Preparation and application of ferulic acid-biotin conjugate

Abstract

The invention provides a preparation method and application of a ferulic acid-biotin conjugate, and relates to the field of cancer treatment. According to the invention,-COOH of ferulic acid and-OH or-NH2 of a connexon are esterified or amidated, and then esterified or amidated with-COOH of biotin, so that seven micromolecular prodrug conjugates are prepared. The reaction conditions are mild, and the prepared prodrug conjugate enhances the targeting property of ferulic acid, enhances the uptake rate of ferulic acid by tumor cells and improves the toxicity of ferulic acid to tumor cells, so that the prodrug conjugate has wide application prospects in the field of anti-tumor treatment.

Description

Technical Field

[0001] The invention relates to the field of anticancer pharmaceutical chemicals and relates to the preparation and application of ferulic acid-biotin conjugates. Background Art

[0002] Ferulic acid (Ferulic Acid, structural formula is as formula A) chemical name is 4-hydroxy-3-methoxycinnamic acid, molecular formula is C 10 H 10 O4 is a hydroxycinnamic acid widely found in the plant kingdom. One of the most common natural sources is bran and the outer layer of grains, such as wheat, rice and oats, citrus fruits and plants such as spinach. It is also one of the active ingredients of Chinese herbal medicines such as Angelica sinensis and Chuanxiong. Ferulic acid is divided into two structures, cis and trans, and is one of the byproducts of phenylalanine and tyrosine metabolism. Ferulic acid has good anti-inflammatory, analgesic, immune-enhancing, antioxidant and anticancer effects, and the anticancer effect is mainly exerted by inducing apoptosis and cell cycle arrest. Compared with cis-ferulic acid, trans-ferulic acid is easier to obtain in natural plants, and its pharmacological effects such as anti-inflammatory, antioxidant and anticancer are stronger than cis-ferulic acid, especially in terms of anticancer effect, trans-ferulic acid shows good effects in inhibiting the proliferation of tumors such as lung cancer, colorectal cancer, cervical cancer, etc., migration, etc., and has the effect of inducing apoptosis of cancer cells. However, ferulic acid has poor targeting to tumor cells, and there is no obvious difference in its uptake between tumor cells and normal cells, which causes ferulic acid to produce certain toxic effects on normal cells during anticancer treatment, thus limiting its application.

[0003] Biotin (Biotin, structural formula is as formula B) is a water-soluble vitamin, also known as vitamin B7 or vitamin H. Biotin participates in the metabolism of the three major nutrients in the body and is an important nutrient that is indispensable to the animal body. A large number of studies have shown that biotin has significant advantages as a targeting molecule and can improve the targeting of drug molecules. This is because the transmembrane transport of biotin often requires the participation of a sodium-dependent multivitamin transporter (SMVT), and the expression of this receptor has been found in human epithelial cells, endothelial cells and cancer cells. Some of these receptors have a high affinity for biotin, also known as biotin receptors (BR). There are overexpressed BRs on the surface of many cancer cells, such as MCF-7 and MBA-MB-231 human breast cancer cells, human cervical cancer Hela cells, etc., so biotin can specifically bind to these cells. When drug molecules bind to biotin to form biotin conjugates, the drug molecules can be specifically located on the surface of tumor cells through the mediation of biotin receptors. The prodrug conjugate formed by coupling biotin and ferulic acid can enhance the targeting of the drug and increase the uptake rate of tumor cells, increase the accumulation of the drug in tumor tissue, improve the efficacy, and reduce the toxicity to normal tissues.

[0004] The ideal prodrug remains stable in the circulatory system, can specifically release the drug after reaching the lesion site, increase the accumulation of the drug in the lesion site, and has only minimal or no toxic side effects on normal tissues. The concentration of intracellular glutathione (GSH) is 100-1000 times higher than that outside the cell, and the GSH in tumor cells is 2-5 times higher than that in normal cells. GSH is reducing, which can reduce disulfide bonds and thioether bonds to sulfhydryl groups, prompting the rapid release of drugs. In addition, many tumor cells have high levels of reactive oxygen species (ROS), which can promote the degradation of sulfur-containing (disulfide bonds and thioether bonds) linkers, accelerate the release of active drugs, and exert anti-tumor effects. This study designed to couple ferulic acid with biotin through linkers containing disulfide bonds or thioether bonds to form a prodrug, so that the drug can be targeted and bound to biotin receptor (BR)-positive cells, and respond to high levels of GSH and ROS in tumor cells, accurately and quickly release the drug, improve the targeting of ferulic acid, enhance the accumulation of drugs in tumor cells, improve the anti-cancer effect, and reduce toxic side effects. Summary of the invention

[0005] Tumor cells have excessive glutathione (GSH), and some tumor cell membranes have high levels of biotin receptor (BR) expression. In addition, the tumor microenvironment presents a higher level of ROS than normal tissues. Therefore, the present invention can better achieve the effect of inhibiting tumors by coupling ferulic acid with a series of redox-sensitive linkers and biotin.

[0006] The present invention provides a method for preparing a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate has structural formulas such as A1, A2, and A3, and intermediates in the reaction process such as B1, B2, B3, B4, and B5.

[0007] Terminology Note:

[0008] A1: Ferulic acid coupled to biotin, linker is HO-RXR-OH (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1, 2, 3, 4, 5, 6)).

[0009] A2: Ferulic acid coupled to biotin, linker is H2N-RXR-NH2 (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1, 2, 3, 4, 5, 6)).

[0010] A3: Ferulic acid coupled to biotin, the linker is NH2-R-OH (R = (-CH2-) n , (n=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)).

[0011] B1: Asafoetida coupled to aliphatic diol, linker is HO-RXR-OH (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1, 2, 3, 4, 5, 6)).

[0012] B2: Ferulic acid coupled with N-hydroxysuccinimide (NHS).

[0013] B3: Ferulic acid coupled to aliphatic diamine, linker is H2N-RXR-NH2 (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1, 2, 3, 4, 5, 6)).

[0014] B4: Biotin coupled with N-hydroxysuccinimide (NHS).

[0015] B5: Ferulic acid coupled to aliphatic amino alcohol, the linker is NH2-R-OH (R = (-CH2-) n , (n=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)).

[0016] The compound numbers of the present invention completely correspond to the structural formula numbers and have the same reference relationship, and are based on the compound structural formula.

[0017] The present invention provides the following technical solutions:

[0018] A method for preparing a ferulic acid-biotin conjugate, wherein the method for preparing a ferulic acid-biotin conjugate shown in A1 comprises the following steps: 1) Ferulic acid and HO-RXR-OH (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1-6)) is dissolved in an inert solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain B1; 2) B1 and biotin are dissolved in an inert solvent, and esterified in the presence of a catalyst and a dehydrating agent to obtain A1.

[0019] The preparation method of the ferulic acid-biotin conjugate shown in A2 comprises the following steps: 1) dissolving ferulic acid and N-hydroxysuccinimide (NHS) in an inert solvent, and esterifying them in the presence of a dehydrating agent to obtain feruloyl-N-hydroxysuccinimide B2; 2) B2 and H2N-RXR-NH2 (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1-6)) is dissolved in an inert solvent and amidated in the presence of an organic base to obtain B3; 3) dissolving biotin and N-hydroxysuccinimide (NHS) in an inert solvent, and esterifying them in the presence of a dehydrating agent to obtain biotin-N-hydroxysuccinimide B4; 4) B3 and B4 are dissolved in an inert solvent and esterified in the presence of an organic base to obtain A2.

[0020] The preparation method of the ferulic acid-biotin conjugate shown in A3 comprises the following steps: 1) dissolving ferulic acid and N-hydroxysuccinimide (NHS) in an inert solvent, and esterifying them in the presence of a dehydrating agent to obtain feruloyl-N-hydroxysuccinimide B2; 2) B7 and NH2-R-OH (R = (-CH2-) n , (n=2-12)) is dissolved in an inert solvent and amidated in the presence of an organic base to obtain B5; 3) B5 and biotin are dissolved in an inert solvent, and esterified in the presence of a catalyst and a dehydrating agent to obtain A3.

[0021] Preferably, the catalyst in step 1) of the reaction of the above-mentioned conjugate A1 is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N The dehydrating agent is selected from one of N, N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N, N'-diisopropylcarbodiimide (DIC), and 1, 1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of ethyl acetate, acetonitrile or N, N-dimethylformamide, but is not limited to these solvents.

[0022] Preferably, the catalyst in step 2) of the reaction of the conjugate A1 is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N The dehydrating agent is selected from one of N, N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N, N'-diisopropylcarbodiimide (DIC), and 1, 1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of N, N-dimethylformamide and tetrahydrofuran, but is not limited to these solvents.

[0023] Preferably, the dehydrating agent in the reaction step 1) of the above-mentioned conjugate A2 is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), and 1,1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of dichloromethane, chloroform or N,N-dimethylformamide, but is not limited to these solvents.

[0024] Preferably, the organic base in the reaction step 2) of the conjugate A2 is selected from one of triethylamine (TEA), pyridine, and N,N-diisopropylethylamine (DIPEA), and can also be carried out in an inorganic weak alkaline solvent, the inorganic weak alkaline solvent is selected from one of sodium bicarbonate (NaHCO3) solution, sodium carbonate (Na2CO3) solution, sodium bisulfite solution (NaHSO3) or disodium hydrogen phosphate (Na2HPO4), but not limited to these solutions; the inert solvent is selected from one of dimethyl sulfoxide or N,N-dimethylformamide, but not limited to these solvents.

[0024] Preferably, the dehydrating agent in the reaction step 3) of the conjugate A2 is selected from one of N, N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N, N'-diisopropylcarbodiimide (DIC), and 1,1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of dichloromethane, chloroform, ethyl acetate or N, N-dimethylformamide, but is not limited to these solvents.

[0025] Preferably, the catalyst of the reaction step 4) of the conjugate A2 is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N'-tetramethyluronium benzotriazole-1-yl hexafluorophosphate (HBTU); the dehydrating agent is selected from N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI); the organic base is selected from one of triethylamine (TEA), pyridine (Pyridine), N,N-diisopropylethylamine (DIPEA), and can also be carried out in an inorganic weak alkaline solvent, the inorganic weak alkaline solvent is selected from one of sodium bicarbonate (NaHCO3) solution, sodium carbonate (Na2CO3) solution, sodium bisulfite solution (NaHSO3) or disodium hydrogen phosphate (Na2HPO4), but not limited to these solutions; the inert solvent is selected from one of acetone, dimethyl sulfoxide or N,N-dimethylformamide, but not limited to these solvents.

[0026] Preferably, the dehydrating agent in the reaction step 1) of the above-mentioned conjugate A3 is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), and 1,1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of dichloromethane, chloroform or N,N-dimethylformamide, but is not limited to these solvents.

[0027] Preferably, the organic base in the reaction step 2) of the conjugate A3 is selected from one of triethylamine (TEA), pyridine, and N,N-diisopropylethylamine (DIPEA), and can also be carried out in an inorganic weak alkaline solvent, the inorganic weak alkaline solvent is selected from one of sodium bicarbonate (NaHCO3) solution, sodium carbonate (Na2CO3) solution, sodium bisulfite solution (NaHSO3) or disodium hydrogen phosphate (Na2HPO4), but not limited to these solutions; the inert solvent is selected from one of dimethyl sulfoxide or N,N-dimethylformamide, but not limited to these solvents.

[0028] Preferably, the catalyst in step 3) of the reaction of the conjugate A3 is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), The dehydrating agent is selected from one of N, N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N, N'-diisopropylcarbodiimide (DIC), and 1,1'-carbonyldiimidazole (CDI); the inert solvent is selected from one of acetone, tetrahydrofuran, dimethyl sulfoxide or N, N-dimethylformamide, but is not limited to these solvents.

[0029] The reaction temperature of the above reaction step is 5 to 85°C.

[0030] Advantageous Effects of the Invention

[0031] The invention provides a preparation method and application of ferulic acid-biotin conjugates. The method esterifies -COOH of ferulic acid with -OH of aliphatic diol through esterification reaction, and then esterifies with biotin; or amidates -COOH of ferulic acid with -NH2 of aliphatic diamine, and then amidates with biotin; or amidates -COOH of ferulic acid with -NH2 of aliphatic amine alcohol, and then esterifies with biotin. Thus, seven small molecule prodrug conjugates are prepared. The prepared small molecule prodrug conjugates have significant beneficial effects. They can specifically target biotin receptors on the surface of tumor cells through the targeting effect of biotin, thereby improving the tumor targeting of ferulic acid. In addition, the ferulic acid-biotin conjugate connected by disulfide bonds and thioether bonds has the characteristics of GSH and ROS stimulation response release, can respond to the high GSH and high ROS environment in tumor cells, quickly release drugs, and enhance the anti-tumor effect of ferulic acid, but the GSH and ROS content in normal cells are relatively low, the drug release is slow, and the toxicity to normal tissues can be reduced. The preparation method of the present invention has mild conditions, can enhance the targeting of ferulic acid, enhance the uptake rate of ferulic acid by tumor cells, improve the inhibition rate of ferulic acid on tumor cells, enhance the specific release in tumor cells, improve the therapeutic effect, and reduce toxic side effects. Therefore, the invention has important application prospects and beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Figure 24-hour cumulative drug release trends of ferulic acid, A1a, A1b, A1c, A2a, A2b, A2c, and A3a in 2 mM (a) and 5 mM (b) GSH environments, 2 mM (c) and 5 mM (d) H2O2 environments, and PBS buffer solution in vitro.

[0033] Figure 2 The cytotoxicity of ferulic acid, A1a, A1b, A1c, A2a, A2b, A2c, A3a at different concentrations (25μM, 50μM, 100μM, 150μM, 200μM, 300μM) on HeLa (a), MCF-7 (b) and HCT-116 (c) cells.

[0034] Figure 3 The cytotoxicity of A1a(a), A1b(b), A1c(c), A2a(d), A2b(e), A2c(f), and A3a(g) on ​​HeLa cells after pretreatment with different concentrations of biotin (0μM, 2μM, 5μM, 10μM).

[0035] Figure 4The cytotoxicity of A1a(a), A1b(b), A1c(c), A2a(d), A2b(e), A2c(f), and A3a(g) on ​​MCF-7 cells after pretreatment with different concentrations of biotin (0μM, 2μM, 5μM, 10μM).

[0036] Figure 5 The cytotoxicity of A1a(a), A1b(b), A1c(c), A2a(d), A2b(e), A2c(f), and A3a(g) on ​​HCT-116 cells after pretreatment with different concentrations of biotin (0μM, 2μM, 5μM, 10μM). DETAILED DESCRIPTION

[0037] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto. Example 1

[0038] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) and dissolve it in ethyl acetate. Take 125 μL (2 eq) of bis(2-hydroxyethyl) disulfide, weigh 73.3 mg (1.2 eq) of 4-dimethylaminopyridine (DMAP), and 154.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide (DCC) and add them to a flask. Add ethyl acetate and stir at 30°C in the dark. Slowly drip the ferulic acid solution into the flask. The process lasts for about 10 minutes. Stir at 30°C for 4 hours. The reaction solution turns from clear to turbid yellow. TLC monitors that the reaction of ferulic acid is complete and the reaction is stopped. Filter the reaction solution and take the filtrate. Purify by silica gel column chromatography (dichloromethane: ethyl acetate = 8:1), and remove the solvent by rotary evaporation to obtain ferulic acid-2-hydroxyethyl dithiomonoester B1a. 2) Weigh 99.2 mg (0.3 mmol) of the above-obtained ferulic acid-2-hydroxyethyl dithiomonoester B1a into a 50 ml round-bottom flask, add 80.6 mg (1.1 eq) of biotin, 55.0 mg (1.5 eq) of 4-dimethylaminopyridine (DMAP), and 69.0 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and then add N,N-dimethylformamide (DMF). Stir at 30°C in the dark for 12 h to obtain a clear and transparent reaction solution. Purify by silica gel column chromatography (dichloromethane: methanol = 15: 1), and remove the solvent by rotary evaporation to obtain ferulic acid-2-hydroxyethyl dithio-biotin conjugate A1a. A1a's NMR data: 1 H NMR (400MHz, DMSO-d6) δ9.64(s,1H),7.59(dd,J=16.1,0.7Hz,1H),7.13(d,J=1.7Hz,1H),7.02–6.97(m,1H),6.86(d,J =8.2Hz,1H),6.40(d,J=16.1Hz,1H),6.24(d,J=5.7Hz,1H),6.14(d,J=5.3Hz,1H),4.46(dt,J=5.7,1.5Hz,1H),4.33(dt ,J=5.7,1.5Hz,1H),4.24(t,J=4.8Hz,2H),4.13(m,2H),3.83(s,2H),3.12(tdd,J=4.8,2.2,0.7Hz,1H),3.05–2.92(m, 4H), 2.84 (dd, J=12.4, 2.1Hz, 1H), 2.73 (d, J=12.4Hz, 1H), 2.32 (t, J=8.3Hz, 2H), 1.63–1.50 (m, 4H), 1.48–1.36 (m, 2H). Example 2

[0039] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) and dissolve it in ethyl acetate. Take 100 μl (2 eq) of 2,2′-dithiodiethanol, weigh 73.3 mg (1.2 eq) of 4-dimethylaminopyridine (DMAP), and 154.7 mg (1.5 eq) of N,N′-dicyclohexylcarbodiimide (DC C) and add them to a flask. Add ethyl acetate and stir at 30°C in the dark. Slowly drip the ferulic acid solution into the flask. The process lasts for about 10 minutes. Stir at 30°C for 4 hours. The reaction solution turns from clear to turbid yellow. TLC monitors that the reaction of ferulic acid is complete and the reaction is stopped. Filter the reaction solution and take the filtrate. Purify by silica gel column chromatography (dichloromethane: ethyl acetate = 8:1), and remove the solvent by rotary evaporation to obtain ferulic acid-2-hydroxyethyl thiomonoester B5a. 2) 89.5 mg (0.3 mmol) of the above-obtained ferulic acid-2-hydroxyethyl thiomonoester B5a was weighed into a flask, and 80.6 mg (1.1 eq) of biotin, 55.0 mg (1.5 eq) of 4-dimethylaminopyridine (DMAP), and 69.0 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added, dissolved in N,N-dimethylformamide (DMF), and stirred at 30°C in the dark for 12 h to obtain a clear and transparent reaction solution, which was purified by silica gel column chromatography (dichloromethane: methanol = 15: 1) to obtain ferulic acid-2-hydroxyethyl thio-biotin conjugate A1b. A3a's NMR data: 1 H NMR(400MHz, DMSO-d6)δ9.64(s,1H),7.59(dd,J=16.1,0.7Hz,1H),7.13(d,J=1.7Hz,1H),7.02–6.97(m,1H),6.86( d,J=8.2Hz,1H),6.41(d,J=16.1Hz,1H),6.24(d,J=5.7Hz,1H),6.14(d,J=5.3Hz,1H),4.46(dt,J=5.7,1.5Hz,1H), 4.33(dt,J=5.7,1.5Hz,1H),4.23–4.17(m,4H),3.83(s,2H),3.12(tdd,J=4.8,2.2,0.7Hz,1H),3.05–2.92(m,4H), 2.84(dd,J=12.4,2.1Hz,1H),2.73(d,J=12.4Hz,1H),2.32(t,J=8.3Hz,2H),1.63–1.50(m,4H),1.48–1.36(m,2H). Example 3

[0040] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) and dissolve it in ethyl acetate. Weigh 118.2 mg (2 eq) of 1,6-hexanediol, 73.3 mg (1.2 eq) of 4-dimethylaminopyridine (DMAP), and 154.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide (DCC) and add them to a flask, add ethyl acetate, and stir at 30°C in a dark place. Slowly drip the ferulic acid solution into the flask, and the process lasts for about 10 minutes. Stir at 30°C for 4 hours. The reaction solution turns from clear to turbid yellow. TLC monitors that the reaction of ferulic acid is complete and the reaction is stopped. Filter the reaction solution and take the filtrate. Purify by silica gel column chromatography (dichloromethane: ethyl acetate = 8:1), and remove the solvent by rotary evaporation to obtain ferulic acid-hexanediol monoester B7a. 2) 88.3 mg (0.3 mmol) of the ferulic acid-hexanediol monoester B1c obtained above was weighed into a flask, and 80.6 mg (1.1 eq) of biotin, 55.0 mg (1.5 eq) of 4-dimethylaminopyridine (DMAP), and 69.0 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added, and then N,N-dimethylformamide (DMF) was added. The mixture was stirred at 30°C in the dark for 12 h to obtain a clear and transparent reaction solution, which was purified by silica gel column chromatography (dichloromethane: methanol = 15: 1) to obtain ferulic acid-hexanediol-biotin conjugate A1c. A1c NMR data: 1H NMR(400MHz,Chloroform-d)δ7.64(d,J=16.0Hz,1H),7.59(dd,J=16.1,0.7Hz,1H),7.13(d,J=1.7Hz,1H) ,7.02–6.97(m,1H),6.86(d,J=8.2Hz,1H),6.40(d,J=16.1Hz,1H),6.24(d,J=5.7Hz,1H),4.98(d,J=5.7H z,1H),4.66(dt,J=5.7,1.5Hz,1H),4.36(dd,J=5.3,2.1Hz,1H),4.21(t,J=6.3H z,2H),3.83(s,2H),3.66(t,J=6.2Hz,2H),3.20(tdd,J=4.8,2.2,0.7Hz,1H),2.94(dd,J=12.4,2.1 Hz,1H),2.74(d,J=12.4Hz,1H),2.62(t,J=7.2Hz,2H),1.83-1.50(m,9H),1.45(m,2H),1.29(m,4H). Example 4

[0041] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) and dissolve it in ethyl acetate. Weigh 63.3 mg (1.1 eq) of N-hydroxysuccinimide (NHS) and 154.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide (DCC) and add them to a bottle, and stir at 30°C in the dark. When the ferulic acid reaction is complete as monitored by TLC, the reaction is stopped, the reaction solution is filtered, and the filtrate is slowly dripped into a large amount of cold ether while stirring. A large amount of white precipitate is seen to precipitate, and stirring is continued for 30 min. Filter and dry the solid to obtain feruloyl-N-hydroxysuccinimide B2. 2) Weigh 101.9 mg (0.35 mmol) of the above-obtained feruloyl-N-hydroxysuccinimide B2 into a flask, add 157.6 mg (2 eq) of cystamine dihydrochloride (CYS), 70.9 mg (1.5 eq) of 11-hydroxybenzotriazole (HOBt), 110.6 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), add N,N-dimethylformamide (DMF), and then add 200 μl of dehydrated triethylamine, stir at 30°C in the dark for 12 h, slowly drop the reaction solution into ice water, stir while dropping, and continue stirring for 30 min until a light yellow precipitate is precipitated. Filter and dry the solid to obtain ferulic acid-cystamine conjugate B3a. 3) Weigh 122.2 mg (0.5 mmol) of biotin, 63.3 mg (1.1 eq) of N-hydroxysuccinimide (NHS), and 154.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide (DCC) into a flask, add 2 ml of N,N-dimethylformamide (DMF), and stir at 55°C for 4 h. When the biotin reaction is complete by TLC, stop the reaction, filter the reaction solution, slowly drop the filtrate into a large amount of cold ether, continue stirring, and a large amount of white precipitate can be seen. Continue stirring for 30 min. Filter and dry the solid to obtain biotin-N-hydroxysuccinimide B4. 4) Weigh 85.2 mg (0.25 mmol) of biotin-N-hydroxysuccinimide B4 and dissolve it in 1 ml of N,N-dimethylformamide (DMF). Weigh 82.1 mg of B3a and dissolve it in 2 ml of NaHCO3 (1 mmol / ml). Then add the B3a solution to the B4 solution and stir at 30°C for 24 hours. Slowly drip the reaction solution into a large amount of citric acid aqueous solution (100 g / L) while stirring. Adjust the temperature to 60°C and continue stirring for 30 minutes. A large amount of white precipitate can be seen. Filter and dry the solid to obtain ferulic acid-cystamine-biotin conjugate A2a. A2a's NMR data: 1H NMR (400MHz, DMSO-d6) δ 9.64 (s, 1H), 7.59 (dd, J = 16.1, 0.7Hz, 1H), 7.28 (s, J = 1.7Hz, 1H), 7.21 (dt, 1H), 7. 12(d,J=1.7Hz,1H),6.87(d,J=8.2Hz,1H),6.74(tdd,J=8.2Hz,1H),6.54(d,J=16.1Hz,1H), 5.84(d,J=5.7H z,2H),5.74(d,J=5.3Hz,2H),4.46(dt,J=5.7,1.5Hz,1H),4.33(dt,J=5.7,1.5Hz,1H),3.83(s,3H),3.45–3 .30(m,4H),3.12(tdd,J=4.8,2.2,0.7Hz,1H),2.88–2.72(m,6H),2.18(t,J=8.2Hz,2H),1.66–1.38(m,6H). Example 5

[0042] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Repeat step 1) of Example 4. 2) Weigh 1.9 mg (0.35 mmol) of the above-obtained feruloyl-N-hydroxysuccinimide B210 into a flask, add 85 μl (2 eq) of 2-aminoethyl sulfide, 70.9 mg (1.5 eq) of 11-hydroxybenzotriazole (HOBt), 110.6 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), add N,N-dimethylformamide (DMF), and then add 200 μl of dehydrated triethylamine, stir at 30°C in the dark for 12 h, slowly add the reaction solution dropwise into 30 ml of ice water, continue stirring, and a light yellow precipitate is visible, and continue stirring for 30 min. Filter and dry the solid to obtain feruloyl-ethyl sulfide ethylamine B3b. 3) Repeat step 3) of Example 4. 4) Weigh 85.2 mg (0.25 mmol) of biotin-N-hydroxysuccinimide B4 and dissolve it in 1 ml of N,N-dimethylformamide (DMF). Weigh 74.1 mg of B6a and dissolve it in 2 ml of NaHCO3 (1 mmol / ml). Then add the B6a solution to the B4 solution and stir at 30°C for 24 h. Slowly drop the reaction solution into 15 ml of citric acid aqueous solution (100 g / L) and continue stirring. Adjust the temperature to 60°C and continue stirring for 30 min. A large amount of white precipitate can be seen. Filter and dry the solid to obtain ferulic acid-aminoethyl sulfide-biotin conjugate A2b. A2b's NMR data: 1 H NMR (400MHz, DMSO-d6) δ 9.64 (s, 1H), 7.59 (dd, J = 16.1, 0.7Hz, 1H), 7.28 (s, J = 1.7Hz, 1H), 7.21 (dt, 1H), 7. 12(d,J=1.7Hz,1H),6.87(d,J=8.2Hz,1H),6.73(tdd,J=8.2Hz,1H),6.54(d,J=16.1Hz,1H), 5.84(d,J=5.7H z,2H),5.74(d,J=5.3Hz,2H),4.46(dt,J=5.7,1.5Hz,1H),4.26(dt,J=5.7,1.5Hz,1H),3.82(s,3H),3.45–3 .30(m,4H),3.12(tdd,J=4.8,2.2,0.7Hz,1H),2.88–2.72(m,6H),2.18(t,J=8.2Hz,2H),1.66–1.38(m,6H). Example 6

[0043] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) in a flask and dissolve in anhydrous acetone. Weigh 63.3 mg (1.1 eq) of N-hydroxysuccinimide (NHS) and 154.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide (DCC) in a flask, stir at 30°C in the dark, and react overnight. After the ferulic acid reaction is complete, add 116.2 mg (2 eq) of 1,6-hexanediamine to the reaction, and continue the reaction for 24 h. Slowly add the reaction solution dropwise to 30 ml of ice water, continue stirring, and a light yellow precipitate is visible. Continue stirring for 30 min. Filter and dry the solid to obtain feruloyl-hexane monoamide B3c. 2) Repeat step 3) of Example 2. 3) Weigh 85.2 mg (0.25 mmol) of biotin-N-hydroxysuccinimide B4 and dissolve it in N,N-dimethylformamide (DMF). Weigh 73.1 mg of B8a and dissolve it in 2 ml of NaHCO3 (1 mmol / ml). Then add the B8a solution to the B4 solution and stir at 30°C for 24 hours. Slowly drop the reaction solution into a large amount of citric acid aqueous solution (100 g / L) and continue stirring. Adjust the temperature to 60°C and continue stirring for 30 minutes. A large amount of white precipitate can be seen. Filter and dry the solid to obtain feruloyl-adipamide-biotin conjugate A2c. A2c's NMR data: 1 H NMR (400MHz, DMSO-d6) δ9.64 (s, 1H), 7.59 (dd, J = 16.1, 0.7Hz, 1H), 7.28 (s, J = 1.7Hz, 1H), 7.21 (dt, 1H), 7.12 (d, J = 1. 7Hz, 1H), 6.87 (d, J = 8.2Hz, 1H), 6.64 (tdd, J = 8.2Hz, 1H), 6.48 (d, J = 16.1Hz, 1H), 5.84 (d, J = 5.7Hz, 2H), 5.74 (d, J = 5.3 Hz,2H),4.46(dt,J=5.7,1.5Hz,1H),4.26(dt,J=5.7,1.5Hz,1H),3.83(s,3H),3.18–3.12(m,5H),2.88–2.72(m,2H), 2.18(t,J=8.2Hz,2H),1.66–1.58(m,2H),1.57–1.52(m,2H),1.52–1.49(m,4H),1.49–1.43(m,2H),1.38–1.27(m,4H). Example 7

[0044] A preparation method and application of a ferulic acid-biotin conjugate, wherein the ferulic acid-biotin conjugate comprises the following steps: 1) Weigh 97.1 mg (0.5 mmol) of ferulic acid (FA) in a flask, add 58.6 mg (1 eq) of 6-amino-1-hexanol dissolved in N,N-dimethylformamide (DMF), then add 200 μl of dehydrated triethylamine, stir at 55°C in the dark for 6 h, stop the reaction when TLC detects that FA is completely reacted, remove most of the solvent by evaporation under reduced pressure, slowly add the reaction solution dropwise into a large amount of ice water, continue stirring, and a light yellow precipitate is visible, and continue stirring for 30 min. Filter and dry the solid to obtain feruloyl-hexanol B5a. 2) Weigh 88.1 mg (0.3 mmol) of the ferulic acid-hexanol B5a obtained above into a flask, add 80.6 mg (1.1 eq) of biotin, 55.0 mg (1.5 eq) of 4-dimethylaminopyridine (DMAP), 69.0 mg (1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and then add N,N-dimethylformamide (DMF). Stir at 30°C in the dark for 12 h to obtain a clear and transparent reaction solution. Purify by silica gel column chromatography (dichloromethane: methanol = 15: 1), and remove the solvent by rotary evaporation to obtain ferulic acid-hexanediol-biotin conjugate A3a. A3a's NMR data: 1 H NMR (400MHz, DMSO-d6) δ9.64 (s, 1H), 7.59 (dd, J = 16.1, 0.7Hz, 1H), 7.28 (s, J = 1.7Hz, 1H), 7.21 (dt, 1H), 7.12 (d, J = 1.7Hz, 1H), 6. 87(d,J=8.2Hz,1H),6.73(tdd,J=8.2Hz,1H),6.35(d,J=16.1Hz,1H),5.84(d,J=5.7Hz,2H),5.74(d,J=5.3Hz,2H),4.46(dt,J=5.7 ,1.5Hz,1H),4.26(dt,J=5.7,1.5Hz,1H),4.02(td,J=6.2,0.8Hz,2H),3.83(s,3H),3.24–3.14(m,2H),3.12(tdd,J=4.8,2.2,0.7 Hz,1H),2.84–2.73(m,2H),2.88–2.72(m,2H),2.32(t,J=8.2Hz,2H),2.18(t,J=8.2Hz,2H),1.67–1.46(m,8H),1.45–1.33(m,6H). Example 8

[0045] In vitro release assay The redox responsiveness of the conjugate was studied by direct release method. PBS buffer solution containing 30% ethanol was used as the release medium. A1a, A1b, A1c, A2a, A2b, A2c and A3a were dissolved in 40mL of release medium: GSH (2mM or 5mM), H2O2 (2mM or 5mM), and PBS solution was used as control, and shaken at 37℃ (100rpm). 100μL of release solution was taken out at 0min, 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 16h, 24h, and the same volume of fresh release medium was added. The drug content was determined by high performance liquid chromatography. Example 9

[0046] Cytotoxicity assay HeLa cells were seeded in 96-well plates at a density of 5 × 10 3 cells, each well containing 100 μL of culture medium, and incubated for 24 hours. Subsequently, ferulic acid (FA), conjugates A1a, A1b, A1c, A2a, A2b, A2c, A3a were prepared using DMEM culture medium containing 10% fetal bovine serum and added to a 96-well plate, 100 μL of drug culture medium mixture per well, and the final drug concentration in the well was 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 3 replicates were set, and a blank control group was set. Subsequently, the 96-well plate was placed in a CO2 incubator at 37°C and incubated for 24 hours. After the incubation, 20 μL of MTT (concentration of 5 mg / ml) was added to each well and continued to incubate at 37°C for 4 hours. Then, the culture medium was removed, 150 μL of DMSO was added, and the absorbance was measured at a wavelength of 570 nm using an enzyme reader to calculate the cell viability. The cytotoxicity of MCF-7 cells and HCT-116 cells was determined in the same manner. To further verify the tumor targeting of biotin, HeLa cells were seeded in 96-well plates at a seeding density of 5 × 10 3cells, each well containing 100 μL of culture medium, and incubated for 24 hours. Different concentrations of biotin (0, 20, 50, 100 μM) were prepared with DMEM culture medium and added to 96-well plates, 20 μL per well. The cells were then cultured for 2 hours under 5% CO2 and 37°C to allow the biotin receptors on the surface of the cancer cells to bind to different concentrations of biotin. Subsequently, ferulic acid (FA), conjugates A1a, A1b, A1c, A2a, A2b, A2c, A3a were prepared with DMEM culture medium and added to 96-well plates, 80 μL of drug culture medium mixture per well, and the final drug concentrations in the wells were 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, and the biotin concentrations were 0, 2, 5, and 10 mM, respectively. Three replicate wells were set up, and a control group was set up. Then continue to culture for 24 hours. The absorbance was measured at 570 nm using an ELISA reader to calculate cell viability. MCF-7 and HCT-116 cells were assayed using the same method.

[0047] according to Figure 1 The results of a and b show that the release of A1a in 2mM GSH for 24 hours is 78.93%, while in 5mM GSH, the complete release is achieved in 16 hours; the release of A1b in 2mM GSH for 24 hours is 18.33%, while in 5mM GSH, the release is 26.37%; the release of A1c in 2mM GSH for 24 hours is 10.83%, while in 5mM GSH, the release is 12.73%; the release of A2a in 2mM GSH for 24 hours is The release of A1a, A1b, A2a and A2b in 2mM GSH was 88.13%, while it was completely released in 12h in 5mM GSH; the release of A2b in 2mM GSH was 19.67% in 24h, while it was 29% in 5mM GSH; the release of A2c in 2mM GSH was 11.4% in 24h, while it was 13.57% in 5mM GSH; the release of A3a in 2mM GSH was 12.45% in 24h, while it was 16.37% in 5mM GSH. Sulfur-containing A1a, A1b, A2a and A2b can be rapidly released in GSH and show dependence on GSH concentration, indicating that the sulfur in the linker makes the prodrug reduction-sensitive. A1a containing a disulfide bond is released faster than A1b containing a thioether bond, and A2a containing a disulfide bond is also released faster than A2b containing a thioether bond, indicating that disulfide bonds are more sensitive to GSH. Figure 1The results of c and d show that the release of A1a in 2mM H2O2 for 24 hours was 44.1%, while that in 5mM H2O2 for 24 hours was 54.07%; the release of A1b in 2mM H2O2 for 24 hours was 61.9%, while that in 5mM H2O2 could be completely released within 24 hours; the release of A1c in 2mM H2O2 for 24 hours was 10.37%, while that in 5mM H2O2 for 24 hours was 15.07%; the release of A2a in 2mM H2O2 for 24 hours was 4 9.9% in 24h in 5mM H2O2, while the release in 24h in 24h in 5mM H2O2 was 61.73%; the release in 24h in 2mM H2O2 of A2b was 71.07%, while it was completely released in 16h in 5mM H2O2; the release in 24h in 2mM H2O2 of A2c was 11.61%, while it was 16.55% in 24h in 5mM H2O2; the release in 24h in 2mM H2O2 of A3a was 14.95% in 24h in 2mM H2O2, while it was 21.4% in 24h in 5mM H2O2. Sulfur-containing A1a, A1b, A2a, and A2b can be rapidly released in the environment of H2O2 and show dependence on H2O2 concentration, indicating that the sulfur in the linker makes the prodrug oxidatively sensitive. A1b containing a thioether bond is released faster than A1a containing a disulfide bond, and A2b containing a thioether bond is also released faster than A2a containing a disulfide bond, indicating that the thioether bond is more sensitive to H2O2. Figure 1 e is the release trend of the prodrug in PBS, the release amount of A1a in 24h is 7.1%; the release amount of A1b in 24h is 9.84%; the release amount of A1c in 24h is 5.48%; the release amount of A2a in 24h is 7.33%; the release amount of A2b in 24h is 10.2%; the release amount of A2c in 24h is 5.9%; the release amount of A3a in 24h is 6.57%. The release rate of the same prodrug in GSH and H2O2 is significantly higher than that in the PBS control group. It can be seen that the sulfur-containing conjugate prodrug is oxidatively and reductively sensitive, and can achieve rapid release under the conditions of high GSH and ROS in the tumor microenvironment or tumor cells, promote the accumulation of drugs in cells, and better exert the efficacy. However, it is slowly released or not released under physiological conditions or in normal tissues, thereby reducing its side effects on normal tissues.

[0048] according to Figure 2 As shown in Table 1, at the same concentration, the survival rates of all tumor cells treated with conjugates were significantly lower than those treated with ferulic acid (FA). In HeLa cells, in the group treated with conjugates containing a diester bond, the survival rates of cells treated with A1a and A1b were significantly lower than those treated with A1c. The IC 50 The value was 262.5 μM, while the IC 50The values ​​were only 226.0 μM, which were 36.4% and 31.3% of those in the FA group, respectively. In the groups treated with the conjugates containing double amide bonds, the cell survival rates after treatment with A2a and A2b were significantly lower than those in the A2c group. The IC 50 The value was 281.8 μM, while the IC 50 The value was only 244.0 μM, which was 39.1% and 33.8% of the FA group, respectively. This indicates that the presence of sulfur in the linker has the effect of enhancing the cytotoxicity of the drug. In the MCF-7 cell treatment group, the cell survival rate after treatment with A1a and A1b was significantly lower than that of the A1c treatment group. The IC 50 The value was 245.3 μM, while the IC 50 The value was only 201.6μM, which was 26.4% and 21.7% of that in the FA group, respectively. The cell survival rates after treatment with A2a and A2b were significantly lower than those after treatment with A2c. The IC 50 The value was 258.4 μM, while the IC 50 The value was only 220.7 μM, which was 27.8% and 23.7% of the FA group, respectively. In the HCT-116 cell treatment group, the cell survival rate after treatment with A1a and A1b was significantly lower than that in the A1c treatment group. The IC 50 The value was 175.5 μM, while the IC 50 The value was only 155.6 μM, which was 40.7% and 36.1% of that in the FA group, respectively. The cell survival rates after treatment with A2a and A2b were significantly lower than those after treatment with A2c. The IC 50 The value was 201.7 μM, while the IC 50The value was only 185.8 μM, which was 46.8% and 43.1% of that in the FA group, respectively. These results indicate that the presence of sulfur in the linker has the effect of enhancing the cytotoxicity of the drug. In particular, the cell survival rate of the A1b group containing a thioether bond was lower than that of the A1a group containing a disulfide bond, and the cell survival rate of the A2b group containing a thioether bond was lower than that of the A2b group containing a disulfide bond, indicating that the prodrug conjugates connected by thioether bonds have stronger cytotoxic effects than those connected by disulfide bonds. In addition, there was no significant difference in cell survival rate between the A1a and A2a groups, and between the A1b and A2b groups, indicating that ester bonds or amide bonds are not the key to affecting cell survival rate. Among all three types of cells, in the treatment group without sulfur in the linker, the cell survival rate of the A3a group was slightly lower than that of the A1c and A2c groups, indicating that the simultaneous presence of ester bonds and amide bonds can enhance the toxic effects of the drug to a certain extent. Among the three cell lines, HCT-116 cells were the most sensitive to the original drug ferulic acid, but the prodrug conjugate had the least effect on its cytotoxicity, which may be due to the lack of biotin receptor (BR) on the cell surface; the conjugate prodrug had a greater effect on the survival rate of MCF-7 cells than HeLa cells, which may be due to the fact that MCF-7 cells express BR more abundantly than HeLa cells. Table 1. IC50 of ferulic acid (FA) and prodrug conjugates A1a, A1b, A1c, A2a, A2b, A2c, A3a on HeLa, MCF-7 and HCT-116 cells 50 (μM)

[0049] Figure 3 The effects of the prodrug conjugates on the viability of HeLa cells under the inhibition of different concentrations of biotin were clearly demonstrated. The results showed that the viability of cells pretreated with biotin was higher than that of cells not pretreated with biotin, and showed a certain concentration dependence. With the gradual increase of biotin concentration, the viability of HeLa cells treated with A1a, A1b, A1c, A2a, A2b, A2c, and A3a showed an upward trend. The same situation also occurred in MCF-7 cells, such as Figure 4 As shown, cell viability increased with increasing concentrations of pre-treatment biotin. Figure 5In the study, there was no significant difference between the survival rate of cells pre-treated with biotin and those without biotin pretreatment in HCT-116 cells, and there was no significant difference in the effect of the same prodrug conjugate pre-treated with different concentrations of biotin on cell survival. This may be because HeLa cells and MCF-7 cells are biotin receptor (BR)-positive cells. After pre-treatment of cells with biotin, the biotin receptor (BR) on the cell surface is occupied, thereby reducing the uptake of drugs carrying biotin, and the drugs cannot quickly enter the cells and exert toxic effects. However, there are very few or no biotin receptors on the surface of HCT-116 cells, so the presence or absence of biotin has little effect on the uptake of drugs by HCT-116 cells, so the survival rate of cells does not change significantly. Figure 5 HCT-116 cells showed reduced cell viability in response to the prodrug conjugates. Figure 2 , probably because it has a strong sensitivity to ferulic acid (FA) itself, and the presence of sulfur enhances the cytotoxic effect of the drug. In general, the ferulic acid (FA) prodrug conjugates linked to biotin: A1a, A1b, A1c, A2a, A2b, A2c and A3a showed obvious biotin-dependent tumor targeting.

Claims

1. A preparation of a ferulic acid-biotin conjugate, characterized in that The conjugate is composed of -COOH of ferulic acid and a linker HO-RXR-OH (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n = 1 to 6)) -OH esterification, or with the linker H2N-RXR-NH2 (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1~6))'s -NH2 is amidated and then reacted with the -COOH of biotin to obtain products A1 and A2. Preparation of a ferulic acid-biotin conjugate, the conjugate is composed of -COOH of ferulic acid and a linker NH2-R-OH (R=(-CH2-) n , (n=2~12))'s -NH2 is amidated and then esterified with the -COOH of biotin to obtain A3.

2. The method for preparing a ferulic acid-biotin conjugate according to claim 1, characterized in that The preparation method of A1 comprises the following steps: 1) Ferulic acid and HO-RXR-OH (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1-6)) is dissolved in an inert solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain B1; 2) B1 and biotin are dissolved in an inert solvent, and esterified in the presence of a catalyst and a dehydrating agent to obtain A1.

3. The method for preparing a ferulic acid-biotin conjugate according to claim 1, characterized in that The preparation method of A2 comprises the following steps: 1) dissolving ferulic acid and N-hydroxysuccinimide (NHS) in an inert solvent, and subjecting them to amidation in the presence of a dehydrating agent to obtain feruloyl-N-hydroxysuccinimide B2; 2) B2 and H2N-RSSR-NH2 (X = -SS-, -S-, -CH2-, R = (-CH2-) n , (n=1-6)) is dissolved in an inert solvent and amidated in the presence of an organic base to obtain B3; 3) dissolving biotin and N-hydroxysuccinimide (NHS) in an inert solvent, and subjecting them to amidation in the presence of a dehydrating agent to obtain biotin-N-hydroxysuccinimide ester B4; 4) B3 and B4 are dissolved in an inert solvent, and amidated in the presence of a catalyst, a dehydrating agent and an organic base to obtain A2.

4. The method for preparing a ferulic acid-biotin conjugate according to claim 1, characterized in that The preparation method of A3 comprises the following steps: 1) dissolving ferulic acid and N-hydroxysuccinimide (NHS) in an inert solvent, and subjecting them to amidation in the presence of a dehydrating agent to obtain feruloyl-N-hydroxysuccinimide B2; 2) B2 and NH2-R-OH (R = (-CH2-) n , (n=2-12)) is dissolved in an inert solvent and amidated in the presence of an organic base to obtain B5; 3) B5 and biotin are dissolved in an inert solvent, and esterified in the presence of a catalyst and a dehydrating agent to obtain A3.

5. The preparation method according to claim 2, characterized in that: The catalyst is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N'-tetramethyluronium benzotriazole-1-yl hexafluorophosphate (HBTU); the dehydrating agent is selected from N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI); The preparation method according to claim 3, characterized in that: the catalyst is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N ,N,N',N'-tetramethylurea benzotriazole-1-yl hexafluorophosphate (HBTU); the dehydrating agent is selected from N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI); the organic base is selected from triethylamine (TEA), pyridine (Pyridi ne), N, N-diisopropylethylamine (DIPEA); the preparation method according to claim 4, characterized in that: the catalyst is selected from 4-dimethylaminopyridine (DMAP), 4-pyridinecarboxaldehyde (PPY), 1,8-diazabicycloundec-7-ene (DBU), 11-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(7-azabenzotriazole-1-yl)-N , one of N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N,N',N'-tetramethyluronium benzotriazole-1-yl hexafluorophosphate (HBTU); the dehydrating agent is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), and 1,1'-carbonyldiimidazole (CDI).

6. The preparation method according to claim 2, 3, or 4, characterized in that: The inert solvent is selected from one of ethyl acetate, dichloromethane, chloroform, acetonitrile, acetone, tetrahydrofuran, dimethyl sulfoxide or N,N-dimethylformamide, but is not limited to these solvents.

7. The method according to steps 2) and 4) of claim 3 and step 2) of claim 4, characterized in that: The organic base in the reaction is selected from one of triethylamine (TEA), pyridine, and N,N-diisopropylethylamine (DIPEA), and can also be carried out in an inorganic weak alkaline solvent, wherein the inorganic weak alkaline solvent is selected from one of sodium bicarbonate (NaHCO3) solution, sodium carbonate (Na2CO3) solution, sodium bisulfite solution (NaHSO3) or disodium hydrogen phosphate (Na2HPO4), but is not limited to these solutions.

8. The preparation method according to claims 2, 3 and 4, characterized in that: The reaction temperature of the step is 5-85°C.

9. The ferulic acid-biotin conjugate prepared according to claim 1 enhances the targeting of ferulic acid, enhances the uptake rate of ferulic acid by tumor cells, and improves the inhibition rate of ferulic acid on tumor cells; and has the characteristics of redox-sensitive release, enhances the specific release ability in tumor tissue, improves the anti-tumor effect, and can reduce the toxicity to normal tissues.

10. All the ferulic acid-biotin conjugates prepared by the present invention are used in the field of tumor treatment.

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