A self-assembled nanoformulation of a carbamate prodrug, its preparation method and application

Abstract

This invention belongs to the field of novel excipients and dosage forms for pharmaceutical preparations, and relates to the construction of four cabazitaxel prodrugs and their self-assembled nanoparticles. Specifically, it involves the synthesis of four different cabazitaxel prodrugs using three different disulfide bonds as connecting bonds and 9-fluorenylmethanol or cyclopentylmethanol as side chains, further preparing self-assembled nanoparticles, and exploring their application in drug delivery systems. The prodrugs are prepared by forming anhydrides from dithiodiacetic acid, 3,3'-dithiodipropionic acid, or 4,4'-dithiodibutyric acid, followed by esterification with 9-fluorenylmethanol or cyclopentylmethanol to obtain intermediate products. These intermediate products then undergo esterification with cabazitaxel to obtain the cabazitaxel prodrugs. Self-assembled nanoparticles of the cabazitaxel prodrugs are obtained through a one-step nanoprecipitation method. The prepared prodrug self-assembled nanoparticles can rapidly release the parent drug at the tumor site, exerting their tumor-killing effect and demonstrating excellent anti-tumor efficacy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to a preparation method and application of four cabazitaxel prodrugs and self-assembled nano-preparations thereof, in particular to a preparation of four cabazitaxel prodrugs with different positions of disulfide bonds as connecting bonds and 9-fluorenylmethanol or cyclopentylmethanol as side chains and self-assembled nano-preparations thereof and application of the same in a drug delivery system. BACKGROUND

[0002] Cabazitaxel (CTX) is an important member of the taxane family, and a solution has been marketed and applied in clinical treatment. Compared with other taxane drugs, cabazitaxel has lower affinity to P-glycoprotein, effectively hinders drug efflux, and thus has lower drug resistance risk. At present, cabazitaxel is often used in combination with prednisone in clinical treatment of prostate cancer. Cabazitaxel also plays a high therapeutic effect on patients with metastatic castration-resistant prostate cancer. In summary, CTX is a chemotherapy drug with promising clinical application prospects.

[0003] However However, cabazitaxel also shows serious toxic side effects in clinical application. On the one hand, due to uneven distribution in the body, it is easy to accumulate in intestinal cells and cause gastrointestinal toxicity such as diarrhea. On the other hand, due to the addition of a large amount of ethanol and Tween 80 for solubilization, the patient tolerance is poor, which greatly restricts its wide use. Moreover, free drugs are easily removed in the body, and lack of tumor targeting, with low delivery efficiency. These shortcomings greatly limit the clinical application of cabazitaxel. Therefore, how to reduce the toxic side effects of cabazitaxel and improve its delivery efficiency is crucial.

[0004] The prodrug strategy has a significant effect on reducing the toxic side effects of drugs. After structural modification, the drug activity and toxicity are greatly reduced, and by introducing a target site-specific corresponding connecting bond, the drug can be released specifically at the target site, reducing damage to other tissues and organs. In addition, in recent years, nano-drug delivery systems have attracted much attention. Nano-drug delivery systems can improve the pharmacokinetics of drugs, prolong the in vivo circulation time of chemotherapy drugs, and improve the bioavailability and stability of drugs. Among them, some nano-drug delivery systems have been used in clinical use, such as doxorubicin liposomes paclitaxel micelles and paclitaxel albumin nanoparticles and the like. These traditional nano-drug delivery systems generally use non-covalent physical embedding to load drugs, and thus have some shortcomings. For example: carrier-related toxicity, drug leakage, low drug loading capacity (usually less than 10%), etc.

[0005] Compared with the shortcomings of traditional nanocarriers, the self-assembled nanodelivery system based on small molecule prodrugs has obvious advantages. Specifically, the drug is modified by simple chemical modification to obtain inactive or low pharmacological activity prodrug, and then the physical and chemical properties are improved. Even stable and uniform nanometer preparation can be formed in aqueous solution by relying on molecular interaction. It has the advantages of no carrier material, high drug loading, good stability, high tumor accumulation capacity, low toxicity and side effects, and rapid release of drug efficacy at tumor site. SUMMARY

[0006] In order to solve the high toxicity of cabazitaxel and the low delivery efficiency in vivo, the present application provides three different disulfide bonds as connecting bonds, 9-fluorenylmethanol or cyclopentylmethanol as side chain, four different cabazitaxel prodrugs are synthesized, and further prepared into carrier-free self-assembled nanometer preparation by one-step nanometer precipitation method, and the application in the preparation of drug delivery system is explored. The preparation process of the present application is very simple, the particle size is small and uniform, the drug loading is higher than 40%, and the stability is good. In vitro experiments show that the introduction of disulfide bond can greatly improve the release rate of prodrug in reducing environment, and has good cytotoxicity effect. Compared with solution, the cell uptake efficiency of self-assembled nanometer preparation is higher. In vivo studies show that the blood circulation time of prodrug self-assembled nanoparticles in vivo is greatly prolonged, the tumor accumulation is obviously improved, and the anti-tumor effect is significantly improved.

[0007] The present application realizes the above-mentioned purposes through the following technical solutions:

[0008] The present application provides a cabazitaxel prodrug with tumor reduction response characteristics, which is connected by a tumor-sensitive connecting bond between an antitumor therapeutic agent and a group or a cyclic group with conjugated structure.

[0009] Further, the antitumor therapeutic agent is an anticancer therapeutic agent containing an active hydroxyl group, selected from cabazitaxel and its derivatives, anthracycline compounds or camptothecin compounds, the tumor-specific response connecting bond includes disulfide bond, monosulfide bond, trisulfide bond, monoselenide bond, diselenide bond, ester bond or amide bond, and the compound with conjugated structure is 9-fluorenylmethanol, 2-(9-fluorenyl)ethanol and 9-fluorenyl acetic acid. The compound of the cyclic group is cyclopentylmethanol, cyclohexylmethanol and cycloheptylmethanol.

[0010] Specifically, the present application provides the cabazitaxel prodrug, which is connected by (a) dithiodiacetic acid, (b) 3,3'-dithiodipropionic acid, (c) 4,4'-dithiodibutyric acid, respectively, with cabazitaxel as a model drug and 9-fluorenylmethanol (Fmoc) as a side chain; and (d) 4,4'-dithiodibutyric acid, with cabazitaxel as a model drug and cyclopentylmethanol (COPL) as a side chain, the structural formula is as follows:

[0011]

[0012] Further, the present application provides a synthesis method of the four cabazitaxel prodrugs, comprising the following steps:

[0013] (1) Dehydrate dithiodiacetic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid into anhydride by acetic anhydride, respectively;

[0014] (2) Esterify the obtained anhydride with 9-fluorenylmethanol (Fmoc) or cyclopentylmethanol (COPL), respectively (esterification reaction occurs), to obtain an intermediate product;

[0015] (3) Esterify the obtained intermediate product with cabazitaxel (CTX), respectively, and finally obtain the end product after separation.

[0016] The reaction formula is as follows:

[0017]

[0018] Further, in step (1), the reaction temperature is 15-60℃, and the time is 2-6 hours.

[0019] Further, in step (2), the solvent is dichloromethane, the molar ratio of anhydride to 9-fluorenylmethanol (Fmoc) or cyclopentylmethanol (COPL) is 1:1-5:1, the catalyst is 4-dimethylaminopyridine (DMAP), the molar ratio of 9-fluorenylmethanol (Fmoc) or cyclopentylmethanol (COPL) to the catalyst 4-dimethylaminopyridine (DMAP) is 1:0.05-0.8, preferably 1:0.1; the reaction temperature is 20-50℃, preferably room temperature, and the time is 4-24 hours, preferably overnight.

[0020] Further, in step (3), the solvent is dichloromethane, the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP), the molar ratio of cabazitaxel, the intermediate product, EDCI, HOBt and DMAP is 1:0.2-5:0.5-4:0.5-4:0.05-0.8, preferably 1:1:2:2:0.2; the reaction temperature is 15-40℃, preferably 25℃, and the time is 24-60 hours, preferably 48-60 hours.

[0021] Specifically, the synthesis method of the four cabazitaxel prodrugs comprises the following steps:

[0022] (1) Dissolve dithiodiglycolic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid in excess acetic anhydride, protect with nitrogen, react at room temperature for 2-4 hours, and then remove the excess acetic anhydride;

[0023] (2) Dissolve the product obtained in step (1) in dichloromethane, and add 9-fluorenylmethanol or cyclopentylmethanol, and add 4-dimethylaminopyridine (DMAP) as a catalyst, stir overnight at room temperature, and after the reaction is completed, separate the intermediate product by column chromatography using a dichloromethane-methanol elution system;

[0024] (3) Dissolve the intermediate product, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP) in anhydrous dichloromethane, activate in an ice bath for 2-4 hours, then add docetaxel, protect with nitrogen, and react at 25°C for 48-60 hours, and separate and purify the obtained product by preparative liquid chromatography.

[0025] The present application also provides a self-assembled nano-preparation of the cabazitaxel prodrug, which can be a non-PEGylated cabazitaxel prodrug nano-preparation, a cabazitaxel prodrug nano-preparation modified by a PEG modifier, or a cabazitaxel prodrug nano-preparation loaded with a fluorescent substance or a hydrophobic drug; the PEG modifier is TPGS (vitamin E polyethylene glycol succinate), PLGA-PEG (poly lactic acid-glycolic acid copolymer-polyethylene glycol), PE-PEG (polyethylene-polyethylene glycol), DSPE-PEG-AA (distearoyl phosphatidyl ethanolamine-polyethylene glycol-arachidonic acid), or DSPE-PEG (distearoyl phosphatidyl ethanolamine-polyethylene glycol), and the weight ratio of the cabazitaxel prodrug to the PEG modifier is 90:10-50:50; the fluorescent substance or the hydrophobic drug is at least one of coumarin-6, rhodamine, DiR, DiI, Cy-5 and Cy-7, and the weight fraction of the fluorescent substance or the hydrophobic drug in the cabazitaxel prodrug is 1%-30%.

[0026] Further, the molecular weight of the PEG in the PEG modifier is 200-4000.

[0027] The present application also provides a preparation method of the cabazitaxel prodrug self-assembled nano-preparation:

[0028] A certain amount of the cabazitaxel prodrug or a mixture of the cabazitaxel prodrug and the PEG modifier, or a mixture of the cabazitaxel prodrug, the PEG modifier, the fluorescent substance or the hydrophobic drug is dissolved in a proper amount of anhydrous ethanol, and the anhydrous ethanol solution is slowly dropped into water under the stirring of a magnetic stirrer, and the prodrug can be spontaneously assembled into nano-particles with uniform particle size. Specifically,

[0029] (1) The preparation method of non-PEGylated nanoparticles: a certain amount of cabazitaxel prodrug is dissolved in a proper amount of anhydrous ethanol, and the anhydrous ethanol solution is slowly added to deionized water under the stirring of a magnetic stirrer, and the prodrug can spontaneously assemble into nanoparticles with uniform particle size, and the anhydrous ethanol in the preparation is removed by a rotary evaporation method under reduced pressure to obtain a nanoparticle preparation without organic solvents.

[0030] (2) The preparation method of PEGylated nanoparticles: a certain amount of cabazitaxel prodrug and PEG modifier are dissolved in a proper amount of anhydrous ethanol, and the anhydrous ethanol solution is slowly added to deionized water under the stirring of a magnetic stirrer, and the prodrug can assemble into PEG-modified nanoparticles with uniform particle size, and the anhydrous ethanol in the preparation is removed by a rotary evaporation method under reduced pressure to obtain PEGylated nanoparticles without organic solvents, and the PEG modifier is TPGS, PLGA-PEG, PE-PEG, DSPE-PEG-AA or DSPE-PEG.

[0031] (3) The preparation method of small molecule prodrug self-assembled nanoparticles loaded with fluorescent substances or hydrophobic drugs: a certain amount of fluorescent substances (such as coumarin-6, rhodamine, DiR, DiI, Cy-5 or Cy-7) or hydrophobic drugs (such as paclitaxel, docetaxel, SN-38, camptothecin), cabazitaxel prodrug and PEG modifier are dissolved in a proper amount of ethanol, and the ethanol solution is slowly added to water under stirring, and the prodrug spontaneously forms uniform nanoparticles. The ethanol in the preparation is removed by rotary evaporation under reduced pressure to obtain a nanoparticle colloidal solution without any organic solvents.

[0032] The application also provides the use of the cabazitaxel prodrug or the cabazitaxel prodrug self-assembled nanoparticles in a drug delivery system.

[0033] The application also provides the use of the cabazitaxel prodrug or the cabazitaxel prodrug self-assembled nanoparticles in the preparation of an antitumor drug.

[0034] The application also provides the use of the cabazitaxel prodrug or the cabazitaxel prodrug self-assembled nanoparticles in an injection, oral administration or topical administration system.

[0035] The application has the following beneficial effects:

[0036] (1) the cabazitaxel is prepared into a prodrug, which can greatly alleviate the strong toxic side effects of cabazitaxel, and further prepared into a self-assembled nano preparation through one-step nano precipitation method, which can avoid the use of carrier materials and cosolvents, and further reduce the toxic side effects; (2) the nano preparation is prepared through one-step nano precipitation method, and the preparation process is extremely simple, and easy to scale production of the preparation; (3) the nano particle has small and uniform particle size, and through PEG modification, the circulation time of the drug in the blood can be prolonged, and the drug is easy to enrich in the tumor site through the EPR effect; (5) the prodrug introduces a tumor-specific response disulfide bond, has a tumor reduction sensitive drug release property, can realize specific and rapid release of the parent drug in the intracellular high reduction environment of the tumor site, and play a tumor killing role, and has excellent antitumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum.

[0038] Figure 2 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum.

[0039] Figure 3 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum.

[0040] Figure 4 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum. 1 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum.

[0041] Figure 5 The figure is the mass spectrum of the cabazitaxel prodrug γ-Fmoc-SS-CTX in the embodiment 1 of the present application and the HNMR spectrum, wherein A is the mass spectrum, B is the HNMR spectrum.

[0042] Figure 6Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0043] Figure 7 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0044] Figure 8 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0045] Figure 9 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0046] Figure 10 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0047] Figure 11 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0048] Figure 12 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0049] Figure 13 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0050] Figure 14 Pictures of appearance of three PEG modified cabazitaxel prodrugs self-assembly nanoparticles of embodiment 3 of the present application, wherein a is α-Fmoc-SS-CTX, b is β-Fmoc-SS-CTX, and c is γ-Fmoc-SS-CTX.

[0051] Figure 15In vivo drug distribution of PEG modified prodrug self-assembled nanoparticles of Example 9 of the present application, wherein the left picture is in vivo fluorescence picture, and the right picture is ex vivo fluorescence picture, a is DiR / α-Fmoc-SS-CTX nanoparticles, b is DiR / β-Fmoc-SS-CTX nanoparticles, c is DiR / γ-Fmoc-SS-CTX nanoparticles, d is DiR solution, H: heart, Li: liver, S: spleen, L: lung, K: kidney, T: tumor.

[0052] Figure 16 Fluorescence quantification of in vivo drug distribution of PEG modified prodrug self-assembled nanoparticles of Example 9 of the present application.

[0053] Figure 17 Pharmacodynamics of PEG modified prodrug self-assembled nanoparticles of Example 10 of the present application, wherein A is tumor growth curve, B is tumor burden, C is tumor picture, D is body weight change. DETAILED DESCRIPTION

[0054] The present application is further illustrated by the following examples without limiting the application to the described examples.

[0055] Example 1: Synthesis of four cabazitaxel prodrugs (γ-Fmoc-SS-CTX, γ-CPOL-SS-CTX, α-Fmoc-SS-CTX, β-Fmoc-SS-CTX)

[0056] (1) Synthesis of γ-Fmoc-SS-CTX

[0057] Into a 100 mL round bottom flask, 4,4'-dithiodibutyric acid (499.9 mg, 2.1 mmol) was added, and reacted at 25°C for 2 h in 5 mL acetic anhydride as solvent. After the reaction was completed, the reaction was distilled under reduced pressure to remove acetic anhydride in the system, and 10 mL of toluene was added to the system in three portions, and dried under reduced pressure. The obtained product was dissolved in 25 mL of dichloromethane, and Fmoc (412.1 mg, 2.1 mmol) and DMAP (25.1 mg, 0.21 mmol) were added, and stirred at 25°C for 12 h. The intermediate product (230.0 mg, yield 46.0%) was separated and purified by column chromatography.

[0058] The intermediate product (230.0 mg, 0.48 mmol), EDCI (184.0 mg, 0.96 mmol), HOBT (127.8 mg, 0.96 mmol) and DMAP (5.9 mg, 0.048 mmol) were dissolved in 30 mL of dichloromethane, activated in an ice water bath for 2 h, cabazitaxel (409.9 mg, 0.48 mmol) was added after the activation was completed, stirred at 25 °C for 48 h, and the final product (120 mg, yield 29.1%) was purified by preparative liquid separation. The above reactions were carried out under nitrogen protection.

[0059] The structure of the prodrug in Example 1 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrum, and the results are shown in Figure 1 The nuclear magnetic hydrogen spectrum analysis results are as follows, respectively:

[0060] 1 H NMR (600 MHz, DMSO-d6) δ 7.99-7.95 (m, 2H), 7.90 (d, J = 7.4 Hz, 2H), 7.74 (s, 1H), 7.69-7.63 (m, 4H), 7.42 (t, J = 7.3 Hz, 4H), 7.38-7.31 (m, 4H), 5.76 (s, 1H), 5.36 (d, J = 7.1 Hz, 1H), 5.09 (d, J = 8.1 Hz, 1H), 4.68 (s, 1H), 4.49 (s, 1H), 4.45 (d, J = 6.4 Hz, 2H), 4.27 (s, 1H), 4.01 (s, 2H), 2.69 (t, J = 7.1 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.53 (d, J = 7.3 Hz, 1H), 2.41 (t, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.92 (t, J = 7.2 Hz, 2H), 1.79 (d, J = 6.6 Hz, 6H), 1.50 (s, 3H), 1.37 (s, 9H), 1.23 (s, 2H), 0.96 (d, J = 9.8 Hz, 6H).

[0061] (2) Synthesis of γ-CPOL-SS-CTX

[0062] 4,4'-Dithiodibutyric acid (518.30 mg, 2.2 mmol) was added to a 100 mL round-bottom flask, and the mixture was reacted at 25 °C for 2 h in 5 mL of acetic anhydride as solvent. After the reaction was complete, the acetic anhydride in the reaction mixture was removed by vacuum distillation. Then, 10 mL of toluene was added to the mixture in three portions, and the mixture was dried by vacuum distillation. The resulting product was dissolved in 25 mL of dichloromethane, and CPOL (234.7 μL, 2.2 mmol) and DMAP (26.8 mg, 0.22 mmol) were added. The mixture was stirred at 25 °C for 12 h, and the intermediate product (270.0 mg, yield 52.0%) was purified by column chromatography.

[0063] The intermediate (150.0 mg, 0.48 mmol), EDCI (184.0 mg, 0.96 mmol), HOBT (127.8 mg, 0.96 mmol), and DMAP (6.1 mg, 0.05 mmol) were dissolved in 30 mL of dichloromethane and activated in an ice-water bath for 2 h. After activation, cabazitaxel (412.5 mg, 0.48 mmol) was added, and the mixture was stirred at 25 °C for 48 h. The final product (90 mg, yield 21.8%) was purified by preparative liquid chromatography. All reactions were carried out under nitrogen protection.

[0064] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown. The results of the 1H NMR spectrum analysis are as follows:

[0065] 1 H NMR (600MHz, DMSO-d6) δ7.99-7.95(m,2H),7.89(d,J=7.6Hz,3H),7.74(s,1H),7.66(t,J=7.6Hz,4H),7.42(t,J=7.4Hz ,4H),7.38-7.31(m,4H),5.36(d,J=7.1Hz,1H),5.09(d,J=8.0Hz,1H),4.68(s,1H),4.49(s,1H),4.45(d,J=6.4Hz,2H) ,4.27(s,1H),4.01(s,2H),2.69(t,J=7.1Hz,2H),2.59(d,J=7.2Hz,1H),2.53(d,J=7.1Hz,1H),2.41(d,J=7.2Hz,2H), 2.23(s,3H),1.94-1.89(m,2H),1.79(d,J=6.7Hz,6H),1.50(s,4H),1.37(s,9H),1.24(s,3H),0.96(d,J=10.1Hz,7H).

[0066] (3) Synthesis of α-Fmoc-SS-CTX

[0067] 2,2'-Dithiodiacetic acid (495.5 mg, 2.7 mmol) was added to a 100 mL round-bottom flask, and the mixture was reacted at 25 °C for 2 h in 5 mL of acetic anhydride as solvent. After the reaction was complete, the acetic anhydride in the reaction mixture was removed by vacuum distillation. Then, 10 mL of toluene was added to the mixture in three portions, and the mixture was dried by vacuum distillation. The resulting product was dissolved in 25 mL of dichloromethane, and Fmoc (529.8 mg, 2.7 mmol) and DMAP (11.7 mg, 0.1 mmol) were added. The mixture was stirred at 25 °C for 12 h, and the intermediate product (230.0 mg, yield 46.0%) was purified by column chromatography.

[0068] The intermediate (230.0 mg, 0.64 mmol), EDCI (245.4 mg, 1.28 mmol), HOBT (170.6 mg, 1.28 mmol), and DMAP (7.8 mg, 0.064 mol) were dissolved in 30 mL of dichloromethane and activated in an ice-water bath for 2 h. After activation, cabazitaxel (546.5 mg, 0.64 mmol) was added, and the mixture was stirred at 25 °C for 48 h. The final product (135.0 mg, yield 24.7%) was purified by preparative liquid chromatography. All reactions were carried out under nitrogen protection.

[0069] The structure of the prodrug in Example 1 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are shown in the figure. Figure 3 As shown. The results of the 1H NMR spectrum analysis are as follows:

[0070] 1H NMR(600MHz, DMSO-d6)δ8.00-7.95(m,1H),7.90(dd,J=11.9,7.5Hz,3H),7.73(s,1H),7.76-7.63(m,5H),7.46-7.39(m,4H),7.41-7.34(m,3H ),7.37-7.28(m,2H),7.16(t,J=8.0Hz,1H),5.81(t,J=9.4Hz,1H),5.76(s,1H),5.07(t,J=8.6Hz,1H),4.94(dd,J=15.2,10.1Hz,1H),4.68(d, J=16.0Hz,1H),4.48(s,1H),4.42(dd,J=14.3,7.0Hz,2H),4.28(dt,J=14.6,7.0Hz,1H),4.01(d,J=5.0Hz,2H),3.85(d,J=6.0Hz,3H),3.80(s ,1H),3.16(s,2H),2.24(d,J=10.1Hz,2H),1.80(d,J=5.1Hz,4H),1.50(d,J=6.5Hz,4H),1.37(d,J=11.0Hz,9H),0.96(dd,J=11.0,7.5Hz,4H).

[0071] (4) Synthesis of β-Fmoc-SS-CTX

[0072] 3,3'-Dithiodipropionic acid (492.7 mg, 2.3 mmol) was added to a 100 mL round-bottom flask, and the mixture was reacted at 25 °C for 2 h in 5 mL of acetic anhydride as solvent. After the reaction was complete, the acetic anhydride in the reaction mixture was removed by vacuum distillation. Then, 10 mL of toluene was added to the mixture in three portions, and the mixture was dried by vacuum distillation. The resulting product was dissolved in 25 mL of dichloromethane, and Fmoc (451.4 mg, 2.3 mmol) and DMAP (28.1 mg, 0.23 mmol) were added. The mixture was stirred at 25 °C for 12 h, and the intermediate product (200.0 mg, yield 40.6%) was purified by column chromatography.

[0073] The intermediate (200.0 mg, 0.46 mmol), EDCI (176.3 mg, 0.92 mmol), HOBT (122.6 mg, 0.92 mmol), and DMAP (5.6 mg, 0.046 mmol) were dissolved in 30 mL of dichloromethane and activated in an ice-water bath for 2 h. After activation, cabazitaxel (392.7 mg, 0.46 mmol) was added, and the mixture was stirred at 25 °C for 48 h. The final product (95 mg, yield 11.7%) was purified by preparative liquid chromatography. All reactions were carried out under nitrogen protection.

[0074] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 Figure 4 As shown. The results of the 1H NMR spectrum analysis are as follows:

[0075] 1 H NMR (600MHz, DMSO-d6) δ7.90 (d, J=7.5Hz, 3H), 7.69-7.64 (m, 4H), 7.42 (d, J= 6.9Hz,3H),7.34(d,J=1.1Hz,1H),5.76(s,2H),5.09(s,1H),4.43(d,J=6.8H z,2H),4.01(s,2H),3.19(s,3H),2.92(s,1H),2.85(s,1H),2.75(s,1H),2.2 4(s,3H),1.81-1.78(m,4H),1.50(s,3H),1.37(s,9H),0.96(d,J=9.1Hz,7H).

[0076] Example 2: Preparation of non-PEGylated γ-Fmoc-SS-CTX and γ-CPOL-SS-CTX prodrug nanoparticles

[0077] Accurately weigh 0.2 mg each of the γ-Fmoc-SS-CTX and γ-CPOL-SS-CTX prodrugs prepared in Example 1, and dissolve them separately in 100 μL of anhydrous ethanol. Vortex sonication is used to ensure complete dissolution of the prodrugs in the organic solvents. Under magnetic stirring, the prodrugs dissolved in anhydrous ethanol are slowly added dropwise to 1.0 mL of deionized water. After the addition is complete, stirring is continued for 3 min. At 25 °C, the organic solvent in the nanoparticles is removed by vacuum distillation, and deionized water is added to bring the volume to 1 mL to obtain non-PEGylated nanoparticles with a prodrug concentration of 0.2 mg / mL. The results are as follows: Figure 5 As shown, the γ-CPOL-SS-CTX prodrug cannot self-assemble into nanoparticles and precipitates immediately after being dropped into water. Figure 5 a), while the γ-Fmoc-SS-CTX prodrug can self-assemble into uniform nanoparticles ( Figure 5 b) The particle size is 152.40±5.46 nm. The above results demonstrate that the Fmoc group we introduced can promote the nano-assembly of the prodrug.

[0078] Example 3: Preparation of PEGylated Fmoc prodrug nanoparticles

[0079] Accurately weigh the α-Fmoc-SS-CTX, β-Fmoc-SS-CTX, γ-Fmoc-SS-CTX prodrugs prepared in Example 1, and DSPE-PEG. 2K(Aivita Pharmaceutical Technology Co., Ltd.) 1.0 mg of each was dissolved in 100 μL of anhydrous ethanol, and the solution was vortexed and sonicated to ensure complete dissolution, yielding three anhydrous ethanol solutions of the prodrugs, and DSPE-PEG. 2K A mixed solution of anhydrous ethanol. Add 25.0 μL of DSPE-PEG to the anhydrous ethanol solution of the precursor drug. 2K Anhydrous ethanol solution was mixed and then slowly added dropwise to 1.0 mL of deionized water under magnetic stirring. After the addition was complete, stirring was continued for 3 min. The organic solvent in the nano-formulation was removed by vacuum distillation at 25 °C, and the volume was adjusted to 1 mL with deionized water to obtain PEGylated prodrug nanoparticles with a prodrug concentration of 1 mg / mL.

[0080] In previous experiments, we demonstrated that introducing Fmoc groups into the prodrug promotes its nanoassembly. However, the γ-CPOL-SS-CTX prodrug without a π-conjugated structure cannot self-assemble into stable nanoparticles; therefore, the γ-CPOL-SS-CTX prodrug will not be further investigated. Furthermore, to improve nanoparticle stability, we prepared PEGylated Fmoc prodrug nanoparticles (α-Fmoc-SS-CTX NPs, β-Fmoc-SS-CTX NPs, and γ-Fmoc-SS-CTX NPs) for subsequent in vitro and in vivo formulation evaluation. The appearance of the formulations is shown below. Figure 6 As shown: All prodrugs in DSPE-PEG 2K With modification, all three types of nanoparticles can be assembled into nanoparticles. The particle size measurement results are shown in Table 1. The particle size of the three types of nanoparticles is about 90 nm, the PDI is less than 0.2, the Zeta potential is about -25 mV, and the drug loading is greater than 50%.

[0081] The morphology of the prepared nanoparticles was observed using transmission electron microscopy, and the results are as follows: Figure 7 As shown, the prodrug nanoparticles are uniform spherical particles.

[0082] Table 1. Particle size, PDI, Zeta potential, and drug loading of PEG-modified cabazitaxel predrug self-assembled nanoparticles

[0083]

[0084] Example 4: Colloidal stability of PEGylated carbapata predrug self-assembled nanoparticles

[0085] The PEGylated cabazitaxel pre-race drug self-assembled nanoparticles (α-Fmoc-SS-CTX NPs, β-Fmoc-SS-CTX NPs, γ-Fmoc-SS-CTX NPs) prepared in Example 3 were mixed with PBS (pH 7.4) or PBS containing 10% fetal bovine serum (pH 7.4) (the ratio of PEGylated cabazitaxel pre-race drug self-assembled nanoparticles to PBS (pH 7.4) or PBS containing 10% fetal bovine serum (pH 7.4) was 1:20, v / v), and then placed in a constant temperature shaker (37°C, 120 rpm). The particle size was measured at 0, 2, 4, 6, 8, and 12 hours. Figure 8 As shown, the three prodrug self-assembled nanoparticles exhibited good stability in PBS (pH 7.4) and PBS containing 10% fetal bovine serum (pH 7.4), with no significant change in particle size. Furthermore, the long-term stability of the three cabazitaxel nanoparticles (1 mg / mL) was investigated by storing them at 4°C. The results are as follows. Figure 9 As shown, the long-term stability of the three carbamazepine predrug nanoparticles was good, and the particle size did not change significantly.

[0086] Example 5: In vitro release experiment of PEGylated carbapata predrug self-assembled nanoparticles

[0087] Using PBS (pH 7.4, containing 30% ethanol (v / v)) as the release medium, the release of CTX from prodrug nanoparticles (α-Fmoc-SS-CTX NPs, β-Fmoc-SS-CTX NPs, γ-Fmoc-SS-CTX NPs) was investigated with or without the addition of dithiothreitol (DTT). The specific experimental method was as follows: 30 mL of the release medium was added to an Erlenmeyer flask, followed by 200 μL of the PEGylated prodrug nanoparticles (1 mg / mL) prepared in Example 3. The flask was immediately transferred to a 37°C constant-temperature shaker. Samples were collected at pre-set time points, and the amount of CTX released from the prodrug nanoparticles was determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 10 As shown, the release rate order of the three prodrug nanoparticles is: α-Fmoc-SS-CTX NPs > γ-Fmoc-SS-CTX NPs > β-Fmoc-SS-CTX NPs. When the DTT concentration is 5 mM, α-Fmoc-SS-CTX NPs released about 90% of the CTX within 2 h, γ-Fmoc-SS-CTX NPs released about 50% of the CTX within 8 h, and β-Fmoc-SS-CTX NPs released the least CTX within 8 h, only about 20%.

[0088] Example 6: Cellular uptake of PEGylated carbapata prodrug self-assembled nanoparticles

[0089] Preparation method of coumarin 6 (C-6) labeled PEGylated nanoparticles: α-Fmoc-SS-CTX, β-Fmoc-SS-CTX, γ-Fmoc-SS-CTX prodrugs, C-6, and DSPE-PEG were accurately weighed separately. 2K 1.0 mg of each was dissolved in 100 μL of anhydrous ethanol and vortexed and sonicated to ensure complete dissolution, yielding three anhydrous ethanol solutions of the prodrugs, a C-6 anhydrous ethanol solution, and a DSPE-PEG solution. 2K A mixed solution of anhydrous ethanol. Add 10.0 μL of C-6 anhydrous ethanol solution and 25.0 μL of DSPE-PEG to the anhydrous ethanol solution of the precursor drug. 2K Anhydrous ethanol solution was mixed and then slowly added dropwise to 1.0 mL of deionized water under magnetic stirring. After the addition was complete, stirring was continued for 3 min. The organic solvent in the nano-formulation was removed by vacuum distillation at 25 °C, and the volume was adjusted to 1 mL with deionized water to obtain C-6-labeled PEGylated prodrug nanoparticles with a prodrug concentration of 1 mg / mL and a C-6 concentration of 0.1 mg / mL.

[0090] Confocal qualitative observation of cell uptake: Healthy 4T1 cells were digested and prepared into a cell suspension (2×10⁻⁶). 4 Sterile climbing smears were placed in 24-well cell culture plates beforehand, and the above cell suspension was added to the wells (500 μL / well). The plates were then placed in a cell culture incubator (37℃, 5% CO2) to allow the cells to adhere fully (culture for 24 h). Coumarin solution or coumarin-labeled self-assembled nanoparticles (1 mg / mL) were diluted 10-fold using fresh culture medium, with a C-6 concentration of 200 ng / mL. Remove the 24-well plate from the cell culture incubator, discard the old culture medium, and add fresh culture medium containing coumarin C-6 solution or C-6-labeled prodrug nanoparticles (1 mL / well). Incubate the cell culture plate in a cell culture incubator (37°C, 5% CO2) at the set times (0.5 h, 2 h). After incubation, remove the cell culture plate, discard the culture medium, stop cell uptake with ice-cold PBS (pH 7.4), and wash away any remaining unuptaken drug. Fix the cells with tissue fixation solution, wash three times with PBS (pH 7.4), stain the cell nuclei with Hoechst 33342, and observe and photograph under a confocal microscope. Results are as follows. Figure 11 As shown, cells exhibit higher uptake efficiency of nanoparticles compared to solutions. There was no significant difference in cellular uptake among the three types of nanoparticles, likely due to their similar shapes, particle sizes, and PEGylated shells.

[0091] Semi-quantitative flow cytometry assay of cell uptake: Healthy 4T1 cells were digested and prepared into a cell suspension, which was then added to 12-well plates, 10 cells per well. 5 Cells were cultured for 24 hours to ensure full adhesion. Coumarin solution or coumarin-labeled self-assembled nanoparticles were diluted 10-fold with fresh culture medium, with a C-6 concentration of 200 ng / mL. The 12-well plates were removed from the incubator, the old culture medium was discarded, and fresh drug-containing culture medium was added. The plates were then returned to the incubator and cultured for another 0.5 or 2 hours. Immediately afterward, the drug-containing culture medium was discarded, and the cells were washed away with PBS (pH 7.4) to remove any untaken drug. 300 μL of trypsin was added to each well, and the cells were pipetted off. 1 mL of culture medium was added to stop digestion. After centrifugation at 4°C, the culture medium was discarded, and the cells were resuspended in 1 mL of PBS (pH 7.4). Cell uptake was immediately measured by flow cytometry. Results are as follows: Figure 12 As shown, cells exhibit higher uptake efficiency of nanoparticles compared to solutions. There was no significant difference in cellular uptake among the three types of nanoparticles, which is attributed to their similar shapes, particle sizes, and PEGylated shells, consistent with confocal results.

[0092] Example 7: Cytotoxicity of self-assembled nanoparticles of PEGylated carbapata prodrug

[0093] Using 4T1, RM-1, and L02 cells as cell models, the MTT assay was used to determine and compare the antitumor cell proliferation activity of CTX solution and three PEGylated Fmoc-CTX prodrugs (α-Fmoc-SS-CTX, β-Fmoc-SS-CTX, and γ-Fmoc-SS-CTX) self-assembled nanoparticles prepared in Example 3. The specific steps are as follows: Cell plating: Cells in good condition and with a confluence of approximately 80% were transferred to a clean bench. The old culture medium was discarded, and PBS (pH 7.4) was added to wash away residual culture medium to prevent interference with trypsin digestion. Then, 1 mL of trypsin solution (0.25%) was added to digest the cells for 3 min. Immediately, the cells were gently pipetted down to a suspended and dispersed state. 5 mL of culture medium was added to stop the digestion, and the cell suspension containing trypsin was transferred to a 15 mL centrifuge. After low-speed centrifugation (1000 rpm, 5 min), the supernatant was discarded, and an appropriate amount of culture medium was added to prepare a cell suspension (concentration of 1×10⁻⁶). 4Cells were cultured in 96-well plates (100 μL / well) and then incubated for 12 h. Drug addition and assay: Fresh culture medium was used to dilute CTX solution and three Fmoc-CTX prodrug self-assembled nanoparticles to different concentrations of drug-containing medium. The 96-well plates were removed from the cell culture incubator, the old medium was discarded, and 200 μL of drug-containing medium was added to each well. Drug-free medium was added as a control well, and fresh medium was added to cell-free wells as zeroing wells. Six wells were added for each concentration. Sterile PBS (pH 7.4) was added to the wells around the perimeter of the 96-well plate to reduce evaporation. The plates were then incubated for 48 h. After incubation, MTT solution (25 μL / well) was added to the wells, and the plates were incubated for another 4 h. After incubation, remove the cell culture plate and discard the supernatant. Add DMSO (200 μL / well) to the wells to dissolve the blue-purple formazan. After dissolution, measure the absorbance of each well using a microplate reader. Use the absorbance of wells containing drug-free culture medium as a control (100% viability). Calculate the cell viability of the drug-treated wells. Experimental results are as follows: Figure 13 As shown, in 4T1( Figure 13 A) and RM-1 Figure 13 B) In the tumor cell model, the three prodrug self-assembled nanoparticles showed slightly inferior anti-tumor cell proliferation activity compared to CTX solution. This is because the prodrugs must release the parent CTX drug from tumor cells to effectively exert their anti-tumor cell proliferation effect. Furthermore, we found that different disulfide bonds also affect the anti-tumor cell proliferation activity of Fmoc-CTX prodrug self-assembled nanoparticles, with the order of activity being: α-Fmoc-SS-CTX NPs > γ-Fmoc-SS-CTX NPs > β-Fmoc-SS-CTX NPs. This is consistent with the release rate of the parent drug by various prodrugs in in vitro release experiments, verifying our hypothesis that the release rate of the parent drug directly affects its anti-tumor cell proliferation toxicity. In L02 ( Figure 13 C) In a cell model, the IC50 of three prodrug self-assembled nanoparticles 50 The value was significantly higher than that of CTX solution, showing a good toxicity reduction effect, which is consistent with our expected design goal: designing CTX in the form of a prodrug can greatly reduce its systemic toxicity.

[0094] Example 8: Pharmacokinetics of self-assembled nanoparticles of PEGylated carbapoxetine predrug

[0095] Sprague-Dawley (SD) rats, fasted for 12 hours prior to administration, were randomly divided into four groups of six rats each (weighing 180–220 g). CTX solution and three different types of Fmoc-CTX prodrug nanoparticles with disulfide bonds at different positions (CTX equivalent dose of 5 mg / kg) prepared in Example 3 were injected via tail vein. At predetermined time points (0.03, 0.08, 0.25, 0.5, 1, 2, 4, 8, and 12 h), 0.5 mL of blood was collected from the orbital venous plexus of the rats and placed into EP tubes. After high-speed centrifugation (10000 rpm, 5 min), 200 μL of the supernatant serum was collected and placed into EP tubes, which were immediately stored at -80°C for analysis.

[0096] Add 50 μL of plasma and 20 μL of 50% acetonitrile-water solution (diazepam, 20.0 ng / mL) to a 1.1 mL 12-tube EP tube, and shake for 3 min. Add 150 μL of acetonitrile to all samples, shake for 10 min (shaker setting 1200), and centrifuge for 10 min (horizontal rotor 4000 rpm, 4℃).

[0097] CTX processing method: Transfer 50 μL of supernatant to a clean 96-well plate, add 200 μL of aqueous solution to the CTX supernatant, shake and mix for 10 min (shaker setting 1200), and inject 5 μL of sample for analysis.

[0098] Prodrug preparation: Transfer 50 μL of supernatant to a clean 96-well plate, add 50 μL of 90% acetonitrile-water solution to all supernatants, shake to mix for 10 min (shaker setting 1200), and inject for analysis. The injection volume is 8 μL.

[0099] Plasma samples collected at different time points were analyzed using liquid chromatography-mass spectrometry to determine the drug concentration in the plasma. The results are as follows: Figure 14 As shown. Pharmacokinetic parameters (AUC0-12 h, t) of CTX solution and three PEG-modified Fmoc-CTX prodrug nanoformulations were calculated using DAS software. 1 / 2The results of the CTX nanoparticles (including the prodrug and parent drug) are shown in Table 2. It can be seen that the CTX solution injected into rats via the tail vein is rapidly cleared, thus hindering the drug's reach to the tumor site via blood circulation. The three CTX prodrug nanoparticles exhibited higher AUCs, ensuring the tumor-targeted accumulation of CTX. Simultaneously, different disulfide bonds significantly affected the pharmacokinetic behavior of the prodrug nanoparticles. Compared to the CTX solution, the total AUC (sum of prodrug and parent drug) of the three CTX nanoparticles increased by: 10.22 times (α-Fmoc-SS-CTX NPs), 83.94 times (β-Fmoc-SS-CTX NPs), and 106.18 times (γ-Fmoc-SS-CTX NPs). The order of AUC h for CTX release from the prodrug nanoparticles was: α-Fmoc-SS-CTX NPs > β-Fmoc-SS-CTX NPs > γ-Fmoc-SS-CTX NPs. This indicates that the AUC of prodrug nanoparticles is related to their reductive drug release sensitivity; the faster the drug is released, the less stable the prodrug nanoparticles are, and the lower the AUC.

[0100] Table 2. Pharmacokinetic parameters of cabazitaxel solution and cabazitaxel prodrug nanoparticles (n=6).

[0101]

[0102] Example 9: In vivo distribution of PEGylated carbapata predrug self-assembled nanoparticles

[0103] Preparation method of DiR-labeled PEGylated nanoparticles: Accurately weigh α-Fmoc-SS-CTX, β-Fmoc-SS-CTX, γ-Fmoc-SS-CTX prodrugs, DiR, and DSPE-PEG, respectively. 2K 1.0 mg of each was dissolved in 100 μL of anhydrous ethanol and vortexed and sonicated to ensure complete dissolution, yielding three prodrug anhydrous ethanol solutions, a DiR solution, and a DSPE-PEG solution. 2K A mixed solution of anhydrous ethanol. Add 10.0 μL of DiR anhydrous ethanol solution and 25.0 μL of DSPE-PEG to the anhydrous ethanol solution of the precursor drug. 2K Anhydrous ethanol solution was mixed and then slowly added dropwise to 1.0 mL of deionized water under magnetic stirring. After the addition was complete, stirring was continued for 3 min. The organic solvent in the nano-formulation was removed by vacuum distillation at 25 °C, and the volume was adjusted to 1 mL with deionized water to obtain DiR-labeled PEGylated prodrug nanoparticles with a prodrug concentration of 1 mg / mL and a DiR concentration of 0.1 mg / mL.

[0104] Collect 4T1 cells that have grown to 80%–90% confluence. In a clean bench, discard the culture medium from the culture dish, add 2 mL of PBS (pH 7.4), remove the remaining medium, and then add 1 mL of trypsin solution (0.25%) to the culture dish to digest the cells for 3 minutes. Gently pipette the adherent cells off the culture dish, immediately add 5 mL of culture medium to stop the digestion, transfer to a centrifuge, centrifuge at low speed to discard the trypsin-containing medium, and add an appropriate amount of PBS (pH 7.4) to resuspend the cells (concentration 5 × 10⁻⁶). 7 (cells / mL). To avoid interference from mouse hair on in vivo imaging results, the backs of BALB / c mice were pre-haired using depilatory cream. 100 μL of the cell suspension was injected subcutaneously into the right posterior back of the hairless mouse using an insulin syringe. The mice were then fed until the tumor reached 500 mm². 3 Mice were randomly divided into four groups of three each. Each group received a DiR solution or DiR / α-Fmoc-SS-CTX NPs, DiR / β-Fmoc-SS-CTX NPs, or DiR / γ-Fmoc-SS-CTX NPs via tail vein injection. The equivalent dose of DiR was 1 mg / kg. Mice were anesthetized at 2, 4, and 6 hours, and fluorescence accumulation at tumor sites was observed using a small animal in vivo imaging system. Finally, at 6 hours, the mice were euthanized by dislocation, and major organs and tumors were isolated. Fluorescence accumulation in different organs and tumor sites was recorded using a small animal in vivo imaging system and quantitative analysis was performed.

[0105] The results are as follows Figure 15 and 16 As shown, the DiR solution, lacking tumor targeting and rapidly cleared from the bloodstream, primarily distributes in organs such as the spleen, failing to reach the tumor site and accumulating minimally there. In contrast, the three prodrug nanoparticles, due to their prolonged blood circulation time, higher AUC, and passive tumor targeting based on the EPR effect, exhibit higher accumulation at the tumor site. Furthermore, the distribution patterns of the different prodrug nanoparticles in vivo vary; DiR / γ-Fmoc-SS-CTX NPs, with a slower elimination rate, accumulate more at the tumor site, consistent with pharmacokinetic results.

[0106] Example 10: Pharmacodynamics of self-assembled nanoparticles of PEGylated carbapoxetine predrug

[0107] When the tumor on the back of the BALB / c mouse grows to 100mm 3Around 10:00 AM, mice were randomly divided into 5 groups of 5 mice each. Each group received intravenous injection of saline, CTX solution, or α-Fmoc-SS-CTX NPs, β-Fmoc-SS-CTX NPs, or γ-Fmoc-SS-CTX NPs prepared in Example 3, respectively. Mice were weighed before administration. The CTX dosage was 5 mg / kg, and the prodrug dosage was converted to the CTX dosage. Administered the drugs every two days for a total of 9 days (3 administrations). Before each administration, the mice were weighed, and the length and width of the tumor were measured with calipers. At the end of the treatment period, the mice were euthanized by dislocation, and the tumors were dissected, photographed, and weighed.

[0108] The efficacy results of Fmoc-CTX prodrug nanoparticles with disulfide bonds at different positions in the 4T1 tumor-bearing mouse model are as follows: Figure 17 As shown, since saline has no therapeutic effect, tumor cells in the saline group could not be inhibited, and the tumor volume increased rapidly. Although tumor growth was somewhat inhibited in the CTX solution group, it still showed considerable toxic side effects in mice. The weight of mice in the CTX solution group decreased by about 20% of their pre-treatment weight on the ninth day, but the weight of mice in the three prodrug nanoparticle groups did not change significantly.

[0109] Furthermore, the three Fmoc-CTX prodrug nanoparticles exhibited different antitumor effects: γ-Fmoc-SS-CTX NPs > α-Fmoc-SS-CTX NPs > β-Fmoc-SS-CTX NPs. γ-CTX-SS-Fmoc prodrugs accumulated the most at the tumor site and had a larger AUC, thus exhibiting the strongest antitumor effect, while β-CTX-SS-Fmoc NPs showed the weakest antitumor effect.

Claims

1. A prodrug of cabazitaxel, characterized in that, The anti-tumor therapeutic agent is connected with a group or a cyclic group with a conjugate structure through a tumor-sensitive connecting bond, and the structure is one of the following a-d:

2. The method of synthesis of a cabazitaxel prodrug according to claim 1, wherein, The method comprises the following steps: firstly, dithiodiglycolic acid, 3,3'-dithiodipropionic acid or 4-4'-dithiodibutyric acid is dehydrated into an anhydride by acetic anhydride, and then esterification is carried out with 9-fluorenylmethyl alcohol or cyclopentylmethanol to obtain an intermediate product; and then esterification is carried out between the intermediate product and cabazitaxel to obtain the cabazitaxel prodrug.

3. The method of synthesis of a cabazitaxel prodrug according to claim 2, wherein, In the reaction of dehydrating dithiodiglycolic acid, 3,3'-dithiodipropionic acid or 4-4'-dithiodibutyric acid into an anhydride by acetic anhydride, the reaction temperature is 15-60 DEG C, and the reaction time is 2-6 hours; In the reaction of the anhydride and 9-fluorenylmethyl alcohol or cyclopentylmethanol, the molar ratio of the anhydride and 9-fluorenylmethyl alcohol or cyclopentylmethanol is 1:1-5:1, the catalyst is 4-dimethylaminopyridine, the molar ratio of 9-fluorenylmethyl alcohol or cyclopentylmethanol and the catalyst 4-dimethylaminopyridine is 1:0.05-0.8, the reaction temperature is 20-50 DEG C, and the reaction time is 4-24 hours; In the esterification reaction of the intermediate product and cabazitaxel, the solvent is dichloromethane, the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and 4-dimethylaminopyridine, the molar ratio of cabazitaxel, the intermediate product, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and 4-dimethylaminopyridine is 1:0.2-5:0.5-4:0.5-4:0.05-0.8, the reaction temperature is 15-40 DEG C, and the reaction time is 24-60 hours.

4. The self-assembled nanoparticle of a protease of Cabazitaxel according to claim 1, characterized in that, The self-assembled nanoparticles are non-PEGylated cabazitaxel prodrug nanoparticles, PEG modifier modified cabazitaxel prodrug nanoparticles, or cabazitaxel prodrug self-assembled nanoparticles loaded with a fluorescent substance or a hydrophobic drug.

5. The method for preparing self-assembled nanoparticles of cabazitaxel predrug as described in claim 4, characterized in that, The preparation process is as follows: The cabazitaxel prodrug, or the cabazitaxel prodrug and the PEG modifier, or the cabazitaxel prodrug, the PEG modifier, the fluorescent substance or the hydrophobic drug are dissolved in anhydrous ethanol to obtain an anhydrous ethanol solution, the anhydrous ethanol solution is added dropwise into water under stirring, the prodrug spontaneously forms uniform nanoparticles, and finally, anhydrous ethanol in the preparation is removed by vacuum distillation to obtain a nanopreparation free of any organic solvent.

6. The production method according to claim 5, wherein The PEG modifier is at least one of TPGS, DSPE-PEG, PLGA-PEG, PE-PEG and DSPE-PEG-AA, and the weight ratio of the cabazitaxel prodrug and the PEG modifier is 90:10-50:50; the fluorescent substance or the hydrophobic drug is at least one of coumarin-6, rhodamine, DiR, DiI, Cy-5 and Cy-7, and the weight fraction of the fluorescent substance or the hydrophobic drug in the cabazitaxel prodrug is 1%-30%.

7. The cabazitaxel prodrug of claim 1 or the cabazitaxel prodrug self-assembled nanoparticles of claim 4 are used in a drug delivery system.

8. The cabazitaxel prodrug of claim 1 or the cabazitaxel prodrug self-assembled nanoparticles of claim 4 are used in the preparation of an anti-tumor drug.

9. Use of the cabazitaxel prodrug of claim 1 or the cabazitaxel prodrug self- assembled nanoparticles of claim 4 in an injectable, oral or topical drug delivery system.

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