Vermilionate and its preparation method and application

Abstract

The present invention relates to a salt of cinnamate, its preparation method, and its application, and belongs to the field of chemical medicine technology. The cinnamate salt of the present invention comprises an alkaline salt and an acidic salt. The cinnamate salt is prepared using cinnamate acid through a one-step reaction, with a purity of ≥95% and a yield of 70% or more. The reaction conditions are not demanding, the equipment requirements are simple, and there is no reliance on special equipment. The production process is simple and controllable, making it more suitable for industrial production. The cinnamate salt of the present invention has anti-inflammatory activity and a significant inhibitory effect on the inflammatory response of microglia. It also has anti-tumor activity and can significantly inhibit the proliferation of large cell lung cancer cells, triple-negative breast cancer cells, colon cancer cells, and prostate cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicine and relates to vermilionate and a preparation method and application thereof. Background Art

[0002] In recent years, with the continuous development of drug molecular entities, drug molecular structures have become increasingly complex, and drug solubility has also deteriorated. A recently developed synthesis process for cinnamic acid has been reported, but the finished product has relatively poor solubility and is difficult to dissolve in water, with a solubility of less than 10 mg / 100 mL.

[0003] Drug salt formation is an important means of improving solubility and bioavailability, and is a common approach used by researchers for final structural optimization of drug candidates. Drug salt formation can also enhance drug stability and hygroscopicity, increase melting points, and improve crystal forms, playing a crucial role in the development of further dosage forms. Furthermore, drug salt formation offers numerous benefits, including masking drug taste, improving patient compliance, and extending drug shelf life.

[0004] Therefore, studying the salt forms of cinnamic acid compounds is of great practical significance. Salt formation can improve some undesirable physical, chemical or biopharmaceutical properties of drugs, such as changing the solubility or dissolution rate of drugs, polymorphism, etc.

[0005] The synthesis process of cinquefoil acid has been reported recently, but the synthesis process and application of cinquefoil acid salts have not been reported yet. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides vermilionate and a preparation method and application thereof, wherein the vermilionate has anti-inflammatory and anti-tumor activities.

[0007] The technical solutions of the present invention are as follows:

[0008] Vermilionate, whose structural formula is shown in Formula I or Formula II:

[0009]

[0010] Where R1 is Li + 、Na + , K + 、Zn 2+ 、Cs + or Ca 2+ ;

[0011] R2 is hydrochloric acid, acetic acid, trifluoroacetic acid, hydrobromic acid, hydroiodic acid, sulfuric acid, maleic acid, fumaric acid, phosphoric acid, tartaric acid, lactic acid, L-pyroglutamic acid, malic acid, citric acid or methanesulfonic acid.

[0012] The preparation method of the vermilionate represented by the above formula I comprises the following steps:

[0013] Vermilion acid is added to an organic solvent (1), and a salt-forming reagent is added to carry out a salt-forming reaction. After the reaction is completed, activated carbon is added for decolorization, and then the organic solvent (1) is removed by concentration to dryness. An organic solvent (2) and deionized water are added to dissolve the solution, and then a crystallization reagent is added to carry out crystallization. After filtering and drying, vermilionate is obtained.

[0014] According to the preferred embodiment of the present invention, the organic solvent 1 is methanol, ethanol or isopropanol, and the amount of the organic solvent 1 is 5-20 times the weight of cinquefoil.

[0015] According to the present invention, the salt-forming agent is preferably lithium hydroxide, sodium methoxide, sodium hydroxide, sodium carbonate, potassium carbonate, potassium hydroxide, cesium carbonate, zinc hydroxide or calcium hydroxide, and the molar ratio of the salt-forming agent to vermilion acid is (0.9-1.5):1.

[0016] More preferably, when the salt-forming reagent is lithium hydroxide, cesium carbonate or calcium hydroxide, the salt-forming reaction conditions are: reaction at 23-27° C. under shaking conditions for 0.5-0.7 h.

[0017] More preferably, when the salt-forming reagent is sodium hydroxide, the salt-forming reaction conditions are: stirring until the system is clear.

[0018] More preferably, when the salt-forming reagent is potassium hydroxide, the salt-forming reaction conditions are: ultrasonic vibration until the system is clear.

[0019] More preferably, when the salt-forming reagent is zinc hydroxide, the salt-forming reaction conditions are: reaction at 33-37° C. under shaking conditions for 1-1.5 hours.

[0020] According to the preferred embodiment of the present invention, the concentration condition is: concentration to dryness at 35-55°C.

[0021] According to the preferred embodiment of the present invention, the second organic solvent is dichloromethane, chloroform or dichloroethane, and the amount of the second organic solvent is 2-3 times the weight of vermilionic acid.

[0022] According to the preferred embodiment of the present invention, the amount of deionized water used is 5% to 10% of the weight of vermilionic acid.

[0023] According to the present invention, the crystallization agent is preferably ethyl acetate, butyl acetate, amyl acetate, methyl formate, isoamyl acetate, isoamyl isovalerate, ethyl benzoate or ethyl butyrate, and the amount of the crystallization agent is 10 to 20 times the weight of vermilionic acid.

[0024] According to the preferred embodiment of the present invention, the crystallization treatment condition is: crystallization at 5-10°C.

[0025] According to the preferred embodiment of the present invention, the drying treatment condition is: drying at 45-50° C. under vacuum conditions.

[0026] The preparation method of the vermilionate represented by the above formula II comprises the following steps:

[0027] Dissolve cinnamic acid in solvent I, maintain the temperature at -10 to 10°C, add the corresponding acid dropwise, and solid will precipitate continuously until the solid is completely precipitated. Then stir until the solid particles turn into fine needle-shaped crystals. After filtration and drying, cinnamic acid salt is obtained.

[0028] According to the present invention, preferably, the solvent I is one or more of methanol, ethanol, isopropanol, ethyl acetate, butyl acetate, amyl acetate, methyl formate, isoamyl acetate, isoamyl isovalerate, ethyl benzoate, and ethyl butyrate, and the amount of the solvent I is 5-10 times the weight of cinquefoil.

[0029] According to the present invention, preferably, the acid is hydrochloric acid, acetic acid, trifluoroacetic acid, hydrobromic acid, hydroiodic acid, sulfuric acid, maleic acid, fumaric acid, phosphoric acid, tartaric acid, lactic acid, L-pyroglutamic acid, malic acid, or citric acid, and the molar ratio of the acid to vermilion acid is (1-4):1.

[0030] More preferably, the acid is dissolved in an alcohol solvent to form an alcohol solution of the acid, which is then added dropwise to the reaction system.

[0031] More preferably, the alcohol solvent is one or more of methanol, ethanol or isopropanol.

[0032] More preferably, the concentration of the acid in the alcoholic acid solution is 6 to 50 mol / L.

[0033] Preferably, according to the present invention, in order to better precipitate the solid, refrigeration crystallization is performed before stirring and / or solvent II is added before stirring, and the solvent II is acetone, methyl isobutyl ketone, anhydrous ether, isopropyl ether or methyl tert-butyl ether.

[0034] More preferably, the amount of solvent II is 2-10 times the weight of cinquefoilic acid.

[0035] The use of the above-mentioned vermilionate in the preparation of drugs for treating neuroinflammation.

[0036] The application of the above-mentioned vermilionate in the preparation of drugs for treating tumors.

[0037] Preferably according to the present invention, the tumors include: large cell lung cancer, triple-negative breast cancer, colon cancer, and prostate cancer.

[0038] Beneficial effects:

[0039] 1. The cinnamate salt in the present invention includes an alkaline salt and an acidic salt. The cinnamate salt is prepared by a one-step reaction using cinnamic acid, with a purity of ≥95% and a yield of more than 70%. The reaction conditions are not demanding, the equipment requirements are simple, there is no dependence on special equipment, the production process is simple and controllable, and it is more suitable for industrial production.

[0040]

[0041] 2. The cinnamate of the present invention has anti-inflammatory activity and has a significant inhibitory effect on the inflammatory response of microglia. Among them, the anti-inflammatory effect of alkaline cinnamate is better than that of acidic cinnamate, but the anti-inflammatory effect of both is better than resveratrol, and the best anti-inflammatory effect is cesium cinnamate. The cinnamate of the present invention also has anti-tumor activity and can significantly inhibit the proliferation of large cell lung cancer cells, triple-negative breast cancer cells, colon cancer cells and prostate cancer cells. Among them, cesium cinnamate has the best effect on triple-negative breast cancer cells. The above results show that the cinnamate of the present invention can be used for the research and development and preparation of drugs for treating neuroinflammation and treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the hydrogen spectrum of vermilionic acid.

[0043] Figure 2 HPLC chromatogram of lithium salt of cinnamic acid.

[0044] Figure 3 This is the hydrogen spectrum of lithium salt of vermilionate. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited thereby.

[0046] The reagents and materials involved in the examples are all common commercial products unless otherwise specified. Vermilion acid can be prepared according to existing technology, such as the preparation described in Example 1 of patent document CN 118440025 A. The hydrogen spectrum of vermilion acid is as follows: Figure 1 shown.

[0047] Example 1:

[0048] 200 mg of cinnabaric acid was added to 4.7 mL of methanol, and 17 mg of lithium hydroxide was added. The system was placed in a constant temperature oscillator (shaker) JCOZ-300J, the reaction temperature was set to 25°C, and the reaction was carried out for 0.5 h. Activated carbon was added for decolorization, and the mixture was filtered. The filtrate was concentrated to dryness at 35-40°C, 0.5 g of dichloroethane and 10 mg of deionized water were added to dissolve the material, and then 4 g of amyl acetate was added to crystallize at 5-10°C, filtered, and dried at 45-50°C under vacuum to obtain 194 mg of cinnabaric acid lithium salt, with a yield of 95% and a purity of 97%. The HPLC chromatogram of the obtained cinnabaric acid lithium salt is shown below. Figure 2 As shown, the hydrogen spectrum is Figure 3 shown.

[0049] Example 2:

[0050] 200 mg of cinnamic acid was added to 5 mL of methanol, followed by 28 mg of sodium hydroxide. The mixture was stirred until clear, decolorized with activated carbon, and filtered. The filtrate was concentrated to dryness at 40-45°C. 0.6 g of dichloromethane and 20 mg of deionized water were added to dissolve the material. 3 g of ethyl acetate was then added to crystallize at 5-10°C, filtered, and dried under vacuum at 45-50°C to obtain 206 mg of cinnamic acid sodium salt, with a yield of 96% and a purity of 98%. The solubility of cinnamic acid sodium salt was greatly improved, making it highly soluble in water. At room temperature and normal pressure, the solubility in water was approximately 10 g / 100 mL.

[0051] Example 3:

[0052] 200 mg of cinnabaric acid was added to 4.5 mL of ethanol, and 40 mg of potassium hydroxide was added. The mixture was ultrasonically shaken until clear, and activated carbon was added for decolorization. The mixture was filtered, and the filtrate was concentrated to dryness at 40-45°C. 0.5 g of dichloromethane and 15 mg of deionized water were added to dissolve the material. Then, 4 g of butyl acetate was added to crystallize at 5-10°C, filtered, and dried under vacuum at 45-50°C to obtain 218 mg of cinnabaric acid potassium salt with a molar yield of 96% and a purity of 97%.

[0053] Example 4:

[0054] 200 mg of cinnabaric acid was added to 4.7 mL of isopropanol, and 68 mg of zinc hydroxide was added. The system was placed in a constant temperature oscillator (shaker) JCOZ-300J, the reaction temperature was set to 35°C, and the reaction was carried out for 1 hour. Activated carbon was added for decolorization, and the mixture was filtered. The filtrate was concentrated to dryness at 40-55°C, 0.5 g of chloroform and 14 mg of deionized water were added to dissolve the material, and then 4 g of amyl acetate was added to crystallize at 5-10°C. The mixture was filtered and dried at 45-50°C under vacuum to obtain 212 mg of cinnabaric acid zinc salt with a yield of 96% and a purity of 95%.

[0055] Example 5:

[0056] According to the preparation method of Example 1, cesium carbonate and calcium hydroxide were used as salt-forming reagents to prepare cesium cinnamate and calcium cinnamate respectively.

[0057] Example 6:

[0058] 2 g of vermilionic acid was dissolved in 20 mL of ethyl acetate, and the reaction system was placed in an ice-salt bath at 0-5°C. 1.5 mL of a methanolic hydrochloric acid solution was added dropwise to the system, wherein the hydrochloric acid concentration in the methanolic hydrochloric acid solution was 6 mol / L. Solids were continuously precipitated. To better precipitate the solids, 5 g of acetone was added and stirred for 1-2 hours. The product was filtered and dried to obtain vermilion hydrochloride with a yield of 70% and a purity of 95%.

[0059] Example 7:

[0060] 1.5 g of cinnabaric acid was dissolved in 17 mL of butyl acetate, and the reaction system was placed in an ice-salt bath at -10 to -5°C. 1.6 mL of ethanolic hydrochloric acid solution was added dropwise to the system to adjust the pH of the system to 1.0 to 2.0, wherein the concentration of hydrochloric acid in the ethanolic hydrochloric acid solution was 6 mol / L. Solids were continuously precipitated, and the reaction mixture was placed in a freezer and refrigerated for crystallization. 15 g of anhydrous ether was added, and stirred for 1-2 hours. The mixture was filtered and dried to obtain cinnabaric hydrochloride with a yield of 72% and a purity of 96%.

[0061] Example 8:

[0062] 2 g of cinnabaric acid was dissolved in 24 mL of a mixed solution of methanol and ethanol (1:1, v / v). The reaction system was placed in an ice-salt bath at -5 to 0°C, and 0.5 mL of a methanol solution of trifluoroacetic acid was added dropwise to the system. The concentration of trifluoroacetic acid in the methanol solution was 50 mol / L. Solids were continuously precipitated. The solution was placed in a freezer and refrigerated for crystallization. 10 g of methyl isobutyl ketone was then added and stirred. The mixture was filtered and dried to obtain cinnabaric acid trifluoroacetate. The yield was 73% and the purity was 95%.

[0063] Example 9

[0064] The preparation methods of cinnamic acid methanesulfonate, cinnamic acid hydrobromide, cinnamic acid hydroiodide, cinnamic acid acetate, cinnamic acid sulfate, cinnamic acid maleate, cinnamic acid fumarate, cinnamic acid phosphate, cinnamic acid tartrate, cinnamic acid lactate, cinnamic acid L-pyroglutamate, and cinnamic acid malate are the same as those in Example 7.

[0065] The cinnamate prepared above or the cinnamate prepared using the above method are summarized and numbered as shown in the following table:

[0066] Table 1. Summary of vermilionate in this patent

[0067] Compound number Salt-forming reagent Vermilionate 1 lithium hydroxide Lithium cinnamate 2 Sodium hydroxide Vermilionic acid sodium salt 3 potassium hydroxide Potassium salt of vermilionate 4 zinc hydroxide Vermilionic acid zinc salt 5 Cesium carbonate Cesium vermilionate 6 calcium hydroxide Vermilionic acid calcium salt 7 Methanesulfonic acid Vermilionic acid methanesulfonate 8 hydrochloric acid Vermilion hydrochloride 9 hydrobromic acid Vermilionic acid hydrobromide 10 hydroiodic acid Vermilionic acid hydroiodide 11 acetic acid Vermilionic acid acetate 12 trifluoroacetic acid Vermilionic acid trifluoroacetate 13 sulfuric acid Vermilionic acid sulfate 14 Maleic acid Vermilionic acid maleate 15 Fumaric acid Vermilionic acid fumarate 16 phosphoric acid Vermilionic acid phosphate 17 tartaric acid Vermilionic acid tartrate 18 lactic acid Vermilionic acid lactate 19 L-Pyroglutamate Vermilionic acid L-pyroglutamate 20 Malic acid Vermilionic acid malate

[0068] Experimental Example 1: Anti-inflammatory activity

[0069] The cinnamate prepared in the examples was applied to BV2 cells (mouse microglia, purchased from Shanghai Lianmai Biotechnology Co., Ltd. (LMAl Bio)). Lipopolysaccharide (LPS) was used to induce cellular inflammation, and the anti-inflammatory activity of different cinnamate salts was tested. The specific steps are as follows:

[0070] 1. NO generation rate detection

[0071] BV2 cells were seeded in 96-well culture plates using DMEM medium and cultured in a 37°C incubator with 5% CO2. The cells were divided into a drug-treated group, a positive control group, and an LPS model group. After 24 hours of culture, the drug-treated group was treated with the corresponding concentrations (1 μM or 10 μM) of vermilionate prepared in the example, and the positive control group was treated with the corresponding concentration (20 μM) of resveratrol. After 2 hours of incubation, LPS (purchased from Sigma-Aldrich) was added to the drug-treated group, the positive control group, and the LPS model group to induce a cellular inflammatory response. Culture was continued in the incubator for 24 hours. The supernatant from each group was reacted with an equal volume of Griess buffer (purchased from Sigma-Aldrich), and the OD value of each group was measured at a wavelength of 540 nm using a microplate reader (BioTek). The NO production rate in the LPS model group was set to 100% (n=3), and the NO production rate of the other groups was calculated based on this.

[0072] 2. Cell Viability Detection

[0073] BV2 cells were seeded on 96-well culture plates using DMEM medium and cultured in a 37°C constant temperature incubator containing 5% CO2. The cells were divided into a drug-treated group and an LPS model group. After culturing for 24 hours, the corresponding concentration (1 μM or 10 μM) of vermilionate prepared in the example was added to the drug-treated group. After incubation for 2 hours, LPS was added to the drug-treated group and the LPS model group at a final concentration of 100 ng / mL to induce a cellular inflammatory response. Culture was continued for 24 hours, and a final concentration of 0.5 mg / mL MTT (purchased from Sigma-Aldrich) was added to each well for live cell staining. After incubation in the incubator, the culture medium was discarded, 100 μL DMSO was added to each well, and the plates were shaken on a shaker to fully dissolve them. The OD value of each group at a wavelength of 490 nm was detected by a microplate reader (BioTek). The cell survival rate of the LPS model group was set to 100% (n=3), and the cell survival rate of the other groups was calculated based on this.

[0074] 3. Test results

[0075] (1) As shown in Table 2, the results of the NO generation rate test show that alkaline vermilionate salts have a very significant effect on inhibiting NO generation. It was further discovered that this is because the carboxyl group has a stronger chelating ability with metals under alkaline conditions. By chelating with the ferrous ions of heme in the IDO1 target, it inhibits IDO1 activity, thereby reducing inflammation and inhibiting NO release. The results show that alkaline vermilionate salts are more effective than acidic vermilionate salts and better than resveratrol. The higher the concentration of vermilionate salts, the stronger the NO inhibition effect. The best is cesium vermilionate salt.

[0076] Table 2. NO generation rate test results

[0077]

[0078]

[0079] (2) The results of cell viability test are shown in Table 3. It can be found that compared with LPS, alkaline vermilionate is basically non-toxic to BV2 cells, and its cell viability is higher than 100%. However, the cell viability of the acidic vermilionate treatment group is lower than 100%. In short, the cytotoxicity of alkaline vermilionate is weaker than that of LPS, which is weaker than that of acidic vermilionate. The lower the concentration of vermilionate, the weaker the cytotoxicity. Among them, cesium vermilionate is the best.

[0080] Table 3. Cell viability test results. Results of the toxic effects of the compounds on BV2 cells.

[0081]

[0082] Experimental Example 2: Antitumor Activity

[0083] The anti-tumor activity of the cinnamate prepared in the example was determined using four cell lines: H460 cells (human large cell lung cancer cells), MDB-MA-231 cells (human triple-negative breast cancer cells), Caco-2 cells (human colon cancer cells), and PC-3 cells (prostate cancer cells), all purchased from Sigma-Aldrich. The specific steps are as follows:

[0084] (1) Cell culture: Tumor cells were cultured using RPMI-1640 medium (complete medium) containing 10% fetal bovine serum (FBS) in a 10-cm-diameter culture dish. 10 mL of complete medium was added to each dish and the cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0085] (2) Cell passaging: When the cell density reaches 80%, discard the old culture medium and wash the cells twice with 10 mL of PBS buffer each time; add 1 mL of trypsin to digest the cells and observe under a microscope. When the cells become round, discard the trypsin and add a certain amount of fresh complete culture medium. Beat the cells into single cells. Then, split the cell suspension into two and add them to two new 10 cm cell culture dishes. Finally, fill the complete culture medium to 10 mL and culture in a 37°C, 5% CO2 cell culture incubator.

[0086] (3) Determination of cell number: When the cells are subcultured to a density of 80%, wash, digest, and pipette the cells according to step (2) to prepare a cell suspension, inoculate the cell suspension into a 96-well plate at 100 μL / well, pre-incubate the 96-well plate in a saturated humidity incubator (37°C, 5% CO2) for 60 min, add 10 μL of CCK8 solution to each well of the 96-well plate, continue to incubate the culture plate in the incubator for 1 to 4 hours, and use a microplate reader to measure the absorbance at 450 nm to determine the cell concentration of the cell suspension;

[0087] (4) Cell proliferation and cytotoxicity assay: Adjust the cell concentration of the cell suspension to 10 3 ~10 4 Cells / well were seeded in a 96-well plate, 100 μL / well, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The cells were divided into a drug-treated group and a positive control group. The drug-treated group was divided into a positive control group according to the IC 50 The cinnamate prepared in the example was added to a 96-well plate, 5-fluorouracil (1 μM) was added to the positive control group, and 6 replicates were performed for each group. The 96-well plate was incubated in an incubator for another 24 hours. 10 μL of CCK8 solution was added to each well of the 96-well plate using a repeating pipette, and the 96-well plate was incubated in the incubator for another 4 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0088] The cell survival rate results are shown in Table 4. The cinnamate salt of the present invention exhibited a good effect of inhibiting cell proliferation in all four tumor cell lines, and was superior to cinnamic acid. Among them, cesium cinnamate salt had the best effect on triple-negative breast cancer.

[0089] Table 4. Cell viability results

[0090]

[0091]

[0092] The present invention improves the solubility of the natural product in water by forming salts of different vermilionic acids, which is beneficial to the transmembrane transport and drug delivery of the compound. The activity of the compound on inflammatory cells and tumor cells is better than that of the natural product.

Claims

1. Use of a salt of vermilionate in the preparation of a drug for treating neuroinflammation, wherein the structural formula of the salt of vermilionate is shown in Formula I: Formula I in, R1 is Li + 、Na + , K + or Cs + .

2. Use of vermilionate in the preparation of a drug for treating tumors, wherein the structural formula of the vermilionate is shown in Formula I: Formula I in, R1 is Cs + ; The tumors are: large cell lung cancer, triple negative breast cancer, colon cancer, and prostate cancer.

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