Substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative as well as preparation method and application thereof

Abstract

The invention discloses a substituted benzoic acid trifluoroalkyl sulfo-alkyl thioester derivative which has a structure as shown in a formula (I), R1 and R2 are OH, OAc or H, and X and Y are straight chain or branched chain alkyl with the carbon atom number of 1-9. The invention also discloses a preparation method of the substituted trifluoroalkyl thioalkyl benzoate thioester derivative, which comprises the following steps: adding NaH into an anhydrous DMF (Dimethyl Formamide) solution containing mercaptan at the temperature of below 0 DEG C, heating to room temperature, stirring and reacting for 10-30 minutes, adding a brominated trifluoroalkyl compound and TBAI (Tetrabutyl Ammonium Imide) at the temperature of below 0 DEG C, and reacting to obtain the substituted trifluoroalkyl thioalkyl benzoate thioester derivative. After the reaction is finished, an organic phase purified product reacts with EDC.HCl, substituted benzoic acid and DMAP, and then separation and purification are performed. The substituted benzoic acid trifluoroalkyl thioalkyl thioester derivative has anti-inflammatory activity and neuroprotective activity, and can be used for preparation and application of anti-inflammatory, neuroprotective and anti-neurodegenerative disease related drugs, foods or health care products. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a substituted benzoic acid trifluoroalkylthioalkylthioester derivative and a preparation method and application thereof. Background Art

[0002] 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.

[0003] 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.

[0004] Patent document with announcement number CN111777588B 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. It can be used to prepare drugs related to inflammation.

[0005] 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.

[0006] Although the existing technology has conducted a lot of research on anti-inflammatory drugs, it is still important to find some novel structural types of drugs with significant anti-inflammatory effects and good safety. The study found that 2,3-dihydroxybenzoic acid thioester compounds have a very significant anti-inflammatory effect on BV-2 microglia. On this basis, the design and synthesis of a series of substituted benzoic acid trifluoroalkylthioalkylthioester derivatives with better activity for the preparation of anti-inflammatory and neuroprotective drugs has important application prospects. Summary of the invention

[0007] The first object of the present invention is to provide a trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid. This thioester derivative has very significant anti-inflammatory and neuroprotective effects on BV-2 microglial cells. Its anti-inflammatory activity is comparable to that of the positive drug butylphthalide, and the active concentration is much lower than that of the positive drug. It can be used in the preparation and application of anti-inflammatory and neuroprotective drugs.

[0008] A trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid, whose structure is shown in formula (I):

[0009]

[0010] Among them, R1 and R2 are OH, OAc or H, and X and Y are straight-chain or branched-chain alkyl groups with 1 to 9 carbon atoms.

[0011] The second object of the present invention is to provide a preparation method of the above-mentioned trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid, including the following steps:

[0012] (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 10 - 30 min, then add bromotrifluoroalkyl compound and TBAI for reaction below 0 °C. Detect the end point of the reaction by TLC. After the reaction is completed, extract the reaction product to obtain the target product;

[0013] (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 end point of the reaction by TLC. After the reaction is completed, separate and purify the reaction product to obtain the trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid.

[0014] Preferably, in step (1), the thiol is a dithiol with 1 to 10 carbon atoms.

[0015] More preferably, the thiol is 1,2-ethanedithiol, 1,4-butanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol or 1,10-decanedithiol.

[0016] Preferably, in step (1), the solvent includes DMF.

[0017] 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.

[0018] 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.

[0019] Preferably, in step (2), the molar ratio of EDC·HCl to thiol is 1-4:1; the molar ratio of the substituted benzoic acid to thiol is 1-10:1; the molar ratio of DMAP to thiol is 0.1-4:1.

[0020] The third object of the present invention is to provide the application of the above-mentioned substituted benzoic acid trifluoroalkylthioalkyl thioester derivative in the preparation of drugs, health products or foods for preventing and / or treating inflammation. The substituted benzoic acid trifluoroalkylthioalkyl thioester derivative of the present invention has significant anti-inflammatory activity in the in vitro screening model BV-2 cells, and can reduce the over-activation of microglia and astrocytes. This thioester derivative can be used as an active ingredient, and a pharmaceutically acceptable carrier, diluent, etc. are added to prepare drugs for preventing and treating inflammation-related diseases.

[0021] The fourth object of the present invention is to provide the application of the above-mentioned substituted benzoic acid trifluoroalkylthioalkyl thioester derivative in neuroprotective drugs, health products or foods. The substituted benzoic acid trifluoroalkylthioalkyl thioester derivative of the present invention has the effect of reducing neuronal injury and death. By adding a pharmaceutically acceptable carrier, diluent, etc., it can be used for the preparation of neuroprotective drugs.

[0022] The fifth object of the present invention is to provide the application of the above-mentioned substituted benzoic acid trifluoroalkylthioalkyl thioester derivative in the preparation of drugs, health products or foods for preventing and / or treating neurodegenerative diseases. The substituted benzoic acid trifluoroalkylthioalkyl thioester derivative of the present invention can be used for preventing and / or treating neurodegenerative diseases, especially Alzheimer's disease, etc.

[0023] The sixth object of the present invention is to provide a drug composition, health product or food for preventing and / or treating inflammation, neuroprotection and neurodegenerative diseases, wherein the drug composition, health product or food contains the substituted benzoic acid trifluoroalkylthioalkyl thioester derivative described in claim 1.

[0024] The pharmaceutically acceptable carrier refers to a conventional drug 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, microcrystalline silica, polyethylene glycols, etc.; absorption promoters such as polysorbate, lecithin, etc., and surfactants such as sorbitan fatty acid esters, poloxamer, etc. In addition, other adjuvants such as sweeteners and flavoring agents can also be added to the drug composition.

[0025] The trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid of the present invention can be administered in unit dosage forms, and the administration routes are enteral administration or parenteral administration, including oral administration, intravenous injection, intramuscular injection, subcutaneous injection, transdermal administration, nasal administration, etc.

[0026] The dosage forms of the drugs of the present invention can be solid preparations, semi-solid 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 various dosage forms can be prepared by conventional methods, for example, by mixing the active ingredient with one or more carriers and then making it into the required dosage form.

[0027] Compared with the prior art, the present invention has at least the following advantages:

[0028] (1) The trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid 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. The introduction of the trifluoromethyl group has been widely used in non-steroidal anti-inflammatory drugs, including marketed drugs such as cyclooxygenase (COXs) inhibitors flufenamic acid and celecoxib.

[0029] (2) The trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid of the present invention can significantly reduce the activation ratio of LPS-induced BV-2 cells at a relatively low concentration (0.03 - 0.3 μM), and its activities of neuroprotection and anti-inflammatory are comparable to those of the positive drug. Description of the Drawings

[0030] Figure 1 The LPS-induced cell activation rate after the trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid prepared in Examples 1 - 8 act on BV-2 microglial cells for 24 h.

[0031] Figure 2 The influence of the trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid prepared in Example 3 on the activation of microglial cells in the cerebral cortex and hippocampus of mice modeled with high-fat diet, where Figure 2 A shows the influence on the activation of microglial cells in the cerebral cortex; Figure 2 B shows the influence on the activation of microglial cells in the hippocampus.

[0032] Figure 3 The influence of the trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid prepared in Example 3 on the activation of astrocytes in the cerebral cortex and hippocampus of mice modeled with high-fat diet, where Figure 3 A shows the influence on the activation of astrocytes in the cerebral cortex; Figure 3Effect of B on the activation of astrocytes in the hippocampus.

[0033] Figure 4 Effect of the trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid prepared in Example 3 on the expression level of inducible nitric oxide synthase (INOS) protein, an inflammation-related index, in the cerebral cortex and hippocampus of mice with high-fat diet-induced models, where Figure 4 A is the effect on the expression level of inducible nitric oxide synthase (INOS) protein, an inflammation-related index, in the cerebral cortex; Figure 4 B is the effect on the expression level of inducible nitric oxide synthase (INOS) protein, an inflammation-related index, in the hippocampus.

[0034] Figure 5 Anti-inflammatory efficacy evaluation results of the trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid prepared in Example 3, where Figure 5 A is the effect on the activation of microglia in the cerebral cortex of naturally aged mice; Figure 5 B is the effect on the activation of astrocytes in the cerebral cortex of naturally aged mice; Figure 5 C is the effect on the expression level of inducible nitric oxide synthase (INOS) protein, an inflammation-related index, in the cerebral cortex of naturally aged mice.

[0035] Figure 6 Neuroprotective efficacy evaluation results of the trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid prepared in Example 3, where Figure 6 A is the effect on the number of mature neurons in the cerebral cortex of naturally aged mice; Figure 6 B is the effect on the number of mature neurons in the hippocampus of naturally aged mice. Detailed implementation manners

[0036] The following further details the above content of the present invention in conjunction with the accompanying drawings and embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0037] Example 1

[0038] The preparation method of the trifluoroalkylthioalkyl thioester derivative of substituted benzoic acid in this example is as follows:

[0039] (1) 1,2-Ethanedithiol (188.0 mg, 2.0 mmol) was dissolved in 10 ml of dry anhydrous DMF. At 0 °C, NaH (60% dispersed in mineral oil, 96.0 mg, 2.4 mmol) was added. After the temperature was raised to room temperature and the reaction was stirred for 30 min, 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 carried out overnight. The reaction endpoint was detected by TLC (n-hexane:ethyl acetate = 20:1). After the reaction was completed, it was diluted with ethyl acetate and washed successively with 1N HCl solution, water, saturated sodium bicarbonate solution and NaCl solution, and then extracted. The obtained organic phase was dried over sodium sulfate, filtered, and then concentrated.

[0040] (2) The product obtained in step (1) was dissolved in 10 ml of dry dichloromethane. 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 carried out at room temperature overnight. The reaction endpoint was detected by TLC (n-hexane:ethyl acetate = 5:1). After the reaction was completed, it was washed successively with 1N HCl solution, water, saturated sodium bicarbonate solution and NaCl solution, and then extracted. The organic layer was dried and concentrated, 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.

[0041] Analysis of HR ESI-MS and 1 1H NMR data determined the structure of Compound 1. HR ESI-TOF-MS m / z 363.0316, calcd. for C 13 1H 15 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 shown below:

[0042]

[0043] Example 2

[0044] In this example, 1,4-butanedithiol (244.0 mg, 2.0 mmol) was used to prepare the trifluoroalkylthioalkylthioester derivative of substituted benzoic acid. The rest was the same as in Example 1, and the obtained product was designated as Compound 2.

[0045] Analyze HR ESI-MS and 1 1H NMR data to determine the structure of Compound 2. HR ESI-TOF-MS m / z 391.0625, calcd. for C 15 1H 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 shown below:

[0046]

[0047] Example 3

[0048] In this example, 1,6-hexanedithiol (301.0 mg, 2.0 mmol) was used to prepare the 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 designated as Compound 3.

[0049] Analyze HR ESI-MS and 1 1H NMR data to determine the structure of Compound 3. HR ESI-TOF-MS m / z 419.0933, calcd. for C 17 1H 23 F3O3S2Na (M+Na) + 419.0933. 11H 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 as follows:

[0050]

[0051] Example 4

[0052] 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.

[0053] Analysis of HR ESI-MS and 1 1H NMR data determined the structure of compound 4. HR ESI-TOF-MS m / z 447.1257, calcd. for C 19 1 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 as follows:

[0054]

[0055] Example 5

[0056] In this example, 1,10-decanedithiol (413.0 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 5.

[0057] Analysis of HR ESI-MS and 1The structure of compound 5 was determined by \(^{1}\)H NMR data. HR ESI-TOF-MS m / z 475.1544, calcd. for C 21 H 31 F3O3S2Na (M+Na) + 475.1559. 1 \(^{1}\)H 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:

[0058]

[0059] Example 6

[0060] Considering the potential influence of the two hydroxyl groups on the benzene ring of compound 3 on the penetration of compound 3 through the blood-brain barrier, in this example, based on the prepared compound 3, the acetylation reaction of compound 3 was carried out to synthesize the prodrug compound 6 of compound 3. The steps are as follows: At room temperature, acetic anhydride (0.47 mL, 5.0 mmol) was added to an 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.

[0061] Analysis of HR ESI-MS and 1 The structure of compound 6 was determined by \(^{1}\)H NMR data. HR ESI-TOF-MS m / z 481.1327, calcd. for C 21 H 28 F3O5S2 (M+H) + 481.1325. 11H 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:

[0062]

[0063] Example 7

[0064] 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, and the obtained product was denoted as compound 7.

[0065] 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 1H 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:

[0066]

[0067] Example 8

[0068] 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 trifluoroalkylthioalkylthioester derivative of substituted benzoic acid, and the rest was the same as in Example 1. The obtained product was designated as Compound 8.

[0069] Analyze HR ESI-MS and 1 The structure of Compound 8 was determined by 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:

[0070]

[0071] Application Example

[0072] Determination of the BV-2 cell activity of the trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid prepared in Examples 1 - 8

[0073] 1. Preparation of culture medium

[0074] RPMI 1640 basal medium: Add 5 mL of double antibiotics (10000 U / mL penicillin and 10 mg / mL streptomycin) to 500 mL of RPMI 1640 medium, mix well, and store in a 4°C refrigerator for later use.

[0075] RPMI 1640 complete medium: Add 5 mL of double antibiotics (10000 U / mL penicillin and 10 mg / mL streptomycin) to 500 mL of RPMI 1640 medium, mix well, take out 50 mL of the medium as the above 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 later use.

[0076] 2. Steps for determining the anti-inflammatory activity of BV-2 cells

[0077] When the BV-2 cells cover approximately 70-80% of the culture dish, subculture begins. In a 24-well cell culture plate, add 1 mL of RPMI 1640 complete medium to each well and add 50,000 cells. Place it in a CO2 incubator and incubate for 24 hours before adding samples. After treating the cells in each well with 500 μL of RPMI 1640 basal medium containing the sample for 2 hours, add 500 μL of medium containing LPS (1 μg / mL) and treat for 24 hours.

[0078] After 24 hours, aspirate the medium. Wash each well 3 times with PBS, and fix with 4% paraformaldehyde at room temperature for 20 minutes. Discard the fixative, wash each well 3 times with PBS, and add the immunostaining blocking solution to block for 60 minutes. Discard the blocking solution, add the diluted primary antibody anti-Iba1 to each well, and incubate overnight at 4°C. The next day, discard the supernatant, wash each well 3 times with PBS, 5 minutes each time, and add the diluted secondary antibody goat anti-rabbit IgG H&L (Alexa 488), and incubate at room temperature for 1 hour. Then stain the cell nuclei with diluted DAPI for 5-10 minutes, and observe the fluorescence under a fluorescence inverted microscope. Randomly select 3 areas for observation and photography, and finally analyze the images using Image J software. The data in the experiment were processed using one-way ANOVA in the prism5.0 statistical software, and the statistical results were expressed as mean ± standard error (X+SEM).

[0079] 3. Analysis of test results

[0080] Figure 1 The activation rate of LPS-induced BV-2 cells after the trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid (compounds 1-8) prepared in Examples 1-8 acted on BV-2 cells for 24 hours at different concentrations. Among them, 0.5% DMSO was used as the negative control (C), LPS (1 μg / mL) was used as the experimental control, and butylphthalide (10 μM) was used as the positive control. *P<0.05, **P<0.01, ***P<0.001, ### P<0.001. ### P<0.001 indicates that BV-2 cells were significantly activated after being treated with LPS (1 μg / mL) alone, and ***P<0.001 indicates that compounds 1-8 can all significantly inhibit the activation of BV-2 cells. The results show that compounds 1-8 have the best effects at a concentration of 0.3 μM, and compounds 3, 4, and 5 have the most significant effects.

[0081] Anti-inflammatory pharmacodynamic evaluation of trifluoroalkylthioalkylthioester derivatives of substituted benzoic acid at the animal level (taking compound 3 as an example)

[0082] 1. Animal grouping

[0083] Grouping of mice modeled with high-fat diet: 60 ICR white mice, 3 - 4 weeks old, 10 - 15 g, male, were randomly and evenly divided into six groups, with 10 mice in each group.

[0084] Grouping of naturally aged model mice: 40 C57BL / 6J black mice, among which 10 were 8 weeks old, about 25 g, female, were divided into one group (young group); another 30 were 18 months old, about 30 g, naturally aged, female, and were randomly and evenly divided into three groups (naturally aged group), with 10 mice in each group.

[0085] 2. Administration method

[0086] All mice were administered by oral gavage.

[0087] Mice modeled with high-fat diet: The normal diet group was administered soybean oil as a blank control group (Control); the first group fed with high-fat diet was administered soybean oil as a negative control group (HFD); the second group fed with high-fat diet was administered metformin with an anti-AD effect at a dose of 140 mg / kg as a positive control group (Met); the third, fourth, and fifth groups fed with high-fat diet were administered compound 3 at doses of 0.1, 5, and 20 mg / kg respectively as experimental groups. Each mouse was administered 150 μL per day for two consecutive months.

[0088] Naturally aged model mice: The young group was administered 0.5% DMSO as a blank control group; the first group of the naturally aged group was administered 0.5% DMSO as a negative control group; the second group of the naturally aged group was administered donepezil, a first-line drug for anti-AD, at a dose of 3 mg / kg as a positive control group; the third group of the naturally aged group was administered compound 3 at a dose of 5 mg / kg as an experimental group. Each mouse was administered 150 μL per day for three consecutive months.

[0089] 3. Analysis of test results

[0090] Mice modeled with high-fat diet: As Figures 2 to 4 shown, the results indicated that compared with the negative control group HFD, the inflammation-related indicators (Iba1 protein, GFAP protein, INOS protein) in the experimental groups of compound 3 at 5 mg / kg and 20 mg / kg were significantly decreased; compared with the blank control group of normal diet group (Control), the activation ratios of microglia (Iba1 protein) and astrocytes (GFAP protein) in the cerebral cortex and hippocampus of the brain and the expression level of INOS protein were comparable.

[0091] Naturally aged model mice: As Figure 5As shown, the results indicate that compared with the negative control group (aging group), the experimental group (aging group + compound dosage of 5 mg / kg) had significantly lower inflammation-related indicators (Iba1 protein, GFAP protein, INOS protein); compared with the blank control group (young group), the activation ratios of microglia (Iba1 protein) and astrocytes (GFAP protein) in the cerebral cortex of the brain and the expression level of INOS protein were comparable.

[0092] As Figure 6 shown, the results indicate that compared with the negative control group (aging group), the number of mature neurons (NeuN protein) in the experimental group (aging group + compound dosage of 5 mg / kg) was significantly increased; compared with the blank control group (young group), the number of mature neurons in the cerebral cortex and hippocampus of the brain was comparable, that is, compound 3 has significant neuroprotective activity.

[0093] The present invention provides substituted benzoic acid trifluoroalkylthioalkyl thioester derivatives and their preparation methods. Through the evaluation of the BV-2 cell bioactivity system, significant anti-inflammatory activity was found, and further verified by animal experiments for its anti-inflammatory and neuroprotective efficacy, which can be used for the preparation and application of drugs related to anti-inflammation and neuroprotection. This research provides a basis for the new drug development and basic research of inflammation-related diseases and has important significance.

Claims

1. A substituted benzoic acid trifluoroalkylthioalkylthioester derivative having a structure shown in formula (I): 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. The method for preparing a substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1, 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.

3. The preparation method 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.

4. The preparation method according to claim 2, 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.

5. The preparation method according to claim 2, characterized in that: 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.

6. The preparation method according to claim 5, 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.

7. 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 inflammation.

8. Use of the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1 in the preparation of neuroprotective preventive and / or therapeutic drugs, health products or foods.

9. 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 neurodegenerative diseases.

10. A pharmaceutical composition, health product or food for preventing and / or treating inflammation, neuroprotection and neurodegenerative diseases, characterized in that: The pharmaceutical composition, health product or food contains the substituted benzoic acid trifluoroalkylthioalkylthioester derivative according to claim 1.

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