WO2025131037A1 - Substituted trifluoroalkyl thioalkyl benzothioate derivative, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed in the present invention are a substituted trifluoroalkyl thioalkyl benzothioate derivative, and a preparation method therefor and the use thereof. The derivative has a structure as shown in a formula (I). Further disclosed in the present invention is the preparation method for the above-mentioned substituted trifluoroalkyl thioalkyl benzothioate derivative. The substituted trifluoroalkyl thioalkyl benzothioate derivative of the present invention has anti-inflammatory activity and neuroprotective activity, and can be used in the preparation and application of pharmaceuticals, foods or health care products related to anti-inflammation, neuroprotection, and anti-neurodegenerative diseases. The substituted trifluoroalkyl thioalkyl benzothioate derivative can also delay the aging process, prevent aging-related diseases, also has the effect of increasing the rate and density of hair growth, and thus can be used for developing pharmaceuticals, foods, health care products or personal care products, etc. aimed at anti-aging, and the prevention and/or treatment of alopecia.

Description

Substituted benzoic acid trifluoroalkylthioalkylthioester derivatives and preparation method and application thereof Technical Field

The invention belongs to the field of medical technology, and specifically relates to substituted benzoic acid trifluoroalkylthioalkylthioester derivatives and preparation methods and applications thereof.

Background Art

Inflammation is a major cause of many diseases and is also a major factor affecting age-related diseases. BV-2 microglia are a common cell model for studying inflammation. When brain damage such as ischemia occurs, microglia are the first to respond to the damage and transform into an active state. Activated microglia can differentiate into different phenotypes, including M1 that causes neurological damage and M2 phenotypes that have neuroprotective effects.

Lipopolysaccharide (LPS) can induce BV-2 microglia to activate into M1 phenotype and promote the release of various pro-inflammatory mediators, thus causing inflammatory response. BV-2 microglia have simple culture conditions, can be cultured continuously, are easy to operate and have low cost. By constructing an LPS-induced BV-2 microglia activation model, lead compounds with anti-inflammatory activity can be screened out by high-throughput. In addition, the brains of mice fed a high-fat diet and normal aging mice are also affected by inflammation, which in turn causes various neurological diseases.

Aging is broadly defined as the time-related functional decline that affects most organisms. Aging increases susceptibility to age-related chronic diseases, such as cancer, metabolic, cardiovascular, musculoskeletal, and neurodegenerative diseases. The main cause of aging in organisms is cellular senescence, which is a relatively stable state that cells enter after they irreversibly exit the cell cycle and lose their ability to proliferate. Currently, there are three main causes of cellular senescence: replicative senescence, oncogene-induced premature senescence, and stress-induced premature senescence. Various stress stimuli include oxidative stress, carcinogenic stress, and chemotherapeutic drug toxicity stress. Etoposide, as a type of chemotherapeutic drug, can inhibit topoisomerase, disrupt the reconnection of DNA supercoils after unwinding, cause DNA damage, and lead to cell senescence.

One of the most prominent features of human aging is graying and hair loss, and the morphology of hair depends on the growth and development of hair follicles. In human adulthood, the cycle of hair follicles is irregular, and it mainly depends on the proliferation and differentiation of hair follicle stem cells. It is generally believed that under the influence of the aging process, the ability of hair follicle stem cells to proliferate, differentiate and maintain their own stable state is impaired, resulting in a progressive decrease and thinning of the number of hair follicles and hair diameter during the growth phase, and a prolonged resting phase of the hair cycle, resulting in sparse hair. Aging-related graying of hair may be related to melanocyte damage induced by ultraviolet rays and reactive oxygen free radicals (ROS). With age, melanocytes age and melanin production is impaired, leading to the generation of aging-related gray hair.

Patent document with publication number CN111777588A discloses a phenylpropanoid compound from Pseudostinex and its application. The invention extracts a new type of phenylpropanoid compound from Pseudostinex. The new type of phenylpropanoid compound exhibits an inhibitory effect on the inflammatory mediator NO produced by LPS-induced BV-2 cells, has a significant anti-inflammatory effect, and is non-toxic to cells. The compound can be used to prepare drugs related to inflammation.

Patent document with publication number CN111574581A discloses low-toxicity, anti-inflammatory ursolic acid derivatives and their preparation methods and applications. The invention uses ursolic acid as a lead compound for structural modification to obtain a low-toxicity, anti-inflammatory new ursolic acid derivative. The derivative has significant inhibitory activity on lipopolysaccharide-induced NO release in RAW264.7 cells and has extremely low cytotoxicity.

Patent document with publication number CN113952362A discloses the use of inducible extracellular vesicles in the preparation of a preparation for prolonging the life span of a mammal or treating or preventing aging. The inducible extracellular vesicles are produced from stem cells or by inducing apoptosis of mesenchymal stem cells by adding staurosporine, ultraviolet irradiation, starvation, or heat stress or a combination thereof. The inducible extracellular vesicles have anti-aging effects, can prolong the life span of a mammal, and have the effect of alleviating hair loss in the elderly.

Although the prior art has conducted much research on anti-inflammatory, anti-aging or anti-hair loss products, it is still of great significance to discover some novel structural types of drugs with significant anti-inflammatory, anti-aging or anti-hair loss effects and good safety.

Summary of the invention

The first object of the present invention is to provide a substituted benzoic acid trifluoroalkylthioalkylthioester derivative, which has a very significant anti-inflammatory and neuroprotective effect on BV-2 microglia, and the anti-inflammatory activity is equivalent to that of the positive drug butylphthalide, and the active concentration is much lower than that of the positive drug, and can be used for the preparation and application of anti-inflammatory and neuroprotective drugs.

A substituted benzoic acid trifluoroalkylthioalkylthioester derivative, whose structure is shown in formula (I):

Here, R ₁ and R ₂ are OH, OAc or H, and X and Y are linear or branched alkyl groups having 1 to 9 carbon atoms.

The present invention also provides a method for preparing the above-mentioned substituted benzoic acid trifluoroalkylthioalkylthioester derivative, comprising the following steps:

(1) adding NaH (60% dispersed in mineral oil) 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 endpoint 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.

Preferably, in step (1), the thiol is a dithiol having 1 to 10 carbon atoms.

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

Preferably, in step (1), the solvent comprises DMF.

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

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.

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

The present invention also provides the use of the above-mentioned substituted benzoic acid trifluoroalkyl thioalkyl thioester derivatives in the preparation of drugs, health products or foods for preventing and/or treating inflammation. The substituted benzoic acid trifluoroalkyl thioalkyl thioester derivatives of the present invention have significant anti-inflammatory activity in the in vitro screening model BV-2 cells, and can reduce the excessive activation of microglia and astrocytes. The thioester derivatives can be used as active ingredients, and pharmaceutically acceptable carriers, diluents, etc. are added to prepare drugs for preventing and treating inflammation-related diseases.

The present invention also provides the use of the above-mentioned substituted benzoic acid trifluoroalkyl thioalkyl thioester derivatives in neuroprotective drugs, health products or foods. The substituted benzoic acid trifluoroalkyl thioalkyl thioester derivatives of the present invention have the effect of reducing neuronal damage and death, and can be used for the preparation of neuroprotective drugs by adding pharmaceutically acceptable carriers, diluents, etc.

The present invention also provides the use of the above-mentioned substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative in the preparation of a drug, health product or food for preventing and/or treating neurodegenerative diseases. The substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative of the present invention can be used to prevent and/or treat neurodegenerative diseases, especially Alzheimer's disease, etc.

The present invention also provides the use of the above-mentioned substituted benzoic acid trifluoroalkylthioalkyl thioester derivatives in the preparation of anti-aging drugs, health products or foods for prevention and/or treatment. The substituted benzoic acid trifluoroalkylthioalkyl thioester derivatives of the present invention have significant activity in alleviating the generation of aging markers in the senescent PC12 cells induced by etoposide, an in vitro screening model of cell senescence. An effective dose of the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative can be used as an active ingredient, and a pharmaceutically acceptable carrier, diluent, etc. are added to prepare drugs for preventing and alleviating aging and aging-related diseases.

The present invention also provides the use of the above-mentioned substituted benzoic acid trifluoroalkyl sulfonated alkyl thioester derivative in the preparation of a drug, health product or food for preventing and/or treating hair loss. The substituted benzoic acid trifluoroalkyl sulfonated alkyl thioester derivative of the present invention can achieve a hair growth effect similar to that of minoxidil at a dosage several times lower than that of minoxidil. The substituted benzoic acid trifluoroalkyl sulfonated alkyl thioester derivative is used as an active ingredient, and a pharmaceutically acceptable carrier, diluent, etc. are added to prepare a drug related to preventing and/or treating hair loss.

Preferably, the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is administered by skin application at an amount of 0.25 times that of minoxidil to achieve a hair growth effect similar to that of minoxidil. For example, 0.50% of the compound 3 in the embodiment of the present invention and 2.0% of the minoxidil group have similar hair growth length and density.

Preferably, when the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is used to prevent and/or treat hair loss, the oral dosage of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is 1 to 25 mg/kg body weight, where the body weight refers to human body weight.

The present invention provides a pharmaceutical composition, health product or food for neuroprotection, prevention and/or treatment of inflammation and neurodegenerative diseases. The pharmaceutical composition, health product or food contains the substituted benzoic acid trifluoroalkylthioalkylthioester derivative.

The present invention also provides an anti-aging and hair loss prevention and/or treatment product, wherein the anti-aging and hair loss prevention and/or treatment product comprises the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1.

Preferably, the product of the present invention is an anti-aging and hair loss prevention and/or treatment drug, wherein the anti-aging, hair loss prevention and/or treatment drug contains the above-mentioned substituted benzoic acid trifluoroalkylthioalkylthioester derivative as an active ingredient.

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 carrier acceptable to the food or health product.

Preferably, the product of the present invention is a toiletries for preventing and/or treating hair loss, wherein the toiletries are composed of the above-mentioned substituted benzoic acid trifluoroalkylthioalkylthioester derivative and an acceptable carrier in the toiletries.

The pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, including fillers such as sucrose, starch, microcrystalline cellulose, inorganic salts, etc.; binders such as cellulose derivatives, starch slurry, povidone, gelatin, etc.; wetting agents such as distilled water, ethanol, etc.; lubricants such as magnesium stearate, micropowder silica gel, polyethylene glycols, etc.; absorption enhancers such as polysorbate, lecithin, etc., surfactant fatty acids such as sorbitan, poloxamer, etc. In addition, other adjuvants such as sweeteners, flavoring agents, etc. can also be added to the pharmaceutical composition.

The substituted benzoic acid trifluoroalkylthioalkylthioester derivatives of the present invention can be administered in the form of a unit dose, and the administration route is enteral administration or non-enteral administration, including oral administration, intravenous injection, intramuscular injection, subcutaneous injection, skin administration, nasal administration, etc.

The dosage form of the drug of the present invention can be solid preparations, semisolid preparations, liquid preparations, 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-mentioned various dosage forms can be prepared by conventional methods, for example, by mixing the active ingredient with one or more carriers and then preparing them into the desired dosage form.

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

(1) The substituted benzoic acid trifluoroalkylthioalkylthioester derivatives of the present invention contain a thioester group, an alkyl chain sulfur atom and a trifluoromethyl group. The introduction of these groups can increase the stability and metabolic process of the drug in the body and is a candidate compound for improving bioavailability. Among them, the introduction of the trifluoromethyl group is widely used in non-steroidal anti-inflammatory drugs, including the marketed drugs cyclooxygenase (COXs) inhibitors flufenamic acid and celecoxib.

(2) The substituted benzoic acid trifluoroalkylthioalkylthioester derivatives of the present invention can significantly reduce the LPS-induced BV-2 cell activation ratio at a relatively low concentration (0.03-0.3 μM), and their neuroprotective and anti-inflammatory activities are comparable to those of positive drugs.

(3) The substituted benzoic acid trifluoroalkylthioalkylthioester derivatives of the present invention can alleviate the oxidative stress level, cell cycle arrest, aging marker SA-β-Gal and lipofuscin production of senescent cells, and are potential anti-aging active compounds that can delay the aging process, prevent aging-related diseases, and prolong the healthy lifespan of living organisms. They have broad application prospects in anti-aging products.

(4) The substituted benzoic acid trifluoroalkylthioalkylthioester derivatives of the present invention can promote hair growth and increase hair density, and have good application prospects in anti-hair loss drugs, health products and toiletries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the effects of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives prepared in Examples 1 to 8 on LPS-activated BV-2 microglial cells after 24 hours of action.

Figure 2 shows the effects of the substituted benzoic acid trifluoroalkylthioalkylthioester derivatives prepared in Example 3 on the activity of microglia and astrocytes in the cerebral cortex and hippocampus of mice fed a high-fat diet, and the expression of the inflammation-related indicator inducible nitric oxide synthase (INOS) protein, wherein Figure 2A-B shows the effects on the activity of microglia in the cerebral cortex and hippocampus; Figure 2C-D shows the effects on the activity of astrocytes in the cerebral cortex and hippocampus; Figure 2E-F shows the effects on the expression of INOS protein in the cerebral cortex and hippocampus.

Figure 3 shows the anti-inflammatory and neuroprotective efficacy evaluation results of the substituted benzoic acid trifluoroalkylthioalkylthioester derivatives prepared in Example 3, wherein Figures 3A-C show the effects on the activity of microglia and astrocytes in the cerebral cortex of naturally aged mice and the expression of the inflammation-related indicator inducible nitric oxide synthase (INOS) protein; Figures 3D-E show the effects on the number of mature neurons in the cerebral cortex and hippocampus of naturally aged mice.

Figure 4 shows the effect of compound 3 on cognitive function of lipopolysaccharide (LPS)-induced neuroinflammatory mice, wherein Figure 4A-B shows the effect of compound 3 on the alternating arm entry rate and total number of arm entries in the Y-maze of neuroinflammatory mice, Figure 4C-D shows the effect of compound 3 on target recognition and discrimination indices in the training and testing phases of the novel object recognition test in neuroinflammatory mice, and Figure 4E-G shows the effect of compound 3 on the escape latency and number of platform crossings in the training and testing phases of the water maze test in neuroinflammatory mice.

FIG5 shows the effects of compounds 1 to 5 on etoposide-induced senescent PC12 cells.

Figure 6 shows the effects of compound 3 on the cell survival rate, oxidative stress level, cell proliferation level, and aging marker - lipofuscin of etoposide-induced senescent PC12 cells, wherein Figure 6A shows the effect on the cell survival rate of etoposide-induced senescent PC12 cells, Figure 6B shows the effect on the reactive oxygen level of PC12 cells after etoposide treatment, Figure 6C shows the effect on the proportion of proliferating cells with EdU incorporation, and Figure 6D shows the effect on the production of lipofuscin, a marker of senescence induced by etoposide.

Figure 7 shows the effects of compound 3 on the hair loss area, hair growth length and hair growth density of aged mice, wherein Figure 7A shows the effect on the percentage of hair loss area of aged mice, Figure 7B shows the effect on the hair length of hair loss rats, and Figure 7C shows the effect on the hair growth density of hair loss rats.

FIG8 shows the effects of compounds 1 and 5 on the length and density of hair growth in rats, wherein FIG8A shows the effects on the hair length of depilated rats, and FIG8B shows the effects on the relative hair growth density of rats.

Figure 9 shows the effects of compounds 1, 2, 4 and 5 on the hair growth length and hair growth density of mice, wherein Figure 9A shows the effect on the relative growth density of mouse hair, and Figure 9B shows the effect on the hair growth length of mice.

DETAILED DESCRIPTION

The above contents of the present invention are further described in detail below in conjunction with the drawings and embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

Example 1

The preparation method of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative in this embodiment is as follows:

(1) 1,2-ethanedithiol (188.0 mg, 2.0 mmol) was dissolved in 10 ml of dry anhydrous DMF, and NaH (60% dispersed in mineral oil, 96.0 mg, 2.4 mmol) was added at 0°C. After warming to room temperature, the mixture was stirred for 30 min. Then, 1-bromo-4,4,4-trifluorobutane (0.3 mL, 2.0 mmol) and TBAI (74.0 mg, 0.2 mmol) were added at 0°C and the reaction was allowed to proceed overnight. The end point of the reaction was detected by TLC (n-hexane: ethyl acetate = 20:1). After the reaction was completed, ethyl acetate was added for dilution, and the mixture was washed and extracted with 1N HCl solution, water, saturated sodium bicarbonate solution, and NaCl solution, respectively. The obtained organic phase was dried over sodium sulfate, filtered, and then concentrated.

(2) The product obtained in step (1) was dissolved in 10 ml of dry dichloromethane, and 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) were added, and the reaction was allowed to react at room temperature overnight. The end point of the reaction was detected by TLC (n-hexane: ethyl acetate = 5:1). After the reaction was completed, the product was washed with 1N HCl solution, water, saturated sodium bicarbonate solution and NaCl solution, respectively, and extracted. The organic layer was dried and concentrated, and purified by silica gel column chromatography (n-hexane: ethyl acetate = 80:1), and then purified by ODS open column (methanol: water = 75:25) to obtain compound 1.

The structure of compound 1 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 363.0316, calcd. for C ₁₃ H ₁₅ F ₃ O ₃ S ₂ Na(M+Na) ⁺ 363.0307. ¹ H NMR (500 MHz, CDCl ₃ ): 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 compound 1 is shown below:

Example 2

In this example, 1,4-butanedithiol (244.0 mg, 2.0 mmol) was used to prepare substituted benzoic acid trifluoroalkylthioalkylthioester derivatives. The rest was the same as in Example 1. The obtained product was recorded as Compound 2.

The structure of compound 2 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 391.0625, calcd. for C ₁₅ H ₁₉ F ₃ O ₃ S ₂ Na(M+Na) ⁺ 391.0620. ¹ H NMR (500 MHz, CDCl ₃ ): 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 compound 2 is shown below:

Example 3

In this example, 1,6-hexanedithiol (301.0 mg, 2.0 mmol) was used to prepare substituted benzoic acid trifluoroalkylthioalkylthioester derivatives. The eluent used for ODS open column purification was MeOH/H ₂ O with a volume ratio of 80:20. The rest was the same as in Example 1. The obtained product was recorded as Compound 3.

The structure of compound 3 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 419.0933, calcd. for C ₁₇ H ₂₃ F ₃ O ₃ S ₂ Na(M+Na) ⁺ 419.0933. ¹ H NMR (500MHz, CDCl ₃ ):δ＝11.24(1H,s),7.41(1H,dd,J＝1.4,8.0Hz),7.11(1H,dd,J＝1.4,8.0Hz),6.82(1H,t,J＝8.0Hz),5.67(1H,s),3.07(2H,t,J＝7.3Hz),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:

Example 4

In this example, 1,8-octanedithiol (357.0 mg, 2.0 mmol) was used to prepare substituted benzoic acid trifluoroalkylthioalkylthioester derivatives. The rest was the same as in Example 3. The obtained product was recorded as Compound 4.

The structure of compound 4 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 447.1257, calcd. for C ₁₉ H ₂₇ F ₃ O ₃ S ₂ Na(M+Na) ⁺ 447.1246. ¹ H NMR (500MHz, CDCl ₃ ):δ＝11.25(1H,s),7.41(1H,dd,J＝1.0,8.0Hz),7.10(1H,dd,J＝1.0,8.0Hz),6.81(1H,t,J＝8.0Hz),5.68(1H,s),3.06(2H,t,J＝7.3Hz),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:

Example 5

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

The structure of compound 5 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 475.1544, calcd. for C ₂₁ H ₃₁ F ₃ O ₃ S ₂ Na(M+Na) ⁺ 475.1559. ¹ H NMR (500MHz, CDCl ₃ ):δ＝11.26(1H,s),7.41(1H,dd,J＝1.4,8.0Hz,),7.10(1H,dd,J＝1.4,8.0Hz),6.81(1H,t,J＝8.0Hz),5.71(1H,s),3.06(2H,t,J＝7.4Hz),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:

Example 6

Considering that the two hydroxyl groups on the benzene ring of compound 3 have a potential effect on the penetration of compound 3 through the blood-brain barrier, this example performs an acetylation reaction on compound 3 based on the prepared compound 3 to synthesize the prodrug compound 6 of compound 3. The steps are as follows: acetic anhydride (0.47 mL, 5.0 mmol) is added to an anhydrous pyridine solution (1.0 mL) of compound 3 (200.0 mg, 0.5 mmol) at room temperature, the reaction is stirred and then quenched with 1N HCl solution, vacuum concentrated, the concentrate is extracted with EtOAc, the organic phase is dried over Na ₂ SO ₄ , filtered and concentrated, and the rest is the same as Example 3, and the obtained product is recorded as compound 6.

The structure of compound 6 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 481.1327, calcd. for C ₂₁ H ₂₈ F ₃ O ₅ S ₂ (M+H) ⁺ 481.1325. ¹ H NMR (500 MHz, CDCl ₃ ):δ＝7.79(1H,dd,J＝1.7,7.9Hz),7.37(1H,dd,J＝1.7,7.9Hz),7.32(1H,t,J＝7.9Hz),3.01(2H,t,J＝7.5Hz),2.57(2H,t,J＝7.1Hz),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 shown below:

Example 7

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 substituted benzoic acid trifluoroalkylthioalkylthioester derivatives. The rest was the same as in Example 3. The obtained product was recorded as Compound 7.

The structure of compound 7 was determined by analyzing HR ESI-MS and ¹ H NMR data. HR ESI-TOF-MS m/z 387.1022, calcd. for C ₁₇ H ₂₃ F ₃ OS ₂ Na(M+Na) ⁺ 387.1035. ¹ H NMR (500MHz, CDCl ₃ ):δ＝7.97(2H,dd,J＝1.2,7.9Hz),7.56(1H,t,J＝7.9Hz),7.45(2H,t,J＝7.9Hz),3.07(2H,t,J＝7.5Hz),2.57(2H,t,J＝7.0Hz),2.51(2H,t,J＝7.3Hz),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 shown below:

Example 8

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 substituted benzoic acid trifluoroalkylthioalkylthioester derivatives. The rest was the same as in Example 1. The obtained product was recorded as Compound 8.

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 F ₃ O ₃ S ₂ (M+H) ⁺ 383.0957. ¹ H NMR (500 MHz, CDCl ₃ ): 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 compound 8 is shown below:

Application Example 1

BV-2 cell activity assay of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives prepared in Examples 1 to 8

1. Culture medium preparation

RPMI 1640 basal medium: Add 5 mL of dual antibody (10,000 U/mL penicillin and 10 mg/mL streptomycin) to 500 mL of RPMI 1640 medium, mix well, and store in a 4°C refrigerator until use.

RPMI 1640 complete medium: add 5 mL of dual antibody (10000 U/mL penicillin and 10 mg/mL streptomycin) to 500 mL of RPMI 1640 medium, mix well, take out 50 mL of medium as the above-mentioned RPMI 1640 basal medium, add 50 mL of fetal bovine serum to the remaining 450 mL of medium, mix well, and store in a 4°C refrigerator for use.

2. BV-2 cell anti-inflammatory activity assay steps

When BV-2 cells covered 70-80% of the culture dish, subculture began. In a 24-well cell plate, 1 mL of RPMI 1640 complete medium and 50,000 cells were added to each well, and the plate was placed in a CO ₂ incubator for 24 hours before adding the sample. After each well was treated with 500 μL of RPMI 1640 basal medium containing the sample for 2 hours, 500 μL of medium containing LPS (1 μg/mL) was added for 24 hours.

After 24 hours, the culture medium was aspirated, each well was washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The fixative was discarded, each well was washed 3 times with PBS, and immunostaining blocking solution was added to block for 60 minutes. The blocking solution was discarded, and the diluted primary antibody anti-Iba1 was added to each well and incubated at 4°C overnight. The next day, the supernatant was discarded, each well was washed 3 times with PBS, each time for 5 minutes, and the diluted secondary antibody goat anti-rabbit IgG H&L (Alexa 488) and incubated at room temperature for 1 hour. Then, the cell nuclei were stained with diluted DAPI for 5-10 minutes, and the fluorescence was observed under an inverted fluorescence microscope. Three locations were randomly selected for observation and photography, and the images were finally analyzed using Image J software. The data in the experiment were processed using one-way ANOVA in prism5.0 statistical software, and the statistical results were expressed as mean ± standard error (X+SEM).

3. Test results analysis

Figure 1 shows the LPS-induced cell activation rate of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives (compounds 1 to 8) prepared in Examples 1 to 8 after acting on BV-2 cells for 24 hours at different concentrations. Among them, 0.5% DMSO is a negative control (C), LPS (1 μg/mL) is an experimental control, and butylphthalide (10 μM) is a positive control. *P<0.05, **P<0.01, ***P<0.001, ^### P<0.001. ^### P<0.001 indicates that BV-2 cells are significantly activated after LPS (1 μg/mL) treatment alone, and ***P<0.001 indicates that compounds 1 to 8 can significantly inhibit BV-2 cell activation. The results show that compounds 1 to 8 have the best effect at a concentration of 0.3 μM, and compounds 3, 4 and 5 have the most significant effects.

Evaluation of the anti-inflammatory efficacy of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives at the animal level (taking compound 3 as an example)

1. Animal grouping

Grouping of high-fat diet model mice: 60 ICR white mice, 3-4 weeks old, 10-15g, male, were randomly divided into six groups, with 10 mice in each group.

Grouping of mice of natural aging model: 40 C57BL/6J black mice, 10 of which were 8 weeks old, about 25g, female, were divided into one group (young group); the other 30 mice were 18 months old, about 30g, naturally aged, female, and randomly divided into three groups (natural aging groups), with 10 mice in each group.

2. Dosage method

All mice were given the drug orally by gavage.

High-fat diet modeling mice: the normal diet group was given soybean oil as a blank control group (Control); the first group fed with a high-fat diet was given soybean oil as a negative control group (HFD); the second group fed with a high-fat diet was given metformin, which has anti-Alzheimer's disease efficacy, at 140 mg/kg, as a positive control group (Met); the third, fourth, and fifth groups fed with a high-fat diet were given compound 3 at doses of 0.1, 5, and 20 mg/kg, respectively, as experimental groups. Each mouse was given 150uL per day for two consecutive months.

Natural aging model mice: the young group was given 0.5% DMSO as a blank control group; the first group of the natural aging group was given 0.5% DMSO as a negative control group; the second group of the natural aging group was given donepezil, a first-line clinical drug for Alzheimer's disease, at 3 mg/kg as a positive control group; the third group of the natural aging group was given compound 3 at a dose of 5 mg/kg as an experimental group. Each mouse was given 150uL per day for three consecutive months.

3. Test result analysis

High-fat diet model mice: As shown in Figure 2A-F, the results showed that compared with the negative control group HFD group, the 5 mg/kg and 20 mg/kg compound 3 experimental groups had significantly reduced inflammation-related indicators (Iba1 protein, GFAP protein, INOS protein); compared with the blank control group normal diet group (Control), the activation ratio of microglia (Iba1 protein) and astrocytes (GFAP protein) in the cerebral cortex and hippocampus and the expression of INOS protein were comparable.

Naturally aged model mice: As shown in Figure 3A-C, the results showed that the experimental group (aging group + compound administration dose of 5 mg/kg) was significantly reduced in inflammation-related indicators (Iba1 protein, GFAP protein, INOS protein) compared with the negative control group (aging group); compared with the blank control group (young group), the activation ratio of microglia (Iba1 protein) and astrocytes (GFAP protein) in the cerebral cortex and the expression level of INOS protein were comparable.

As shown in Figure 3D and E, the results showed that the number of mature neurons (NeuN protein) in the experimental group (aging group + compound administration dose of 5 mg/kg) was significantly increased compared with the negative control group (aging group); compared with the blank control group (young group), the number of mature neurons in the cerebral cortex and hippocampus in the brain was comparable, that is, compound 3 has significant neuroprotective activity.

The present invention provides a substituted benzoic acid trifluoroalkylthioalkylthioester derivative and a preparation method thereof. The anti-inflammatory activity is found to be significant through the evaluation of the BV-2 cell biological activity system, and the anti-inflammatory and neuroprotective effects are further verified through animal experiments, and the derivative can be used for the preparation and application of anti-inflammatory and neuroprotective related drugs. The research provides a basis for the development of new drugs and basic research on inflammation-related diseases, which is of great significance.

Application Example 2

Evaluation of the cognitive function activity of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives (Compound 3) on lipopolysaccharide (LPS)-induced neuroinflammation mice

The effect of compound 3 on cognitive function of LPS-induced neuroinflammatory mice was studied by animal behavior experiments. Experimental method: 50 of 60 C57BL/6 mice were randomly selected and continuously intraperitoneally injected with LPS (1 mg/kg) for 30 days, and the remaining 10 were blank groups (injected with the same volume of PBS). Compound 3 and positive control galanthamine (GA) were dissolved in soybean oil. Subsequently, the LPS modeling mice were randomly divided into 5 groups, and compound 3 (1 mg/kg, 5 mg/kg, 10 mg/kg), galanthamine (4 mg/kg), and soybean oil (control group) were given oral gavage for 2 months. The Y maze and new object recognition test experiments were performed on each group of mice to evaluate the working memory and short-term memory of the mice. In addition, impaired spatial memory and long-term memory are prominent features of cognitive decline, so the water maze experiment was used to evaluate the spatial memory and long-term memory of mice, so as to comprehensively evaluate the improvement effect of compound 3 on the cognitive function of LPS-induced neuroinflammatory mice.

As shown in Figures 4A and B, in the Y-maze, the alternating arm entry rate and total arm entry number of LPS-induced mice were significantly reduced compared with the blank control group mice. In contrast, the arm entry rate and total arm entry number of mice treated with compound 3 (10 mg/kg) and GA were higher than those of the LPS control group. As shown in Figures 4C and D, in the novel object recognition test, there were no significant differences in the target recognition and discrimination indices of the groups during the training phase. However, the recognition and discrimination indexes of the LPS control group were lower, while those of the groups treated with compound 3 (5 mg/kg) and GA were higher than those of the LPS control group. These results indicate that both compound 3 and GA can improve the working memory and short-term memory of LPS-induced neuroinflammatory mice. As shown in Figures 4E-G, during the four-day training phase, the escape latency of the LPS control group was significantly greater than that of the blank control group, while the escape latency of the GA group and compound 3 group was significantly shorter than that of the LPS control group. In the test phase on the fifth day, changes in escape latency similar to those in the training phase were observed in the LPS control group, GA group, and compound 3 treatment group. In addition, the number of times the LPS control group crossed the platform was significantly lower than that of the blank control group. However, in the GA and compound 3 (10 mg/kg) groups, these parameters returned to normal levels. Therefore, it was confirmed that compound 3 can improve cognitive dysfunction in LPS-induced neuroinflammation mice and has anti-Alzheimer's disease efficacy.

Application Example 3

Evaluation of in vitro anti-aging biological activities of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives (Compounds 1-5) prepared in Examples 1-5

Determination of the activity of compounds 1 to 5 in alleviating senescence markers in etoposide-induced senescent NIH3T3 cells: (1) Effects of compounds 1 to 5 on senescence-related β-galactosidase activity in etoposide-induced senescent NIH3T3 cells. Experimental method:

Place a 12mm round glass slide in each well of the 24-well cell plate, add 250μL 100μg/mL L-polylysine for coating, incubate overnight, rinse with PBS 3 times, and add samples after culturing in a _CO2 incubator for 24 hours. Cell sample treatment: The sample to be tested is prepared with DMSO to the required concentration for determination. The prepared sample to be tested is added to EM to prepare 1mL of the sample solution of a certain concentration. Replace the CM in the 24-well plate with the above-mentioned sample solution to be tested, the positive control is 500nM rapamycin (Rapa), and the negative control is 0.5% DMSO (C). After 24 hours of action, the culture medium is removed, and 1mL of CM culture medium containing 0.3μM etoposide (Eto) is added to each well except the negative control group. The negative control group is treated with CM culture medium containing the same volume of DMSO and continued to incubate in a _CO2 incubator for 48 hours. Staining treatment: The cells were treated with cell senescence β-galactosidase staining reagent (Shanghai Biotech Co., Ltd., product number C0602), then washed with 70% ethanol solution, sliced and sealed, and observed under an ordinary optical microscope. The proportion of cells stained blue (i.e., cells positive for cell senescence β-galactosidase) in all cells in the field of view was calculated.

As shown in Figure 5, etoposide significantly caused NIH3T3 cell senescence. After adding different concentrations (0.1 μM, 0.3 μM, 1 μM) of compounds 1 to 5, the proportion of positive cells decreased significantly, indicating that pretreatment with compounds 1 to 5 can alleviate etoposide-induced cell senescence, and as the chain length of the compound increases, the alleviation effect is stronger.

(2) Effect of compound 3 on cell survival in etoposide-induced senescent PC12 cells

Experimental method: 200 μL CM was added to each well of a 96-well cell plate and 7,000 cells were inoculated. The cells were cultured in a CO ₂ incubator for 24 hours before adding the sample. The cell sample addition treatment method in this application example is the same as that in application example 1. After that, each well was replaced with 100 μL EM solution containing 200 μg/mL thiazolyl blue tetrazolium bromide (MTT). After incubation for 2 hours, 100 μL DMSO was added to each well, shaken at room temperature for 10 minutes, and the absorbance value of each well at 570 nm was measured using an enzyme reader.

As shown in Figure 6A, etoposide significantly reduced the cell survival rate of PC12 cells. Pretreatment with the positive drug 500nM rapamycin and 0.003-0.3μM compound 3 for 24h had no significant effect on the cell survival rate of etoposide-induced senescent PC12 cells, while pretreatment with 1μM compound 3 could reduce the cell survival rate of etoposide-induced senescent PC12 cells (P=0.02).

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

Experimental method: The cell culture and sample addition methods in this application example are the same as those in application example 1. After the cells are added to the 24-well plate, 200 μL of 10 μM reactive oxygen probe DCFH-DA (Shanghai Biotech Biotechnology Co., Ltd., product number S0033) is added to each well, incubated in the dark for 20 minutes, then washed three times with EM solution, and photographed using an inverted fluorescence microscope. Image J software is used to quantitatively analyze the fluorescence intensity values in the photos.

As shown in Figure 6B, etoposide treatment can significantly increase the reactive oxygen levels of PC12 cells, while pretreatment with rapamycin and 0.003-1 μM compound 3 can significantly reduce the abnormal reactive oxygen levels induced by etoposide, and as the concentration of compound 3 increases, the ability to reduce abnormal reactive oxygen levels becomes stronger.

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

Experimental method: The cell culture and sample addition methods in this application example are the same as those in application example 1. After the cells are added to the 24-well plate, the cells are treated with EdU cell proliferation detection reagent (Shanghai Bio-Tech Biotechnology Co., Ltd., product number C0071S), and finally, the slides are prepared and sealed and photographed using an upright fluorescence microscope. The ratio of cells with green fluorescence (i.e., proliferating cells with EdU embedded in DNA) to blue fluorescent cells (all cells) in the field of view is calculated.

As shown in Figure 6C, 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 as the concentration of compound 3 increased, the ability to increase the proportion of proliferating cells with EdU incorporation became stronger, indicating that compound 3 could alleviate the cell cycle arrest of senescent cells.

(5) Effect of compound 3 on lipofuscin in etoposide-induced senescent PC12 cells

Experimental method: The cell culture and sample addition methods in this application example are the same as those in application example 1. After the cells are added and fixed in the 24-well plate, they are washed with PBS three times and then stained with 0.2 ml of Sudan black staining solution. After washing, they are counterstained with nuclear fast red staining solution, washed, and then sliced and sealed. The cells are observed and photographed under an ordinary optical microscope, and the proportion of cells stained blue-black (i.e., lipofuscin-positive cells) in the field of view is calculated.

As shown in Figure 6D, etoposide significantly induced the production of lipofuscin, a marker of aging. After adding different concentrations of compound 3, the proportion of cells containing lipofuscin decreased to varying degrees, among which 0.3 μM compound 3 had the best effect.

Application Example 4

Evaluation of the in vivo hair growth promoting activity of the substituted benzoic acid trifluoroalkylthioalkylthioester derivatives 1 to 5 prepared in Examples 1 to 5

(1) Study on the effect of compound 3 on hair loss in aged mice

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

Dosage: All three groups of mice were given oral administration. The first group of the natural aging group was given 0.5% DMSO as a blank control group; the second group of the natural aging group was given 5 mg/kg of compound 3 as an experimental group; the young group was given 0.5% DMSO. Each mouse was given 150uL per day for three consecutive months.

As shown in Figure 7A, the blank control group had more severe hair loss than the young group; the experimental group had significantly improved hair loss compared to the blank control group, and the area and length of new hair grown were comparable to those of the young group. (2) Study on the effect of different doses of compound 3 on promoting hair growth in rats

Experimental method: Establishment of a rat hair removal experimental animal model: 20 white SD rats, 6-8 weeks old, weighing about 250g, female; 1.0% sodium pentobarbital anesthesia, that is, 1g sodium pentobarbital dissolved in 100mL of 0.9% saline, a dose of 30mg/kg, shaved with an electric shaver, and no remaining hair in the 4cm×5cm area on the back of the rat, that is, the model was successfully established.

Animal grouping: The SD rats with successful modeling were randomly divided into 5 groups, with 4 rats in each group.

Dosage method: The 4 groups of rats were sprayed on the hair removal area with the following drugs: 0.02% compound 3 (i.e. 0.02g compound 3 in 100mL carrier solvent; 0.4mg/kg), 0.10% compound 3 (2.0mg/kg), 0.50% compound 3 (10.0mg/kg), 2.0% minoxidil (40.0mg/kg). The 5th group of rats served as blank controls and were sprayed with an equal volume of mixed solvent (water: ethanol: monoacetin = 0.4: 0.2: 0.4). The volume of each administration was 0.5mL, and the administration was done once a day for four weeks.

Experimental results: a. Hair growth length

Four areas were randomly selected in the rat dosing area every week, and five hairs were randomly plucked in each area to measure and record the hair length. The results are shown in Figure 7B. Compared with the blank control group, the hair length of the experimental groups with different concentrations of compound 3 increased. The hair growth length of the experimental group with 0.50% compound 3 and the group with 2.0% minoxidil was comparable, and both increased significantly compared with the blank control group.

b. Hair growth density

Image J was used to analyze the hair density in the drug-treated area of the rats every week. The results are shown in Figure 7C. Compared with the blank control group, the hair growth density of the experimental groups treated with different concentrations of compound 3 increased. The hair growth density of the experimental group treated with 0.50% compound 3 was equivalent to that of the 2.0% minoxidil group, and was significantly increased compared with the blank control group. After 4 weeks, the hair growth density of the experimental groups treated with different concentrations of compound 3 increased significantly compared with the blank control group.

(3) Study on the effect of compounds 1 and 5 on promoting hair growth in rats

Experimental method: Establishment of a rat hair removal experimental animal model: 16 white SD rats, 6-8 weeks old, weighing approximately 250 g, female; anesthesia with 1.0% sodium pentobarbital, dose 30 mg/kg, shaved with an electric shaver, the model was successfully established when there was no remaining hair in the 4 cm × 5 cm area on the back of the rat.

Animal grouping: The SD rats with successful modeling were randomly divided into 4 groups, with 4 rats in each group.

Administration method: The three groups of rats were sprayed on the hair removal area with the following: 0.50% compound 1 (10.0 mg/kg), 0.50% compound 5 (10.0 mg/kg), and 2.0% minoxidil (40.0 mg/kg). The fourth group of rats served as blank controls and were sprayed with an equal volume of a mixed solvent (water: ethanol: glycerol monoacetate = 0.4: 0.2: 0.4). The volume of each administration was 0.5 mL, and the administration was performed once a day for four weeks.

Experimental results: a. Hair growth length

The results are shown in FIG8A . From the second week, the hair of the rats in the compound 1 group showed significant growth compared with the blank control group. From the third week, the hair of the rats in the compound 5 group showed significant growth compared with the blank control group.

b. Hair growth density

The results are shown in FIG8B . From the first week, the hair density of the rats in the compound 1 group increased significantly compared with the blank control group. From the third week, the hair density of the rats in the compound 5 group increased significantly compared with the blank control group.

(4) Study on the effect of compounds 1, 2, 4 and 5 on promoting hair growth in mice

Experimental method: Establishment of mouse hair removal experimental animal model: 50 black C57 mice, 6-8 weeks old, weighing about 20g, male; 0.3% sodium pentobarbital anesthesia, dose 40mg/kg, hair removal with depilatory cream, the model was successfully established when there was no remaining hair in the 3cm×4cm area on the back of the mouse.

Animal grouping: The successfully modeled C57 mice were randomly divided into 10 groups, with 5 mice in each group.

Dosage method: 9 groups of mice were sprayed on the formed hair removal area with the following drugs: 2.0% minoxidil (100.0 mg/kg), 0.08% compound 1 (4.0 mg/kg), 0.25% compound 1 (12.5 mg/kg), 0.08% compound 2 (4.0 mg/kg), 0.25% compound 2 (12.5 mg/kg), 0.08% compound 4 (4.0 mg/kg), 0.25% compound 4 (12.5 mg/kg), 0.08% compound 5 (4.0 mg/kg), 0.25% compound 5 (12.5 mg/kg), and the 10th group of mice was sprayed with an equal volume of mixed solvent (water: ethanol: monoacetin = 0.4: 0.2: 0.4) as blank control. The volume of each administration was 0.1 mL, and the drug was administered once a day for three weeks.

Experimental results: a. Hair growth density

The results are shown in FIG9A . From the second week onwards, the hair of mice in compound 2, 4 and 5 groups showed significant growth compared with the blank control group. From the third week onwards, the hair of mice in compound 1 group showed significant growth compared with the blank control group.

b. Hair growth length

The results are shown in Figure 9B. Starting from the first week, the hair density of mice in the compound 5 group increased significantly compared with the blank control group. Starting from the second week, the hair density of mice in the compound 1, 2 and 4 groups increased significantly compared with the blank control group.

Claims (12)

A substituted benzoic acid trifluoroalkylthioalkylthioester derivative having a structure shown in formula (I):
Here, R ₁ and R ₂ are OH, OAc or H, and X and Y are linear or branched alkyl groups having 1 to 9 carbon atoms. The method for preparing a substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1, characterized in that it comprises the following steps: (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. The method for preparing a substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 2, characterized in that in step (1), the thiol is a dithiol having 1 to 10 carbon atoms, including but not limited to 1,2-ethanedithiol, 1,4-butanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol or 1,10-decanedithiol; In step (2), the substituted benzoic acid includes but is not limited to 2,3-dihydroxybenzoic acid, 2,3-diacetoxybenzoic acid, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 2-acetoxybenzoic acid or 3-acetoxybenzoic acid; 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; In step (2), the molar ratio of EDC·HCl, substituted benzoic acid, DMAP and thiol is 1-4:1-10:0.1-4:1. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of medicines, health products or foods for preventing and/or treating inflammation. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of drugs, health products or foods for neuroprotection, prevention and/or treatment. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of medicines, health products or foods for preventing and/or treating neurodegenerative diseases. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of drugs, health products or foods for preventing and/or treating anti-aging. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of drugs, health products, foods or toiletries for preventing and/or treating hair loss. The use according to claim 8 is characterized in that the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is administered by skin application in an amount of 0.25 times that of minoxidil to achieve a hair growth effect similar to that of minoxidil. The use according to claim 8 is characterized in that, when used, the oral dose of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative is 1 to 25 mg/kg body weight. A pharmaceutical composition, health product or food for neuroprotection, prevention and/or treatment of inflammation and neurodegenerative diseases, characterized in that the pharmaceutical composition, health product or food contains the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1. 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 the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1.