Application of substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative in prevention and / or treatment of aging resistance and alopecia resistance

Abstract

The invention discloses an application of a substituted trifluoroalkyl thioalkyl benzoate derivative in preparation of anti-aging drugs, food or health care products. The structure of the substituted trifluoroalkyl thioalkyl benzoate derivative is shown as a formula (I). The invention also discloses an application of the substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative in medicines, foods, health products or wash supplies for preventing and / or treating alopecia. The substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative can delay the senescence process and prevent senescence-related diseases, also has the effect of increasing the hair growth speed and density, and can be used for developing anti-senescence and alopecia prevention and / or treatment medicines, foods, health care products or wash supplies and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of trifluoroalkylthioalkyl thioester derivatives of substituted benzoic acid in the prevention and / or treatment of anti-aging and anti-hair loss. Background Art

[0002] Aging is broadly defined as a time-related decline in function that affects most organisms. Aging increases susceptibility to age-related chronic diseases such as cancer, metabolism, cardiovascular, musculoskeletal, and neurodegenerative diseases. The main cause of organismal aging is cellular senescence, which is a relatively stable state that cells enter after irreversibly exiting the cell cycle and losing their proliferative capacity. Currently, the causes of cellular senescence are mainly divided into three types: replicative senescence, oncogene-induced premature senescence, and stress-induced premature senescence. Among them, various stress stimuli include oxidative stress, carcinogenic stress, chemotherapy drug toxicity stress, etc. Etoposide, as a type of chemotherapy drug, can inhibit topoisomerase, disrupt the reconnection after DNA supercoil unwinding, cause DNA damage, and lead to cellular senescence.

[0003] One of the most prominent features of human aging is graying and loss of hair. The morphology of hair depends on the growth and development of hair follicles. In adulthood, the cyclic replacement of hair follicles is irregular and mainly depends on the proliferation and differentiation of hair follicle stem cells. It is generally believed that affected by the aging process, the ability of hair follicle stem cells to proliferate, differentiate, and maintain their own state stability is impaired, resulting in a progressive decrease in the number and diameter of anagen hair follicles, thinning of hair, and an extended telogen phase of the hair cycle, leading to hair loss. Aging-related gray hair may be related to melanocyte damage induced by ultraviolet light and reactive oxygen species (ROS). With age, melanocytes age and melanin production is impaired, resulting in the production of aging gray hair.

[0004] The patent document with the publication number CN114874306B discloses an eggshell membrane polypeptide composition, a preparation method, and its application in anti-wrinkle and anti-hair loss. The composition includes at least one of the polypeptides shown in SEQ ID NO.1-11. This polypeptide composition has an obvious promoting effect on proliferation and migration activity, has an inhibitory effect on the contraction function of human skeletal muscle cells, promotes hair follicle regeneration, and has the function of preventing hair loss, and has application prospects in anti-wrinkle, anti-aging, and anti-hair loss cosmetics or cosmetics.

[0005] The patent document with the publication number CN113952362A discloses the application of inducible extracellular vesicles in the preparation of preparations for extending the lifespan of mammals or treating or preventing aging. The inducible extracellular vesicles are produced from stem cells or induced by adding staurosporine, ultraviolet irradiation, starvation method, or heat stress method or a combination thereof to induce apoptosis of mesenchymal stem cells. The inducible extracellular vesicles have anti-aging effects, can extend the lifespan of mammals, and have the effect of reducing senile alopecia.

[0006] Although the prior art has conducted extensive research on anti-aging products, it is still of great significance to find some new drugs, health products, etc. to prevent and alleviate cell aging and extend the healthy lifespan of living organisms. Summary of the Invention

[0007] The first object of the present invention is to provide the application of a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of a drug for preventing and / or treating anti-aging,

[0008]

[0009] wherein, R1 and R2 are OH, OAc or H, and X and Y are linear or branched alkyl groups with 1 to 9 carbon atoms.

[0010] The substituted benzoic acid trifluoroalkylthioalkylthioester derivative of the present invention has a significant activity of alleviating the generation of senescence markers in etoposide-induced senescent PC12 cells, an in vitro screening model of cell aging. An effective dose of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative can be used as an active ingredient, and a pharmaceutically acceptable carrier, diluent, etc. can be added to prepare a drug for preventing, alleviating aging and aging-related diseases.

[0011] The second object of the present invention is to provide the application of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of a drug for preventing and / or treating alopecia. The substituted benzoic acid trifluoroalkylthioalkylthioester derivative of the present invention can achieve a hair growth effect similar to that of minoxidil at a dose several times lower than minoxidil. Using the substituted benzoic acid trifluoroalkylthioalkylthioester derivative as an active ingredient, a pharmaceutically acceptable carrier, diluent, etc. can be added to prepare a drug for preventing and / or treating alopecia-related diseases.

[0012] Preferably, the substituted benzoic acid trifluoroalkylthioalkylthioester derivative achieves a hair growth effect similar to that of minoxidil at a dose of 0.25 times that of minoxidil by transdermal administration. For example, the hair growth length and density of the compound 3 in the embodiment of the present invention at 0.50% are comparable to those of the minoxidil group at 2.0%.

[0013] Preferably, when the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative is used for preventing and / or treating hair loss, the oral dose of the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative is 1 to 25 mg / kg body weight, and the body weight refers to the body weight of a human.

[0014] The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, including fillers such as sucrose, starch, microcrystalline cellulose, inorganic salts, etc.; binders such as cellulose derivatives, starch paste, polyvinylpyrrolidone, gelatin, etc.; wetting agents such as distilled water, ethanol, etc.; lubricants such as magnesium stearate, colloidal silicon dioxide, polyethylene glycols, etc.; absorption promoters such as polysorbate, lecithin, etc., surfactants such as sorbitan fatty acid esters, poloxamer, etc. In addition, other adjuvants such as sweeteners, flavoring agents, etc. can be added to the composition.

[0015] The substituted benzoic acid trifluoroalkylthioalkyl thioester derivative described in the present invention can be administered in unit dosage form, and the administration route is enteral administration or parenteral administration, including oral administration, intravenous injection, intramuscular injection, subcutaneous injection, etc.

[0016] The dosage form of the drug can be solid preparation, semi-solid preparation, liquid preparation, etc., including tablets, pills, powders, dispersible tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft capsules, hard capsules, sterile injections, liniments, suppositories, etc. The above various dosage forms can be prepared by conventional methods, for example, mixing the active ingredient with one or more carriers and then making it into the required dosage form.

[0017] The third object of the present invention is to provide the use of the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative shown in the above formula (I) in the preparation of anti-aging and food or health products for preventing and / or treating hair loss.

[0018] The fourth object of the present invention is to provide the use of the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative shown in the above formula (I) in the preparation of hair care products for preventing and / or treating anti-aging and hair loss.

[0019] The fifth object of the present invention is to provide an anti-aging and product for preventing and / or treating hair loss, and the product includes the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative shown in the above formula (I).

[0020] Preferably, the product of the present invention is an anti-aging and drug for preventing and / or treating hair loss, and the anti-aging, drug for preventing and / or treating hair loss uses the above-mentioned substituted benzoic acid trifluoroalkylthioalkyl thioester derivative as an active ingredient.

[0021] Preferably, the product of the present invention is an anti-aging and hair loss prevention and / or treatment food or health product, wherein the food or health product is composed of the above-mentioned substituted benzoic acid trifluoroalkylthioalkylthioester derivative and a food or health product acceptable carrier.

[0022] Preferably, the product of the present invention is a hair care product for hair loss prevention and / or treatment, wherein the hair care product is composed of the above-mentioned substituted benzoic acid trifluoroalkylthioalkylthioester derivative and a hair care product acceptable carrier.

[0023] The sixth object of the present invention is to provide a preparation method of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative shown in the above formula (I), comprising the following steps:

[0024] (1) Add NaH (60% dispersed in mineral oil) to a solvent containing thiol below 0 °C, raise the temperature to room temperature and stir for reaction for 10 - 30 min, then add bromotrifluoroalkyl compound and TBAI for reaction below 0 °C, detect the reaction end point by TLC, and after the reaction is completed, extract the reaction product to obtain the target product.

[0025] (2) Dissolve the target product obtained in step (1) in an organic solvent, add EDC·HCl, substituted benzoic acid and DMAP, and carry out the reaction at room temperature. Detect the reaction end point by TLC. After the reaction is completed, separate and purify the reaction product to obtain the substituted benzoic acid trifluoroalkylthioalkylthioester derivative.

[0026] Preferably, in step (1), the thiol is a dithiol having 1 - 10 carbon atoms.

[0027] More preferably, the thiol is 1,2 - ethanedithiol, 1,4 - butanedithiol, 1,6 - hexanedithiol, 1,8 - octanedithiol or 1,10 - decanedithiol.

[0028] Preferably, in step (1), the solvent includes DMF.

[0029] Preferably, in step (1), the molar ratio of NaH to thiol is 1 - 2:1; the molar ratio of bromotrifluoroalkyl compound to thiol is 1:1; the molar ratio of TBAI to thiol is 0.1 - 1:1.

[0030] Preferably, the substituted benzoic acid includes 2,3 - dihydroxybenzoic acid, 2,3 - diacetoxybenzoic acid, 2 - hydroxybenzoic acid, 3 - hydroxybenzoic acid, 2 - acetoxybenzoic acid or 3 - acetoxybenzoic acid.

[0031] Preferably, in step (2), the molar ratio of EDC·HCl to thiol is 1-4:1; the molar ratio of substituted benzoic acid to thiol is 1-10:1; the molar ratio of DMAP to thiol is 0.1-4:1.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] (1) The substituted benzoic acid trifluoroalkylthioalkylthioester derivative of the present invention can alleviate the oxidative stress level, cell cycle arrest, senescence markers SA-β-Gal and lipofuscin production in senescent cells, and is a potential anti-aging active compound, which can delay the aging process, prevent aging-related diseases, extend the healthy lifespan of living organisms, and has broad application prospects in anti-aging products.

[0034] (2) The substituted benzoic acid trifluoroalkylthioalkylthioester derivative of the present invention can promote hair growth and increase hair density, and has good application prospects in anti-hair loss drugs, health products and washing and care products. Description of the Drawings

[0035] Figure 1 Shows the effects of Compounds 1-8 on the senescence marker - senescence-associated β-galactosidase in etoposide-induced senescent PC12 cells.

[0036] Figure 2 Shows the effect of Compound 3 on the cell viability of etoposide-induced senescent PC12 cells.

[0037] Figure 3 Shows the effect of Compound 3 on the oxidative stress level of etoposide-induced senescent PC12 cells.

[0038] Figure 4 Shows the effect of Compound 3 on the cell proliferation level of etoposide-induced senescent PC12 cells.

[0039] Figure 5 Shows the effect of Compound 3 on the senescence marker - lipofuscin in etoposide-induced senescent PC12 cells.

[0040] Figure 6 Shows the effect of Compound 3 on the hair removal area of aged mice.

[0041] Figure 7 Shows the effect of Compound 3 on the hair growth length of rats.

[0042] Figure 8 Shows the effect of Compound 3 on the hair growth density of rats.

[0043] Figure 9 Shows the effects of Compounds 1 and 5 on the hair growth length of rats.

[0044] Figure 10 Effects of Compounds 1 and 5 on the Hair Growth Density of Rats Detailed Implementation Modes

[0045] The present invention will be further described in detail below in combination with the accompanying drawings and embodiments. However, the protection scope of the present invention is not limited to the described content. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. For the reagents and methods used in the following embodiments, unless otherwise specified, conventional reagents and methods are usually used.

[0046] Example 1

[0047] The preparation method of the trifluoroalkylthioalkylthioester derivative of substituted benzoic acid in this example is as follows:

[0048] (1) Dissolve 1,2-ethanedithiol (188.0 mg, 2.0 mmol) in 10 ml of dry anhydrous DMF. Add NaH (60% dispersed in mineral oil, 96.0 mg, 2.4 mmol) at 0 °C. After raising the temperature to room temperature and stirring for 30 min, add 1-bromo-4,4,4-trifluorobutane (0.3 mL, 2.0 mmol) and TBAI (74.0 mg, 0.2 mmol) at 0 °C, and react overnight. Detect the reaction end point by TLC (n-hexane:ethyl acetate = 20:1). After the reaction is completed, dilute with ethyl acetate, and wash with 1N HCl solution, water, saturated sodium bicarbonate solution and NaCl solution respectively, and extract. The obtained organic phase is dried with sodium sulfate, filtered, and then concentrated.

[0049] (2) Dissolve the product obtained in step (1) in 10 ml of dry dichloromethane, add EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 382.0 mg, 2.0 mmol), 2,3-dihydroxybenzoic acid (154.0 mg, 1.0 mmol) and DMAP (4-dimethylaminopyridine, 122.0 mg, 1.0 mmol), and react at room temperature overnight. Detect the reaction end point by TLC (n-hexane:ethyl acetate = 5:1). After the reaction is completed, wash with 1N HCl solution, water, saturated sodium bicarbonate solution and NaCl solution respectively, extract, dry and concentrate the organic layer, purify by silica gel column chromatography (n-hexane:ethyl acetate = 80:1), and then purify by ODS open column (methanol:water = 75:25) to obtain Compound 1.

[0050] Analyze HR ESI-MS and 1 H NMR data to determine the structure of Compound 1. HR ESI-TOF-MS m / z 363.0316, calcd. for C 13 H15 F3O3S2Na(M+Na) + 363.0307. 1 1H NMR(500 MHz, CDCl3): δ = 11.08 (1H, s), 7.39 (1H, dd, J = 1.3, 8.0 Hz), 7.12 (1H, dd, J = 1.3, 8.0 Hz), 6.83 (1H, t, J = 8.0 Hz), 5.69 (1H, s), 3.27 (2H, m), 2.79 (2H, m), 2.71 (2H, t, J = 7.1 Hz), 2.25 (2H, m), 1.93 (2H, m). The structure of the obtained compound 1 is as follows:

[0051]

[0052] Example 2

[0053] In this example, 1,4-butanedithiol (244.0 mg, 2.0 mmol) was used to prepare a trifluoroalkylthioalkylthioester derivative of substituted benzoic acid. The rest was the same as in Example 1, and the obtained product was denoted as compound 2.

[0054] Analysis of HR ESI-MS and 1 1H NMR data determined the structure of compound 2. HR ESI-TOF-MS m / z 391.0625, calcd. for C 15 H 19 F3O3S2Na(M+Na) + 391.0620. 1 1H NMR(500 MHz, CDCl3): δ = 11.19 (1H, s), 7.41 (1H, dd, J = 1.4, 8.0 Hz), 7.11 (1H, dd, J = 1.4, 8.0 Hz), 6.82 (1H, t, J = 8.0 Hz), 5.69 (1H, s), 3.09 (2H, t, J = 7.1 Hz), 2.57 (4H, m), 2.22 (2H, m), 1.83 (4H, m), 1.73 (2H, m). The structure of the obtained compound 2 is as follows:

[0055]

[0056] Example 3

[0057] In this example, 1,6-hexanedithiol (301.0 mg, 2.0 mmol) was used to prepare a trifluoroalkylthioalkylthioester derivative of substituted benzoic acid. The eluent used for purification by ODS open column was MeOH / H2O with a volume ratio of 80:20. The rest was the same as in Example 1, and the obtained product was denoted as compound 3.

[0058] Analysis of HR ESI-MS and 1 H NMR data to determine the structure of compound 3. HR ESI-TOF-MS m / z 419.0933, calcd. for C 17 H 23 F3O3S2Na (M+Na) + 419.0933. 1 1H NMR (500 MHz, CDCl3): δ = 11.24 (1H, s), 7.41 (1H, dd, J = 1.4, 8.0 Hz), 7.11 (1H, dd, J = 1.4, 8.0 Hz), 6.82 (1H, t, J = 8.0 Hz), 5.67 (1H, s), 3.07 (2H, t, J = 7.3 Hz), 2.50 - 2.59 (4H, m), 2.22 (2H, m), 1.85 (2H, m), 1.69 (2H, m), 1.61 (2H, m), 1.45 (4H, m). The structure of the obtained compound 3 is shown below:

[0059]

[0060] Example 4

[0061] In this example, 1,8-octanedithiol (357.0 mg, 2.0 mmol) was used to prepare the trifluoroalkylthioalkylthioester derivative of substituted benzoic acid, and the rest was the same as in Example 3. The obtained product was designated as compound 4.

[0062] Analysis of HR ESI-MS and 1 H NMR data to determine the structure of compound 4. HR ESI-TOF-MS m / z 447.1257, calcd. for C 19 H 27 F3O3S2Na (M+Na) + 447.1246. 1 1H NMR (500 MHz, CDCl3): δ = 11.25 (1H, s), 7.41 (1H, dd, J = 1.0, 8.0 Hz), 7.10 (1H, dd, J = 1.0, 8.0 Hz), 6.81 (1H, t, J = 8.0 Hz), 5.68 (1H, s), 3.06 (2H, t, J = 7.3 Hz), 2.54 (4H, m), 2.22 (2H, m), 1.85 (2H, m), 1.68 (2H, m), 1.60 (2H, m), 1.32 - 1.47 (8H, m). The structure of the obtained compound 4 is shown below:

[0063]

[0064] Example 5

[0065] In this example, 1,10-decanedithiol (413.0 mg, 2.0 mmol) was used to prepare the trifluoroalkylthioalkylthioester derivative of substituted benzoic acid. The rest was the same as in Example 3, and the obtained product was designated as Compound 5.

[0066] Analysis of HR ESI-MS and 1 1H NMR data determined the structure of Compound 5. HR ESI-TOF-MS m / z 475.1544, calcd. for C 21 1H 31 F3O3S2Na (M+Na) + 475.1559. 1 1H NMR (500 MHz, CDCl3): δ = 11.26 (1H, s), 7.41 (1H, dd, J = 1.4, 8.0 Hz,), 7.10 (1H, dd, J = 1.4, 8.0 Hz), 6.81 (1H, t, J = 8.0 Hz), 5.71 (1H, s), 3.06 (2H, t, J = 7.4 Hz), 2.57 (2H, m), 2.50 (2H, m), 2.22 (2H, m), 1.85 (2H, m), 1.68 (2H, m), 1.57 (2H, m), 1.43 (2H, m), 1.38 (2H, m), 1.25 - 1.45 (8H, m). The structure of the obtained Compound 5 is shown below:

[0067]

[0068] Example 6

[0069] Considering that the two hydroxyl groups on the benzene ring of Compound 3 may have a potential impact on the penetration of Compound 3 through the blood-brain barrier, in this example, based on the prepared Compound 3, an acetylation reaction was carried out on Compound 3 to synthesize the prodrug Compound 6 of Compound 3 to improve the anti-brain aging efficacy of Compound 3. The steps are as follows: Acetic anhydride (0.47 mL, 5.0 mmol) was added to the anhydrous pyridine solution (1.0 mL) of Compound 3 (200.0 mg, 0.5 mmol). After stirring the reaction, the reaction was quenched with 1N HCl solution, concentrated in vacuo, the concentrate was extracted with EtOAc, the organic phase was dried over Na2SO4, filtered and concentrated. The rest was the same as in Example 3, and the obtained product was designated as Compound 6.

[0070] Analysis of HR ESI-MS and 1 1H NMR data determined the structure of Compound 6. HR ESI-TOF-MS m / z 481.1327, calcd. for C 21 1H 28F3O5S2(M+H) + 481.1325. 1 1H NMR(500 MHz, CDCl3): δ = 7.79 (1H, dd, J = 1.7, 7.9 Hz), 7.37 (1H, dd, J = 1.7, 7.9 Hz), 7.32 (1H, t, J = 7.9 Hz), 3.01 (2H, t, J = 7.5 Hz), 2.57 (2H, t, J = 7.1 Hz), 2.50 (2H, m), 2.33 (3H, s), 2.30 (3H, s), 2.20 (2H, m), 1.84 (2H, m), 1.66 (2H, m), 1.60 (2H, m), 1.43 (4H, m). The structure of the obtained compound 6 is as follows:

[0071]

[0072] Example 7

[0073] In this example, 1,10-decanedithiol (413.0 mg, 2.0 mmol) and benzoic acid (122.0 mg, 1.0 mmol) were used to prepare a trifluoroalkylthioalkylthioester derivative of substituted benzoic acid. The rest was the same as in Example 3. The obtained product was denoted as compound 7.

[0074] Analyze HR ESI-MS and 1 1H NMR data to determine the structure of compound 7. HR ESI-TOF-MS m / z 387.1022, calcd. for C 17 H 23 F3OS2Na(M+Na) + 387.1035. 1 1H NMR(500 MHz, CDCl3): δ = 7.97 (2H, dd, J = 1.2, 7.9 Hz), 7.56 (1H, t, J = 7.9 Hz), 7.45 (2H, t, J = 7.9 Hz), 3.07 (2H, t, J = 7.5 Hz), 2.57 (2H, t, J = 7.0 Hz), 2.51 (2H, t, J = 7.3 Hz), 2.22 (2H, m), 1.85 (2H, m), 1.69 (2H, m), 1.60 (2H, m), 1.45 (4H, m). The structure of the obtained compound 7 is as follows:

[0075]

[0076] Example 8

[0077] In this example, 1,4-butanedithiol (244.0 mg, 2.0 mmol) and 1-bromo-5,5,5-trifluoropentane (0.3 mL, 2.0 mmol) were used to prepare a trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid. The rest was the same as in Example 1, and the obtained product was designated as Compound 8.

[0078] The structure of Compound 8 was determined by analyzing HR ESI-MS and 1 H NMR data. HR ESI-TOF-MS m / z 383.0959, calcd. for C 16 H 22 F3O3S2 (M + H) + 383.0957. 1 H NMR (500 MHz, CDCl3): δ = 11.20 (1H, s), 7.40 (1H, dd, J = 1.2, 8.0 Hz,), 7.11 (1H, dd, J = 1.2, 8.0 Hz), 6.82 (1H, t, J = 8.0 Hz), 5.70 (1H, s), 3.10 (2H, t, J = 7.0 Hz), 2.55 (4H, m), 2.09 (2H, m), 1.65 - 1.84 (8H, m). The structure of the obtained Compound 8 is shown below:

[0079]

[0080] Application Example 1: In vitro anti-aging bioactivity evaluation of trifluoroalkylthioalkyl thioester derivatives (Compounds 1 - 8) of substituted benzoic acid prepared in Examples 1 - 8

[0081] Determination of the alleviating activity of Compounds 1 - 8 on senescence markers in etoposide-induced senescent PC12 cells:

[0082] (1) Effects of Compounds 1 - 8 on the activity of senescence-related β-galactosidase in etoposide-induced senescent PC12 cells

[0083] Experimental method:

[0084] Place a 12-mm round glass slide in each well of a 24-well cell culture plate, add 250 μL of 100 μg / mL L-polylysine for coating. After overnight incubation, rinse 3 times with PBS. Culture in a CO₂ incubator for 24 hours and then add samples. Cell sample addition treatment: Prepare the test sample to the required concentration with DMSO. Add the prepared test sample to EM to prepare a 1-mL test sample solution at a certain concentration. Replace the CM in the 24-well plate with the above test sample solution. The positive control is 500 nM rapamycin (Rapa), and the negative control is 0.5% DMSO (C). After 24 hours of treatment, aspirate the culture medium. Add 1 mL of CM medium containing 7.5 μM etoposide (Eto) to each well except the negative control group. The negative control group is treated with CM medium containing the same volume of DMSO. Continue to incubate in a CO₂ incubator for 48 hours. Staining treatment: Treat the cells with a cell senescence β-galactosidase staining reagent (Shanghai Beyotime Biotechnology Co., Ltd., product number C0602), then wash with 70% ethanol solution, prepare slides, mount the slides, observe the cells under an ordinary optical microscope, and calculate the proportion of cells stained blue (i.e., cells positive for cell senescence β-galactosidase) in all cells in the field of view.

[0085] As Figure 1 shown, etoposide significantly induced cell senescence. After adding compounds 1-8 at different concentrations (0.01 μM, 0.1 μM, 1 μM), the proportion of cells containing cell senescence-related β-galactosidase decreased significantly, indicating that pretreatment with compounds 1-8 could alleviate etoposide-induced PC12 cell senescence, and the alleviating effect was stronger with the increase in the chain length of the compounds.

[0086] (2) Effect of compound 3 on cell viability in etoposide-induced senescent PC12 cells

[0087] Experimental method:

[0088] Add 200 μL of CM to each well of a 96-well cell culture plate and inoculate 7,000 cells. Culture in a CO₂ incubator for 24 hours and then add samples. The method for adding cell samples in this example is the same as that in Example 1. Then replace each well with 100 μL of EM solution containing 200 μg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After incubation for 2 hours, add 100 μL of DMSO to each well, shake at room temperature for 10 minutes, and measure the absorbance value of each well at 570 nm using a microplate reader.

[0089] As Figure 2As shown, etoposide significantly reduced the cell viability of PC12 cells, while pretreatment with 500 nM rapamycin, a positive drug, and 0.003 - 0.3 μM compound 3 for 24 h had no significant effect on the cell viability of etoposide-induced senescent PC12 cells, while pretreatment with 1 μM compound 3 could reduce the cell viability of etoposide-induced senescent PC12 cells (P = 0.02).

[0090] (3) Effect of compound 3 on reactive oxygen species in etoposide-induced senescent PC12 cells

[0091] Experimental method:

[0092] In this example, the cell culture and sample addition methods were the same as those in Example 1. After the cells in the 24-well plate were treated with samples, 200 μL of the solution containing 10 μM reactive oxygen species probe DCFH-DA (Shanghai Beyotime Biotechnology Co., Ltd., product number S0033) was added to each well, and the cells were incubated in the dark for 20 min. Then, they were washed 3 times with EM solution, and photographed using an inverted fluorescence microscope. The fluorescence intensity values in the photos were quantitatively analyzed using Image J software.

[0093] As Figure 3 shown, etoposide treatment could significantly increase the reactive oxygen species level in PC12 cells, while pretreatment with rapamycin and 0.003 - 1 μM compound 3 could significantly reduce the abnormal reactive oxygen species level induced by etoposide, and the ability to reduce the abnormal reactive oxygen species level became stronger as the concentration of compound 3 increased.

[0094] (4) Effect of compound 3 on cell proliferation in etoposide-induced senescent PC12 cells

[0095] Experimental method:

[0096] In this example, the cell culture and sample addition methods were the same as those in Example 1. After the cells in the 24-well plate were treated with samples, the cells were treated with an EdU cell proliferation detection reagent (Shanghai Beyotime Biotechnology Co., Ltd., product number C0071S). Finally, after making the slides and mounting them, they were photographed using an upright fluorescence microscope. The proportion of cells with green fluorescence (i.e., proliferating cells with EdU incorporated into DNA) in the blue fluorescence cells (all cells) in the field of view was calculated.

[0097] As Figure 4 shown, etoposide significantly reduced the proportion of proliferating cells with EdU incorporation, while pretreatment with rapamycin and 0.1 - 1 μM compound 3 could significantly increase the proportion of proliferating cells with EdU incorporation, and the ability to increase the proportion of proliferating cells with EdU incorporation became stronger as the concentration of compound 3 increased. Compound 3 could be used to relieve the cell cycle arrest of senescent cells.

[0098] (5) Effect of Compound 3 on Lipofuscin in Etoposide-Induced Senescent PC12 Cells

[0099] Experimental method:

[0100] In this example, the cell culture and sample addition methods were the same as those in Example 1. After the cells in the 24-well plate were treated with samples and fixed, they were washed 3 times with PBS, then 0.2 mL of Sudan Black staining solution was added for staining. After washing, Nuclear Fast Red staining solution was used for counterstaining. After washing, the specimens were prepared and sealed. The cells were observed and photographed under an ordinary optical microscope, and the proportion of cells stained blue-black (i.e., lipofuscin-positive cells) in all cells in the field of view was calculated.

[0101] As Figure 5 shown, etoposide significantly induced the production of the senescence marker lipofuscin. After adding different concentrations of Compound 3, the proportion of cells containing lipofuscin decreased to varying degrees, and the effect of 0.3 μM Compound 3 was the best.

[0102] In-Vivo Hair Growth Promoting Activity Evaluation of Substituted Benzoic Acid Trifluoroalkylthioalkylthioester Derivatives 1 - 5 Prepared in Examples 1 - 5 of Application Example 2

[0103] (1) Study on the Hair Loss Situation of Aged Mice by Compound 3

[0104] Animal grouping: 20 C57BL / 6J black mice, 18 months old, about 30 g, naturally senescent, female, randomly and evenly divided into two groups; 10 C57BL / 6J black mice, 8 weeks old, about 25 g, female, divided into one group.

[0105] Drug administration method: All three groups of mice were administered by oral gavage. The first group of the naturally senescent group was administered 0.5% DMSO as the blank control group; the second group of the naturally senescent group was administered 5 mg / kg Compound 3 as the experimental group; the young group was administered 0.5% DMSO. Each mouse was administered 150 μL per day for three consecutive months.

[0106] As Figure 6 shown, compared with the young group, the blank control group had severe hair loss; compared with the blank control group, the experimental group had significantly improved hair loss and new hair growth.

[0107] (2) Study on the Effect of Different Doses of Compound 3 on Promoting Hair Growth in Rats

[0108] Experimental method: Establish an animal model of rat hair loss experiment: 20 white SD rats, 6 - 8 weeks old, weighing about 250 g, female; anesthetized with 1.0% sodium pentobarbital, that is, 1 g of sodium pentobarbital was dissolved in 100 mL of physiological saline with a concentration of 0.9%, at a dose of 30 mg / kg, and the hair was shaved with an electric hair clipper. When there was no remaining hair in the 4 cm × 5 cm area on the back of the rat, the model was successfully established.

[0109] Animal grouping: The successfully modeled SD rats were randomly and evenly divided into 5 groups, with 4 rats in each group.

[0110] Administration method: Four groups of rats were respectively administered by spraying and smearing in the formed hairless area: 0.02% of Compound 3 (i.e., 0.02 g of Compound 3 in 100 mL of carrier solvent; 0.4 mg / kg), 0.10% of Compound 3 (2.0 mg / kg), 0.50% of Compound 3 (2.0 mg / kg), 2.0% of minoxidil (40.0 mg / kg). The rats in the fifth group were used as blank controls and smeared with a mixed solvent of equal volume (water: ethanol: glycerol monoacetate = 0.4:0.2:0.4). The volume of each administration was 0.5 mL, and the administration was carried out once a day for four weeks.

[0111] Experimental results:

[0112] a. Hair growth length

[0113] Four regions were randomly selected within the administration area of the rats every week, and 5 hairs were randomly plucked from each region to measure and record the hair length. The results are as Figure 7 shown. Compared with the blank control group, the hair length increased in the experimental groups with different concentrations of Compound 3. The hair growth lengths in the experimental group of 0.50% Compound 3 and the 2.0% minoxidil group were comparable and were significantly increased compared with the blank control group.

[0114] b. Hair growth density

[0115] The hair density within the administration area of the rats was analyzed by Image J every week. The results are as Figure 8 shown. Compared with the blank control group, the hair growth density increased in the experimental groups with different concentrations of Compound 3. The hair growth densities in the experimental group of 0.50% Compound 3 and the 2.0% minoxidil group were comparable and were significantly increased compared with the blank control group. Moreover, after 4 weeks, the experimental groups with different concentrations of Compound 3 were significantly increased compared with the blank control group.

[0116] (3) Study on the effect of Compound 1 and 5 on promoting hair growth in rats

[0117] Experimental method:

[0118] An experimental animal model of rat hair removal was established: 16 white SD rats, 6 - 8 weeks old, with a body weight of about 250 g, female; anesthetized with 1.0% sodium pentobarbital at a dose of 30 mg / kg, and shaved with an electric hair clipper. When there was no remaining hair in the 4 cm × 5 cm area on the back of the rat, the modeling was successful.

[0119] Animal grouping: The successfully modeled SD rats were randomly and evenly divided into 4 groups, with 4 rats in each group.

[0120] Administration method: The rats in the 3 groups were respectively sprayed and applied with drugs in the depilated areas: 0.50% of Compound 1 (10.0 mg / kg), 0.50% of Compound 5 (10.0 mg / kg), 2.0% of minoxidil (40.0 mg / kg). The rats in the 4th group were used as blank controls and were applied with a mixed solvent (water: ethanol: glycerol monoacetate = 0.4: 0.2: 0.4) in an equal volume. The volume of each drug administration was 0.5 mL, and the drug was administered once a day for four weeks.

[0121] Experimental results:

[0122] a. Hair growth length

[0123] The results were as Figure 9 shown. Starting from the second week, compared with the blank control group, the hair of the rats in the Compound 1 group showed a significant increase in growth. Starting from the third week, compared with the blank control group, the hair of the rats in the Compound 5 group showed a significant increase in growth.

[0124] b. Hair growth density

[0125] The results were as Figure 10 shown. Starting from the first week, compared with the blank control group, the hair density of the rats in the Compound 1 group showed a significant increase. Starting from the third week, compared with the blank control group, the hair density of the rats in the Compound 5 group showed a significant increase.

Claims

1. Use of a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of a drug for preventing and / or treating anti-aging, in, R1 and R2 are OH, OAc or H, and X and Y are straight-chain or branched-chain alkyl groups having 1 to 9 carbon atoms.

2. Use of a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of a drug for preventing and / or treating hair loss.

3. The use according to claim 2, characterized in that: The substituted benzoic acid trifluoroalkylthioalkylthioester derivative can achieve a hair growth effect similar to that of minoxidil by applying the drug to the skin in an amount of 0.25 times that of minoxidil.

4. The use according to claim 2, characterized in that: When used, the oral dosage of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is 1 to 25 mg / kg body weight.

5. Use of a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of anti-aging and hair loss prevention and / or treatment foods or health products.

6. Use of a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I) in the preparation of a preventive and / or therapeutic anti-aging and hair loss care product.

7. An anti-aging and hair loss prevention and / or treatment product, characterized in that: The anti-aging and hair loss prevention and / or treatment product comprises a substituted benzoic acid trifluoroalkylthioalkylthioester derivative represented by formula (I).

8. The method for preparing a substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) adding NaH to a thiol-containing solvent at a temperature below 0° C., heating the mixture to room temperature and stirring the mixture for 10 to 30 minutes, then adding a bromotrifluoroalkyl compound and TBAI at a temperature below 0° C. to react, detecting the reaction end point by TLC, and extracting the reaction product after the reaction is completed to obtain the target product; (2) dissolving the target product obtained in step (1) in an organic solvent, adding EDC·HCl, substituted benzoic acid and DMAP, reacting at room temperature, detecting the reaction end point by TLC, and separating and purifying the reaction product after the reaction is completed to obtain the substituted benzoic acid trifluoroalkylthioalkylthioester derivative.

9. The preparation method according to claim 8, characterized in that: In step (1), the solvent is DMF; the molar ratio of NaH, bromotrifluoroalkyl compound, TBAI and thiol is 1-2:1:0.1-1:

1.

10. The preparation method according to claim 8, characterized in that: In step (2), the molar ratio of EDC·HCl, substituted benzoic acid, DMAP and thiol is 1-4:1-10:0.1-4:1.

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