Preparation and application of a chiral modified paclitaxel prodrug compound and its self-assembled nanoformulation

Abstract

This invention belongs to the field of pharmaceutical formulation technology, and relates to the preparation of a chiral modified paclitaxel prodrug compound and its self-assembled nanoparticles, as well as their application in antitumor therapy. The compound is shown in general formula (I), and further self-assembled nanoparticles are formed. The resulting chiral modified paclitaxel prodrug self-assembled nanoparticles with different configurations exhibit differences in self-assembly ability, cytotoxicity, and antitumor activity, but all demonstrate highly efficient and low-toxicity antitumor effects. Simultaneously, through in vitro and in vivo studies, chiral modified paclitaxel prodrug self-assembled nanoparticles with the optimal therapeutic index were obtained, representing a highly efficient and precise drug delivery technology for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to the preparation of a chiral modified paclitaxel prodrug compound and its self-assembled nanoparticles and their application in antitumor therapy. Background Technology

[0002] Chirality describes spatial structures that cannot be perfectly superimposed on their mirror images. Studies show that almost all amino acids that make up living organisms are left-handed (L-amino acids), while the monosaccharides that make up nucleic acids are all right-handed (D-monosaccharides). Therefore, living organisms are typical chiral systems, and chiral biorecognition is a fundamental behavior regulating physiological processes. The human body's unique chiral environment allows for precise chiral recognition of drugs; drugs can only exert their effects when chirality matches, while chiral mismatch can reduce efficacy or even produce toxic side effects. The most representative case is the "thalidomide" incident in the 1960s, where the failure to remove the teratogenic S-configuration of thalidomide through chiral resolution led to a large number of phocomelia. Since then, the US FDA has required all racemic drugs marketed in the US to describe the pharmacological effects, toxicity, and clinical efficacy of their enantiomers, and to determine the absolute configuration of the marketed drug. In 2006, China issued the "Technical Guidelines for Quality Control Research of Chiral Drugs" to supervise and manage research on chiral drugs. Furthermore, among the more than 220 small molecule new drugs approved by the US FDA from 2014 to 2020, chiral drugs accounted for as much as 40%, making them an important component of clinical drugs.

[0003] However, the development of existing chiral drugs faces the following problems: (1) Synthetic dilemma: The synthesis of some chiral drugs requires the participation of chiral catalysts. Although many chiral catalysts have been developed, the catalytic efficiency and enantioselectivity of the catalysts are still insufficient for the synthesis of some complex chiral molecules. (2) Chiral drug purification dilemma: Although commonly used chiral separation methods (such as chiral chromatography) can separate chiral enantiomers, the separation efficiency is often low, requiring a long separation time and a large amount of solvent. (3) The effectiveness of chiral drugs: Drugs with different chiral configurations have certain differences in pharmacological activity, and the mechanism of action of some chiral drugs is still not fully understood.

[0004] Compared to chiral drugs, chiral nanomedicine delivery systems combine the advantages of both chiral and nanotechnology, enabling the biorecognition and delivery of chiral drugs at the nanoscale. Currently, most extensive research on chiral nanoformulations focuses on the surface chiral modification of inorganic nanoparticles. While the introduction of chirality can provide inorganic nanoformulations with tunable optical activity and chiral recognition capabilities, the non-degradable toxicity and potential immunogenicity of inorganic nanoparticles hinder the clinical development and application of chiral nanoformulations.

[0005] In contrast, the self-assembly technology of small molecule prodrugs, which has emerged in recent years, provides an effective strategy to overcome the above-mentioned shortcomings. Small molecule prodrug self-assembled nanoparticles involve designing and synthesizing small molecule prodrugs to improve the physicochemical properties of the parent drug and enabling the prodrugs to self-assemble into nanosystems under certain conditions. Small molecule prodrug self-assembled nanodelivery systems integrate prodrug strategies and nanotechnology, offering advantages such as simple preparation processes, good stability, high drug loading capacity, and good safety. Therefore, to overcome the aforementioned shortcomings, this invention constructs a simple and efficient method for the synthesis and purification of chiral paclitaxel prodrugs, and for the first time combines small molecule prodrug self-assembly nanotechnology with chiral technology to construct a small molecule prodrug self-assembled nanodelivery system with biorecognition capabilities. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention provides a chiral modified paclitaxel prodrug compound, its preparation method, and its application in antitumor therapy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A chiral modified paclitaxel prodrug compound, the compound being of general formula (I),

[0009]

[0010] in,

[0011] X and R can be the same or different for Se, S, and C;

[0012] n can be 0, 1, 2, 3, or 4.

[0013] The compound is a chiral cyclic diselenyl-paclitaxel prodrug S-(+)-CSEP:

[0014]

[0015] Chiral cyclic diselenyl-paclitaxel prodrug R-(-)-CSEP:

[0016]

[0017] A method for synthesizing the chiral modified paclitaxel prodrug compound:

[0018] (1) Synthesis of 1,2-diselenopentane-3-carboxylic acid: under nitrogen protection, selenium powder and sodium borohydride were reacted, and then selenium powder was added again. Subsequently, it was reacted with 2,4-dibromobutyric acid. Post-treatment yielded 1,2-diselenopentane-3-carboxylic acid.

[0019] (2) Synthesis of chiral modified paclitaxel prodrug: Under nitrogen protection, 1,2-diselenopentane-3-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel were mixed and subjected to esterification reaction, and then purified to obtain the chiral modified paclitaxel prodrug compound shown in general formula (I).

[0020] In step (1), selenium powder and sodium borohydride are reacted, and then selenium powder is added and reacted at 70-110℃ for 0.5-3 hours to obtain an intermediate product. The intermediate product is reacted with 2,4-dibromobutyric acid at 15-35℃ for 12-24 hours, and after treatment, 1,2-diselenopentane-3-carboxylic acid is obtained. In the synthesis of 1,2-diselenopentane-3-carboxylic acid, the molar ratio of selenium powder to sodium borohydride is 1:1-50, and the molar ratio of intermediate product to 2,4-dibromobutyric acid is 1:0.5-3.

[0021] In step (2), 1,2-diselenopentane-3-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel are mixed and stirred at 15-35°C for 8-24 hours for esterification reaction; wherein the molar ratio of paclitaxel, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) is 1:(0.5-3):(0.1-1).

[0022] The purification method involves preparative liquid-phase separation of the obtained product using a conventional octadecylsilane-bonded silica gel packed column. Depending on the retention time, chiral compounds can be separated. The mobile phase consists of acetonitrile and water in a ratio of (50-80):(50-20).

[0023] A chiral modified paclitaxel prodrug self-assembled nanoparticle

[0024] The nanoparticles are non-PEGylated prodrug self-assembled nanoparticles of chiral modified paclitaxel prodrug compounds;

[0025] The above-mentioned prodrug self-assembled nanoparticles are obtained by PEG modification / active targeting modification; or, the above-mentioned prodrug self-assembled nanoparticles are obtained by encapsulating hydrophobic fluorescent substances / drugs; wherein the PEG is TPGS, DSPE-PEG, PLGA-PEG, PE-PEG or DSPE-PEG-FA.

[0026] A method for preparing self-assembled chiral modified paclitaxel prodrug nanoparticles,

[0027] When the self-assembled nanoparticles are non-PEGylated prodrug self-assembled nanoparticles, the preparation method is as follows: dissolve the prodrug in an organic solvent, add the solution dropwise to water while stirring, and the prodrug spontaneously forms uniform nanoparticles; remove the organic solvent in the formulation by vacuum rotary evaporation to obtain a nanocolloidal solution without any organic solvent, which is the non-PEGylated prodrug self-assembled nanoparticles.

[0028] When the self-assembled nanoparticles are PEG-modified / actively targeting group-modified prodrug self-assembled nanoparticles, the preparation method is as follows: the PEG modifier / actively targeting modifier and the prodrug are dissolved in an organic solvent, and the solution is added dropwise to water while stirring. The prodrug spontaneously forms uniform nanoparticles. The organic solvent in the formulation is removed by vacuum rotary evaporation to obtain a nanocolloidal solution free of any organic solvent, which is the PEG-modified / actively targeting group-modified prodrug self-assembled nanoparticle. The mass ratio of the prodrug to the PEG modifier / actively targeting modifier is 1:(0.1-1). The PEG modifier is TPGS, DSPE-PEG, PLGA-PEG, PE-PEG or DSPE-PEG-FA, and the active targeting modifier is an antibody, sugar residue, hormone, receptor or ligand.

[0029] When the self-assembled nanoparticles are prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances / drugs, the preparation method is as follows: dissolve the PEG modifier, the hydrophobic fluorescent substance / drug, and the prodrug in an organic solvent, and add the solution dropwise to water while stirring. The prodrug spontaneously forms uniform nanoparticles. Remove the organic solvent from the formulation by vacuum rotary evaporation to obtain a nanocolloidal solution without organic solvent, which is the prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances / drugs. The mass ratio of the prodrug to the PEG modifier and the hydrophobic fluorescent substance / drug is 1:(0.1-1):(0.1-1).

[0030] A pharmaceutical composition comprising the chiral modified paclitaxel prodrug compound.

[0031] The application of the chiral modified paclitaxel prodrug compound, the self-assembled nanoparticles, or the pharmaceutical composition thereof, and the application of the chiral modified paclitaxel prodrug compound, the self-assembled nanoparticles, or the pharmaceutical composition thereof in a drug delivery system.

[0032] The application of the chiral modified paclitaxel prodrug compound, the self-assembled nanoparticles, or the pharmaceutical composition thereof, and the application of the chiral modified paclitaxel prodrug compound, the self-assembled nanoparticles, or the pharmaceutical composition thereof in the preparation of antitumor drugs.

[0033] The application of the chiral modified paclitaxel prodrug or the self-assembled nanoparticles in the preparation of antitumor drugs, injection administration, oral administration or local administration systems.

[0034] Application of the chiral modified paclitaxel prodrug or the self-assembled nanoparticles in the preparation of drug delivery systems that improve efficacy and reduce toxicity.

[0035] The beneficial effects of this invention are:

[0036] (1) This invention designs and synthesizes a chiral modified paclitaxel prodrug and prepares chiral small molecule prodrug self-assembled nanoparticles for the first time. The prepared prodrug self-assembled nanoparticles have uniform particle size, extremely high drug loading, and are simple and easy to prepare. (2) The in vivo and in vitro fate of the chiral prodrug self-assembled nanoparticles was investigated at the formulation, cellular, and animal levels. The results showed that there was no significant difference in particle size distribution among the chiral prodrug self-assembled nanoparticles with different configurations, but the stability of the cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles with the S-(+) configuration was slightly better. In terms of cytotoxicity, the chiral nanoparticles with the S-(+) configuration showed stronger cytotoxicity. In addition, the chiral nanoparticles with the S-(+) configuration had better pharmacokinetics than the chiral nanoparticles with the R-(-) and RS-(±) configurations. In terms of antitumor activity, the antitumor effects of the prodrug self-assembled nanoparticles with different chiral configurations also differed, and the chiral nanoparticles with the S-(+) configuration were able to inhibit tumors more effectively.

[0037] The nanoparticles prepared using the chiral-modified paclitaxel prodrug compound of this invention are self-assembled paclitaxel prodrug nanoparticles with different chiral configurations. These nanoparticles possess advantages such as uniform particle size, high drug loading, good stability, chiral biorecognition ability, and good antitumor efficacy. Furthermore, the chirality of the self-assembled prodrug nanoparticles significantly influences their in vivo and in vitro fate. Experimental results show that chiral configuration affects the stability, cytotoxicity, pharmacokinetics, and antitumor efficacy of the self-assembled prodrug nanoparticles. Attached Figure Description

[0038] Figure 1 The S-(+) configuration cyclodiseleno-paclitaxel prodrug of Example 1 of this invention 1 H NMR spectrum and high-resolution mass spectrum.

[0039] Figure 2 The R-(-) configuration cyclodiseleno-paclitaxel prodrug of Example 1 of this invention 1 H NMR spectrum and high-resolution mass spectrum.

[0040] Figure 3 This is an image of the appearance of the non-PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles of Example 2 of the present invention.

[0041] Figure 4 This is a transmission electron microscope image of the PEGylated chiral cyclodiselement-paclitaxel prodrug self-assembled nanoparticles of Example 3 of the present invention.

[0042] Figure 5 This is a stability diagram of the PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles of Example 3 of the present invention in 4°C, room temperature and phosphate buffer containing 10% fetal bovine serum.

[0043] Figure 6 This is a redox release curve of the PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles from Example 3 of the present invention.

[0044] Figure 7 This is a tumor growth curve of PEGylated chiral diselenyl-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) in vivo anti-4T1 breast cancer experiment mice, as shown in Example 3 of the present invention.

[0045] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0046] Figure 8 This is a tumor burden diagram of mice bearing 4T1 breast cancer in vivo using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) as an anti-4T1 breast cancer experiment according to Example 3 of the present invention.

[0047] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0048] Figure 9 This is a graph showing the weight change of tumor-bearing mice in an in vivo anti-4T1 breast cancer experiment using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) as described in Example 3 of the present invention.

[0049] Figure 10 This is a graph showing the blood routine analysis results of mice bearing 4T1 breast cancer in vivo using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) as an anti-4T1 breast cancer experiment in Example 3 of the present invention.

[0050] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0051] Figure 11 This is a tumor growth curve in vivo in vivo anti-Lewis lung cancer experiment of self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) of PEGylated chiral cyclodiselement-paclitaxel prodrug in Example 3 of the present invention.

[0052] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0053] Figure 12 This image shows the tumor weight of mice in vivo during an anti-Lewis lung cancer experiment using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg). (Example 3 of this invention)

[0054] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0055] Figure 13 This is a graph showing the weight change of tumor-bearing mice in an in vivo anti-Lewis lung cancer experiment using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 10 mg / kg) as described in Example 3 of this invention.

[0056] Figure 14 This is a tumor growth curve in vivo in vivo anti-Lewis lung cancer experiment of self-assembled nanoparticles (paclitaxel equivalent dose 20 mg / kg) of PEGylated chiral cyclodiselement-paclitaxel prodrug in Example 3 of the present invention in tumor-bearing mice.

[0057] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0058] Figure 15 This image shows the tumor weight in vivo in vivo anti-Lewis lung cancer experiment of self-assembled nanoparticles (paclitaxel equivalent dose 20 mg / kg) of PEGylated chiral cyclodiseleno-paclitaxel prodrug in Example 3 of the present invention, in mice bearing tumors.

[0059] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0060] Figure 16 This is a graph showing the weight change of tumor-bearing mice in an in vivo anti-Lewis lung cancer experiment using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 20 mg / kg) as described in Example 3 of this invention.

[0061] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests)

[0062] Figure 17The image shows the liver and kidney function test results of in vivo anti-Lewis lung cancer experiments in tumor-bearing mice using PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles (paclitaxel equivalent dose 20 mg / kg) as described in Example 3 of this invention.

[0063] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all are two-tailed t-tests) Detailed Implementation

[0064] The present invention will now be described in detail with reference to the embodiments.

[0065] This invention obtains chiral small molecule prodrugs and combines them with nanotechnology to construct a highly efficient and precise nanodelivery platform with chiral biorecognition capabilities. The self-assembled chiral prodrug nanoparticles with specific configurations have outstanding effects in formulation properties, cellular level, and in vivo antitumor efficacy. In terms of delivery technology, the selected PEG surface modifier is a medical material approved by the FDA with good biocompatibility and safety, and the presence of PEG can prevent the nanoparticles from being phagocytosed by the reticuloendothelial system, thereby improving the stability of the nanoparticles.

[0066] Example 1: Synthesis of chiral cyclic diselenyl-paclitaxel prodrugs (S-(+)-CSEP and R-(-)-CSEP)

[0067] Synthesis of 1,2-diselenopentane-3-carboxylic acid: Selenium powder (1.58 g, 20 mmol) and 30 mL of anhydrous ethanol were added to a 250 mL round-bottom three-necked flask. Sodium borohydride (1.51 g, 40 mmol) was dissolved in 40 mL of anhydrous ethanol and placed in a 50 mL dropping funnel. Under nitrogen protection, the ethanol solution containing sodium borohydride was slowly added dropwise to the three-necked flask. After stirring in an ice bath for 0.5 hours, a second portion of selenium powder (1.97 g, 25 mmol) was added, and the reaction was carried out at 80 °C for 0.5 hours. Subsequently, 2,4-dibromo-butyric acid (4.92 g, 20 mmol) was added, and the mixture was stirred overnight at 25 °C. After the reaction was complete, solid impurities were removed by filtration. The filtrate was diluted with a large amount of water and then extracted with ethyl acetate to obtain the target compound, 1,2-diselenopentane-3-carboxylic acid. Finally, the ethyl acetate layer was dried over anhydrous magnesium sulfate, and organic reagents were removed by rotary evaporation to obtain a dark reddish-brown solid.

[0068] Synthesis of the prodrug: The 1,2-diselenopentane-3-carboxylic acid obtained above and paclitaxel (molar ratio = 1:1) were dissolved in 20 mL of dichloromethane. Then, 10 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolved in dichloromethane (2 equivalents) and 0.2 equivalents of 4-dimethylaminopyridine were added. The mixture was stirred at room temperature for 12 hours. The entire reaction was carried out under N2 protection to obtain the reaction product. The obtained product was purified by preparative liquid chromatography. The mobile phase was acetonitrile:water = 60:40. The chromatographic column was a YMC-pack ODS-A (250*20mm 5μm) preparative C18 column with a flow rate of 5 mL / min. The peak times of the two prodrugs were approximately 62 min (S-(+)-CSEP) and 71 min (R-(-)-CSEP), and the yields were approximately 10% (S-(+)-CSEP) and 9% (R-(-)-CSEP).

[0069] The structure of the prodrug was confirmed using proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, with the following results: Figure 1 and Figure 2 As shown, the spectral analysis results are as follows:

[0070] S-(+)-CSEP:

[0071] 1 H NMR(400MHz,Chloroform-d)δ8.14(d,2H,o-2O-ArH),7.77(d,2H,o-3'NH-ArH),7.62(t,1H,Ar-H),7.54-7.34(m,10H,Ar-H),6.99(d,1H,3'-N H-),6.29-6.25(m,2H,10-H,13-H),5.96(dd,1H,3'-H),5.69(d,1H,2-H),5.50(d,1H,2'-H),4.98(dd,1H,5-H),4.47-4.42(m,2H,CH2CH2SeSe CH CO,7-H),4.32(d,1H,20-αH),4.21(d,1H,20-βH),3.82(d,1H,3-H),3.68-3.60(m,1H, CH2 CH2SeSeCHCO),3.51-3.46(m,1H,CH2CH2SeSeCHCO),3.30-3.23(m,1H,CH2 CH 2SeSeCHCO),2.8-2.52(m,1H,6-αH),2.49-2.48(m,1H,CH2 CH2SeSeCHCO),2.46(s,3H,4O-Ac-H),2.39(dd,1H,14-αH),2.22(s,dd,4H,10O-Ac-H,14-βH),1.92(s,3H,18-CH3),1.89-1.85(m,1H,6-βH),1.68(s,3H,19-CH3),1.23(s,3H,16-CH3),1.14(s,3H,17-CH3).

[0072] HRMS of C 51 H 55 NO 15 Se2,(ESI)m / z[M+Na] + =1104.18065,[M+K] + =1120.15411.

[0073] R-(-)-CSEP:

[0074] 1 H NMR(400MHz,Chloroform-d)δ8.14(d,2H,o-2O-ArH),7.76(d,2H,o-3’NH-ArH),7.62(t,1H,Ar-H),7.56-7.34(m,10H,Ar-H),6.88(d,1H,3’-NH-),6.30-6.23(m,2H,10-H,13-H),6.00(dd,1H,3’-H),5.69(d,1H,2-H),5.47(d,1H,2’-H),4.97(dd,1H,5-H),4.54(t,1H,CH2CH2SeSe CH CO),4.47-4.42(m,1H,7-H),4.32(d,1H,20-αH),4.20(d,1H,20-βH),3.81(d,1H,3-H),3.62-3.47(m,2H, CH2 CH2SeSeCHCO),3.25-3.18(m,1H,CH2 CH 2SeSeCHCO),2.56(m,1H,6-αH),2.6-2.48(m,3H,6-αH,7-OH,CH2 CH2SeSeCHCO),2.44(s,3H,4O-Ac-H),2.37(dd,1H,14-αH),2.23(s,4H,10O-Ac-H),2.17(dd,1H,14-βH),1.9 5(s,3H,18-CH3),1.92-1.85(m,1H,6-βH),1.68(s,3H,19-CH3),1.23(s,3H,16-CH3),1.14(s,3H,17-CH3).

[0075] HRMS of C 51 H 55 NO 15 Se2,(ESI)m / z[M+Na] + =1104.18135,[M+K] + =1120.11533.

[0076] Example 2: Preparation and characterization of self-assembled nanoparticles of non-PEGylated chiral diselenyl-paclitaxel prodrug

[0077] Weigh 2 mg of the prodrug (S-(+)-CSEP, R-(-)-CSEP, or a mixture thereof prepared in Example 1) and dissolve it in anhydrous ethanol. While stirring, add the drug-containing ethanol solution dropwise to 1 mL of deionized water to prepare non-PEGylated nanoparticles with concentrations of 0.4 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively, representing non-S-(+)-CSEPNs, non-R-(-)-CSEPNs, and non-RS-(±)-CSEPNs (a mixture of S-(+)-CSEP and R-(-)-CSEP in a 1:1 mass ratio). Figure 3 As shown in Tables 1-3, the particle size of the three types of nanoparticles gradually increased with increasing drug concentration, and all showed varying degrees of precipitation at 1.0 mg / mL. Among them, non-S-(+)-CSEPNs had the smallest particle size and the least precipitation.

[0078] Table 1. Particle size and polydispersity index of unPEG-modified prodrug self-assembled nanoparticles (0.4 mg / mL)

[0079]

[0080] Table 2. Particle size and polydispersity index of unPEG-modified prodrug self-assembled nanoparticles (0.8 mg / mL)

[0081]

[0082] Table 3. Particle size and polydispersity index of unPEG-modified prodrug self-assembled nanoparticles (1.0 mg / mL)

[0083]

[0084] Example 3: Preparation and characterization of PEGylated chiral cyclic diselenyl-paclitaxel prodrug self-assembled nanoparticles

[0085] Weigh out DSPE-PEG 2000 2 mg of the drug and 8 mg of the prodrug (S-(+)-CSEP, R-(-)-CSEP, or a mixture thereof prepared in Example 1) were dissolved in anhydrous ethanol to obtain a solution. Under stirring, 1.6 mL of the solution was added dropwise to 8 mL of deionized water, spontaneously forming uniform nanoparticles, namely S-(+)-CSEPNs, R-(-)-CSEPNs, and RS-(±)-CSEPNs (S-(+)-CSEP and R-(-)-CSEP were mixed in a 1:1 mass ratio). The three types of nanoparticles had uniform particle size distribution, all around 80-100 nm, with polydispersity indices less than 0.2 and surface charges around -20 mV (Table 4). Figure 4 As shown, transmission electron microscopy (TEM) images reveal that the nanoparticles are uniformly spherical. Furthermore, the stability of the nanoparticles was evaluated at 4°C, room temperature, and in phosphate-buffered saline (PBS, pH 7.4) containing 10% fetal bovine serum. Figure 5 As shown in figures ab, the particle size of the three formulations showed no significant change after storage at 4°C and room temperature for more than 11 days, indicating that the nanoparticles have good long-term storage stability. Figure 5 As shown in c, when the self-assembled nanoparticles of the current drug are incubated with phosphate buffer containing 10% fetal bovine serum, the particle size fluctuation range of S-(+)-CSEPNs and RS-(±)-CSEPNs is smaller, indicating better stability.

[0086] Table 4. Particle size, polydispersity index, surface charge, and drug loading of PEGylated nanoparticles

[0087]

[0088] Example 4: In vitro release of chiral cyclic diselenyl-paclitaxel prodrug self-assembled nanoparticles

[0089] To meet the release conditions, a phosphate-buffered saline (PBS) solution with pH 7.4 containing 10% anhydrous ethanol and 1% sodium dodecyl sulfate was used as the release medium. 200 μL of PEGylated chiral diselenyl-paclitaxel prodrug self-assembled nanoparticles prepared in Example 3 were uniformly mixed with release media containing different concentrations of hydrogen peroxide or dithiothreitol, and incubated at 37°C for 24 hours. Samples were taken at preset time points, and the concentration of paclitaxel was determined using high-performance liquid chromatography. Figure 6As shown, the release of paclitaxel from the three types of nanoparticles was concentration-dependent under both oxidative (H2O2, 1mM, 10mM and 30mM) and reducing (DTT, 1mM, 10mM and 30mM) conditions. The higher the concentration of the oxidizing agent, the more paclitaxel was released, and there was no significant difference in the in vitro release of nanoparticles with different chiral configurations.

[0090] Example 5: Cytotoxicity of self-assembled chiral cyclic diselenyl-paclitaxel prodrug nanoparticles

[0091] To investigate the in vitro antitumor activity and safety of the prodrug self-assembled nanoparticles, the cytotoxicity of the prodrug self-assembled nanoparticles against three types of tumor cells (mouse breast cancer cells (4T1), mouse melanoma cells (B16-F10), and human lung cancer cells (A549)) and one type of normal cell (mouse fibroblasts (3T3)) was determined using the MTT assay. First, cells in good condition were digested with trypsin, centrifuged, and then uniformly dispersed in culture medium and seeded into 96-well plates. After incubating the cells at 37°C for 12 hours, the original culture medium was discarded, and the cells were incubated with a series of concentrations of paclitaxel solution (Taxol) and the PEGylated chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles prepared in Example 3. Forty-eight hours after drug administration, the drug-containing culture medium was removed, and the cells were incubated for another 4 hours with 200 μL of 0.5 mg / mL MTT solution. After incubation, discard the MTT solution and invert the 96-well plate onto filter paper to remove residual liquid. Add DMSO and shake on a shaker for 10 minutes to dissolve formazan in the live cells. Measure the absorbance at 490 nm or 570 nm using a microplate reader. In the cytotoxicity assay, wells without cells were used as zeroing wells, and wells with cells but without the drug were used as control wells.

[0092] As shown in Table 5, the paclitaxel group exhibited the strongest cytotoxicity, followed by S-(+)-CSEPNs, RS-(±)-CSEPNs, and R-(-)-CSEPNs. Due to the delayed release of paclitaxel from the prodrug self-assembled nanoparticles, the cytotoxicity of the prodrug self-assembled nanoparticles was weaker than that of paclitaxel. Compared to R-(-)-CSEPNs and RS-(±)-CSEPNs, S-(+)-CSEPNs showed stronger cytotoxicity, which may be related to the high selective recognition of S-(+)-CSEPNs by the cell membrane.

[0093] Table 5. Half-maximal inhibitory concentrations (IC50) of paclitaxel and three prodrug-based self-assembled nanoparticles 50 (unit nM)

[0094]

[0095] Example 6: Pharmacokinetic Study of Self-Assembled Nanoparticles of Chiral Cyclic Diseleno-Taxol Prodrug

[0096] Healthy Sprague-Dawley rats, weighing 180-220g, were randomly divided into four groups. They were fasted for 12 hours before administration but had free access to water. DiR solution and three types of PEGylated chiral cyclic diselenyl-paclitaxel prodrug self-assembled nanoparticles (obtained in Example 3 above) labeled with DiR were injected via tail vein. The dosage was 1 mg / kg (calculated as DiR). Blood samples were collected at predetermined time points. After light-protected protein precipitation, the samples were analyzed using an ELISA reader. As shown in Table 6, the DiR solution was rapidly metabolized and cleared in vivo after administration, with a smaller area under the curve (AUC). In contrast, the DiR-labeled prodrug nanoparticles showed a significant improvement in pharmacokinetic parameters after administration. The overall AUC of S-(+)-CSEPNs, R-(-)-CSEPNs, and RS-(±)-CSEPNs are also shown. 0-24 h These values ​​were 17.0, 6.5, and 10.7 times that of the DiR solution, respectively. Among them, S-(+)-CSEPNs exhibited the best pharmacokinetics, possibly due to their unique chiral configuration, which makes them difficult to clear in vivo.

[0097] Table 6. Pharmacokinetic parameters of DiR solution and DiR co-assembled prodrug nanoparticles

[0098]

[0099] Example 7: In vivo anti-breast cancer study of self-assembled chiral cyclic diselenyl-paclitaxel prodrug nanoparticles.

[0100] 100 μL of mouse breast cancer cells (4T1, 4 × 10⁻⁶) were added. 6 Cells were subcutaneously inoculated into the right back of female BALB / c mice with PBS solution. The tumors were cultured until they reached 100 mm². 3 Around 10 days after administration, mice were randomly divided into 6 groups of 5 mice each. Each group received paclitaxel, paclitaxel (albumin-bound) nanoparticles, and the S-(+)-CSEPNs, R-(-)-CSEPNs, and RS-(±)-CSEPNs obtained in Example 3 above. Each group received an intravenous injection (calculated as paclitaxel) of 10 mg / kg every two days, for a total of 5 administrations per group. A saline group was set up as a negative control group. After administration, the tumor volume and body weight of the mice were recorded daily. On day 10, complete blood count and liver and kidney function analyses were performed, and the tumor tissue was dissected and weighed. Figure 7-8 This indicates that tumors in the saline group mice continued to grow rapidly, while tumor growth in all drug-treated groups was significantly inhibited. The S-(+)-CSEPNs group showed the best tumor inhibition effect. Regarding safety, there was no significant fluctuation or decrease in body weight among the groups. Figure 9 However, mice in the paclitaxel group showed a decrease in blood cells (leukocytes, monocytes, and neutrophils), indicating its systemic toxicity. Figure 10 The blood routine indicators of mice in the prodrug self-assembled nanoparticle group showed no significant changes compared with those in the saline group, indicating good safety. Therefore, chiral-modified cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles achieve highly efficient and low-toxicity antitumor therapy.

[0101] Example 8: In vivo anti-lung cancer study of self-assembled chiral cyclic diselenyl-paclitaxel prodrug nanoparticles

[0102] 100 μL of a solution containing 4 × 10⁴ Lewis lung cells was added. 6 Cells were subcutaneously inoculated into the right back of C57BL / 6 mice using PBS solution. The tumors were allowed to grow to 100 mm². 3 Around 10:00 AM, mice were randomly divided into 6 groups of 4 mice each. Each group received paclitaxel, paclitaxel (albumin-bound) nanoparticles, and S-(+)-CSEPNs, R-(-)-CSEPNs, and RS-(±)-CSEPNs obtained in Example 3 above. Each component (based on paclitaxel) was administered at either a high dose (20 mg / kg) or a low dose (10 mg / kg). The administration was intravenous every two days, with a total of 5 administrations per group. A saline group was set up as a negative control group. Tumor volume and body weight were recorded daily after administration. On day 10, complete blood count and liver and kidney function analyses were performed, and tumor tissue was dissected and weighed. At the low dose (10 mg / kg), tumor growth in the saline group mice continued to grow rapidly, while tumor growth in the administered groups was significantly inhibited. Figure 11-12 ).Depend on Figure 13 It can be seen that there was no significant change in body weight among the mice in each group at a dose of 10 mg / kg.

[0103] At high doses (20 mg / kg), the antitumor effect of the prodrug self-assembled nanoparticles showed a concentration-dependent effect; the inhibitory effect on tumors became more significant with increasing dosage. Figure 14-15 Regarding safety, mice in both the paclitaxel group and the paclitaxel (albumin-bound) nanoparticle group showed significant weight loss at a dose of 20 mg / kg, indicating that paclitaxel and paclitaxel (albumin-bound) nanoparticles have certain toxic side effects. While the weight of mice in the prodrug self-assembled nanoparticle group fluctuated, no significant weight loss was observed, suggesting a certain level of safety. Figure 16 ).

[0104] also, Figure 17The results showed that mice treated with high doses of paclitaxel (albumin-bound) nanoparticles exhibited increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating strong hepatotoxicity after treatment. However, the hematological analysis and liver and kidney function indicators of mice in the prodrug self-assembled nanoparticle group showed no significant changes compared to the saline group, suggesting good in vivo safety. In summary, compared to paclitaxel solution (Taxol) and paclitaxel (albumin-bound) nanoparticles, chiral cyclodiseleno-paclitaxel prodrug self-assembled nanoparticles, while possessing potent antitumor activity, did not cause significant systemic toxicity, making them a safe and efficient chemotherapy drug delivery system. Furthermore, experiments demonstrated that the chiral configuration of the nanoparticles affects their antitumor efficacy.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A chiral modified paclitaxel prodrug compound, characterized in that, The compound is shown in general formula (I). in, X and R can be the same or different for Se, S, and C; n can be 0, 1, 2, 3, or 4.

2. The chiral modified paclitaxel prodrug compound according to claim 1, characterized in that, The compound is a chiral cyclic diselenyl-paclitaxel prodrug S-(+)-CSEP: Chiral cyclic diselenyl-paclitaxel prodrug R-(-)-CSEP:

3. The method for synthesizing the chiral modified paclitaxel prodrug compound according to claim 2, characterized in that: (1) Synthesis of 1,2-diselenopentane-3-carboxylic acid: under nitrogen protection, selenium powder and sodium borohydride were reacted, and then selenium powder was added again. Subsequently, it was reacted with 2,4-dibromobutyric acid. Post-treatment yielded 1,2-diselenopentane-3-carboxylic acid. (2) Synthesis of chiral modified paclitaxel prodrug: Under nitrogen protection, 1,2-diselenopentane-3-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel were mixed and subjected to esterification reaction, and then purified to obtain the chiral modified paclitaxel prodrug compound shown in general formula (I).

4. The method for synthesizing the chiral modified paclitaxel prodrug compound according to claim 3, characterized in that, In step (1), selenium powder and sodium borohydride are reacted, and then selenium powder is added and reacted at 70-110℃ for 0.5-3 hours to obtain an intermediate product. The intermediate product is reacted with 2,4-dibromobutyric acid at 15-35℃ for 12-24 hours, and after treatment, 1,2-diselenopentane-3-carboxylic acid is obtained. In the synthesis of 1,2-diselenopentane-3-carboxylic acid, the molar ratio of selenium powder to sodium borohydride is 1:1-50, and the molar ratio of intermediate product to 2,4-dibromobutyric acid is 1:0.5-3.

5. The method for synthesizing the chiral modified paclitaxel prodrug compound according to claim 3, characterized in that, In step (2), 1,2-diselenopentane-3-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel are mixed and stirred at 15-35°C for 8-24 hours for esterification reaction; wherein the molar ratio of paclitaxel, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) is 1:(0.5-3):(0.1-1).

6. The method for synthesizing the chiral modified paclitaxel prodrug compound according to claim 3, characterized in that, The purification method involves preparative liquid-phase separation of the obtained product using a conventional octadecylsilane-bonded silica gel column to obtain the chiral compound; wherein the mobile phase is acetonitrile and water in a ratio of (50-80):(50-20).

7. A chiral modified paclitaxel prodrug self-assembled nanoparticle, characterized in that: The nanoparticles are non-PEGylated prodrug self-assembled nanoparticles containing the chiral modified paclitaxel prodrug compound of claim 1; The above-mentioned prodrug self-assembled nanoparticles are obtained by PEG modification / active targeting modification; or, the above-mentioned prodrug self-assembled nanoparticles are obtained by encapsulating hydrophobic fluorescent substances / drugs; wherein the PEG is TPGS, DSPE-PEG, PLGA-PEG, PE-PEG or DSPE-PEG-FA.

8. A pharmaceutical composition, characterized in that: The composition comprises the chiral modified paclitaxel prodrug compound of claim 1.

9. The application of the chiral modified paclitaxel prodrug compound of claim 1, the self-assembled nanoparticles of claim 7, or the pharmaceutical composition of claim 8, characterized in that: The application of the chiral modified paclitaxel prodrug compound, self-assembled nanoparticles, or the pharmaceutical composition in a drug delivery system.

10. The application of the chiral modified paclitaxel prodrug compound of claim 1, the self-assembled nanoparticles of claim 7, or the pharmaceutical composition of claim 8, characterized in that: The use of the chiral modified paclitaxel prodrug compound, self-assembled nanoparticles, or the pharmaceutical composition in the preparation of antitumor drugs.

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