Cysteine-doxorubicin conjugate, and preparation method and application thereof
By preparing a disulfide-linked conjugate of cysteine and docetaxel, the complexity and toxicity issues in the preparation of docetaxel nanocarriers were resolved, achieving efficient tumor cell uptake and drug release, and improving antitumor activity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHANGZHOU UNIV
- Filing Date
- 2024-02-29
- Publication Date
- 2026-04-28
AI Technical Summary
Existing docetaxel nanocarriers suffer from complex preparation processes, poor reproducibility, high costs, and carrier-related toxicity risks, as well as insufficient uptake and drug release efficiency within tumor cells.
The preparation process is simple: cysteine and docetaxel are linked by disulfide bonds to form a conjugate. The conjugate is formed by the reaction of docetaxel ester of 3-(2-pyridinedithio)propionate and cysteine.
It achieves efficient tumor cell uptake and reduction-responsive drug release, improves the inhibitory effect on prostate and breast cancer cells, and avoids carrier-related toxicity issues.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically relates to a cysteine-docetaxel conjugate, its preparation method, and its application. Background Technology
[0002] Paclitaxel is a natural product extracted from the yew tree and can be used to treat tumors such as breast cancer, prostate cancer, ovarian cancer, and non-small cell lung cancer. Docetaxel is a semi-synthetic taxane-based antitumor chemotherapy drug, also known as docetaxel. It has slightly higher water solubility and slightly better antitumor activity than paclitaxel. Nevertheless, docetaxel still faces challenges such as drug resistance in clinical use.
[0003] To address this, researchers have developed polymer-based docetaxel nanocarriers to improve the antitumor activity of docetaxel, achieving some success. However, these nanocarriers still suffer from challenges such as complex preparation processes, poor reproducibility, high costs, and carrier-related toxicity risks. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a cysteine-docetaxel conjugate with the following molecular structure:
[0005] .
[0006] The present invention also provides a method for preparing the above-mentioned cysteine-docetaxel conjugate.
[0007] (1) Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate
[0008] Docetaxel and 3-(2-pyridinedithio)propionic acid were dissolved in dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and stirred at room temperature (25°C, the same below) to dissolve the solution. The mixture was then heated to 40°C and stirred to react. After the reaction was completed, the solvent was evaporated to dryness, and the solution was purified by column chromatography to obtain docetaxel ester of 3-(2-pyridinedithio)propionic acid.
[0009] The molar ratio of docetaxel, 3-(2-pyridinedithio)propionic acid, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 1:1–2:5–15, and the reaction time was 12–48 hours.
[0010] The preparation reaction formula is:
[0011]
[0012] (2) Preparation of disulfide-linked cysteine-docetaxel conjugates
[0013] The 3-(2-pyridinedithio)propionic acid docetaxel ester prepared in step (1) was dissolved in N,N-dimethylformamide. Cysteine was added and stirred at room temperature to dissolve the ester. The mixture was then heated to 50°C and stirred for 24 hours. The reaction solution was cooled to room temperature and then added to a dialysis bag. The mixture was dialyzed in ultrapure water for 3 days and then freeze-dried to obtain a disulfide-linked cysteine-docetaxel conjugate.
[0014] The molar ratio of docetaxel 3-(2-pyridinedithio)propionic acid to cysteine is 1:1 to 2.
[0015] The preparation reaction formula is:
[0016] .
[0017] The present invention also provides an application of the above-mentioned cysteine-docetaxel conjugate in the field of antitumor therapy.
[0018] The beneficial effects of the present invention are as follows: the chemical structure of the docetaxel conjugate provided by the present invention is determined, the preparation process is simple, it has high tumor cell uptake and reduction-responsive drug release behavior, and it is superior to single docetaxel or other modified docetaxel conjugates in inhibiting prostate cancer cells 22Rv1 and breast cancer cells MCF-7, and it can avoid carrier-related toxicity problems. Attached Figure Description
[0019] Figure 1 The 1H NMR spectrum of docetaxel 3-(2-pyridinedithio)propionic acid ester prepared in Example 1;
[0020] Figure 2 The 1H NMR spectrum of the disulfide-linked cysteine-docetaxel conjugate prepared in Example 4;
[0021] Figure 3 High-resolution mass spectrum of the disulfide-linked cysteine-docetaxel conjugate prepared in Example 4;
[0022] Figure 4 The proton NMR spectrum of the disulfide-linked glutathione-docetaxel conjugate prepared in Comparative Example 1;
[0023] Figure 5 High-resolution mass spectrum of the disulfide-linked glutathione-docetaxel conjugate prepared for Comparative Example 1. Detailed Implementation
[0024] A method for preparing the above-mentioned cysteine-docetaxel conjugate:
[0025] (1) Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate
[0026] Docetaxel and 3-(2-pyridinedithio)propionic acid were dissolved in dichloromethane at a molar ratio of 1:1–2. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a molar ratio of 5–15:1 to docetaxel was added. After dissolution at room temperature (25°C), the mixture was heated to 40°C and stirred for 12–48 hours. After the reaction was complete, the solvent was evaporated to dryness, and the product was purified by column chromatography to obtain docetaxel ester of 3-(2-pyridinedithio)propionic acid. The reaction formula is as follows:
[0027]
[0028] (2) Preparation of disulfide-linked cysteine-docetaxel conjugates
[0029] The 3-(2-pyridinedithio)propionic acid docetaxel ester prepared in step (1) was dissolved in N,N-dimethylformamide. Following a molar ratio of 3-(2-pyridinedithio)propionic acid docetaxel ester to cysteine of 1:1-2, cysteine was added and stirred at room temperature until dissolved. The mixture was then heated to 50°C and stirred for 24 hours. The reaction solution was cooled to room temperature and then transferred to a dialysis bag. Dialysis was performed in ultrapure water for 3 days. After freeze-drying, a disulfide-linked cysteine-docetaxel conjugate was obtained. The reaction formula is as follows:
[0030] .
[0031] Example 1
[0032] Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate
[0033] Docetaxel (80.8 mg, 0.1 mmol) and 3-(2-pyridinedithio)propionic acid (21.5 mg, 0.1 mmol) were dissolved in 5 mL of dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (95.9 mg, 0.5 mmol) was added to the solution and stirred at room temperature. The solution was then heated to 40 °C and stirred for 48 h. After stirring, the solvent was evaporated to dryness and purified by column chromatography using a methanol / dichloromethane gradient elution to give docetaxel 3-(2-pyridinedithio)propionic acid ester in 78% yield (moles of docetaxel 3-(2-pyridinedithio)propionic acid ester obtained ÷ moles of docetaxel reactants (0.1 mmol) × 100%, the same below).
[0034] The docetaxel ester of 3-(2-pyridinedithio)propionic acid synthesized in Example 1 was characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Figure 1As shown, in addition to the hydrogen signal of docetaxel, the characteristic signal of pyridine hydrogen (8.5 ppm) also appeared in the 1H NMR spectrum, indicating that 3-(2-pyridinedithio)propionic acid and docetaxel ester were successfully condensed.
[0035] Example 2
[0036] Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate
[0037] Docetaxel (80.8 mg, 0.1 mmol) and 3-(2-pyridinedithio)propionic acid (32.3 mg, 0.15 mmol) were dissolved in 5 mL of dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (153.4 mg, 0.8 mmol) was added to the solution. After stirring and dissolving at room temperature, the solution was heated to 40 °C and stirred for 24 h. After stirring, the solvent was evaporated to dryness, and the solution was purified by column chromatography using a methanol / dichloromethane gradient elution to give docetaxel 3-(2-pyridinedithio)propionic acid ester in 81% yield.
[0038] Example 3
[0039] Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate
[0040] Docetaxel (80.8 mg, 0.1 mmol) and 3-(2-pyridinedithio)propionic acid (43 mg, 0.2 mmol) were dissolved in 5 mL of dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (191.7 mg, 1 mmol) was added to the solution. After stirring and dissolving at room temperature, the mixture was heated to 40 °C and stirred for 12 h. After stirring, the solvent was evaporated to dryness, and the mixture was purified by column chromatography using a methanol / dichloromethane gradient elution to give docetaxel 3-(2-pyridinedithio)propionic acid ester in 83% yield.
[0041] Example 4
[0042] Preparation of cysteine-docetaxel conjugates linked by disulfide bonds
[0043] The 3-(2-pyridinedithio)propionic acid docetaxel ester (100.5 mg, 0.1 mmol) prepared in Example 1 was dissolved in 5 mL of N,N-dimethylformamide, and then cysteine (12.2 mg, 0.1 mmol) was added. After stirring and dissolving at room temperature, the mixture was heated to 50 °C and stirred for 24 hours. After stirring, the reaction solution was cooled to room temperature and then added to a dialysis bag. After dialyzing in sufficient ultrapure water for 3 days, the retentate in the dialysis bag was freeze-dried to obtain the cysteine-docetaxel conjugate linked by disulfide bonds, with a yield of 90% (moles of cysteine-docetaxel conjugate obtained ÷ moles of 3-(2-pyridinedithio)propionic acid docetaxel ester reactant (0.1 mmol) × 100%, the same below).
[0044] The cysteine-docetaxel conjugates synthesized in Example 4, linked by disulfide bonds, were characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Figure 2 As shown, the characteristic signal of pyridine hydrogen disappeared in the 1H NMR spectrum, and the characteristic signal of cysteine (3-4 ppm) appeared, indicating that cysteine successfully substituted pyridine in docetaxel 3-(2-pyridinedithio)propionate.
[0045] like Figure 3 As shown, the high-resolution mass spectrometry revealed a [M+H] cysteine-docetaxel conjugate linked by disulfide bonds. + The characteristic peaks indicate that the cysteine-docetaxel conjugate linked by disulfide bonds in Example 4 was successfully synthesized.
[0046] Example 5
[0047] Preparation of cysteine-docetaxel conjugates linked by disulfide bonds
[0048] Based on Example 4, the amount of cysteine reactant was increased, and all other operations were the same as in Example 4:
[0049] The 3-(2-pyridinedithio)propionic acid docetaxel ester (100.5 mg, 0.1 mmol) prepared in Example 1 was dissolved in 5 mL of N,N-dimethylformamide, and then cysteine (18.3 mg, 0.15 mmol) was added. After stirring and dissolving at room temperature, the mixture was heated to 50 °C and stirred for 24 hours. After stirring, the reaction solution was cooled to room temperature and then added to a dialysis bag. After dialyzing in sufficient ultrapure water for 3 days, the retentate in the dialysis bag was freeze-dried to obtain the cysteine-docetaxel conjugate linked by disulfide bonds, with a yield of 92%.
[0050] Example 6
[0051] Preparation of cysteine-docetaxel conjugates linked by disulfide bonds
[0052] Based on Example 5, the amount of cysteine reactant was further increased, while the rest of the operations were the same as in Example 4:
[0053] The 3-(2-pyridinedithio)propionic acid docetaxel ester (100.5 mg, 0.1 mmol) prepared in Example 1 was dissolved in 5 mL of N,N-dimethylformamide, and then cysteine (24.4 mg, 0.2 mmol) was added. After dissolving by stirring at room temperature, the mixture was heated to 50 °C and stirred for 24 hours. After stirring, the reaction solution was cooled to room temperature and then added to a dialysis bag. After dialyzing in sufficient ultrapure water for 3 days, the retentate in the dialysis bag was freeze-dried to obtain the cysteine-docetaxel conjugate linked by disulfide bonds, with a yield of 89%.
[0054] Comparative Example 1
[0055] Preparation of glutathione-docetaxel conjugates linked by disulfide bonds
[0056] Based on Example 4, the reactant "cysteine" was replaced with an equimolar amount of "glutathione", and all other operations were the same as in Example 4:
[0057] The 3-(2-pyridinedithio)propionic acid docetaxel ester (100.5 mg, 0.1 mmol) prepared in Example 1 was dissolved in 5 mL of N,N-dimethylformamide, and then glutathione (30.7 mg, 0.1 mmol) was added. After dissolving by stirring at room temperature, the mixture was heated to 50 °C and stirred for 24 hours. After stirring, the reaction solution was cooled to room temperature and then added to a dialysis bag. After dialyzing in sufficient ultrapure water for 3 days, the retentate in the dialysis bag was freeze-dried to obtain the glutathione-docetaxel conjugate linked by disulfide bonds, with a yield of 85%.
[0058] The glutathione-docetaxel conjugate synthesized in Comparative Example 1 via disulfide bonds was characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Figure 4 As shown, the characteristic signal of pyridine hydrogen disappeared in the 1H NMR spectrum, and the characteristic signal of glutathione (3-4 ppm) appeared, indicating that glutathione successfully substituted pyridine in docetaxel 3-(2-pyridinedithio)propionate.
[0059] like Figure 5 As shown, the high-resolution mass spectrometry revealed a [M+H] glutathione-docetaxel conjugate linked by disulfide bonds. + The characteristic peaks indicate that the glutathione-docetaxel conjugate linked by disulfide bonds in Comparative Example 1 was successfully synthesized.
[0060] Comparative Example 2
[0061] Preparation of iRGD peptide-docetaxel conjugates linked by disulfide bonds
[0062] Based on Example 4, the reactant "cysteine" was replaced with an equimolar amount of "thiolized iRGD peptide", and all other operations were the same as in Example 4:
[0063] The docetaxel ester (100.5 mg, 0.1 mmol) of 3-(2-pyridinedithio)propionate prepared in Example 1 was dissolved in 5 mL of N,N-dimethylformamide. Then, thiolized iRGD peptide (107.9 mg, 0.1 mmol) was added to the solution and stirred at room temperature to dissolve. The solution was then heated to 50 °C and stirred for 24 hours. After stirring, the reaction solution was cooled to room temperature and then added to a dialysis bag. After dialyzing in sufficient ultrapure water for 3 days, the retentate in the dialysis bag was freeze-dried to obtain the iRGD peptide-docetaxel conjugate linked by disulfide bonds, with a yield of 71%.
[0064] In vitro drug release assay:
[0065] Preparation of pH 7.4 phosphate buffer: Add 2.16g of disodium hydrogen phosphate dodecahydrate, 0.26g of potassium dihydrogen phosphate, and 10g of Tween-80 to 100g of ultrapure water, stir and dissolve at room temperature, and bring the volume to 1000mL. Then adjust the pH to 7.4 with a 17% hydrochloric acid solution to obtain a pH 7.4 phosphate buffer.
[0066] Preparation of pH 7.4 phosphate buffer containing dithiothreitol: 2.16 g of disodium hydrogen phosphate dodecahydrate, 0.26 g of potassium dihydrogen phosphate, 10 g of Tween-80, and 1.54 g of dithiothreitol were added to 100 g of ultrapure water, stirred and dissolved at room temperature, and the volume was adjusted to 1000 mL. Then, the pH was adjusted to 7.4 with a 17% dilute hydrochloric acid aqueous solution to obtain the pH 7.4 phosphate buffer containing dithiothreitol.
[0067] The cysteine-docetaxel conjugates prepared in Example 4 were added to the above-mentioned pH 7.4 phosphate buffer and the above-mentioned pH 7.4 phosphate buffer containing dithiothreitol at a concentration of 50 μM, respectively, and stirred at room temperature to dissolve. 2 mL × 18 samples were taken from each group and placed in a shaker at 37°C with continuous shaking at 150 rpm. At time points of 1, 2, 4, 8, 12, and 24 hours, 3 samples were taken from each group and fully extracted with methanol. The concentrations of thiolated docetaxel and the total concentration of docetaxel were determined by high performance liquid chromatography, and the cumulative release rate was calculated (the number of moles of released docetaxel structure ÷ the total number of moles of docetaxel structure × 100%, the same below). The average value was taken, and the specific results are shown in Table 1.
[0068] The glutathione-docetaxel conjugates prepared in Comparative Example 1 were added to the above-mentioned pH 7.4 phosphate buffer and the above-mentioned pH 7.4 phosphate buffer containing dithiothreitol at a concentration of 50 μM, respectively, and stirred at room temperature to dissolve. 2 mL × 18 samples were taken from each group and placed in a shaker at 37℃ with continuous shaking at 150 rpm. At time points of 1, 2, 4, 8, 12, and 24 hours, 3 samples were taken from each group and fully extracted with methanol. The total concentration of thiolated docetaxel and docetaxel was determined by high performance liquid chromatography, and the cumulative release rate (average) was calculated. The specific results are shown in Table 1.
[0069] The iRGD peptide-docetaxel conjugates prepared in Comparative Example 2 were added to the above-mentioned pH 7.4 phosphate buffer and the above-mentioned pH 7.4 phosphate buffer containing dithiothreitol at a concentration of 50 μM, respectively, and stirred at room temperature to dissolve. 2 mL × 18 samples were taken from each group and placed in a shaker at 37℃ with continuous shaking at 150 rpm. At time points of 1, 2, 4, 8, 12, and 24 hours, 3 samples were taken from each group and fully extracted with methanol. The total concentration of thiolated docetaxel and docetaxel was determined by high performance liquid chromatography, and the cumulative release rate (average) was calculated. The specific results are shown in Table 1.
[0070] Table 1
[0071]
[0072] As shown in Table 1, cysteine-docetaxel conjugate, glutathione-docetaxel conjugate, and iRGD peptide-docetaxel conjugate were all relatively stable in phosphate buffer at pH 7.4, with a cumulative drug release of less than 1% over 24 hours. However, in phosphate buffer containing dithiothreitol at pH 7.4, the drug release of these three docetaxel conjugates was significantly accelerated. The cumulative drug release of cysteine-docetaxel conjugate was close to 90% over 24 hours, while the cumulative drug release of glutathione-docetaxel conjugate and iRGD peptide-docetaxel conjugate was more than 60% over 24 hours. It is evident that the reductive response release rate of cysteine-docetaxel conjugates is higher than that of glutathione-docetaxel conjugates and iRGD peptide-docetaxel conjugates. This indicates that, despite both conjugates containing disulfide bonds, the molecular structure of cysteine-docetaxel conjugates is significantly more sensitive to reduction, enabling them to respond more quickly to the reductive microenvironment within cells and effectively release the drug.
[0073] Cellular uptake experiment:
[0074] Docetaxel, the cysteine-docetaxel conjugate prepared in Example 4, the glutathione-docetaxel conjugate prepared in Comparative Example 1, and the iRGD peptide-docetaxel conjugate prepared in Comparative Example 2 were each fully dissolved in dimethyl sulfoxide to a concentration of 10 mM, which were used as stock solutions. The stock solutions were then diluted with 1640 medium to a concentration of 50 μM, which were used as test solutions.
[0075] Prostate cancer cells 22Rv1 were injected at a rate of 1×10⁻⁶ per well. 5 Cells were seeded at a density of 100 cells / well in 12-well plates and cultured in a CO2 incubator for 24 hours to allow adherence. Then, the prepared test solutions were incubated with the 22Rv1 cells for 6 hours. The test solutions were aspirated, and the cells were washed three times with phosphate-buffered saline (PFS). Cells were lysed with 1% Triton solution for 1 hour. The resulting cell lysates were mixed with methanol at a 1:1 ratio (v / v), and the concentration of docetaxel in each mixture was determined by high-performance liquid chromatography (HPLC), and the cell uptake (per 100 cells / well) was calculated. 5 The amount of docetaxel taken up by each cell (hereinafter the same). Three parallel experiments were set up for each test solution sample, and the average was taken. The specific results are shown in Table 2:
[0076] MCF-7 breast cancer cells were stored at 1×10⁻⁶ cells per well. 5Cells were seeded at a density of [number] cells per well into 12-well plates and cultured in a CO2 incubator for 24 hours to allow adherence. Then, the prepared test solutions were incubated with MCF-7 breast cancer cells for 6 hours. After aspirating the test solutions, the cells were washed three times with phosphate-buffered saline (PFS), and lysed with 1% Triton solution for 1 hour. The resulting cell lysates were mixed with methanol at a 1:1 ratio (v / v), and the concentration of docetaxel in each mixture was determined by high-performance liquid chromatography (HPLC), and the cell uptake was calculated. Three parallel experiments were conducted for each test solution sample, and the average results were taken. Specific results are shown in Table 2.
[0077] Table 2
[0078]
[0079] As shown in Table 2, the tumor cell uptake of cysteine-docetaxel conjugate was significantly higher than that of docetaxel alone and glutathione-docetaxel conjugate. Among them, the tumor cell uptake of glutathione-docetaxel conjugate was comparable to that of docetaxel alone, indicating that conjugation with glutathione did not increase the tumor cell uptake of docetaxel.
[0080] Although the iRGD peptide coupled in Comparative Example 2 is a known peptide structure with targeting ability against cancer cells, and the coupling did indeed improve the uptake of cancer cells, the cysteine-docetaxel conjugate prepared in this method still has a significantly higher uptake effect on cancer cells. This may be because tumor cells can more effectively take up the cysteine-docetaxel conjugate through cysteine transporters.
[0081] In vitro antitumor experiments:
[0082] Docetaxel, the cysteine-docetaxel conjugate prepared in Example 4, the glutathione-docetaxel conjugate prepared in Comparative Example 1, and the iRGD peptide-docetaxel conjugate prepared in Comparative Example 2 were each fully dissolved in dimethyl sulfoxide at a concentration of 10 mM to serve as the mother liquor.
[0083] The above-mentioned docetaxel stock solution was diluted with 1640 medium to prepare docetaxel test solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.
[0084] The above-mentioned cysteine-docetaxel conjugate stock solution was diluted with 1640 medium to prepare cysteine-docetaxel conjugate test solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.
[0085] Dilute the above glutathione-docetaxel conjugate stock solution with 1640 medium to prepare glutathione-docetaxel conjugate test solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM respectively.
[0086] Dilute the above iRGD peptide-docetaxel conjugate stock solution with 1640 medium to prepare iRGD peptide-docetaxel conjugate test solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM respectively.
[0087] Seed prostate cancer cells 22Rv1 into a 96-well plate at a density of 5×10 3 cells per well, and culture them in a carbon dioxide incubator for 24 hours to allow them to adhere. Then, incubate the above-prepared test solutions with the 22Rv1 cells for 48 hours respectively, and detect the cell viability by the standard MTT method. Calculate the half-inhibitory concentration of single docetaxel, cysteine-docetaxel conjugate, and glutathione-docetaxel conjugate against prostate cancer cells 22Rv1 respectively. Set 6 parallel experiments for each test solution, repeat the experiment three times, and take the average. The specific results are shown in Table 3:
[0088] Seed breast cancer cells MCF-7 into a 96-well plate at a density of 5×10 3 cells per well, and culture them in a carbon dioxide incubator for 24 hours to allow them to adhere. Then, incubate the above-prepared test solutions with the breast cancer cells MCF-7 for 48 hours respectively, and detect the cell viability by the standard MTT method. Calculate the half-inhibitory concentration of single docetaxel, cysteine-docetaxel conjugate, and glutathione-docetaxel conjugate against breast cancer cells MCF-7 respectively. Set 6 parallel experiments for each test solution, repeat the experiment three times, and take the average. The specific results are shown in Table 3:
[0089] Table 3
[0090]
[0091] As shown in Table 3, the half-inhibitory concentration of each compound against 22Rv1 cells and MCF-7 cells is as follows: cysteine-docetaxel conjugate < iRGD peptide-docetaxel conjugate < docetaxel < glutathione-docetaxel conjugate, indicating that the anti-tumor activity of cysteine-docetaxel conjugate is higher than that of single docetaxel, glutathione-docetaxel conjugate, and iRGD peptide-docetaxel conjugate. This is mainly because the cysteine-docetaxel conjugate has better reduction-responsive drug release characteristics and a high tumor cell uptake.
[0092] The antitumor activity of glutathione-docetaxel conjugate is lower than that of docetaxel alone, mainly due to the weaker reduction-responsive drug release characteristics of glutathione-docetaxel conjugate. At the same time, its cellular uptake is not superior to that of docetaxel alone.
[0093] While the antitumor activity of iRGD peptide conjugation has been significantly improved, it still shows a considerable gap compared with the cysteine-docetaxel conjugate prepared by this method.
Claims
1. A cysteine-docetaxel conjugate, characterized in that: The molecular structure of the coupling compound is as follows: 。 2. A method for preparing the cysteine-docetaxel conjugate as described in claim 1, characterized in that: The preparation method is as follows: (1) Preparation of docetaxel ester of 3-(2-pyridinedithio)propionate Docetaxel and 3-(2-pyridinedithio)propionic acid were dissolved in dichloromethane, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and stirred at room temperature to dissolve. The mixture was then heated and stirred to react. After the reaction was completed, the solvent was evaporated to dryness, and the 3-(2-pyridinedithio)propionic acid docetaxel ester was obtained by column chromatography. (2) Preparation of cysteine-docetaxel conjugate The 3-(2-pyridinedithio)propionic acid docetaxel ester prepared in step (1) was dissolved in N,N-dimethylformamide, cysteine was added and stirred at room temperature to dissolve, the reaction was heated and stirred, the reaction solution was cooled to room temperature, and then added to a dialysis bag for dialyzing in ultrapure water. After freeze-drying, the cysteine-docetaxel conjugate was obtained.
3. The method for preparing the cysteine-docetaxel conjugate as described in claim 2, characterized in that: In step (1), the molar ratio of docetaxel, 3-(2-pyridinedithio)propionic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1 to 2:5 to 15.
4. The method for preparing the cysteine-docetaxel conjugate as described in claim 2, characterized in that: The heating and stirring reaction described in step (1) is a stirring reaction at 40°C for 12 to 48 hours.
5. The method for preparing the cysteine-docetaxel conjugate as described in claim 2, characterized in that: In step (2), the molar ratio of docetaxel 3-(2-pyridinedithio)propionate to cysteine is 1:1 to 2.
6. The method for preparing the cysteine-docetaxel conjugate as described in claim 2, characterized in that: The heating and stirring reaction described in step (2) is a stirring reaction at 50°C for 24 hours.
7. The use of the cysteine-docetaxel conjugate as described in claim 1 in the preparation of an antitumor drug.
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