Liver-targeted sorafenib prodrug as well as preparation method and application thereof

Abstract

The invention discloses a liver-targeted sorafenib prodrug as well as a preparation method and application thereof, and relates to the field of chemical pharmacy, the liver-targeted sorafenib prodrug is simple to prepare, sorafenib is selected as a core drug component, and the sorafenib is covalently linked with beta-galactoside. Due to the hydrophilicity of the beta-galactoside and the existence of a plurality of hydroxyl groups, after the sorafenib which is originally poor in solubility in water is covalently connected with the beta-galactoside, the solubility of the sorafenib is greatly improved. Due to the characteristic, the preparation process of the medicine is simplified, and a good foundation is laid for application of subsequent medicine in a solution system and in-vivo transportation and absorption. The molecular structure of the prepared liver targeting sorafenib prodrug S-Gal contains a galactoside bond. When the S-Gal enters a body, the S-Gal can be actively taken by ASGPR on the surface of a hepatoma carcinoma cell, and is hydrolyzed under the action of beta-Gal over-expressed in the cell to release a sorafenib raw drug, so that the liver targeting of the drug is enhanced, and the systemic toxicity is reduced.

Description

Technical Field

[0001] The present invention relates to the field of chemical pharmacy, and in particular to a liver-targeted sorafenib prodrug and a preparation method and application thereof. Background Art

[0002] Cancer has always been a major threat to human health. Despite significant progress in immunotherapy and targeted therapy in recent years, chemotherapy remains an important means of clinical cancer treatment. However, traditional chemotherapy drugs generally suffer from poor water solubility, significant toxic side effects, and low bioavailability, which severely limit their therapeutic effectiveness.

[0003] Sorafenib is currently the only molecularly targeted drug approved by the US FDA for the treatment of advanced hepatocellular carcinoma (HCC). It exerts anti-tumor effects by inhibiting the RAF / MEK / ERK signaling pathway and the vascular endothelial growth factor receptor (VEGFR). However, sorafenib also faces challenges such as poor water solubility, poor targeting, damage to normal tissues, low bioavailability, toxic side effects due to nonspecific distribution, and the development of drug resistance after long-term use. Therefore, how to improve the efficacy of sorafenib and reduce its side effects through rational drug delivery strategies has become a key scientific issue that needs to be addressed. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a liver-targeted sorafenib prodrug capable of improving the water solubility of sorafenib and achieving tumor microenvironment-responsive release, as well as a preparation method and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: Provided is a method for preparing a liver-targeted sorafenib prodrug, comprising the following specific steps: S1: 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was dissolved in acetonitrile, and then silver oxide and 4-hydroxybenzaldehyde were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was filtered and the precipitate was washed with ethyl acetate. The filtrate and washing solution were combined, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 1; S2: Compound 1 was dissolved in a solvent with a volume ratio of chloroform to isopropanol of 25:8, and sodium borohydride was added at 0°C. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, the mixture was washed with 10% aqueous citric acid solution, 10% aqueous sodium bicarbonate solution, and water, and then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 2; S3: Compound 2 was dissolved in dichloromethane, and triethylamine was added. 4-nitrophenyl chloromethyl ester-dichloromethane solution was slowly added dropwise at 0°C. After the addition was complete, the mixture was stirred and reacted at room temperature for 20 hours. The mixture was washed with 5% sodium bicarbonate aqueous solution and water in sequence, and then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography to obtain compound 3; S4: After mixing sorafenib with tetrahydrofuran, n-butyl lithium was slowly added dropwise under a nitrogen environment at -78°C. After stirring for 2 hours, compound 3 was dissolved in tetrahydrofuran and slowly added dropwise to the reaction solution. After stirring for 24 hours, the reaction was quenched with a saturated sodium chloride solution, concentrated under reduced pressure, extracted with dichloromethane, dried over anhydrous sodium sulfate, and filtered; and then concentrated under reduced pressure to obtain a mixture; S5: The mixture was mixed with methanol, and then sodium methoxide was added at 0°C and stirred for 4 hours. After the reaction was completed, ion exchange resin was added, and the pH of the reaction solution was adjusted to neutral. The ion exchange resin was filtered out, and the solution was concentrated under reduced pressure. The liver-targeted sorafenib prodrug S-Gal was obtained by purification using silica gel column chromatography.

[0006] Furthermore, in step S1, the molar ratio of 2,3,4,6-tetra-0-acetyl-α-D-galactopyranosyl bromide, silver oxide and 4-hydroxybenzaldehyde is 12 mmol: 24 mmol: 13 mmol; and petroleum ether: ethyl acetate with a volume ratio of 7:3 is used as the eluent during silica gel column chromatography purification.

[0007] Furthermore, in step S2, the mass ratio of compound 1 to sodium borohydride is 1 g:183 mg; and during purification by silica gel column chromatography, petroleum ether:ethyl acetate in a volume ratio of 1:1 is used as the eluent.

[0008] Furthermore, in step S3, the mass ratio of compound 2, triethylamine and 4-nitrophenyl chloromethyl ester is 944.8 mg:420 mg:461 mg; and petroleum ether:ethyl acetate with a volume ratio of 2:1 is used as the eluent during purification by silica gel column chromatography.

[0009] Furthermore, in step S4, the mass ratio of sorafenib, n-butyllithium and compound 3 is 240 mg:350 mg:800 mg.

[0010] Furthermore, in step S5, the ratio of the mixture, methanol and sodium methoxide is 341 mg:20 mL:86 mg; and during purification by silica gel column chromatography, dichloromethane:methanol in a volume ratio of 6:1 is used as the eluent.

[0011] The present invention also provides a liver-targeted sorafenib prodrug, which is prepared by the above-mentioned method to achieve covalent bonding of sorafenib and β-galactoside, and has a structural formula as shown in the following formula I:

[0012] I The liver-targeted sorafenib prodrug has high water solubility, liver targeting and low toxicity.

[0013] The present invention also provides a use of a liver-targeted sorafenib prodrug in the preparation of a drug for improving the delivery efficiency of sorafenib.

[0014] Furthermore, the drug is an injection, comprising a liver-targeted sorafenib prodrug and an injection solvent.

[0015] Furthermore, the injection solvent includes water for injection, 0.9% sodium chloride injection or 5% glucose injection.

[0016] The beneficial effects of the present invention are: The present invention is simple to prepare. Sorafenib is selected as the core drug ingredient and covalently linked to β-galactoside. Due to the hydrophilicity and the presence of multiple hydroxyl groups on β-galactoside, the solubility of sorafenib, which originally had poor solubility in water, is significantly improved after covalently linking to β-galactoside. This property not only simplifies the drug preparation process but also lays a good foundation for subsequent drug application in solution systems and its delivery and absorption in the body.

[0017] The prepared liver-targeted sorafenib prodrug, S-Gal, contains a galactosidic bond in its molecular structure. Once S-Gal enters the body, it is actively taken up by ASGPR on the surface of liver cancer cells and hydrolyzed by overexpressed β-Gal within the cells, releasing the sorafenib prodrug, thereby enhancing the drug's liver targeting and reducing systemic toxicity. This controlled drug release mechanism enables sorafenib to achieve high concentrations precisely at the site of liver cancer, avoiding unnecessary distribution in other normal tissues and organs. This greatly improves the targeting of sorafenib to the liver cancer site and provides strong technical support for the precision treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthetic route of liver-targeted sorafenib prodrug S-Gal; Figure 2 This is a macroscopic comparison of the water solubility of S-Gal; Figure 3 This is a graph showing the experimental results of the effect of β-Gal concentration on the in vitro release of S-Gal in Example 3; Figure 4 This is a graph showing the experimental results of the effect of time on the in vitro release of S-Gal in Example 4; Figure 5 This is a comparison of the inhibition of S-Gal on 4T1 and HepG2 tumor cells; Figure 6 This is a graph showing the results of the in vivo anticancer activity test of S-Gal in Example 6; Figure 7 This is a diagram of the biosafety test results in Example 6; Figure 8 This is the H&E staining image of the five internal organs of the mouse in Example 6. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0020] The human hepatocellular carcinoma cells (HepG2), mouse breast cancer cells (4T1), and mouse hepatocellular carcinoma cells (H22) used in the examples of this invention were purchased from the National Laboratory Cell Resource Sharing Platform. Kunming (KM) mice were obtained from Hunan Slake Jingda Laboratory Animal Co., Ltd. The sources of the materials used in the examples are shown in Table 1 below; the sources of the instruments and equipment are shown in Table 2 below. Table 1

[0021] Table 2

[0022] Example 1 Preparation of liver-targeted sorafenib prodrug S-Gal The specific steps are as follows: S1: 5 g (12.0 mmol) of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was weighed into a 250 mL round-bottom flask and dissolved in 125 mL of acetonitrile. 24 mmol of silver oxide and 13 mmol of 4-hydroxybenzaldehyde were then added to the reaction solution. The reaction was stirred at room temperature for 16 h. After the reaction was complete, the solution was filtered and the precipitate was washed with ethyl acetate. The filtrate and washings were combined and concentrated under reduced pressure to remove the solvent. The solution was purified by silica gel column chromatography using petroleum ether:ethyl acetate (volume ratio:7:3) as eluent to obtain 3.27 g of compound 1 as a white solid in a yield of 60%. Compound 1 was subjected to nuclear magnetic resonance detection, and the test results were: 1 H NMR (400MHz, CDCl3) δ 9.89 (s, 1H), 7.92 – 7.71 (m, 2H), 7.18 – 6.92 (m, 2H), 5.63 – 5.35 (m, 2H), 5.24 – 5.03 (m, 2H), 4.35 – 4.01 (m, 2H), 2.16 (s, 3H), 2.04 (s, 6H), 1.99 (s, 3H). 13C NMR (101MHz, CDCl3) δ 190.79, 171.30 – 167.54 (m), 161.23, 132.92 – 129.47 (m), 116.67, 98.52, 78.64 – 74.64(m), 71.25, 70.62(d, J=13.1Hz), 68.31(d, J=4.1Hz), 66.72(d, J=7.2Hz), 61.35, 20.65(q, J=7.3, 6.4Hz). According to the results, the structure of compound 1 is shown in Formula II below;

[0023] II S2: 1 g of compound 1 was weighed into a 100 mL round-bottom flask and dissolved in a solvent with a volume ratio of chloroform:isopropanol of 25:8. The reaction flask was placed at 0°C and 183 mg of sodium borohydride was added. The reaction flask was then moved to room temperature and stirred for 3 h. The reaction was monitored by thin-layer chromatography. After completion of the reaction, the mixture was washed once with 50 mL of a 10% aqueous citric acid solution, three times with 50 mL of a 10% aqueous sodium bicarbonate solution, and once with 50 mL of ultrapure water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The mixture was purified by silica gel column chromatography with a volume ratio of petroleum ether:ethyl acetate of 2:1 as the eluent to obtain 944.8 mg of compound 2 as a white solid in a yield of 94%. Compound 2 was subjected to nuclear magnetic resonance detection, and the test results were: 1 H NMR (400MHz, CDCl3) δ 7.22 (d, J=8.4Hz, 1H), 6.98 – 6.88 (m, 1H), 5.38 (d, J=3.1Hz, 1H), 5.09 – 4.97 (m, 1H), 4.20 – 3.97(m, 2H), 2.11(s, 2H), 1.99(d, J=5.8Hz, 3H), 1.95(s, 2H). 13 C NMR (101MHz, CDCl3) δ 170.56 – 170.15 (m), 169.52, 156.22, 136.03, 128.35, 116.81, 99.60, 78.40 – 75.89(m), 70.75(d, J=16.1Hz), 68.58(d, J=7.4Hz), 66.87(d, J=10.2Hz), 65.32 – 63.36(m), 61.33, 20.63(q, J=7.0, 6.2Hz). According to the results, the structure of compound 2 is shown in Formula III below;

[0024] III S3: 944.8 mg of compound 2 was weighed into a 100 mL round-bottom flask, 20 mL of dichloromethane was added to dissolve it, and then 420 mg of triethylamine was added. The reaction flask was placed at 0°C, 461 mg of 4-nitrophenyl chloromethyl ester was weighed and dissolved in 5 mL of dichloromethane and slowly added dropwise to the above reaction solution. After the addition was complete, the reaction flask was moved to room temperature and stirred for 20 h. Dichloromethane was added to dilute the mixture, and the mixture was washed once with 5% sodium bicarbonate aqueous solution and once with ultrapure water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The mixture was purified by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 2:1 as eluent to obtain 741 mg of yellow solid compound 3 in a yield of 57%; Compound 3 was subjected to nuclear magnetic resonance detection, and the test results were: 1 H NMR (400MHz, CDCl3) δ 8.32 – 8.24 (m, 1H), 7.38 (t, J=9.4Hz, 2H), 7.03 (d, J=8.3Hz, 1H), 5.54 – 5.44(m, 1H), 5.24(s, 1H), 5.07(d, J=8.0Hz, 1H), 4.28 – 4.03(m, 1H), 2.19(s, 2H), 2.07(t, J=1.3Hz, 4H), 2.02(s, 2H). 13 C NMR (101MHz, CDCl3) δ 170.60 –169.89(m), 169.44, 157.35, 155.43, 152.40, 145.33, 130.58, 129.01, 125 .30(d, J=4.5Hz), 121.76(d, J=9.5Hz), 116.97, 99.28(d, J=17.1Hz), 77.48 – 76.70(m), 71.35 – 69.86(m), 68.52, 66.78(d, J=7.3Hz), 61.32, 20.68(q, J=7.3Hz), 14.18. According to the results, the structure of compound 3 is shown in Formula IV below;

[0025] IV S4: Weigh 240 mg of sorafenib into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran, protect with nitrogen, and place in a low-temperature reactor to cool to -78°C, then slowly add 350 mg of n-butyllithium dropwise, stir and react for 2 h, weigh 800 mg of compound 3, dissolve in 20 mL of tetrahydrofuran, and slowly add dropwise to the reaction solution, stir and react for 24 h, quench the reaction with saturated sodium chloride solution, concentrate under reduced pressure to remove the organic solvent, and then extract three times with dichloromethane, followed by drying with anhydrous sodium sulfate and filtering; and then concentrate under reduced pressure to obtain 341 mg of the mixture; S5: 341 mg of the mixture was placed in a 100 mL round-bottom flask, 20 mL of methanol was added, the reaction flask was placed at 0°C, 86 mg of sodium methoxide was added and stirred for 4 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, ion exchange resin was added, the pH of the reaction solution was adjusted to neutral, the ion exchange resin was filtered to remove the ion exchange resin, and the solution was concentrated under reduced pressure. Silica gel column chromatography with a volume ratio of dichloromethane: methanol of 6:1 as the eluent was used to purify the solution to obtain 140 mg of liver-targeted sorafenib prodrug S-Gal as a white solid.

[0026] S-Gal was tested by nuclear magnetic resonance, and the test results were: 1 H NMR (400MHz, Methanol-d4) δ 8.44 (d, J=5.6Hz, 1H), 7.59 – 7.41 (m, 3H), 7.42 – 7.28 (m, 2H), 7.17 – 7.06 (m, 4H), 7.02 (dd, J=5.6, 2.6Hz, 1H), 5.13 (s, 2H), 4.58 (s, 3H), 3.90 (d, J=3.4Hz, 1H), 3.84 – 3.73(m, 3H), 3.69(d, J=6.2Hz, 1H), 3.59(d, J=3.4Hz, 1H). 13 C NMR (101MHz, Methanol-d4) δ 166.65, 165.42, 157.50, 154.47, 151.72, 149.97, 148.68, 130.24, 129.5 3, 121.17, 120.33, 116.49, 113.74, 109.92, 101.30 (d, J=18.7Hz), 77.97 – 76.97(m), 75.36, 73.40, 70.87, 68.54, 66.20, 49.30 – 47.34(m). According to the results, the structure of S-Gal is shown in Formula I below:

[0027] I The synthetic route of liver-targeted sorafenib prodrug S-Gal is as follows Figure 1 shown.

[0028] In specific implementation, the auxiliary organic solvents dichloromethane, tetrahydrofuran and methanol used in the preparation process will be removed during the reduced pressure concentration, so when the auxiliary organic solvent is used, the amount of the auxiliary organic solvent can be flexibly adjusted.

[0029] Example 2 Characterization of the liver-targeted sorafenib prodrug S-Gal Take the S-Gal prepared in Example 1 to visually observe the improvement of the water solubility and dispersibility of the liver-targeted drug S-Gal compared to the original chemotherapy drug sorafenib and its clinical drug sorafenib tosylate. Specifically, weigh S-Gal containing 1 mg of sorafenib, sorafenib (SF) and sorafenib tosylate (SF-TSA), respectively, and place them in 1.5 mL EP tubes. Then add 500 μL of pure water to each tube to dissolve them, and leave them for 2 days. Take photos and record the phenomena every day. The results are as follows Figure 2 As shown by Figure 2 It can be seen that through direct observation with the naked eye, it was found that the water solubility and dispersibility of S-Gal were significantly better than those of sorafenib and sorafenib tosylate.

[0030] Example 3 Effect of β-galactosidase (β-Gal) concentration on the in vitro release of S-Gal The in vitro release of S-Gal is affected by the concentration of β-galactosidase (β-Gal). β-Gal specifically catalyzes the hydrolysis of 4-methylumbelliferyl-β-D-pyranoside (4-MUG), breaking it down to form 4-methylumbelliferone and the corresponding sugar. 4-Methylumbelliferone fluoresces at a wavelength of 323 nm, with a sharp increase in fluorescence at 450 nm. β-Galase activity can be quantitatively analyzed by measuring fluorescence intensity. Within a given enzyme dosage and reaction time, higher enzyme activity results in more 4-methylumbelliferone hydrolyzed and a higher fluorescence intensity. However, the liver-targeted drug S-Gal itself, sorafenib, does not exhibit fluorescence absorption at 450 nm when excited by 323 nm light. Therefore, during the experiment, β-Gal and S-Gal were reacted at varying mass ratios. After the reaction, 4-MUG was added, and the remaining β-Gal in the reaction system was measured to determine if S-Gal was completely released. Specifically: 1 μg / μL liver-targeted sorafenib prodrug S-Gal was mixed with β-Gal solutions at concentrations of 1, 2, 3, 5, 10, 15, and 20 μg / μL (i.e., the mass ratios of S-Gal to β-Gal were 1:1, 1:2, 1:3, 1:5, 1:10, 1:15, and 1:20, respectively), and incubated at 37°C for 12 hours. After the incubation, 10 μL of 4-methylumbelliferyl-β-D-pyranoside (4-MUG) solution at a concentration of 0.5 μg / μL was added to react with the above mixed solutions. After standing for 5 minutes, the mixtures were observed under 365 nm ultraviolet light and photographed. A fluorescence spectrophotometer was then used to detect the fluorescence intensity of the mixed solutions of S-Gal and β-Gal at different mass ratios at 450 nm under an excitation wavelength of 323 nm. The results are shown in Figure 2. Figure 3 As shown, Figure 3 a is the 365nm UV absorption graph of the release of reactions with different mass ratios, Figure 3 b is the fluorescence intensity comparison diagram at 450nm for the release of different mass ratio reactions. Figure 3 As can be seen, with increasing β-Gal concentration, the fluorescence intensity under 365nm UV irradiation continued to increase, and the fluorescence at 450nm also continued to increase. When the mass ratio of S-Gal to β-Gal was 1:1, there was no absorption under 365nm UV light, and the fluorescence at 450nm was the same as that of S-Gal itself, with no absorption, indicating that S-Gal was completely hydrolyzed by β-Gal. This shows that S-Gal can respond to the overexpression of β-Gal in liver cancer sites, causing the galactosidic bond in its structure to break, releasing sorafenib and improving its targeting.

[0031] Example 4 Effect of time on S-Gal release It can be seen from Example 3 that the mass ratio of S-Gal and β-Gal for complete reaction is 1:1. This example also uses the mass ratio of S-Gal to β-Gal of 1:1 to explore the effect of time on the release of S-Gal. Specifically: prepare equal amounts of mixed solutions of S-Gal and β-Gal and incubate them at 37°C for 1, 3, 4, 5, 7, 9, 10, 11, and 12 hours, respectively. After the incubation is completed, add 10 μL of 4-MUG solution with a concentration of 0.5 μg / μL to react with the above mixed solution. After standing for 5 minutes, observe under 365nm ultraviolet light and take pictures. Then use a fluorescence spectrophotometer to detect the fluorescence intensity of mixed solutions of S-Gal and β-Gal with different mass ratios at 450nm. The results are as follows. Figure 4 As shown, Figure 4 a is the 365nm ultraviolet absorption graph of the release of S-Gal and β-Gal at a mass ratio of 1:1 over the reaction time. Figure 4b is the release of S-Gal and β-Gal at a mass ratio of 1:1 over the reaction time. The fluorescence intensity at 450 nm is shown. Figure 4 c is the statistical graph of the release of S-Gal and β-Gal with a mass ratio of 1:1 over the reaction time; Figure 4 It can be seen that when the mass ratio of S-Gal to β-Gal is 1:1, the light intensity under 365nm UV light and the fluorescence intensity at 450nm gradually decrease with the extension of incubation time. When the incubation time is 11 hours, there is no fluorescence at 365nm, and the fluorescence intensity at 450nm also basically disappears, indicating that when the mass ratio of S-Gal to β-Gal is 1:1, S-Gal can be completely hydrolyzed by the action of β-galactosidase after incubation at 37°C for 11 hours.

[0032] Example 5 Cytotoxicity Experiment of S-Gal The CCK-8 assay was used to verify the cytotoxicity of S-Gal against 4T1 and HepG2 tumor cells, with sorafenib and sorafenib tosylate used as controls. Since sorafenib and sorafenib tosylate are nearly insoluble in water, dimethyl sulfoxide (DMSO) was used for solubilization.

[0033] The experimental steps are as follows: (1) Count the cell suspension using a hemocytometer and inoculate the cell suspension into a 96-well plate to a cell density of 5,000 cells / well in a volume of 100 μL. After inoculation, gently shake the plate to evenly distribute the cells, then place the plate in a 37°C, 5% CO2 incubator for incubation.

[0034] (2) After 12 hours of cell culture, place the 96-well plate in a clean bench, remove the original culture medium with a pipette, and add 100 μL of PBS buffer to each well for two washes. Add DMEM culture medium containing S-Gal, sorafenib, and sorafenib toluenesulfonic acid, respectively, to make the final concentration of sorafenib 10, 20, 40, 80, and 100 μg / mL (set 4 replicates for each concentration). At the same time, set up a blank control group without drug and a zero adjustment group containing only culture medium. After adding drugs, continue to culture the culture plate in the incubator. Place the 96-well plate in an incubator at 37°C and 5% CO2 for culture.

[0035] (3) After the cells have been cultured for 48 hours, observe the cell survival status using an inverted microscope. Then use a pipette to aspirate the culture medium in the 96-well plate, wash each well twice with 100 μL of PBS buffer, and then wash once with 100 μL of culture medium. Add 10 μL of CCK-8 and 100 μL of culture medium mixed solution to each well, and then place the 96-well plate in a 37°C, 5% CO2 incubator for 4 hours. After incubation for 4 hours, take out the 96-well plate and use a microplate reader to measure the absorbance (OD) value of each well at an excitation wavelength of 450 nm. Calculate the cell survival rate of each group. The cell survival rate is calculated according to the following formula: ; The results are as follows Figure 5 As shown, Figure 5 a is the effect of S-Gal, SF and SF-TSA on the survival rate of 4T1 cells, Figure 5 b shows the effects of S-Gal, SF and SF-TSA on the survival rate of HepG2 cells. Figure 5 As can be seen, for 4T1 cells, the cell-killing effect of S-Gal gradually increased with increasing concentration, indicating that the drug has dose-dependent cytotoxicity on the tested cells, and the cell-killing effect of S-Gal can reach approximately 70%. For HepG2 cells, the cell-killing effect of S-Gal can reach approximately 70% with increasing concentration. This shows that the liver-targeted prodrug S-Gal has a certain inhibitory effect on the growth of 4T1 and HepG2 cells.

[0036] Example 6 In vivo anticancer activity experiment of S-Gal Female KM mice aged 6 to 8 weeks were purchased and placed in a feeding room for one week. At the same time, H22 cells were placed in DMEM complete medium and cultured in an incubator at 37°C and 5% CO2. They were passaged regularly to ensure that the cells were always in the logarithmic growth phase to maintain good cell activity and proliferation. After completing the cell count using a hemocytometer, the cells were resuspended in PBS buffer and then subcutaneously inoculated into each female Kunming mouse with 1×10 6 H22 cells. The mice that have been inoculated were returned to the cage and raised in the usual way. At regular intervals, the tumor size was measured with a vernier caliper and the tumor volume was calculated according to the formula: tumor volume (mm 3 ) = (long diameter × short diameter) 2 / 2, calculate the tumor volume. During the experiment, pay close attention to the tumor growth rate, morphological changes, etc., and record the relevant data in detail. When the tumor volume grows to 100mm 3 When the time is about 2 hours, subsequent experiments can be carried out.

[0037] When the tumor volume reaches 100 mm 3The mice were randomly divided into 4 groups (5 in each group), and the groups were set as follows: normal saline (negative control), SF (positive control), and S-Gal as the experimental group. All groups were administered via the tail vein, once every 2 days, for a total of 5 times. The intravenous injection dose of sorafenib for each group of mice was set at 9 mg / kg. During the administration period, the tumor volume and body weight of the mice were measured regularly every day, and the physical condition and tumor changes of the mice were closely monitored. After the administration cycle, the experimental mice were euthanized by cervical dislocation. Subsequently, the tumor tissues of the mice were quickly separated and collected, and the tumors were photographed and weighed. The collected tumors were fixed with paraformaldehyde for subsequent sectioning and hematoxylin and eosin (H&E) staining analysis, so as to deeply explore the effects of the drug on the tumor from a histological perspective. The results are shown in Figure 6 and Figure 7 As shown, Figure 6 a is a schematic diagram of the time of tumor implantation and drug administration in mice. Figure 6 b is the growth curve of tumor volume in each group of mice. Figure 6 c is the changes in body weight of mice in each group during the treatment period; Figure 7 a is the statistical graph of the average weight of the tumor in each group of mice. Figure 7 b is the tumor photos of mice in each group. Figure 7 c is the H&E staining image of the tumor sections of mice in each group; Figure 6 As can be seen, the tumors in mice injected with saline showed rapid growth. In sharp contrast, tumor growth was effectively slowed in the experimental groups that received intravenous injections of SF, S-Gal, and S-PTX. There was no significant difference in body weight between the experimental and control groups, indicating that the experimental groups effectively inhibited tumor growth without significantly adversely affecting the mice's normal physiological metabolism and nutrient absorption.

[0038] Depend on Figure 7As can be seen, the tumor weights of mice treated with SF and S-Gal showed a significant decrease compared to those in the saline group. This data further strongly confirms that our S-Gal has a significant inhibitory effect on liver cancer. From the captured tumor images, it can be visually observed that the tumors of mice treated with S-Gal are significantly different from those in the saline group in both size and morphology, which fully confirms the excellent effect of the above-mentioned drug in treating liver cancer. In addition, H&E staining images of tumor sections from each group of mice show that the tumor cells in the saline control group are densely distributed and the tumor tissue is intact and free of necrosis, while the S-Gal-treated group caused significant necrosis in the tumor tissue of mice. In summary, through the intuitive display of tumor weight measurement data, tumor images, and H&E staining images of tumor sections, it is fully confirmed that our S-Gal has extremely broad application prospects in the field of liver cancer treatment, providing a solid experimental foundation for subsequent in-depth research and clinical translation.

[0039] To investigate whether the prepared liver-targeted sorafenib prodrug S-Gal has systemic toxicity or biosafety, mice were killed by cervical dislocation, and then the heart, liver, spleen, lung, kidney and other major organs of mice in different treatment groups were removed for preparation of histological sections and H&E staining analysis. Figure 8 As shown, compared to the five internal organs of mice in the saline control group, the liver-targeted sorafenib prodrugs S-Gal and S-PTX caused negligible damage to major organs such as the heart, liver, spleen, lungs, and kidneys. In stark contrast, mice in the intravenous SF injection group showed signs of lung inflammation and liver damage. Specifically, lung inflammation and liver damage were manifested by the presence of numerous neutrophils, fibrin, and other exudates within the alveolar cavities, congestion and edema of the alveolar walls, and infiltration of inflammatory cells. In the liver, hepatocyte swelling, severe granular deformation, congestion, and microcystic fatty degeneration were observed. This further demonstrates that the liver-targeted sorafenib prodrug S-Gal prepared in this study possesses tumor targeting and tissue specificity. Its liver targeting enhances the efficacy of sorafenib and significantly reduces damage to normal tissues, providing new avenues for the treatment of liver cancer.

Claims

1. A method for preparing a liver-targeted sorafenib prodrug, characterized in that: The specific steps include: S1: 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was dissolved in acetonitrile, and then silver oxide and 4-hydroxybenzaldehyde were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was filtered and the precipitate was washed with ethyl acetate. The filtrate and washing solution were combined, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 1; S2: Compound 1 was dissolved in a solvent with a volume ratio of chloroform to isopropanol of 25:8, and sodium borohydride was added at 0°C. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, the mixture was washed with 10% aqueous citric acid solution, 10% aqueous sodium bicarbonate solution, and water, and then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 2; S3: Compound 2 was dissolved in dichloromethane, and triethylamine was added. 4-nitrophenyl chloromethyl ester-dichloromethane solution was slowly added dropwise at 0°C. After the addition was complete, the mixture was stirred and reacted at room temperature for 20 hours. The mixture was washed with 5% sodium bicarbonate aqueous solution and water in sequence, and then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography to obtain compound 3; S4: After mixing sorafenib with tetrahydrofuran, n-butyl lithium was slowly added dropwise under a nitrogen environment at -78°C. After stirring for 2 hours, compound 3 was dissolved in tetrahydrofuran and slowly added dropwise to the reaction solution. After stirring for 24 hours, the reaction was quenched with a saturated sodium chloride solution, concentrated under reduced pressure, extracted with dichloromethane, dried over anhydrous sodium sulfate, and filtered; and then concentrated under reduced pressure to obtain a mixture; S5: The mixture was mixed with methanol, and then sodium methoxide was added at 0°C and stirred for 4 hours. After the reaction was completed, ion exchange resin was added, and the pH of the reaction solution was adjusted to neutral. The ion exchange resin was filtered out, and the solution was concentrated under reduced pressure. The liver-targeted sorafenib prodrug S-Gal was obtained by purification using silica gel column chromatography.

2. The preparation method according to claim 1, characterized in that In step S1, the molar ratio of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, silver oxide, and 4-hydroxybenzaldehyde is 12 mmol:24 mmol:13 mmol; and during purification by silica gel column chromatography, petroleum ether:ethyl acetate in a volume ratio of 7:3 is used as the eluent.

3. The preparation method according to claim 2, characterized in that In step S2, the mass ratio of compound 1 to sodium borohydride is 1 g:183 mg; and during purification by silica gel column chromatography, petroleum ether:ethyl acetate in a volume ratio of 1:1 is used as the eluent.

4. The preparation method according to claim 3, characterized in that In step S3, the mass ratio of compound 2, triethylamine and 4-nitrophenyl chloromethyl ester is 944.8 mg:420 mg:461 mg; and petroleum ether:ethyl acetate with a volume ratio of 2:1 is used as the eluent during purification by silica gel column chromatography.

5. The preparation method according to claim 4, characterized in that In step S4, the mass ratio of sorafenib, n-butyllithium and compound 3 is 240 mg:350 mg:800 mg.

6. The preparation method according to claim 5, characterized in that In step S5, the ratio of the mixture, methanol and sodium methoxide is 341 mg:20 mL:86 mg; and during purification by silica gel column chromatography, dichloromethane:methanol in a volume ratio of 6:1 is used as the eluent.

7. A liver-targeted sorafenib prodrug, characterized in that: The liver-targeted sorafenib prodrug is prepared by the method according to claim 6 to achieve covalent linkage between sorafenib and β-galactoside, and its structural formula is shown in Formula I below: I The liver-targeted sorafenib prodrug has high water solubility, liver targeting and low toxicity.

8. Use of liver-targeted sorafenib prodrug in the preparation of drugs for improving the delivery efficiency of sorafenib.

9. The use according to claim 8, characterized in that The drug is an injection, comprising a liver-targeted sorafenib prodrug and an injection solvent.

10. The use according to claim 9, characterized in that Injection solvents include water for injection, 0.9% sodium chloride injection or 5% glucose injection.

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