increased (P < 0.05). Furthermore, derivatives 1-8 all showed good performance, indicating that the dihydromyricetin derivative of this invention is beneficial in reducing Aβ. 1-42 The deposition exerts a neuroprotective effect on AD and improves cognitive function in rats.

[0117] d. Brain tissue specimen preparation: After the water maze test, mice in each group were randomly selected and deeply anesthetized by intraperitoneal injection of 3.5% chloral hydrate at a rate of 3 ml / kg. The mice were then perfused with physiological saline at the apex of the heart, and brain tissue was rapidly harvested by decapitation on ice. Half of the brain tissue was placed in an ultra-low temperature freezer at -80℃ for subsequent molecular biology protein detection. The other half of the brain was fixed in 4% paraformaldehyde solution and dehydrated overnight at 4℃ using a gradient of 10%, 20%, and 30% sucrose solutions.

[0118] e. ELISA detection: Select mouse brain tissue from each group, add protein extraction buffer from the ELISA kit according to the instructions, grind evenly, let stand for 3 hours to allow the residue to precipitate, select the supernatant to detect the concentration, dilute with EIA buffer as needed, label with antibody, prepare standards, construct standard curve, load samples, incubate at 4℃ for 12 hours, wash the plate, add TMB chromogen solution, develop color, and detect with an ELISA reader.

[0119] (1) Detection of Aβ in rat brain by ELISA 1-42 The result is as follows Figure 1 As shown, Aβ in the brains of group 2 rats... 1-42 The level of Aβ in the brains of rats in groups 3-10 was significantly higher than that in group 1; while the level of Aβ in the brains of rats in groups 3-10 was significantly higher than that in group 1. 1-42 The expression of these markers was significantly lower in group 1 than in group 2.

[0120] f. Semi-quantitative detection of protein levels (Western Blotting, WB)

[0121] (1) Protein extraction

[0122] Take about 200mg of each group of brain tissue prepared in the ultra-low temperature freezer, weigh them separately, and add them to each group according to the ratio of 10ml / g protein lysis buffer and 20μl / g PMSF. Mix well and use a hand homogenizer to grind the tissue into a homogenized suspension without particles. Let it stand on ice for 10min, and then centrifuge it in a 4℃ low temperature high speed centrifuge at 12000rpm for 15min. Collect the supernatant and store it.

[0123] (2) Protein concentration detection: The concentration of each group of proteins was detected using the Beyotime BCA kit, and the protein concentration of each group was balanced.

[0124] (3) Protein denaturation: Mix the protein sample and SDS-PAGE loading buffer at a ratio of 4:1, heat in a constant temperature metal bath at 100℃ for 5 minutes, cool naturally, and then store in a -20℃ refrigerator for later use.

[0125] (4) Prepare the separating gel: Prepare the required concentration of separating gel according to the instructions.

[0126] (5) Electrophoresis: Fix the prepared separating gel onto the gel casting plate, place it in the electrophoresis tank, add the electrophoresis solution, take out the comb, add the protein sample for loading, cover the lid, adjust the voltage to 80V, start electrophoresis, observe the marker protein separation, adjust the voltage to 120V, observe the target protein range separation, pause and prepare for electrophoresis.

[0127] (6) Electrophoresis: Cut the gel strip within the required protein range according to the marker markings, place it on the transfer rack, and then tightly attach it with a PVDF membrane. Place it in the electrophoresis tank, adjust the current to 250mA, and the time is determined by the molecular weight of the target protein.

[0128] (7) Sealing: After the electroporation is completed, take out the PVDF membrane strip and place it in 5% BSA sealing solution at 37°C for 2 hours.

[0129] (8) Incubation with primary antibody: Place the band in a uniformly diluted primary antibody and incubate at 4°C for 12 hours.

[0130] (9) Incubation with secondary antibody: Take out the band, wash it 3 times with 1×PBST for 10 min each time, and incubate it in the corresponding species secondary antibody at 37℃ for 1 h.

[0131] (10) Development: Take out the strip, wash it 3 times with 1×PBST for 20 min each time, add ECL ultrasensitive developer (A solution: B solution = 1:1), and use Image Lab software to detect the gray value of the strip.

[0132] Effects of DHM on autophagy-related proteins in rats

[0133] Beclin1 plays a central role in autophagy, being an essential molecule for autophagosome formation. It recruits various autophagy-related proteins to regulate autophagosome formation and maturation, reflecting the overall level of autophagy. LC3 and p62 are typical markers of autophagy, crucial in autophagy initiation, nucleation, and expansion. LC3-II is a structural protein of autophagosomes, and its level reflects the number of activated autophagosomes; the LC3-II / I ratio is commonly used to compare autophagy levels. p62 is one of the marker proteins for autophagy. During autophagy, p62 protein is continuously degraded in the cytoplasm. When autophagy activity is weakened or autophagy function is defective, p62 protein accumulates in the cytoplasm, and its content reflects the level of autolysosome clearance. LAMP1 is a lysosomal membrane protein, and its expression level reflects the number of lysosomes. Cathepsin D is one of the most important proteolytic enzymes in lysosomes, degrading autolysosomes to maintain cell stability.

[0134] The results are as follows Figure 2-6As shown, the detection revealed that compared with group 1, group 2 showed no significant change in the expression of the autophagy-regulating gene Beclin1 (P>0.05), a significantly decreased LC3-II / I ratio (P<0.001), significantly increased expression of autophagy substrate-associated protein p62 and lysosome-associated membrane protein LAMP1 (P<0.001), and increased expression of cathepsin D (P<0.05). Compared with group 2, group 3-10 showed increased Beclin1 expression (P<0.01), no significant change in LC3-II / I expression (P>0.05), significantly decreased expression of p62 and LAMP1 (P<0.001), and decreased expression of cathepsin D (P<0.01).

[0135] The above results indicate that autophagic flux initiation and autolysosomal degradation functions are impaired in AD rats. Dihydromyricetin intervention effectively improved the autophagic flux disorder in AD rats, and significantly improved the autolysosomal degradation function. In summary, the dihydromyricetin derivative of this invention can promote the degradation of autophagic substrates, prevent the accumulation of autolysosomes, and thus restore the patency of autophagic flux, thereby reducing Aβ deposition in the brains of AD mice and ultimately playing a therapeutic role in AD.

[0136] Example 6 Anti-aging Experiment

[0137] One hundred aged male C57BL / 6 mice (22-24 months old) were acclimatized in an animal facility for two weeks, with light levels adjusted to a 12-hour day / night cycle. During this period, the mice had free access to water and food, and the ambient temperature was maintained at 20℃-22℃, with a relative humidity of 50%-60%.

[0138] Two weeks later, the animals were divided into two groups: a control group and experimental groups 1-9, with 10 animals in each group. The experimental groups were injected with dihydromyricetin and its derivatives 1-8 at a dose of 2 mg / kg / day, while the control group was given an equal volume of physiological saline via intraperitoneal injection. Each group was injected once daily for 8 consecutive weeks, and their body weight and water intake were monitored three times a week.

[0139] Eight weeks later, mice were sacrificed, and liver tissue samples were dissected and weighed. A certain amount of PBS was added, pH 7.4. The samples were then rapidly frozen in liquid nitrogen for later use. After thawing, the samples were kept at 2-8°C. A certain amount of PBS (pH 7.4) was added, and the samples were thoroughly homogenized manually or using a homogenizer. The samples were centrifuged for approximately 20 minutes (2000-3000 rpm). The supernatant was carefully collected. One aliquot was used for testing, and the remainder was frozen for later use. SIRT3 was measured according to the operating procedures of the Mouse Silent Regulatory Protein 3 (SIRT3) ELISA Kit (Abcam, USA).

[0140] SIRT3 protein expression improvement rate = (Experimental group expression level - Control group expression level) / Control group expression level * 100%

[0141] Table 8. Improvement rate of SIRT3 protein expression

[0142]

[0143]

[0144]

[0145] Sirtuins, known as "longevity factors," are NAD+-dependent deacetylases that play a crucial role in cellular metabolism and environmental stress. Upregulation of the SIRT3 gene expression can enhance cellular energy while protecting mitochondria from oxidative stress, thus delaying cellular aging. The dihydromyricetin derivative prepared in this invention can improve SIRT3 protein expression in tissues and is significantly superior to the efficacy of dihydromyricetin alone.

[0146] While specific embodiments have been described above with reference to disclosed examples and embodiments, such embodiments are merely illustrative and do not limit the scope of the invention. Changes and modifications can be made by those skilled in the art without departing from the broader aspects of the invention as defined in the appended claims.

[0147] All publications, patents, and patent documents are incorporated herein by reference as if they were individually incorporated herein by reference. This should not be construed as limiting any aspects inconsistent with this disclosure. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. A dihydromyricetin derivative or a pharmaceutically acceptable salt thereof, characterized in that, The dihydromyricetin derivative is a compound as shown in Formula I: Formula I Among them, R1 is independently selected from C 1-6 Alkyl or H; L is selected from -(CR3R4)m-, where m is selected from integers from 0 to 6; R3 and R4 are independently selected from H or C. 1-6 alkyl; R2 is selected from 3-12 membered heterocyclic groups; the heterocyclic group is selected from tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, or piperazineyl; the heterocyclic group is optionally C-shaped. 1-6 Alkyl or hydroxyl substitution; Or R2 can be selected from -NR5R6; R5 and R6 are independently selected from H and C. 1-6 Alkyl or -C(=NH)NH2.

2. The dihydromyricetin derivative as described in claim 1, characterized in that, The C 1-6 The alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl.

3. The dihydromyricetin derivative as described in claim 1 or 2, characterized in that, The compound shown in formula (I) is selected from the following structures: 。 4. A method for preparing a dihydromyricetin derivative as described in any one of claims 1-3, characterized in that, Includes the following steps: In this compound, the compound of formula II reacts with the compound of formula III to generate the compound of formula I, wherein X1 is selected from Br, I or OTf.

5. A pharmaceutical composition, characterized in that, This includes dihydromyricetin derivatives or pharmaceutically acceptable salts thereof as described in any one of claims 1-3.

6. A pharmaceutical preparation, characterized in that, Includes dihydromyricetin derivatives or pharmaceutically acceptable salts thereof as described in any one of claims 1-3, and pharmaceutically acceptable carriers.

7. The pharmaceutical formulation as described in claim 6, characterized in that, The pharmaceutically acceptable carriers include pharmaceutically acceptable excipients.

8. The pharmaceutical formulation as described in claim 7, characterized in that, The excipients are selected from one or more of the following: fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, humectants, pH adjusters, solubilizers or cosolvents or osmotic pressure regulators.

9. The composition according to claim 5, characterized in that, The composition also contains one or more therapeutic agents.

10. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of a medicament for neurodegenerative diseases; wherein the neurodegenerative diseases are selected from neuroinflammation, Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

11. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of a medicament for treating immune system diseases; wherein the immune system diseases are selected from rheumatoid arthritis, muscular dystrophy, systemic lupus erythematosus, chordal syndrome, multiple sclerosis, systemic sclerosis, scleroderma, small cell lung cancer syndrome, renal tuberculosis, lymphoma, hepatitis B, or chronic lymphocytic leukemia.

12. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of an anti-aging drug.

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