Application of benzoic acid derivative

Abstract

The invention belongs to the technical field of medicines, and relates to application of benzoic acid derivatives (benzoic acid compounds and salts thereof, corresponding chiral isomers and racemates). The benzoic acid derivative is applied to preparation of drugs for preventing and treating various cardiovascular diseases and metabolic diseases caused by lipid imbalance. The benzoic acid derivative has a good lipid-lowering effect, can effectively prevent and treat various cardiovascular diseases caused by lipid imbalance, is extremely low in toxicity, and is expected to be developed into a first-line medicine for preventing and treating cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of a benzoic acid derivative (benzoic acid compounds and their salts and corresponding chiral isomers and racemates). Background Technology

[0002] Lipid accumulation and metabolic disorders are the common pathological basis of many metabolic and chronic diseases, harming multiple organs and systems. Their complications are mainly divided into two categories: cardiovascular diseases and metabolic diseases, such as atherosclerosis, hyperlipidemia, and non-alcoholic fatty liver disease. Among these, hyperlipidemia has become a global health problem with an increasing prevalence. Hyperlipidemia is not only an independent chronic disease but also a contributing factor to numerous health problems, including cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, stroke, and hypertension, as well as metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease.

[0003] Currently used lipid-lowering drugs include statins, fibrates, and niacin. Atorvastatin and simvastatin are effective in lowering cholesterol levels, while fenofibrate and bezafibrate can significantly reduce triglyceride levels. However, these drugs all have serious muscle toxicity, hepatotoxicity, and drug interactions. Therefore, developing low-toxicity, highly effective lipid-lowering drugs remains a current research hotspot.

[0004] 2-Lactylaminobenzoic acid is a novel compound extracted and isolated from the metabolites of microorganisms. Chinese patents CN103864638A and CN106831475A disclose methods for preparing 2-lactylaminobenzoic acid and its chiral isomers. These patents only describe the corresponding compounds as having analgesic, anti-inflammatory, antiplatelet aggregation, and antithrombotic effects. Summary of the Invention

[0005] The purpose of this invention is to provide an application of benzoic acid derivatives (benzoic acid compounds and their salts and corresponding chiral isomers and racemates).

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of a benzoic acid derivative: The application of benzoic acid derivatives in the preparation of drugs for the prevention and treatment of various cardiovascular and metabolic diseases caused by lipid imbalance.

[0008] The benzoic acid derivatives are the compounds shown in Formula 1, their salts, and corresponding chiral isomers and racemates;

[0009]

[0010] In the formula,

[0011] R1 and R2 may be the same or different and are selected from H and C1-C4 alkyl groups;

[0012] R3, R4, and R5 can be the same or different and can be selected from H or halogens.

[0013] Preferably, the benzoic acid derivatives are compounds of Formula 1, their salts, and corresponding chiral isomers and racemates;

[0014] In the formula, R1 represents H, Na, or —(CH2). n —CH3 (n=0~3);

[0015] R2 can be H, CH3, or (CH2). n —CH3 (n=0~2);

[0016] R3 can be H, F, Cl, or Br;

[0017] R4 can be H, F, Cl, or Br;

[0018] R5 can be H, F, Cl, or Br.

[0019] More preferably, the benzoic acid derivative is 2-lactamide benzoic acid and its salts and corresponding chiral isomers and racemates, as shown in the following structural formula;

[0020]

[0021] Furthermore, the benzoic acid derivatives are S-2-lactamidobenzoic acid, R-2-lactamidobenzoic acid, and S / R-2-lactamidobenzoic acid.

[0022] The 2-lactamidobenzoic acid was isolated from the metabolites of Penicillium chrysogenum or P. notatum strains, and its preparation method is as follows:

[0023] (1) Prepare solid culture medium and fermentation culture medium;

[0024] (2) Inoculate Penicillium chrysogenum or P. notatum strains onto solid culture medium, incubate at 28℃ for 5 days, then transfer to fermentation culture medium and incubate at 28℃ for 14 days to obtain fermentation broth.

[0025] (3) After extraction with an extraction solvent, the fermentation broth was concentrated under reduced pressure to obtain a paste-like crude extract.

[0026] (4) The paste-like crude extract was dissolved in methanol, mixed with octadecylsilane-bonded silica gel (ODS), loaded onto a column, and eluted with a gradient of 40% to 100% methanol. The fractions Fr1 to Fr4 obtained were separated by preparative liquid chromatography [column: octadecylsilane-bonded silica gel (ODS); mobile phase: methanol-water (40:60 to 80:20)] to obtain the new compound 2-lactamide benzoic acid.

[0027] Meanwhile, 2-lactamide benzoic acid can also be obtained through chemical synthesis. The specific synthetic route is as follows: The raw materials, intermediates, and reaction routes involved in the above synthetic method are as follows:

[0028]

[0029] (1) Lactic acid (II) reacts with acetyl chloride (III) in anhydrous tetrahydrofuran or acetic anhydride to obtain intermediate (IV), with a reaction temperature of 10-40℃ and a reaction time of 2-15h;

[0030] (2) Intermediate (IV) reacts with thionyl chloride to obtain intermediate (V), with a reaction temperature of 40-80℃ and a reaction time of 1-4h;

[0031] (3) Intermediate (V) reacts with methyl anthranilate (VI) in anhydrous diethyl ether to generate intermediate (VII), with a reaction temperature of 10-40℃ and a reaction time of 1-24h;

[0032] (4) Intermediate (VII) reacts with potassium carbonate solution in methanol to obtain intermediate (VIII), and intermediate (VIII) reacts with sodium hydroxide solution in tetrahydrofuran to obtain 2-lactamide benzoic acid (I); or intermediate (VII) reacts with sodium hydroxide solution in tetrahydrofuran and methanol to directly obtain 2-lactamide benzoic acid (I); the former reaction temperature is 15-35℃ and the reaction time is 1-20h, and the latter reaction temperature is 10-40℃ and the reaction time is 1-24h.

[0033] The drug is administered via any one or a combination of the following routes of administration: intravenous, submuscular, subcutaneous, intradermal, intraperitoneal, oral, rectal, or a combination thereof.

[0034] The lipid-regulating drugs mentioned are those used to treat diseases caused by lipid accumulation or lipid metabolism disorders, including atherosclerosis, coronary heart disease, hypertension, obesity, type 2 diabetes, non-alcoholic fatty liver disease, hyperlipidemia, gout, pancreatitis, and chronic kidney disease.

[0035] This invention uses two cellular hyperlipidemic models: one is a human liver cancer cell HepG2 hyperlipidemic cell model induced by sodium oleate, and the other is a mouse embryonic fibroblast 3T3-L1 hyperlipidemic cell model induced by dexamethasone (Dex), bovine insulin (INS), and 3-isobutyl-1-methylxanthine (IBMX).

[0036] Cellular assays showed that S-2-lacticaminobenzoic acid, R-2-lacticaminobenzoic acid, and S / R-2-lacticaminobenzoic acid all exhibited extremely low cytotoxicity, significantly reduced triglyceride (TG) and total cholesterol (TC) levels in hyperlipidemic cell models, reduced lipid accumulation, and demonstrated excellent lipid-lowering effects.

[0037] The animal high-fat model used in this invention is a zebrafish high-fat model induced by egg yolk feeding.

[0038] Zebrafish experiments showed that 2-lactamide benzoic acid (2-LABA) significantly reduced lipid, triglyceride (TG), and total cholesterol (TC) levels in a high-fat zebrafish model. Furthermore, 2-LABA also improved the reduction of erythrocytes in the zebrafish heart caused by high-fat diets, inhibited tail thrombosis, and regulated the levels of thrombotic factors COX-1, TXB2, and 6-keto-PGF1α.

[0039] The above results indicate that 2-lactamide benzoic acid and its racemic mixture both have good lipid-lowering effects and can effectively prevent and treat various cardiovascular diseases caused by lipid imbalance. Moreover, they have extremely low toxicity and are expected to be developed into first-line drugs for the prevention and treatment of cardiovascular diseases. Attached Figure Description

[0040] Figure 1 The figure shows the cytotoxicity of 2-lactamide benzoic acid to HepG2 cells (n=3).

[0041] Figure 2 The effect of 2-lactamide benzoic acid on lipid, TG, and TC content in the HepG2 high-fat model is shown in the figure (n=3, *p<0.05, **p<0.01, ***p<0.001VS Mod).

[0042] Figure 3 The figure shows the cytotoxicity of 2-lactylaminobenzoic acid to 3T3-L1 cells (n=3).

[0043] Figure 4 The effect of 2-lactamide benzoic acid on the content of lipids, TG, and TC in the 3T3-L1 high-fat model is shown in the figure (n=3, *p<0.05, **p<0.01, ***p<0.001VS Mod).

[0044] Figure 5The effect of 2-lactamide benzoic acid on the content of lipids (n=6) and TG and TC (n=3) in a high-fat zebrafish model (*p<0.05, **p<0.01, ***p<0.001VS Mod).

[0045] Figure 6 The effect of 2-lactamide benzoic acid on cardiac erythrocytes, tail thrombi (n=6), COX-1, TXB2, and 6-keto-PGF1α in a zebrafish high-fat model (*p<0.05, **p<0.01, ***p<0.001 vs Mod). Detailed Implementation

[0046] The following is a further explanation and description of the technical solution of the present invention; however, the scope of protection of the present invention is not limited to the following embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0047] This invention uses cellular high-fat models and zebrafish high-fat models to determine the lipid-lowering effect of 2-lactamide benzoic acid, which is isolated from microbial metabolites or obtained through chemical synthesis.

[0048] The following examples describe how benzoic acid derivatives were obtained, as described above or in CN103864638A and CN106831475A.

[0049] Example 1.2 Cytotoxicity assay of 2-lactylaminobenzoic acid against HepG2 cells

[0050] 1.1 Experimental cell line: HepG2

[0051] 1.2 Experimental Groups: Simvastatin (0, 10, 50, 100, 150, 200, 250 μmol / L) group, S-2-lacticaminobenzoic acid, R-2-lacticaminobenzoic acid, and S / R-2-lacticaminobenzoic acid (0, 250, 500, 750, 1000, 1250, 1500 μmol / L) groups, for a total of 4 groups. The corresponding concentrations for each group were prepared using DMEM complete medium.

[0052] 1.3 Experimental Methods: HepG2 cells in the logarithmic growth phase were cultured and collected using standard methods. The cell suspension concentration was adjusted with DMEM complete medium (DMEM basal medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody). HepG2 cells were then cultured at a density of 5 × 10⁶ cells / year. 3Cells were seeded at a density of 100 μL / well in 96-well plates, with the edge wells filled with PBS. The plates were gently shaken to mix and then incubated at 37°C with 5% CO2. After 24 hours, the old medium in the 96-well plates was discarded. According to the groupings, 200 μL of DMEM complete medium containing different concentrations of simvastatin, S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid were added to each well, with three replicates for each concentration. After 24 hours of incubation, 110 μL of the drug-containing medium was discarded from each well, and then 10 μL of CCK-8 solution was added to each well, followed by incubation for 1 hour. The absorbance of each well at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0053] 1.4 Experimental Results: The experimental results are shown in [link to results]. Figure 1 At concentrations below 1250 μmol / L in the S-2-lacticaminobenzoic acid, R-2-lacticaminobenzoic acid, and S / R-2-lacticaminobenzoic acid groups, cell viability was above 80%, while in the positive control group simvastatin, cell viability was above 80% at concentrations below 50 μmol / L. These results indicate that the chiral isomers and racemic mixtures of 2-lacticaminobenzoic acid have significantly lower cytotoxicity than simvastatin.

[0054] Example 2.2 Study on the lipid-lowering activity of lactoylaminobenzoic acid in a HepG2 hyperlipidemic cell model

[0055] 2.1 Cell line used in experiments: HepG2

[0056] 2.2 Experimental Groups: The experiment included a control group, a model group, a positive control group (50 μmol / L simvastatin), and low, medium, and high dose groups of S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid (10, 50, and 100 μmol / L), for a total of 12 groups. Each group was prepared with the corresponding concentration using DMEM complete medium containing 400 μmol / L sodium oleate.

[0057] 2.3 Experimental Methods:

[0058] 2.3.1 HepG2 hyperlipidemic cell model induction protocol: HepG2 cells were induced with 400 μmol / L sodium oleate for 24 h to establish a hyperlipidemic model.

[0059] 2.3.2 Lipid content determination in HepG2 cells: HepG2 cells were divided into groups of 5 × 10⁻⁶ cells. 4Cells were seeded at a density of 500 μL per well in 24-well plates. After gentle shaking to mix, the plates were incubated at 37°C with 5% CO2. Cells were divided into 12 groups: a control group, a model group, a positive control group, and low-, medium-, and high-dose treatment groups for S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid. After 24 hours, the old culture medium in the 24-well plates was discarded. The control group received 500 μL of DMEM complete medium, while the other 11 groups received 500 μL of DMEM complete medium containing 400 μmol / L sodium oleate. Simultaneously, the positive control group received 50 μmol / L simvastatin, and the treatment groups received low-, medium-, and high-dose (10, 50, and 100 μmol / L) doses of S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid, respectively, with three replicates for each concentration. After 24 hours, discard the culture medium, wash once with PBS, and perform Oil Red O staining according to the kit instructions. Finally, add 300 μL of isopropanol to each well, incubate at 37°C for 30 min, and shake for 5 min. Transfer 150 μL from each well to a 96-well plate, measure the absorbance at 510 nm, and calculate the lipid content.

[0060] 2.3.3 Determination of TG and TC content in HepG2 cells: HepG2 cells were divided into groups of 2 × 10⁻⁶ cells. 5 Cells were seeded at a density of 1 cell / well in 6-well plates, with 2 mL added to each well. After gentle shaking to mix, the plates were incubated at 37°C with 5% CO2. Cells were divided into 12 groups: a control group, a model group, a positive control group, and low-, medium-, and high-dose treatment groups for S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid. After 24 hours, the old culture medium in the 6-well plates was discarded. 2 mL of DMEM complete medium was added to the control group, and 2 mL of DMEM complete medium containing 400 μmol / L sodium oleate was added to the remaining 11 groups. Simultaneously, 50 μmol / L simvastatin was added to the positive control group, and low-, medium-, and high-dose (10, 50, and 100 μmol / L) S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid were added to each treatment group, with 3 replicates for each concentration. After 24 hours, the culture medium was discarded, the cells were washed once with PBS, and the TG and TC contents were measured according to the kit instructions.

[0061] 2.4 Experimental Results: The experimental results are shown in [the table below]. Figure 2Different doses of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid significantly reduced lipid, TG, and TC levels in the HepG2 hyperlipidemic cell model in a dose-dependent manner. Furthermore, the lipid-lowering effects of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid at the same dose were similar, with no significant difference. In addition, at the same dose, the lipid-lowering effects of these three were similar to those of simvastatin, with no significant difference. These results indicate that 2-lactic acid has excellent lipid-regulating effects, and its chiral structure has no significant impact on its lipid-lowering efficacy.

[0062] Example 3.2 Cytotoxicity assay of 2-lactylaminobenzoic acid against 3T3-L1 cells

[0063] 3.1 Cell line used in experiments: 3T3-L1

[0064] 3.2 Experimental Groups: Simvastatin (0, 50, 75, 100, 125, 150, 175 μmol / L), S-2-lacticaminobenzoic acid, R-2-lacticaminobenzoic acid, and S / R-2-lacticaminobenzoic acid (0, 500, 750, 1000, 1250, 1500, 1750 μmol / L), for a total of 4 groups. Each group was prepared with the corresponding concentration using DMEM complete medium.

[0065] 3.3 Experimental Methods: 3T3-L1 cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted with DMEM complete medium. 3T3-L1 cells were then cultured at a concentration of 5 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of 100 μL / well in 96-well plates, with the edge wells filled with PBS. The plates were gently shaken to mix and then incubated at 37°C with 5% CO2. After 24 hours, the old medium in the 96-well plates was discarded. According to the groupings, 200 μL of DMEM complete medium containing different concentrations of simvastatin, S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid were added to each well, with three replicates for each concentration. After 24 hours of incubation, 110 μL of the drug-containing medium was discarded from each well, and then 10 μL of CCK-8 solution was added to each well, followed by incubation for 1 hour. The absorbance of each well at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0066] 3.4 Experimental Results: The experimental results are shown in [link to results]. Figure 3At concentrations below 1750 μmol / L, S-2-lactylaminobenzoic acid, R-2-lactylaminobenzoic acid, and S / R-2-lactylaminobenzoic acid showed no cytotoxicity to 3T3-L1 cells, while in the positive control group (simvastatin), at concentrations below 100 μmol / L, cell viability remained above 80%. These results indicate that the chiral isomers and racemic mixtures of 2-lactylaminobenzoic acid exhibited significantly lower cytotoxicity than simvastatin.

[0067] Example 4.2 Study on the lipid-lowering activity of 2-lactylaminobenzoic acid in a 3T3-L1 hyperlipidemic cell model

[0068] 4.1 Cell line used in experiments: 3T3-L1

[0069] 4.2 Experimental groups: control group, model group, positive drug group (50 μmol / L simvastatin), and low, medium and high dose groups of S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid (10, 50, 100 μmol / L), for a total of 12 groups.

[0070] 4.3 Experimental Methods:

[0071] 4.3.1 Induction protocol for 3T3-L1 hyperlipidemic cell model: Inducer 1 and Inducer 2 were prepared using dexamethasone (Dex), bovine insulin (INS), and 3-isobutyl-1-methylxanthine (IBMX) to establish a 3T3-L1 hyperlipidemic cell model.

[0072] Preparation of induction solution 1: Induction solution 1 is a DMEM complete culture medium solution containing a final concentration of 1 μmol / L Dex, 0.5 mmol / L IBMX, and 10 μg / mL INS.

[0073] Preparation of Induction Solution 2: Induction Solution 2 is DMEM complete culture medium containing INS at a final concentration of 10 μg / mL; Induction Agent 1 and Induction Agent 2 are prepared fresh for use.

[0074] Induction protocol: After 3T3-L1 cells reached 100% confluence, they were cultured in DMEM complete medium until cell contact inhibition was achieved for 48 hours. After 48 hours of cell contact inhibition, inducer 1 was added and cultured for 2 days, followed by inducer 2 for another 2 days. Then, the cells were cultured in DMEM complete medium for 2 days, for a total of 8 days of induction. The observation of obvious lipid droplet formation indicated successful differentiation induction.

[0075] 4.3.2 Determination of lipid content in 3T3-L1 cells: 3T3-L1 cells were divided into groups of 5 × 10⁻⁶ cells. 4Cells were seeded at a density of 500 μL / well in 24-well plates. After gentle shaking to mix, the plates were incubated at 37°C with 5% CO2. Cells were divided into 12 groups: control group, model group, positive control group, and low-, medium-, and high-dose groups treated with S-2-lactic-aminobenzoic acid (SLA), R-2-lactic-aminobenzoic acid (RLA), and S / R-2-lactic-aminobenzoic acid (S / R-2-lactic-aminobenzoic acid). Timing began when the 3T3-L1 cell density reached 100%, and contact inhibition lasted for 48 hours. After contact inhibition, the old culture medium in the 24-well plates was discarded, and the inducing agents were added to establish a high-lipidemia model. The control group received 500 μL of DMEM complete culture medium, while the other 11 groups received inducing agent one for 2 days, inducing agent two for 2 days, and DMEM complete culture medium for 2 days. Simultaneously, the positive control group received 50 μmol / L simvastatin, while the drug administration groups received low, medium, and high doses (10, 50, and 100 μmol / L) of S-2-lactamide benzoic acid, R-2-lactamide benzoic acid, and S / R-2-lactamide benzoic acid, respectively, with three replicates for each concentration. After induction, the culture medium was discarded, and the samples were washed once with PBS and stained with Oil Red O according to the kit instructions. Finally, 300 μL of isopropanol was added to each well, and the mixture was incubated at 37°C for 30 min, followed by shaking for 5 min. 150 μL from each well was transferred to a 96-well plate, and the absorbance was measured at 510 nm to calculate the lipid content.

[0076] 4.3.3 Determination of TG and TC content in 3T3-L1 cells: 3T3-L1 cells were prepared at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 3T3-L1 cells / well in 6-well plates, with 2 mL added to each well. After gentle shaking to mix, the plates were incubated at 37°C with 5% CO2. Cells were divided into 12 groups: control group, model group, positive control group, and low-, medium-, and high-dose groups treated with S-2-lactic-aminobenzoic acid (SLA), R-2-lactic-aminobenzoic acid (RLA), and S / R-2-lactic-aminobenzoic acid (S / R-2-lactic-aminobenzoic acid). Timing began when the 3T3-L1 cell density reached 100%, and contact inhibition lasted for 48 hours. After contact inhibition, the old culture medium in the 6-well plates was discarded, and induction agents were added to establish a high-lipidemia model. The control group received 2 mL of DMEM complete culture medium, while the other 11 groups received induction agent one for 2 days, induction agent two for 2 days, and DMEM complete culture medium for 2 days. Simultaneously, the positive control group was given 50 μmol / L simvastatin, while the treatment groups were given low, medium, and high doses (10, 50, and 100 μmol / L) of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid, respectively, with three replicates for each concentration. After induction, the culture medium was discarded, the cells were washed once with PBS, and the cells were collected. The TG and TC contents were measured according to the kit instructions.

[0077] 4.4 Experimental Results: The experimental results are shown in [link to results]. Figure 4Different doses of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid significantly reduced lipid, TG, and TC levels in the 3T3-L1 hyperlipidemic cell model in a dose-dependent manner. The lipid-lowering effects of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid at the same dose were similar, with no significant difference. Furthermore, at the same dose, the lipid-lowering effects of these three drugs were similar to those of simvastatin, with no significant difference. These results are consistent with those of the HepG2 hyperlipidemic cell model, further demonstrating the excellent lipid-regulating effect of 2-lactic acid.

[0078] Example 5.2 Study on the lipid-lowering activity of 2-lactylaminobenzoic acid in a zebrafish high-fat model

[0079] 5.1 Experimental fish: WT(AB) zebrafish

[0080] 5.2 Experimental Groups: The experiment included a control group, a model group, a positive control group (50 μmol / L simvastatin), and low-, medium-, and high-dose groups of S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid (10, 50, and 250 μmol / L), for a total of 12 groups. Each group was prepared with the corresponding concentration using E3 medium containing 2% egg yolk.

[0081] 5.3 Experimental Methods:

[0082] 5.3.1 Lipid content determination: Zebrafish at 5 dpf were randomly selected and cultured in 6-well plates, 10 fish per well. The control group was cultured in normal E3 medium. The model group, positive drug group, and S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid administration groups were fed with 2% egg yolk in addition to normal feeding. The positive drug group was exposed to 50 μmol / L simvastatin while on a high-fat diet. The administration groups were exposed to different concentrations of the sample (low, medium, and high concentrations of 10, 50, and 250 μmol / L, respectively) while on a high-fat diet. The medium was changed daily for all groups until 9 dpf. Six fish were randomly selected from each group, and Oil Red O staining was performed according to the kit instructions. The optical density (IOD) of lipid droplets was calculated using Image (grayscale value) analysis.

[0083] 5.3.2 Determination of TG and TC content: Zebrafish 5 days post-fertilization (dpf) were randomly selected and cultured in 90 mm petri dishes, with 50 fish per dish. The control group was cultured in normal E3 medium. The model group, positive control group, and S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid administration groups were fed with 2% egg yolk in addition to normal feeding. The positive control group was exposed to 50 μmol / L simvastatin while on a high-fat diet. The administration groups were exposed to different concentrations of the sample (low, medium, and high concentrations of 10, 50, and 250 μmol / L, respectively) while on a high-fat diet. The medium was changed daily for all groups. After 9 days post-fertilization, the samples were collected, and the TG and TC content was determined according to the kit instructions, with 3 replicates for each concentration.

[0084] 5.4 Experimental Results: The experimental results are shown in [the table below]. Figure 5 Different doses of S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid all significantly reduced lipid levels and TG and TC content in a high-fat zebrafish model, exhibiting a dose-dependent effect. At the same dose, the three drugs showed similar activities with no significant differences, and their effects were similar to those of simvastatin at the same dose, demonstrating good lipid-lowering effects.

[0085] Example 6.2 Study on the antithrombotic activity of 2-lactic acid on a zebrafish high-fat model

[0086] 6.1 Experimental fish: WT(AB) zebrafish

[0087] 6.2 Experimental Groups: The experiment included a control group, a model group, a positive control group (50 μmol / L aspirin), and low, medium, and high dose groups of S-2-lactic-aminobenzoic acid, R-2-lactic-aminobenzoic acid, and S / R-2-lactic-aminobenzoic acid (10, 50, and 250 μmol / L), for a total of 12 groups. Each group was prepared with the corresponding concentration using E3 medium containing 2% egg yolk.

[0088] 6.3 Experimental Methods:

[0089] 6.3.1 Cardiac Erythrocyte Assay: Five-day-fleshed zebrafish of uniform development were randomly selected and placed in 6-well plates, 10 fish per well. The control group was cultured in normal E3 medium. The model group, positive control group, and low-, medium-, and high-dose S-2-lactic-aminobenzoic acid (SLA), R-2-lactic-aminobenzoic acid (RLA), and S / R-2-lactic-aminobenzoic acid (S / R-2-lactic-aminobenzoic acid) groups were fed a normal diet supplemented with 2% egg yolk. The positive control group was given 50 μmol / L aspirin exposure during a high-fat diet. The drug administration groups were given different concentrations of SLA, R-2-lactic-aminobenzoic acid (RLA), and S / R-2-lactic-aminobenzoic acid (RLA) (low, medium, and high concentrations of 10, 50, and 250 μmol / L, respectively) exposure during a high-fat diet. The medium was changed daily for all groups until 9 days post-flop (dpf). Six fish from each group were randomly selected and placed in 24-well plates. The culture medium was removed, and o-anisidine staining solution was added for staining in the dark for 10-15 minutes. Remove the 24-well plate, quickly aspirate the staining solution, and wash several times with DMSO. Finally, observe under a microscope and photograph the cardiac area. Use Image software to analyze the staining intensity (SI) of cardiac erythrocytes to calculate the thrombosis rate and the effect of drugs in preventing thrombosis.

[0090] 6.3.2 Tail Thrombosis Measurement: Ten zebrafish of uniform development (5 dpf) were randomly selected and placed in each well of a 6-well plate. The control group was cultured in normal E3 medium. The model group, positive control group, and low-, medium-, and high-dose S-2-lactic-aminobenzoic acid (S-2-lactic-aminobenzoic acid) groups were fed a diet supplemented with 2% egg yolk on top of their normal diet. The positive control group was exposed to 50 μmol / L aspirin during a high-fat diet. The drug control groups were exposed to different concentrations of the sample (low, medium, and high concentrations of 10, 50, and 250 μmol / L, respectively) during a high-fat diet. The medium was changed daily for all groups until 9 dpf. Six fish from each group were randomly selected and placed in a 24-well plate. The culture medium was removed, and o-anisidine staining solution was added for staining in the dark for 10-15 min. The 24-well plates were then removed, the staining solution was quickly aspirated, and the plates were washed several times with DMSO. Finally, the thrombus was observed under a microscope and photographed. The length of the thrombus was analyzed using Image software to calculate the thrombus formation rate and the effect of drug in preventing thrombosis.

[0091] 6.3.3 Determination of COX-1, TXB2, and 6-keto-PGF1α content: Zebrafish 5 days post-fertilization (dpf) were randomly selected and cultured in 90 mm petri dishes, with 50 fish per dish. The control group was cultured in normal E3 medium. The model group, positive control group, and S-2-lactic acid, R-2-lactic acid, and S / R-2-lactic acid administration groups were fed with 2% egg yolk in addition to normal feeding. The positive control group was exposed to 50 μmol / L aspirin while on a high-fat diet. The administration groups were exposed to different concentrations of samples (low, medium, and high concentrations of 10, 50, and 250 μmol / L, respectively) while on a high-fat diet. The medium was changed daily for all groups. After 9 days post-fertilization (dpf), samples were collected, and the contents of COX-1, TXB2, and 6-keto-PGF1α were determined according to the kit instructions, with 3 replicates for each concentration.

[0092] 6.4 Experimental Results: The experimental results are shown in [link to experimental results]. Figure 6 S-2-lactamide benzoic acid, R-2-lactamide benzoic acid, and S / R-2-lactamide benzoic acid can all significantly improve the reduction of erythrocytes in the heart of zebrafish caused by high lipid intake and inhibit the formation of thrombi in the tail. Their antithrombotic effects are similar to those of the positive control drug aspirin. Furthermore, at concentrations of 50 and 250 μmol / L, they can significantly regulate the levels of COX-1, TXB2, and 6-keto-PGF1α, exhibiting excellent antithrombotic activity.

[0093] The experimental results above indicate that S-2-lactamide benzoic acid, R-2-lactamide benzoic acid, and S / R-2-lactamide benzoic acid all have excellent lipid-lowering effects and can effectively prevent and treat thrombotic diseases caused by lipid disorders.

Claims

1. An application of a benzoic acid derivative, characterized in that: Application of benzoic acid derivatives in the preparation of drugs for the prevention and treatment of various cardiovascular and metabolic diseases caused by lipid imbalance.

2. The application of the benzoic acid derivatives according to claim 1, characterized in that: The benzoic acid derivatives are the compounds shown in Formula 1, their salts, and corresponding chiral isomers and racemates; In the formula, R1 and R2 may be the same or different and are selected from H and C1-C4 alkyl groups; R3, R4, and R5 can be the same or different and can be selected from H or halogens.

3. The application of the benzoic acid derivatives according to claim 2, characterized in that: The benzoic acid derivatives are the compounds shown in Formula 1, their salts, and corresponding chiral isomers and racemates; In the formula, R1 represents H, Na, or —(CH2). n —CH3 (n=0~3); R2 can be H, CH3, or (CH2). n —CH3 (n=0~2); R3 can be H, F, Cl, or Br; R4 can be H, F, Cl, or Br; R5 can be H, F, Cl, or Br.

4. The application of the benzoic acid derivatives according to claim 3, characterized in that: The benzoic acid derivatives are 2-lactamide benzoic acid and its salts and corresponding chiral isomers and racemates, as shown in the following structural formula; 5. The application of the benzoic acid derivatives according to any one of claims 1-4, characterized in that: The drug is administered via any one or a combination of the following routes of administration: intravenous, submuscular, subcutaneous, intradermal, intraperitoneal, oral, rectal, or a combination thereof.

6. The application of the benzoic acid derivatives according to any one of claims 1-4, characterized in that: The drugs mentioned are for the prevention and treatment of various cardiovascular and metabolic diseases caused by lipid imbalance, including atherosclerosis, coronary heart disease, hypertension, obesity, type 2 diabetes, non-alcoholic fatty liver disease, hyperlipidemia, gout, pancreatitis, and chronic kidney disease, which are caused by lipid accumulation or lipid metabolism disorders.

Images