Vanillic acid derivatives and preparation methods and applications thereof

Abstract

The invention discloses a series of vanillic acid derivatives, which are synthesized by vanillic acid derivatization. Through performance experiments, novel vanillic acid derivatives are obtained, which are ineffective against Candida albicans ATCC 36082 when used alone, but can double the effect when used in combination with amphotericin B or fluconazole. The novel vanillic acid derivatives have great potential as synergists for antibacterial drugs such as amphotericin B and fluconazole. The invention also discloses a preparation method and application of the vanillic acid derivatives.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial drugs, and in particular to vanillic acid derivatives and preparation methods and applications thereof. Background Art

[0002] Amphotericin B and fluconazole are broad-spectrum antifungal drugs of the polyene and triazole classes used to treat infections caused by Candida species. Amphotericin B binds to ergosterol (5,7-dienoxysterol) and alters the fluidity of fungal membranes, leading to plasmolysis and cell death. Azoles inhibit cell membrane synthesis by inhibiting the conversion of lanosterol to ergosterol. However, the limited therapeutic efficacy of amphotericin B due to dose-dependent nephrotoxicity and post-infusion reactions has hampered its clinical importance (Clinical Infectious Diseases, 2019, 68, S4: S260). Similarly, the development of resistance of Candida species to azole antifungal drugs has also limited treatment options. In addition, the physicochemical properties of antifungal drugs, including low water solubility and selectivity, reduce the efficacy of these drugs (AAPS PharmSciTech, 2022, 23, 6: 206). Exploring newly designed small molecule drug delivery and synergistic combination therapies may improve therapeutic efficacy and overcome drug delivery barriers. In a synergistic approach, the activity of a drug may be increased due to molecular interactions with synergistic molecules, and the combined effect of the two drugs may be greater than the sum of each drug molecule (Elife, 2020, 9: e54160). This application explores the synthesis of compounds that have antibacterial synergistic effects in combination with fluconazole and amphotericin B. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide a vanillic acid derivative, which greatly helps to improve the density and formation enthalpy through the introduction of nitro group, improves the high detonation characteristics of the energetic compound, and improves the stability of the energetic compound through the introduction of amino group; the introduction of coordinated oxygen effectively improves the density of the energetic compound.

[0005] Another object of the present invention is to provide a method for preparing vanillic acid derivatives, which starts from known available raw materials and realizes the synthesis of new vanillic acid derivatives through multi-step reactions, providing a new idea for synthesizing new high-energy density energetic compounds in energetic materials.

[0006] In order to achieve these purposes and other advantages according to the present invention, a vanillic acid derivative is provided, which has the structure of the following formula (I):

[0007]

[0008] Wherein, R is hydrogen or an alkyl group, and n is an even number less than 20.

[0009] Preferably, R is hydrogen and n is 12, 14, 16 or 18.

[0010] Preferably, R is methyl and n is 8, 10, 12, 14, 16 or 18.

[0011] Preferably, R is ethyl, n-propyl or isopropyl, and n is 2, 4, 6, 8, 10, 12, 14, 16 or 18.

[0012] Preferably, R is isopropyl and n=14.

[0013] The object of the present invention can be further achieved by a method for preparing a vanillic acid derivative, comprising: using vanillic acid as a raw material, obtaining the corresponding target compound through a methyl esterification reaction and a hydroxyalkylation reaction, or further an ester hydrolysis and esterification reaction under alkaline conditions.

[0014] Preferably, in the methyl esterification reaction of vanillic acid, p-toluenesulfonic acid is used as a catalyst, and the added molar amount thereof is 10% of the vanillic acid.

[0015] Preferably, the alkylating agent used in the hydroxyalkylation reaction is an alkyl bromide.

[0016] Preferably, the ester hydrolysis condition under alkaline conditions is 1 M sodium hydroxide aqueous solution.

[0017] The purpose of the present invention can be further achieved by using the vanillic acid derivative in the preparation of amphotericin B and fluconazole synergistic antibacterial drug synergist.

[0018] The present invention has at least the following beneficial effects:

[0019] 1. The present invention obtains a novel vanillic acid derivative which is ineffective against Candida albicans ATCC 36082 when used alone, but can double the effect when used in combination with amphotericin B or fluconazole. The novel vanillic acid derivative has great potential as a synergist of antibacterial drugs such as amphotericin B and fluconazole, and has positive significance for the research and development of synergistic antibacterial drugs.

[0020] 2. The preparation method of the present invention starts from the known available raw material vanillic acid and optimizes the synthesis route to achieve high-yield synthesis of vanillic acid derivatives, thereby providing a new idea for synthesizing vanillic acid derivatives.

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a time-killing curve of the compound 5g in Example 5 of the present invention in combination with fluconazole against Candida albicans ATCC 36082;

[0023] Figure 2 This is a time-killing curve of the compound 5g in Example 5 of the present invention in combination with amphotericin B against Candida albicans ATCC 36082;

[0024] Figure 3 It is the hemolytic toxicity graph of compound 5g, fluconazole, amphotericin B, fluconazole / 5g, and amphotericin B / 5g in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0027] <Example 1>

[0028] Compounds 3a-3i have the following general structural formulas:

[0029]

[0030] The specific synthetic route is as follows:

[0031]

[0032] The specific synthesis steps are as follows:

[0033] Compound 3: There are two preparation methods: method A and method B:

[0034] Method A: Thionyl chloride (0.86 mL, 11.9 mmol) was slowly added to ice-cooled methanol (25 mL) containing vanillic acid (1 g, 5.95 mmol) and triethylamine (1.24 mL, 8.9 mmol). After 48 hours, the reaction mixture was evaporated under reduced pressure, extracted with ethyl acetate (3×), and the organic layer was dried under reduced pressure and purified by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to give 0.64 g of methyl 4-hydroxy-3-methoxybenzoate (Compound 3).

[0035] Method B: A solution of vanillic acid (1 g, 5.95 mmol) in methanol (50 mL) was refluxed, and then p-toluenesulfonic acid (0.1 g, 0.59 mmol) was added. After 24 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate. The mixture was extracted with dichloromethane (3×), and the organic layer was evaporated to dryness under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1.05 g (>99%) of methyl 4-hydroxy-3-methoxybenzoate (3).

[0036] Methyl 4-hydroxy-3-methoxybenzoate (3) white solid; yield: 61% (method A), 91.8% (method B); 1 H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.3, 1.9Hz, 6-CH), 7.53 (d, 1H, J = 1.9Hz, 2-CH), 6 .92(d,1H,J=8.4Hz,5-CH),3.93(s,3H,9-CH3),3.87(s,3H,8-CH3); LR-EI-MS(m / z):182.1[M] + .

[0037] Methyl 4-ethoxy-3-methoxybenzoate (3a) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of bromoethane (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to afford 3a.

[0038] Methyl 4-ethoxy-3-methoxybenzoate (3a): white powder; yield: 78%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.4, 2.1Hz, 6-CH), 7.52 (d, 1H, J = 1.9Hz, 2-CH), 6.86 (d, 1H, J = 8.4Hz, 5-CH), 4.1 4(q,2H,J=6.8Hz,10-CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.47(t,2H,J=7.0Hz,11-CH); LR-EI-MS(m / z):210.1[M] + .

[0039] 4-Butoxy-3-methoxybenzoate 3b was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of bromobutyl (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to afford 3b.

[0040] 4-Butoxy-3-methoxybenzoic acid methyl ester (3b): white powder; yield: 78%; mp: 45-47°C; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.1Hz, 6-CH), 7.52 (d, 1H, J = 2 .0Hz,2-CH),6.85(d,1H,J=8.4Hz,5-CH),4.04(t,2H,J=6.8Hz,10-CH2),3.89( s,3H,9-CH3),3.87(s,3H,8-CH3),1.83(quin,2H,J=7.2Hz,11-CH2),1.48(sex t,2H,J=7.6Hz,12-CH2),1.83(t,3H,J=7.6,13-CH3); LR-EI-MS(m / z):238.1[M] + .

[0041] 4-Hexyloxy-3-methoxybenzoate 3c was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of bromohexane (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1), afforded 3c.

[0042] 4-Hexyloxy-3-methoxybenzoic acid methyl ester (3c): white powder; yield: 75%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40-1.20(overlapped,8H,11–14-CH2),0.86(t,3H,J=5.2Hz,15-CH3); LR-EI-MS(m / z):266.1[M] + .

[0043] Methyl 4-octyloxy-3-methoxybenzoate (3d) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of bromooctane (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×), and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to give 3d.

[0044] 4-Octyloxy-3-methoxybenzoic acid methyl ester (3d): white powder; yield: 74%; mp: 33-35°C; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J=6.8Hz,10-CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40-1.20(overlapped,12H,11–16-CH2),0.86(t,3H,J=5.2Hz,17-CH3), 13 CNMR (100MHz,CDCl3): 13C NMR (400MHz, CDCl3) δppm 166.9(C-7),152.6(C-4),148.8(C-3),123.5(C-6),122.3(C-1),112.3(C-2),111.4(C-5),111.4(C-5),69.0(C-10),56.0(C-9), 51.9(C-8),31.8,(C-11),29.3(C-12),29.2(C-13),29.0(C-14),25.9(C-15),22.6(C-16),14.1(C-17).LR-EI-MS(m / z):294.0[M] + ; HR-EI-MS (m / z) calcd.for C 17 H 26 O4[M] + :294.1831,found:294.1835.

[0045] Methyl 4-decyloxy-3-methoxybenzoate (3e) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of bromodecane (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1), afforded 3e.

[0046] 4-Decyloxy-3-methoxybenzoic acid methyl ester (3e): white powder; yield: 85%; mp: 35-37°C; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40–1.20(overlapped,16H,11–18-CH2),0.86(t,3H,J=6.4Hz,19-CH3); LR-EI-MS(m / z):322.3[M] + ; HR-EI-MS (m / z) calcd.for C 19 H 30 O4[M] +:322.2144,found:322.2145.

[0047] Methyl 4-dodecyloxy-3-methoxybenzoate (3f) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL) and then adding dodecane bromide (1.1 equivalents) dropwise. The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to give 3f.

[0048] 4-Dodecyloxy-3-methoxybenzoic acid methyl ester (3f): white powder; yield: 75%; mp: 40-42°C; 1 HNMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40–1.20(overlapped,20H,11–20-CH2),0.86(t,3H,J=6.4Hz,21-CH3); LR-EI-MS(m / z):350.2[M] + ; HR-EI-MS (m / z) calcd.for C 21 H 34 O4[M] + :350.2457,found:350.2449.

[0049] Methyl 4-tetradecyloxy-3-methoxybenzoate (3 g) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL) and then adding tetradecane bromide (1.1 equivalents) dropwise. The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to give 3 g.

[0050] 4-Tetradecyloxy-3-methoxybenzoic acid methyl ester (3g): white powder; yield: 72%; mp: 49-51°C; 1 HNMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40–1.20(overlapped,24H,11–22-CH2),0.86(t,3H,J=6.4Hz,23-CH3); LR-EI-MS(m / z):378.2[M] + ; HR-EI-MS (m / z) calcd.for C 23 H 38 O4[M] + :378.2770,found:378.2772.

[0051] Methyl 4-hexadecyloxy-3-methoxybenzoate (3h) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of hexadecane bromide (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification was performed by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 3h.

[0052] 4-Hexadecyloxy-3-methoxybenzoic acid methyl ester (3h): white powder; yield: 84%; mp: 56-58°C; 1 HNMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40–1.20(overlapped,28H,11–24-CH2),0.86(t,3H,J=6.4Hz,25-CH3); LR-EI-MS(m / z):406.2[M] +; HR-EI-MS (m / z) calcd.for C 25 H 42 O4[M] + :406.3083,found:406.3092.

[0053] Methyl 4-octadecyloxy-3-methoxybenzoate (3i) was prepared by treating methyl 4-hydroxy-3-methoxybenzoate (3) (0.1 g, 0.55 mmol) with a solution of potassium carbonate (0.15 g, 1.1 mmol) in acetone (5 mL), followed by the dropwise addition of octadecane bromide (1.1 equivalents). The reaction mixture was refluxed for 16-30 hours and monitored by TLC until all substrate was consumed. The cooled mixture was extracted with ethyl acetate (3×) and the solvent was dried under reduced pressure. Further purification by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1), afforded 3i.

[0054] 4-Octadecyloxy-3-methoxybenzoic acid methyl ester (3i): white powder; yield: 86%; mp: 60-62°C; 1 HNMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 4.04 (t, 2H, J = 6.8Hz, 10- CH2),3.89(s,3H,9-CH3),3.87(s,3H,8-CH3),1.40–1.20(overlapped,32H,11–26-CH2),0.86(t,3H,J=6.4Hz,27-CH3); LR-EI-MS(m / z):434.2[M] + ; HR-EI-MS (m / z) calcd.for C 27 H 46 O4[M] + :434.3396,found:434.3406.

[0055] <Example 2>

[0056] Compounds 1a-1i have the following structural formulas:

[0057]

[0058] The specific synthetic route is as follows:

[0059]

[0060] The specific synthesis steps are as follows:

[0061] There are two methods for preparing 4-ethoxy-3-methoxybenzoic acid (1a): Method A and Method B:

[0062] Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then ethyl bromide (1.1 equivalents) was added dropwise, and the reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. 1a was further purified by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent.

[0063] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3a (100 mg, 0.476 mmol) to deprotect methyl 4-ethoxy-3-methoxybenzoate (3a) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to give 1a in quantitative yield.

[0064] 4-Ethoxy-3-methoxybenzoic acid (1a): white powder; yield: 51.4% (method A), >99% (method B); 1 H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, J=8.3, 1.9Hz, 1H, H-6), 7.53 (d, J=1.9Hz, 1H, H-2), 6.91 (d, J=8.3Hz, 1 H,H-5),4.33(q,J=7.1Hz,2H,H-9),3.93(s,3H,H-8),1.36(t,J=7.1Hz,3H,H-10); LR-EI-MS(m / z):196.1[M] + .

[0065] 4-Butoxy-3-methoxybenzoic acid (1b) can be prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then bromobutyl (1.1 equivalents) was added dropwise. The reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1b.

[0066] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3b (100 mg, 0.420 mmol) to deprotect methyl 4-butoxy-3-methoxybenzoate (3b) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to give 1b in quantitative yield.

[0067] 4-Butoxy-3-methoxybenzoic acid (1b): white powder; yield: 60.0% (method A), >99% (method B); 1 H-NMR (400MHz, CDCl3) δ (ppm): 7.70 (dd, J=8.5, 2.0Hz, 1H, H-6), 7.56 (d, J=2.0Hz, 1H, H-2), 6.88 (d, J=8.5Hz, 1H, H-5), 4.07 (t, J=6.8 Hz,2H,H-9),3.90(s,3H,H-8),1.81(p,J=7.0Hz,2H,H-10),1.49(m,2H,H-11),0.97(t,J=7.4Hz,3H,H-12); LR-EI-MS(m / z):224.1[M] + .

[0068] 4-Hexyloxy-3-methoxybenzoic acid (1c) was prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then bromohexane (1.1 equivalents) was added dropwise. The reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. 1c was further purified by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1c.

[0069] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3c (100 mg, 0.376 mmol) to deprotect methyl 4-hexyloxy-3-methoxybenzoate (3c) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to afford 1c in quantitative yield.

[0070] 4-Hexyloxy-3-methoxybenzoic acid (1c): white powder; yield: 54.6% (method A), >99% (method B); 1HNMR (400MHz, CDCl3) δ (ppm): 7.72 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.57 (d, J=2.0Hz, 1H, H-2), 6.88 (d, J=8.5Hz, 1H, H-5), 4.07 (t, J=6.9Hz, 2H, H-9 ),3.91(s,3H,H-8),1.86(p,J=7.3Hz,2H,H-10),1.47–1.31(overlapped,6H,H-11–13),0.89(t,J=6.9Hz,3H,H-14); LR-EI-MS(m / z):252.1[M] + .

[0071] 4-Octyloxy-3-methoxybenzoic acid (1d) was prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then bromooctane (1.1 equivalents) was added dropwise. The reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to give 1d.

[0072] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3d (100 mg, 0.340 mmol) to deprotect methyl 4-octyloxy-3-methoxybenzoate (3d) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to afford 1d in quantitative yield.

[0073] 4-Octyloxy-3-methoxybenzoic acid (1d): white powder; yield: 52.9% (method A), >99% (method B); 1 HNMR (400MHz, CDCl3) δ (ppm): 7.73 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.58 (d, J=2.0Hz, 1H, H-2), 6.89 (d, J=8.6Hz, 1 H,H-5),4.07(t,J=6.9Hz,2H,H-9),3.91(s,3H,H-8),1.86(p,2H,J=7.0Hz,H-10),1.44(p,J=7.5Hz,2H ,H-11),1.37–1.19(overlapped,8H,H-12–15),0.87(t,J=6.7Hz,3H,H-116); LR-EI-MS(m / z):280.2[M] + .

[0074] 4-Decyloxy-3-methoxybenzoic acid (1e) was prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then bromodecane (1.1 equivalents) was added dropwise, and the reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1e.

[0075] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3e (100 mg, 0.310 mmol) to deprotect methyl 4-decyloxy-3-methoxybenzoate (3e) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to give 1e in quantitative yield.

[0076] 4-Decyloxy-3-methoxybenzoic acid (1e): white powder; yield: 54.56% (method A), >99% (method B); 1 H NMR (400MHz, CDCl3) δ (ppm): 7.72 (dd, J=8.0, 2.1Hz, 1H, H-6), 7.57 (d, J=2.0H z,1H,H-2),6.89(d,J=8.6Hz,1H,H-5),4.07(t,J=6.9Hz,2H,H-9),3.91(s,3H, H-8),1.86(p,J=7.0Hz,2H,H-10),1.44(p,J=7.2Hz,2H,H-11)1.36–1.24(over lapped,14H,H-12–17),0.86(t,J=6.7Hz,3H,H-18); LR-EI-MS(m / z):308.1[M] + .

[0077] 4-Dodecyloxy-3-methoxybenzoic acid (1f) was prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then dodecane bromide (1.1 equivalents) was added dropwise. The reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1f.

[0078] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3f (100 mg, 0.286 mmol) to deprotect methyl 4-dodecyloxy-3-methoxybenzoate (3f) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to afford 1f in quantitative yield.

[0079] 4-Dodecyloxy-3-methoxybenzoic acid (1f): white powder; yield: 40.1% (method A), >99% (method B); 1 H NMR (400MHz, CDCl3) δ (ppm): 7.72 (dd, J=8.0, 4.0Hz, 1H, H-6), 7.57 (s, 1H, H-2), 6.88 (d, J=8.0Hz, 1H, H-5), 4.06 (t, J=8.0Hz, 2H, H-9), 3.90 (s, 3H, H-8),1.86(p,J=8.0Hz,2H,H-10),1.44(m,2H,H-11)1.36–1.24(overlapped,16H,H-12–19),0.86(t,J=6.7Hz,3H,H-20); LR-EI-MS(m / z):336.2[M] + ; HR-EI-MS (m / z) calcd.for C 20 H 32 O4[M] + :336.2010,found:336.2289.

[0080] 4-Tetradecyloxy-3-methoxybenzoic acid (1 g) was prepared in two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 eq.) in ethanol (5 mL), and then tetradecane bromide (1.1 eq.) was added dropwise, and the reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1 g.

[0081] Method B: Methyl 4-tetradecyloxy-3-methoxybenzoate (3 g) was deprotected by alkaline hydrolysis of the ester by adding aqueous sodium hydroxide solution (1 M, 1 mL) dropwise to an ice-cooled solution of 3 g (100 mg, 0.264 mmol) of methanol. The reaction mixture was neutralized with 1 M hydrochloric acid to give 1 g in quantitative yield.

[0082] 4-Tetradecyloxy-3-methoxybenzoic acid (1 g): white powder; yield: 54.7% (method A), >99% (method B);1 H NMR (400MHz, CDCl3) δ (ppm): 7.72 (d, J = 8.0, 2.0Hz, 1H, H-6), 7.57 (s, 1H, H- 2),6.88(d,J=8.0Hz,1H,H-5),4.06(t,J=8.0Hz,2H,H-9),3.90(sd,3H,H-8) ,1.86(p,J=8.0Hz,2H,H-10),1.45(p,J=8.0Hz,2H,H-11)1.35–1.24(overlapped,20H,H-12–21),0.86(t,J=6.4Hz,3H,H-22); LR-EI-MS(m / z):364.1[M] + ; HR-EI-MS (m / z) calcd.for C 22 H 36 O4[M] + :364.2614,found:364.2611.

[0083] 4-Hexadecyloxy-3-methoxybenzoic acid (1h) was prepared in two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then hexadecane bromide (1.1 equivalents) was added dropwise, and the reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. 1h was further purified by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluents.

[0084] Method B: Methyl 4-hexadecyloxy-3-methoxybenzoate (3h) was deprotected by alkaline hydrolysis of the ester by adding aqueous sodium hydroxide solution (1M, 1 mL) dropwise to an ice-cooled methanol solution of 3h (100 mg, 0.246 mmol). The reaction mixture was neutralized with 1M hydrochloric acid to give 1h in quantitative yield.

[0085] 4-Hexadecyloxy-3-methoxybenzoic acid (1h): white powder; yield: 45.1% (method A), >99% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.72 (d, J = 8.0, 2.0Hz, 1H, H-6), 7.57 (d, J = 2.0Hz ,1H,H-2),6.88(d,J=8.8Hz,1H,H-5),4.06(t,J=6.8Hz,2H,H-9),3.90(s,3H,H -8),1.86(p,J=7.2Hz,2H,H-10),1.45(p,J=8.0Hz,2H,H-11)1.35–1.24(over lapped,24H,H-12–23),0.86(t,J=6.4Hz,3H,H-24); LR-EI-MS(m / z):392.1[M] + ; HR-EI-MS (m / z) calcd.forC 24 H 40 O4[M] + :392.2927,found:392.2918.

[0086] 4-Octadecyloxy-3-methoxybenzoic acid (1i) was prepared by two options: Method A and Method B. Method A: Vanillic acid (200 mg, 1.190 mmol) was refluxed with aqueous potassium hydroxide solution (2 equivalents) in ethanol (5 mL), and then octadecane bromide (1.1 equivalents) was added dropwise, and the reaction progress was monitored by TLC. After 8-12 hours, the cooled reaction mixture was extracted with ethyl acetate (3×), the organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 1i.

[0087] Method B: Aqueous sodium hydroxide solution (1 M, 1 mL) was added dropwise to an ice-cooled methanol solution of 3i (100 mg, 0.230 mmol) to deprotect methyl 4-octadecyloxy-3-methoxybenzoate (3i) by alkaline hydrolysis of the ester. The reaction mixture was neutralized with 1 M hydrochloric acid to afford 1i in quantitative yield.

[0088] 4-Octadecyloxy-3-methoxybenzoic acid (1i): white powder; yield: 47.2% (method A), >99% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.72 (d, J = 8.4, 2.0Hz, 1H, H-6), 7.57 (d, J = 2.0Hz ,1H,H-2),6.88(d,J=8.4Hz,1H,H-5),4.06(t,J=7.2Hz,2H,H-9),3.90(s,3H,H -8),1.86(p,J=7.2Hz,2H,H-10),1.45(p,J=8.0Hz,2H,H-11)1.48–1.23(over lapped,28H,H-12–25),0.86(t,J=6.4Hz,3H,H-26); LR-EI-MS(m / z):420.4[M] + ; HR-EI-MS (m / z) calcd.forC 26 H 44 O4[M] + :420.3240,found:420.3218.

[0089] <Example 3>

[0090] Compounds 2a-2i have the following structural formulas:

[0091]

[0092] The specific synthetic route is as follows:

[0093]

[0094] The specific synthesis steps are as follows:

[0095] Ethyl 4-ethoxy-3-methoxybenzoate (2a) was prepared by refluxing 1a (25 mg, 0.127 mmol) in ethanol and then adding a catalytic amount of sulfuric acid. After 24-36 hours, the reaction mixture was cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2a.

[0096] 4-Ethoxy-3-methoxybenzoic acid ethyl ester (2a): white powder; yield 56.8%, mp.: 45-47℃, 1HNMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.53 (d, J=2.0Hz, 1H, H-2), 6.85 (d, J=8.4Hz, 1H, H-5), 4.33 (q, J=7.1Hz, 2 H,H-9),4.14(q,J=7.0Hz,2H,H-11),3.90(s,3H,H-8),1.47(t,J=7.0Hz,3H,H-12),1.36(t,J=7.1Hz,3H,H-10); LR-EI-MS(m / z):224.1[M] + ; HR-EI-MS (m / z) calcd.for C 12 H 16 O4[M] + :224.1049,found:224.1050.

[0097] The preparation method of ethyl 4-butoxy-3-methoxybenzoate (2b) is as follows: 1b (25 mg, 0.112 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (volume ratio = 3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2b.

[0098] Ethyl 4-butoxy-3-methoxybenzoate (2b): colorless liquid; yield: 59.2%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.5, 2.0Hz, 1H, H-6), 7.52 (d, J=2.0H z,1H,H-2),6.85(d,J=8.5Hz,1H,H-5),4.33(q,J=7.1Hz,2H,H-9),4.06(t,J=6 .8Hz,2H,H-11),3.89(s,3H,H-8),1.83(p,J=6.9Hz,2H,H-12),1.49–1.32(ove rlapped,4H,H-10,13),0.96(t,J=7.4Hz,3H,H-14); LR-EI-MS(m / z):252.1[M] + ; HR-EI-MS (m / z) calcd.for C 12 H 16 O4[M] + :252.1362,found:252.1350.

[0099] The preparation method of ethyl 4-hexyloxy-3-methoxybenzoate (2c) is as follows: 1c (25 mg, 0.099 mmol) was refluxed in ethanol, and then a catalytic amount of sulfuric acid was added. After 24-36 hours, the reaction mixture was cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2c.

[0100] 4-Hexyloxy-3-methoxybenzoic acid ethyl ester (2c): white powder; yield: 67.5%; mp: 32-33°C; 1 HNMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.52 (d, J=2.1Hz, 1H, H -2),6.85(d,J=8.4Hz,1H,H-5),4.33(t,J=7.2Hz,2H,H-9),4.05(t,J=6.9Hz,2H,H-11) ,3.89(s,3H,H-8),1.84(p,J=7.0Hz,2H,H-12),1.45(m,2H,H-13),1.47–1.29(overla pped,6H,H-10,H-14–15–H-9′),0.88(t,J=7.0Hz,3H,H-16); LR-EI-MS(m / z):280.2[M] + ; HR-EI-MS (m / z) calcd.for C 16 H 24 O4[M] + :280.1675,found:280.1673.

[0101] The preparation method of ethyl 4-octyloxy-3-methoxybenzoate (2d) is as follows: 1d (25 mg, 0.089 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (volume ratio = 3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2d.

[0102] 4-Octyloxy-3-methoxybenzoic acid ethyl ester (2d): white powder, yield 60.0%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.52 (d, J=2.1Hz, 1H,H-2),6.85(d,J=8.5Hz,1H,H-5),4.33(q,J=7.1Hz,2H,H-9),4.04(t,J=6.9H z,2H,H-11),3.90(s,3H,H-8),1.84(t,J=7.0Hz,3H,H-12),1.52–1.14(overlap ped,12H,H-10,H-13–17),0.85(t,J=7.8Hz,3H,H-18); LR-EI-MS(m / z):308.2[M] + ; HR-EI-MS (m / z) calcd.for C 18 H 28 O4[M] + :308.1988,found:308.1989.

[0103] The preparation method of ethyl 4-decyloxy-3-methoxybenzoate (2e) is as follows: 1e (25 mg, 0.081 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2e.

[0104] 4-Decyloxy-3-methoxybenzoic acid ethyl ester (2e): white powder, yield 67.2%; mp: 33-34℃, 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.1Hz, 1H, H-6), 7.52 (d, J=1.9Hz, 1H,H-2),6.85(d,J=8.5Hz,1H,H-5),4.33(q,J=7.1Hz,2H,H-9),4.04(t,J=6.9H z,2H,H-11),3.89(s,3H,H-8),1.84(p,J=7.0Hz,2H,H-12),1.42–1.18(overlap ped,14H,H-10,H-13–19),0.86(t,J=7.2Hz,3H,H-20); LR-EI-MS(m / z):336.2[M] + ; HR-EI-MS (m / z) calcd.forC 20 H 32 O4[M]+ :336.2301,found:336.2303.

[0105] The preparation method of ethyl 4-dodecyloxy-3-methoxybenzoate (2f) is as follows: 1f (25 mg, 0.074 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2f.

[0106] 4-Dodecyloxy-3-methoxybenzoic acid ethyl ester (2f): white powder, yield: 50.2%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.52 (d, J=2.0Hz, 1H,H-2),6.85(d,J=8.4Hz,1H,H-5),4.33(q,J=7.2Hz,2H,H-9),4.04(t,J=7.0H z,2H,H-11),3.89(s,3H,H-8),1.84(p,J=7.0Hz,2H,H-12),1.47–1.24(overlap ped,20H,H-10,H-13–21),0.86(t,J=7.0Hz,3H,H-22); LR-EI-MS(m / z):364.3[M] + ; HR-EI-MS (m / z) calcd.forC 22 H 36 O4[M] + :364.2614,found:364.2601.

[0107] The preparation method of ethyl 4-tetradecyloxy-3-methoxybenzoate (2 g) is as follows: 1 g (25 mg, 0.069 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (volume ratio = 3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2 g.

[0108] 4-Tetradecyloxy-3-methoxybenzoic acid ethyl ester (2g): white powder, yield: 55.2%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.52 (d, J=2.0Hz, 1H,H-2),6.85(d,J=8.4Hz,1H,H-5),4.33(q,J=7.0Hz,2H,H-9),4.04(t,J=6.9H z,2H,H-11),3.89(s,3H,H-8),1.84(p,J=7.0Hz,2H,H-12),1.45–1.35(overlap ped,24H,H-10,H-13–23),0.86(t,J=7.0Hz,3H,H-24); LR-EI-MS(m / z):392.3[M] + .

[0109] The preparation method of ethyl 4-hexadecyloxy-3-methoxybenzoate (2h) is as follows: 1h (25 mg, 0.064 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2h.

[0110] 4-Hexadecyloxy-3-methoxybenzoic acid ethyl ester (2h): white powder, yield: 40.0%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.4, 2.0Hz, 1H, H-6), 7.52 (d, J=2.0Hz, 1H,H-2),6.85(d,J=8.4Hz,1H,H-5),4.33(q,J=7.0Hz,2H,H-9),4.04(t,J=6.9H z,2H,H-11),3.89(s,3H,H-8),1.84(p,J=7.0Hz,2H,H-12),1.45–1.35(overlap ped,28H,H-10,H-13–25),0.86(t,J=7.0Hz,3H,H-26); LR-EI-MS(m / z):420.2[M] + ; HR-EI-MS (m / z) calcd.forC 26 H 44 O4[M] + :420.3240,found:420.3257.

[0111] The preparation method of ethyl 4-octadecyloxy-3-methoxybenzoate (2i) is as follows: 1i (25 mg, 0.060 mmol) is refluxed in ethanol, and then a catalytic amount of sulfuric acid is added. After 24-36 hours, the reaction mixture is cooled and neutralized with a 10% sodium bicarbonate aqueous solution, and then extracted with ethyl acetate / water (3:1, 3×). The organic layer is dried under reduced pressure and purified by silica gel chromatography using n-hexane and ethyl acetate (volume ratio = 9:1) as eluent to obtain 2i.

[0112] Ethyl 4-octadecyloxy-3-methoxybenzoate (2i): white powder, yield: 40.1%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, J=8.0, 2.0Hz, 1H, H-6), 7.52 (d, J=2.0Hz, 1H,H-2),6.85(d,J=8.0Hz,1H,H-5),4.33(q,J=8.0Hz,2H,H-9),4.04(t,J=8.0H z,2H,H-11),3.89(s,3H,H-8),1.84(p,J=8.0Hz,2H,H-12),1.45–1.24(overlap ped,32H,H-10,H-13–27),0.86(t,J=8.0Hz,3H,H-28); LR-EI-MS(m / z):448.2[M] + ; HR-EI-MS (m / z) calcd.forC 28 H 48 O4[M] + :448.3553,found:448.3554.

[0113] <Example 4>

[0114] Compounds 4a-4i have the following structural formulas:

[0115]

[0116] The specific synthetic route is as follows:

[0117]

[0118] The specific synthesis steps are as follows:

[0119] Propyl 4-ethoxy-3-methoxybenzoate (4a) was prepared by refluxing 1a (25 mg, 0.127 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography, with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4a.

[0120] 4-Ethoxy-3-methoxybenzoic acid propyl ester (4a): oily substance; yield: 84%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5Hz, 5-CH), 4.23 (t, 2H, J = 6.7Hz, 9-CH) 2),4.14(q,2H,J=7.0Hz,12-CH2),3.90(s,3H,8-CH3),1.76(h,J=7.3Hz,2H,10-CH2),1.47(t,3H,J=7.0Hz,11-CH3),1.00(t,3H,J=7.4Hz,13-CH3); 13 C NMR(100MHz,CDCl3)δppm 166.5(C-7),152.3(C-4),148.8(C-3),128.4(C-6),122.8(C-1),112.2(C-2),111.2(C-5),66.3 (C-9),64.4(C-12),56.0(C-8),22.2(C-10),14.6(C-13),10.5(C-11).LR-EI-MS(m / z):238.2[M] + ; HR-EI-MS (m / z) calcd.for C 13 H 18 O4[M] + :238.1205,found:238.1201.

[0121] Propyl 4-butoxy-3-methoxybenzoate (4b) was prepared by refluxing 1b (25 mg, 0.112 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with a 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4b.

[0122] 4-Butoxy-3-methoxybenzoic acid propyl ester (4b): oily substance; yield: 83%; 1 H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d ,1H,J=8.5Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.05(t,2H,J=6.8Hz,12-CH2),3.89(s,3H,8-CH3 ),1.83(quin.,J=6.9Hz,2H,13-CH2),1.76(sextet,J=7.5Hz,2H,10-CH2),1.48(sextet,2H,J=6.9H z,14-CH2),1.00(t,3H,J=7.5Hz,11-CH3).0.89(t,3H,J=7.4Hz,15-CH3); LR-EI-MS(m / z):266.2[M] + ,HR-EI-MS(m / z)calcd.for C 15 H 22 O4[M] + :266.1518,found:266.1529.

[0123] Propyl 4-hexyloxy-3-methoxybenzoate (4c) was prepared by refluxing 1c (25 mg, 0.099 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with a 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4c.

[0124] 4-Hexyloxy-3-methoxybenzoic acid propyl ester (4c): oily substance; yield: 81%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.3, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2- CH),6.85(d,1H,J=8.5Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12 -CH2),3.90(s,3H,8-CH3),1.76(quin.,J=6.9Hz,2H,13-CH2),1.44(quin,2H,J=7.6Hz ,14-CH2),1.32(sextet,J=6.8Hz,2H,10-CH2),1.38–1.26(overlapped,4H,15-CH2,and 16-CH2), 1.00 (t, 3H, J = 7.4Hz, 11-CH3), 0.88 (t, 3H, J = 6.4Hz, 17-CH3); LR-EI-MS (m / z): 294.2[M] + ,HR-EI-MS(m / z)calcd.for C 17 H 25 O4[M] + :294.1831,found:294.1833.

[0125] The preparation method of 4-octyloxy-3-methoxybenzoic acid propyl ester (4d) is as follows: 1d (25 mg, 0.089 mmol) is refluxed in propanol, and then a catalytic amount of p-toluenesulfonic acid is added. After 36-72 hours, the reaction mixture is cooled and neutralized with a 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer is dried under reduced pressure and further purified by silica gel column chromatography, with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4d.

[0126] 4-Octyloxy-3-methoxybenzoic acid propyl ester (4d): oily substance; yield: 85%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.3, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.24(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.90(s,3H,8-CH3),1.84(sextet,J=6 .9Hz,2H,10-CH2),1.76(quin.,J=7.0Hz,2H,13-CH2),1.44(quin,2H,J=7.0Hz,14-CH2),1.36–1.21(overlap ped,8H,15–18-CH2),1.01(t,3H,J=7.4Hz,11-CH3),0.86(t,3H,J=6.9Hz,19-CH3); LR-EI-MS(m / z):322.2[M] + ,HR-EI-MS(m / z)calcd.forC 19 H 30 O4[M] + :322.2144,found:322.2155.

[0127] Propyl 4-decyloxy-3-methoxybenzoate (4e) was prepared by refluxing 1e (25 mg, 0.081 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography, with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4e.

[0128] 4-Decyloxy-3-methoxybenzoic acid propyl ester (4e): oily substance; yield: 85%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.90(s,3H,8-CH3),1.85(sextet,J=6 .9Hz,2H,10-CH2),1.77(quin.,J=7.0Hz,2H,13-CH2),1.44(quin,2H,J=7.0Hz,14-CH2),1.38–1.18(overlap ped,12H,15–20-CH2),1.01(t,3H,J=7.5Hz,11-CH3),0.86(t,3H,J=6.9Hz,21-CH3); LR-EI-MS(m / z):350.2[M] + ,HR-EI-MS(m / z)calcd.forC 21 H 34 O4[M] + :350.2457,found:350.2467.

[0129] Propyl 4-dodecyloxy-3-methoxybenzoate (4f) was prepared by refluxing 1f (25 mg, 0.074 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4f.

[0130] 4-Dodecyloxy-3-methoxybenzoic acid propyl ester (4f): oily substance; yield: 80%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.90(s,3H,8-CH3),1.84(sextet,J=7 .0Hz,2H,10-CH2),1.75(quin.,J=7.0Hz,2H,13-CH2),1.43(quin,2H,J=7.0Hz,14-CH2),1.38–1.20(overlap ped,16H,15–22-CH2),1.00(t,3H,J=7.5Hz,11-CH3),0.86(t,3H,J=7.2Hz,23-CH3); LR-EI-MS(m / z):378.2[M] + ,HR-EI-MS(m / z)calcd.for C 23 H 38 O4[M] + :378.2770,found:378.2761.

[0131] Propyl 4-tetradecyloxy-3-methoxybenzoate (4 g) was prepared by refluxing 1 g (25 mg, 0.069 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% sodium bicarbonate aqueous solution and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4 g.

[0132] 4-Tetradecyloxy-3-methoxybenzoic acid propyl ester (4g): oily substance; yield: 76%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.90(s,3H,8-CH3),1.84(sextet,J=7 .0Hz,2H,10-CH2),1.75(quin.,J=7.0Hz,2H,13-CH2),1.44(quin,2H,J=7.0Hz,14-CH2),1.38–1.20(overlap ped,20H,15–24-CH2),1.00(t,3H,J=7.5Hz,11-CH3),0.86(t,3H,J=6.9Hz,25-CH3); LR-EI-MS(m / z):406.3[M] + ,HR-EI-MS(m / z)calcd.for C 25 H 42 O4[M] + :406.3083,found 406.3081.

[0133] The preparation method of 4-hexadecyloxy-3-methoxybenzoic acid propyl ester (4h) is as follows: 1h (25 mg, 0.064 mmol) is refluxed in propanol, and then a catalytic amount of p-toluenesulfonic acid is added. After 36-72 hours, the reaction mixture is cooled and neutralized with a 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer is dried under reduced pressure and further purified by silica gel column chromatography, and eluted with a gradient of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4h.

[0134] 4-Hexadecyloxy-3-methoxybenzoic acid propyl ester (4h): oily substance; yield: 80%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.89(s,3H,8-CH3),1.85(sextet,J=7 .0Hz,2H,10-CH2),1.76(quin.,J=7.0Hz,2H,13-CH2),1.46(quin,2H,J=7.0Hz,14-CH2),1.40–1.22(overlap ped,24H,15–26-CH2),1.00(t,3H,J=7.5Hz,11-CH3),0.86(t,3H,J=6.9Hz,27-CH3); LR-EI-MS(m / z):434.3[M] + ,HR-EI-MS(m / z)calcd.for C 27 H 46 O4[M] + :434.3396,found:434.3392.

[0135] Propyl 4-octadecyloxy-3-methoxybenzoate (4i) was prepared by refluxing 1i (25 mg, 0.060 mmol) in propanol and then adding a catalytic amount of p-toluenesulfonic acid. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% sodium bicarbonate aqueous solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and further purified by silica gel column chromatography, with a gradient elution of n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 4i.

[0136] 4-Octadecyloxy-3-methoxybenzoic acid propyl ester (4i): oily substance; yield: 84%; 1H NMR (400MHz, CDCl3) δ (ppm): 7.64 (dd, 1H, J = 8.5, 2.0Hz, 6-CH), 7.53 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.5 Hz,5-CH),4.23(t,2H,J=6.7Hz,9-CH2),4.04(t,2H,J=6.8Hz,12-CH2),3.90(s,3H,8-CH3),1.83(sextet,J=7 .0Hz,2H,10-CH2),1.76(quin.,J=7.0Hz,2H,13-CH2),1.44(quin,2H,J=7.0Hz,14-CH2),1.38–1.18(overlap ped,28H,15–28-CH2),1.00(t,3H,J=7.5Hz,11-CH3),0.85(t,3H,J=6.9Hz,29-CH3); LR-EI-MS(m / z):462.3[M] + ,HR-EI-MS(m / z)calcd.for C 29 H 50 O4[M] + :462.3709,found:462.3713.

[0137] <Example 5>

[0138] Compounds 5a-5i have the following structural formulas:

[0139]

[0140] The specific synthetic route is as follows:

[0141]

[0142] The specific synthesis steps are as follows:

[0143] Preparation of isopropyl 4-ethoxy-3-methoxybenzoate (5a) (A): 1a (25 mg, 0.127 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5a.

[0144] Isopropyl 4-ethoxy-3-methoxybenzoate (5a): white powder; yield: 79.8% (method A); 1H NMR (400MHz, CDCl3) δ (ppm): 7.63 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85 (d, 1H, J = 8.4Hz, 5-CH), 5.21 (sept, 1H, J=6.5Hz,9-CH),4.14(q,2H,J=7.0Hz,11-CH2),3.91(s,3H,8-CH3),1.34(d,6H,J=6.5Hz,10,10′-CH3,),1.47(t,3H,J=7.0Hz,12-CH3); 13 C NMR(100MHz,CDCl3)δppm 166.0(C-7),152.2(C-4),148.7(C-3),123.4(C-6),123.3(C-1),112.2(C-2),111.2(C-5) ,68.1(C-9),64.4(C-11),56.0(C-8),22.0(C-10),14.6(C-12); LR-EI-MS(m / z):238.1[M] + ,HR-EI-MS(m / z)calcd.for C 13 H 18 O4[M] + :238.1205,found:238.1202.

[0145] Preparation of isopropyl 4-butoxy-3-methoxybenzoate (5b) (A): 1b (25 mg, 0.112 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5b.

[0146] Isopropyl 4-butoxy-3-methoxybenzoate (5b): oil: 84.6% (method A); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.85(d,1H,J=8.4Hz,5-CH),5.20(sept,1H,J=6.2Hz,9-CH),4.05(q,2H,J=6.7Hz,11-CH2),3 .89(s,3H,8-CH3),1.83(quin.,2H,J=6.8Hz,12-CH2),1.48(sextet.,2H,J=7.2Hz,13-CH2), 1.34(d,6H,J=6.2Hz,10,10′-CH3),0.96(t,3H,J=7.3Hz,14-CH3); LR-EI-MS(m / z):266.1[M] + ,HR-EI-MS(m / z)calcd.for C 15 H 22 O4[M] + :266.1518),found:266.1525.

[0147] Preparation of isopropyl 4-hexyloxy-3-methoxybenzoate (5c) (A): 1c (25 mg, 0.099 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with a 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5c.

[0148] Isopropyl 4-hexyloxy-3-methoxybenzoate (5c): oil yield: 76.5% (method A); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.84 (d, 1H, J=8.4Hz,5-CH),5.20(sept,1H,J=6.2Hz,9-CH),4.04(q,2H,J=6.7Hz,11-CH2),3.89(s,3H,8-CH3),1.84 (quin.,2H,J=7.0Hz,12-CH2),1.44(quin.,2H,J=6.3Hz,13-CH2),1.34(d,6H,J=6.2Hz,10,10′-CH3),1. 31–1.25(overlapped,4H,14-CH2,and15-CH2),0.89(t,3H,J=7.0Hz,16-CH3); LR-EI-MS(m / z):294.2[M] + ,HR-EI-MS(m / z)calcd.forC 17 H 26 O4[M] + :294.1831),found:294.1835.

[0149] Preparation of isopropyl 4-octyloxy-3-methoxybenzoate (5d) (A): 1d (25 mg, 0.089 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio 9:1) as eluent to obtain 5d.

[0150] Isopropyl 4-octyloxy-3-methoxybenzoate (5d): oil (80%); yield: 80% (method A); 1HNMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 1.9Hz, 2-CH), 6.84 (d, 1 H,J=8.4Hz,5-CH),5.21(sept,1H,J=6.2Hz,9-CH),4.04(q,2H,J=6.7Hz,11-CH2),3.89(s,3H,8-CH3), 1.84(quin.,2H,J=7.0Hz,12-CH2),1.44(quin.,2H,J=7.7Hz,13-CH2),1.34(d,6H,J=6.2Hz,10,10′-C H3),1.30–1.23(overlapped,8H,14–17-CH2),0.86(t,3H,J=5.4Hz,18-CH3); LR-EI-MS(m / z):322.0[M] + ; HR-EI-MS (m / z) calcd.forC 19 H 30 O4[M] + :322.2144),found:322.2157.

[0151] Preparation of isopropyl 4-decyloxy-3-methoxybenzoate (5e) (A): 1e (25 mg, 0.081 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel column chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5e.

[0152] Isopropyl 4-decyloxy-3-methoxybenzoate (5e): oil (83.2%); yield: 83.2% (method A); 1HNMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.51 (d, 1H, J = 1.9Hz, 2-CH), 6.84 (d, 1 H,J=8.5Hz,5-CH),5.20(sept,1H,J=6.2Hz,9-CH),4.04(q,2H,J=6.7Hz,11-CH2),3.89(s,3H,8-CH3),1 .84(quin.,2H,J=7.0Hz,12-CH2),1.43(quin.,2H,J=7.8Hz,13-CH2),1.33(d,6H,J=6.2Hz,10,10′-CH 3),1.29–1.23(overlapped,12H,14–19-CH2),0.85(t,3H,J=7.0Hz,20-CH3); LR-EI-MS(m / z):350.1[M] + .

[0153] Preparation of isopropyl 4-dodecyloxy-3-methoxybenzoate (5f) Method A: 1f (25 mg, 0.074 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5f.

[0154] Preparation method B: N, N'-dicyclohexylcarbodiimide (1.2 equivalents) was added to an ice-cold dichloromethane solution of 4-dodecyloxy-3-methoxybenzoic acid (1f) containing 4-dimethylaminopyridine (0.1 equivalents). The mixture was stirred at room temperature for reaction. After 24 hours, the urea precipitate was filtered out. The filtrate was dried under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5f.

[0155] Isopropyl 4-dodecyloxy-3-methoxybenzoate (5f): white powder; yield: 10.1% (method A), 44% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.4, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 1.9Hz, 2-CH), 6.84 (d, 1H ,J=8.5Hz,5-CH),5.21(sept,1H,J=6.7Hz,9-CH),4.04(q,2H,J=6.7Hz,11-CH2),3.89(s,3H,8-CH3),1 .84(quin.,2H,J=7.0Hz,12-CH2),1.43(quin.,2H,J=7.6Hz,13-CH2),1.34(d,6H,J=6.2Hz,10,10′-CH 3),1.31–1.23(overlapped,16H,14–21-CH2),0.86(t,3H,J=6.9Hz,22-CH3); LR-EI-MS(m / z):378.3[M] + ; HR-EI-MS (m / z) calc.for C 23 H 38 O4[M] + :378.2770,found:378.2762.

[0156] Preparation of isopropyl 4-tetradecyloxy-3-methoxybenzoate (5 g) Method A: 1 g (25 mg, 0.069 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5 g.

[0157] Preparation method B: N, N'-dicyclohexylcarbodiimide (1.2 equivalents) was added to an ice-cold solution of 4-tetradecyloxy-3-methoxybenzoic acid (1 g) in dichloromethane containing 4-dimethylaminopyridine (0.1 equivalents). The mixture was stirred at room temperature for reaction. After 24 hours, the urea precipitate was filtered out. The filtrate was dried under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5 g.

[0158] Isopropyl 4-tetradecyloxy-3-methoxybenzoate (5 g): white powder, yield: 17% (method A), 56% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.3, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.84 (d, 1 H,J=8.5Hz,5-CH),5.20(sept,1H,J=6.2Hz,9-CH),4.0(t,2H,J=6.9Hz,11-CH2),3.89(s,3H,8-CH3),1 .84(quin.,2H,J=7.0Hz,12-CH2),1.43(quin,2H,J=6.7Hz,13-CH2),1.33(d,6H,J=6.2Hz,10,10′-CH3 ),1.29–1.31(overlapped,20H,14–23-CH2),0.86(t,3H,J=6.7Hz,24-CH3); LR-EI-MS(m / z):406.3[M] + ,HR-EI-MS(m / z)calc.forC 25 H 42 O4[M] + :406.3083),found:406.3080.

[0159] Preparation of isopropyl 4-hexadecyloxy-3-methoxybenzoate (5h) Method A: 1h (25 mg, 0.064 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5h.

[0160] Preparation method B: N, N'-dicyclohexylcarbodiimide (1.2 equivalents) was added to an ice-cold dichloromethane solution of 4-hexadecyloxy-3-methoxybenzoic acid (1h) containing 4-dimethylaminopyridine (0.1 equivalents). The mixture was stirred at room temperature for reaction. After 24 hours, the urea precipitate was filtered out. The filtrate was dried under reduced pressure, and the residue was purified by silica gel column chromatography, eluted with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5h.

[0161] Isopropyl 4-hexadecyloxy-3-methoxybenzoate (5h): white powder, yield: 25.8% (method A), 43% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.3, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.84 (d, 1H ,J=8.4Hz,5-CH),5.21(sept,1H,J=6.2Hz,9-CH),4.0(t,2H,J=6.9Hz,11-CH2),3.89(s,3H,8-CH3),1. 84(quin.,2H,J=7.0Hz,12-CH2),1.43(quin.,2H,J=6.7Hz,13-CH2),1.34(d,6H,J=6.2Hz,10,10′-CH3 ),1.29–1.31(overlapped,24H,14–25-CH2),0.86(t,3H,J=6.6Hz,26-CH3); LR-EI-MS(m / z):434.3[M] + ,HR-EI-MS(m / z)calc.forC 27 H 46 O4[M] + :434.3396,found:434.3410.

[0162] Preparation of isopropyl 4-octadecyloxy-3-methoxybenzoate (5i) Method A: 1i (25 mg, 0.060 mmol) was refluxed in isopropanol, and then a catalytic amount of p-toluenesulfonic acid was added. After 36-72 hours, the reaction mixture was cooled and neutralized with 10% aqueous sodium bicarbonate solution, and then extracted with dichloromethane (3×). The organic layer was dried under reduced pressure and purified by silica gel chromatography using n-hexane / ethyl acetate (volume ratio = 9:1) as eluent to obtain 5i.

[0163] Preparation method B: N,N'-dicyclohexylcarbodiimide (1.2 equivalents) was added to an ice-cold dichloromethane solution of 4-octadecyloxy-3-methoxybenzoic acid (1i) containing 4-dimethylaminopyridine (0.1 equivalents). The mixture was stirred at room temperature for reaction. After 24 hours, the urea precipitate was filtered out. The filtrate was dried under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with n-hexane / ethyl acetate (volume ratio = 9:1) to obtain 5i.

[0164] Isopropyl 4-octadecyloxy-3-methoxybenzoate (5i): white powder; yield: 16% (method A), 55% (method B); 1H NMR (400MHz, CDCl3) δ (ppm): 7.62 (dd, 1H, J = 8.3, 2.0Hz, 6-CH), 7.52 (d, 1H, J = 2.0Hz, 2-CH), 6.84 (d, 1H ,J=8.4Hz,5-CH),5.21(sept,1H,J=6.2Hz,9-CH),4.0(t,2H,J=6.9Hz,11-CH2),3.89(s,3H,8-CH3),1. 84(quin.,2H,J=7.5Hz,12-CH2),1.43(quin.,2H,J=8.1Hz,13-CH2),1.34(d,6H,J=6.2Hz,10,10′-CH3 ),1.23–1.30(overlapped,28H,14–27-CH2),0.88(t,3H,J=7.5Hz,28-CH3); LR-EI-MS(m / z):462.2[M] + ,HR-EI-MS(m / z)calcd.forC 29 H 50 O4[M] + :462.3709,found:462.3697.

[0165] <Example 6>

[0166] Performance experiment:

[0167] 1. The antifungal and synergistic effects of the vanillic acid derivatives in Examples 1-5 against Candida albicans ATCC 36082 were measured by inhibition zone (mm) in a disc diffusion assay.

[0168] Experimental Procedure: Disk diffusion assays were performed as described in the NCCLS Guidelines 2004 to determine the antifungal susceptibility (zone of inhibition) of fluconazole (10 μg / mL), amphotericin B (5 μg / mL) and compounds (5 μg / mL) alone or in combination to observe the synergistic effect of all compounds with both fungal drugs. Briefly, C. albicans ATCC 36082 was cultured on Sabouraud dextrose agar and the inoculum was adjusted to 0.5 McFarland units using phosphate buffer. Sterile Whatman filter paper disks (6 mm in diameter) soaked with fungicidal drugs, compounds and combinations of drugs and compounds (fluconazole 10 μg / mL + compound 5 μg / mL, amphotericin B 5 μg / mL + compound 5 μg / mL) were placed on the lawn of C. albicans ATCC 36082. The diameter of the zone of inhibition was measured after incubation at 35°C for 24 h. Disk diffusion assays were performed in triplicate and the average values ​​were calculated.

[0169] The experimental results are shown in Table 1 below. The experimental results show that among the 37 synthesized vanillic acid derivatives, 11 showed intrinsic bactericidal activity at a concentration of 5 μg / mL, and the diameter of the inhibition zone around the sample to inhibit Candida albicans ATCC 36082 ranged from 5.0 to 11 mm. The breakpoint evaluation of amphotericin B and fluconazole (Table 1) showed that at a concentration of 5.0 μg / mL, the inhibition zone of amphotericin B was 19.5±0.5 mm; at a concentration of 10.0 μg / mL, the inhibition zone of fluconazole was 13.3±0.6 mm. The inhibitory activity of compounds 2c, 4c, 5a and 5e was improved after combination with fluconazole, increasing the inhibition zone to 17.7–23.1 mm. Although compound 5g showed no inhibitory effect against Candida albicans ATCC 36082, it exhibited significant synergistic effects with fluconazole (inhibition zone = 27.1 ± 0.3 mm) and amphotericin B (30.0 ± 0.5 mm).

[0170] Table 1. Inhibitory effects of the compounds and their combination with amphotericin B or fluconazole on Candida albicans ATCC 36082 (expressed in millimeters of the diameter of the inhibition zone)

[0171]

[0172]

[0173] 2. Experimental process:

[0174] The antifungal index is the ratio of the inhibition zones produced by the compound (used alone or in combination), fluconazole, and amphotericin B. The antifungal index of each compound was calculated by the following formula:

[0175] AFI = zone of inhibition (ZOI) of the compound (used alone or in combination) / ZOI of the standard fungicide.

[0176] AFI values ​​> 1 indicate relative effectiveness of the test compound.

[0177] Percent inhibition of fluconazole and amphotericin B and synergistic inhibition in combination with compounds were determined by broth microdilution assay at concentrations used in the disk diffusion assay according to the guidelines in CLSI document M27-Ed4 (Table 3).

[0178] Experimental results: The relative synergistic antibacterial effects and inhibition percentages of the tested compounds are shown in Tables 2 and 3. The comparative analysis of the antifungal index in Table 2 showed that the antifungal index of compound / amphotericin B was lower than that of fluconazole / amphotericin B (0.68); the antifungal index of compound / fluconazole was lower than that of amphotericin B / fluconazole (1.46). The synergistic antifungal index between compound + fluconazole / fluconazole was greatly improved (0.70–2.04) compared with the compound / fluconazole range (0.34–0.83). Similarly, the antifungal index of the combination of compound and amphotericin B was 0.82–1.79 compared with the antifungal index of compound / amphotericin B (0.26–0.56). The growth inhibition percentages of Candida albicans ATCC 36082 using amphotericin B (5 μg / mL) and fluconazole (10 μg / mL) were 84.1% and 64.1%, respectively (Table 3). Compounds 2e (68.3%), 4c (72.1%), 5e (73.1%) and 5g (85.7%) showed synergistic inhibitory effects with fluconazole, respectively. 5g had significant synergistic activity with fluconazole. Except for 5g, most of the tested compounds had antagonistic effects on the inhibition of amphotericin B.

[0179] Table 2. Antifungal index of the compounds compared with amphotericin B or fluconazole, and the combination of amphotericin B or fluconazole and the compounds compared with the single use

[0180]

[0181] Note: Antifungal index > 1 indicates that the sample is effective; NA indicates no antifungal activity. The antifungal index can indicate the breakthrough of drug resistance after the sample is combined with amphotericin B (5 μg / mL) or fluconazole (10 μg / mL). The combined dose of the compound is 5 μg / mL.

[0182] Table 3. Inhibitory effect of the combination of compound (5 μg / mL) with amphotericin B (5 μg / mL) or fluconazole (10 μg / mL) on Candida albicans ATCC 36082 (%)

[0183]

[0184]

[0185] Note: - indicates no inhibition rate.

[0186] <Example 7>

[0187] Further experiments were conducted on compound 5g, which showed significant synergistic activity with fluconazole:

[0188] 1. Time-kill curve of compound 5g combined with fluconazole against Candida albicans ATCC 36082

[0189] Experimental procedure: C. albicans ATCC 36082 was cultured in yeast potato dextrose broth to a density of approximately 10 5 –10 6 CFU / mL (OD 0.1-0.5@600nm). Aliquots of medium (final volume 200 μL in microtiter plates) containing yeast cells and MIC concentrations of 0.5×, 1×, and 2× fluconazole, as well as fluconazole and 5g (5 μg / mL), were prepared in triplicate. Growth reduction was observed at 0, 6, 12, 24, and 48 hours for each treatment, as well as positive and negative controls (medium inoculated with and without viable cells). 100 μL of inoculum from each dilution was plated on yeast potato dextrose agar to determine viable colony counts (CFU / mL).

[0190] A reduction of ≥2 log CFU / mL after treatment with 5 g / fluconazole of the starting inoculum and the standard drug was considered a synergistic effect.

[0191] Experimental results:

[0192] Compound 5g did not directly inhibit the growth of Candida albicans ATCC 36082, but had a potential synergistic effect with fluconazole. The calculated minimum inhibitory concentration (MIC) of fluconazole was ≥32 μg / mL, showing an inhibition rate of more than 80%, and the synergistic effect of 5g reduced the dose required to achieve 80% or higher inhibition of Candida albicans ATCC 36082. When Candida albicans ATCC 36082 cells were co-treated with 5 μg / mL of 5g, the observed MIC of fluconazole was ≥5 μg / mL. Figure 1 Time-kill curves for fluconazole and compound 5g are illustrated. Compounds at a concentration of 5 μg / mL reduced logarithmic growth of Candida albicans ATCC 36082 by two-fold when administered together with fluconazole (0.5-, 1-, and 2-fold of their synergistic MIC). Fluconazole at MIC values ​​≥32 μg / mL required longer times (12–48 h) to achieve a ≥3 log CFU / mL reduction. However, a sudden arrest of cell division and a significant reduction in growth were observed within the first 3 h at 2.5, 5.0, and 10 μg / mL of fluconazole in synergy with 5 μg / mL of 5g, followed by a sharp decrease in the rate of cell division. Log colony forming units ( Figure 1 ).

[0193] 2. Time-kill curve of compound 5g combined with amphotericin B against Candida albicans ATCC 36082

[0194] Experimental procedure: C. albicans ATCC 36082 was cultured in yeast potato dextrose broth to a density of approximately 105 –10 6 CFU / mL (OD 0.1-0.5@600nm). Aliquots of medium (final volume 200 μL in microtiter plates) containing yeast cells and MIC concentrations of 0.5×, 1× and 2× amphotericin B, as well as amphotericin B and 5g (5 μg / mL), were prepared in triplicate. The reduction in growth at 0, 6, 12, 24 and 48 hours for each treatment was observed, as well as positive and negative controls (medium inoculated with and without viable cells). 100 μL of the inoculum from each dilution was plated on yeast potato dextrose agar to determine the viable colony count (CFU / mL).

[0195] A reduction of ≥2 log CFU / mL after treatment with 5 g / mL of the starting inoculum and standard drug was considered a synergistic effect.

[0196] Experimental results:

[0197] Compound 5g did not directly inhibit the growth of C. albicans ATCC 36082, but had a potential synergistic effect with amphotericin B. The calculated minimum inhibitory concentration (MIC) of amphotericin B was ≥4 μg / mL, showing an inhibition rate of more than 80%, and the synergistic effect of 5g reduced the dose required to achieve 80% or higher inhibition of C. albicans ATCC 36082. When C. albicans ATCC 36082 cells were co-treated with 5 μg / mL of 5g, the observed MIC of amphotericin B was ≥0.5 μg / mL. Figure 2 Time-kill curves for amphotericin B and compound 5g are illustrated. Amphotericin B (≥5 μg / mL) began to inhibit growth after 8 hours; however, it decreased to ≥3 log CFU / mL ( Figure 2 ). In addition, a four-fold decrease in amphotericin B concentration resulted in 80% inhibition of C. albicans ATCC 36082. The observed time-inactivation assay results were complemented by plating the inoculum on SDA agar plates, which showed no growth after 3–48 h of treatment.

[0198] 3. Hemolytic toxicity

[0199] Experimental Procedure: For hemolysis assay, 5 mL of fresh human blood was drawn from healthy individuals and placed in lithium heparin vacuum blood collection tubes. Concentrated red blood cells were separated by centrifugation for 10 minutes and the supernatant was discarded. The red blood cells were washed three times with phosphate buffered saline (pH 7.4), centrifuged and resuspended in a ratio of 1:10 red blood cells: phosphate buffered saline (weight / volume). The percentage of hemolysis was observed using fluconazole (10 μg / mL), amphotericin B (5 μg / mL), 5g (5 μg / mL), fluconazole / 5g and amphotericin B / 5g, DMSO (5%), phosphate buffered saline (negative control), Triton X-100 and sodium dodecyl sulfate (1%, positive control). Aliquots of 190 μL of phosphate buffered saline diluted red blood cell suspension were distributed in duplicate in microtiter plates at 37°C. RBCs were treated with two-fold concentrations of 5g, fluconazole and amphotericin B alone or in combination for 30 minutes at physiological temperature. The amount of hemoglobin released from ruptured erythrocytes was estimated at 576 nm and converted to percentage hemolysis.

[0200] Experimental results:

[0201] Compounds or drugs may cause toxic hemolysis at higher concentrations. According to the US FDA, in vitro hemolysis experiments should be performed to determine the toxicity of the compound at the expected dosing concentration [International Journal of Toxicology, 2011, 30, 6: 600.]. In vitro membrane lysis assays using washed red blood cells at physiological pH, temperature and ionic strength can quickly disseminate the level of drug-induced cytotoxicity and the dose-dependency of the test compound [Molecules, 2022, 28, 1: 212]. Figure 3 In the assay, 5 μg / mL of compound 5g synergized with fluconazole and amphotericin B, showing no hemolysis after a 50-min incubation period, while 5% DMSO showed 10–20% lysis of erythrocytes during the assay period. Furthermore, a two-fold increase in 5g concentration did not show increased hemolysis. However, triton X-100 and SDS showed synergistic lytic behavior and lysed 100% of erythrocytes within a 5–15-min incubation time.

[0202] The invention synthesizes a series of vanillic acid derivatives and conducts corresponding experiments to obtain that the compound 4-tetradecyloxy-3-methoxybenzoic acid isopropyl ester (5 g) is ineffective against Candida albicans ATCC 36082 when used alone, but can double the effect and reduce the dosage when used in combination with amphotericin B or fluconazole, and can be a synergist of antibacterial drugs such as amphotericin B and fluconazole.

[0203] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A vanillic acid derivative having the structure of the following formula (I): （I） in, R is isopropyl, n=14.

2. A method for preparing the vanillic acid derivative according to claim 1, comprising the following steps Using vanillic acid as a raw material, adding p-toluenesulfonic acid to prepare methyl 4-hydroxy-3-methoxybenzoate through methyl esterification reaction, neutralization, extraction, and silica gel column purification, treating methyl 4-hydroxy-3-methoxybenzoate with an acetone solution of potassium carbonate, adding tetradecane bromide dropwise, refluxing for hydroxyalkylation reaction, extracting with ethyl acetate, drying under reduced pressure, and purifying with a silica gel column to obtain methyl 4-tetradecyloxy-3-methoxybenzoate; 1M sodium hydroxide aqueous solution was added dropwise to an ice-cooled methanol solution of 4-tetradecyloxy-3-methoxybenzoic acid, ester hydrolysis was performed under alkaline conditions, and then neutralized with 1M hydrochloric acid to obtain 4-tetradecyloxy-3-methoxybenzoic acid; N,N'-dicyclohexylcarbodiimide is added to an ice-cold dichloromethane solution of 4-tetradecyloxy-3-methoxybenzoic acid containing 4-dimethylaminopyridine, reacted at room temperature for 24 hours, filtered, the filtrate is dried under reduced pressure, and the residue is purified by silica gel column chromatography to obtain a compound of formula (I).

3. The method of claim 2, wherein: In the methyl esterification reaction of vanillic acid, p-toluenesulfonic acid is used as a catalyst, and the added molar amount thereof is 10% of the vanillic acid.

4. Use of the vanillic acid derivative according to claim 1 in the preparation of amphotericin B and fluconazole synergistic antibacterial drug synergist.

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