Preparation and application of podophyllotoxin-biotin conjugate and clathrate compound

Abstract

The invention provides a biotin-podophyllotoxin conjugate and a preparation method and application of a clathrate compound, and relates to the field of cancer treatment. The preparation method comprises the following steps: reacting-COOH of biotin with-OH or-NH2 of a connexon through acylation reaction, and coupling with-OH of podophyllotoxin, so as to prepare a plurality of biotin-podophyllotoxin conjugates, and preparing the biotin-podophyllotoxin conjugate inclusion compounds from the biotin-podophyllotoxin conjugates by using a solvent evaporation method. The reaction conditions are mild, the prepared biotin-podophyllotoxin conjugate and clathrate have specific reductive release or oxidative release characteristics in tumor cells, the targeting of podophyllotoxin is enhanced, the uptake rate of podophyllotoxin by tumor cells is enhanced, the toxicity of podophyllotoxin to tumor cells is improved, and the biotin-podophyllotoxin conjugate and clathrate have broad application prospects. Therefore, the compound has a wide application prospect in the field of anti-tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemicals, specifically to the preparation and application of podophyllotoxin-biotin conjugates and their inclusion compounds. Background Technology

[0002] Podophyllotoxin (PPT, structural formula A) is a lignan-based antitumor component extracted from the roots and stems of *Podophyllum heliotropium* (also known as podophyllin), a plant belonging to the genus *Podophyllum* in the family Berberidaceae. Podophyllotoxin effectively inhibits the assembly of microtubules in tumor cells, thereby inhibiting the structure of the mitotic spindle, thus exhibiting potent antitumor activity. However, the poor water solubility and lack of selectivity of natural podophyllotoxin, along with its high toxicity to both normal and tumor cells, limit its application in cancer treatment. To overcome these drawbacks, researchers have designed and developed numerous podophyllotoxin derivatives. For example, using podophyllotoxin and 5-carboxylic acid indole as raw materials, different indole substituents were introduced at the C-4 position of the podophyllotoxin core, resulting in the design and synthesis of a series of indole podophyllotoxin derivatives. Many studies have shown that the synthesized derivatives exhibit higher in vitro antitumor activity and lower toxicity compared to the original podophyllotoxin. However, these derivatives still suffer from poor water solubility, bone marrow suppression, and poor selectivity for tumor tissues, which limits the clinical application of podophyllotoxin. Therefore, improving the water solubility of podophyllotoxin, enhancing its selectivity for tumor cells, and reducing its toxic side effects on normal cells remain urgent problems to be solved. Stimulus-responsive drug carriers designed based on the tumor microenvironment have attracted widespread attention in antitumor drug delivery systems. By utilizing the characteristic signals of the tumor microenvironment as stimuli, various responsive drug delivery systems can be constructed to achieve targeted drug delivery to tumor sites. Studies have shown that disulfide bonds and thioether bonds can respond to high levels of reduced glutathione (GSH) and reactive oxygen species (ROS) in tumor cells, respectively, thereby triggering specific drug release, enhancing drug accumulation in tumor cells, and improving drug targeting.

[0003] Biotin (structural formula B) is a water-soluble vitamin, also known as vitamin H or coenzyme R. It plays a vital biological role in many organisms, being essential for the metabolism of carbohydrates, fats, and proteins, and a necessary nutrient for maintaining normal growth, development, and health. Research has found that the biotin receptor (BR) is a glycoprotein overexpressed on the surface of various tumor cells, while its expression is low or absent in normal cells. This characteristic allows biotin and its derivatives to act as targeting ligands, achieving specific recognition and drug delivery to tumor cells through high-affinity interaction with the biotin receptor. In recent years, numerous studies have shown that biotin can selectively deliver anticancer drugs to cancer cells with overexpressed biotin receptors, such as ovarian cancer, colon cancer, and lung cancer, thereby exerting its anticancer activity. When drug molecules bind to biotin to form biotin conjugates, the drug molecules can be specifically localized to the surface of tumor cells through the mediation of the biotin receptor. Conjugating biotin to podophyllotoxin can significantly enhance drug targeting and increase drug uptake by tumor cells. Summary of the Invention

[0004] Due to the overexpression of biotin receptors on the surface of certain tumor cells, biotin can serve as a targeted ligand for drug carriers, achieving targeted drug delivery through biotin-receptor interactions. Furthermore, many tumor cells exhibit higher levels of reducing GSH and ROS; designing GSH and ROS-sensitive chemical bonds can enhance drug release and accumulation in tumor cells. Based on these characteristics, this invention has developed a series of podophyllotoxin conjugates that are GSH and ROS-sensitive and coupled to linkers (including disulfide bonds, thioether bonds, and carbon bonds), improving the targeting of podophyllotoxin. These conjugates are then formulated with cyclodextrin to further enhance the targeting of podophyllotoxin, increase its water solubility, and better achieve tumor-inhibiting effects.

[0005] This invention provides a method for preparing a biotin-podophyllotoxin conjugate, wherein the biotin-podophyllotoxin conjugate has the structural formulas A1, A2, and A3.

[0006] Terminology Explanation: A1: Biotin-conjugated podophyllotoxin, linker is HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n , (n=2, 3, 4, 5, 6)).

[0007] A2: Biotin-conjugated podophyllotoxin, linker is XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n, (n=2, 3, 4, 5, 6)).

[0008] A3: Biotin-conjugated podophyllotoxin, linker is HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n , (n=2, 3, 4, 5, 6)).

[0009] B1: Podophyllotoxin coupled with an aliphatic carboxylic acid, with linkers of HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-). n , (n=2, 3, 4, 5, 6)).

[0010] B2: Podophyllotoxin conjugated with succinic anhydride (SAA).

[0011] B3: Podophyllotoxin conjugate, linker is XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n , (n=2, 3, 4, 5, 6)).

[0012] B4: Podophyllotoxin coupled with an aliphatic dicarboxylic acid, with the linker being HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-)). n , (n=2, 3, 4, 5, 6)).

[0013] B5: Biotin conjugate XRX (X=(-OH, -NH2), R=(-CH2-) n , (n=2, 3, 4, 5, 6)).

[0014] In this invention, the compound numbers and structural formula numbers correspond completely and have the same referential relationship, based on the compound structural formula.

[0015] This invention provides the following technical solutions: A method for preparing a biotin-podophyllotoxin conjugate and its inclusion complex, wherein the method shown in A1 includes the following steps: (1) Combine biotin with HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B1; (2) B1 was dissolved in a reaction solvent and reacted with podophyllotoxin in the presence of a catalyst and a dehydrating agent to obtain A1, with the following structural formula:

[0016] A1 (3) The inclusion complex was prepared by solvent evaporation. First, cyclodextrin (CD) was dissolved in anhydrous ethanol and stirred at 50°C for 20 min. Then, A1 with a molar amount of 0.5 times that of cyclodextrin was weighed and added to it. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A1 / CD inclusion complex.

[0017] Preferably, the catalyst in reaction steps (1) to (2) of the above reaction is one or more of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT) or N-methylpyrrolidone (NMP) (but not limited to the above three); the dehydrating agent is one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or 1-(tert-butoxycarbonyl)-2-(1H-1,2,3-triazol-4-yl)-ethane (HATU) (but not limited to the above three).

[0018] Preferably, the reaction steps (1) to (2) described above are carried out in an inert solvent; the inert solvent is any one of dichloromethane, chloroform, dioxane, ethyl acetate, acetonitrile or N,N-dimethylformamide, but is not limited to these solvents.

[0019] Preferably, the method for preparing the biotin-podophyllotoxin conjugate inclusion complex in reaction step (3) of the above reaction is characterized in that: the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, sulfobutyl-α-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl-γ-cyclodextrin or derivatives of cyclodextrin, but is not limited to the above.

[0020] Preferably, the reaction temperature of the reaction steps (1) to (2) described above is 0 to 80°C.

[0021] The preparation method of the biotin-podophyllotoxin conjugate and inclusion complex shown in A2 includes the following steps: (1) Podophyllotoxin and succinic anhydride are dissolved in a reaction solvent and reacted in the presence of a catalyst to obtain B2; (2) B2 reacts with XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B3; (3) B3 is dissolved in the reaction solvent with biotin and reacted in the presence of a catalyst and a dehydrating agent to obtain A2;

[0022] A2 (4) The inclusion complex was prepared by solvent evaporation. First, cyclodextrin (CD) was dissolved in anhydrous ethanol and stirred at 50°C for 20 min. Then, A2 with a molar amount of 0.5 times that of cyclodextrin was weighed and added to it. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A2 / CD inclusion complex.

[0023] Preferably, the catalyst in reaction steps (1) to (2) of the above reaction is one or more of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT) or N-methylpyrrolidone (NMP) (but not limited to the above three); the dehydrating agent is one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or 1-(tert-butoxycarbonyl)-2-(1H-1,2,3-triazol-4-yl)-ethane (HATU) (but not limited to the above three).

[0024] Preferably, the reaction steps (1) to (2) described above are carried out in an inert solvent; the inert solvent is any one of dichloromethane, chloroform, dioxane, ethyl acetate, acetonitrile or N,N-dimethylformamide, but is not limited to these solvents.

[0025] Preferably, the method for preparing the biotin-podophyllotoxin conjugate inclusion complex in reaction step (3) of the above reaction is characterized in that: the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, sulfobutyl-α-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl-γ-cyclodextrin or derivatives of cyclodextrin, but is not limited to the above.

[0026] Preferably, the reaction temperature of the reaction steps (1) to (2) described above is 0 to 80°C.

[0027] The preparation method of the biotin-podophyllotoxin conjugate and inclusion complex shown in A3 includes the following steps: (1) Podophyllotoxin is reacted with HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B4; (2) React biotin with HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst to obtain B5; (3) Dissolve B4 and B5 in the reaction solvent and react them in the presence of a catalyst and a dehydrating agent to obtain A3:

[0028] A3 (4) The inclusion complex was prepared by solvent evaporation. First, cyclodextrin (CD) was dissolved in anhydrous ethanol and stirred at 50°C for 20 min. Then, A3 with a molar amount of 0.5 times that of cyclodextrin was weighed and added to it. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A3 / CD inclusion complex.

[0029] Preferably, the catalyst in reaction steps (1) to (3) of the above reaction is one or more of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT) or N-methylpyrrolidone (NMP) (but not limited to the above three); the dehydrating agent is one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or 1-(tert-butoxycarbonyl)-2-(1H-1,2,3-triazol-4-yl)-ethane (HATU) (but not limited to the above three).

[0030] Preferably, the reactions in steps (1) to (3) of the above reaction are carried out in an inert solvent; the inert solvent is any one of dichloromethane, trichloromethane, dioxane, ethyl acetate, acetonitrile or N,N-dimethylformamide, but is not limited to these solvents.

[0031] Preferably, the method for preparing the biotin-podophyllotoxin conjugate inclusion complex in reaction step (4) of the above reaction is characterized in that: the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, sulfobutyl-α-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl-γ-cyclodextrin or derivatives of cyclodextrin, but is not limited to the above.

[0032] Preferably, the reaction temperature of the reaction steps (1) to (3) described above is 0 to 80°C.

[0033] Beneficial effects of the invention: This invention provides a method for preparing biotin-podophyllotoxin conjugates and inclusion complexes, as well as their applications. The method for preparing the biotin-podophyllotoxin conjugates and inclusion complexes is characterized by the conjugate being composed of the -OH group of podophyllotoxin and the linker HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-)). n (n=2, 3, 4, 5, 6)) reacts to generate B1, which then reacts with biotin to obtain A1; or podophyllotoxin first reacts with succinic anhydride to obtain B2, which then reacts with the linker XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6)) reacts to generate B3, and finally B3 reacts with biotin to obtain A2; or podophyllotoxin reacts with the linker HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6)) react to produce B4, while biotin first reacts with XRX (X=(-OH, -NH2), R=(-CH2-)) nThe reaction (n=2, 3, 4, 5, 6) generates B5, and the final reaction of the two yields A3. Then, the biotin-podophyllotoxin conjugate is prepared into a biotin-podophyllotoxin conjugate inclusion complex using a solvent evaporation method. The prepared biotin-podophyllotoxin conjugate and its inclusion complex exhibit significant beneficial effects. The biotin-podophyllotoxin conjugate contains disulfide or thioether bonds, which can respond to the high GSH and ROS environments within tumor cells to release the drug, respectively, thereby increasing drug release and accumulation within tumor cells. The inclusion complex of the biotin-podophyllotoxin conjugate and cyclodextrin improves the drug's water solubility. By specifically targeting biotin receptors on the surface of tumor cells through the targeting action of biotin, the selectivity of the conjugate and its corresponding inclusion complex for tumor cells is improved, enhancing anti-tumor effects and reducing toxicity to normal tissues. Furthermore, the preparation method of this invention is mild and easy for industrial production. Therefore, this invention has significant application prospects and beneficial effects. Attached Figure Description

[0034] Figure 1 Release curves of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD in PBS (containing 30% ethanol) containing 1 mM DTT are shown in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0035] Figure 2 Release curves of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD in PBS (containing 30% ethanol) with a concentration of 10 mM H2O2 are shown in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0036] Figure 3 Release curves of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 in PBS (containing 30% ethanol), i.e., in an environment free of DTT and H2O2.

[0037] Figure 4The effects of increasing concentrations of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, A3c / HP-β-CD, and podophyllotoxin on HeLa cell survival in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0038] Figure 5 The effects of biotin-inhibiting concentrations (0, 2, 5, 10 μM) on the survival rate of HeLa cells in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 were investigated.

[0039] Figure 6 The uptake of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, A3c / HP-β-CD, and podophyllotoxin in HeLa cells over time is shown in Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9. Detailed Implementation

[0040] The present invention will be further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0041] Example 1 (1) Preparation of B1a: 1 mmol β-mercaptoethylamine and 1 mmol mercaptoacetic acid were dissolved in ethyl acetate, and then 1.2 mmol H2O2 and 0.02 mmol potassium iodide were added. The reaction was allowed to proceed at room temperature for 2 h, and the reaction was quenched with a saturated solution (10 ml) of sodium thiosulfate. The organic layer was washed with brine and dried with anhydrous sodium sulfate. The liquid was collected by vacuum filtration. Then, the liquid was purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and methanol (V1:V2=10:1). The liquid was collected and concentrated under reduced pressure. Product B1a was obtained in a yield of 81.6%.

[0042]

[0043] B1a B1a NMR data: ¹H NMR (400 MHz, DMSO) δ 11.0 (t, ¹H), 3.65 (s, 2H), 3.09 (tt, J = 6.0, 3.7 Hz, 2H), 2.80 (t, J = 3.7 Hz, 2H), 1.89–1.79 (m, ¹H), 1.72 (dt, J = 6.7, 6.0 Hz, 1H). (2) Preparation of B2a: 1 mmol of B1a and 1 mmol of podophyllotoxin were dissolved in ethyl acetate, and then 2.5 mmol of DMAP (4-dimethylaminopyridine) was added. The reaction was carried out at room temperature for 12 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, the filtrate was collected, and the product was purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of petroleum ether and ethyl acetate (V1:V2=3:1). The liquid was collected and concentrated under reduced pressure. B2a was obtained in a yield of 72.3%.

[0044]

[0045] B2a (3) Preparation of A1a: 1 mmol B2a, 1 mmol biotin, and 2.5 mmol DMAP (4-dimethylaminopyridine) were dissolved in 6 mL of ethyl acetate and reacted overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the mixture, and the liquid was collected by vacuum filtration. The liquid was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. Product A1a was obtained in 74.4% yield.

[0046]

[0047] A1a NMR data for A1a: ¹H NMR (400 MHz, DMSO) δ 6.94 – 6.84 (m, 2H), 6.34 (dd, J = 9.5, 0.6 Hz, 3H), 5.96 (d, J = 1.6 Hz, 2H), 5.94 – 5.87 (m, 1H), 5.84 (d, J = 5.7 Hz, 1H), 5.74 (d, J = 5.4 Hz, 1H), 4.60 (ddt, J = 7.3, 3.3, 0.7 Hz, 1H), 4.49 – 4.42 (m, 1H), 4.28 – 4.23 (m, 2H), 4.20 (dd, J = 12.5, 2.6 Hz, 1H). 3.84 (s, 6H), 3.79 (s, 3H), 3.65 (d, J = 2.9 Hz, 2H), 3.62 – 3.53 (m,1H), 3.38 – 3.23 (m, 3H), 3.12 (tdd, J = 4.7, 2.2, 0.7 Hz, 1H), 2.89 – 2.70 (m, 4H), 2.18 (t, J = 8.2 Hz, 2H), 1.67 – 1.38 (m, 6H). (4) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A1a was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A1a / HP-β-CD inclusion complex.

[0048] Example 2 (1) Preparation of B4a: 1 mmol of B3a (3-((2-aminoethyl)thio)propionic acid) and 1 mmol of podophyllotoxin were dissolved in ethyl acetate, and then 2.5 mmol of DMAP (4-dimethylaminopyridine) was added. The reaction was carried out at room temperature for 12 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, the filtrate was collected, and the product was purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of petroleum ether and ethyl acetate (V1:V2=3:1). The liquid was collected and concentrated under reduced pressure. B4a was obtained in a yield of 63.2%.

[0049]

[0050] B3a

[0051] B4a (2) Preparation of A1b: 1 mmol B4a, 1 mmol biotin, and 2.5 mmol DMAP (4-dimethylaminopyridine) were dissolved in 6 mL ethyl acetate and reacted overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the mixture, and the liquid was collected by vacuum filtration. The liquid was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. Product A1b was obtained in a yield of 68.3%.

[0052]

[0053] A1b A1b NMR data: 1 H NMR (400 MHz, DMSO) δ 6.92 (d, J = 0.6 Hz, 1H), 6.73(t, J = 4.2 Hz, 1H), 6.34 (dd, J = 9.8, 0.6 Hz, 3H), 5.96 (d, J = 1.6 Hz, 2H), 5.87– 5.78 (m, 2H), 5.74 (d, J = 5.4 Hz, 1H), 4.60 (ddq, J = 8.0, 3.9, 0.7 Hz, 1H), 4.49 – 4.42 (m, 1H), 4.29 – 4.23 (m, 2H), 4.20 (dd, J = 12.5, 2.6 Hz, 1H), 3.84(s, 6H), 3.79 (s, 3H), 3.58 (ddd, J = 9.2, 8.0, 2.5 Hz, 1H), 3.38 – 3.26 (m,1H), 3.24 (td, J = 4.2, 1.8 Hz, 2H), 3.12 (tdd, J = 4.7, 2.2, 0.6 Hz, 1H), 2.92 –2.76 (m, 3H), 2.73 (d, J = 12.4 Hz, 1H), 2.68 – 2.57 (m, 4H), 2.18 (t, J = 8.2Hz, 2H), 1.67 – 1.38 (m, 6H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A1b was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A1b / HP-β-CD inclusion complex.

[0054] Example 3 (1) Preparation of B6a: 1 mmol of B5a (7-aminoheptaanoic acid) and 1 mmol of podophyllotoxin were dissolved in ethyl acetate, and then 2.5 mmol of DMAP (4-dimethylaminopyridine) was added. The reaction was carried out at room temperature for 12 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, the filtrate was collected, and the product was purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of petroleum ether and ethyl acetate (V1:V2=3:1). The liquid was collected and concentrated under reduced pressure. B6a was obtained in a yield of 78.5%.

[0055]

[0056] B5a

[0057] B6a (2) Preparation of A1c: 1 mmol B6a, 1 mmol biotin, and 2.5 mmol DMAP (4-dimethylaminopyridine) were dissolved in 6 mL ethyl acetate and reacted overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum. The liquid was then collected and purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. Product A1c was obtained in a yield of 55.6%.

[0058]

[0059] A1c A1c NMR data: 1 H NMR (400 MHz, DMSO) δ 6.92 (d, J = 0.7 Hz, 1H), 6.39 –6.30 (m, 4H), 5.96 (d, J = 1.6 Hz, 2H), 5.91 – 5.81 (m, 2H), 5.74 (d, J = 5.4 Hz, 1H), 4.60 (ddt, J = 7.3, 3.3, 0.7 Hz, 1H), 4.46 (dd,J = 5.8, 2.2 Hz, 1H), 4.29 –4.23 (m, 2H), 4.20 (dd, J = 12.5, 2.6 Hz, 1H), 3.84 (s, 6H), 3.79 (s, 3H), 3.58(ddd, J = 9.2, 8.0, 2.5 Hz, 1H), 3.31 (tddd, J = 9.2, 4.4, 3.8, 2.6 Hz, 1H), 3.17– 3.08 (m, 3H), 2.85 (d, J = 12.4 Hz, 1H), 2.72 (dd, J = 12.4, 2.1 Hz, 1H), 2.42– 2.33 (m, 2H), 2.18 (t, J = 8.1 Hz, 2H), 1.64 – 1.34 (m, 11H), 1.34 – 1.23 (m, 4H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A1c was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A1c / HP-β-CD inclusion complex.

[0060] Example 4 (1) Preparation of B1b: 1 mmol β-mercaptoethylamine and 1 mmol 2-mercaptoethanol were dissolved in 6 mL ethyl acetate, and then 1.2 mmol H2O2 and 0.02 mmol potassium iodide were added. The reaction mixture was allowed to stand at room temperature for 2 h. The reaction was quenched with a saturated solution (10 mL) of sodium thiosulfate, the organic layer was washed with brine, and dried with anhydrous sodium sulfate. The mixture was then filtered under vacuum, and the liquid was collected. The product was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and methanol (V1:V2=10:1). The liquid was collected and concentrated under reduced pressure. Product B1b was obtained in a yield of 76.6%.

[0061]

[0062] B1b The NMR data for B1b are as follows: 1 H NMR (400 MHz, DMSO) δ 3.76 (dt, J = 5.3, 4.3 Hz, 4H), 3.24 (t,J = 5.3 Hz, 2H), 3.08 (tt, J = 6.0, 3.7 Hz, 4H), 2.85 (t, J = 4.3 Hz, 4H), 2.77 (t, J = 3.7 Hz, 4H), 1.89 – 1.79 (m, 2H), 1.72 (dt, J = 6.7, 6.0 Hz, 2H). (2) Preparation of B2b: 1 mmol of podophyllotoxin and 1 mmol of succinic anhydride were dissolved in 10 mL of dichloromethane. After complete dissolution, 1.2 mmol of DMAP (4-dimethylaminopyridine) was added, and the mixture was refluxed at room temperature overnight. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mmol / L) (20 mL × 3), and the organic phase was washed with deionized water (20 mL × 3). The organic phase was dried with anhydrous sodium sulfate, filtered under vacuum, and the liquid was collected and concentrated under reduced pressure to obtain product B2b, with a yield of 60.7%.

[0063]

[0064] B2b (3) Preparation of B3b: 1 mmol of B2b and 1 mmol of B1b were dissolved in 10 mL of dichloromethane. After complete dissolution, 1.2 mmol of DMAP (4-dimethylaminopyridine) and 1.2 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added, and the reaction was carried out overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum and the liquid was collected. The solution was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. B3b was obtained in a yield of 53.4%.

[0065]

[0066] B3b (4) Preparation of A2a: 1 mmol B3b and 1 mmol biotin were dissolved in 10 mL dichloromethane. After complete dissolution, 1.2 mmol DMAP (4-dimethylaminopyridine) and 1.2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added. The reaction was carried out overnight at 30°C, and the reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum. The liquid was then collected and purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and methanol (V1:V2=10:1). The liquid was collected and concentrated under reduced pressure. A2a was obtained in a yield of 50.6%.

[0067]

[0068] A2a A2a NMR data: 1 H NMR (400 MHz, Chloroform- d ) δ 6.94 – 6.84 (m, 4H),6.34 (dd, J = 9.8, 0.6 Hz, 6H), 5.96 (d, J = 1.6 Hz, 4H), 5.91 – 5.84 (m, 3H), 5.83 (s, 1H), 5.74 (d, J = 5.3 Hz, 2H), 4.64 – 4.56 (m, 2H), 4.49 – 4.42 (m,2H), 4.32 – 4.16 (m, 10H), 3.84 (s, 12H), 3.79 (s, 6H), 3.58 (ddd, J = 9.2,8.0, 2.5 Hz, 2H), 3.36 – 3.23 (m, 6H), 3.12 (tdd, J = 4.7, 2.2, 0.7 Hz, 2H), 3.07 – 2.92 (m, 4H), 2.89 – 2.61 (m, 16H), 2.18 (t, J = 8.1 Hz, 4H), 1.64 –1.38 (m, 12H). (5) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A2a was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A2a / HP-β-CD inclusion complex.

[0069] Example 5 (1) Preparation of B5b: 1 mmol B2b and 1 mmol B4b (2-[(2-aminoethyl)sulfonyl]ethanol) were dissolved in 10 mL dichloromethane. After complete dissolution, 1.2 mmol DMAP (4-dimethylaminopyridine) and 1.2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added, and the reaction was carried out overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum. The liquid was then collected and purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. B5b was obtained in a yield of 45.1%.

[0070]

[0071] B4b

[0072] B5b (2) Preparation of A2b: 1 mmol B5b and 1 mmol biotin were dissolved in 10 mL dichloromethane. After complete dissolution, 1.2 mmol DMAP (4-dimethylaminopyridine) and 1.2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added. The reaction was carried out overnight at 30°C, and the reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum and the liquid was collected. The solution was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and methanol (V1:V2=10:1). The liquid was collected and concentrated under reduced pressure. A2b was obtained in a yield of 65.2%.

[0073]

[0074] A2b A2b NMR data: 1 H NMR (400 MHz, Chloroform- d ) δ 6.92 (d, J = 0.6 Hz, 2H), 6.77 (t, J = 4.2 Hz, 2H), 6.34 (dd, J = 9.8, 0.6 Hz, 6H), 5.96 (d, J = 1.6 Hz,4H), 5.91 – 5.81 (m, 4H), 5.74 (d, J= 5.3 Hz, 2H), 4.67 – 4.59 (m, 2H), 4.49 –4.42 (m, 2H), 4.32 – 4.16 (m, 10H), 3.84 (s, 12H), 3.79 (s, 6H), 3.58 (ddd, J =9.2, 8.0, 2.5 Hz, 2H), 3.36 – 3.24 (m, 4H), 3.23 (dd, J = 4.2, 1.5 Hz, 2H), 3.12 (tdd, J = 4.7, 2.2, 0.7 Hz, 2H), 2.86 (d, J = 2.1 Hz, 1H), 2.83 – 2.77 (m,2H), 2.77 – 2.55 (m, 18H), 2.18 (t, J = 8.2 Hz, 4H), 1.67 – 1.38 (m, 12H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A2b was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A2b / HP-β-CD inclusion complex.

[0075] Example 6 (1) Preparation of B7b: 1 mmol B2b and 1 mmol B6b (6-amino-1-hexanol) were dissolved in 10 mL dichloromethane. After complete dissolution, 1.2 mmol DMAP (4-dimethylaminopyridine) and 1.2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added, and the reaction was carried out overnight at 30°C. The reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was filtered under vacuum. The liquid was then collected and purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and ethyl acetate (V1:V2=5:1). The liquid was collected and concentrated under reduced pressure. B7b was obtained in a yield of 77.5%.

[0076]

[0077] B6b

[0078] B7b (2) Preparation of A2c: 1 mmol B7b and 1 mmol biotin were dissolved in 10 mL dichloromethane. After complete dissolution, 1.2 mmol DMAP (4-dimethylaminopyridine) and 1.2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added. The reaction was carried out overnight at 30°C, and the reaction progress was monitored by TLC. Anhydrous sodium sulfate was added to dry the solution, and the mixture was vacuum filtered and the liquid was collected. The solution was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and methanol (V1:V2=10:1). The liquid was collected and concentrated under reduced pressure. A2c was obtained in a yield of 64.7%.

[0079]

[0080] A2c A2c NMR data: 1 H NMR (400 MHz, Chloroform- d ) δ 6.92 (d, J = 0.6 Hz,2H), 6.39 – 6.30 (m, 8H), 5.96 (d, J = 1.6 Hz, 4H), 5.91 – 5.84 (m, 3H), 5.83(s, 1H), 5.74 (d, J = 5.3 Hz, 2H), 4.60 (ddq, J = 7.9, 3.9, 0.6 Hz, 2H), 4.49 –4.42 (m, 2H), 4.29 – 4.23 (m, 3H), 4.20 (dd, J = 12.5, 2.6 Hz, 2H), 4.06 (t, J =6.2 Hz, 4H), 3.84 (s, 11H), 3.79 (s, 6H), 3.58 (ddd, J = 9.2, 8.0, 2.5 Hz, 2H),3.30 (tddd, J = 9.3, 4.5, 3.7, 2.7 Hz, 2H), 3.17 – 3.08 (m, 6H), 2.85 (d, J =12.4 Hz, 2H), 2.79 – 2.68 (m, 6H), 2.68 – 2.61 (m, 4H), 2.18 (t, J = 8.1 Hz, 4H), 1.69 – 1.28 (m, 27H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A2c was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A2c / HP-β-CD inclusion complex.

[0081] Example 7 (1) Preparation of B2c: 1 mmol of podophyllotoxin and 2 mmol of B1c were dissolved in 10 mL of dichloromethane, followed by the addition of 1 mmol of DMAP (4-dimethylaminopyridine) and 1 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). The mixture was refluxed at 40°C overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mmol / L) (20 mL × 3), and the organic phase was washed with deionized water (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered under vacuum, and the liquid was collected. The liquid was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and ethyl acetate (V1:V2 = 1:1). The liquid was collected and concentrated under reduced pressure. Product B2c was obtained in 45.8% yield.

[0082]

[0083] B1c

[0084] B2C (2) Preparation of B3c: 1 mmol of activated biotin was dissolved in 6 mL of DMF, and 1 mmol of ethanolamine was added dropwise. The reaction was carried out overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the product was purified by column chromatography. Column chromatography conditions: elution was first performed with a mixed solution of dichloromethane, methanol, and acetic acid (V1:V2:V3=8:1:0.1), followed by elution with a mixed solution of dichloromethane and methanol (V1:V2=4:1). The liquid was collected and concentrated under reduced pressure. Product B3c was obtained with a yield of 89.3%.

[0085]

[0086] B3c (3) Preparation of A3a: Dissolve 1 mmol B2c and 1 mmol B3c in 6 mL DMF. Add 2 mmol DMAP (4-dimethylaminopyridine) and 2 mmol EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and react overnight at 30°C. Monitor the reaction progress by TLC. After the reaction is complete, purify by column chromatography. Column chromatography conditions: Elute with a mixture of dichloromethane and methanol (V1:V2=16:1). Collect the liquid and concentrate under reduced pressure. Obtain product A3a in 48.4% yield.

[0087]

[0088] A3a NMR data for A3a: ¹H NMR (400 MHz, DMSO) δ 7.92 (d, J = 5.2 Hz, 1H), 6.88 (s, 1H), 6.55 (d, J = 8.9 Hz, 3H), 6.41 (s, 1H), 6.35 (d, J = 7.1 Hz, 1H), 6.00 (d, J = 7.5 Hz, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.45 (dd, J = 9.4, 6.9 Hz, 1H), 4.33 - 4.27 (m, 3H), 4.14 (s, 1H), 4.02 (d, J = 5.9 Hz, 2H), 3.73 (d, J = 3.1 Hz, 6H), 3.65 (s, 3H), 3.67 - 3.55 (m, 2H), 3.27 (s, 1H), 3.10(s, 1H), 3.02 (s, 1H), 2.91 (dt, J = 13.6, 7.1 Hz, 2H), 2.84 (s, 2H), 2.85 –2.77 (m, 1H), 2.75 – 2.63 (m, 3H), 2.58 (d, J = 12.3 Hz, 1H), 2.07 –1.24 (m,8H). (4) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A3a was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the A3a / HP-β-CD inclusion complex.

[0089] Example 8 (1) Preparation of B5c: 1 mmol of podophyllotoxin and 2 mmol of B4c were dissolved in 20 mL of dichloromethane, and then 1 mmol of DMAP (4-dimethylaminopyridine) and 1 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added. The mixture was refluxed at 40°C overnight, and the reaction was monitored by TLC. After the reaction was completed, the mixture was washed with dilute hydrochloric acid (1 mmol / L) (20 mL × 3), and the organic phase was washed with deionized water (20 mL × 3). The organic phase was dried with anhydrous sodium sulfate, filtered under vacuum, and the liquid was collected. The liquid was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixed solution of dichloromethane and ethyl acetate (V1:V2=1:1). The liquid was collected and concentrated under reduced pressure. Product B5c was obtained in a yield of 47.6%.

[0090]

[0091] B4c

[0092] B5c (2) Preparation of A3b: Dissolve 1 mmol of B3c and B5c in 6 mL of DMF. Add 1 mmol of DMAP (4-dimethylaminopyridine) and 1 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and react overnight at 30°C. Monitor the reaction progress by TLC. After the reaction is complete, purify by column chromatography. Column chromatography conditions: Elute with a mixture of dichloromethane and methanol (V1:V2=16:1). Collect the liquid and concentrate under reduced pressure. Obtain product A3b in 51.8% yield.

[0093]

[0094] A3b A3b NMR data: 1 H NMR (400 MHz, DMSO) δ 6.94 – 6.83 (m, 2H), 6.34 (dd, J = 9.6, 0.6 Hz, 3H), 5.96 (d, J = 1.6 Hz, 2H), 5.87 – 5.78 (m, 2H), 5.74 (d, J=5.3 Hz, 1H), 4.67 – 4.59 (m, 1H), 4.49 – 4.42 (m, 1H), 4.28 – 4.23 (m, 2H), 4.23 – 4.13 (m, 3H), 3.84 (s, 6H), 3.79 (s, 3H), 3.58 (ddd, J = 9.2, 8.0, 2.5Hz, 1H), 3.42 – 3.33 (m, 2H), 3.33 – 3.26 (m, 1H), 3.12 (tdd, J = 4.7, 2.2, 0.7Hz, 1H), 2.95 – 2.77 (m, 5H), 2.73 (d, J = 12.4 Hz, 1H), 2.61 (dt, J = 9.1, 6.8Hz, 4H), 2.19 (t, J = 8.2 Hz, 2H), 1.68 – 1.38 (m, 6H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A3b was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A3b / HP-β-CD inclusion complex.

[0095] Example 9 (1) Preparation of B7c: 1 mmol of podophyllotoxin and 1 mmol of B6c were dissolved in 20 mL of dichloromethane, followed by the addition of 1 mmol of DMAP (4-dimethylaminopyridine) and 1 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). The mixture was refluxed at 40°C overnight, and the reaction was monitored by TLC. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mmol / L) (20 mL × 3), and the organic phase was washed with deionized water (20 mL × 3). The organic phase was dried with anhydrous sodium sulfate, filtered under vacuum, and the liquid was collected. The liquid was then purified by column chromatography. Column chromatography conditions: elution was performed using a mixture of dichloromethane and ethyl acetate (V1:V2 = 1:1). The liquid was collected and concentrated under reduced pressure. Product B7c was obtained in a yield of 53.6%.

[0096]

[0097] B6c

[0098] B7c (2) Preparation of A3c: Dissolve 1 mmol of B3c and B7c in 2 mL of DMF. Add 1 mmol of DMAP (4-dimethylaminopyridine) and 1 mmol of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and react overnight at 30°C. Monitor the reaction progress by TLC. After the reaction is complete, purify by column chromatography. Column chromatography conditions: Elute with a mixture of dichloromethane and methanol (V1:V2=16:1). Collect the liquid and concentrate under reduced pressure. Obtain product A3c in 42.6% yield.

[0099]

[0100] A3c A3c NMR data: 1 H NMR (400 MHz, DMSO) δ 6.94 – 6.83 (m, 2H), 6.34 (dd, J = 9.8, 0.6 Hz, 3H), 5.96 (d, J = 1.6 Hz, 2H), 5.87 – 5.78 (m, 2H), 5.74 (d, J =5.3 Hz, 1H), 4.67 – 4.59 (m, 1H), 4.46 (dd, J = 5.8, 2.2 Hz, 1H), 4.29 – 4.23(m, 2H), 4.23 – 4.13 (m, 3H), 3.84 (s, 6H), 3.79 (s, 3H), 3.58 (ddd, J = 9.2,8.0, 2.5 Hz, 1H), 3.40 (t, J = 4.9 Hz, 2H), 3.37 – 3.31 (m, 1H), 3.31 – 3.25(m, 1H), 3.12 (tdd, J = 4.7, 2.2, 0.7 Hz, 1H), 2.85 (d, J = 12.4 Hz, 1H), 2.72(dd, J = 12.4, 2.1 Hz, 1H), 2.42 – 2.26 (m, 4H), 2.19 (t, J = 8.1 Hz, 2H), 1.66 –1.58 (m, 3H), 1.57 (dt, J= 1.9, 1.0 Hz, 2H), 1.56 – 1.50 (m, 3H), 1.50 – 1.42(m, 3H), 1.42 – 1.35 (m, 3H), 1.35 – 1.29 (m, 1H). (3) The inclusion complex was prepared by solvent evaporation. First, 2 mmol of HP-β-CD was dissolved in 20 mL of anhydrous ethanol and stirred at 50°C for 20 min. Then, 1 mmol of A3c was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by vacuum concentration to obtain the A3c / HP-β-CD inclusion complex.

[0101] Water solubility evaluation Excess podophyllotoxin, A1a, A2a, A3a, A1b, A2b, A3b, A1c, A2c, A3c, A1a / HP-β-CD, A2a / HP-β-CD, A3a / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD were dissolved in 1 mL of deionized water and stirred at 37°C for 24 h. The solutions were then centrifuged (8000 rpm / 5 min), and the supernatants were collected. The absorbance was measured at 291 nm using a UV spectrophotometer, and the concentrations of these substances were calculated based on their UV standard curves to assess their water solubility at 37°C.

[0102] In vitro release experiment Compared to normal cells, tumor cells contain higher levels of GSH and ROS. Disulfide bonds are sensitive to reducing GSH, while thioether bonds are sensitive to ROS oxidation. Therefore, the conjugates containing disulfide bonds in this invention can be reduced by GSH to release the drug, and the conjugates containing thioether bonds can be oxidized to release the drug. DTT, as a strong reducing agent, has a stronger reducing ability than GSH. The release behavior of the inclusion complexes in PBS solution (containing 30% ethanol) was studied using DTT. 10 mg of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A2c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD were added to 40 mL of release medium (PBS solution containing 30% ethanol), respectively. The DTT concentration in the release medium was 1 mM. Figure 1 In addition, a control group without DTT was prepared. Figure 3The release medium was placed in a shaker at 37°C (150 rpm) and shaken. 200 μL of the release medium was collected at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, and 200 μL of fresh release medium was added. The release rate was detected and calculated using HPLC.

[0103] The release behavior of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A2c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD in PBS solution (containing 30% ethanol) with H2O2 was investigated to simulate a ROS oxidation environment. 10 mg of the inclusion complex was added to 40 mL of the release medium (PBS solution containing 30% ethanol). The concentration of H2O2 in the release medium was 10 mM. Figure 2 In addition, a control group without H2O2 was prepared. Figure 3 The release medium was placed in a shaker at 37°C (150 rpm) and shaken. 200 μL of the release medium was collected at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, and 200 μL of fresh release medium was added. The release rate was detected and calculated using HPLC.

[0104] Cytotoxicity assay HeLa cells (positive for biotin receptor expression) were seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 3 Cells were incubated for 24 hours. Podophyllotoxin (PPT) and its inclusion complexes A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A2c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD were added to 96-well plates using 10% fetal bovine serum DMEM medium. 100 μL of the drug-medium mixture was added to each well, resulting in final drug concentrations of 1 µM, 10 µM, 20 µM, 40 µM, 80 µM, and 160 µM. Three replicates were performed for each concentration. A control group was included, containing no drug-containing medium. The plates were incubated at 37°C in a CO2 incubator for 24 h. Then, 20 μL of MTT (5 mg / mL) was added to each well, and incubation continued for another 4 h. The medium was then removed, and 150 μL of MTT was added to each well. DMSO was used, and then the absorbance was measured at 570 nm to calculate cell viability.

[0105] To further verify the tumor-targeting properties of biotin, HeLa cells were seeded in 96-well plates at a density of 5 × 10⁶ cells per well. 3 Cells were incubated in 96-well plates with 100 μL of culture medium per well for 24 hours. Different concentrations of biotin (0, 20, 50, and 100 μM) were prepared using DMEM medium and added to each well at 20 μL. Cells were then cultured at 37°C with 5% CO2 for 2 hours to allow biotin receptors on the cancer cell surface to bind to different concentrations of biotin. Subsequently, podophyllotoxin (PPT), conjugates A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A2c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD were prepared using DMEM medium and added to each well at 80 μL of the drug-medium mixture, resulting in a final drug concentration of 160 µM and biotin concentrations of 0, 2, 5, and 10 μM. Three replicates were set up, and a control group was included. Cells were then cultured for another 24 hours. Absorbance was measured at 570 nm using a microplate reader to calculate cell viability.

[0106] Cellular uptake assay Trypsin was added to HeLa cells with 80% cell coverage in culture dishes to detach the two cell types from the surface of the dishes. The cells were then dispersed in 2 mL of culture medium and seeded into six-well plates. 1 mL of cell suspension was added to each well, followed by 1 mL of culture medium. The cell density in the six-well plates was 3 × 10⁶ cells per well. 5 Cells were then incubated overnight in a 37°C CO2 incubator containing 5% CO2 until approximately 80% confluence. Podophyllotoxin, A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD were diluted to a final concentration of 3 μg / mL using DMEM medium. The original medium was aspirated and replaced with 2 mL of drug-containing medium. Cells were then incubated at 37°C CO2 for 0.5 h, 1 h, and 3 h, respectively. After culturing for 0.5 h, 1 h, and 3 h, 200 μL of culture medium was aspirated, and the protein was precipitated with 400 μL of acetonitrile. The mixture was vortexed for 3 min and then centrifuged. The supernatant was then used to determine the drug content in the culture medium by high performance liquid chromatography.

[0107]

[0108] The results are shown in Tables 1, 2, and 3. At 37°C, the solubility of the A1a / HP-β-CD inclusion complex was 16.67 times that of A1a and 5.22 times that of podophyllotoxin. The solubility of the A2a / HP-β-CD inclusion complex was 35.33 times that of A2a and 12.23 times that of podophyllotoxin. The solubility of the A3a / HP-β-CD inclusion complex was 22.83 times that of A3a and 5.68 times that of podophyllotoxin. The solubility of the A1b / HP-β-CD inclusion complex was 13.5 times that of A1b and 3.62 times that of podophyllotoxin. The solubility of the A2b / HP-β-CD inclusion complex was 32.35 times that of A2b and 9.99 times that of podophyllotoxin. The solubility of the A3b / HP-β-CD inclusion complex was 14.86 times that of A3b and 4.25 times that of podophyllotoxin; the solubility of the A1c / HP-β-CD inclusion complex was 15.89 times that of A1c and 5.62 times that of podophyllotoxin; the solubility of the A2c / HP-β-CD inclusion complex was 55.18 times that of A2c and 12.59 times that of podophyllotoxin; and the solubility of the A3c / HP-β-CD inclusion complex was 16.05 times that of A3c and 5.90 times that of podophyllotoxin. These results indicate that all three prepared inclusion complexes improved the water solubility of the included guest molecules. Furthermore, compared to the parent drug podophyllotoxin, the water solubility of the inclusion complexes was also improved to some extent. This suggests that using HP-β-CD to form inclusion complexes with prodrugs can improve the solubility of conjugates in water.

[0109] Table 1. Solubility (μg / mL) of podophyllotoxin (PPT) and its conjugate prodrugs A1a, A2a, A3a, A1a / HP-β-CD, A2a / HP-β-CD and A3a / HP-β-CD in deionized water at 37°C.

[0110] Table 2. Solubility (μg / mL) of podophyllotoxin (PPT) and its conjugate prodrugs A1b, A2b, A3b, A1b / HP-β-CD, A2b / HP-β-CD and A3b / HP-β-CD in deionized water at 37°C.

[0111] Table 3. Solubility (μg / mL) of podophyllotoxin (PPT) and its conjugate prodrugs A1c, A2c, A3c, A1c / HP-β-CD, A2c / HP-β-CD and A3c / HP-β-CD in deionized water at 37°C.

[0112] from Figure 1 , 2As can be seen from Figure 3, within 24 hours, the release rates of inclusion complexes A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A2c / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, and A3c / HP-β-CD in PBS solution (containing 30% ethanol) were all between 20% and 30%, indicating that the amount of drug released by the inclusion complexes was very small under conditions without external stimulation. In the presence of 1 mM DTT, the release rates of A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD within 24 h were all around 25%, while the release rates of A1a / HP-β-CD, A2a / HP-β-CD, and A3a / HP-β-CD inclusion complexes within 24 h were all around 70%. This is due to the reducing sensitivity of disulfide bonds.

[0113] In a 10 mM H2O2 environment, the release rates of inclusion complexes A1a / HP-β-CD, A2a / HP-β-CD, and A3a / HP-β-CD were only about 40% after 24 h. However, the release rates of inclusion complexes containing thioether bonds (A1b / HP-β-CD, A2b / HP-β-CD, and A3b / HP-β-CD) all reached 75% after 24 h in a 10 mM H2O2 environment, indicating that inclusion complexes containing thioether bonds have a more significant sensitivity to H2O2 compared to those without H2O2. Figure 3 Compared with the drug release of H2O2, the release effect is more obvious. This is because the sulfide bond cleavage caused by oxidizing H2O2 accelerates the drug release.

[0114] In vitro release results showed that, without reducing and oxidizing stimulation, both reduction-sensitive and oxidation-sensitive biotin-podophyllotoxin conjugates remained stable, preventing premature drug release into the bloodstream and reducing toxic side effects on normal tissues. In the tumor microenvironment with high concentrations of DTT or H2O2, the drug was rapidly released, exerting its anti-tumor effect and enhancing therapeutic efficacy.

[0115] according to Figure 4As shown in Table 4, all drugs exhibited strong cytotoxicity in HeLa cells within a drug concentration range of 1–160 μM. However, at high concentrations, the cytotoxicity of the inclusion complexes A1a / HP-β-CD, A2a / HP-β-CD, A3a / HP-β-CD, A1b / HP-β-CD, A2b / HP-β-CD, A3b / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD was significantly stronger than that of free podophyllotoxin. This may be due to the high expression of BR on the surface of HeLa cells, which enhances the uptake of the inclusion complexes by HeLa cells, thereby enhancing the cytotoxicity of the inclusion complexes on HeLa cells. Furthermore, with increasing drug concentration, the cytotoxic effects and IC50 values ​​of A1b / HP-β-CD, A2b / HP-β-CD, and A3b / HP-β-CD on HeLa cells were observed to decrease. 50 The values ​​were all superior to those of A1a / HP-β-CD, A2a / HP-β-CD, and A3a / HP-β-CD. This may be due to the different drug release mechanisms caused by disulfide bonds and thioether bonds, which differ in their redox responsiveness. Disulfide bonds are widely used in the design of smart drug delivery systems due to their high reduction responsiveness, while thioether bonds are ROS-sensitive. Furthermore, the drug release of disulfide bond prodrugs is highly dependent on the reactivity between the disulfide bond and the thiol, while thioether bonds are broken by ROS-triggered reactions, exhibiting different reaction kinetics and resulting in faster or more efficient drug release within cells. The cytotoxicity of the A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD groups was significantly lower than other groups. This is because these conjugates do not contain redox-sensitive chemical bonds, preventing rapid or complete drug release and thus reducing cytotoxicity.

[0116] Table 4. Half-maximal inhibitory concentrations (IC50) of podophyllotoxin (PPT) and its conjugate prodrugs PPT, A1a / HP-β-CD, A2a / HP-β-CD, A3a / HP-β-CD, A1b / HP-β-CD, A2b / HP-β-CD, A3b / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD on HeLa cells. 50 Value (μM)

[0117] like Figure 5The results show the effect of prodrug conjugates on HeLa cell survival under different biotin concentrations at the same drug concentration. The results indicate that the survival rate of cells pretreated with biotin was higher than that of untreated cells, and this effect was concentration-dependent; the inhibitory effect became more pronounced with increasing biotin concentration. We can see that the biotin-linked podophyllotoxin (PPT) prodrug conjugate inclusion complexes A1a / HP-β-CD, A2a / HP-β-CD, A3a / HP-β-CD, A1b / HP-β-CD, A2b / HP-β-CD, A3b / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD exhibit significant biotin-dependent tumor targeting.

[0118] like Figure 6 As shown, within 0.5 h and 1 h, the uptake rates of A1a / HP-β-CD, A1b / HP-β-CD, A1c / HP-β-CD, A2a / HP-β-CD, A2b / HP-β-CD, A3a / HP-β-CD, A3b / HP-β-CD, A1c / HP-β-CD, A2c / HP-β-CD, and A3c / HP-β-CD in HeLa cells were significantly higher than those of podophyllotoxin. These data indicate that the uptake of tumor cell conjugate inclusion complexes is significantly higher relative to the parent drug. This is because HeLa cells have an overexpressed biotin receptor (BR) on their surface, allowing the inclusion complexes to specifically recognize BR, promote binding to tumor cells, and enter the cells via endocytosis. In contrast, podophyllotoxin enters cells via free diffusion, resulting in a slower cellular entry rate.

Claims

1. Preparation of a biotin-podophyllotoxin conjugate and its inclusion complex, characterized in that... The conjugate is composed of the -OH group of podophyllotoxin and the linker HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-). n Podophyllotoxin (n=2, 3, 4, 5, 6) reacts with the -COOH group to generate B1, which then reacts with the -COOH group of biotin to give A1; or podophyllotoxin first reacts with succinic anhydride to give B2, which then reacts with the linker XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-)). n The -OH group of podophyllotoxin (n=2, 3, 4, 5, 6) reacts to generate B3, which then reacts with biotin to give A2; or the -OH group of podophyllotoxin reacts with the linker HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-)). n The -COOH group of (n=2, 3, 4, 5, 6) reacts to form B4, while biotin first reacts with XRX (X=(-OH, -NH2), R=(-CH2-)). n (n=2, 3, 4, 5, 6)) react to generate B5, which then reacts with B4 to obtain A3. Biotin-podophyllotoxin conjugates (A1, A2 and A3) are then prepared into inclusion complexes with cyclodextrin or cyclodextrin derivatives. ； A1 ； A2 ； A3。 2. The method for preparing the biotin-podophyllotoxin conjugate according to claim 1, wherein the linker is HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6)), characterized in that The preparation methods of B1 and A1 include the following steps: (1) Connect podophyllotoxin with the linker HOOC-RYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B1; ； B1 (2) B1 reacts with biotin in a reaction solvent in the presence of a catalyst and a dehydrating agent to obtain A1.

3. The method for preparing a biotin-podophyllotoxin conjugate according to claim 1, wherein the linker is XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6)), characterized in that The preparation methods of B2 and A2 include the following steps: (1) Podophyllotoxin first reacts with succinic anhydride to give B2, and then reacts with linkers XRYRX (X=(-OH, -NH2), Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B3; ； B2 ； B3 (2) B3 reacts with biotin in a reaction solvent in the presence of a catalyst and a dehydrating agent to obtain A2.

4. The method for preparing a biotin-podophyllotoxin conjugate according to claim 1, wherein the linker is HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6)), characterized in that The preparation methods for B4, B5, and A3 include the following steps: (1) Podophyllotoxin first binds to the linker HOOC-RYR-COOH (Y=(-SS-, -S-, -CH2-), R=(-CH2-) n (n=2, 3, 4, 5, 6) are dissolved in the reaction solvent and reacted in the presence of a catalyst and a dehydrating agent to obtain B4; ； B4 ； B5 (2) Biotin and XRX (X=(-OH, -NH2), R=(-CH2-) n (n=2, 3, 4, 5, 6)) react to produce B5; finally, it reacts with B4 to produce A3.

5. The preparation method according to claims 2, 3, and 4, characterized in that: The catalyst in steps (1) to (2) of claims 2, 3, and 4 is one or more of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), or N-methylpyrrolidone (NMP) (but not limited to the above three); the dehydrating agent in steps (1) to (2) of claims 2, 3, and 4 is one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (but not limited to the above three). The reactions in steps (1) to (2) of claims 2, 3, and 4 are carried out in an inert solvent; the inert solvent is any one of dichloromethane, chloroform, dioxane, methanol, ethyl acetate, and N,N-dimethylformamide, but is not limited to the above solvents.

6. The preparation method according to claims 2, 3, and 4, characterized in that: The coupling reaction temperature in steps (1) to (2) of claims 2, 3, and 4 is 0 to 80°C.

7. The preparation of the biotin-podophyllotoxin conjugate and inclusion complex according to claim 1, characterized in that... The inclusion complexes were all prepared by solvent evaporation, as follows: First, cyclodextrin (CD) was dissolved in anhydrous ethanol and stirred at 50°C for 20 min. Then, biotin-podophyllotoxin conjugate, with a molar amount equal to 0.5 times that of cyclodextrin, was weighed and added to the solution. After complete dissolution, stirring was continued at 50°C for 2 hours. The solvent was removed by concentration under reduced pressure to obtain the biotin-podophyllotoxin inclusion complex.

8. The method for preparing the biotin-podophyllotoxin conjugate inclusion complex according to claim 7, characterized in that: Cyclodextrins are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, sulfobutyl-α-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl-γ-cyclodextrin, or derivatives of cyclodextrins, but are not limited to the above.

9. The method for preparing the biotin-podophyllotoxin conjugate inclusion complex according to claim 7, characterized in that: The organic solvent is any one of ethyl acetate, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide, but is not limited to these solvents. The aqueous solution can be any one of pure water, PBS buffer, physiological saline, or 5% glucose solution, but is not limited to these aqueous solutions.

10. The biotin-podophyllotoxin conjugate and inclusion complex prepared in this invention are applied in the field of tumor treatment, which enhances the targeting of podophyllotoxin, improves the water solubility of podophyllotoxin, enhances the uptake of podophyllotoxin by tumor cells, and improves the toxicity of podophyllotoxin to tumor cells.

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