Small molecule compound with sulfonic acid or sulfinic acid lactone structure, composition and application thereof

Abstract

The invention discloses a small molecule compound with a sulfonic acid or sulfinic acid lactone structure, a composition and application of the small molecule compound. The invention provides a small molecule compound with a sulfonic acid or sulfinic acid lactone structure, which is verified to have extremely low irritation to skin, excellent infiltration capacity and relatively high stability, not only can remarkably remove active oxygen and reduce oxidative stress, but also can relieve skin inflammation, has very good skin cell protecting and repairing capacity, and can be used for preparing skin care products. The composition can be used as a new cosmetic raw material.

Description

[0001] Cross Reference Statement

[0002] This patent application claims priority to the Chinese patent application filed on November 1, 2023, with application number 2023114494701 and invention name “Small molecule compounds, compositions and applications having sulfonic acid or sulfinic acid lactone structure”. The full text of the above application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biochemistry, and in particular to a small molecule compound with a sulfonic acid or sulfenic acid lactone structure, a composition and application thereof. Background Art

[0004] Protecting and repairing damaged skin is usually an important function that skin care products or cosmetics need to have. Factors that cause skin damage include hormone levels, excessive exposure to the sun, haze, and improper diet. Inflammation and damage to the skin can easily lead to rough skin, pigmentation, reduced collagen, and decreased skin antioxidant capacity, which can easily accelerate skin aging in the long term. Studies have shown that quenching ROS and removing harmful oxygen free radicals can relieve skin inflammation and repair damaged skin. In existing cosmetics, although traditional antioxidants such as vitamin C, vitamin E, niacinamide and arbutin have certain ROS scavenging capabilities, they still have serious irritation, weak stability, and poor skin permeability.

[0005] Therefore, it is of great value to develop new cosmetic raw materials that are highly permeable, less irritating, and capable of quenching ROS, scavenging harmful oxygen free radicals, and alleviating skin inflammation. Summary of the invention

[0006] The object of the present invention is to provide a small molecule compound having a sulfonic acid or sulfenic acid lactone structure.

[0007] Another object of the present invention is to provide a composition.

[0008] Another object of the present invention is to provide a method for alleviating skin inflammation.

[0009] Another object of the present invention is to provide a method for reducing active oxygen in skin cells.

[0010] Another object of the present invention is to provide uses of the above compound or composition.

[0011] In order to solve the above technical problems, the present invention provides, in a first aspect, a compound having a structure shown in the following general formula I, a salt, a stereoisomer or a solvate thereof,

[0012]

[0013] Wherein, X is oxygen or nothing;

[0014] R 1 and R 2 are independently hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 、-OCH2OC(O)R 1-4 , halogen, cyano, nitro, -C(O)R 1-5 or-C(O)OR 1-6 ;

[0015] R 1-1 , R 1-2 , R 1-3 , R 1-4 , R 1-5 and R 1-6 are independently hydrogen, C 1-6 Alkyl, halogen substituted C 1-6 Alkyl or -NR a R b ;

[0016] R a and R b are independently hydrogen, C 1-6 Alkyl or halogen substituted C 1-6 alkyl.

[0017] In some preferred embodiments, X is oxygen.

[0018] In some preferred embodiments, R 1 and R 2 are independently hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 or -OCH2OC(O)R 1-4 .

[0019] In some preferred embodiments, R 1 and R 2 are independently hydroxyl, C 1-6 Alkoxy,

[0020] In some preferred embodiments, the C 1-6 Alkyl is C 1-4 More preferably, the C 1-4 The alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0021] In some preferred embodiments, the C 1-6 Alkoxy is C 1-4 Alkoxy; More preferably, C 1-4 Alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy.

[0022] In some preferred embodiments, the halogen is fluorine, chlorine, bromine or iodine.

[0023] In some preferred embodiments, the halogen-substituted C 1-6 Alkyl is halogen substituted C 1-4 Alkyl; more preferably halogen-substituted methyl, halogen-substituted ethyl, halogen-substituted n-propyl, halogen-substituted isopropyl, halogen-substituted n-butyl, halogen-substituted isobutyl or halogen-substituted tert-butyl.

[0024] In some preferred embodiments, R 1 is hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, More preferably, R 1 Hydroxyl, methoxy,

[0025] In some preferred embodiments, R 2 is hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, More preferably, R 2 Hydroxyl, methoxy,

[0026] In some preferred embodiments, R 1-3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or More preferably, R 1-3 is isopropyl, tert-butyl or

[0027] In some preferred embodiments, R 1-4 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or More preferably, R 1-4 It is tert-butyl.

[0028] In some preferred embodiments, the compound is selected from any one of the following:

[0029]

[0030]

[0031]

[0032]

[0033] In some preferred embodiments, the compound is not

[0034] The second aspect of the present invention provides a composition, which comprises the compound described in the first aspect of the present invention, its salt, stereoisomer or solvate; and cosmetically acceptable excipients.

[0035] The third aspect of the present invention provides use of the compound described in the first aspect of the present invention, its salt, stereoisomer or solvate, or the composition described in the second aspect of the present invention in the preparation of cosmetics.

[0036] The fourth aspect of the present invention provides a method for relieving skin inflammation, the method comprising the steps of: administering the compound, salt, stereoisomer or solvate thereof described in the first aspect of the present invention to a subject; or, administering the composition described in the second aspect of the present invention to a subject.

[0037] The fifth aspect of the present invention provides a method for reducing reactive oxygen in skin cells, the method comprising the steps of: administering the compound, salt, stereoisomer or solvate thereof described in the first aspect of the present invention to a subject; or, administering the composition described in the second aspect of the present invention to a subject.

[0038] Compared with the prior art, the present invention has at least the following advantages:

[0039] The present invention provides a class of small molecule compounds with a sulfonic acid or sulfinic acid lactone structure, which have been verified to have extremely low irritation to the skin, excellent penetration ability and high stability, can not only significantly remove active oxygen and reduce oxidative stress, but also relieve skin inflammation, have a good ability to protect and repair skin cells, and can be used as a new raw material for cosmetics.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0042] Figure 1 is a behavior trajectory diagram of a representative zebrafish selected from each group according to an embodiment of the present invention;

[0043] Figure 2 is a statistical diagram of the total movement distance of each group of zebrafish within 20 minutes according to an embodiment of the present invention;

[0044] Figure 3 is a statistical graph of the average movement speed of each group of zebrafish within 20 minutes according to an embodiment of the present invention;

[0045] Figure 4 is a representative picture of inflammatory cell staining at the neuromast of each group of zebrafish according to the embodiment of the present invention;

[0046] Figure 5 is a statistical diagram of the number of inflammatory cells in each group of zebrafish neuromasts according to an embodiment of the present invention;

[0047] Figure 6 is the effect of I-8 on zebrafish wound repair according to the embodiment of the present invention;

[0048] Figure 7 is the statistics of the tail fin areas of each group of zebrafish in the embodiment of the present invention;

[0049] Figure 8 This is a graph showing the effect of I-8 on the number of inflammatory cells in the wound of zebrafish according to an embodiment of the present invention;

[0050] Fig. 9 It is a statistical diagram of the number of inflammatory cells in each group of wounds according to an embodiment of the present invention.

[0051] Figure 10 is According to the ROS content in zebrafish in the embodiment of the present invention, the white line area is the zebrafish yolk sac (ROS quantification area), and the value in brackets is the I-8 concentration, in μg / mL;

[0052] Figure 11 is Statistics of ROS content in zebrafish according to the embodiment of the present invention;

[0053] Figure 12 is According to the β-galactosidase activity picture in zebrafish in the embodiment of the present invention, the tissue appears blue-green after β-galactosidase staining. Comparing the tissues pointed by arrows on the dorsal fin, the more arrows there are, the darker the blue-green color is, and the higher the β-galactosidase activity is;

[0054] Figure 13 isAccording to the statistics of β-galactosidase activity in zebrafish in the embodiment of the present invention;

[0055] Figure 14 is Statistics of the expression of genes related to antioxidant and anti-aging in zebrafish according to the embodiments of the present invention;

[0056] Figure 15 is According to the embodiment of the present invention, compound I-8 can down-regulate TNF-α of HaCaT;

[0057] Figure 16 is According to the embodiment of the present invention, compound I-8 can down-regulate IL-8 of HaCaT;

[0058] Figure 17 is According to the examples of the present invention, compound I-8 can downregulate IL-1β of HaCaT;

[0059] Figure 18 is According to the embodiment of the present invention, compound I-8 can down-regulate EP2 of HaCaT;

[0060] Figure 19 is According to the embodiment of the present invention, compound I-8 can down-regulate TRPV1 of HaCaT;

[0061] Figure 20 is According to the example of the present invention, compound I-8 removes ROS;

[0062] Figure 21 is According to the embodiment of the present invention, compound I-8 increases the secretion of type I collagen;

[0063] Figure 22 is According to the examples of the present invention, compound I-8 up-regulates COL3A1 gene expression;

[0064] Figure 23 is According to the examples of the present invention, compound I-8 up-regulates COL4A1 gene expression;

[0065] Figure 24 is According to the examples of the present invention, compound I-8 up-regulates COL7A1 gene expression;

[0066] Figure 25 is According to the examples of the present invention, compound I-8 up-regulates LAMA5 gene expression. DETAILED DESCRIPTION

[0067] Due to the poor stability of traditional cosmetic antioxidants, the application conditions are demanding, such as decomposition by light during the day, and can only be applied at night; poor skin permeability, often need high concentration and large amount of application to be effective, some individuals often have no effect; in addition, these antioxidants are also highly irritating, and some fragile skins need to strictly control the dosage, otherwise it is easy to cause serious allergies and inflammatory reactions. The inventors have developed a class of small molecule polyphenol compounds with sulfonic acid or sulfenic acid lactone structure through extensive and in-depth research, these compounds have low irritation, good permeability, high stability, and can significantly quench reactive oxygen species (ROS), remove harmful oxygen free radicals, eliminate oxidative stress, relieve skin inflammation and soothe and repair damaged skin cells. Such compounds and their salts, stereoisomers or solvates can be used as new raw materials for cosmetics, with the potential to replace existing antioxidants.

[0068] Compound

[0069] The present invention relates to a class of small molecule compounds having a sulfonic acid or sulfenic acid lactone structure, the structure of which is shown in the following general formula I:

[0070]

[0071] In the above general formula I, X is oxygen or none. Based on the beneficial effect of further improving the stability and permeability of the compound, X is oxygen.

[0072] R 1 and R 2

[0073] R 1 The number of is 0-4, for example, 0, 1, 2, 3 or 4. 1 , which can be hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 、-OCH2OC(O)R 1-4 , halogen, cyano, nitro, -C(O)R 1-5 or -COOR 1-6 ; and, R 1-1 , R 1-2 , R 1-3 , R 1-4 , R 1-5 and R 1-6 are independently hydrogen, C 1-6 Alkyl, halogen substituted C 1-6 Alkyl or -NR a Rb ; R a and R b are independently hydrogen, C 1-6 Alkyl or halogen substituted C 1-6 In a preferred embodiment, R 1 Hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 or -OCH2OC(O)R 1-4 .

[0074] Likewise, R 2 The number of is 0-4, for example, 0, 1, 2, 3 or 4. 2 , which can be hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 、-OCH2OC(O)R 1-4 , halogen, cyano, nitro, -C(O)R 1-5 or -COOR 1-6 ; and, R 1-1 , R 1-2 , R 1-3 , R 1-4 , R 1-5 and R 1-6 are independently hydrogen, C 1-6 Alkyl, halogen substituted C 1-6 Alkyl or -NR a R b ; R a and R b are independently hydrogen, C 1-6 Alkyl or halogen substituted C 1-6 In a preferred embodiment, R 2 Hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3or -OCH2OC(O)R 1-4 .

[0075] In a more preferred embodiment of the present invention, the above R 1-3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or More preferably, R 1-3 isopropyl, tert-butyl or R 1-4 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or More preferably, R 1-4 It is tert-butyl.

[0076] R 1 and R 2 The number of can be equal or different. In a preferred embodiment, R 1 and R 2 The number of is equal.

[0077] R 1 and R 2 The substitution position is not limited, but in a preferred embodiment, R 1 and R 2 The positions of the biphenyl rings are respectively 4 and 4' (as shown in I-8 in a specific embodiment of the present invention). As used in the present invention, the position numbering rule of the biphenyl ring is: And in the sulfonic acid or sulfinic acid lactone structure, the O atom is covalently bonded to the carbon on 2', the S atom is covalently bonded to the carbon on 2, and the oxygen atom and the sulfur atom are covalently bonded to each other to form a lactone structure.

[0078] Based on the excellent effect of promoting the compound to clear active oxygen and alleviate verification, in a more preferred embodiment, R 1 and R 2 At least one of them has the ability to donate electrons to the parent ring, and preferably both have the ability to donate electrons to the target. 1 and R 2 are independently hydroxyl, C 1-6 Alkoxy, More preferably, R 1 and R 2 is hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, In a specific embodiment of the present invention, R 1 Hydroxyl, methoxy, In a specific embodiment of the present invention, R 2 Hydroxyl, methoxy,

[0079] In certain embodiments of the present invention, the specific structures of the compounds are shown in the following table, wherein compounds I-1 to I-12, I-17 and I-18 have better penetration ability than I-13 to I-16.

[0080] surface

[0081]

[0082]

[0083]

[0084]

[0085] Composition

[0086] The present invention also relates to a composition containing the compound of the present invention, its salt, stereoisomer or solvate. The composition of the present invention may be a cosmetic composition or an external preparation.

[0087] As used in the present invention, the term "cosmetics" refers to products that are applied to the human body surface (such as the epidermis, hair, lips, etc.) by spreading, spraying or other similar methods to clean, maintain, beautify, or eliminate bad odors, and the product has a soothing effect on the application site.

[0088] When used as a cosmetic composition, the composition of the compound, its salt, stereoisomer or solvate of the present invention is used as a cosmetic benefit agent in the cosmetic composition. In addition, the composition also includes a cosmetically acceptable medium for diluting, dispersing or using as a carrier of the cosmetic benefit agent to promote its distribution when the composition is applied to the skin.

[0089] These media can be aqueous, anhydrous or emulsions, and oily carriers can form emulsion systems as carriers in the presence of water and emulsifiers. Preferably, the composition is aqueous or emulsion, especially water-in-oil or oil-in-water emulsions, preferably oil-in-water emulsions. Cosmetic compositions are generally in the form of, but not limited to, liquids, creams or emulsions. In a preferred embodiment, water is used as a carrier to form a cosmetic composition. In a preferred embodiment, the cosmetic composition also includes a carrier other than water, such as oil, fat, wax oily base (such as coconut oil, palm oil, olive oil, castor oil, mink oil, snake oil, silicone oil and its derivatives, tallow, lanolin and its derivatives, carnauba wax, spermaceti, beeswax, liquid paraffin, vaseline, microcrystalline wax, squalane, fatty acids, fatty alcohols and esters, etc.), powdery base (such as talc, kaolin, zinc white, titanium dioxide, bentonite, magnesium stearate, zinc stearate, calcium carbonate, magnesium carbonate, calcium hydrogen phosphate, etc.), and solvent base (such as alcohols, small molecule ketones, ethers, small molecule esters).

[0090] In the cosmetic composition of the present invention, cosmetically acceptable auxiliary materials may also be added. The term "cosmetically acceptable auxiliary materials" refers to substances that play a role in the formation, stability or imparting color, fragrance and other characteristics of cosmetics. For example, preservatives (such as benzoic acid and its derivatives, chlorobutanol, chloroxylenol, salicylic acid and its derivatives, sorbic acid and its derivatives, imidazolidinyl urea, phenylethanol, etc.), antioxidants (such as butylated hydroxyanisole, tert-butylated hydroxyanisole, vitamin E, propyl gallate, etc.), moisturizers (such as glycerol, propylene glycol, sorbitol, polyethylene glycol, lactic acid, sodium lactate, sodium pyrrolidonecarboxylate, hyaluronic acid, hydrolyzed collagen, chitin and its derivatives, glucose esters), sunscreens (titanium dioxide, zinc oxide, aminobenzoic acid esters and their derivatives, salicylic acid and its derivatives, benzophenone, etc.), surfactants (lecithin, saponin glycosides, alkyl glycosides, etc.), antioxidants, colorants (such as organic synthetic pigments azo series, anthraquinone series, etc., or inorganic pigments such as zinc oxide, titanium dioxide, ferric oxide, ferrous hydroxide, chromium trioxide, ferric oxide, etc., or natural pigments capsaicin, sorghum red, anthocyanins, etc.), flavors (natural or blended flavors such as ambergris, castoreum, musk, civet, rose oil, peppermint oil, spearmint oil, lavender oil, fennel oil, etc.), water-soluble polymers, chelating agents (such as disodium ethylenediaminetetraacetic acid and its derivatives) and film agents (such as polyvinyl acetate, polyacrylate emulsion, hyaluronic acid, polyglyceryl-2 isostearate, etc.), etc.

[0091] In the cosmetic composition of the present invention, there is no restriction on the amount of each raw material and auxiliary material added.

[0092] Use of the compound or composition

[0093] The present invention also relates to the use of the above-mentioned compound, its salt, stereoisomer or solvate, or a composition containing the above-mentioned compound, its salt, stereoisomer or solvate for: (i) preparing cosmetics; (ii) relieving skin inflammation; (iii) reducing active oxygen in skin cells; and / or (iv) repairing damaged skin.

[0094] As used herein, the term "skin" includes the skin on the face, neck, chest, back, arms, axillae, arms, hands, legs, and scalp. As used herein, a cosmetic benefit agent is meant to include a component that (a) improves a facial or body feature, such as a skin feature, after topical application, (b) benefits a facial or body feature, such as a skin feature, or (c) both (a) and (b). In a preferred embodiment, the compound or cosmetic composition is for topical application.

[0095] Method for relieving skin inflammation and / or reducing active oxygen in skin cells and / or repairing damaged skin

[0096] The present invention also relates to a method for (a) relieving skin inflammation and / or (b) reducing active oxygen in skin cells and / or (c) repairing damaged skin, comprising the steps of administering the above-mentioned compound, its salt, stereoisomer or solvate to a subject; or administering the above-mentioned composition to a subject. The administration method is parenteral administration; preferably external use, such as smearing, external application, and applying an appropriate amount locally to the skin, mucous membrane, etc.

[0097] the term

[0098] As used herein, the term "alkyl" refers to a linear or branched saturated monovalent hydrocarbon group, wherein the alkyl group may be optionally substituted with one or more substituents. In a specific embodiment, the alkyl group is a group having 1 to 20 (C 1-20 ), 1 to 15 (C 1-15 ), 1 to 12 (C 1-12 ), 1 to 10 (C 1-10 ) or 1 to 6 (C 1-6 ) carbon atoms, or a linear saturated monovalent hydrocarbon group having 3 to 20 (C 3-20 ), 3 to 15 (C 3-15 ), 3 to 12 (C 3-12 ), 3 to 10 (C 3-10 ) or 3 to 6 (C 3-6 ) carbon atoms. The linear C 1-6 and branched C 3-6 Alkyl groups are also referred to as "lower alkyl groups". Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms), n-propyl, isopropyl, butyl (including all isomeric forms), n-butyl, isobutyl, tert-butyl, pentyl (including all isomeric forms) and hexyl (including all isomeric forms). For example, C 1-6 Alkyl refers to a linear saturated monovalent hydrocarbon group having 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon group having 3 to 6 carbon atoms. In one embodiment, the alkyl group is an optionally substituted alkyl group described elsewhere herein. In some embodiments, C 1-6 Alkyl is C 1-4 Alkyl, C 1-4 Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl. 1-6 Any one or more hydrogen atoms in the alkyl group are replaced by halogen atoms. 1-6 Alkyl is halogen substituted C 1-4 In other embodiments, the halogen-substituted C 1-4The alkyl group is halogen-substituted methyl, halogen-substituted ethyl, halogen-substituted n-propyl, halogen-substituted isopropyl, halogen-substituted n-butyl, halogen-substituted isobutyl or halogen-substituted tert-butyl.

[0099] As used herein, the term "alkoxy" refers to a stable straight or branched chain, or cyclic hydrocarbon group, or a combination thereof, consisting of the indicated number of carbon atoms and one or more (in one embodiment, one to three) O atoms. Examples of alkoxy include, but are not limited to -O-CH3, -O-CH2-CH3, -O-CH2-CH2-CH3, -O-CH-(CH3)2, and -O-CH2-CH2-O-CH3. In one embodiment, the alkoxy is an optionally substituted alkoxy described elsewhere herein. In some embodiments, the alkoxy is C 1-6 In some embodiments, the alkoxy group is C 1-4 In some embodiments, the alkoxy group is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy. In other embodiments, C 1-6 Any one or more hydrogen atoms in the alkoxy group are replaced by halogen atoms. 1-6 The alkoxy group is a halogen-substituted methoxy group, a halogen-substituted ethoxy group, a halogen-substituted n-propoxy group, a halogen-substituted isopropoxy group, a halogen-substituted n-butoxy group, a halogen-substituted isobutoxy group or a halogen-substituted tert-butoxy group.

[0100] As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0101] As used in the present invention, "(O)" is a =O structure, such as -NH-C(O)- is an amide group, -OC(O)- is an acyloxy group, and -C(O)- is a carbonyl group.

[0102] As used herein, the term "hydrogen" includes protons (1H), deuterium (2H), tritium (3H) and / or mixtures thereof. In the compounds described herein, one or more positions occupied by hydrogen may be enriched with deuterium and / or tritium. Such isotopically enriched analogs may be prepared by appropriately isotopically labeled starting materials obtained from commercial sources or by known literature procedures.

[0103] As used herein, the term "hydroxy" refers to -OH.

[0104] As used herein, the term "amino" refers to -NH2.

[0105] As used herein, the term "cyano" refers to -CN.

[0106] As used herein, the term "nitro" refers to -NO2.

[0107] The term "substituted" refers to moieties having substituents replacing a hydrogen or one or more non-hydrogen atoms on the molecule.

[0108] As used herein, the term "solvate" refers to a compound formed by the interaction of a solvent with a compound provided herein or a salt thereof.

[0109] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0110] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0111] General Synthesis Step 1

[0112]

[0113] General Synthesis Step 2

[0114]

[0115] General Synthesis Step 3

[0116]

[0117] General Synthesis Step 4

[0118]

[0119] Example 1: Synthesis and characterization of compound I-8

[0120]

[0121] Synthesis of intermediate 2Dissolve 2-iodo-5-methoxyphenol (0.73 g, 2.92 mmol, 1.2 eq) and DMAP (0.03 g, 0.24 mmol, 0.1 eq) in DCM (5.00 mL) and pyridine (10.00 mL), cool to 0°C and stir evenly, then dissolve compound 1 (0.50 g, 2.43 mmol, 1 eq) in DCM (5.00 mL), then slowly drop the solution into the reaction system, control the temperature at 0°C, let the system naturally warm up to room temperature after the addition is complete, and react overnight. LC-MS shows that the raw material has reacted completely, stop stirring, add water and DCM to extract, separate the organic phase, concentrate, and purify on a silica gel column (PE: EA = 10: 1) to obtain intermediate 2 (0.96 g, 2.23 mmol, 94.61% yield, 93.42% purity) as a yellow oil. LC-MS: [M+1] + =421.

[0122] Synthesis of intermediate 3 (i.e. I-17) Compound 2 (0.2 g, 0.48 mmol, 1 eq), Pd(Pivate)2 (0.015 g, 0.048 mmol, 0.1 eq), TBAB (0.18 g, 0.57 mmol, 1.2 eq), potassium acetate (0.14 g, 1.43 mmol, 3 eq) were dissolved in DMAc (2.00 mL), the atmosphere was fully replaced with nitrogen, the temperature was raised to 60 ° C, and the reaction was stirred overnight under nitrogen protection. The reaction was completed after LC-MS monitoring. The mixture was filtered, extracted with EA, concentrated, and purified by silica gel column (PE: EA = 1: 1) to obtain intermediate 3 (0.12 g, 0.42 mmol, 88.49% yield, 93.32% purity) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ7.76(d,J=2.8Hz,1H),7.74(d,J=2.7Hz,1H),7.43(d,J=2.7Hz,1H),7.28– 7.23(m,1H),6.93(dd,J=8.8,2.6Hz,1H),6.84(d,J=2.6Hz,1H),3.91(s,3H),3.87(s,3H).LC-MS: [M+1] + =293.

[0123] Synthesis of Compound I-8Compound 3 (1.00 g, 3.42 mmol, 1 eq) was dissolved in DCM (20.00 mL), the temperature was cooled to 0°C, and BBr3 (1.0 M in DCM, 13.68 mmol, 13.68 mL, 4 eq) was slowly added dropwise under nitrogen protection. The temperature was controlled at 0°C. After the addition was completed, the system was allowed to naturally warm to room temperature and reacted overnight. The reaction was monitored by LC-MS. The reaction was quenched with ice water, extracted with EA and DCM, concentrated, and purified by silica gel column (PE: EA = 1: 1) to obtain compound I-8 (0.25 g, 0.95 mmol, 27.78% yield, 100% purity) as a light yellow solid. 1H NMR (400MHz, DMSO-d6) δ7.96(d,J=8.5Hz,1H),7.92(d,J=8.8Hz,1H),7.27–7.22(m,2H),6.87(dd,J=8.6,2.4Hz,1H),6.78(d,J=2.4Hz,1H).LC-MS: [M-1] - =263.

[0124] Example 2: Synthesis and characterization of compound I-11

[0125]

[0126] In Example 1, the reaction of synthesizing compound 3 from compound 2 also produces compound 3'. Compound 3' can be subjected to the same demethylation step to obtain compound I-11. LC-MS: [M-1] - =263.

[0127] Example 3: Synthesis and characterization of compound I-13

[0128]

[0129] Compound I-8 (130 mg, 0.49 mmol), DMAP (6 mg, 0.05 mmol) and DIEA (254 mg, 1.97 mmol) were dissolved in THF (5 mL), cooled to 0°C, and then dimethylaminoformyl chloride (212 mg, 1.97 mmol) was slowly added dropwise to the reaction system, and the temperature was controlled at 0°C. After the addition was completed, the system was allowed to naturally warm to room temperature, reacted overnight, concentrated, and purified on a silica gel column (DCM: EA = 100: 5) to obtain I-13 (90 mg, yield 45.01%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ8.33(d,J=8.8Hz,1H),8.30(d,J=8.8Hz,1H),7.92(d,J=2.4Hz,1H),7.74(dd,J=8.7,2.5Hz,1H) ,7.45(d,J=2.3Hz,1H),7.34(dd,J=8.7,2.4Hz,1H),3.08(d,J=6.2Hz,6H),2.94(d,J=2.8Hz,6H).LCMS:m / z=407.1(M+H + ,ESI).

[0130] Example 4: Synthesis and characterization of compounds I-14 and I-15

[0131] Using the synthesis method in Example 3, I-8 was used as the raw material, and dimethylaminoformyl chloride was replaced by isobutyryl chloride or pivaloyl chloride to obtain I-14 and I-15, both of which were white solids. I-14: LCMS: m / z=405 (M+H + ,ESI).I-15: 1 HNMR (400MHz, DMSO-d6) δ8.24(d,J=8.8Hz,1H),8.07(dd,J=7.7,1.3Hz,1H),7.83(t,J=8.0Hz,1H),7.75(dd,J=8 .3,1.3Hz,1H),7.54(d,J=2.4Hz,1H),7.38(dd,J=8.8,2.4Hz,1H),1.35(s,9H),1.32(s,9H).LCMS:m / z=433(M+H + ,ESI).

[0132] Example 5: Synthesis and characterization of compound I-1

[0133] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-1 was prepared: LCMS: m / z=247 (MH + ,ESI).

[0134] Example 6: Synthesis and characterization of compound I-2

[0135] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-2 was prepared: LCMS: m / z=295 (MH + ,ESI).

[0136] Example 7: Synthesis and characterization of compound I-3

[0137] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-3 was prepared: LCMS: m / z=247 (MH + ,ESI).

[0138] Example 8: Synthesis and characterization of compound I-4

[0139] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-1 was prepared: LCMS: m / z=311 (MH + ,ESI).

[0140] Example 9: Synthesis and characterization of compound I-5

[0141] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-5 was prepared: LCMS: m / z=279 (MH + ,ESI).

[0142] Example 10: Synthesis and characterization of compound I-6

[0143] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-6 was prepared: LCMS: m / z=295 (MH + ,ESI).

[0144] Example 11: Synthesis and characterization of compound I-7

[0145] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-7 was prepared: LCMS: m / z=295 (MH + ,ESI).

[0146] Example 12: Synthesis and characterization of compound I-9

[0147] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-9 was prepared: LCMS: m / z=263 (MH + ,ESI).

[0148] Example 13: Synthesis and characterization of compound I-10

[0149] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-10 was prepared: LCMS: m / z=279 (MH + ,ESI).

[0150] Example 14: Synthesis and characterization of compound I-12

[0151] According to the general synthesis steps 1 and 2, the synthesis method in Example 1 was used to prepare compound As raw material, compound I-12 was prepared: LCMS: m / z=247 (MH + ,ESI).

[0152] Example 15: Synthesis and characterization of compound I-16

[0153] According to the general synthesis step 4, compound I-3 and As raw material, compound I-16 was prepared: LCMS: m / z=493 (M+H + ,ESI).

[0154] Example 16: Synthesis and characterization of compound I-18

[0155] According to the general synthetic step 1, compound As raw material, intermediate 4 was prepared:

[0156]

[0157] Intermediate 4 was hydrogenated to remove the benzyl protection to prepare compound I-18: LCMS: m / z=293 (MH + ,ESI).

[0158] Example 17: Synthesis and characterization of compound I-19

[0159] According to the general synthetic step 1, compound As raw materials, compound I-19 was prepared: 1HNMR (400MHz, DMSO-d6) δ10.95(s,1H),10.67(s,1H),8.58(d,J=8.9Hz,1H),7.32–7.29(m,1H),7.28–7. 26(m,1H),7.26–7.23(m,1H),6.98(dd,J=8.4,1.2Hz,1H),6.90(dd,J=8.1,1.2Hz,1H).LCMS:m / z=263(MH + ,ESI).

[0160] Example 18: Synthesis and characterization of compound I-20

[0161] According to the general synthetic step 1, compound As raw materials, compound I-20 was prepared: 1 H-NMR(400MHz,DMSO-d6)δ10.77(s,1H),10.46(s,1H),10.22(s,1H),8.45–8.43(d,1H),7.2 5-7.24(d,1H),7.21–7.18(dd,1H),6.45–6.44(d,1H),6.28-6.27(d,1H).LCMS: m / z=279(MH + ,ESI).

[0162] Example 19: Synthesis and characterization of compound I-21

[0163] According to the general synthetic step 1, compound As raw material, intermediate 5 was prepared:

[0164]

[0165] Intermediate 5 was hydrogenated to remove the benzyl protection to prepare compound I-21: 1 H-NMR (400MHz, DMSO-d6) δ10.48(s,1H),8.07(d,J=8.7Hz,1H),7.99(d,J=8.7Hz,1H),7.48–7 .41(m,2H),6.89(dd,J=8.7,2.4Hz,1H),6.81(d,J=2.4Hz,1H),3.90(s,3H).LCMS:m / z=277(MH + ,ESI).

[0166] Example 20: Synthesis and characterization of compound I-22

[0167] According to the general synthetic step 1, compound As raw material, intermediate 6 was prepared:

[0168]

[0169] Intermediate 6 is hydrogenated to remove the benzyl protection to prepare compound I-22: 1 H-NMR (400MHz, DMSO-d6) δ11.04(s,1H),8.67(d,J=9.0Hz,1H),7.49(d,J=2.8Hz,1H),7.44(dd,J=9.1,2.9Hz,1H ),7.30(t,J=8.2Hz,1H),7.01(dd,J=8.3,1.2Hz,1H),6.92(dd,J=8.1,1.1Hz,1H),3.91(s,3H).LCMS:m / z=277(MH + ,ESI).

[0170] Example 21: Synthesis and characterization of compound I-23

[0171] According to the general synthetic step 1, compound As raw material, compound I-23 was prepared: LCMS: m / z=277 (MH + ,ESI).

[0172] Example 22: Synthesis and Characterization of Compound I-24

[0173] According to the general synthetic step 1, compound As raw material, compound I-24 was prepared: LCMS: m / z=281 (MH + ,ESI).

[0174] Example 23: Synthesis and characterization of compound I-25

[0175] According to the general synthetic step 1, compound As raw material, compound I-25 was prepared: LCMS: m / z=297 (MH + ,ESI).

[0176] Example 24: Synthesis and Characterization of Compound I-26

[0177] According to the general synthetic step 1, compound As raw material, compound I-26 was prepared: LCMS: m / z=277 (MH + ,ESI).

[0178] Example 25: Synthesis and characterization of compound I-27

[0179] According to the general synthetic step 1, compound As raw material, compound I-27 was prepared: LCMS: m / z=281 (MH + ,ESI).

[0180] Example 26: Synthesis and Characterization of Compound I-28

[0181] According to the general synthetic step 1, compound As raw material, compound I-28 was prepared: LCMS: m / z=297 (MH + ,ESI).

[0182] Test Example 1: Zebrafish model to evaluate the efficacy of compounds in relieving pain and relieving inflammation

[0183] Experimental animals: healthy wild AB zebrafish at 3 dpf (days post fertilization)

[0184] Evaluation of the efficacy of relieving stinging pain: Healthy AB zebrafish were taken and randomly divided into blank control, modeling, positive control and sample groups. The zebrafish in the blank control group were treated with embryo culture water; the zebrafish in the modeling group were treated with modeling drugs (100μMSDS, sodium dodecyl sulfate); the positive control zebrafish were treated with both modeling drugs and positive drugs (200μM asiatica glycoside solution); the zebrafish in the sample group were treated with both modeling drugs and test samples of different concentrations (high, medium and low concentrations) at the same time. After a certain period of continuous action, the behavior trajectory of the zebrafish was recorded using a behavior analyzer, and the changes in the total movement distance and speed were statistically analyzed. The results are presented in Figures 2 to 4 SDS causes pain to zebrafish, prompting them to move violently. Therefore, compared with the blank control group, the total movement distance and average speed of the model group increased significantly. Figure 2 In the behavior trajectory diagram, the black lines are the behavior trajectories of the zebrafish in the hole. The more black lines there are and the more chaotic they are, the more intense the movement of the zebrafish is.

[0185] Evaluation of soothing and anti-inflammatory efficacy: As described above, the modeling drug was 40 μM copper sulfate, the positive drug was 200 μM asiaticoside, and the I-8 concentration was 66.6, 22.2, and 7.4 μg / mL. After zebrafish were treated as described above, the inflammatory cells in the body were stained, the number of inflammatory cells in the neuromasts was counted, and statistical analysis was performed. The results are presented in Figures 5-6 middle.

[0186] Conclusion: When the concentration of I-8 is 22.2, 66.6 and 200 μg / mL, it has a certain effect of relieving stinging pain. When the concentration of I-8 is 7.4, 22.2 and 66.6 μg / mL, it has a certain effect of relieving inflammation.

[0187] Test Example 2: Evaluation of the ability of compounds to penetrate into the skin using a Franz diffusion cell

[0188] Experimental materials: one-month-old Bama Xiang pig back skin, thickness 0.8-1.0 mm; 0.1 mg / mL test compound solution, solvent 50 mM Sodium Phosphate Buffer (PB) buffer solution, containing 20% ​​ethanol.

[0189] Experimental process:

[0190] 1. Pipette 400uL of the test compound solution and add it to the supply pool. Repeat three times.

[0191] 2. Add about 7600uL 50mM PB buffer solution to the corresponding receiving pool

[0192] 3. Permeate the test compound at 32°C for 2h and 6h

[0193] 4. At 2h and 6h, wash the skin with 50mM PB buffer solution containing 20% ​​ethanol, remove the skin of the infiltrated area and homogenize it, add acetonitrile solution containing internal standard

[0194] 5. Centrifuge, take 100uL of the supernatant and add it to 100uL of pure water, mix well and use LC-MS / MS for quantitative analysis

[0195] The quantitative test results of the example compounds penetrating into the skin are shown in Table 2 below:

[0196] Table 2

[0197]

[0198] Compound I-8 has good skin penetration ability and can reach a penetration amount of 10ug / g within 6 hours.

[0199] Test Example 3: Zebrafish Model to Evaluate the Repair Efficacy of Compounds

[0200] Experimental animals: healthy wild AB zebrafish at 3 dpf (days post fertilization)

[0201] Evaluation of wound repair efficacy: Healthy AB zebrafish at 3 dpf were used as experimental animals and randomly divided into blank control group, model group, positive control group and sample group. Zebrafish in the blank control group were treated with culture water; zebrafish in the model group were treated with culture water after cutting the tail fin; zebrafish in the positive control and sample groups were treated with 600 μg / mL hyaluronic acid and different concentrations of I-8 (1, 3, 9 μg / mL) after cutting the tail fin. The above groups of fry were placed in a constant temperature incubator at 28.5°C and kept away from light for 48 hours, with the liquid changed every 24 hours. Image-Pro Plus software was used to calculate the tail fin area of ​​zebrafish in each group, and GraphPad software was used to perform statistical analysis of the data. The results are presented in Figures 7-8 middle.

[0202] Evaluation of the efficacy of repair and anti-inflammatory: As described above, each group of fry was placed in a constant temperature incubator at 28.5℃ and kept away from light for 24 hours. Neutral red was used to mark the inflammatory cells in the fish, and the number of inflammatory cells at the tail was counted. GraphPad software was used to perform statistical analysis on the data. The results are presented in Figures 9-10 .

[0203] Conclusion: If Figures 7-8 As shown in the figure, compared with the blank control group, the tail fin of zebrafish in the model group regenerated, but the area was significantly reduced. When the zebrafish tail amputation model was treated with I-8 at concentrations of 1, 3, and 9 μg / mL, the area of ​​the zebrafish tail fin increased compared with the model group, and it was statistically significant. This result suggests that when the concentration of I-8 is 1, 3, and 9 μg / mL, it can further promote the regeneration of tail fin tissue and has a certain effect on repairing wounds. Figures 9-10 The results showed that compared with the blank control group, the number of inflammatory cells in the zebrafish tail amputation site in the model group increased significantly. When the zebrafish tail amputation model was treated with I-8 at concentrations of 1, 3, and 9 μg / mL, the number of inflammatory cells in the wound was reduced compared with the model group, and it was statistically significant. This result suggests that when the concentration of I-8 is 1, 3, and 9 μg / mL, it has a certain effect on repairing inflammation.

[0204] Test Example 4: Comparison of kinetic solubility in aqueous buffer with Urolithin A

[0205] Experimental method: Take 30μL of 10mM DMSO stock solution of the test compound and place it in the solubility sample plate (two replicates), then add 970μL of aqueous buffer to the above well plate, seal the plate with a plate sealer, and shake the plate at 1100rpm for 2h at 25℃. Transfer the sample in the solubility well plate to the filter plate and filter to obtain the filtrate. Transfer 10μL of the filtrate and 10μL DMSO to 980μL methanol, then dilute 10 times with methanol: water (1:1), and analyze the sample by LC-MS / MS to obtain Area (filtered). Take another 10mM DMSO stock solution of the test compound, dilute it to 300μM with DMSO, transfer 10μL of this 300μM DMSO solution and 10μL aqueous buffer to 980μL methanol, then dilute 10 times with methanol: water (1:1), and analyze the sample by LC-MS / MS to obtain Area (std). The kinetic solubility was calculated according to the following formula:

[0206]

[0207] Where DF is the dilution factor.

[0208] Compound Kinetic solubility (μM) Urolithin A 98.76 I-8 >300*

[0209] *The exact kinetic solubility value cannot be measured when the actual solubility is greater than 300μM

[0210] Conclusion: Compared with the control compound Urolithin A, I-8 has better solubility, overcoming the problem of poor water solubility of Urolithin A and the difficulty in developing topical preparations.

[0211] Test Example 5: Using the zebrafish model to evaluate the anti-oxidative efficacy of compounds

[0212] Experimental animals: healthy wild AB zebrafish at 48 hpf (hours post fertilization)

[0213] Evaluation of anti-oxidative efficacy: Wild AB zebrafish at 48 hpf were used as experimental animals, 4 μM menadione was used as a modeling drug, and a zebrafish oxidative aging model was constructed by chemical mutagenesis. Fucoxanthin was used as a positive control compound, and different concentrations of I-8 were used to act on the zebrafish oxidative aging model for 22 hours. The content of reactive oxygen species (ROS), β-galactosidase activity, and the expression changes of three antioxidant and anti-aging related genes (Cu / Zn-sod, Mn-sod, ampk) in the fish were detected and statistically analyzed.

[0214] During the experiment, the modeling drug, positive compound and I-8 were dispersed together in the culture water of zebrafish, and the above-mentioned groups of larvae were placed in a constant temperature incubator at 28.5℃ and kept away from light for 22 hours. TM Deep Red fluorescent probe and β-galactosidase staining kit were used to specifically label ROS and β-galactosidase in the fish, respectively, and the staining intensity was quantified using Image-Pro Plus software. After extracting the RNA of the whole fish, the expression changes of the three genes Cu / Zn-sod, Mn-sod, and ampk in the fish were detected at the mRNA level using fluorescent real-time quantitative PCR (Quantitative Real-time PCR, RT-qPCR) technology. The above data were statistically analyzed using GraphPad software. The results are presented in Figures 10 to 14.

[0215] Conclusion: As shown in Figures 10 to 13, compared with the blank control group, after continuous treatment of zebrafish with menadione for 22 hours, the ROS content and β-galactosidase activity in the fish increased significantly, and there was a certain statistical significance. Since menadione is an oxidant, it can produce unstable semiquinones through the intracellular reductase system, thereby generating a large amount of ROS, accelerating the aging of the body, and β-galactosidase is a hydrolase in the cell lysosome, and its increased activity is a significant feature of cell aging. When the zebrafish oxidative aging model was treated with I-8 at a concentration of 22.2μg / mL, the ROS content and β-galactosidase activity in the fish were downregulated compared with the modeling group, and there was a significant statistical significance. The results suggest that when the concentration of I-8 is 22.2μg / mL, it has a certain antioxidant effect.

[0216] like Figure 14 The results showed that when the zebrafish oxidative aging model was treated with I-8 at concentrations of 2.5, 7.5 and 22.2 μg / mL, the expression of Cu / Zn-sod and Mn-sod genes was upregulated compared with the model group, and it was statistically significant. When the zebrafish oxidative aging model was treated with I-8 at a concentration of 2.5 μg / mL, the expression of ampk gene was upregulated compared with the model group, and it was statistically significant.

[0217] The above results suggest that when the concentration of I-8 is 2.5, 7.5 and 22.2 μg / mL, it can reverse the abnormal expression of Cu / Zn-sod and Mn-sod genes induced by menadione; when the concentration is 2.5 μg / mL, it can reverse the abnormal expression of ampk gene induced by menadione.

[0218] Inflammation is a physiological response that protects the body from various injuries, such as physical injury, pathogens, exposure to toxic chemicals, and ultraviolet radiation. The main manifestations of inflammation in the early stage are capillary dilation, hyperpermeability, and edema. Various inflammatory mediators play a key role in the occurrence of acute and chronic inflammation, such as interleukin-1α (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2). PGE2 is the most produced prostaglandin in the body, and its production begins with arachidonic acid. Arachidonic acid is first converted to prostaglandin H2 (PGH2) by cyclooxygenase (COX), and then further catalyzed by prostaglandin E synthase to produce PGE2. Ultimately, PGE2 exerts biological functions mainly based on inflammation by acting on four E-type prostaglandin (EP) receptors EP1-4. Among them, EP2 receptors are coupled to Gs proteins and mainly send signals through the cAMP-PKA-CREB pathway triggered by adenylate cyclase. Studies have also shown that selective small molecule antagonists can be developed by targeting EP2 receptors to alleviate downstream pathological processes mediated by EP2 receptors, thereby developing a new generation of anti-inflammatory therapies.

[0219] In addition, TRPV1 receptor is a nociceptor that can be activated by a variety of factors, such as chemicals (capsaicin), noxious heat stimulation and acidification. TRPV1 is widely present in C-type sensory nerve afferent fibers and keratinocytes. After TRPV1 receptor is activated, monovalent and divalent cations (mainly Ca 2+ ) enters the cell, triggers action potentials, and is transmitted to the higher central nervous system, producing a burning pain sensation. Therefore, after the test substance acts, blocking or inhibiting the expression of TRPV1 receptor protein helps to relieve the burning pain and achieve the purpose of soothing. Therefore, by detecting the expression of TRPV1 gene in keratinocytes after sample treatment, this dimensional indicator can be used to preliminarily determine whether the sample has a soothing effect.

[0220] In the following test examples 6-1 and 6-2, a model was established using immortalized keratinocytes (HaCaT) and UVB, and the expression levels of TNF-α, IL-8, IL-1β and EP2 were detected after the test compound was acted on HaCaT; a model was established using immortalized keratinocytes (HaCaT) and capsaicin, and the expression level of TRPV1 was detected after the test compound was acted on HaCaT, so as to conduct a multi-dimensional evaluation of the soothing effect of the test substance.

[0221] Test Example 6-1: Detection of TNF-α, IL-8, IL-1β and EP2 gene expression in HaCaT after UVB irradiation

[0222] 1) Cell inoculation: HaCaT cells were inoculated at 6×105 cells / well in a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 12 h. A normal control group (0 mJ / cm2 + vehicle control), a model control group (80 mJ / cm2 + vehicle control), a low-concentration group (80 mJ / cm2 + low-concentration compound), a medium-concentration group (80 mJ / cm2 + medium-concentration compound), and a high-concentration group (80 mJ / cm2 + high-concentration compound) were set up.

[0223] 2) UVB modeling: Before UVB irradiation, HaCaT was washed 3 times with D'Hanks, and 1 mL of D'Hanks was added to the wells to immerse the cells. The control group was wrapped with tin foil and placed in a dark place. According to the experimental grouping, UVB modeling (80 mJ / cm2) was performed for the groups that needed irradiation;

[0224] 3) Administration: DMEM medium containing different concentrations and compounds was added to each test group and cultured for 24 hours;

[0225] 4) After the culture was completed, the cells were sampled, the total RNA of each experimental group was extracted, cDNA was synthesized, and the gene expression of β-actin and the target gene was detected by q-PCR.

[0226] 5) Using β-actin as the internal reference of gene expression, calculate the relative RNA expression of the target gene.

[0227] Relative RNA expression = 2 △△C(t)

[0228]

[0229] △C(t)=C(t) 目的基因 -C(t) β-actin

[0230] Test Example 6-2: Detection of TRPV1 gene expression in HaCaT after capsaicin stimulation

[0231] 1) Cell inoculation: HaCaT cells were inoculated at 6×105 cells / well in a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 12 h. A normal control group (0 μM capsaicin + vehicle control), a model control group (0 μM capsaicin + vehicle control), a low-concentration group (15 μM capsaicin + low-concentration compound), a medium-concentration group (15 μM capsaicin + medium-concentration compound), and a high-concentration group (15 μM capsaicin + high-concentration compound) were set up.

[0232] 2) Induction and administration: Discard the culture medium in the 6-well plate and carry out the administration. Add the culture medium containing the test substance and the capsaicin stock solution according to the above drug groups, 1 mL per well. After the administration, place the 24-well plate in an incubator (37°C, 5% CO2) and culture for 24h±2h.

[0233] 3) After the incubation, the cells were gently washed once or twice with D-Hanks, fresh culture medium was added to the normal control group, and fresh culture medium containing the corresponding concentration of compound was added to the sample group, and cultured at 37° C., 5% CO 2 for 24 h.

[0234] 4) After the culture was completed, the cells were sampled, the total RNA of each experimental group was extracted, cDNA was synthesized, and the gene expression of β-actin and the target gene was detected by q-PCR.

[0235] 5) Using β-actin as the internal reference of gene expression, calculate the relative RNA expression of the target gene.

[0236] Relative RNA expression = 2 △△C(t)

[0237]

[0238] △C(t)=C(t) 目的基因 -C(t) β-actin

[0239] like Figure 15~ Figure 19 The results showed that compound I-8 could down-regulate the expression of TNF-α, IL-8, IL-1β, EP2 and TRPV1 in a dose-dependent manner, indicating that compound I-8 has a soothing effect.

[0240] Test Example 7: Evaluation of the antioxidant efficacy of compounds using HaCaT immortalized human keratinocytes

[0241] Oxidative reactions such as respiratory metabolism in organisms will produce active free radicals. Under normal conditions, free radicals have a stable scavenging system, which keeps the free radical content in the body at a low concentration. Oxidative stress (OS) refers to the imbalance between oxidation and antioxidant effects in the body due to endogenous and / or exogenous stimulation, resulting in excessive free radicals. Excessive free radicals produce a series of negative effects in the body, which can oxidatively damage biological molecules and further cause cell death and tissue damage. In organisms, except for a very small amount of ROS used by the body, almost all ROS should be removed in time. Medium-wave erythema-effect ultraviolet (UVB) (280-319nm) radiation damage is the most important factor causing skin photoaging, mainly damaging skin keratinocytes (HaCaT), which is specifically manifested as accumulation of photoproducts, increased ROS and increased oxidative damage. Organisms have formed a complete set of antioxidant systems in the long-term evolution process, which makes the production and elimination of free radicals in dynamic balance. It is an adaptive mechanism of organisms. The main members include antioxidant enzymes, antioxidants, and proteins that separate transition metals, etc. They can specifically limit the oxidative damage of the body. Therefore, the antioxidant efficacy of the test compound can be evaluated by detecting the ROS clearance rate in keratinocytes after UVB radiation. The following test example 7 uses HaCaT human immortalized keratinocytes to evaluate the antioxidant efficacy of the compound.

[0242] Test Example 7: Detection of ROS content in HaCaT after UVB irradiation

[0243] 1) Cell seeding: HaCaT cells were plated at 2×10 4 Each well was inoculated into a 96-well plate and cultured in an incubator (37°C, 5% CO2) for 12 h. A control group (120 mJ / cm 2 + solvent control), low concentration group (120mJ / cm 2 + low concentration compounds), medium concentration group (120mJ / cm 2 + medium concentration compound), high concentration group (120mJ / cm 2 + high concentration compounds);

[0244] 2) UVB modeling: Before UVB irradiation, HaCaT was washed 3 times with D'Hanks, and 50 μL of D'Hanks was added to the wells to immerse the cells. The control group was wrapped with tin foil and placed in a dark place. According to the experimental grouping, UVB modeling was performed for the groups that needed irradiation; 3) Drug administration: DMEM culture medium containing different concentrations of compounds was added to each experimental group and cultured for 24 hours;

[0245] 4) ROS fluorescent probe reprinting: According to the dilution ratio of 1:1000, use PBS to dilute DCFH-DA to a final concentration of 10μmol / L. Except for the bare cell wells, discard the culture medium in the remaining wells, wash with PBS three times, add 200μLDCFH-DA working solution to each well, incubate in CO2 incubator for 30min, and after the incubation, wash the cells in each well three times with PBS;

[0246] 5) Fluorescence analysis: Place the 96-well plate to be tested on the detection platform of the fluorescence microplate reader, set the incident light wavelength to 525nm, the excitation light wavelength to 488nm, and read the results for analysis.

[0247] Experimental results: Figure 20 The results showed that compound I-8 could scavenge ROS in a dose-dependent manner, indicating that compound I-8 has antioxidant effect.

[0248] As we age, human skin gradually becomes atrophic (thinning), fragile, poorly pigmented, and has delayed wound healing. Skin fragility is partly attributed to changes in hemidesmosomes and downregulation of various collagens (Collagen Ⅰ, Ⅲ, Ⅳ, Ⅶ) or laminins (such as LN-5) at the dermal-epidermal junction. LN-5 has been shown to be a component of anchoring fibers in the basement membranes of the skin, cornea, conjunctiva, and other tissues. LN-5 participates in cell-to-cell interactions through the mediation of integrins and proteoglycans, thus playing a vital role in cell adhesion, growth, migration, and differentiation. The appearance of skin wrinkles is closely related to the normal synthesis and expression of collagen and laminin. Therefore, the increase in the above-mentioned collagen content and LN-5 can achieve a certain effect in resisting the formation of wrinkles, and plays an important role in the process of skin aging. Therefore, the protein level of Collagen I in epidermal cells (fibroblasts) was detected by ELISA, and the transcription level of epidermal cells (keratinocytes) III, IV, VII and LN-5 was detected by Real-Time PCR to evaluate the firming and anti-wrinkle effect of the test compound. The following test examples 8-1 and 8-2 used HaCaT human immortalized keratinocytes and HSF human skin fibroblasts to evaluate the anti-wrinkle effect of the compound.

[0249] Test Example 8-1: Detection of Type I Collagen Expression in HSF Human Skin Fibroblasts

[0250] 1) Cell seeding: 2×10 cells were seeded in a 96-well plate. 4 cells / well (37°C, 5% CO2) and cultured for 24 hours.

[0251] 2) Administration: Discard the culture medium in the 96-well plate and carry out the administration operation. Add the culture medium containing the compound to the sample group and add the cell culture medium without the compound to the control group, 200 μL per well. After the administration, place the 96-well plate in an incubator (37°C, 5% CO2) and culture for 24h±2h.

[0252] 3) Type I collagen detection: After the incubation, the cell supernatant was collected and the type I collagen content was determined using a human type I collagen enzyme-linked immunosorbent assay kit.

[0253] Test Example 8-2: Detection of Collagen III, IV, VII and LN-5 gene expression levels in HaCaT human immortalized keratinocytes

[0254] 1) Cell seeding: Epidermal cells were seeded at 1×10 6 Cells / well were inoculated in a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 12 h. A control group (0% compound), a low concentration group (low concentration compound), a medium concentration group (medium concentration compound) and a high concentration group (high concentration compound) were set up respectively.

[0255] 2) Administration: according to the experimental groups, add DMEM medium containing different concentrations and compounds and continue culturing for 24 hours;

[0256] 3) Collecting cells: After the culture is completed, collect cell samples for subsequent detection of Collagen III, IV, VII and LN-5 expression.

[0257] like Figure 21~ Figure 25 The results showed that compound I-8 could increase the secretion of type I collagen and up-regulate the gene expressions of COL3A1, COL4A1, COL7A1 and LAMA5 in a dose-dependent manner, indicating that compound I-8 has anti-wrinkle effects.

[0258] Test Example 9: Detection and calculation of the ROS clearance rate of each compound on HaCaT cells after UVB irradiation

[0259] Using the experimental method in Test Example 7, the ROS scavenging rate of each compound on HaCaT cells after UVB radiation was calculated according to the following formula, and the results are listed in Table 1.

[0260]

[0261] Where S: fluorescence intensity

[0262] Table 1.

[0263] Compound (content) ROS clearance rate - % Compound (content) ROS clearance rate - % I-1(0.0025％) 5 I-14(0.0025％) 29 I-2(0.0025％) 41 I-15(0.0025％) 27 I-3(0.0025％) 6 I-16(0.0025％) 38 I-4(0.0025％) 37 I-17(0.0025％) 2 I-5(0.0025％) 31 I-18(0.0025％) 21 I-6(0.0025％) 28 I-19(0.0025％) 19 I-7(0.0025％) 40 I-20(0.0025％) 37 I-8(0.00015625％) 12 I-21(0.0025％) 8 I-8(0.000625％) 22 I-22(0.0025％) 9 I-8(0.0025％) 26 I-23(0.0025％) 34 I-9(0.0025％) 11 I-24(0.0025％) 31 I-10(0.0025％) 15 I-25(0.0025％) 29 I-11(0.0025％) 12 I-26(0.0025％) 36 I-12(0.0025％) 33 I-27(0.0025％) 35 I-13(0.0025％) 35 I-28(0.0025％) 30

[0264] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A compound having a structure as shown in the following general formula I, a salt, a stereoisomer or a solvate thereof, in, X is oxygen or none; R 1 and R 2 are independently hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 、-OCH2OC(O)R 1-4 , halogen, cyano, nitro, -C(O)R 1-5 or -COOR 1-6 ; R 1-1 , R 1-2 , R 1-3 , R 1-4 , R 1-5 and R 1-6 are independently hydrogen, C 1-6 Alkyl, halogen substituted C 1-6 Alkyl or -NR a R b ; R a and R b are independently hydrogen, C 1-6 Alkyl or halogen substituted C 1-6 alkyl.

2. The compound, salt, stereoisomer or solvate thereof according to claim 1, characterized in that X is oxygen.

3. The compound, salt, stereoisomer or solvate thereof according to claim 1, characterized in that The C 1-6 Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; and / or, the C 1-6 Alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy; and / or, the halogen is fluorine, chlorine, bromine or iodine; and / or, the halogen-substituted C 1-6 The alkyl group is halogen-substituted methyl, halogen-substituted ethyl, halogen-substituted n-propyl, halogen-substituted isopropyl, halogen-substituted n-butyl, halogen-substituted isobutyl or halogen-substituted tert-butyl.

4. The compound, salt, stereoisomer or solvate thereof according to claim 1, characterized in that R 1 and R 2 are independently hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, amino, -NC(O)R 1-1 、-NR a R b 、-CH2C(O)OR 1-2 , phenyl, -OC(O)R 1-3 or -OCH2OC(O)R 1-4 .

5. The compound, salt, stereoisomer or solvate thereof according to claim 1, characterized in that R 1 and R 2 are independently hydrogen, hydroxyl, C 1-6 Alkoxy, Among them, R 1-3 and R 1-4 are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or 6. The compound, salt, stereoisomer or solvate thereof according to claim 1, characterized in that R 1 and R 2 are independently hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, 7. The compound, salt, stereoisomer or solvate thereof according to any one of claims 1 to 6, characterized in that The compound is selected from any one of the following:

8. A composition, characterized in that The composition comprises the compound according to any one of claims 1 to 7, its salt, stereoisomer or solvate; and cosmetically acceptable excipients.

9. Use of the compound according to any one of claims 1 to 7, its salt, stereoisomer or solvate, or the composition according to claim 8 in the preparation of cosmetics.

10. A method for relieving skin inflammation and / or reducing active oxygen in skin cells, characterized in that: The method comprises the steps of: The compound, salt, stereoisomer or solvate thereof according to any one of claims 1 to 7 is administered to the subject; or the composition according to claim 8 is administered to the subject.

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