Dihydromyricetin derivative as well as preparation method and application thereof

Abstract

The invention relates to the field of chemical synthesis, in particular to a dihydromyricetin derivative and a preparation method and application thereof. The invention discloses structures of different dihydromyricetin derivatives, and solves the problems that dihydromyricetin is poor in stability and cannot stably play a role in a human body. The dihydromyricetin derivative disclosed by the invention can be stably stored under the conditions that the temperature is 25-45 DEG C and the pH value is less than or equal to 9, can regulate the immune function of an organism, has a treatment effect on degenerative diseases, can stably play a role in a human body, is suitable for drug development of the human body, and has an important application prospect.

Description

Technical Field

[0001] The present invention relates to the field of chemical synthesis, and in particular to dihydromyricetin derivatives and preparation methods and applications thereof. Background Art

[0002] Dihydromycetin (DHM, DMY), also known as dihydromyricetin, dihydromyricetin, and ampelopsin, was first isolated by Kotake and Kubota in 1940 from the leaves of A. meliaefolia, a plant of the genus Ampelops in the Vitaceae family. It exists in plants such as the Vitaceae, Ericaceae, Oleaceae, Garciniaaceae, and Salicaceae, especially in A. tengcha, where its content is relatively high.

[0003] Existing research shows that dihydromyricetin has anticancer, antioxidant, anti-inflammatory, and antibacterial effects, as well as blood pressure and blood sugar lowering, lipid-lowering, and cardiovascular protective properties. Furthermore, dihydromyricetin has a protective and mitigating effect on the progression of diseases such as Alzheimer's disease, brain aging, and muscular atrophy. Dihydromyricetin itself has a large π-bond conjugated system and a strongly coordinated oxygen atom. Its suitable spatial structure allows for chelation with metal ions. The resulting dihydromyricetin metal complex can be added to pharmaceuticals, foods, and cosmetics to exert its antibacterial, antioxidant, and anti-aging effects. Furthermore, dihydromyricetin contains six hydroxyl groups and possesses a certain degree of weak acidity. Esterification can improve its solubility and thus enhance its antioxidant activity in oils and fats. Dihydromyricetin esters can be used in cosmetics to enhance antioxidant properties, prevent skin aging, and reduce the need for antibacterial preservatives.

[0004] However, dihydromyricetin, due to its o-triphenolic hydroxyl structure, is easily oxidized and has poor stability. pH, temperature, and metal ions all have a certain impact on its stability. Studies have shown that dihydromyricetin is stable under weakly acidic conditions with a pH value of ≤4, but increasing the pH value accelerates its oxidation rate. When heated at a temperature not exceeding 100°C for a time not exceeding 30 minutes, the chemical structure of dihydromyricetin remains stable, but as the temperature increases, its stability deteriorates and irreversible oxidation reactions may even occur. Due to its inherent instability, dihydromyricetin decomposes and loses its medicinal properties during administration, limiting its medical use.

[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 problem of dihydromyricetin's inherent poor stability and inability to exert its efficacy stably in the human body, the present invention provides a class of dihydromyricetin derivatives. While maintaining the pharmacological activity of dihydromyricetin, the physicochemical properties of the compound are improved and modulated, thereby enhancing efficacy, safety, and stability.

[0007] Specifically, the present invention provides dihydromyricetin derivatives, wherein the dihydromyricetin derivatives have the following formula (I) compounds and pharmaceutically acceptable salts thereof:

[0008]

[0009] wherein R1 is independently selected from C 1-6 Alkyl, C 1-6 Alkylcarbonyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylsulfinyl 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-; X is selected from O, S, NH; n and m are each independently selected from an integer of 0 to 6;

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

[0013] R2 is selected from 3-12 membered heterocyclic groups; the heterocyclic groups are optionally substituted by C 1-6 Alkyl, halogen, hydroxy, -NR5R6, -CN, -SF5, or C 1-6 Alkoxy substitution;

[0014] or R2 is selected from NR5R6;

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

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

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

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

[0019] As a further preferred embodiment, 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 further preferred, the heterocyclic group is selected from azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl or diazepanyl.

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

[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 the compound of formula (I), comprising the following steps:

[0025]

[0026] Wherein, 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 by a route selected from the group consisting of oral, parenteral, rectal, nasal, pulmonary, topical, buccal 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 may be any oral dosage form known in the art, preferably including tablets, capsules, suspensions, oral solutions, and other oral dosage forms known in the art. When used as an oral dosage form, the dosage standard used is, for example, 500-1500 mg / day, preferably 700-1200 mg / day, preferably 800-1000 mg / day, and most preferably 1000 mg / day.

[0029] The duration of administration of the pharmaceutical composition of the present invention may depend on the severity of the disease, and is preferably at least 1 month, for example, 1, 2, 3, 4, 5 or 6 months, and may be lifelong depending on the disease condition.

[0030] According to an embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient; preferably, the excipient is selected from at least one of the following excipients, including but not limited to: a filler, a disintegrant, a binder, a lubricant, a surfactant, a flavoring agent, a wetting agent, a pH adjuster, a solubilizer or cosolvent, and an osmotic pressure regulator. Those skilled in the art can easily determine how to select the appropriate excipient and its corresponding dosage based on the requirements of the specific dosage form.

[0031] According to an embodiment 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 compound or pharmaceutically acceptable salt of formula (I), and the use of the pharmaceutical composition in the preparation of a medicament. The drug 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.

[0033] The present invention also provides at least one of the compounds represented by formula (I) or pharmaceutically acceptable salts thereof, and use of the pharmaceutical composition in preparing a medicament, wherein the use includes use in treating immune system diseases. The 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.

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

[0035] Beneficial effects

[0036] (1) The dihydromyricetin derivatives of the present invention can be stably stored at 25°C-45°C and pH ≤ 9, and are suitable for drug development for human use;

[0037] (2) The dihydromyricetin derivatives of the present invention can regulate the body's immune function, and their effects are significantly stronger than dihydromyricetin;

[0038] (3) The dihydromyricetin derivatives of the present invention have therapeutic effects on degenerative diseases and can stably exert their effects in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Detection results of Aβ1-42 in rat brain;

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

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

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

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

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

[0045] Definitions and Explanations of Terms

[0046] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The group definitions and compound structures resulting from such combinations and couplings should be understood to be within the scope of this specification and / or claims.

[0047] Unless otherwise indicated, numerical ranges recited in this specification and claims are equivalent to recite at least each specific integer value therein. For example, the numerical range "0-6" is equivalent to recite each integer value within the numerical range "0-6," namely, 0, 1, 2, 3, 4, 5, and 6. When the value is 0, such as in -(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 may be described as "halogen" in this specification.

[0049] The case of being optionally substituted by a substituent includes the case of being unsubstituted as well as being substituted by one or more substituents. For example, "optionally substituted by one, two or more R" means that it may be unsubstituted by R (unsubstituted) or substituted by one, two or more R.

[0050] The term "alkyl" means a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, free of unsaturated bonds, having, for example, 1 to 6 carbon atoms and connected to the rest of the molecule by a single bond. 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 may be unsubstituted or substituted with one or more suitable substituents. Alkyl groups may also be isotopomers of naturally abundant alkyl groups enriched in isotopes of carbon and / or hydrogen (i.e., deuterium or tritium).

[0051] When used alone or as part of another substituent, the term "C 1-6 “Alkyl” is understood to mean a linear or branched, saturated, monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl radical 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 heterocyclyl" 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 (e.g., fused, bridged, spiro) 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 also be optionally oxidized to various oxidation states to form nitrogen oxides, -S(O)- or -S(O)2- states. Preferably, the heterocyclyl may be selected from "3-10 membered heterocyclyl". The term "3-10 membered heterocyclyl" means a saturated or unsaturated non-aromatic ring or ring system, and contains at least one heteroatom selected from O, S and N. The heterocyclyl may be attached to the rest of the molecule via any one of the carbon atoms or the nitrogen atom (if present). The heterocyclic group may include fused or bridged rings and spirocyclic rings. In particular, the heterocyclic group may include, but is not limited to, a 4-membered ring such as azetidinyl, oxetane; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclic group may be bicyclic, such as, but not limited to, a 5,5-membered ring such as hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5,6-membered bicyclic ring such as hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl ring. The heterocyclic group may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 1,2,3,5-tetrahydrooxazolyl, or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. When the 3-12 membered heterocyclic group is linked to another group to form the compound of the present invention, the linking may be to a carbon atom of the 3-12 membered heterocyclic group or to a heterocyclic atom on the 3-12 membered heterocyclic group ring. For example, when the 3-12 membered heterocyclic group is selected from piperazinyl, the linking may be to a nitrogen atom on the piperazinyl ring and to a carbon atom at the para position thereof. Alternatively, when the 3-12 membered heterocyclic group is selected from piperidinyl, the linking may be to a nitrogen atom on the piperidinyl ring and to a carbon atom at the para position thereof.

[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 those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0054] References in the specification to "one embodiment," "an embodiment," etc. 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 referenced in other parts of the specification. Furthermore, when a particular aspect, feature, structure, unit, or characteristic is described in conjunction with an embodiment, whether or not explicitly described, it is within the knowledge of those skilled in the art to influence or relate that aspect, feature, structure, unit, or characteristic to other embodiments.

[0055] As will be understood by those skilled in the art, all numerical values, including those expressing the amounts of ingredients, properties such as molecular weight, reaction conditions, etc., are approximate and are understood to be optionally modified in all instances by the term "about". These values may vary depending on the desired properties sought to be obtained by those skilled in the art utilizing the teachings described herein. It will also be understood that these values inherently contain variability necessarily resulting from the standard deviation found in their respective testing measurements.

[0056] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition or produce the effect described. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptom being treated.

[0057] The determination of an "effective amount" or a therapeutically effective amount is well within the capabilities of those skilled in the art. This includes an amount of a compound described herein, or an amount of a combination of compounds described herein, for example, to treat or prevent a disease or disorder in a host, or to treat a symptom of a disease or disorder. Thus, an "effective amount" generally refers to an amount that provides the desired effect.

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

[0059] The term "treating" includes (i) preventing the occurrence of a disease, pathology, or medical condition (e.g., prophylaxis); (ii) inhibiting the disease, pathology, or medical condition or arresting its development; (iii) alleviating the disease, pathology, or medical condition; and / or (iv) alleviating the symptoms associated with the disease, pathology, or medical condition. Thus, the term "treating" may extend to prophylaxis and may include preventing, prophylactically, preventing, reducing, halting, or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treating" may include pharmaceutical, therapeutic, nutritional, and / or prophylactic administration, as appropriate.

[0060] As used herein, "subject" or "patient" refers to an individual who has symptoms or is at 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 "model systems" for research purposes, such as the mouse model described herein. Similarly, a patient. In addition, a patient can refer to any living organism, which may include adults or adolescents (such as children), preferably mammals (e.g., human or non-human), who can benefit from the administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the class mammals: humans, non-human primates, such as chimpanzees, and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, 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 "providing" and "administering" are used interchangeably herein and refer to placing the disclosed compound into the subject's body by a method or route that causes the compound to be at least partially localized to the desired site. The compound may be administered by any appropriate route so as to deliver it to the desired location in the subject.

[0061] The compounds and compositions described herein can be administered with other compositions to prolong the stability and activity of the compositions, or in conjunction with other supplements, nutraceuticals, therapeutic agents, or drugs.

[0062] The term "inhibit" refers to slowing, stopping, or reversing the growth or progression of a disease, infection, condition, or cell population. For example, the inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to the growth or progression that would occur in the absence of treatment or contact.

[0063] Whenever the term "comprising" is used herein, the option of using the term "consisting of," "comprising or consisting essentially of," is contemplated. 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, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel characteristics of the aspect. In each instance herein, any one of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms. The disclosure illustratively described herein may be appropriately implemented without any element or elements not specifically disclosed herein. DETAILED DESCRIPTION

[0064] The following examples are intended to illustrate the above invention and should not be construed as narrowing its scope. Those skilled in the art will readily appreciate that the embodiments have proposed many other ways of implementing the present invention. It should be understood that many changes and modifications may be made while remaining within the scope of the present invention.

[0065] Example 1

[0066]

[0067] Under N2, 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 mixture was reacted at 40°C for 24 h, and then 1 mL of acetic acid was added. The mixture was filtered, evaporated to dryness under reduced pressure, the solvent was recovered, and the mixture was purified by silica gel column chromatography (V 石油醚 :V 乙酸乙酯 =6:1) to obtain 7,3',4',5'-tetramethoxydihydromyricetin intermediate with a yield of 26.2%.

[0068] Example 2

[0069]

[0070] To a vial containing a degassed (3× vacuum / Ar) mixture of Br-L-R2 (4.11 mmol), bis(pinacolato)diboron (1.25 g, 4.94 mmol) and potassium acetate (1.21 g, 12.3 mmol) in dioxane (10 mL) was added PdCl2(dppf)CH2Cl2 adduct (0.090 g, 0.123 mmol). The reaction mixture was degassed, sealed, and heated at 110°C for 16 hours. The mixture was diluted with water and then extracted with EtOAc. The organic phase was concentrated and initially purified via flash chromatography (EtOAc / hexane) to give the intermediate with the structure shown in the table below.

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

[0072]

[0073] Example 3

[0074]

[0075] At room temperature, the compound of Formula II (0.3 mmol) was dissolved in 1,4-dioxane (2 mL) and water (0.4 mL). Compounds of Formula III (L2-1 to L2-8, 1.05 mmol), cesium carbonate (1.07 mmol), and Pd(PPH3)4 (0.036 mmol) were added sequentially. The reaction solution was stirred at 95°C overnight under a nitrogen atmosphere. After completion of the reaction, the mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (3 × 20 mL). 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 resulting crude products were separated and purified by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:2) to obtain compounds L3-1 to L3-8 as yellow oils. Their structures are shown in Table 2.

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

[0077]

[0078]

[0079] Example 4 Stability Test

[0080] This example investigates the stability of dihydromyricetin and its derivatives under different temperatures and light conditions.

[0081] The experimental method is as follows:

[0082] Stability of dihydromyricetin and its derivatives at different temperatures: 1 mL of a 1 mg / mL solution of D-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 mixed solution was placed in a water bath at different temperatures (25, 37, and 45°C), sealed, and placed under natural scattered light for 1 hour, 2 hours, or 4 hours. The retained amount 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. The mixture was placed at room temperature (25°C) and samples were taken after 8 hours to determine the DMY retention using HPLC.

[0084] HPLC analysis conditions: An Agilent 1260 HPLC system equipped with a photodiode array detector, autosampler, and a Symmetric C18 column (4.6 mm × 250 mm, 5.0 μm; Waters, USA) was used. The mobile phase consisted of 25% acetonitrile and 75% aqueous solution (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 the stability study 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 above 37°C, dihydromyricetin undergoes auto-oxidation to generate other components. Therefore, if administered to the human body, dihydromyricetin will quickly lose its pharmaceutical activity. However, the derivatives of the present invention are less affected by temperature, and the retained amount hardly changes, making them 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 under acidic conditions, both dihydromyricetin and the derivatives of the present invention can exist stably. Under neutral conditions, dihydromyricetin will undergo structural changes and cannot exist stably, while the derivatives of the present invention can also exist relatively stably under neutral conditions, which provides possibilities for administration such as injection and oral administration. Under alkaline conditions, all dihydromyricetin undergoes structural changes, and the components of dihydromyricetin can no longer be detected in samples stored in alkaline solutions, while the derivatives of the present invention can still exist relatively stably. Many parts of the human body are in neutral to alkaline conditions, and the derivatives of the present invention provide possibilities for application in various scenarios. Selecting the treatment for 8 hours simulates the situation of the drug in the human body, and 8 hours is sufficient for the human body to complete absorption.

[0092] Example 5: Immunomodulation related

[0093] a. Take 110 SPF male mice and group them according to the table below. Group 1 is the blank group (normal mice). Groups 2-10 are all injected intraperitoneally with cyclophosphamide 80 mg / kg / day for 3 consecutive days to establish the model. The treated mice are called model mice. The blank group mice are injected intraperitoneally with an equal volume of normal saline for 3 consecutive days during the same period. After modeling, groups 3-10 are injected with dihydromyricetin and derivatives 1-8 at 2 mg / kg / day, and groups 1 and 2 are injected intraperitoneally with an equal volume of normal saline. Each group is injected once a day for 17 consecutive days. After the intraperitoneal injection, fast for 24 hours, measure body weight (body weight) using an electronic balance, remove eyeballs to collect blood, and sacrifice by cervical dislocation. Take the spleen and thymus tissue and place them in a -80℃ refrigerator for storage.

[0094] b. Immune organ index was calculated by weighing. After extracting the thymus and spleen, the connective tissue and adipose tissue were removed, rinsed with saline, and dried with filter paper. The thymus and spleen (immune organ mass) of the mouse were weighed using a semi-micro analytical balance to calculate the thymus index and spleen index. 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: in groups 3-11, compared with group 2, the spleen index and thymus index of the model mice were increased, among which the dihydromyricetin derivatives had a significantly higher increase than dihydromyricetin, indicating that both dihydromyricetin and dihydromyricetin derivatives can enhance the body's immune function, but compared with dihydromyricetin, the dihydromyricetin derivatives of the present invention can effectively antagonize the immunosuppression caused by cyclophosphamide, and the enhancement effect is stronger.

[0098] c. The spleens of mice in each group were weighed and ground, and single spleen cells were isolated using lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd., mouse spleen lymphocyte separation medium kit). Based on the cell count results, the concentration was 1×10 4 Take 50 μL of the pre-prepared single cell suspension and add anti-mouse CD3 + 、CD4 + 、CD8 + 1 μL of fluorescent antibody was added. After incubation at 4°C in the dark for 30 min, cells were removed and washed once with PBS buffer. The cells were centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. 500 μL of PBS buffer was added to the pellet, and the mixture was gently mixed. The proportion of each cell subpopulation was then determined using a flow cytometer (Accui C5, BD Biosciences, USA).

[0099] Here are the results:

[0100] Table 5 T lymphocyte subset indicators

[0101]

[0102] From the above results, it can be seen that the proportions of CD3+, CD4+, and CD8+ T lymphocyte subsets and CD4+ / CD8+ values in the spleen of model mice (groups 2-11) in group 3-11 were higher than those in group 2, indicating that dihydromyricetin and the dihydromyricetin derivatives of the present invention have an improving effect on the spleen T lymphocyte subset indicators of immunosuppressive model mice, and the dihydromyricetin derivatives have a more obvious improving effect.

[0103] d. Biological index detection: On the 17th day of the experiment, the eyeballs of mice in each group were removed and blood was collected. The blood was allowed to stand for 2-3 hours and centrifuged at 3500 rpm for 15 minutes. The upper serum was aspirated and 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 Levels of TNF-α, IL-6, and IL-8 in mouse serum

[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, and compared with group 2, the levels of TNF-α, IL-6, and IL-8 in groups 3-10 were significantly reduced. TNF-α, IL-6, and IL-8 are all indicators of inflammation. Immunosuppression is an abnormal state in which collective immune function is impaired and the ability to respond to antigens is reduced. After the model mice were injected with cyclophosphamide, the levels of TNF-α, IL-6, and IL-8 increased significantly, indicating that cyclophosphamide caused damage to the mouse immune system. After adding the dihydromyricetin derivative of the present invention, the levels were significantly reduced, which played a role in regulating the body's immune function.

[0108] Example 5 Degenerative Disease Related

[0109] Alzheimer's disease, commonly known as senile dementia, occurs mostly in elderly people over 60 years old. Its clinical features mainly include spatial cognition, executive dysfunction, and memory loss. It is a common neurodegenerative disease. In this example, AD mice were used to explore the therapeutic effect of the compounds of the present invention on neurodegenerative diseases.

[0110] According to current research, excessive deposition of Aβ is the main cause of AD. 1-42 The AD model was induced by microinjection into the bilateral hippocampus of rats.

[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 added to a 1 mg Aβ 1-42 Add 200 μL of HFIP to the EP tube containing monomer powder, seal it, vortex to mix, and incubate it at room temperature for 60 minutes until the liquid becomes clear. This will give 1 mmol / L of Aβ. 1-42 -HFIP solution. Then Aβ 1-42 - Place the HFIP solution back on ice for 5 minutes. Take 4 sterile EP tubes and dispense Aβ 1-42 55 μL of each HFIP solution was added and HFIP was evaporated in a fume hood to obtain colorless and transparent Aβ 1-42 Peptide film, stored in a -20 ℃ refrigerator. Before use, take a packaged EP tube, operate on an ice box, add DMSO 11μL, water bath ultrasonic (300W, 35Hz) for 10min, then add PBS solution 539μL, vortex mix, and place it in a 4 ℃ refrigerator for 1d. After the incubation, place the EP tube in a centrifuge, centrifuge at 1000r / min for 10min at 4 ℃, take the supernatant, and you can get 100μmol / L Aβ 1-42 oligomers.

[0112] b. 110 healthy male SD rats aged 8 to 10 weeks (normal rats) with a body weight of (20±20) g were randomly divided into groups according to Table 7. 1-42 After the rats were anesthetized, the bilateral hippocampus was located according to the rat brain atlas. The bregma was taken as the zero point, 2.4 mm lateral to the midline, 3.8 mm posterior to the bregma, and the needle was inserted to a depth of 3.0 mm. Aβ was injected into the bilateral hippocampus of the rats. 1-42 The rats treated with 5μg / μL and 2μL were called AD rats. The + dihydromyricetin derivative group was treated with 120mg / kg dihydromyricetin derivative, while the control group was gavaged with the same amount of normal saline for 4 weeks.

[0113] c. Morris Water Maze: Five days after treatment, all rats underwent a navigation test. Rats were placed into the water pool 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 rats were guided to the platform and rested for 10 seconds before the next experiment. One day afterward, the platform was hidden for a spatial exploration test. Rats in 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 they 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 the "normal rats + normal saline", the "AD rats + normal saline" group had a significantly prolonged escape latency after the fourth day of positioning navigation training (P < 0.05), and the number of times the original platform was crossed and the proportion of time spent in the target quadrant was significantly reduced (P < 0.01). Compared with the "AD rats + dihydromyricetin derivatives" group, the escape latency was significantly shortened (P < 0.05), the number of times the original platform was crossed and the proportion of time spent in the target quadrant was significantly increased (P < 0.05), and the performance effects of derivatives 1-8 were all good, indicating that the dihydromyricetin derivatives of the present invention are beneficial to reduce Aβ 1-42 Deposition exerts a neuroprotective effect against AD and improves cognitive function in rats.

[0117] d. Brain Tissue Preparation: After the water maze test, mice from each random group were deeply anesthetized with 3.5% chloral hydrate injected intraperitoneally at a standard dose of 3 ml / kg. Perfused apically with normal saline, the brains were quickly decapitated on ice and the brains were obtained. Half of the brain tissue was stored in a -80°C ultra-low temperature freezer for subsequent molecular biology protein analysis. Half of the brain was fixed in 4% paraformaldehyde solution and refrigerated at 4°C overnight. The cells were then dehydrated using a gradient of 10, 20, and 30% sucrose solutions.

[0118] e. ELISA assay: Select brain tissue from each group of mice and add the protein extraction buffer in the ELISA kit according to the instructions. Grind evenly and let it stand for 3 h to allow the residue to settle. Select the supernatant for concentration detection and dilute with EIA buffer as needed. Label with antibodies, prepare standards, generate a standard curve, load the sample, incubate at 4°C for 12 h, wash the plate, add TMB chromogen solution, develop color, and detect with a microplate reader.

[0119] (1) ELISA method for detecting Aβ in rat brain 1-42 The results are as follows Figure 1 As shown, the Aβ level in the brain of rats in group 2 1-42 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 TNF-α was significantly lower than that of group 2.

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

[0121] (1) Protein extraction

[0122] About 200 mg of prepared brain tissue from each group was taken out of the ultra-low temperature refrigerator, weighed separately, and added to each group at a ratio of 10 ml / g protein lysis buffer and 20 μl / g PMSF. The tissue was ground into a particle-free and homogenous suspension using a handheld homogenizer. The suspension was placed on ice for 10 min and centrifuged at 12,000 rpm in a 4°C low-temperature high-speed centrifuge for 15 min. The supernatant was aspirated and stored.

[0123] (2) Protein concentration detection: The BCA kit was used to detect the concentration of each group of proteins and balance the proteins of each group.

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

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

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

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

[0128] (7) Blocking: After electroporation, remove the PVDF membrane strips and place them in 5% BSA blocking solution at 37°C for 2 h.

[0129] (8) Incubation with primary antibody: Place the strips in evenly diluted primary antibody and incubate at 4°C for 12 h.

[0130] (9) Incubation with secondary antibody: Remove the strips, wash with 1× PBST three times, 10 min each time, and incubate with the corresponding secondary antibody at 37°C for 1 h.

[0131] (10) Development: The strips were removed and washed three times with 1×PBST for 20 min each time. ECL supersensitive developer (solution A:solution B = 1:1) was added dropwise, and the grayscale value of the strips was detected using Image Lab software.

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

[0133] Beclin1 plays a central role in the process of cellular autophagy and is an essential molecule for the formation of autophagosomes. It can recruit multiple autophagy-related proteins to regulate the formation and maturation of autophagosomes, reflecting the level of autophagy. LC3 and p62 are typical markers of autophagy and are crucial in the initiation, nucleation, and expansion of autophagy. LC3-II is a structural protein of the autophagosome, and its level reflects the number of activated autophagosomes. The LC3-II / I ratio is usually used to compare the level of autophagy. p62 is one of the marker proteins that reflects autophagy. When autophagy occurs, p62 protein is continuously degraded in the cytoplasm. When autophagy activity is weakened and autophagy function is defective, p62 protein will continue to accumulate in the cytoplasm, and its content briefly reflects the level of autophagic lysosome clearance. LAMP1 is a lysosomal membrane protein, and its expression level reflects the number of lysosomes. Cathepsin D is one of the most important lysosomal proteases, which can degrade autophagic lysosomes to maintain cell stability.

[0134] The results are as follows Figure 2-6As shown in the results, compared with group 1, the expression of the autophagy regulatory gene Beclin1 in group 2 did not change significantly (P>0.05), the autophagy-specific marker molecule LC3-Ⅱ / Ⅰ ratio was significantly decreased (P<0.001), the expression of the autophagy substrate-related protein p62 and the lysosome-related membrane protein LAMP1 was significantly increased (P<0.001), and the expression of the cathepsin Cathepsin D was increased (P<0.05). Compared with group 2, the expression of Beclin1 in groups 3-10 increased (P<0.01), the expression of LC3-Ⅱ / Ⅰ did not change significantly (P>0.05), the expression of p62 and LAMP1 was significantly decreased (P<0.001), and the expression of Cathepsin D was decreased (P<0.01).

[0135] The above results show that the autophagic flux initiation and autophagolysosomal degradation function of AD rats are impaired. After intervention with dihydromyricetin, the autophagic flux disorder of AD rats is effectively improved, and the degradation function of autophagolysosomes is significantly improved. In summary, the dihydromyricetin derivatives of the present invention can promote the degradation of autophagic substrates, prevent the accumulation of autophagolysosomes, and then restore the patency of the autophagic flux, thereby reducing the deposition of Aβ in the brain of AD mice and ultimately playing a role in treating AD.

[0136] Example 6 Anti-aging experiment

[0137] One hundred 22-24 month old C57BL / 6 male mice were acclimated in an animal room for two weeks, with light intensity adjusted to a 12-hour day / night rhythm. During this period, the mice had free access to water and food, and the ambient temperature was maintained at 20°C-22°C and relative humidity at 50%-60%.

[0138] After two weeks, the animals were divided into groups: a control group and experimental groups 1-9, with 10 animals in each group. The experimental groups received a daily injection of dihydromyricetin and derivatives 1-8 at 2 mg / kg / day, while the control group received an equal volume of saline intraperitoneally. Each group received injections once daily for eight consecutive weeks, and body weight and water intake were monitored three times a week.

[0139] After 8 weeks, the mice were killed, and the liver tissue specimens were cut and weighed. A certain amount of PBS, pH 7.4 was added. The specimens were quickly frozen with liquid nitrogen for later use. After the specimens were thawed, the temperature was still maintained at 2-8°C. A certain amount of PBS (PH 7.4) was added, and the specimens were thoroughly homogenized by hand or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). The supernatant was carefully collected. One portion was packaged for testing, and the rest 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 - control group expression) / control group expression * 100%

[0141] Table 8 Improvement rate of SIRT3 protein expression

[0142]

[0143]

[0144]

[0145] Sirtuins, the "longevity factor," are NAD+-dependent deacetylases that play a key role in cellular metabolism and environmental stress. Upregulation of SIRT3 gene expression can increase cellular energy while protecting mitochondria from oxidative stress, thus slowing cellular aging. The dihydromyricetin derivatives prepared in the examples of the present invention can improve SIRT3 protein expression in tissues and are significantly more effective than dihydromyricetin.

[0146] Although specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are merely illustrative and do not limit the scope of the invention. Changes and modifications may 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 individually incorporated herein by reference. No limitations inconsistent with the present disclosure should be construed thereby. The present invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the present 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: wherein R1 is independently selected from C 1-6 Alkyl, C 1-6 Alkylcarbonyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylsulfinyl or H; L is selected from -(CR3R4)nC(=X)-, -C(=X)-(CR3R4)n-, -(CR3R4)m-, -(CR3R4)nC(=O)-NH-, -(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- or OC(=O)-(CR3R4)n-; X is selected from O, S or NH; n and m are each independently selected from an integer of 0 to 6; R3, R4 are independently selected from H, C 1-6 Alkyl, halogen, hydroxy, -NR5R6 or -CN; R2 is selected from 3-12 membered heterocyclic groups; the heterocyclic groups are optionally substituted by C 1-6 Alkyl, halogen, hydroxy, -NR5R6, -CN, -SF5, or C 1-6 Alkoxy substitution; or R2 is selected from -NR5R6; R5, R6 are independently selected from H, C 1-6 Alkyl or -C(=NH)NH2.

2. The dihydromyricetin derivative according to claim 1, wherein The R1 is independently selected from C 1-6 Alkyl or H; L is selected from -(CR3R4)m-, wherein R3, R4 are independently selected from H, C 1-6 alkyl.

3. The dihydromyricetin derivative according to claim 1 or 2, wherein The R2 is selected from 5-10 membered heterocyclic groups, the heterocyclic groups are optionally C 1-6 Alkyl, halogen, hydroxy, -NR5R6, -CN, or C 1-6 Alkoxy substitution; Or, said R2 is selected from NR5R6; R5, R6 are independently selected from H, C 1-6 Alkyl, C(=NH)NH2.

4. The dihydromyricetin derivative according to claim 3, wherein 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.

5. The dihydromyricetin derivative according to claim 3, wherein The heterocyclic group is selected from azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl or diazepanyl; more preferably, the heterocyclic group is selected from tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl or piperazinyl.

6. The dihydromyricetin derivative according to any one of claims 1 to 5, wherein The compound represented by formula (I) is selected from the following structures:

7. A method for preparing a dihydromyricetin derivative according to any one of claims 1 to 6, characterized in that: The steps include: Wherein, 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.

8. A pharmaceutical composition, characterized in that The invention comprises a dihydromyricetin derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical preparation, characterized in that The invention comprises the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

10. The pharmaceutical preparation according to claim 9, characterized in that The pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients.

11. The pharmaceutical preparation according to claim 9, wherein The auxiliary material is selected from one or more of a filler, a disintegrant, a binder, a lubricant, a surfactant, a flavoring agent, a wetting agent, a pH regulator, a solubilizer or a cosolvent or an osmotic pressure regulator.

12. The composition according to claim 8, wherein The compositions also contain one or more therapeutic agents.

13. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a medicament for treating insomnia, sleep disorders, anxiety, cognitive decline, memory impairment and neurodegenerative diseases.

14. The use according to claim 13, characterized in that The neurodegenerative disease includes neuroinflammation, Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

15. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of a medicament for treating immune system diseases.

16. The use according to claim 14, characterized in that The immune system disease includes rheumatoid arthritis, muscular dystrophy, systemic lupus erythematosus, cord syndrome, multiple sclerosis, systemic sclerosis, scleroderma, small cell lung cancer syndrome, renal tuberculosis, lymphoma, hepatitis B or chronic lymphocytic leukemia.

17. Use of the dihydromyricetin derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the preparation of anti-aging drugs.

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