Galactosamine-doxetaxel conjugate, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a galactosamine-doxetaxel conjugate as well as a preparation method and application thereof. First, doxetaxel and 3,3'-diseleno-dipropionic acid are used as raw materials, and after heating reaction, 3,3'-diseleno-dipropionic acid doxetaxel is obtained. Then, the obtained 3,3'-diseleno-dipropionic acid doxetaxel and galactosamine are used as raw materials, and after heating reaction, galactosamine-3,3'-diseleno-dipropionic acid doxetaxel conjugate is obtained through column chromatography separation and purification. The obtained conjugate can be specifically recognized by endogenous lectin receptors (such as asialoglycoprotein receptor ASGPR) which are highly expressed on the surface of hepatoma cells, so as to be taken up by hepatoma cells through a receptor-mediated pathway, and has application prospect in the direction of hepatoma targeted treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a galactosamine-docetaxel conjugate, its preparation method, and its application. Background Technology

[0002] Docetaxel is a semi-synthetic chemotherapy drug commonly used to treat various cancers, including breast cancer, lung cancer, ovarian cancer, prostate cancer, gastric cancer, and head and neck cancer, and has potential applications in cancer treatment. However, due to the low water solubility, non-selective biodistribution, systemic toxicity, and severe allergic reactions of free docetaxel, its therapeutic efficacy is limited in clinical practice, resulting in serious toxic side effects.

[0003] Improving the tumor targeting of docetaxel through conjugate targeted drug delivery systems is currently a hot research topic, but it still faces challenges such as low drug loading capacity and complex drug loading steps. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a galactosamine-docetaxel conjugate with the following chemical structural formula:

[0005]

[0006] This invention also provides a method for preparing a galactosamine-docetaxel conjugate:

[0007] (1) Preparation of 3,3'-diselenodipropionated docetaxel

[0008] Using docetaxel and 3,3'-diselenodipropionic acid as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as condensing agent and 4-dimethylaminopyridine (DMAP) as catalyst, the reaction was carried out in a solvent under heat. After cooling to room temperature, the resulting reaction system was evaporated to dryness, dissolved in DMSO, transferred to ultrapure water for dialyzing, and then freeze-dried to obtain 3,3'-diselenodipropionic acid-modified docetaxel.

[0009] (2) Using 3,3'-diselenodipropionated docetaxel and galactosamine (D(+)-Galactosamine hydrochloride) prepared in step (1) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as condensing agent and 4-dimethylaminopyridine (DMAP) as catalyst, the reaction was heated in a solvent and then cooled to room temperature. The resulting reaction system was transferred to ultrapure water for dialyzing and then freeze-dried. The freeze-dried product was purified by column chromatography to obtain galactosamine-3,3'-diselenodipropionated docetaxel conjugate.

[0010] As a preferred embodiment, in step (1), the molar ratio of docetaxel, 3,3'-diselenopropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:8:10:1.5.

[0011] Preferably, in step (1), the solvent is dichloromethane.

[0012] As a preferred option: in step (1), the heating reaction is a reflux reaction at 40°C for 24 hours.

[0013] As a preferred embodiment, in step (2), the molar ratio of 3,3'-diselenodipropionated docetaxel, galactosamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:8:10:2.

[0014] Preferably, in step (2), the solvent is dimethyl sulfoxide or N,N-dimethylformamide.

[0015] As a preferred option: in step (2), the heating reaction is a reflux reaction at 50°C for 24 hours.

[0016] The present invention also provides an application of the above-mentioned galactosamine-docetaxel conjugate in targeted therapy for liver cancer.

[0017] The beneficial effects of this invention are as follows: The diselenylene bond conjugate prepared by this invention has dual-response drug release characteristics of oxidation and reduction. Due to the vigorous metabolism of tumor cells, the oxidation and reduction levels in tumor cells are higher than those in normal cells. Therefore, the dual-response drug release characteristics of this diselenylene bond conjugate are more conducive to its selective release of drugs in tumor cells, resulting in a higher efficacy in killing tumor cells. At the same time, the galactose ligand of the diselenylene bond conjugate prepared by this invention can be specifically recognized by endogenous lectin receptors (such as desialyl glycoprotein receptor ASGPR) highly expressed on the surface of liver cancer cells, thereby being specifically taken up by liver cancer cells through a receptor-mediated pathway, achieving the effect of selectively killing liver cancer cells. Attached Figure Description

[0018] Figure 1 The 1H NMR spectrum of the product prepared in step (1) of Example 1;

[0019] Figure 2 The 1H NMR spectrum of the product prepared in step (2) of Example 1;

[0020] Figure 3 The 1H NMR spectrum of the product prepared in step (1) of Example 2;

[0021] Figure 4The 1H NMR spectrum of the product prepared in step (2) of Example 2;

[0022] Figure 5 The 1H NMR spectrum of the product prepared in step (1) of Example 3;

[0023] Figure 6 The 1H NMR spectrum of the product prepared in step (2) of Example 3;

[0024] Figure 7 The infrared spectra of the products prepared in Examples 1, 2, and 3 are shown. Detailed Implementation

[0025] Example 1: Preparation of galactosamine-3,3'-diselenopropionic acid docetaxel conjugate

[0026] (1) Preparation of 3,3'-diselenodipropionated docetaxel

[0027] Add 6 mL of dichloromethane to a 25 mL round-bottom flask. While maintaining continuous magnetic stirring, add docetaxel (200 mg, 0.248 mmol) and 3,3'-diselenopropionic acid (603 mg, 1.984 mmol). After dissolving at room temperature (25 °C), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (475.42 mg, 2.48 mmol) and 4-dimethylaminopyridine (45.5 mg, 0.372 mmol). Then heat to 40 °C and reflux with stirring. After 24 hours, allow the flask to cool naturally to room temperature. Centrifuge and collect the supernatant, then evaporate to dryness (a small amount of black solid byproduct appears during the reaction; centrifugation removes this byproduct). Dissolve the byproduct in DMSO, transfer it to a dialysis bag (molecular weight cutoff 800 Da), and dialyze it in sufficient ultrapure water for three days. Freeze-dry the precipitate in the dialysis bag to obtain 282.37 mg of white powdered solid 3,3'-diselenodipropionated docetaxel, yield 82.5% (moles of 3,3'-diselenodipropionated docetaxel ÷ moles of docetaxel × 100%). The preparation reaction formula is as follows:

[0028]

[0029] The 3,3'-diselenodipropionated docetaxel synthesized in step (1) was characterized by 1H NMR spectroscopy and high performance liquid chromatography (HPLC). As shown in the figure, compared with free docetaxel, the 1H NMR spectrum of 3,3'-diselenodipropionated docetaxel showed characteristic peaks (1-8) of the 3,3'-diselenodipropionate group, indicating the successful synthesis of 3,3'-diselenodipropionated docetaxel. The peak elution time of 3,3'-diselenodipropionated docetaxel was 19.490 min and the purity was 84%, as determined by HPLC (mobile phase acetonitrile / water = 60:40, v / v; flow rate 1 mL / min; detection wavelength 227 nm).

[0030] (2) Preparation of docetaxel conjugate with galactosamine-3,3'-diselenodipropionate

[0031] Add 4 mL of N,N-dimethylformamide to a 10 mL round-bottom flask. While continuously stirring with a magnetic stirrer, add galactosamine (250 mg, 1.16 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (278 mg, 1.45 mmol), 4-dimethylaminopyridine (35 mg, 0.29 mmol), and docetaxel 3,3'-diselenopropionate prepared in step (1) (200 mg, 0.145 mmol). After dissolving by stirring at room temperature, heat to 50 °C and reflux. After stirring for 48 hours, the mixture was allowed to cool naturally to room temperature in the flask. The supernatant was obtained by centrifugation and transferred to a dialysis bag (molecular weight cutoff 800 Da). The mixture was dialyzed in sufficient ultrapure water for three days. The retained solution in the dialysis bag was then freeze-dried. The freeze-dried product was purified by column chromatography to obtain 198 mg (purity 87%) of a white powder solid galactosamine-3,3'-diselenopropionic acid docetaxel conjugate, with a yield of 80% (moles of galactosamine-3,3'-diselenopropionic acid docetaxel conjugate ÷ moles of 3,3'-diselenopropionic acid docetaxel × 100%). The preparation reaction formula is as follows:

[0032]

[0033] The synthesized galactosamine-3,3'-diselenodipropionated docetaxel conjugate was characterized by 1H NMR and IR spectroscopy. As shown in the figure, compared with the 3,3'-diselenodipropionated docetaxel obtained in step (1), the 1H NMR spectrum of the galactosamine-3,3'-diselenodipropionated docetaxel conjugate showed the characteristic peak of galactosamine (1-22), indicating that the formula was successfully synthesized; as shown in the figure, compared with free docetaxel, the 1657 cm⁻¹ infrared spectrum of the galactosamine-3,3'-diselenodipropionated docetaxel conjugate showed a higher peak at 1657 cm⁻¹. -1The enhanced peak is attributed to the C=O stretching vibration in the amide bond formed by the reaction of galactosamine with 3,3'-diselenopropionic acid-modified docetaxel, 1154 cm⁻¹. -1 The enhanced peak at 1115 cm⁻¹ is attributed to the COC stretching vibration in the ester bond formed by the reaction of 3,3'-diselenodipropionic acid and docetaxel. -1 The enhanced peak is attributed to the CO stretching vibration in galactosamine.

[0034] Example 2: Preparation of galactosamine-succinatedocetoxetine conjugate

[0035] (1) Preparation of succinate-modified docetaxel

[0036] Add 6 mL of dichloromethane to a 25 mL round-bottom flask. While continuously stirring with a magnetic stirrer, add docetaxel (200 mg, 0.248 mmol) and succinic anhydride (372.3 mg, 3.72 mmol). After dissolving at room temperature, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (475.42 mg, 2.48 mmol) and 4-dimethylaminopyridine (45.5 mg, 0.248 mmol). The mixture was heated to 40°C and refluxed with stirring for 24 hours. After cooling naturally to room temperature in the flask, the mixture was evaporated to dryness, and DMSO was added. The solution was then transferred to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in sufficient ultrapure water for three days. The retentate in the dialysis bag was freeze-dried to obtain 237.8 mg of a white powdery solid docetaxel succinate, with a yield of 95.3% (moles of docetaxel succinate ÷ moles of docetaxel × 100%).

[0037] The synthesized docetaxel succinate was characterized by 1H NMR spectroscopy and high performance liquid chromatography (HPLC). As shown in the figure, compared with free docetaxel, the 1H NMR spectrum of docetaxel succinate showed characteristic peaks of succinic anhydride (1-4 and 5-6), indicating the successful synthesis of docetaxel succinate. The elution time of docetaxel succinate was determined to be 7.405 min by HPLC (mobile phase acetonitrile / water = 60:40, v / v; flow rate 1 mL / min; detection wavelength 227 nm), and the purity was 87%.

[0038] (2) Preparation of galactosamine-succinate-docetaxel conjugate

[0039] Add 4 mL of N,N-dimethylformamide to a 10 mL round-bottom flask. While continuously stirring with a magnetic stirrer, add galactosamine (342 mg, 1.584 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (380 mg, 1.98 mmol), 4-dimethylaminopyridine (48 mg, 0.396 mmol), and docetaxel succinate prepared in step (1) (200 mg, 0.198 mmol). Stir at room temperature until dissolved. After decomposition, the mixture was heated to 50°C and refluxed with stirring for 48 hours. It was then allowed to cool naturally to room temperature in the flask, transferred to a dialysis bag (molecular weight cutoff 800 Da), and dialyzed in sufficient ultrapure water for three days. The retentate in the dialysis bag was then freeze-dried. The freeze-dried product was purified by column chromatography to obtain 224 mg (91% purity) of a white powder solid galactosamine-succinate-docetaxel conjugate, with a yield of 85% (moles of galactosamine-succinate-docetaxel conjugate ÷ moles of succinate-docetaxel × 100%).

[0040] The synthesized galactosamine-succinate-docetaxel conjugate was characterized by 1H NMR and IR spectra. As shown in the figure, compared with the succinate-docetaxel prepared in step (1), the characteristic peak of galactosamine (1-22) appeared in the 1H NMR spectrum of the galactosamine-succinate-docetaxel conjugate, indicating that the galactosamine-succinate-docetaxel conjugate was successfully synthesized. As shown in the figure, compared with free docetaxel, the 1657 cm⁻¹ infrared spectrum of the galactosamine-succinate-docetaxel conjugate showed a higher peak. -1 The enhanced peak at 1154 cm⁻¹ is attributed to the C=O stretching vibration in the amide bond formed by the reaction of galactosamine and succinate-docetaxel. -1 The enhanced peak at 1115 cm⁻¹ is attributed to the COC stretching vibration in the ester bond formed by the reaction of succinic anhydride and docetaxel. -1 The enhanced peak is attributed to the CO stretching vibration in galactosamine.

[0041] The preparation reaction formula for Example 2 is as follows:

[0042]

[0043] Example 3: Preparation of galactosamine-3,3'-dithiodipropionic acid-modified docetaxel conjugate

[0044] (1) Preparation of 3,3'-dithiodipropionic acid-modified docetaxel

[0045] Add 6 mL of dichloromethane to a 25 mL round-bottom flask. While continuously stirring with a magnetic stirrer, add docetaxel (200 mg, 0.248 mmol) and 3,3'-dithiodipropionic acid (416 mg, 1.984 mmol). After dissolving by stirring at room temperature, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (475.42 mg, 2.48 mmol) and 4-dimethylaminopyridine (45.5 mg, 0.372 mmol). The mixture was heated to 40°C and stirred under reflux for 24 hours. After the mixture was allowed to cool naturally to room temperature, DMSO was added and the solution was transferred to a dialysis bag (molecular weight cutoff 800 Da). The solution was then dialyzed in sufficient ultrapure water for three days. The precipitate in the dialysis bag was freeze-dried to obtain 263.48 mg of white powdered docetaxel 3,3'-dithiodipropionated, with a yield of 89.1% (moles of docetaxel 3,3'-dithiodipropionated ÷ moles of docetaxel × 100%).

[0046] The preparation reaction formula is as follows:

[0047]

[0048] The synthesized docetaxel 3,3'-dithiodipropionate was characterized by 1H NMR spectroscopy and high performance liquid chromatography (HPLC). As shown in the figure, compared with free docetaxel, the 1H NMR spectrum of 3,3'-dithiodipropionate showed characteristic peaks (1-8 and 9-10) of 3,3'-dithiodipropionate, indicating that the synthesis of 3,3'-dithiodipropionate docetaxel was successful. The elution time of 3,3'-dithiodipropionate docetaxel was 24.629 min and the purity was 93%, as determined by HPLC (mobile phase acetonitrile / water = 60:40, v / v; flow rate 1 mL / min; detection wavelength 227 nm).

[0049] (2) Preparation of docetaxel conjugate with galactosamine-3,3'-dithiodipropionic acid

[0050] Add 4 mL of N,N-dimethylformamide to a 10 mL round-bottom flask. While continuously stirring with a magnetic stirrer, add galactosamine (290 mg, 1.344 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (322 mg, 1.68 mmol), 4-dimethylaminopyridine (41 mg, 0.336 mmol), and docetaxel 3,3'-dithiodipropionate prepared in step (1) (200 mg, 0.168 mmol). After dissolving by stirring at room temperature, heat to 50 °C. After reflux and stirring at ℃ for 48 hours, the mixture was allowed to cool naturally to room temperature in the flask. It was then transferred to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed against sufficient ultrapure water for three days. The retentate in the dialysis bag was freeze-dried, and the freeze-dried product was purified by column chromatography to obtain 254 mg (purity 93%) of a white powder solid galactosamine-3,3'-dithiodipropionated docetaxel conjugate, with a yield of 86% (moles of galactosamine-3,3'-dithiodipropionated docetaxel conjugate ÷ moles of 3,3'-dithiodipropionated docetaxel × 100%). The preparation reaction formula is as follows:

[0051]

[0052] The synthesized galactosamine-3,3'-dithiodipropionated docetaxel conjugate was characterized by 1H NMR and IR spectroscopy. As shown in the figure, compared with the 3,3'-dithiodipropionated docetaxel prepared in step (1), the 1H NMR spectrum of the galactosamine-3,3'-dithiodipropionated docetaxel conjugate showed the characteristic peaks of galactosamine (1-22), indicating the successful synthesis of the galactosamine-3,3'-dithiodipropionated docetaxel conjugate. As shown in the figure, compared with free docetaxel, the 1657 cm⁻¹ infrared spectrum of the galactosamine-3,3'-dithiodipropionated docetaxel conjugate showed a higher peak at 1657 cm⁻¹. -1 The enhanced peak is attributed to the C=O stretching vibration in the amide bond formed by the reaction of galactosamine with 3,3'-dithiodipropionic acid-modified docetaxel, 1154 cm⁻¹. -1 The enhanced peak at 1115 cm⁻¹ is attributed to the COC stretching vibration in the ester bond formed by the reaction of 3,3'-dithiodipropionic acid and docetaxel. -1 The enhanced peak is attributed to the CO stretching vibration in galactosamine.

[0053] In vitro drug release assay:

[0054] The conjugates prepared in Examples 1-3 were sequentially dispersed at a concentration of 0.1 mM in release solution I (10 mM phosphate buffer containing 1% (mass percentage, the same below) Tween 80, pH 7.4), release solution II (10 mM phosphate buffer containing 1% Tween 80, 10 mM glutathione, pH 7.4), and release solution III (10 mM phosphate buffer containing 1% Tween 80, 0.1% hydrogen peroxide, pH 7.4). The solutions were then placed in a shaker at 37°C with continuous shaking (150 rpm). 1 mL of the release solution was taken at 1, 2, 4, 8, 12, and 24 hours and thoroughly mixed with 1 mL of acetonitrile. The concentration of docetaxel and its derivatives released was determined by HPLC, and the cumulative release amount was calculated. Three parallel determination experiments were performed at each time point, and the average cumulative release amount was taken, as shown in Table 1(1).

[0055] Table 1(1): Cumulative release (%) of the conjugates prepared in Examples 1-3

[0056]

[0057] As shown in Table 1(1), the diselenylene conjugate prepared in Example 1 exhibits both oxidation- and reduction-responsive drug release characteristics, while the disulfide conjugate prepared in Example 3 only exhibits reduction-responsive drug release characteristics. The conjugate prepared in Example 2 is insensitive to both oxidation and reduction environments. In terms of reduction responsiveness, the diselenylene conjugate prepared in Example 1 is superior to the disulfide conjugate prepared in Example 3. Because tumor cells have a high metabolic rate, resulting in higher oxidation and reduction levels than normal cells, the oxidation- and reduction-responsive drug release characteristics of the diselenylene conjugate prepared in Example 1 are more conducive to its selective drug release in tumor cells, leading to a higher efficacy in killing tumor cells.

[0058] In vitro drug release comparison experiment:

[0059] The 3,3'-diselenodipropionated docetaxel prepared in step (1) of Example 1, the succinic acid-modified docetaxel prepared in step (1) of Example 2, and the 3,3'-dithiodipropionated docetaxel prepared in step (1) of Example 3 were sequentially dispersed in release solution I (10 mM phosphate buffer containing 1% Tween 80, pH 7.4), release solution II (10 mM phosphate buffer containing 1% Tween 80, 10 mM glutathione, pH 7.4), and release solution III (10 mM phosphate buffer containing 1% Tween 80, 0.1% hydrogen peroxide, pH 7.4), respectively. 7.4 phosphate buffer) and then placed in a shaker at 37°C with continuous shaking (150 rpm). 1 mL of the release solution was taken at 1, 2, 4, 8, 12, and 24 hours, and thoroughly mixed with 1 mL of acetonitrile. The concentration of docetaxel and its derivatives released was determined by HPLC, and the cumulative release amount was calculated. Three parallel determination experiments were performed at each time point, and the average cumulative release amount was taken as shown in Table 1(2):

[0060] Table 1(2): Cumulative release (%) of the conjugates prepared in step (1) of Examples 1-3

[0061]

[0062]

[0063] As shown in Table 1(2), compared with the disulfide bond conjugate prepared in step (1) of Example 1, the diselenide bond conjugate still has the dual-response drug release characteristics of oxidation and reduction, while the disulfide bond conjugate prepared in step (1) of Example 3 still only has the reduction-responsive drug release characteristics. However, in terms of reduction responsiveness, the diselenide bond conjugate prepared in step (1) of Example 1 is significantly inferior to the disulfide bond conjugate prepared in step (1) of Example 3.

[0064] This is because the conjugates prepared in steps (1) of Example 1 and (1) of Example 3 do not contain galactose ligand end-capping, resulting in a smaller influence of the structure of the prepared product molecules on the diselenyl and disulfide bonds. Since sulfur has a stronger electron-accepting ability than selenium, the disulfide bond's advantage of being more sensitive to reduction conditions is evident here. In contrast, in the final product molecule structure of step (2) of Example 1, it is likely that both the galactose ligand and docetaxel influence the diselenyl bond, making its oxidation and reduction responses superior to those of the disulfide bond, thus giving it a greater advantage in killing tumor cells.

[0065] Evaluation of fibroblast toxicity in mice and evaluation of liver cancer cell killing effect:

[0066] (1) Preparation of test solution

[0067] Using dimethyl sulfoxide as solvent, 5 mM free docetaxel basic solutions, galactosamine-3,3'-diselenopropionic acid docetaxel conjugate basic solutions prepared in Example 1, galactosamine-succinate docetaxel conjugate basic solutions prepared in Example 2, and galactosamine-3,3'-dithiopropionic acid docetaxel conjugate basic solutions prepared in Example 3 were prepared. These basic solutions were then diluted with DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) to prepare test solutions of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM, respectively.

[0068] Prepare a 5 mg / mL phosphate buffer solution of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) (pH 7.4), filter it through a 0.22 μm filter membrane, and use it as the MTT solution.

[0069] (2) Evaluation of mouse fibroblast toxicity

[0070] Mouse fibroblast NIH3T3 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight for adhesion. The culture medium was then replaced with different concentrations of the test solution prepared in step (1) above (n=5). The cells were cultured at 37°C for 24 hours, 48 ​​hours, and 72 hours. After the culture cycle was completed, 20 μL of the MTT solution prepared in step (1) above was added, and the cells were cultured at 37°C for 4 hours. The culture medium was then removed, and 200 μL of dimethyl sulfoxide was added to dissolve the cells and produce purple crystals. The absorbance of each well at 492 nm was then measured using a microplate reader. Cells cultured in blank medium (containing only docetaxel or its conjugates) were considered to have 100% survival rate. The average cell survival rate of each experimental group was calculated to obtain the half-maximal inhibitory concentration (WMC) of mouse fibroblasts.

[0071] (3) Evaluation of the killing effect on liver cancer cells

[0072] HepG2 liver cancer cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight for adhesion. The culture medium was then replaced with different concentrations of the test solution prepared in step (1) above (n=5). The cells were cultured at 37°C for 24 hours, 48 ​​hours, and 72 hours. After the culture cycle was completed, 20 μL of the MTT solution prepared in step (1) above was added, and the cells were cultured at 37°C for 4 hours. The culture medium was then removed, and 200 μL of dimethyl sulfoxide was added to dissolve the cells and produce purple crystals. The absorbance of each well at 492 nm was then measured using a microplate reader. Cells cultured in blank medium (containing only docetaxel or its conjugates) were considered to have 100% survival rate. The average survival rate of cells in each experimental group was calculated to obtain the half-maximal inhibitory concentration (WMC) of HepG2 liver cancer cells.

[0073] Table 2: Half-inhibitory concentrations of each compound on NIH3T3 cells

[0074]

[0075] Table 3: Half-inhibitory concentrations of each compound on HepG2 cells

[0076]

[0077] Table 4: Ratio of the half-maximal inhibitory concentration (WMC) of each compound against NIH3T3 cells to the half-maximal inhibitory concentration (WMC) against HepG2 cells

[0078]

[0079] As shown in Tables 2 and 3, the liver cancer cell killing activity and cytotoxicity of the galactosamine-3,3'-diselenopropionic acid-modified docetaxel conjugate prepared in Example 1, the galactosamine-succinate-modified docetaxel conjugate prepared in Example 2, the galactosamine-3,3'-dithiopropionic acid-modified docetaxel conjugate prepared in Example 3, and free docetaxel were all time- and concentration-dependent. After incubation for 24 hours, 48 ​​hours, and 72 hours, the killing effect on HepG2 cells was: Example 1 > Example 3 > Example 2, all weaker than free docetaxel. Similarly, the cytotoxicity against NIH3T3 cells was: Example 1 > Example 3 > Example 2, all weaker than free docetaxel.

[0080] To compare the selective killing effects of the prepared conjugates and free docetaxel on HepG2 cells, the ratio of the half-maximal inhibitory concentration (WMC) of each compound against NIH3T3 cells to the WMC against HepG2 cells was calculated. A higher ratio indicates better selective killing of HepG2 cells. As shown in Table 4, the ratios of the WMC of the conjugates prepared in Examples 1-3 against NIH3T3 cells to the WMC against HepG2 cells were all higher than those of free docetaxel, thus demonstrating selective killing effects on HepG2 cells. The order was: Example 1 > Example 3 > Example 2 > Free docetaxel.

[0081] The above results indicate that the galactosamine-3,3'-diselenodipropionated docetaxel conjugate prepared in Example 1 exhibited the highest selective killing effect on HepG2 cells. This may be due to the dual-responsive drug release characteristics of the diselenobinate bond (the high oxidation and reduction environment of tumor cells allows the diselenobinate bond to break more rapidly and release the drug) and the targeting effect of galactose on liver cancer cells. The disulfide bond has a certain reduction responsiveness, therefore the galactosamine-3,3'-diselenodipropionated docetaxel conjugate prepared in Example 3 was the second most effective. Succinic acid has no responsiveness, therefore the galactosamine-succinodipropionated docetaxel conjugate prepared in Example 2 had a lower selective killing effect on HepG2 cells than the former two.

[0082] Although the three compounds prepared in Examples 1, 2, and 3 showed lower killing effects on HepG2 cells than free docetaxel, all three compounds could target liver cancer cells through conjugated galactose ligands. That is, the galactose ligands of the conjugates could be specifically recognized by endogenous lectin receptors (such as desialyl glycoprotein receptor ASGPR) highly expressed on the surface of liver cancer cells, thereby being specifically taken up by liver cancer cells through a receptor-mediated pathway, achieving the effect of selectively killing liver cancer cells. As a result, the conjugates prepared in Examples 1-3 showed higher selective killing effects on HepG2 cells than free docetaxel.

Claims

1. A galactosamine-docetaxel conjugate, characterized in that: The chemical structural formula of the coupling compound is as follows. 。 2. A method for preparing the galactosamine-docetaxel conjugate as described in claim 1, characterized in that: The preparation method is as follows: (1) Preparation of 3,3'-diselenopropionic acid-modified docetaxel Using docetaxel and 3,3'-diselenodipropionic acid as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent and 4-dimethylaminopyridine as a catalyst, the reaction was carried out in a solvent by heating, then cooled to room temperature, the resulting reaction system was evaporated to dryness, dissolved in DMSO, then transferred to ultrapure water for dialyzing, and finally freeze-dried to obtain the 3,3'-diselenodipropionic acid-modified docetaxel. (2) Using 3,3'-diselenodipropionated docetaxel and galactosamine prepared in step (1) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as condensing agent and 4-dimethylaminopyridine as catalyst, the reaction was heated in a solvent and then cooled to room temperature. The resulting reaction system was transferred to ultrapure water for dialyzing and then freeze-dried. The freeze-dried product was purified by column chromatography to obtain galactosamine-docetaxel conjugate.

3. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (1), the molar ratio of docetaxel, 3,3'-diselenopropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:8:10:1.

5.

4. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (1), the solvent is dichloromethane.

5. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (1), the heating reaction is a reflux reaction at 40°C for 24 hours.

6. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (2), the molar ratio of 3,3'-diselenopropionated docetaxel, galactosamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:8:10:

2.

7. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (2), the solvent is dimethyl sulfoxide or N,N-dimethylformamide.

8. The method for preparing the galactosamine-docetaxel conjugate as described in claim 2, characterized in that: In step (2), the heating reaction is a reflux reaction at 50°C for 24 hours.

9. The use of the galactosamine-docetaxel conjugate as described in claim 1 in the preparation of targeted therapies for liver cancer.

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