Thiocarbonate bond bridged dimer prodrug as well as self-assembled nanoparticles and application thereof

Abstract

The invention discloses a thiocarbonate bond bridged dimer prodrug as well as self-assembled nanoparticles and application thereof, belongs to the technical field of medicines, and relates to construction of the thiocarbonate bond bridged dimer prodrug as shown in a structural general formula (I) and the self-assembled nanoparticles thereof and application of the thiocarbonate bond bridged dimer prodrug and the self-assembled nanoparticles thereof in drug delivery. The preparation method is simple and easy to implement, the thiocarbonate bond bridged dimer prodrug can be self-assembled to form nanoparticles and has oxidation-reduction dual response drug release capacity, intelligent response activation of the prodrug in tumor cells can be achieved, and the anti-tumor effect and safety of the prodrug are guaranteed. The invention provides a new strategy for developing a high-efficiency and low-toxicity drug delivery system, and meets the urgent demand on high-end chemotherapy preparations in clinic. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a thiocarbonate bond-bridged dimer prodrug and self-assembled nanoparticles thereof and applications thereof, and specifically relates to the construction of a thiocarbonate bond-bridged redox-sensitive paclitaxel dimer prodrug and dimer prodrug self-assembled nanoparticles containing the prodrug, as well as applications thereof in the preparation of anti-tumor drugs. Background Art

[0002] Cancer seriously threatens 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 removed by surgery, and tumors that have metastasized and spread. However, most chemotherapy drugs have disadvantages such as poor targeting, large toxic side effects, and a narrow therapeutic window. Nano drug delivery systems have shown certain advantages in the delivery of chemotherapy drugs, but the vast majority of nano drugs fail to improve patients' overall survival. This is mainly due to the insufficient affinity between the drug and the nanocarrier, resulting in low drug loading, high carrier-related toxicity, sudden or delayed drug release, and poor tumor selectivity.

[0003] In order to meet the above challenges, a carrier-free drug delivery strategy has emerged, namely the small molecule prodrug self-assembly nanodelivery system. Since the prodrug acts as both a carrier and a drug, the small molecule prodrug self-assembly nanoparticles can achieve high drug loading and low carrier-related toxicity. Therefore, a variety of chemotherapy drugs have been applied to the small molecule prodrug self-assembly nanodelivery system to achieve safer and more effective therapeutic effects. In particular, dimer prodrug nanoassemblies, as a promising branch of small molecule prodrug self-assembly nanodelivery systems, have aroused great interest. Dimer prodrugs are composed of two drug molecules connected by a linker, which can further increase the drug loading (more than 60%). The structure of the dimer highlights the key role of the linker in the dimer prodrug nanoassembly, which requires rational design to achieve chemical stability, assembly stability and effective site-specific activation of the prodrug.

[0004] In recent years, sulfur bonds have unique advantages in constructing dimeric prodrug nanoassemblies. On the one hand, sulfur bonds have a bond angle-dihedral angle close to 90°, which can produce "structural defects" to balance the intermolecular forces and effectively improve the self-assembly ability of dimeric prodrugs. Previous studies have found that the number of sulfur atoms has an important influence on the self-assembly of prodrugs. Compared with single sulfur bonds (-S-) and disulfide bonds (-SS-), trisulfide bonds (-SSS-) have more sulfur atoms and sulfur-containing dihedral angles, which can more effectively enhance the self-assembly ability of dimeric prodrugs, thereby improving the stability of the assembly, in vivo circulation time and tumor accumulation. On the other hand, sulfur atoms have a larger atomic radius and lower electronegativity, which makes it easier for the outermost electrons of sulfur atoms to gain or lose, and directly brings about the diversity of sulfur atom valence states (-2 to +6 valence). This also makes the sulfur-containing linker show a unique redox responsiveness, which can respond to reactive oxygen species (ROS) and glutathione (GSH) that are highly expressed specifically in tumor sites, thereby triggering drug release. For example, the valence state of the sulfur atom in a single sulfur bond is -2, which can trigger drug release in response to oxidative stimulation signals. As the number of "series" sulfur atoms in the connecting chain increases, the valence of the sulfur atom increases, making the trisulfide bond have ultra-high reduction sensitivity and weak oxidation sensitivity. However, tumors have a heterogeneous redox microenvironment, and prodrug preparations that can only respond to a single stimulation signal (oxidation or reduction) are difficult to produce a comprehensive and effective therapeutic effect. In addition, although the "series" trisulfide bond can improve the assembly stability of the dimer prodrug to a certain 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 connecting bonds to further improve the assembly stability and activatability of the dimer prodrug without weakening its chemical stability. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the "tandem" trisulfide bond, which has the most promising development and transformation prospects, is modified. A new strategy of connecting three sulfur atoms to the central carbon atom and rearranging the trisulfide bond in a "satellite" distribution is proposed, namely, a trithiocarbonate bond (-SC(S)S-). This strategy does not change the number of sulfur atoms in the connecting bond, but only changes the arrangement of sulfur atoms in the connecting bond. The purpose of the present invention is to design and synthesize a dimeric prodrug containing a thiocarbonate bond bridge, prepare the dimeric prodrug self-assembled nano drug delivery system, and its application in the preparation of anti-tumor drugs. And using trisulfide bonds, sulfur-carbon-sulfur bonds and tricarbon bonds as controls, the effects of different chemical connecting bonds on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of dimeric prodrug self-assembled nanoparticles are explored, and the chemical bridge with the best effect is comprehensively screened out, providing new strategies and more options for the development of tumor microenvironment intelligent responsive drug delivery systems, meeting the urgent needs for efficient chemotherapy preparations in clinical practice.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a thiocarbonate bond-bridged dimer prodrug or a pharmaceutically acceptable salt thereof represented by the general structural formula (I):

[0008]

[0009] Wherein, n=1 to 5;

[0010] Drug is a drug containing hydroxyl, amino or carboxyl groups. The drug containing hydroxyl, amino or carboxyl groups is selected from anti-tumor drugs, antimetabolites and anti-inflammatory drugs. The anti-tumor drugs are selected from taxanes, anthraquinones, nucleosides, camptothecins, platinums, vinblastines, podocaside and artemisinin compounds; the antimetabolites are selected from pyrimidines, purines, ribavirins and folic acids; the anti-inflammatory drugs are selected from halofantrine, griseofulvin, cyclosporin A and its derivatives.

[0011] Further, n = 1 to 3;

[0012] Drug is a taxane or anthraquinone compound.

[0013] Specifically, the thiocarbonate bond-bridged dimer prodrug of the present invention selects paclitaxel as a model drug, and uses 2,2'-trithiocarbonate diacetic acid as a connecting bond. At the same time, the drug is connected through 2,2'-trithiodiacetic acid, 2,2'-methylenedithiodiacetic acid or 1,7-pimelic acid to prepare a trisulfide bond, sulfur-carbon-sulfur bond or tricarbon bond-bridged paclitaxel dimer prodrug as a control compound.

[0014] The paclitaxel dimer prodrug prepared with 2,2'-trithiocarbonate diacetic acid as the connecting bond is named PSC(S)SP, and its structural formula is:

[0015]

[0016] The paclitaxel dimer prodrug prepared with 2,2'-trithiodiacetic acid as the connecting bond is named PSSSP, and its structural formula is:

[0017]

[0018] The paclitaxel dimer prodrug prepared with 2,2'-methylenedithiodiacetic acid as the connecting bond is named PSCSP, and its structural formula is:

[0019]

[0020] The paclitaxel dimer prodrug prepared with 1,7-pimelic acid as the connecting bond is named PCCCP, and its structural formula is:

[0021]

[0022] The present invention provides a method for synthesizing a paclitaxel dimer prodrug containing a thiocarbonate bond, a trisulfide bond, a sulfur-carbon-sulfur bond and a tricarbon bond bridge, comprising the following steps:

[0023] (1) dissolving 2,2'-trithiocarbonate diacetic acid, 2,2'-trithiodiacetic acid, 2,2'-methylenedithiodiacetic acid or 1,7-heptanedioic acid in dichloromethane and stirring evenly; separately dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel in anhydrous dichloromethane and stirring evenly, and then mixing with the above 2,2'-trithiocarbonate diacetic acid, 2,2'-trithiodiacetic acid, 2,2'-methylenedithiodiacetic acid or 1,7-heptanedioic acid solution respectively, and stirring at room temperature for 1-8 hours under N2 protection;

[0024] (2) EDCI and DMAP were added to the mixed solution, and stirring was continued at room temperature for 8-48 h under N2 protection. The obtained product was separated and purified by preparative liquid phase separation.

[0025] In the above preparation method, the paclitaxel can also 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, platinums, vinca alkaloids, podocaside, artemisinins and camptothecins.

[0026] A pharmaceutical composition comprises the thiocarbonate bond-bridged 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 anti-tumor drugs.

[0028] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug or a pharmaceutical composition containing the prodrug in preparing 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 preparing an injection, oral administration or local administration system.

[0030] The present invention also provides the self-assembled nanoparticles of the thiocarbonate bond-bridged dimer prodrug, and the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles include non-PEGylated thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles, PEG-modified thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles, thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances, and thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles encapsulating other drugs. The preparation method is a nanoprecipitation method, including a high-speed stirring method and an ultrasonic method.

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

[0032] When the thiocarbonate bond-bridged dimer prodrug is a non-PEGylated self-assembled nanoparticle, the thiocarbonate bond-bridged dimer prodrug is dissolved in an organic solvent, and the solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent.

[0033] When the PEGylated thiocarbonate-bridged dimer prodrug self-assembles nanoparticles, the thiocarbonate-bridged dimer prodrug and PEG are dissolved in an organic solvent, and the solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent.

[0034] When the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles are used to encapsulate hydrophobic fluorescent substances or other drugs, the thiocarbonate bond-bridged dimer prodrug, the fluorescent substance or the drug, and PEG are dissolved in an organic solvent, and the solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent.

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

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

[0037] The mass ratio of the thiocarbonate bond-bridged dimer prodrug to the PEG modifier is 90:10 to 60:40. Within this range, the prodrug nanoparticles can exert a better anti-tumor effect.

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

[0039] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles in preparing a drug delivery system.

[0040] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug self-assembly nanoparticles in the preparation of anti-tumor drugs.

[0041] The present invention also provides the use of the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles in the preparation of injection, oral administration or local administration systems.

[0042] The technical problem solved by the present invention is to introduce thiocarbonate bonds into dimer prodrugs and self-assembled nanoparticles, design redox-sensitive dimer prodrugs bridged by thiocarbonate bonds, and use the dimer prodrugs for the construction of self-assembled nanoparticles, thereby achieving good chemical stability, high drug loading, good assembly stability, low toxicity and side effects, and tumor site-specific rapid drug release, thereby improving the therapeutic effect. At the same time, using trisulfide bonds, sulfur-carbon-sulfur bonds, and tricarbon bond-bridged dimer prodrugs as controls, the differences in self-assembly, redox-sensitive response, and anti-tumor activity of different chemical bridges are investigated, as well as the effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of prodrug self-assembled nanoparticles.

[0043] The advantages of the present invention are:

[0044] (1) A dimeric prodrug containing a thiocarbonate bond bridge and a control prodrug containing a trisulfide bond, a sulfur-carbon-sulfur bond, and a tricarbon bond bridge were designed and synthesized. The synthesis method is simple and easy;

[0045] (2) Uniform dimer prodrug self-assembled nanoparticles were prepared. The preparation method is simple and easy, and the drug can be loaded efficiently. The drug loading capacity is greater than 70%, and the particle size remains basically unchanged after being placed at 4°C and 25°C for 60 days.

[0046] (3) The differences in self-assembly, redox-sensitive response ability and anti-tumor activity of different chemical bridges were investigated, as well as the effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of prodrug self-assembled nanoparticles. Based on the experimental results, the thiocarbonate bond-bridged prodrug has better chemical stability and higher redox dual hypersensitivity, and can be specifically activated in the tumor redox microenvironment. At the same time, the thiocarbonate bond-bridged prodrug nanoparticles also have the best assembly ability. The present invention provides new strategies and more options for the development of tumor microenvironment intelligent responsive drug delivery systems, meeting the urgent need for efficient chemotherapy preparations in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] A: Mass spectrum of PSSSP.

[0049] B: HPLC purity graph of PSSSP.

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

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

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

[0053] C: HPLC purity graph of PSC(S)SP.

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

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

[0056] B: Mass spectrum of PSCSP.

[0057] C: HPLC purity graph of PSCSP.

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

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

[0060] B: Mass spectrum of PCCCP.

[0061] C: HPLC purity graph of PCCCP.

[0062] Figure 5 The particle size diagram and transmission electron microscope diagram of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 of the present invention.

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

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

[0065] C: Particle size diagram and transmission electron microscopy image of triple carbon bond-bridged paclitaxel dimer prodrug self-assembled nanoparticles.

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

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

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

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

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

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

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

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

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

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

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

[0077] Fig. 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: Blood concentration-time curve of paclitaxel in paclitaxel dimer prodrug self-assembled nanoparticles.

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

[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 the body weight of tumor-bearing mice.

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

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

[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 the body weight of tumor-bearing mice.

[0087] C: Effect of paclitaxel dimer prodrug self-assembled nanoparticles on the tumor-bearing rate of Balb / C mice. DETAILED DESCRIPTION

[0088] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[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, and 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'-trithiodiacetic acid, and stirred at room temperature for 4 hours. Then 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and stirred at room temperature for 24 hours. The above reaction was carried out under N2 protection throughout the process, and the obtained product was separated and purified by preparative liquid phase separation.

[0091] Mass spectrometry was used to determine the structure of the PSSSP prepared in Example 1. The mass spectrum and HPLC spectrum after preparative liquid phase separation and purification are shown in Figure 1The results show that PSSSP has relatively poor chemical stability and will undergo sulfur-sulfur exchange reactions during storage to generate paclitaxel dimer prodrugs bridged by disulfide bonds 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'-trithiocarbonate diacetic acid was dissolved in 5-10 mL of dichloromethane, and 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'-trithiocarbonate diacetic acid, and stirred at room temperature for 4 hours. Then 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and stirred at room temperature for 24 hours. The above reaction was carried out under N2 protection throughout the process, and the obtained product was separated and purified by preparative liquid phase separation.

[0094] The structure of PSC(S)SP prepared in Example 2 was determined by mass spectrometry and nuclear magnetic resonance spectroscopy. Figure 2 shown.

[0095] The results of NMR spectroscopy analysis 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 results are MS (ESI) m / z [M+Na] + =1919.568718,[M+K] + = 1935.541881. The purity results showed that the purity of PSC(S)SP was 99.85%, which met the requirements of subsequent experiments.

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

[0099] 0.2 mmol of 2,2'-methylenedithiodiacetic acid was dissolved in 5-10 mL of dichloromethane, and 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'-methylenedithiodiacetic acid, and stirred at room temperature for 4 hours. Then 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and stirred at room temperature for 24 hours. The above reaction was carried out under N2 protection throughout the process, and the obtained product was separated and purified by preparative liquid phase separation.

[0100] The structure of the PSCSP prepared in Example 3 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy. Figure 3 shown.

[0101] The results of NMR spectroscopy analysis 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=15.0Hz,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 results are MS (ESI) m / z [M+Na] + =1889.614198,[M+K] + =1905.588872. The purity results showed that the purity of PSCSP was 99.77%, which met the requirements of subsequent experiments.

[0104] Example 4: Synthesis of Tri-carbon Bridged Paclitaxel Dimer Prodrug (PCCCP)

[0105] 0.2 mmol of 1,7-heptanediol was dissolved in 5-10 mL of dichloromethane, and 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, mixed with the dichloromethane solution of 1,7-heptanediol, and stirred at room temperature for 4 hours. Then 0.2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.02 mmol of 4-dimethylaminopyridine were added, and stirred at room temperature for 24 hours. The above reaction was carried out under N2 protection throughout the process, and the obtained product was separated and purified by preparative liquid phase separation.

[0106] The structure of PCCCP prepared in Example 4 was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Figure 4 shown.

[0107] The results of NMR spectroscopy analysis 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 result showed that the purity of PCCCP was 99.90%, which met the requirements of subsequent experiments.

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

[0111] Accurately weigh DSPE-PEG 2k 3 mg and 12 mg of the paclitaxel dimer prodrug in Example 2-4 were dissolved in 1 mL of anhydrous ethanol, and the anhydrous ethanol solution was slowly added dropwise to 4 mL of deionized water under stirring to spontaneously form uniform nanoparticles PSC(S)SP NPs, PSCSP NPs and PCCCP NPs. The ethanol was removed by reduced pressure rotary evaporation 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 about 90-110 nm, among which the particle size of PSC(S)SP NPs was the smallest. The particle size distribution of each group was less than 0.2, the surface charge was about -20 mV, and the drug loading was higher than 70%. The particle size and morphology of the above-mentioned paclitaxel dimer prodrug self-assembled nanoparticles were measured by transmission electron microscopy, and the results are shown in Table 1. Figure 5 , Transmission electron microscopy images showed that the drug-loaded nanoparticles were uniform spherical.

[0112] The long-term storage stability of prodrug self-assembled nanoparticles at 4°C and 25°C was investigated using the change in particle size as an indicator. Figure 6 As shown, the prepared paclitaxel dimer prodrug self-assembled nanoparticles were placed for 60 days without significant change in particle size, and had good long-term storage stability at 4°C and 25°C.

[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 test of paclitaxel dimer prodrug self-assembled nanoparticles

[0116] Phosphate buffer (PBS) at pH 7.4 containing 30% acetonitrile was used as the release medium to investigate the in vitro release of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5. 0.2 mL of the prodrug self-assembled nanoparticles prepared in Example 5 (paclitaxel content of 1 mg / mL) was added to 30 mL of the release medium, and a certain concentration of hydrogen peroxide (H2O2, 0.2 mM, 1 mM, 10 mM) or dithiothreitol (DTT, 0.05 mM, 0.5 mM, 2 mM) was added to the release medium. At 37°C, samples were taken at set time points, and the concentration of released paclitaxel was determined by high performance liquid chromatography to investigate the release of the nanoparticles under oxidative and reducing conditions, respectively. The results are shown in FIG. Figure 7As shown, PSC(S)SP NPs exhibited ultrahigh redox dual responsiveness, while PSCSP NPs only showed oxidation sensitivity and PCCCP NPs had no redox responsive drug release ability.

[0117] Example 7: Cytotoxicity of Paclitaxel Dimer Prodrug Self-Assembled Nanoparticles

[0118] The MTT method was used to investigate the toxicity of the paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 to three tumor cells and one normal cell: mouse breast cancer (4T1) cells, mouse melanoma (B16F10) cells, human oral epithelial carcinoma (KB) cells and mouse fibroblast (3T3) cells. First, the cells with good morphology were digested, diluted to 2000 cells / mL with culture medium, and then 200 μL of cell suspension was added to each well of a 96-well plate, and incubated in an incubator for 24 hours to make it adhere to the wall. After the cells adhered to the wall, paclitaxel dimer prodrug self-assembled nanoparticles were added with tadalafil or prepared in Example 5. In this experiment, the preparation and dilution of the drug solution and the nanoparticle preparation were all made with the culture medium of the corresponding cells, and aseptically filtered with a 0.22 μm filter membrane. 200 μL of the test solution was added to each well, and 3 parallel wells were added for each concentration. The control group, that is, no test solution was added, a single 200 μL culture medium was added, and the cells were incubated in an incubator together. 48h after drug addition, the 96-well plate was taken out, 20μL of 5mg / mL MTT solution was added to each well, and the medium was discarded after incubation in an incubator for 4h. The 96-well plate was inverted on filter paper to fully absorb the residual liquid, and 200μL DMSO was added to each well and oscillated on an oscillator for 10min to dissolve the blue-purple crystals. Set A1 well (containing only 200μL DMSO) as the zeroing well. Use an ELISA reader to measure the absorbance value of each well after zeroing at 570nm.

[0119] The results are as follows Figure 8 As shown in the figure, since the prodrug needs to undergo an activation process to exert its effect in cells, the cytotoxicity of the three dimer prodrug nanoassemblies is weaker than that of Taxol. The cytotoxicity of the dimer prodrug nanoassemblies is closely related to their redox activation ability. The order of antitumor activity of the three dimer prodrug nanoassemblies is: PSC(S)SP NPs>PSCSP NPs>PCCCP NPs. PSC(S)SP NPs have dual redox hypersensitivity and can effectively respond to the redox microenvironment of tumor cells, so they show the strongest in vitro antitumor activity.

[0120] Example 8: Pharmacokinetic study of paclitaxel dimer prodrug self-assembled nanoparticles

[0121] SD rats weighing between 180-220 g were randomly divided into groups, fasted for 12 h before administration, and allowed to drink water freely. Taxol, paclitaxel for injection (albumin-bound), and paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 were intravenously injected respectively. The dosage was 5 mg / kg (paclitaxel equivalent). Blood was collected from the eye sockets at the specified time points, and plasma was separated and obtained. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

[0122] The experimental results are as follows Fig. 9 As shown in the figure, paclitaxel in Taxol and Paclitaxel for Injection (Albumin-bound) is rapidly cleared from the blood due to its short half-life. In contrast, the circulation time of paclitaxel dimer prodrug self-assembled nanoparticles is significantly prolonged. At the same time, different chemical linkers have a significant effect on the pharmacokinetic behavior of dimer prodrug nanoparticles. Compared with PSCSP NPs and PCCCP NPs, PSC(S)SP NPs have stronger colloidal stability and prolonged their in vivo retention.

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

[0124] BALB / c mice bearing 4T1 tumors were used as a model. Paclitaxel dimer prodrug self-assembled nanoparticles prepared in Example 5 were administered via tail vein. Taxol, paclitaxel for injection (albumin-bound) and normal saline intravenous injection groups were set as control groups. The dosages were 10 mg / kg and 30 mg / kg (paclitaxel equivalent), respectively. The results are shown in Table 1. Fig.10 and Fig.11 As shown in the figure, all the preparation groups slowed down the tumor growth to some extent compared with the saline group. In contrast, paclitaxel (albumin-bound) for injection and PSC(S)SP NPs showed higher antitumor effects. This is because PSC(S)SP NPs have good colloidal stability, which improves their pharmacokinetic behavior and has a higher area under the curve. At the same time, PSC(S)SP NPs have a faster drug release rate in tumor cells, which improves their cytotoxicity. However, the taxol group and paclitaxel (albumin-bound) for injection group showed obvious weight loss, while the paclitaxel dimer prodrug nanoparticle group had no obvious weight loss, indicating that PSC(S)SP NPs have better safety while having comparable antitumor effects with paclitaxel (albumin-bound) for injection. In summary, the stability, cytotoxicity, pharmacokinetic distribution and tumor site responsive drug release ability of the nanoparticles will affect the final antitumor effect. The above results once again prove the advantages of thiocarbonate-bridged paclitaxel dimer prodrug self-assembled nanoparticles.

Claims

1. A thiocarbonate bond-bridged dimer prodrug or a pharmaceutically acceptable salt thereof, having the following structural formula: in, n＝1～5； Drug is a drug containing hydroxyl, amino or carboxyl groups; the drug containing hydroxyl, amino or carboxyl groups is selected from anti-tumor drugs, anti-metabolism drugs and anti-inflammatory drugs, the anti-tumor drugs are selected from taxanes, anthraquinones, nucleosides, camptothecins, platinums, vinblastines, pocosides, and artemisinin compounds; the anti-metabolism drugs are selected from pyrimidines, purines, ribavirins, and folic acids; the anti-inflammatory drugs are selected from halofantrine, griseofulvin, cyclosporin A and their derivatives.

2. The thiocarbonate bond-bridged dimer prodrug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the structural formula, n=1-3; Drug is a taxane anti-tumor drug.

3. The thiocarbonate bond-bridged dimer prodrug or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The prodrug is specifically a paclitaxel dimer prodrug bridged by a thiocarbonate bond, and the structural formula is as follows:

4. A method for synthesizing the thiocarbonate bond-bridged dimer prodrug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The steps include: (1) dissolving 2,2'-trithiocarbonate diacetic acid in dichloromethane and stirring evenly; separately dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and a drug containing a hydroxyl group, an amino group or a carboxyl group in anhydrous dichloromethane and stirring evenly, and then mixing with the 2,2'-trithiocarbonate diacetic acid solution, and reacting by stirring at room temperature under N2 protection; (2) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added to the mixed solution, and the reaction was continued at room temperature under N2 protection. The obtained product was separated and purified by preparative liquid phase separation.

5. The thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles according to any one of claims 1 to 3, characterized in that: Including non-PEGylated thiocarbonate-bridged dimer prodrug self-assembled nanoparticles, PEG-modified thiocarbonate-bridged dimer prodrug self-assembled nanoparticles, thiocarbonate-bridged dimer prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances, and thiocarbonate-bridged dimer prodrug self-assembled nanoparticles encapsulating other drugs; The preparation method comprises the following steps: When the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles are non-PEGylated, the thiocarbonate bond-bridged dimer prodrug is dissolved in an organic solvent, and the solution is slowly added dropwise to water under stirring, the prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent; When the PEGylated thiocarbonate-bridged dimer prodrug is used to self-assemble nanoparticles, the thiocarbonate-bridged dimer prodrug and PEG are dissolved in an organic solvent, and the solution is slowly added dropwise to water under stirring, the prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent; When the thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles are used to encapsulate hydrophobic fluorescent substances or other drugs, the thiocarbonate bond-bridged dimer prodrug, the fluorescent substance or the drug, and PEG are dissolved in an organic solvent, and the solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms uniform nanoparticles, and the organic solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent.

6. The thiocarbonate bond-bridged dimer prodrug self-assembled nanoparticles according to claim 5, characterized in that: The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG and PE-PEG, and the molecular weight of the PEG is 1000-5000; the solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, and acetone; and the weight ratio of the thiocarbonate bond-bridged dimer prodrug to the PEG modifier is 90:10 to 60:

40.

7. A pharmaceutical composition comprising the thiocarbonate-bridged dimer prodrug according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of the thiocarbonate bond-bridged dimer prodrug according to any one of claims 1 to 3, the dimer prodrug self-assembled nanoparticles according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of a drug delivery system.

9. Use of the thiocarbonate bond-bridged dimer prodrug according to any one of claims 1 to 3, the dimer prodrug self-assembled nanoparticles according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of anti-tumor drugs.

10. Use of the thiocarbonate bond-bridged dimer prodrug according to any one of claims 1 to 3, the dimer prodrug self-assembled nanoparticles according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of an injection, oral administration or topical administration system.

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