1,3-bisbenzylphenol compound, preparation method and application thereof in whitening and anti-aging cosmetics

Abstract

The application belongs to the technical field of cosmetics and medicine, and specifically discloses 1,3-bisbenzyl phenol compounds, a preparation method and application thereof in the preparation of whitening and anti-aging cosmetics. The application takes a diaryl heptenepolyphenol compound with alpha-glucosidase inhibition activity in sea flower as a parent, and first designs and synthesizes 4,4'-(1,3-phenylene bis(methylene)) bis 2-methyl phenol (compound 2) and 3,3'-(1,3-phenylene bis(methylene)) bis 1,2-benzene diol (compound 3). The application discloses that the compounds 1-3 have significant activity in inhibiting alpha-glucosidase and scavenging free radicals, the compound 1, i.e., 4,4'-(1,3-phenylene bis(methylene)) diphenol and the compound 2 have significant activity in inhibiting tyrosinase, the compounds have a higher cell melanin synthesis inhibition rate than a classic whitening ingredient, i.e., phenylethyl resorcinol on the market, and therefore have a good development and application prospect in the field of whitening, lightening and anti-aging cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics and medicine, and particularly relates to a 1,3-bisbenzylphenol compound, a preparation method and an application thereof in the preparation of whitening and anti-aging cosmetics. Background Art

[0002] Oxygen free radicals generated during cellular metabolism can cumulatively damage intracellular biomolecules, leading to cell aging and loss of proliferation. This imbalance in the oxidative and antioxidant systems is considered a major contributing factor to skin aging. Tyrosinase, also known as polyphenol oxidase, is an oxidoreductase widely found in animals, plants, microorganisms, and humans. It is a key rate-limiting enzyme in melanin synthesis and is closely associated with the development of excessive melanin deposition, such as freckles and brown spots on human skin. Its application in medicine and cosmetics has attracted widespread attention both domestically and internationally in recent years. Currently, most skin whitening and brightening agents on the market rely on tyrosinase inhibition, such as α-arbutin, kojic acid, and 4-butylresorcinol, or antioxidants, such as vitamin C, vitamin E, and their derivatives, to achieve their whitening effects. α-arbutin, a hydroquinone derivative, is cytotoxic to melanocytes and a strong skin irritant. Long-term use can cause permanent white spots on the skin, and it has been included in the list of banned cosmetic ingredients in many countries. Kojic acid inhibits tyrosinase's catalytic activity by chelating its copper ions. Its use in commercial skin and hair lighteners can easily lead to contact allergies, skin lesions, and liver cancer. Vitamin C and vitamin E, important antioxidants in the human body, do not directly inhibit tyrosinase but instead reduce the production of colored intermediates in melanin biosynthesis, resulting in only limited whitening and brightening effects. These unfavorable factors limit their widespread use in whitening and anti-freckle cosmetics.

[0003] Previously, Xu Gang's research group isolated a series of diarylheptanoids from Ottelia acuminata var. acuminata, an edible aquatic vegetable of the Bai ethnic group in Dali, Yunnan. These compounds exhibited significant α-glucosidase inhibitory activity and have potential therapeutic value for diabetes (Liu HX, Ma JZ, Ye YS, Zhao JJ, Wan SJ, Hu XY, Xu G. α-Glucosidase inhibitive diarylheptanoids from Ottelia acuminata var. acuminata, a traditional vegetable of Bai Nationality in Yunnan, Natural Products and Bioprospecting 2022, 12:22). Studies have shown that tyrosinase is a protein with sugar chains. During its maturation, its original sugar chains must undergo a series of modifications and cleavages before it can be converted into mature tyrosinase and exert its normal biological function. α-glucosidases I and II are key enzymes in this sugar chain modification and cleavage process. (Mehta A, Zitzmann N, Rudd PM, Block TM, Dwek RA. α-Glucosidase inhibitors as potential broad-based anti-viral agents, FEBS Letters, 1998, 430(1): 17-22). When α-glucosidase is inhibited, the sugar chain modification on the glycoprotein is blocked, and active tyrosinase cannot be produced, which in turn reduces the formation of melanin. (Takahashi H, Parsons PG, Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells, the Journal of Investigative Dermatology, 1992, 98(4): 481-487.). Therefore, inhibiting α-glucosidase to reduce the maturation of tyrosinase and thereby reduce the production of melanin and skin pigmentation is a new possible approach for whitening and removing freckles. Considering that the content of diarylheptene polyphenol compounds from natural sources is low, their structural characteristics are cis- and trans-olefin structures, their molecular stability is poor, they are easily isomerized by double bond shift through ene reactions, they are difficult to synthesize, and their molecules are relatively flexible.To solve the problem of drug source, we use this kind of natural product as a lead compound, and use it as a template to simplify and replace the flexible open-chain molecular structure into rigid benzene ring polyphenol structural analogues by using conformational restriction and bioisosteric replacement strategies. At the same time, in order to improve the antioxidant activity, the phenolic hydroxyl group on the benzene ring is increased to synthesize a series of 1,3-bisbenzylphenol compounds (compounds 1-3), and their tyrosinase inhibitory activity, alpha-glucosidase inhibitory activity, antioxidant activity and cell melanin synthesis inhibitory activity are studied. Among them, compound 1 is a known compound (Larry Q. Reyes, Salumeh Issazadeh, Jane Zhang, Buu Dao, and Russell J. Varley, Synthesis of Tri-Aryl Methane Epoxy Resin Isomers and Their Cure with Aromatic Amines, Macromolecular Materials and Engineering, Macromol. Mater. Eng. 2020, 305(2), 1900546.), but there is no report on its biological activity. The antioxidant activity, tyrosinase inhibitory activity and alpha-glucosidase inhibitory activity of compounds 1, 2 and 3 are first discovered, and compounds 2 and 3 are new compounds. This kind of component has higher cell melanin synthesis inhibition rate than the classic whitening ingredient phenylethyl resorcinol on the market, and has good application prospect in whitening, anti-aging and diabetes treatment. SUMMARY

[0004] In view of the deficiencies in the prior art, the technical problem to be solved by the present application is to provide a 1,3-bisbenzylphenol compound, a preparation method and its application in the preparation of whitening and anti-aging cosmetics.

[0005] To solve the technical problem of the present application, the present application provides the following technical solutions:

[0006] The first aspect of the technical scheme of the present application is to provide a 1,3-bisbenzylphenol compound, whose structural formula is shown in the structural formula of compound 2 and compound 3.

[0007]

[0008] The compound 2 is named 4,4'-(1,3-phenylenebis(methylene))bis2-methylphenol.

[0009] The compound 3 is named 3,3'-(1,3-phenylenebis(methylene))bis1,2-benzenediol.

[0010] The second aspect of the technical solution of the present invention is to provide a preparation method of the above-mentioned 1,3-bisbenzylphenol compound, which comprises the following steps: 1,3-bis(bromomethyl)benzene or 1,3-bis(chloromethyl)benzene is mixed with o-cresol or 1,2-diphenol in a molar ratio of 1:(5-10), aluminum chloride is used as a catalyst, and under nitrogen protection, the mixture is heated at 100-120°C for a period of time (preferably, at 110°C for 2 hours), and then purified by column chromatography to obtain the compound.

[0011] The third aspect of the technical solution of the present invention is to provide the application of the above-mentioned 1,3-bisbenzylphenol compounds. Specifically:

[0012] Application of the above 1,3-bisbenzylphenol compounds in the preparation of α-glucosidase inhibitors.

[0013] Application of the above 1,3-bisbenzylphenol compounds in the preparation of drugs for treating diabetes.

[0014] The above-mentioned 1,3-bisbenzylphenol compounds are used in the preparation of anti-skin oxidation / aging products, wherein the anti-oxidation mainly refers to the removal of intracellular reactive oxygen free radicals.

[0015] The use of the above-mentioned 1,3-bisbenzylphenol compounds in the preparation of skin care products or skin disease treatment drugs for reducing melanin production, preventing or treating pigmentation diseases. The pigmentation diseases are preferably freckles, chloasma, stretch marks, age spots or melanoma.

[0016] In particular, the use of the above compound 2 in the preparation of tyrosinase inhibitors.

[0017] The fourth aspect of the technical solution of the present invention is to provide the application of compound 1. Specifically:

[0018] Use of compound 1 in the preparation of tyrosinase inhibitors.

[0019] Application of compound 1 in the preparation of α-glucosidase inhibitors.

[0020] Application of compound 1 in the preparation of drugs for treating diabetes.

[0021] The application of compound 1 in the preparation of anti-skin oxidation / aging products, wherein the anti-oxidation mainly refers to the scavenging of intracellular reactive oxygen free radicals.

[0022] Use of compound 1 in the preparation of a skin care product or a skin disease treatment drug for reducing melanin production, preventing or treating pigmentation diseases, wherein the pigmentation diseases are preferably freckles, chloasma, stretch marks, age spots and / or melanoma.

[0023] The compound 1 is named 4,4'-(1,3-phenylenebis(methylene))diphenol;

[0024] The compound 1 has the following structural formula:

[0025]

[0026] The fifth aspect of the technical solution of the present invention is to provide a whitening and / or anti-skin oxidation / aging skin care composition or pharmaceutical composition, comprising an effective amount of at least one of the structural formulas of Compound 1, Compound 2, and Compound 3 as an active ingredient, and one or more acceptable carriers in the cosmetics field or the pharmaceutical field.

[0027] The pharmaceutical composition mentioned above refers to a pharmaceutical composition for treating skin diseases.

[0028] The skin care composition or skin disease treatment composition is related to whitening and / or anti-oxidative / aging effects on the skin. Whitening refers to inhibiting tyrosinase activity, α-glucosidase activity, and / or melanin production and pigmentation in cells; and anti-oxidative / aging effects refer to scavenging reactive oxygen free radicals within cells, particularly those generated by ultraviolet light.

[0029] The sixth aspect of the technical solution of the present invention is to provide a skin care composition or pharmaceutical composition comprising an effective amount of at least one of the structural formulas of Compound 1, Compound 2, and Compound 3 as an active ingredient for use in the preparation of whitening and / or anti-aging skin care products or skin disease treatment drugs.

[0030] Compounds 1, 2, and 3 have good skin-lightening and whitening effects (i.e., for example, they have strong α-glucosidase and / or tyrosinase inhibitory effects in an in vitro cell test system, reducing the production of cellular melanin); they also have good antioxidant effects (i.e., for example, they have strong free radical scavenging effects in an in vitro cell test system); specifically, compounds 1 and 2 have high tyrosinase inhibitory activity, α-glucosidase inhibitory activity, and antioxidant activity, and have a cellular melanin synthesis inhibition rate significantly higher than that of the classic whitening ingredient phenylethylresorcinol on the market; compound 3 has extremely excellent α-glucosidase inhibitory activity and antioxidant activity, and has a cellular melanin synthesis inhibition rate slightly better than that of the classic whitening ingredient phenylethylresorcinol on the market. Compounds 1, 2, and 3 can be prepared in a highly pure form; are dermatologically and toxicologically acceptable; and exhibit good stability to light effects, and therefore can be preferably used as active ingredients in whitening and anti-aging cosmetics.

[0031] The skin care or dermatological treatment product is associated with whitening and / or anti-oxidative / anti-aging effects. In this regard, whitening refers to inhibiting tyrosinase activity, and / or inhibiting alpha-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-oxidative / anti-aging refers to scavenging of intracellular reactive oxygen species, particularly those generated by ultraviolet light exposure.

[0032] The skin care or dermatological treatment product is capable of improving the aesthetic and / or cosmetic appearance of the skin. These improvements can be manifested in any of the following: reduction of dermatological signs of aging, which are attributed to, for example, chronological aging, hormonal aging, and / or photoaging; reduction of skin fragility; reduction of pore size; prevention and / or reversal of collagen and / or elastin loss; amelioration of the effects of estrogen imbalance; prevention of skin atrophy; prevention and / or reduction of the appearance and / or depth of lines and / or wrinkles, including fine lines and / or wrinkles; prevention, reduction, and / or treatment of hyperpigmentation; improvement of liver tone, skin tone clarity, and / or firmness; prevention, reduction, and improvement of skin sagging; promotion of antioxidant activity; improvement of skin resiliency, plumpness, suppleness, and / or softness; increase of procollagen and / or collagen production; improvement of skin texture and / or promotion of restoration of original texture; promotion of skin barrier repair and / or function; improvement of skin contour appearance; restoration of skin radiance and / or brightness; minimization of dermatological signs of fatigue and stress, such as skin blotching and / or resistance to stress caused by environmental influences (pollution, temperature changes); replenishment of essential nutrients and / or components in the skin that are diminished due to aging and / or menopause; enhancement of the association between skin cells; increase of cell proliferation and / or multiplication; enhancement of skin cell metabolism that is diminished due to aging and / or menopause; retardation of cell aging; inhibition of enzymes in the skin that accelerate skin cell aging; minimization of skin dryness and / or increase of skin moisture; minimization of skin discoloration, including dark under-eye circles; promotion and / or acceleration of cell turnover; increase of skin thickness; increase of skin elasticity and / or firmness; increase of epidermal exfoliation, with or without the aid of alpha-hydroxy acids or other exfoliants; prevention and reversal of glycosaminoglycan (GAG), collagen, and / or elastin loss; microcirculation improvement; reduction and / or prevention of cellulite formation; reduction of acne formation; use as a skin and hair lightening agent or anti-aging spot agent. In particular, the pharmaceutical composition of the present application is capable of improving the aesthetic appearance, health, and vitality of the skin. Such an improvement can be manifested in at least one of the following: reduction of melanin production, treatment of pigmentation disorders, preferably freckles, chloasma, stretch marks, age spots, or melanoma; prevention and / or reversal of collagen and / or elastin loss; improvement of skin texture; improvement of skin tone, clarity, and / or firmness; promotion / acceleration of cell turnover; and increase of skin thickness.

[0033] The seventh aspect of the technical solution of the present invention is to provide a pharmaceutical composition for treating diabetes, which includes an effective amount of at least one of the compounds 1, 2, and 3 as active ingredients, and one or more acceptable carriers in the pharmaceutical field.

[0034] The diabetes therapeutic drug exerts its effect by inhibiting the activity of α-glucosidase.

[0035] The eighth aspect of the technical solution of the present invention is to provide a pharmaceutical composition comprising an effective amount of at least one of Compound 1, Compound 2, and Compound 3 as an active ingredient for use in the preparation of a drug for treating diabetes.

[0036] The acceptable carriers in the cosmetic field mentioned above refer to conventional cosmetic carriers in the cosmetic field, which include emulsifiers (such as oil-in-water, water-in-oil, water-in-silicone oil, silicone oil-in-water, water-in-oil-in-water, oil-in-water-in-oil, silicone oil-in-water-in-oil, etc.), creams, lotions, liquids (such as aqueous solutions or water-alcohol solutions), anhydrous bases (such as lipsticks or powders, etc.), gels, ointments, milks, ointments, sprays, solid doses, eye creams, etc. The compositions of the present invention can be made into various forms of cosmetics, including sunscreens, sunless tanning products, hair care products (such as shampoos, conditioners, hair dyes, bleaches, hair straighteners, and perms), nail polishes, moisturizers, skin lotions and creams, lipsticks and lip balms, facial cleansers, toners, facial masks, deodorants, antiperspirants, exfoliants, shaving products (creams, aftershaves), wet wipes, tanning lotions, body washes, body oils, foot care products (powders, sprays), foundation creams, rouge, eye shadows and eyeliners, lip glosses, mascara, and baby products (baby skin creams, body oils, shampoos, talcum powders, and wet wipes). In addition, these cosmetics can also be used as leave-on cosmetics or makeup removers.

[0037] The acceptable carriers in the pharmaceutical field as described above include one or more anesthetics, antiallergens, antifungals, antimicrobials, anti-inflammatory agents, antioxidants, preservatives, chelating agents, colorants, decolorizing agents, softeners, emulsifiers, exfoliants, film-forming agents, fragrances, wetting agents, insect repellents, lubricants, moisturizing agents, pharmaceutical agents, light stabilizers, preservatives, skin protectants, skin penetration enhancers, sunscreens, stabilizers, surfactants, thickeners, viscosity regulators, vitamins, or any combination thereof.

[0038] The present invention has the following advantages and beneficial effects:

[0039] 1. Based on the diarylheptene polyphenol compounds with α-glucosidase inhibitory activity in sea lettuce, a series of 1,3-bisbenzylphenol compounds were designed and synthesized for the first time: 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol (Compound 2) and 3,3'-(1,3-phenylenebis(methylene))bis-1,2-benzenediol (Compound 3).

[0040] 2. It was disclosed for the first time that 1,3-bisbenzylphenol compounds 1-3 have significant activity in inhibiting α-glucosidase and scavenging free radicals. Compounds 1 and 2 have significant activity in inhibiting tyrosinase. Compounds 1-3 have a higher cell melanin synthesis inhibition rate than the classic whitening ingredient phenylethylresorcinol on the market.

[0041] 3. Compounds 1-3 reported in the present invention have simple synthesis steps, high yield, low manufacturing cost, good safety and stability, and have good development and application prospects in the field of whitening, brightening and anti-aging cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 :Effects of three compounds and positive drug kojic acid on tyrosinase activity.

[0043] Figure 2 : Inhibitory effects of three compounds and positive acarbose on α-glucosidase activity.

[0044] Figure 3 : Scavenging effects of three compounds and positive drug vitamin E on ABTS free radicals.

[0045] Figure 4 :Effects of three compounds and positive drug phenylethylresorcinol on B16 cell activity.

[0046] Figure 5 : Effects of compounds 1-3 and phenylethylresorcinol on melanin synthesis in B16 cells.

[0047] Figure 6 :Compound 1 1 H NMR spectrum (600 MHz, CD3OD).

[0048] Figure 7 :Compound 1 13 C NMR spectrum (150 MHz, DMSO-d6).

[0049] Figure 8 :Compound 2 1 H NMR spectrum (600 MHz, DMSO-d6).

[0050] Figure 9 :Compound 213 C NMR spectrum (150 MHz, DMSO-d6).

[0051] Figure 10 :Compound 3 1 H NMR spectrum (500 MHz, DMSO-d6).

[0052] Figure 11 :Compound 3 13 C NMR spectrum (125 MHz, DMSO-d6). DETAILED DESCRIPTION

[0053] The following specific examples can further illustrate the present invention, but do not limit the scope of protection requested by the claims of the present invention in any way.

[0054] Example 1: Synthesis of Compounds 1-3

[0055] 1. Synthesis of Compound 1: 1,3-bis(bromomethyl)benzene (1.5 g, 5.73 mmol), phenol (6.2 mL, 57.3 mmol), and aluminum chloride (195 mg, 1.5 mmol) were weighed in sequence and heated at 110° C. under nitrogen protection until the starting materials disappeared. The mixture was reacted for 2 hours, cooled to room temperature, and extracted with saturated aqueous sodium carbonate solution and ethyl acetate three times each. The mixture was then dried over sodium sulfate, filtered and dried by suction, passed through a silica gel column, and purified with ethyl acetate / petroleum ether (EA:PE=1:200, v / v) to give the product, Compound 1 (473.3 mg, 42% yield, white solid).

[0056]

[0057] 4,4'-(1,3-phenylenebis(methylene))diphenol (Compound 1): white solid, 1 H NMR (600MHz, CD3OD, δ, ppm, J / Hz): 9.17 (s, 2H), 7.15 (t, J = 10, 1H), 7.04 (s, 1H), 6.98-6.95 (m, 6H), 6.66-6.64 (m, 4H), 3.76 (s, 4H). 13 C NMR (150MHz, DMSO-d6, δ, ppm): 155.9, 142.4, 131.8, 130.0, 129.3, 128.8, 126.5, 115.6, 40.7. HRMS(ESI-)calcd for C 20 H 17 O2[MH]-=289.1234, found 289.1246. 1 H NMR spectrum, 13C NMR spectrum Figures 6-7 shown.

[0058] 2. Synthesis of Compound 2: 1,3-bis(bromomethyl)benzene (0.3 g, 1.15 mmol), o-cresol (0.6 g, 5.75 mmol), and aluminum chloride (39 mg, 0.29 mmol) were weighed in sequence and heated at 110°C under nitrogen protection until the starting materials disappeared. The mixture was reacted for 2 hours, cooled to room temperature, and directly chromatographed on a silica gel column using ethyl acetate / petroleum ether (EA:PE=1:7.5, v / v) as the eluent to give the product, Compound 2 (40.7 mg, 26% yield, brown solid).

[0059]

[0060] 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol (Compound 2): brown solid, 1 H NMR (600MHz, DMSO-d6, δ, ppm, J / Hz): 9.05 (s, 2H), 7.15 (t, J = 7.6Hz, 1H), 7.05 (s, 1H), 6.97 (d, J = 7. 6Hz, 2H), 6.87 (s, 2H), 6.80 (dd, J = 8.2, 2.3Hz, 2H), 6.67 (d, J = 8.1Hz, 2H), 3.73 (s, 4H), 2.06 (s, 6H). 13 C NMR(150MHz,DMSO-d6,δ,ppm):153.5,142.0,131.3,130.9,128.8,128.3,126.7,126.0,123.6,114.5,40.4,16.0.HRMS(ESI-)calcd for C 22 H 21 O2[MH]-=317.1547, found 317.1561. 1 H NMR spectrum, 13 C NMR spectrum Figures 8-9 shown.

[0061] 3. Synthesis of compound 3: 1,3-bis(bromomethyl)benzene (1.5 g, 5.73 mmol), 1,2-benzenediol (6.3 g, 57.2 mmol), and aluminum chloride (195 mg, 1.5 mmol) were weighed in sequence and heated at 110°C under nitrogen protection until the starting materials disappeared. The mixture was reacted for 2 hours, cooled to room temperature, and directly chromatographed on a silica gel column using ethyl acetate / petroleum ether (EA:PE=1:2, v / v) as the eluent to give the product compound 3 (124.8 mg, 32% yield, light yellow solid).

[0062]

[0063] 3,3'-(1,3-phenylenebis(methylene))bis-1,2-benzenediol (Compound 3): Pale yellow solid, 1 H NMR (500MHz, DMSO-d6, δ, ppm, J / Hz): 8.74 (s, 2H), 8.64 (s, 2H), 7.15 (t, J = 7.6Hz, 1H), 7.01 (s, 1H), 6.95 (dd ,J=7.6,1.7Hz,2H),6.61(d,J=8.0Hz,2H),6.53(d,J=2.1Hz,2H),6.44(dd,J=8.0,2.1Hz,2H),3.69(s,4H). 13 C NMR(125MHz,DMSO-d6,δ,ppm):145.5,143.9,142.4,132.5,129.4,128.7,126.6,119.8,116.5,115.9,41.0.HRMS(ESI)calcd for C 20 H 18 NaO4[M+Na] + =345.1097, found 345.1097. 1 HNMR spectrum, 13 C NMR spectrum Figures 10-11 shown.

[0064] Example 2: Study on the inhibition of tyrosinase activity by compounds 1-3

[0065] 1. Solution preparation

[0066] Phosphate buffer (0.2 M, pH 6.8): Accurately weigh 2.84 g of Na2HPO4 and 2.4 g of NaH2PO4, dissolve them separately in purified water to 100 mL, mix equal volumes of the two, and adjust the pH to 6.8.

[0067] L-DOPA solution: Accurately weigh 3.95 mg of L-DOPA and dissolve it in 10 mL of phosphate buffer to prepare a 2 mM L-DOPA solution.

[0068] Tyrosinase solution: Accurately weigh 1 mg of tyrosinase (enzyme activity of 500 U / mg) and dissolve it in 5 mL of phosphate buffer (0.2 M, pH 6.8) to prepare a tyrosinase solution with an enzyme activity of 100 U / mL.

[0069] Sample working solution: Accurately weigh 10 mg each of compounds 1-3 and kojic acid into 500 μL of ultrapure water. Slowly add 1 M NaOH dropwise until the solution is clear and translucent. Finally, make up to 1 mL with purified water to prepare a 10 mg / mL stock solution. The test compounds and the positive control drug kojic acid are then diluted with the above-mentioned phosphate buffer to sample working solutions of 31.2, 62.5, 125, 250.00, and 500.00 μg / mL.

[0070] 2. Tyrosinase activity assay

[0071] Four experimental groups were set up: sample group A1, sample negative control group A2, enzyme standard group B1, and enzyme negative control group B2; three parallels were set up for each sample, and a 200 μL reaction system was prepared according to Table 1. The corresponding volume of phosphate buffer, the test sample solution of each concentration (final concentration of 3.12, 6.25, 12.5, 25, and 50 μg / mL), tyrosinase solution (final concentration of 20 U / mL), and the reaction substrate L-DOPA (final concentration of 1.2 mM) were added to a 96-well plate in sequence. The plates were incubated at 37°C in a thermostatted microplate shaker for 30 minutes, and the absorbance of each experimental group was then measured at 475 nm in a multifunctional microplate reader (SPARK 10M, TECAN). The inhibition rate of the test compound on tyrosinase was calculated as follows:

[0072] Tyrosinase inhibition rate (%) = [1-(A1-A2) / (B1-B2)] × 100

[0073] Table 1 Tyrosinase activity inhibition test reaction system preparation (volume / μL)

[0074]

[0075] like Figure 1 As shown in Figure 2, compound 1, compound 2 and the positive control drug kojic acid all showed dose-dependent inhibitory effects on tyrosinase activity, with compound 2 having the best inhibitory effect. The half-inhibitory concentrations (IC50) of compound 1, compound 2 and kojic acid on tyrosinase were calculated by nonlinear fitting using Graphpad Prism 8.0 software. 50 The concentrations of compound 3 were 10.43 μg / mL (i.e. 35.96 μM), 4.36 μg / mL (i.e. 13.68 μM) and 18.61 μg / mL (i.e. 131.6 μM), respectively. 50 >50μg / mL). The results showed that compounds 1 and 2 had good inhibitory activity against tyrosinase, with tyrosinase inhibitory effects of 3.7 times and 9.6 times that of kojic acid, respectively, and are expected to be used as browning inhibitors for skin whitening and brightening.

[0076] Example 3: Study on inhibition of alpha-glucosidase activity by compound 1-3

[0077] 1. Solution preparation

[0078] Phosphate buffer (0.2M, pH 6.8): precisely weigh Na2HPO4 2.84g, NaH2PO4 2.4g, respectively, dissolve in purified water to 100mL, mix equal volumes and adjust pH to 6.8.

[0079] Alpha-glucosidase solution: alpha-glucosidase powder from Saccharomyces cerevisiae is prepared into 1U / mL alpha-glucosidase solution with phosphate buffer (0.2M, pH 6.8), and stored at -20℃.

[0080] Substrate PNPG solution: precisely weigh 4-nitrophenyl-α-D-glucopyranoside (PNPG) 211mg with an analytical balance, add 70mL of the above phosphate buffer to dissolve uniformly, prepare 10mM substrate stock solution, and store at -20℃ in the dark.

[0081] Positive control working solution: acarbose is selected as the positive control in this experiment, and acarbose powder 103.3mg is precisely weighed, dissolved and mixed thoroughly with 1mL of the above phosphate buffer to prepare 160mM acarbose stock solution. Then the stock solution is diluted with the above phosphate buffer to concentrations of 2.5, 5, 10, 20, 40, 80 and 160mM of positive control working solution.

[0082] Test compound working solution: precisely weigh 2.90mg of compound 1, 3.19mg of compound 2 and 3.23mg of compound 3, respectively, and place them in 500μL of ultrapure water, slowly add 1M NaOH until the solution is clear and transparent, and finally make up to 1mL with purified water to prepare 10mM compound stock solution. Then dilute it with the above phosphate buffer to concentrations of 0.062, 0.125, 0.25, 0.5, 1 and 2mM of compound 1 working solution, concentrations of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8mM of compound 2 working solution, and concentrations of 3.12, 6.25, 12.5, 25, 50 and 100μM of compound 3 working solution.

[0083] 2. Alpha-glucosidase activity determination

[0084] Four test groups were set up: blank group A, enzyme standard group B, sample negative control group C, and sample group D; 3 parallels were set for each sample. According to Table 2, a 600 μL reaction system was prepared, and the corresponding volume of phosphate buffer, working solution of each concentration of the sample to be tested (the final concentration was 20 times diluted with the working solution of the test compound), α-glucosidase solution (final concentration 0.005U / mL) and reaction substrate PNPG (final concentration 0.5mM) were added to the 96-well plate in sequence and mixed thoroughly. After the reaction system was incubated at 37°C for 50 minutes, 60 μL of 0.1mol / LNa2CO3 solution was added to terminate the reaction. The 96-well plate to be tested was placed in a multifunctional microplate reader (SPARK 10M, TECAN) to measure the absorbance of each test group at 405nm. The inhibition rate of α-glucosidase was calculated as follows:

[0085] α-glucosidase inhibition rate (%) = [1-(DC) / (BA)] × 100

[0086] Table 2 α-glucosidase activity inhibition test reaction system preparation (volume / μL)

[0087]

[0088] like Figure 2 As shown in the figure, the three compounds and the positive control drug acarbose all inhibited the activity of α-glucosidase in a dose-dependent manner. The half-inhibitory concentrations (IC50) of compound 1, compound 2, compound 3 and acarbose on α-glucosidase were calculated by nonlinear fitting using Graphpad Prism 8.0 software. 50 The results showed that all three compounds had good inhibitory activity against α-glucosidase, with the inhibitory effect significantly superior to that of the positive control drug acarbose. These compounds are expected to reduce the production of mature active tyrosinase by effectively inhibiting α-glucosidase, thereby reducing skin pigmentation.

[0089] Example 4: Study on the antioxidant activity of compounds 1-3

[0090] Prepare an ABTS radical stock solution by mixing equal volumes of 7.4 mmol / L ABTS solution and 2.6 mmol / L K2S2O8 solution at room temperature in the dark for 16 hours. Dilute the ABTS radical stock solution with phosphate buffer (pH 7.4, 10 mmol / L) to an absorbance of 0.70 ± 0.05 at 734 nm to prepare an ABTS working solution. Mix 200 μL of the ABTS working solution with 10 μL of phosphate buffer (pH 7.4, 10 mmol / L) and measure the absorbance at 734 nm as A0. Accurately weigh 10 mg of compound 1 and dissolve it in 500 μL of purified water. Slowly add 1 mol / L NaOH dropwise until the solution is clear and translucent. Finally, make up to 1 mL with purified water to prepare a 10 mg / mL stock solution. Dilute the stock solution to be tested to the desired concentration with phosphate buffer (pH 7.4, 10 mmol / L). The positive control drug Trolox (water-soluble vitamin E) was directly prepared in pure water to a 10 mg / mL stock solution, which was then diluted with water to the corresponding concentration. 10 μL of sample solution at various concentrations was mixed with 200 μL of ABTS working solution to achieve final concentrations of 0.9, 1.79, 3.58, 7.15, 10.73, 14.30, 25, and 50 μg / mL for the positive drug vitamin E; 0.62, 1.25, 2.50, 5.00, 10, 20, 40, and 50 μg / mL for compound 1; and 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μg / mL for compounds 2 and 3. The mixed sample solutions were allowed to stand at room temperature for 10 minutes, and the absorbance (Ai) was measured at 734 nm. At the same time, 10 μL of the sample solution of the corresponding concentration was mixed with 200 μL of 10 mmol / L pH 7.4 phosphate buffer solution, and the background absorbance Aj at a wavelength of 734 nm was measured. Three replicates were set for each sample concentration. The formula for calculating the scavenging rate of the sample for ABTS free radicals is as follows:

[0091] ABTS free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100

[0092] like Figure 3 As shown in the figure, within the test concentration range, the tested compounds all showed good scavenging effects on ABTS free radicals. Graphpad Prism 8.0 software was used for nonlinear fitting to calculate the IC values ​​of compounds 1-3 for ABTS free radical scavenging. 50 The half maximal inhibitory concentrations were 6.78 μg / mL (i.e., 23.22 μM), 2.56 μg / mL (i.e., 8.02 μM), and 2.10 μg / mL (i.e., 6.49 μM), respectively. The IC value of the positive control drug vitamin E was50 The value was 4.75 μg / mL (i.e., 11.05 μM). The above experimental results show that compounds 1-3 have good free radical scavenging ability, among which compounds 2 and 3 have better antioxidant activity than the positive control drug vitamin E, and are expected to help cells resist oxidative stress damage and delay skin aging.

[0093] Table 3 Half inhibitory concentration IC of the three compounds 50 value

[0094]

[0095] Example 5: Cell activity test of compounds 1-3

[0096] Treatment of test samples:

[0097] Sample group: Compounds 1-3 and phenylethylresorcinol were dissolved in DMSO and diluted with 1640 culture medium to a stock solution with a concentration of 38.6 μmol / L. The stock solution was then diluted with 1640 culture medium to a series of concentrations of 19.3 μmol / L, 9.65 μmol / L, 4.825 μmol / L, and 2.413 μmol / L for later use.

[0098] Negative control group: 1640 basal culture medium.

[0099] Cell viability assay:

[0100] B16 cells were plated in 96-well plates. After 24 hours, the culture medium was discarded and basal culture medium containing different concentrations of test samples was added. After 24 hours, the OD490 nm was detected by MTT method, and the effect of the test samples on B16 cell activity was analyzed by t test.

[0101] The statistical analysis software was SPSS, and the comparison between the test sample and the negative control was performed using the independent sample t-test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05. *P < 0.05, * indicates that the test sample had a significant difference compared with the negative control group at this concentration. The experimental results are as follows Figure 4 .

[0102] Depend on Figure 4 It can be seen that when the concentration of the three compounds is 19.3 μmol / L, the cell activity is >90% and there is no cytotoxicity.

[0103] Example 6: Compound 1-3 Cell Melanin Synthesis Inhibition Experiment

[0104] Using 19.3 μmol / L compound 1-3 and phenylethyl resorcinol as experimental samples and 1640 basal medium as negative control, B16 cells were plated on a 6-well plate and cultured for 24 hours. The culture medium was then replaced with 1640 basal medium containing different concentrations of the test samples. After two more changes of medium, the cells were washed twice with PBS. 200 μL of 0.25% trypsin was added to each well to digest the cells. The cells were collected into a centrifuge tube and centrifuged for 5 minutes. 200 μL of 1M NaOH containing 10% DMSO was added to each tube to lyse the cells, shaken evenly, and transferred to a 96-well plate. The absorbance of each well at 405 nm was detected by a microplate reader. The formula for calculating the inhibition rate of cell melanin synthesis is:

[0105]

[0106] Where: T—absorbance of test sample well;

[0107] C—the average of three absorbance values ​​of the negative control group;

[0108] The statistical analysis software was SPSS, and the comparison between the test sample and the negative control was performed using the independent sample t-test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05. *P < 0.05; **P < 0.01, * indicates that the test sample had a significant difference compared with the negative control group at this concentration. The results are shown in Figure 2. Figure 5 .

[0109] Depend on Figure 5 It can be seen that the inhibition rates of 19.3 μmol / L compounds 1-3 and phenylethyl resorcinol on B16 cell melanin synthesis are 52.230%, 40.881%, 34.798% and 32.791%, respectively, which have a significant effect on inhibiting cell melanin synthesis (P<0.05). Moreover, the inhibition rate of compound 1-3 on B16 cell melanin synthesis is significantly higher than that of phenylethyl resorcinol, a classic whitening ingredient on the market, indicating that compound 1-3 has excellent whitening effect.

Claims

1. A 1,3-bisbenzylphenol compound, the structural formula of which is shown in the structural formula of the following compound 2 or compound 3:

2. The method for preparing 1,3-bisbenzylphenol compounds according to claim 1, characterized in that: The preparation method comprises the following steps: mixing 1,3-bis(bromomethyl)benzene or 1,3-bis(chloromethyl)benzene with o-cresol or 1,2-diphenol in a substance ratio of 1:(5-10), using aluminum chloride as a catalyst, heating at 100-120° C. for a period of time under nitrogen protection, and then purifying by column chromatography to obtain the product.

3. Use of the 1,3-bisbenzylphenol compound according to claim 1 in the preparation of α-glucosidase inhibitors or drugs for treating diabetes, Application in the preparation of anti-skin oxidation and / or aging products, Application of the present invention in preparing skin care products or drugs for treating skin diseases that reduce melanin production and Application in the preparation of skin care products for preventing or treating pigmentation diseases or in the preparation of skin disease treatment drugs for preventing or treating pigmentation diseases.

4. Use of the compound 2 named 4,4'-(1,3-phenylenebis(methylene))bis-2-methylphenol in claim 1 in the preparation of a tyrosinase inhibitor.

5. Use of compound 1 in the preparation of tyrosinase inhibitors or α-glucosidase inhibitors or drugs for treating diabetes, Application in the preparation of anti-skin oxidation and / or aging products, The use of compound 1 in preparing a skin care product for reducing melanin production or a drug for treating skin diseases that reduces melanin production, as well as in preparing a skin care product for preventing or treating pigmentation diseases or a drug for treating skin diseases that prevent or treat pigmentation diseases, is as follows:

6. A whitening and / or anti-skin oxidation / aging skin care composition or pharmaceutical composition, characterized in that: Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 At least one of, and one or more acceptable carriers in the field of cosmetics.

7. Comprising an effective amount of Compound 1 as an active ingredient Compound 2 Compound 3 Use of at least one of the skin care composition or pharmaceutical composition in the preparation of whitening and / or anti-aging skin care products or in the preparation of skin disease treatment drugs that reduce melanin production or in the preparation of skin disease treatment drugs that prevent or treat pigmentation diseases.

8. A pharmaceutical composition for treating diabetes, characterized in that: Comprising an effective amount of compound 1 as an active ingredient Compound 2 Compound 3 At least one of, and one or more acceptable carriers in the pharmaceutical field.

9. Comprising as an active ingredient an effective amount of Compound 1 Compound 2 Compound 3 Use of at least one pharmaceutical composition in the preparation of a drug for treating diabetes.

10. The whitening and / or anti-skin oxidation / aging skin care composition or pharmaceutical composition according to claim 6, characterized in that: Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells.

11. The use according to claim 7, characterized in that Whitening refers to inhibiting tyrosinase activity, and / or inhibiting α-glucosidase activity, and / or inhibiting cellular melanin production and pigmentation; anti-skin oxidation / aging refers to removing active oxygen free radicals in cells.

12. The use according to claim 3, 5 or 11 or the composition according to claim 10, characterized in that The pigmentation disease is freckles, chloasma, stretch marks, age spots and / or melanoma.

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