Thio carbonate bond bridged dimer prodrugs, self-assembled nanoparticles thereof, and uses thereof

Abstract

The application belongs to the technical field of medicine, and relates to a thio carbonate bond bridged dimer prodrug and self-assembled nanoparticles thereof, and application thereof in drug delivery. The application relates to a thio carbonate bond bridged dimer prodrug as shown in a general structure (I) and construction of self-assembled nanoparticles thereof, and application thereof in drug delivery. The preparation method is simple and easy to implement, the thio carbonate bond bridged dimer prodrug can be self-assembled to form nanoparticles, and has redox dual-response drug release capacity, so that intelligent response activation of the prodrug in tumor cells can be realized, and the anti-tumor effect and safety of the prodrug are ensured. The application provides a new strategy for developing an efficient and low-toxicity drug delivery system, and meets the urgent needs of high-end chemotherapy preparations in clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a thiocarbonate bond-bridged dimer prodrug, its self-assembled nanoparticles, and its applications. Specifically, it relates to the construction of a thiocarbonate bond-bridged redox-sensitive paclitaxel dimer prodrug and self-assembled nanoparticles containing the prodrug, as well as its application in the preparation of antitumor drugs. Background Technology

[0002] Cancer poses a serious threat to human life and health. Currently, chemotherapy is one of the most commonly used and effective strategies for treating cancer, especially for advanced tumors, tumors that cannot be surgically removed, and tumors that have metastasized. However, most chemotherapy drugs suffer from drawbacks such as poor targeting, significant toxic side effects, and a narrow therapeutic window. Nanomedicine delivery systems have shown some advantages in delivering chemotherapy drugs, but the vast majority of nanomedicines have failed to improve patients' overall survival. This is mainly due to insufficient affinity between the drug and the nanocarrier, leading to problems such as low drug loading, high carrier-related toxicity, burst or delayed drug release, and poor tumor selectivity.

[0003] To address these challenges, a carrier-free drug delivery strategy has emerged: small molecule prodrug self-assembled nanodelivery systems. Since the prodrug acts as both a carrier and a drug, small molecule prodrug self-assembled nanoparticles can achieve high drug loading and low carrier-related toxicity. Therefore, several chemotherapeutic drugs have already utilized small molecule prodrug self-assembled nanodelivery systems to achieve safer and more effective therapeutic effects. In particular, dimer prodrug nanoassemblies, as a promising branch of small molecule prodrug self-assembled nanodelivery systems, have attracted considerable interest. Dimeric prodrugs consist of two drug molecules linked by a linker bond, which can further increase drug loading (over 60%). The dimer structure highlights the crucial role of the linker bond in dimer prodrug nanoassemblies, requiring rational design to achieve the prodrug's chemical stability, assembly stability, and effective site-specific activation.

[0004] In recent years, sulfur bonds have demonstrated unique advantages in constructing dimer prodrug nanoassemblies. On one hand, sulfur bonds possess a near 90° bond-dihedral angle, which can generate "structural defects" to balance intermolecular forces, effectively enhancing the self-assembly capability of dimer prodrugs. Previous studies have found that the number of sulfur atoms significantly impacts prodrug self-assembly. Compared to monosulfide bonds (-S-) and disulfide bonds (-SS-), trisulfide bonds (-SSS-) have more sulfur atoms and sulfur-containing dihedral angles, more effectively enhancing the self-assembly capability of dimer prodrugs, thereby improving assembly stability, in vivo circulation time, and tumor accumulation. On the other hand, sulfur atoms have a large atomic radius and low electronegativity, making it easier for sulfur atoms to gain or lose their outermost electrons, directly leading to a diversity of sulfur atom valence states (-2 to +6). This also allows sulfur-containing bonds to exhibit unique redox responsiveness, responding to tumor-specific highly expressed reactive oxygen species (ROS) and glutathione (GSH), thereby triggering drug release. For example, the sulfur atom in a monosulfide bond has a valence of -2, which can trigger drug release in response to oxidative stimuli. As the number of sulfur atoms in a tandem linkage increases, the valence of the sulfur atom rises, resulting in trisulfide bonds with extremely high reduction sensitivity and relatively low oxidative sensitivity. However, tumors possess a heterogeneous redox microenvironment, making prodrug formulations that only respond to a single stimulus (oxidation or reduction) insufficient to produce comprehensive and effective therapeutic effects. Furthermore, although tandem trisulfide bonds can improve the assembly stability of dimer prodrugs to some extent, the occurrence of sulfur-sulfur exchange reactions inevitably limits the chemical stability of the prodrug. Therefore, it is still necessary to develop new sulfur-containing linkages to further enhance the assembly stability and activatability of dimer prodrugs without weakening their chemical stability. Summary of the Invention

[0005] To address the problems of existing technologies, this invention modifies the "tandem" trisulfide bond, which currently holds the most promising potential for research and commercialization. A novel strategy is proposed: linking three sulfur atoms to a central carbon atom, rearranging the trisulfide bonds in a "satellite" distribution, namely, the trithiocarbonate bond (-SC(S)S-). This strategy does not change the number of sulfur atoms in the linking bonds, only their arrangement. The objective of this invention is to design and synthesize dimer prodrugs bridged by thiocarbonate bonds, prepare self-assembled nanoparticle drug delivery systems for these dimer prodrugs, and explore their application in the preparation of antitumor drugs. Using trisulfide bonds, sulfur-carbon-sulfide bonds, and three-carbon bonds as controls, the effects of different chemical linking bonds on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the self-assembled dimer prodrug nanoparticles are investigated. The optimal chemical bridging is comprehensively screened to provide new strategies and more options for developing intelligent responsive drug delivery systems for the tumor microenvironment, meeting the urgent clinical demand for highly effective chemotherapy agents.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a thiocarbonate-linked dimeric prodrug of general structural formula (I) or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Where n = 1 to 5;

[0010] The drug is a drug containing a hydroxyl, amino, or carboxyl group. The drug containing a hydroxyl, amino, or carboxyl group is selected from antitumor drugs, antimetabolites, and anti-inflammatory drugs. The antitumor drugs are selected from taxanes, anthraquinones, nucleosides, camptothecins, platinum compounds, vincristine alkaloids, piosides, and artemisinin compounds; the antimetabolites are selected from pyrimidines, purines, thiabendazoles, and folic acid derivatives; and the anti-inflammatory drugs are selected from halofanthracene, griseofulvin, cyclosporine A, and their derivatives.

[0011] Furthermore, n = 1 to 3;

[0012] Drugs are taxanes or anthraquinones.

[0013] Specifically, the thiocarbonate-bridged dimer prodrug of this invention uses paclitaxel as the model drug, with 2,2'-trithiocarbonate diacetic acid as the linking bond. Simultaneously, a paclitaxel dimer prodrug bridged by trisulfide, sulfur-carbon-sulfur, or three-carbon bonds is prepared by linking the drug with 2,2'-trithiodiacetic acid, 2,2'-methylene dithiodiacetic acid, or 1,7-heptanoic acid as a control compound.

[0014] The paclitaxel dimer prodrug prepared using 2,2'-trithiocarbonate diacetic acid as a linking bond was named PSC(S)SP, and its structural formula is as follows:

[0015]

[0016] The paclitaxel dimer prodrug prepared using 2,2'-trithiodiacetic acid as a linking bond is named PSSSP, and its structural formula is as follows:

[0017]

[0018] The paclitaxel dimer prodrug prepared using 2,2'-methylenedithioacetic acid as a linking bond is named PSCSP, and its structural formula is as follows:

[0019]

[0020] The paclitaxel dimer prodrug prepared using 1,7-heptanoic acid as a linking bond was named PCCCP, and its structural formula is as follows:

[0021]

[0022] This invention provides a method for synthesizing a paclitaxel dimer prodrug containing thiocarbonate bonds, trisulfide bonds, sulfur-carbon-sulfur bonds, and three-carbon bonds, comprising the following steps:

[0023] (1) Dissolve 2,2'-trithiocarbonate diacetic acid, 2,2'-trithiodiacetic acid, 2,2'-methylene dithiodiacetic acid or 1,7-heptane in dichloromethane and stir until homogeneous; separately dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel in anhydrous dichloromethane and stir until homogeneous, then mix with the above solutions of 2,2'-trithiocarbonate diacetic acid, 2,2'-trithiodiacetic acid, 2,2'-methylene dithiodiacetic acid or 1,7-heptane, and stir at room temperature for 1-8 hours under N2 protection;

[0024] (2) Add EDCI and DMAP to the mixed solution, and continue stirring at room temperature for 8-48 hours under N2 protection. The resulting product is purified by preparative liquid phase separation.

[0025] In the above preparation method, the paclitaxel can be replaced by other anticancer drugs containing active hydroxyl, amino, or carboxyl groups. The anticancer drugs containing active hydroxyl, amino, or carboxyl groups are selected from other taxanes, anthraquinones, nucleosides, platinum compounds, vincristine alkaloids, piosides, artemisinin derivatives, and camptothecin derivatives.

[0026] A pharmaceutical composition comprising the thiocarbonate-linked dimer prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and excipient.

[0027] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of an antitumor drug.

[0028] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of a drug delivery system.

[0029] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in the preparation of injectable, oral, or topical delivery systems.

[0030] This invention also provides self-assembled nanoparticles of the aforementioned thiocarbonate-linked dimeric prodrug, comprising non-PEGylated thiocarbonate-linked dimeric prodrug self-assembled nanoparticles, PEG-modified thiocarbonate-linked dimeric prodrug self-assembled nanoparticles, thiocarbonate-linked dimeric prodrug self-assembled nanoparticles loading hydrophobic fluorescent substances, and thiocarbonate-linked dimeric prodrug self-assembled nanoparticles loading other drugs. The preparation method is a nanoprecipitation method, including high-speed stirring and ultrasonic methods.

[0031] The preparation method of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles is as follows:

[0032] When a non-PEGylated thiocarbonate-linked dimer prodrug self-assembles into nanoparticles, the thiocarbonate-linked dimer prodrug is dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0033] When PEGylated thiocarbonate-linked dimer prodrugs self-assemble into nanoparticles, the thiocarbonate-linked dimer prodrug and PEG are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0034] When assembling nanoparticles from thiocarbonate-linked dimer prodrugs loaded with hydrophobic fluorescent substances or other drugs, the thiocarbonate-linked dimer prodrug, fluorescent substance or drug, and PEG are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

[0035] The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG, and PE-PEG, with DSPE-PEG being the preferred PEG modifier. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000, or 5000, and more preferably 2000.

[0036] The solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, and acetone.

[0037] The mass ratio of thiocarbonate-bridged dimer prodrug to PEG modifier is 90:10 to 60:40. Under these conditions, the prodrug nanoparticles can exert a good anti-tumor effect.

[0038] The thiocarbonate bond-bridged dimer self-assembled nanoparticles have a particle size of 80-120 nm, a particle size distribution of less than 0.2, and a drug loading of 70-75%.

[0039] The present invention also provides the application of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of drug delivery systems.

[0040] The present invention also provides the application of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of antitumor drugs.

[0041] The present invention also provides the application of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of injection, oral or topical drug delivery systems.

[0042] The technical problem solved by this invention is to introduce thiocarbonate bonds into dimer prodrugs and self-assembled nanoparticles, design redox-sensitive dimer prodrugs bridged by thiocarbonate bonds, and use these dimer prodrugs in the construction of self-assembled nanoparticles. This achieves good chemical stability, high drug loading, good assembly stability, low toxicity, and rapid drug release specific to tumor sites, thereby improving therapeutic efficacy. Simultaneously, dimer prodrugs bridged by trisulfide bonds, sulfur-carbon-sulfide bonds, and tricarbon bonds are used as controls to investigate the differences in self-assembly, redox-sensitive response, and antitumor activity of different chemical bridging methods, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the prodrug self-assembled nanoparticles.

[0043] The advantages of this invention are:

[0044] (1) A dimer prodrug with thiocarbonate bond bridging and a control prodrug with trisulfide bond, sulfur-carbon-sulfur bond and tricarbon bond bridging were designed and synthesized. The synthesis method is simple and easy to implement.

[0045] (2) Uniform dimer prodrug self-assembled nanoparticles were prepared. The preparation method is simple and easy to implement, achieving efficient drug loading with an ultra-high drug loading of more than 70%. The particle size remained basically unchanged after being placed at 4℃ and 25℃ for 60 days.

[0046] (3) The differences in self-assembly, redox-sensitive response, and antitumor activity of different chemical bridging agents were investigated, as well as their effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of prodrug self-assembled nanoparticles. Based on the experimental results, thiocarbonate-bridged prodrugs exhibit better chemical stability and higher redox-sensitive properties, enabling specific activation in the tumor redox microenvironment. Furthermore, thiocarbonate-bridged prodrug nanoparticles also demonstrate the best assembly ability. This invention provides a new strategy and more options for developing intelligent responsive drug delivery systems for the tumor microenvironment, meeting the urgent clinical need for highly effective chemotherapeutic agents. Attached Figure Description

[0047] Figure 1 This provides structural confirmation of the trisulfide-bridged paclitaxel dimer prodrug (PSSSP) in Example 1 of this invention.

[0048] A: Mass spectrum of PSSSP.

[0049] B: High-performance liquid chromatography purity diagram of PSSSP.

[0050] Figure 2 This provides structural confirmation of the thiocarbonate-bridged paclitaxel dimer prodrug (PSC(S)SP) in Example 2 of this invention.

[0051] A: PSC(S)SP 1 H-NMR spectrum.

[0052] B: Mass spectrum of PSC(S)SP.

[0053] C: High performance liquid chromatography purity chromatogram of PSC(S)SP.

[0054] Figure 3 This provides structural confirmation of the sulfur-carbon-sulfur bond-bridged paclitaxel dimer prodrug (PSCSP) in Example 3 of this invention.

[0055] A: PSCSP 1 H-NMR spectrum.

[0056] B: Mass spectrum of PSCSP.

[0057] C: High-performance liquid chromatography purity diagram of PSCSP.

[0058] Figure 4 This provides structural confirmation of the three-carbon bond-bridged paclitaxel dimer prodrug (PCCCP) in Example 4 of this invention.

[0059] A: PCCCP 1 H-NMR spectrum.

[0060] B: Mass spectrum of PCCCP.

[0061] C: High-performance liquid chromatography purity diagram of PCCCP.

[0062] Figure 5 The images show the particle size distribution and transmission electron microscope (TEM) image of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 of this invention.

[0063] A: Particle size diagram and transmission electron microscope image of self-assembled nanoparticles of thiocarbonate-linked paclitaxel dimer prodrug.

[0064] B: Particle size diagram and transmission electron microscope image of self-assembled nanoparticles of paclitaxel dimer prodrug bridged by sulfur-carbon-sulfur bonds.

[0065] C: Particle size diagram and transmission electron microscope image of self-assembled nanoparticles of paclitaxel dimer prodrugs bridged by three carbon bonds.

[0066] Figure 6 This is a stability diagram of the self-assembled nanoparticles of paclitaxel dimer prodrug prepared in Example 5 of this invention.

[0067] A: Stability of paclitaxel dimer prodrug self-assembled nanoparticles at 4°C.

[0068] B: Stability of paclitaxel dimer prodrug self-assembled nanoparticles at 25°C.

[0069] Figure 7 This is an in vitro release test diagram of the paclitaxel dimer prodrug self-assembled nanoparticles in Example 6 of the present invention.

[0070] A: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.2mM H2O2 conditions.

[0071] B: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 1mM H2O2 conditions.

[0072] C: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 10mM H2O2 conditions.

[0073] D: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.05 mM DTT conditions.

[0074] E: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 0.5 mM DTT conditions.

[0075] F: In vitro release assay of paclitaxel dimer prodrug self-assembled nanoparticles under 2mM DTT conditions.

[0076] Figure 8 This is a cytotoxicity diagram of the paclitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present invention.

[0077] Figure 9 This is a blood drug concentration-time curve of the paclitaxel dimer prodrug self-assembled nanoparticles in Example 8 of the present invention.

[0078] A: Blood concentration-time curve of paclitaxel dimer prodrug self-assembled nanoparticles.

[0079] B: Paclitaxel plasma concentration-time curve of paclitaxel self-assembled nanoparticles of paclitaxel dimer prodrug.

[0080] Figure 10 This is an in vivo antitumor experiment diagram (10 mg / kg) of paclitaxel dimer prodrug self-assembled nanoparticles in Example 9 of the present invention.

[0081] A: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0082] B: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on body weight in tumor-bearing mice.

[0083] C: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on tumor bearing rate in Balb / C mice.

[0084] Figure 11 This is an in vivo antitumor experiment diagram (30 mg / kg) of paclitaxel dimer prodrug self-assembled nanoparticles in Example 9 of the present invention.

[0085] A: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0086] B: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on body weight in tumor-bearing mice.

[0087] C: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on tumor bearing rate in Balb / C mice. Detailed Implementation

[0088] The present invention will be further illustrated by way of embodiments below, but the invention is not limited to the scope of the embodiments described herein.

[0089] Example 1: Synthesis of trisulfide-bridged paclitaxel dimer prodrug (PSSSP)

[0090] 0.2 mmol of 2,2'-trithiodiacetic acid was dissolved in 5-10 mL of dichloromethane. Separately, 0.04 mmol of 4-dimethylaminopyridine (DMAP), 0.4 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 0.4 mmol of paclitaxel were dissolved in 20 mL of anhydrous dichloromethane and mixed with the 2,2'-trithiodiacetic acid solution in dichloromethane. The mixture was stirred at room temperature for 4 h. Then, 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 24 h. The entire reaction was carried out under N2 protection. The resulting product was purified by preparative liquid chromatography.

[0091] The structure of the PSSSP prepared in Example 1 was determined by mass spectrometry. The mass spectrum and high-performance liquid chromatography (HPLC) spectrum after preparative liquid chromatography purification are shown below. Figure 1As shown, the results indicate that PSSSP has relatively poor chemical stability, undergoing a sulfur-sulfur exchange reaction during storage to form a paclitaxel dimer prodrug bridged by disulfide and tetrasulfide bonds. Therefore, it is difficult to obtain high-purity PSSSP for subsequent research.

[0092] Example 2: Synthesis of thiocarbonate-bridged paclitaxel dimer prodrug (PSC(S)SP)

[0093] 0.2 mmol of 2,2'-trithiocarbonyl diacetic acid was dissolved in 5-10 mL of dichloromethane. Separately, 0.04 mmol of 4-dimethylaminopyridine (DMAP), 0.4 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 0.4 mmol of paclitaxel were dissolved in 20 mL of anhydrous dichloromethane and mixed with the dichloromethane solution of 2,2'-trithiocarbonyl diacetic acid. The mixture was stirred at room temperature for 4 h. Then, 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 24 h. The entire reaction was carried out under N2 protection. The resulting product was purified by preparative liquid chromatography.

[0094] The structure of the PSC(S)SP prepared in Example 2 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown.

[0095] The nuclear magnetic resonance spectroscopy analysis results are as follows:

[0096] 1H NMR (600MHz, CDCl3-d) δ8.133(d,J=7.2Hz,4H,Ar-H),7.729(d,J=7.3Hz,4H,Ar-H),7.611(t,J=7.4Hz,2H,Ar-H),7.512(t,J=7.9Hz,6H ,Ar-H),7.455-7.381(m,8H,Ar-H),7.379-7.322(m,6H,Ar-H),6.884(d,J=9.2Hz,2H,7-OH),6.271(s,2H,10-CH),6.254-6.176(m,2H,1 3-CH),5.968(dd,J=9.2,3.2Hz,2H,3'-CH),5.676(d,J=7.0Hz,2H,2-CH),5.492(d,J=3.2Hz,2H,2'-CH),4.963(dd,J=9.6,2.3Hz,2H,5 -CH),4.415(dd,J=11.0,6.6Hz,2H,7-CH),4.312(d,J=8.5Hz,2H,20-CH2-αH),4.226(d,J=16.4Hz,2H,20-CH2-βH),4.209-4.154(m,4H, CH2 SC(S)S CH2 ),3.786(d,J=7.1Hz,2H,3-CH),2.579-2.522(m,2H,6-CH2-αH),2.413(s,6H,4-OAc -CH3),2.346(dd,J=15.3,9.3Hz,2H,14-CH2-αH),2.220(s,6H,10-OAc-CH3),2.171( dd,J=15.4,8.8Hz,2H,14-CH2-βH),1.915-1.879(m,2H,6-CH2-βH),1.872(d,J=6.9 Hz,6H,18-CH3),1.677(s,6H,19-CH3),1.211(s,6H,17-CH3),1.128(s,6H,16-CH3).

[0097] The mass spectrometry result is MS(ESI) m / z [M+Na]. + =1919.568718, [M+K] + =1935.541881. The purity results show that the purity of PSC(S)SP is 99.85%, which meets the requirements for subsequent experiments.

[0098] Example 3: Synthesis of a sulfur-carbon-sulfur bond-bridged paclitaxel dimer prodrug (PSCSP)

[0099] 0.2 mmol of 2,2'-methylenedithiodiacetic acid was dissolved in 5-10 mL of dichloromethane. Separately, 0.04 mmol of 4-dimethylaminopyridine (DMAP), 0.4 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 0.4 mmol of paclitaxel were dissolved in 20 mL of anhydrous dichloromethane and mixed with the 2,2'-methylenedithiodiacetic acid solution in dichloromethane. The mixture was stirred at room temperature for 4 h. Then, 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 24 h. The entire reaction was carried out under N2 protection. The resulting product was purified by preparative liquid chromatography.

[0100] The structure of the PSCSP prepared in Example 3 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 3 As shown.

[0101] The nuclear magnetic resonance spectroscopy analysis results are as follows:

[0102] 1 H NMR (600MHz, CDCl3-d) δ8.139 (d, J=7.5Hz, 4H, Ar-H), 7.725 (d, J=7.6Hz, 4H, Ar- H),7.606(d,J=6.9Hz,2H,Ar-H),7.517(t,J=7.7Hz,4H,Ar-H),7.469(d,J=6.9H z,2H,Ar-H),7.418(t,J=7.6Hz,4H,Ar-H),7.378(dt,J=7.7,3.7Hz,8H,Ar-H),7 .340(t,J=7.2Hz,2H,Ar-H),6.916(d,J=9.3Hz,2H,7-OH),6.297(s,2H,10-CH), 6.255(t,J=9.0Hz,2H,13-CH),6.010(dd,J=9.3,3.2Hz,2H,3'-CH),5.686(d,J= 7.1Hz,2H,2-CH),5.531(d,J=3.2Hz,2H,2'-CH),4.976(dd,J=9.6,2.3Hz,2H,5- CH),4.439(dd,J=10.9,6.7Hz,2H,7-CH),4.320(d,J=8.5Hz,2H,20-CH2-αH),4. 205(d,J=8.5Hz,2H,20-CH2-βH),3.811(d,J=7.0Hz,2H,3-CH),3.707(s,2H,CH2S CH2 SCH2), 3.433 (d, J = 15.0 Hz, 2H, CH2 SCH2S CH2 ), 3.359(d, J=15.1Hz, 2H, CH2 SCH2S CH2 ),2.558(ddd,J=15.4,9.7,6.6Hz,2H,6-CH2-αH),2.451(s,6H,4-OAc-CH3),2 .370(dd,J=15.3,9.3Hz,2H,14-CH2-αH),2.221(s,6H,10-OAc-CH3),2.177(dd ,J=15.4,8.9Hz,2H,14-CH2-βH),1.929(s,6H,18-CH3),1.902-1.855(m,2H,6 -CH2-βH),1.683(s,6H,19-CH3),1.225(s,6H,17-CH3),1.136(s,6H,16-CH3).

[0103] The mass spectrometry result is MS(ESI) m / z [M+Na]. + =1889.614198, [M+K] + =1905.588872. The purity results show that the purity of PSCSP is 99.77%, which meets the requirements for subsequent experiments.

[0104] Example 4: Synthesis of a three-carbon bond-bridged paclitaxel dimer prodrug (PCCCP)

[0105] 0.2 mmol of 1,7-pimecrolic acid was dissolved in 5-10 mL of dichloromethane. Separately, 0.04 mmol of 4-dimethylaminopyridine (DMAP), 0.4 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 0.4 mmol of paclitaxel were dissolved in 20 mL of anhydrous dichloromethane and mixed with the 1,7-pimecrolic acid dichloromethane solution. The mixture was stirred at room temperature for 4 h. Then, 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 24 h. The entire reaction was carried out under N2 protection. The resulting product was purified by preparative liquid chromatography.

[0106] The structure of the PCCCP prepared in Example 4 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 4 As shown.

[0107] The nuclear magnetic resonance spectroscopy analysis results are as follows:

[0108] 11H NMR (600 MHz, CDCl3-d) δ 8.134 (d, J = 6.7 Hz, 4H, Ar-H), 7.725 (d, J = 7.3 Hz, 4H, Ar-H), 7.610 (t, J = 7.4 Hz, 2H, Ar-H), 7.502 (dt, J = 17.5, 7.6 Hz, 6H, Ar-H), 7.429 - 7.349 (m, 12H, Ar-H), 7.340 - 7.295 (m, 2H, Ar-H), 6.924 (d, J = 9.2 Hz, 2H, 7-OH), 6.298 (s, 2H, 10-CH), 6.244 (t, J = 9.1 Hz, 2H, 13-CH), 5.967 (dd, J = 9.3, 3.5 Hz, 2H, 3’-CH), 5.680 (d, J = 7.1 Hz, 2H, 2-CH), 5.515 (d, J = 3.5 Hz, 2H, 2’-CH), 4.973 (d, J = 7.3 Hz, 2H, 5-CH), 4.440 (dd, J = 10.9, 6.6 Hz, 2H, 7-CH), 4.316 (d, J = 8.5 Hz, 2H, 20-CH2-αH), 4.200 (d, J = 8.5 Hz, 2H, 20-CH2-βH), 3.809 (d, J = 7.0 Hz, 2H, 3-CH), 2.554 (ddd, J = 14.5, 9.7, 6.6 Hz, 2H, 6-CH2-αH), 2.450 (s, 6H, 4-OAc-CH3), 2.412 - 2.363 (m, 2H, 14-CH2-αH), 2.323 (td, J = 16.1, 15.6, 7.2 Hz, 4H, CH2 CH2CH2CH2 CH2 ), 2.221 (s, 6H, 20-OAc-CH3), 2.143 (dd, J = 15.4, 8.9 Hz, 2H, 14-CH2-βH), 1.934 (s, 6H, 18-CH3), 1.911 - 1.862 (m, 2H, 6-CH2-βH), 1.682 (s, 6H, 18-CH3), 1.532 (p, J = 7.5 Hz, 4H, CH2 CH2 CH2 CH2 CH2), 1.249 (d, J = 8.1 Hz, 1H, CH2CH2 CH2 CH2CH2), 1.226 (s, 6H, 17-CH3), 1.197 (d, J = 7.1 Hz, 1H, CH2CH2 CH2 CH2CH2), 1.134 (s, 6H, 16-CH3).

[0109] The mass spectrometry result is MS (ESI) m / z [M+Na] +=1853.693735. The purity results show that the PCCCP purity is 99.90%, which meets the requirements for subsequent experiments.

[0110] Example 5: Preparation and characterization of self-assembled nanoparticles of paclitaxel dimer prodrug

[0111] Accurately weigh DSPE-PEG 2k 3 mg of paclitaxel dimer prodrug and 12 mg of the paclitaxel dimer prodrug from Examples 2-4 were dissolved in 1 mL of anhydrous ethanol. The anhydrous ethanol solution was then slowly added dropwise to 4 mL of deionized water with stirring, spontaneously forming uniform nanoparticles PSC(S)SP NPs, PSCSP NPs, and PCCCP NPs. Ethanol was removed by rotary evaporation under reduced pressure to obtain a nanocolloidal solution free of organic reagents. The results are shown in Table 1. The particle size of each group of nanoparticles was approximately 90-110 nm, with PSC(S)SP NPs having the smallest particle size. The particle size distribution of each group was less than 0.2, the surface charge was approximately -20 mV, and the drug loading was higher than 70%. The particle size and morphology of the self-assembled nanoparticles of the above-mentioned paclitaxel dimer prodrug were determined by transmission electron microscopy, and the results are as follows: Figure 5 Transmission electron microscopy images show that the drug-loaded nanoparticles are uniformly spherical.

[0112] The long-term stability of prodrug self-assembled nanoparticles at 4℃ and 25℃ was investigated using particle size change as an indicator. The results are as follows: Figure 6 As shown, the prepared paclitaxel dimer prodrug self-assembled nanoparticles did not show significant changes in particle size after 60 days of storage, exhibiting good long-term stability at 4℃ and 25℃.

[0113] Table 1. Particle size, particle size distribution, surface charge, and drug loading of paclitaxel dimer prodrug self-assembled nanoparticles.

[0114]

[0115] Example 6: In vitro release assay of self-assembled nanoparticles of paclitaxel dimer prodrug

[0116] The in vitro release of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 was investigated using phosphate-buffered saline (PBS) containing 30% acetonitrile (pH 7.4) as the release medium. 0.2 mL of the prodrug self-assembled nanoparticles (paclitaxel content 1 mg / mL) prepared in Example 5 was added to 30 mL of the release medium. Certain concentrations of hydrogen peroxide (H₂O₂, 0.2 mM, 1 mM, 10 mM) or dithiothreitol (DTT, 0.05 mM, 0.5 mM, 2 mM) were added to the release medium. Samples were taken at set time points at 37°C, and the concentration of released paclitaxel was determined by high-performance liquid chromatography (HPLC) to investigate the release of the nanoparticles under oxidizing and reducing conditions. The results are as follows: Figure 7As shown, PSC(S)SP NPs exhibit extremely high redox dual responsiveness, while PSCSP NPs only exhibit oxidation sensitivity, and PCCCP NPs have no redox responsive drug release capability.

[0117] Example 7: Cytotoxicity of self-assembled nanoparticles of paclitaxel dimer prodrug

[0118] The MTT assay was used to investigate the toxicity of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 against three types of tumor cells and one type of normal cell: mouse breast cancer (4T1) cells, mouse melanoma (B16F10) cells, human oral epithelial carcinoma (KB) cells, and mouse fibroblast (3T3) cells. First, morphologically sound cells were digested, diluted with culture medium to 2000 cells / mL, and then 200 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated for 24 h to allow cell adhesion. After cell adhesion, either paclitaxel or the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 were added. In this experiment, the drug solution and nanoparticle formulation were prepared and diluted using the corresponding cell culture medium and aseptically filtered through a 0.22 μm filter membrane. 200 μL of the test solution was added to each well, with three parallel wells for each concentration. The control group, i.e., without the test drug solution, was supplemented with 200 μL of culture medium and incubated with the cells in an incubator. Forty-eight hours after drug addition, the 96-well plate was removed, and 20 μL of 5 mg / mL MTT solution was added to each well. The plate was incubated for 4 hours, then the culture medium was discarded. The 96-well plate was inverted onto filter paper to thoroughly absorb any remaining liquid. Then, 200 μL of DMSO was added to each well, and the plate was shaken for 10 minutes to dissolve the blue-purple crystals. Well A1 (containing only 200 μL of DMSO) was designated as the zeroing well. The absorbance of each well after zeroing was measured at 570 nm using a microplate reader.

[0119] The results are as follows Figure 8 As shown, since the prodrugs require activation to exert their effects in cells, the cytotoxicity of the three dimeric prodrug nanoassemblies was weaker than that of paclitaxel. The cytotoxicity of the dimeric prodrug nanoassemblies was closely related to their redox activation capacity. The order of antitumor activity of the three dimeric prodrug nanoassemblies was: PSC(S)SP NPs > PSCSP NPs > PCCCP NPs. PSC(S)SP NPs, due to their dual redox hypersensitivity, could effectively cope with the redox microenvironment of tumor cells, thus exhibiting the strongest in vitro antitumor activity.

[0120] Example 8: Pharmacokinetic Study of Self-Assembled Nanoparticles of Paclitaxel Dimer Prodrug

[0121] SD rats weighing 180-220g were randomly assigned to groups and fasted for 12 hours before drug administration, but with free access to water. They were intravenously injected with paclitaxel, albumin-bound paclitaxel for injection, or the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5, respectively. The dosage was 5 mg / kg (paclitaxel equivalent). Blood was collected from the orbital sinus at specified time points, and plasma was obtained. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0122] Experimental results are as follows Figure 9 As shown, due to their short half-life, paclitaxel in both paclitaxel and albumin-bound paclitaxel for injection is rapidly cleared from the bloodstream. In contrast, the cycling time of paclitaxel dimer prodrug self-assembled nanoparticles is significantly prolonged. Furthermore, different chemical linkages significantly influence the pharmacokinetic behavior of the dimer prodrug nanoparticles. Compared to PSCSP NPs and PCCCP NPs, PSC(S)SP NPs exhibit stronger colloidal stability, thus prolonging their in vivo retention.

[0123] Example 9: In vivo antitumor experiment of self-assembled nanoparticles of paclitaxel dimer prodrug

[0124] Using 4T1 tumor-bearing BALB / c mice as a model, self-assembled nanoparticles of the paclitaxel dimer prodrug prepared in Example 5 were administered via tail vein. Control groups included Taxol, paclitaxel for injection (albumin-bound), and intravenous saline, with dosages of 10 mg / kg and 30 mg / kg (paclitaxel equivalent), respectively. Results are as follows: Figure 10 and Figure 11 As shown, all formulations slowed tumor growth to some extent compared to the saline group. In contrast, injectable paclitaxel (albumin-bound) and PSC(S)SP NPs exhibited higher antitumor effects. This is because PSC(S)SP NPs possess good colloidal stability, which enhances their pharmacokinetic behavior and results in a higher area under the curve. Simultaneously, PSC(S)SP NPs exhibit a faster drug release rate in tumor cells, improving their cytotoxicity. However, significant weight loss was observed in the paclitaxel group and the injectable paclitaxel (albumin-bound) group, while no significant weight loss was observed in the paclitaxel dimer prodrug nanoparticle group, indicating that PSC(S)SP NPs, while possessing comparable tumor-suppressive effects to injectable paclitaxel (albumin-bound), also offer better safety. In summary, the stability, cytotoxicity, pharmacokinetic distribution, and tumor-site responsive drug release capacity of nanoparticles all influence the final antitumor effect. The above results further demonstrate the advantages of thiocarbonate-bridged paclitaxel dimer prodrug self-assembled nanoparticles.

Claims

1. A thiocarbonate-linked dimer prodrug or a pharmaceutically acceptable salt thereof, characterized in that, The prodrug is specifically a paclitaxel dimer prodrug bridged by thiocarbonate bonds, with the following structural formula: 。 2. A method for synthesizing the thiocarbonate-linked dimer prodrug of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (1) Dissolve 2,2'-trithiocarbonate diacetic acid in dichloromethane and stir until homogeneous; separately dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and drugs containing hydroxyl, amino or carboxyl groups in anhydrous dichloromethane and stir until homogeneous, then mix with the 2,2'-trithiocarbonate diacetic acid solution, and react under N2 protection at room temperature with stirring. (2) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to the mixed solution, and continue stirring the reaction at room temperature under N2 protection. The resulting product is purified by preparative liquid phase separation.

3. The thiocarbonate-linked dimer prodrug self-assembled nanoparticles according to claim 1, characterized in that, These include non-PEGylated thiocarbonate-linked dimeric prodrug self-assembled nanoparticles, PEG-modified thiocarbonate-linked dimeric prodrug self-assembled nanoparticles, thiocarbonate-linked dimeric prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances, and thiocarbonate-linked dimeric prodrug self-assembled nanoparticles loaded with other drugs. The preparation method includes the following steps: When a non-PEGylated thiocarbonate-linked dimer prodrug self-assembles into nanoparticles, the thiocarbonate-linked dimer prodrug is dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent. When PEGylated thiocarbonate-linked dimer prodrugs self-assemble into nanoparticles, the thiocarbonate-linked dimer prodrug and PEG are dissolved in an organic solvent. While stirring, the solution is slowly added dropwise to water. The prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a solvent-free colloidal solution. The PEG is selected from TPGS, DSPE-PEG, PLGA-PEG, and PE-PEG, and the molecular weight of the PEG is 1000-5000. The weight ratio of the thiocarbonate-linked dimer prodrug to PEG is 90:10 to 60:

40. When assembling nanoparticles from thiocarbonate-linked dimer prodrugs loaded with hydrophobic fluorescent substances or other drugs, the thiocarbonate-linked dimer prodrug, fluorescent substance or drug, and PEG are dissolved in an organic solvent. Under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles. The organic solvent is removed by vacuum distillation to obtain a nanocolloidal solution free of organic solvent.

4. The thiocarbonate-linked dimer prodrug self-assembled nanoparticles as described in claim 3, characterized in that, The organic solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, and acetone.

5. A pharmaceutical composition comprising the thiocarbonate-linked dimeric prodrug of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

6. The use of the thiocarbonate-linked dimer prodrug of claim 1, the self-assembled dimer prodrug nanoparticles of claim 3, or the pharmaceutical composition of claim 5 in the preparation of a drug delivery system.

7. The use of the thiocarbonate bond-bridged dimer prodrug of claim 1, the self-assembled nanoparticles of the dimer prodrug of claim 3, or the pharmaceutical composition of claim 5 in the preparation of antitumor drugs.

8. The use of the thiocarbonate bond-bridged dimer prodrug of claim 1, the self-assembled nanoparticles of the dimer prodrug of claim 3, or the pharmaceutical composition of claim 5 in the preparation of injection, oral, or topical drug delivery systems.

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