Phenolic acid-sugar conjugate with blood fat reducing activity as well as preparation method and application of phenolic acid-sugar conjugate

Abstract

The invention relates to the technical field of pharmaceutical compounds, in particular to a phenolic acid-glycoconjugate with hypolipidemic activity as well as a preparation method and application of the phenolic acid-glycoconjugate. The synthesis process has few steps, and the total yield of the product is high (the total yield of the compound 6 is greater than or equal to 75%); the synthesis process is mild in reaction condition, does not need to react at high temperature and high pressure, and is beneficial to large-scale production; the performance of the synthesized product is greatly improved compared with that before synthesis, and a reliable research method is provided for improving the bioavailability of natural polyphenol and developing natural lipid-lowering drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compounds, and in particular to a phenolic acid-sugar conjugate with lipid-lowering activity, and a preparation method and application thereof. Background Art

[0002] The increasing prevalence of overweight and obesity, particularly driven by modern lifestyles and dietary changes, poses a serious threat to global health. Obesity is a complex metabolic disorder closely associated with excessive lipid accumulation in the body. This disorder not only contributes to cardiovascular diseases such as atherosclerosis, hyperlipidemia, and coronary heart disease, but also contributes to chronic conditions such as hypertension, type 2 diabetes, and non-alcoholic fatty liver disease.

[0003] Pancreatic lipase (PL), an enzyme secreted by the pancreas, plays a key role in the digestion of dietary fat, accounting for 50–70% of lipolysis in the human body. Therefore, targeted inhibition of PL has emerged as a promising therapeutic approach for the treatment of obesity-related lipid metabolism disorders. However, orlistat, the only PL inhibitor approved by the US Food and Drug Administration, often causes gastrointestinal discomfort, liver toxicity, and cardiovascular complications with long-term use. Therefore, there is an urgent need for safer and more effective natural compounds that can act as potent PL inhibitors without significant side effects.

[0004] In recent years, plant-derived polyphenolic compounds, such as syringic acid (SA), ferulic acid (FA), and caffeic acid (CA), have garnered significant attention due to their lipid-lowering properties. They inhibit fatty acid synthase (FAS), thereby inhibiting endogenous fatty acid biosynthesis, while activating acetyl-CoA oxidase (ACO), promoting β-oxidation of fatty acids and enhancing lipid catabolism in adipose tissue. Despite their potential as druggable compounds, polyphenolic acids are limited by their poor water solubility, resulting in poor bioavailability and pharmacokinetic parameters in humans, hindering their in-depth medicinal chemistry and clinical research.

[0005] Conjugating bioactive compounds with sugar molecules can improve their solubility, bioavailability, and targeted delivery. Currently, the methods for conjugating bioactive compounds with sugar molecules reported in domestic and international patents (Chinese patent applications CN118666924A, CN118955589A, and CN114790472A; International patent application US2010256345A1) primarily rely on traditional chemical synthesis and enzymatic methods. The traditional chemical synthesis method primarily involves glycosylation through four steps: protection of the phenolic hydroxyl group, introduction of the sugar group, removal of the acetyl group, and deprotection of the phenolic hydroxyl group. The enzymatic method, on the other hand, involves combining a pretreated glycosyl donor, the bioactive compound, and a selected enzyme in an optimized reaction system. The enzyme specifically recognizes specific sites on the glycosyl donor and the bioactive compound, catalyzing the transfer of the sugar groups from the glycosyl donor to the bioactive compound to form a glycosylated product. While existing methods enable the efficient synthesis of phenolic acid-sugar conjugates, these glycoside compounds are susceptible to enzymatic degradation and the acidic environment of the digestive tract, resulting in hydrolysis and reduction to the original phenolic acid. This degradation process results in low solubility and poor bioavailability of the target products, a problem that remains unresolved. Therefore, improving the solubility and bioavailability of phenolic acid compounds through structural modification has become a key technical bottleneck that needs to be overcome to fully realize their lipid-lowering effects. Summary of the Invention

[0006] To address the above problems, the present invention provides a phenolic acid-sugar conjugate with lipid-lowering activity, a preparation method thereof, and an application thereof. The present invention significantly improves the technical problems of low water solubility and poor bioavailability of natural phenolic acids. The phenolic acid-sugar conjugate obtained by this method exhibits excellent lipid-lowering activity as tested by PL inhibition rate, cholesterol micelle binding ability, and bile salt binding rate.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a phenolic acid-sugar conjugate having lipid-lowering activity. The structure of the phenolic acid-sugar conjugate is as follows:

[0009]

[0010] wherein R is hydrogen or β-galactosyl.

[0011] The present invention also provides the use of the phenolic acid-sugar conjugate described in the above technical solution in the preparation of lipid-lowering drugs.

[0012] Preferably, the lipid-lowering activity is enhanced by increasing the PL inhibition rate of the phenolic acid-sugar conjugate.

[0013] Preferably, the lipid-lowering activity is enhanced by increasing the cholesterol micelle binding capacity and bile salt binding capacity of the phenolic acid-sugar conjugate.

[0014] The present invention also provides the use of the phenolic acid-sugar conjugate described in the above technical solution in improving the water solubility of phenolic acid.

[0015] The present invention also provides a method for preparing a phenolic acid-sugar conjugate having lipid-lowering activity, comprising the following steps:

[0016] 1) Compound 1, acetone, potassium carbonate, 18-crown ether-6, and propargyl bromide were mixed and reacted, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined;

[0017] The organic phase is dried, filtered, concentrated and recrystallized in sequence to obtain compound 2;

[0018] The structural formula of the compound 1 is as follows, wherein R 1 is methyl, ethyl or isopropyl;

[0019]

[0020] The structural formula of the compound 2 is as follows:

[0021]

[0022] 2) Compound 3 was mixed with trimethylsilyl azide and tin chloride and reacted, the aqueous phase was extracted with dichloromethane, and the organic phases were combined;

[0023] The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 4;

[0024] The structural formula of the compound 3 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0025]

[0026] The structural formula of the compound 4 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0027]

[0028] 3) mixing the compound 4 obtained in step 2) with a mixed solvent, the compound 2 obtained in step 1), copper sulfate, and sodium L-ascorbic acid, and reacting the mixture; extracting the aqueous phase with ethyl acetate; and combining the organic phases;

[0029] The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 5;

[0030] The structural formula of the compound 5 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0031]

[0032] 4) mixing the compound 5 obtained in step 3) with a mixed solvent and sodium hydroxide and reacting the mixture to obtain a reactant;

[0033] The reactants were acidified with a hydrochloric acid solution until a white precipitate was formed, the suspension was collected, the suspension was filtered, the solid was collected, and the solid was dried to obtain a phenolic acid-sugar conjugate.

[0034] Preferably, in step 1), the mass of compound 1, the volume of acetone, the mass of potassium carbonate, the mass of 18-crown ether-6, and the volume ratio of propynyl bromide are 5.89 g:93 mL:9.6 g:506 mg:6 mL;

[0035] The reaction conditions include: stirring the reaction mixture at 60° C. for 3 h;

[0036] Drying is done using sodium sulfate;

[0037] The filtering conditions include: vacuum filtration;

[0038] The recrystallization conditions include: a mixed solvent of ethyl acetate and petroleum ether, 80° C.; ethanol, 100° C.;

[0039] The volume ratio of the ethyl acetate to petroleum ether is 1:1.

[0040] Preferably, the mass ratio of compound 3 to trimethylsilyl azide and tin chloride in step 2) is 3.90:3.41:7.81;

[0041] The reaction conditions include: stirring in an ice bath for 1 h;

[0042] Drying is done using sodium sulfate;

[0043] The filtering conditions include: vacuum filtration;

[0044] The elution solvents for the silica gel column chromatography purification are petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 1:1.

[0045] Preferably, in step 3), the mass of compound 4 and the volume of the mixed solvent, the mass of compound 2, the mass of copper sulfate, and the mass of sodium L-ascorbic acid salt are 1.3 g:160 mL:0.74 g:0.13 g:0.31 g;

[0046] The mixed solvent is dimethylformamide and water, and the volume ratio of dimethylformamide to water is 6:1;

[0047] The reaction conditions include: stirring at 18°C for 8h;

[0048] Drying with sodium sulfate;

[0049] The filtering conditions include: vacuum filtration;

[0050] The elution solvents for the silica gel column chromatography purification are petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 1:1.

[0051] Preferably, in step 4), the mass of compound 5, the volume of the mixed solvent, and the mass of sodium hydroxide are 7.7 g:124 mL:1.48 g;

[0052] The mixed solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 4:1;

[0053] The reaction conditions include: stirring at 18°C for 12h;

[0054] The concentration of the hydrochloric acid solution is 1 mol / L;

[0055] The drying conditions include: temperature of 40° C. and time of 12 hours.

[0056] The invention activates the anomeric acetyl group of readily available sugar acetate under acidic conditions to synthesize azido sugar, and reacts it with the propargyl group of protected phenolic acid through a click chemistry reaction under copper catalysis to obtain triazole glycoside, which is then deprotected under alkaline conditions to obtain the desired sugar conjugate.

[0057] Beneficial effects of the present invention:

[0058] (1) The synthesis process has few steps and the total yield of the product is high (the total yield of compound 6 is ≥75%);

[0059] (2) The reaction conditions of the synthesis process are mild, and there is no need to react under high temperature and high pressure, which is conducive to large-scale production;

[0060] (3) The performance of the synthesized product is greatly improved compared with that before synthesis, which provides a reliable research method for improving the bioavailability of natural polyphenols and the development of natural lipid-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0062] Figure 1 The synthetic route of the phenolic acid-sugar conjugate of the present invention is shown in FIG. DETAILED DESCRIPTION

[0063] The present invention provides a phenolic acid-sugar conjugate having lipid-lowering activity. The structure of the phenolic acid-sugar conjugate is as follows:

[0064]

[0065] Wherein R is acetyl or tetraacetyl-β-galactosyl.

[0066] The present invention also provides the use of the phenolic acid-sugar conjugate described in the above technical solution in the preparation of a lipid-lowering drug. The present invention preferably enhances the lipid-lowering activity by increasing the PL inhibition rate of the phenolic acid-sugar conjugate. The present invention preferably enhances the lipid-lowering activity by increasing the cholesterol micelle binding capacity and bile salt binding capacity of the phenolic acid-sugar conjugate.

[0067] The present invention also provides the use of the phenolic acid-sugar conjugate described in the above technical solution in improving the water solubility of phenolic acid.

[0068] The present invention provides a method for preparing a phenolic acid-sugar conjugate having lipid-lowering activity, comprising the following steps:

[0069] 1) Compound 1, acetone, potassium carbonate, 18-crown ether-6, and propargyl bromide were mixed and reacted, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined;

[0070] The organic phase is dried, filtered, concentrated and recrystallized in sequence to obtain compound 2;

[0071] The structural formula of the compound 1 is as follows, wherein R 1 is methyl, ethyl or isopropyl;

[0072]

[0073] The structural formula of the compound 2 is as follows:

[0074]

[0075] 2) Compound 3 was mixed with trimethyl azide and tin chloride and reacted, the aqueous phase was extracted with dichloromethane, and the organic phases were combined;

[0076] The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 4;

[0077] The structural formula of the compound 3 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0078]

[0079] The structural formula of the compound 4 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0080]

[0081] 3) mixing the compound 4 obtained in step 2) with a mixed solvent, the compound 2 obtained in step 1), copper sulfate, and sodium L-ascorbic acid, and reacting the mixture; extracting the aqueous phase with ethyl acetate; and combining the organic phases;

[0082] The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 5;

[0083] The structural formula of the compound 5 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl;

[0084]

[0085] 4) mixing the compound 5 obtained in step 3) with a mixed solvent and sodium hydroxide and reacting the mixture to obtain a reactant;

[0086] The reactants were acidified with a hydrochloric acid solution until a white precipitate was formed, the suspension was collected, the suspension was filtered, the solid was collected, and the solid was dried to obtain a phenolic acid-sugar conjugate.

[0087] In the present invention, compound 1, acetone, potassium carbonate, 18-crown-6, and propargyl bromide are mixed and subjected to a propargyl etherification reaction. The aqueous phase is extracted with ethyl acetate, and the organic phases are combined. The organic phases are sequentially dried, filtered, concentrated, and recrystallized to obtain compound 2. In the present invention, the mass ratio of compound 1, volume of acetone, mass of potassium carbonate, mass of 18-crown-6, and volume of propargyl bromide is preferably 5.89 g:93 mL:9.6 g:506 mg:6 mL. In the present invention, the reaction conditions preferably include stirring the reaction mixture at 60°C for 3 hours. Sodium sulfate is preferably used for drying. In the present invention, the filtration conditions preferably include filtering with filter paper followed by vacuum filtration. In the present invention, the recrystallization conditions preferably include a mixed solvent of ethyl acetate and petroleum ether at 80°C; ethanol at 100°C; and the volume ratio of ethyl acetate to petroleum ether is preferably 1:1.

[0088] In the present invention, compound 3 is mixed with trimethylsilyl azide and tin chloride, followed by an azidation reaction. The aqueous phase is extracted with dichloromethane, and the organic phases are combined; the organic phases are sequentially dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 4. In the present invention, the mass ratio of compound 3 to trimethylsilyl azide and tin chloride is preferably 3.90:3.41:7.81. In the present invention, the reaction conditions preferably include: stirring the reaction mixture in an ice bath for 1 hour. In the present invention, sodium sulfate is preferably used for drying. In the present invention, the filtration conditions preferably include: filtering with filter paper followed by vacuum filtration. In the present invention, the elution solvents for the silica gel column chromatography purification are preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 1:1. The present invention does not specifically limit other conditions for the silica gel column chromatography purification; conventional methods can be used.

[0089] In the present invention, the obtained compound 4 is mixed with a mixed solvent, compound 2, copper sulfate, and sodium L-ascorbic acid, followed by a click chemistry reaction. The aqueous phase is extracted with ethyl acetate, and the organic phases are combined. The organic phases are sequentially dried, filtered, concentrated, and purified by silica gel column chromatography to yield compound 5. In the present invention, the mass of compound 4 to the volume of the mixed solvent, the mass of compound 2, the mass of copper sulfate, and the mass of sodium L-ascorbic acid are preferably 1.3 g:160 mL:0.74 g:0.13 g:0.31 g. In the present invention, the mixed solvent is preferably dimethylformamide and water, with the volume ratio of dimethylformamide to water being preferably 6:1. In the present invention, the reaction conditions preferably include stirring the reaction mixture at 18°C for 8 hours. Sodium sulfate is preferably used for drying. In the present invention, the filtration conditions preferably include filtering with filter paper followed by vacuum filtration. In the present invention, the eluting solvent for the silica gel column chromatography purification is preferably petroleum ether and ethyl acetate, with the volume ratio of petroleum ether to ethyl acetate being preferably 1:1. The present invention has no particular limitation on other conditions for silica gel column chromatography purification, and conventional conditions may be used.

[0090] In the present invention, the obtained compound 5 is mixed with a mixed solvent and sodium hydroxide and reacted to obtain a reactant; the reactant is acidified with a hydrochloric acid solution until a white precipitate is formed, the suspension is collected, the suspension is filtered, the solid is collected, and the solid is dried to obtain a phenolic acid-sugar conjugate. In the present invention, the mass of the compound 5, the volume of the mixed solvent, and the mass of the sodium hydroxide are preferably 7.7 g:124 mL:1.48 g. In the present invention, the mixed solvent is preferably water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is preferably 4:1. In the present invention, the reaction conditions preferably include: stirring the reaction mixture at 18°C for 12 hours. In the present invention, the concentration of the hydrochloric acid solution is preferably 1 mol / L. In the present invention, the drying conditions preferably include: a temperature of 40°C and a time of 12 hours.

[0091] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0092] Example 1

[0093] Preparation of compound 1:

[0094] To a solution of syringic acid (5.15 g, 28.3 mmol, 1 equiv) in MeOH (100 mL) was added concentrated sulfuric acid (1 mL, 18.4 mmol, 0.65 equiv) dropwise under an ice bath. After the addition was complete, the ice bath was removed and the mixture was heated to reflux in an oil bath. After reflux for 12 hours, the reaction mixture was cooled to room temperature. The mixture was poured into water, and the aqueous phase was extracted with CH2Cl2. The combined organic layers were washed sequentially with saturated NaHCO3 solution, water, and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated by air separation.

[0095] The structure of compound 1 is as follows, R 1 For methyl:

[0096]

[0097] Preparation and data of compound 2:

[0098] To a solution of compound 1 (5.89 g, 27.77 mmol, 1.0 equiv) in 93 mL of acetone were added KCO (9.60 g, 69.44 mmol, 2.5 equiv), 18-crown-6 (506.0 mg, 1.39 mmol, 0.05 equiv), and propargyl bromide (6.0 mL, 69.44 mmol, 2.5 equiv). The reaction mixture was stirred at 60°C for 3 h, after which the aqueous phase was extracted with ethyl acetate (EtOAc). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was recrystallized to afford compound 2 (6.46 g, 25.83 mmol, 93% yield) as a white crystalline solid. Recrystallization was performed using a mixture of ethyl acetate and petroleum ether in a 4:1 volume ratio at 80°C.

[0099] The structure of compound 2 is as follows:

[0100]

[0101] Other analysis data are as follows:

[0102] 1 H NMR (400MHz, CDCl3) δ7.27 (s, 2H), 4.77 (d, J = 2.5 Hz, 2 H), 3.88(s, 9 H), 2.42 (t, J = 2.4 Hz, 1 H).

[0103] 13 C NMR (101MHz, CDCl3) δ166.5, 153.1, 139.3, 125.8, 106.6, 78.8, 75.1, 59.8, 56.2, 52.2.

[0104] Example 2

[0105] Preparation and data of compound 4a:

[0106] The structure of compound 3a is as follows, where R is an acetyl group:

[0107]

[0108] To a solution of 3a (3.903 g, 10 mmol, 1 equiv) in anhydrous CH2Cl2 (50 mL) were added trimethylsilyl azide (TMSN3) (3.94 mL, 30 mmol, 3 equiv) and tin chloride (3.51 mL, 30 mmol, 3 equiv) dropwise. The reaction mixture was stirred in an ice bath for 1 hour and then poured into water. The aqueous phase was extracted with CH2Cl2. The combined organic layers were washed sequentially with saturated NaHCO3 solution, water, and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated by air separation. The residue was purified by silica gel column chromatography (hexane:EtOAc = 3:1) to yield the target compound 4a (9.92 g, 15.92 mmol, 91%) as a clear gum.

[0109] 3a is glucose pentaacetate purchased from Anaiji Chemical; CAS number: 604-68-2; product number: D100006-50g.

[0110] The structure of compound 4a is as follows:

[0111]

[0112] Other analysis data are as follows:

[0113] 1 H NMR (400MHz, CDCl3) δ7.92 (s, 1 H), 7.27 (d, J = 6.9 Hz, 3 H), 5.88 (d, J = 8.9 Hz, 1 H), 5.48-5.38(m, 2H), 5.30-5.18(m, 3 H), 4.28 (dd, J=12.6, 5.0Hz, 1 H), 4.12 (dd, J=12.6, 2.1Hz, 1 H), 4.01-3.97(m, 1 H), 3.89(s, 3 H), 3.86(s, 6 H), 2.06 (d, J = 5.0 Hz, 6 H), 2.02(s, 3 H), 1.84(s, 3 H).

[0114] 13 C NMR (101MHz, CDCl3) δ170.4, 169.8, 169.3, 168.8, 166.5, 152.9, 145.4, 140.0, 125.6, 12 1.4, 106.5, 85.5, 74.9, 72.7, 70.1, 67.5, 66.0, 61.4, 56.1, 52.2, 20.6, 20.4, 20.4, 20.1.

[0115] Example 3

[0116] The structure of compound 3b is as follows, where R is an acetyl group:

[0117]

[0118] Preparation and data of compound 4b:

[0119] To a solution of 3b (29.9433 g, 10 mmol, 1 equiv) in anhydrous CH2Cl2 (50 mL) were added trimethylsilyl azide TMSN3 (3.94 mL, 30 mmol, 3 equiv) and tin chloride (3.51 mL, 30 mmol, 3 equiv) dropwise. The reaction mixture was stirred in an ice bath for 1 hour and then poured into water. The aqueous phase was extracted with CH2Cl2. The combined organic layers were washed sequentially with saturated NaHCO3 solution, water, and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated by air separation. The residue was purified by silica gel column chromatography (hexane:EtOAc = 1:2) to yield the target compound 4b (3.5918 g, 8.4 mmol, 90%). Other analytical data are as follows:

[0120] 3b is lactose octaacetate purchased from Anaiji Chemical; CAS number: 6291-42-5; A02006330-10g.

[0121] The structure of compound 4b:

[0122]

[0123] 1 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1 H), 7.24 (s, 2 H), 5.81 (d, J = 8.6 Hz, 1 H), 5.44–5.36 (m, 2 H), 5.34 (d, J = 3.2 Hz, 1 H), 5.26–5.17 (m, 2 H), 5.11 (dd, J = 10.4, 7.9 Hz, 1 H), 4.95 (dd, J = 10.4, 3.4 Hz, 1 H), 4.50 (d, J = 7.9 Hz, 1 H), 4.45 (dd, J = 12.3, 1.7 Hz, 1 H), 4.16–4.03 (m, 3 H), 3.97–3.86 (m, 6 H), 3.84 (s, 6 H), 2.14 (s, 3 H), 2.08 (s, 3 H), 2.05 (s, 3 H), 2.04 (s, 3 H), 2.03 (s, 3 H), 1.95 (s, 3 H), 1.82 (s, 3 H).<00其0380>

[0124] 13 13C NMR (101 MHz, CDCl3) δ 170.26, 170.08, 170.01, 169.95, 169.42, 169.03, 168.97, 166.49, 152.92, 145.19, 139.98, 125.61, 121.46, 106.53, 101.01, 85.31, 77.20, 75.69, 75.55, 72.65, 70.79, 70.69, 70.27, 68.89, 66.46, 66.00, 61.67, 60.70, 56.06, 52.16, 20.65, 20.61, 20.56, 20.54, 20.51, 20.40, 20.12.

[0125] Example 4

[0126] Preparation and data of compound 5a:

[0127] To a solution of compound 4a (6.00 g, 16.08 mmol, 1.0 equiv) in a DMF / H₂O mixture (160 mL, v / v = 6:1) were added compound 2 (4.83 g, 19.30 mmol, 1.2 equiv), CuSO₄ (1.03 g, 6.43 mmol, 0.4 equiv), and sodium L-ascorbic acid (2.55 g, 12.86 mmol, 0.8 equiv). After completion of the reaction, the mixture was filtered through Celite, and the filtrate was concentrated. The aqueous phase was then extracted with ethyl acetate, and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated by air separation. The residue was purified by silica gel column chromatography using PE:EA (1:1) as the eluent to obtain the target compound 5a (9.92 g, 15.92 mmol, 99%) as a clear, gelatinous substance.

[0128] The structure of compound 5a is as follows, where R is an acetyl group:

[0129]

[0130] Other analysis data are as follows:

[0131] 1 H NMR (400MHz, CDCl3) δ7.92 (s, 1 H), 7.27 (d, J = 6.9 Hz, 3 H), 5.88 (d, J = 8.9 Hz, 1 H), 5.48-5.38(m, 2 H), 5.30-5.18(m, 3 H), 4.28 (dd, J=12.6, 5.0Hz, 1 H), 4.12 (dd, J=12.6, 2.1Hz, 1 H), 4.01-3.97(m, 1 H), 3.89(s, 3 H), 3.86(s, 6 H), 2.06 (d, J = 5.0 Hz, 6 H), 2.02(s, 3 H), 1.84(s, 3 H).

[0132] 13C NMR (101MHz, CDCl3) δ170.4, 169.8, 169.3, 168.8, 166.5, 152.9, 145.4, 140.0, 125.6, 12 1.4, 106.5, 85.5, 74.9, 72.7, 70.1, 67.5, 66.0, 61.4, 56.1, 52.2, 20.6, 20.4, 20.4, 20.1.

[0133] Example 5

[0134] Preparation and data of compound 5b:

[0135] To a solution of compound 4b (1.30 g, 1.97 mmol, 1.0 equiv) in a DMF / H₂O mixture (160 mL, v / v = 6:1) were added compound 2 (0.74 g, 2.95 mmol, 1.5 equiv), CuSO₄ (0.13 g, 0.79 mmol, 0.4 equiv), and sodium L-ascorbic acid (0.31 g, 1.58 mmol, 0.8 equiv). After completion of the reaction, the mixture was filtered through Celite, and the filtrate was concentrated. The aqueous phase was then extracted with ethyl acetate, and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated by air separation. The residue was purified by silica gel column chromatography using PE:EA (1:1) as the eluent to obtain the target compound 5b (1.65 g, 1.81 mmol, 92%) as a white powder.

[0136] The structure of compound 5b, R is acetyl:

[0137]

[0138] Other analysis data are as follows:

[0139] 1 H NMR (400MHz, CDCl3) δ7.83 (s, 1 H), 7.24(s, 2 H), 5.81 (d, J = 8.6 Hz, 1 H), 5.44-5.36(m, 2 H), 5.34 (d, J = 3.2 Hz, 1 H), 5.26-5.17(m, 2 H), 5.11 (dd, J=10.4, 7.9Hz, 1 H), 4.95 (dd, J=10.4, 3.4Hz, 1 H), 4.50 (d, J = 7.9 Hz, 1 H), 4.45 (dd, J=12.3, 1.7Hz,1 H), 4.16-4.03(m, 3 H), 3.97-3.86(m, 6 H), 3.84(s, 6 H), 2.14(s, 3 H), 2.08(s, 3 H), 2.05(s, 3 H), 2.04(s, 3 H), 2.03(s, 3 H), 1.95(s, 3 H), 1.82(s, 3 H).

[0140] 13 C NMR (101MHz, CDCl3) δ170.26, 170.08, 170.01, 169.95, 169.42, 169.03, 168 .97, 166.49, 152.92, 145.19, 139.98, 125.61, 121.46, 106.53, 101.01, 85.3 1, 77.20, 75.69, 75.55, 72.65, 70.79, 70.69, 70.27, 68.89, 66.46, 66.00, 61.67, 60.70, 56.06, 52.16, 20.65, 20.61, 20.56, 20.54, 20.51, 20.40, 20.12.

[0141] Example 6

[0142] Preparation and data of compound 6a:

[0143] To a solution of compound 5a (7.70 g, 12.36 mmol, 1 equiv) in a mixed solvent of HO / THF (124 mL, v / v = 4:1) was added NaOH (1.48 g, 37.08 mmol, 3 equiv). The reaction mixture was stirred at 18°C for 12 h, then acidified with 1 M HCl solution until a white precipitate was observed. The resulting suspension was then filtered under vacuum, and the collected solid was thoroughly washed with deionized water. Subsequently, the product was dried in a vacuum oven for 12 h to afford the target compound 6a (4.92 g, 11.15 mmol, 90%) as a white powder.

[0144] The structure of compound 6a, R is acetyl:

[0145]

[0146] Other analytical data are as follows: [α]23D = -8.8 (c = 0.09, MeOH);

[0147] 1 H NMR (400MHz, CD3OD) δ8.16 (s, 1 H), 7.25(s, 2 H), 5.56 (d, J = 9.2 Hz, 1 H), 5.10(s, 2 H), 3.90-3.80(m, 2 H), 3.78(s, 6 H), 3.66 (dd, J=12.2, 5.3Hz, 1 H), 3.57-3.42(m, 3 H);

[0148] 13 C NMR (101MHz, MeOD) δ169.4, 154.6, 145.4, 141.3, 127.7, 125.1, 107.9, 89.5, 81.1, 78.5, 74.0, 70.8, 66.4, 62.4, 56.6;

[0149] HRMS(ESI):m / z calcd for C18H22N3O10[MH]–440.1311, found440.1314.

[0150] Example 7

[0151] Preparation and data of compound 6b:

[0152] To a solution of compound 5b (968.2 mg, 1.06 mmol, 1 equiv) in a mixed solvent of HO / THF (124 mL, v / v = 4:1) was added NaOH (425.0 mg, 10.62 mmol, 10 equiv). The reaction mixture was stirred at 18°C for 12 h, then acidified with 1M HCl solution until a white precipitate was observed. The resulting suspension was then filtered under vacuum, and the collected solid was thoroughly washed with deionized water. Subsequently, the product was dried in a vacuum oven for 12 h to afford the target compound 6b (622.3 mg, 1.03 mmol, 97%) as a white powder.

[0153] The structure of compound 6b, R is hydrogen:

[0154]

[0155] Other analytical data are as follows: [α]23D = +20.4 (c = 0.17, H2O);

[0156] 1 H NMR (400MHz, DMSO-d6) δ8.30 (s, 1 H), 7.19(s, 2 H), 5.61 (d, J = 9.3 Hz, 1 H), 5.53 (d, J = 6.1 Hz, 1 H), 5.07 (d, J = 19.2 Hz, 1 H), 5.01(s, 2 H), 4.86(s, 1 H)、4.69-4.60(m, 2 H), 4.51(s, 1 H), 4.23 (d, J = 6.9 Hz, 1 H)、3.88-3.73(m, 8 H)、3.63-3.43(m, 9 H)、3.38-3.34(m, 3 H);

[0157] 13 C NMR (101MHz, DMSO-d6) δ167.67, 153.20, 143.74, 139.84, 127.72, 124.24, 106.89, 104.26, 8 7.41, 80.20, 78.22, 76.06, 75.69, 73.71, 72.21, 71.04, 68.60, 65.75, 60.88, 60.51, 56.41;

[0158] HRMS(ESI):m / z calcd for C24H32N3O15[MH]–602.1839, found602.1943.

[0159] Example 8

[0160] Preparation of compound 7a:

[0161] Compound 6a was added to a dry reaction flask and dissolved in water. Sodium hydroxide was then added to the aqueous solution of compound 6a in a 1:1 mass ratio. The reaction was stirred at room temperature for 30 minutes. After the reaction, the reaction solution was concentrated to obtain a white solid, which was then dried to obtain the sodium salt 7a.

[0162] The structure of compound 7a, R is hydrogen:

[0163]

[0164] Example 9

[0165] Preparation of compound 7b:

[0166] Compound 6b was added to a dry reaction flask and dissolved in water. Sodium hydroxide was then added to the 6b aqueous solution in a 1:1 mass ratio of compound 6b to sodium hydroxide. The reaction was stirred at room temperature for 30 minutes. After the reaction, the reaction mixture was concentrated to obtain a white solid. The reaction mixture was dried to obtain salt 7b.

[0167] The structure of compound 7b, R is hydrogen:

[0168]

[0169] Comparative Example 1

[0170] Unglycosylated syringic acid.

[0171] Comparative Example 2

[0172] Orlistat is the only PL inhibitor approved by the US Food and Drug Administration.

[0173] Effect Example 1

[0174] Evaluation of the solubility and hypolipidemic activity of target compounds

[0175] (1) Solubility determination:

[0176] Accurately weigh 0.20 g each of the target compounds prepared in Examples 6, 7, 8, and 9, and Comparative Example 1, and add them to 10 mL of deionized water. Stir the mixture at room temperature for 2 hours, then centrifuge. Discard the supernatant, freeze-dry the precipitate, and weigh it. The water solubility is calculated as (0.200 g - weight of the precipitate) / 0.200 g × 100%.

[0177] Table 1 Solubility of different sugar-phenolic acid conjugates

[0178] sample Solubility (%) Example 6 97.28％ Example 7 96.27％ Example 8 100％ Example 9 100％ Comparative Example 1 10.64％

[0179] The solubility results are shown in Table 1. The solubility of Comparative Example 1 in water at room temperature was approximately 10.64%, while the solubility of Examples 6, 7, 8, and 9 was significantly improved. These results demonstrate that glycosylation effectively increases the water solubility of phenolic acids. Furthermore, the solubility of Examples 8 and 9 was significantly better than that of Examples 6 and 7, likely due to the presence of sodium, which enhances electrostatic interactions with water molecules, further improving solubility.

[0180] (2) Evaluation of lipid-lowering activity:

[0181] PL inhibition rate: 50 μL of the target compounds prepared in Examples 6, 7, 8, and 9 and the solutions of Comparative Examples 1 and 2 at a concentration of 10 mg / mL, 50 μL of 50 mmol / L PBS (pH 8.0), and 50 μL of 10 mg / mL PL solution were added to each well. The mixture was incubated at 37°C for 10 minutes to allow the enzyme to interact with the ligand. Then, 50 μL of 1 mmol / L 4-nitrobenzene laurate substrate solution was added to initiate the reaction. The reaction was incubated at 37°C in the dark for 120 minutes to ensure the interaction between the enzyme and the substrate. The absorbance was measured at a wavelength of 405 nm using a microplate reader to monitor the hydrolysis of the substrate. The calculation formula for the PL inhibition rate is as follows: PL inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100%

[0182] Wherein, A1 is the absorbance (OD value) of the reaction system containing sample and PL; A2 is the OD value of the reaction system containing sample but no PL; A3 is the OD value of the reaction system containing normal PL; A4 is the OD value of the reaction system containing neither sample nor PL.

[0183] Table 2 PL inhibition rate, cholesterol micelle binding capacity and bile salt binding capacity of different sugar-phenolic acid conjugates

[0184]

[0185] The results of the PL inhibition assay are shown in Table 2. Comparative Example 1 exhibited a 57.40% inhibition rate for PL activity at 10 mg / mL. Examples 6 and 7 exhibited significantly higher inhibition rates than Comparative Example 1. The high inhibition rate of 90.17% achieved by Example 6 is comparable to that of Comparative Example 2, highlighting the feasibility of glycosylation-modified phenolic acids. On the other hand, despite their high water solubility, Examples 8 and 9 exhibited significantly reduced PL inhibition, to 33.5% and 31.9%, respectively. These results suggest that ionic modification may be detrimental to PL inhibitory activity and demonstrate the crucial role of the carboxyl group.

[0186] Cholesterol micelle binding capacity: Oleic acid, taurocholate, cholesterol, PBS (pH 7.4), and sodium chloride were mixed to prepare a cholesterol micelle solution, which was then sonicated for 5 minutes. 5 mL each of the target compounds prepared in Examples 6, 7, 8, and 9, and the solution in Comparative Example 1, at a concentration of 10 mg / mL, was mixed with 3 mL of the cholesterol micelle solution and incubated with shaking at 37°C for 2 hours. The mixture was then incubated at 37°C for 12 hours. The supernatant was collected by centrifugation, and the cholesterol content was determined using a total cholesterol kit.

[0187] Bile Salt Binding Rate: Prepare standard solutions of sodium taurocholate and sodium deoxycholate at concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mmol / L, respectively. Transfer a 2 mL aliquot of each standard solution to a test tube, and then add 6 mL of a 60% aqueous H2SO4 solution. Incubate the solution in a 70°C water bath for 20 minutes. After incubation, cool the solution in an ice bath for 5 minutes and measure the absorbance at 387 nm in a microplate reader. Based on the results, plot a calibration curve: y = 0.1004x + 0.1244, R 2 =0.9957 (sodium taurocholate); y = 0.1239x + 0.0142, R 2 =0.9999 (sodium deoxycholate).

[0188] 4 mL of each of the target compounds prepared in Examples 6, 7, 8, and 9 and the solution in Comparative Example 1 at a concentration of 10 mg / mL was mixed with 4 mL of a 1 mmol / L bile salt solution and incubated at 37°C with shaking for 1 hour. After completion of the incubation, the mixture was centrifuged, the supernatant collected, boiled, cooled in an ice bath, and analyzed at a wavelength of 387 nm. The bile salt binding rate was calculated as follows: Bile salt binding rate (%) = (S1 - S2) / S1 × 100%, where S1 is the total amount of bile salts initially added and S2 is the amount of bile salts remaining in the supernatant.

[0189] The results of cholesterol micelle binding and bile salt binding rate determinations are shown in Table 2. The target compounds prepared in Examples 6, 7, 8, and 9, as well as Comparative Example 1, all showed cholesterol micelle binding ability. The cholesterol micelle binding ability of Comparative Example 1 at a concentration of 10 mg / mL was 27.52%, comparable to that of Examples 8 and 9. However, Examples 6 and 7 exhibited significantly higher cholesterol binding abilities, at 52.62% and 49.21%, respectively, indicating that glycosylation plays a key role in enhancing the micelle binding activity of phenolic acids.

[0190] The target compounds prepared in Examples 6, 7, 8, and 9, as well as Comparative Example 1, all showed binding affinity to deoxycholate and taurocholate. Glycosylation significantly enhanced the binding ability of Examples 6 and 7 compared to Comparative Example 1. Interestingly, the binding ability of Examples 8 and 9 did not significantly increase compared to Comparative Example 1, suggesting that the carboxyl groups in these compounds altered their interactions with bile salts.

[0191] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A phenolic acid-sugar conjugate having lipid-lowering activity, characterized in that: The structure of the phenolic acid-sugar conjugate is as follows: wherein R is hydrogen or β-galactosyl.

2. Use of the phenolic acid-sugar conjugate according to claim 1 in the preparation of a lipid-lowering drug.

3. The use according to claim 2, characterized in that The lipid-lowering activity of phenolic acid-sugar conjugates was enhanced by increasing the PL inhibition rate.

4. The use according to claim 2, characterized in that The lipid-lowering activity is enhanced by improving the cholesterol micelle-binding and bile salt-binding abilities of phenolic acid-glycoconjugates.

5. Use of the phenolic acid-sugar conjugate according to claim 1 in improving the water solubility of phenolic acid.

6. A method for preparing a phenolic acid-sugar conjugate having lipid-lowering activity, characterized in that: The following steps are involved: 1) Compound 1, acetone, potassium carbonate, 18-crown ether-6, and propargyl bromide were mixed and reacted, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined; The organic phase is dried, filtered, concentrated and recrystallized in sequence to obtain compound 2; The structural formula of the compound 1 is as follows, wherein R 1 is methyl, ethyl or isopropyl; The structural formula of the compound 2 is as follows; 2) Compound 3 was mixed with trimethylsilyl azide and tin chloride and reacted, the aqueous phase was extracted with dichloromethane, and the organic phases were combined; The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 4; The structural formula of the compound 3 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl; The structural formula of the compound 4 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl; 3) mixing the compound 4 obtained in step 2) with a mixed solvent, the compound 2 obtained in step 1), copper sulfate, and sodium L-ascorbic acid, and reacting the mixture; extracting the aqueous phase with ethyl acetate; and combining the organic phases; The organic phase was dried, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 5; The structural formula of the compound 5 is as follows, wherein R is acetyl or tetraacetyl-β-galactosyl; 4) mixing the compound 5 obtained in step 3) with a mixed solvent and sodium hydroxide and reacting the mixture to obtain a reactant; The reactants were acidified with a hydrochloric acid solution until a white precipitate was formed, the suspension was collected, the suspension was filtered, the solid was collected, and the solid was dried to obtain a phenolic acid-sugar conjugate.

7. The preparation method according to claim 6, characterized in that In step 1), the mass ratio of compound 1, the volume of acetone, the mass of potassium carbonate, the mass of 18-crown ether-6, and the volume ratio of propargyl bromide is 5.89 g:93 mL:9.6 g:506 mg:6 mL; The reaction conditions include: stirring the reaction mixture at 60° C. for 3 h; Drying is done using sodium sulfate; The filtering conditions include: filtering with filter paper and then filtering under reduced pressure; The recrystallization conditions include: a mixed solvent of ethyl acetate and petroleum ether, 80° C.; ethanol, 100° C.; The volume ratio of the ethyl acetate to petroleum ether is 1:

1.

8. The preparation method according to claim 6, characterized in that In step 2), the mass ratio of compound 3 to trimethylsilyl azide and tin chloride is 3.90:3.41:7.81; The reaction conditions include: stirring in an ice bath for 1 h; Drying is done using sodium sulfate; The filtering conditions include: vacuum filtration; The elution solvents for the silica gel column chromatography purification are petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 1:

1.

9. The preparation method according to claim 6, characterized in that In step 3), the mass of compound 4, the volume of the mixed solvent, the mass of compound 2, the mass of copper sulfate, and the mass of L-ascorbic acid sodium salt are 1.3 g:160 mL:0.74 g:0.13 g:0.31 g; The mixed solvent is dimethylformamide and water, and the volume ratio of dimethylformamide to water is 6:1; The reaction conditions include: stirring at 18°C for 8h; Drying with sodium sulfate; The filtering conditions include: vacuum filtration; The elution solvents for the silica gel column chromatography purification are petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 1:

1.

10. The preparation method according to claim 6, characterized in that In step 4), the mass of compound 5, the volume of the mixed solvent, and the mass of sodium hydroxide are 7.7 g:124 mL:1.48 g; The mixed solvent is water and tetrahydrofuran, and the volume ratio of water to tetrahydrofuran is 4:1; The reaction conditions include: stirring at 18°C for 12h; The concentration of the hydrochloric acid solution is 1 mol / L; The drying conditions include: temperature of 40° C. and time of 12 hours.

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