Paclitaxel prodrugs bridged by cyclic diselenide / dithio / carbon bond and preparation and application of self-assembled nanoparticles thereof

Abstract

The application belongs to the technical field of medicine, and relates to a paclitaxel prodrug antitumor preparation, in particular to a cyclic structure bridged paclitaxel prodrug and construction of self-assembled nanoparticles thereof, and application of the paclitaxel prodrug in a drug delivery system. The application designs and synthesizes a cyclic structure (wherein X is Se, S or CH2; R is Se, S or CH2; n=0-8; m=0-8; the cyclic structure includes a structure containing a saturated or unsaturated bond) bridged paclitaxel prodrug, and self-assembled nanoparticles are prepared. The cyclic structure bridged paclitaxel prodrug self-assembled nanoparticles in the application can effectively improve the curative effect of paclitaxel and reduce the toxic side effects. The cyclic structure can significantly affect the pharmaceutical property, in-vivo fate and antitumor activity of the paclitaxel prodrug self-assembled nanoparticles. The prodrug or a pharmaceutical composition thereof or small molecule prodrug self-assembled nanoparticles can be used for preparing a tumor microenvironment intelligent response type drug delivery system, so as to improve the antitumor effect of the drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a kind of cyclic structure bridged paclitaxel prodrug antitumor preparation, specifically to cyclic diselenium / disulfide / carbon bond bridged paclitaxel prodrug and its self-assembly nanoparticle preparation, and its application in drug delivery system. BACKGROUND

[0002] The incidence and mortality of cancer are increasing year by year, which seriously threatens human health. Chemotherapy is one of the most commonly used and effective strategies in cancer treatment. However, most of the chemotherapy drugs have the disadvantages of poor water solubility, narrow therapeutic window, poor targeting and strong toxic side effects. For example, paclitaxel (PTX) is a classic first-line drug for non-small cell lung cancer and breast cancer in clinical practice. However, paclitaxel has poor water solubility (<0.3 μg / ml), and its commercial injection Taxol needs to use polyoxyethylene castor oil and ethanol as solubilizers, which can cause serious toxic side effects and greatly limit its clinical application. Therefore, it is urgent to explore more efficient drug delivery strategies to improve the adverse physicochemical properties of drugs and reduce the toxic side effects.

[0003] Prodrugs can improve the physicochemical properties and in vivo fate of drugs through structural modification. The application of nanotechnology can prolong the blood circulation time of drugs and improve tumor targeting. The prodrug nanodelivery system combining prodrug strategy with nanotechnology can effectively improve the problems of poor water solubility and strong toxic side effects of paclitaxel. Whether it is a prodrug or a nanodelivery system, the selective release of smart drugs at the target site is very important for the effectiveness and safety of the preparation. Therefore, the intelligent drug delivery system based on tumor microenvironment stimulation-response drug release has become a research hotspot in recent years. Compared with normal cells, there is a higher concentration of reactive oxygen species (ROS) and glutathione (GSH) in tumor cells. This special redox microenvironment has been widely used to design intelligent responsive drug delivery systems to achieve tumor site-specific drug release while reducing the toxic side effects on normal organs and tissues.

[0004] Intelligent responsive prodrugs are usually composed of three parts: parent drug, responsive linker and side chain. The parent drug and side chain are connected by the responsive linker. Among them, the responsive linker and side chain play an important role in the assembly process of nanoparticles. The commonly used responsive linker is a straight chain of single sulfur bond, disulfide bond, single selenium bond and diselenium bond. There is no report on prodrug self-assembled nanoparticles bridged by cyclic diselenium bond and cyclic disulfide bond. We first collected the responsive linker and side chain of paclitaxel prodrug into a cyclic structure, which significantly reduces the structural complexity of the prodrug. We speculate that the cyclic structure can disrupt the close packing of prodrug molecules, which is expected to enhance the self-assembly ability of the prodrug. In addition, previous studies have shown that straight chain disulfide bond and diselenium bond have redox dual sensitive characteristics, which can intelligently respond to the high oxidation-reduction state in tumor cells and release drugs. Different linkers have different element compositions and different redox sensitivities. Therefore, paclitaxel prodrugs modified by different linkers have different pharmaceutical properties, in vivo fates and anti-tumor effects. Therefore, we compared the differences in redox sensitivity between paclitaxel prodrug self-assembled nanoparticles bridged by cyclic diselenium bond and cyclic disulfide bond and investigated the in vivo fates of different prodrug nanoparticles. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a paclitaxel prodrug antitumor preparation bridged by a cyclic structure, in particular a paclitaxel small molecule prodrug bridged by a cyclic structure and the preparation and application of nanoparticles thereof. The nanoparticles are self-assembled nanoparticles, which have the advantages of small particle size, uniform distribution, high drug loading, good stability, good anti-tumor effect and good safety.

[0006] The purpose of the present application is to design and synthesize paclitaxel prodrugs containing different cyclic structures and prepare self-assembled nanoparticles thereof. Experimental results show that the cyclic structure (cyclic diselenium bond, cyclic disulfide bond or cyclic carbon bond) will affect the colloidal stability, redox responsiveness and anti-tumor effect of the prodrug self-assembled nanoparticles. The present application provides more choices for developing new prodrug self-assembled nanodelivery systems.

[0007] The present application achieves the above-mentioned purpose by the following technical solutions:

[0008] The present application provides a paclitaxel prodrug bridged by a cyclic structure represented by general formula (I) or a pharmaceutically acceptable salt thereof, and the structure is as follows:

[0009]

[0010] Wherein, X is Se, S or CH2; R is Se, S or CH2; n=0-8; m=0-8 (for example, the cyclic structure containing X and R elements is a four-membered ring, a five-membered ring, a six-membered ring, a seven-membered ring, an eight-membered ring or a nine-membered ring); the cyclic structure (the cyclic structure containing X and R elements) includes a structure containing saturated or unsaturated bonds.

[0011] The paclitaxel in the cyclic structure bridged paclitaxel prodrug is replaced by an antitumor drug, an antimetabolite, an anti-inflammatory drug or other poorly soluble drugs, the antitumor drug is selected from taxanes, anthraquinones, nucleosides, camptothecins, platinums, vinblastines, podophyllotoxins, artemisinin compounds; the antimetabolite is selected from pyrimidines, purines, thymidines; the anti-inflammatory drug or other poorly soluble drug is selected from halofantrine, griseofulvin, distamycin A, dexamethasone and their derivatives.

[0012] Specifically, the application provides cyclic structure bridged paclitaxel prodrugs, and the prodrugs are respectively named as PTX-Cy-Se, PTX-Cy-S and PTX-Cy-C, and the structural formulas are as follows:

[0013]

[0014] The cyclic structure is 1,2-diselenopentane-4-carboxylic acid or 1,2-dithiopentane-4-carboxylic acid or cyclopentanoic acid.

[0015] The application provides a synthesis method of 1,2-diselenopentane-4-carboxylic acid, which comprises the following steps: first, stirring and reacting the first selenium powder with sodium borohydride under ice bath for 0.5-1.5 hours, then reacting with the second selenium powder at 60-100 DEG C for 0.5-2 hours to obtain an intermediate product. The intermediate product is reacted with 3-bromo-2-bromomethylpropionic acid at 20-40 DEG C for 10-24 hours (preferably, 25 DEG C for 10 hours) to obtain 1,2-diselenopentane-4-carboxylic acid.

[0016] Further, the molar ratio of the first selenium powder to sodium borohydride is 1:1-3, preferably 1:2; the molar ratio of the first selenium powder to the second selenium powder is 1:1-3, preferably 1:1.25; the molar ratio of the intermediate product to 3-bromo-2-bromomethylpropionic acid is 1:1-3, preferably 1:1.

[0017] Further, the reaction solvent in the synthesis of 1,2-diselenopentane-4-carboxylic acid is ethanol.

[0018] Further, the present application provides a synthesis method of the taxol prodrug bridged by the cyclic structure, comprising the following steps: dissolving 1,2-diselena-cyclopentane-4-carboxylic acid or 1,2-dithia-cyclopentane-4-carboxylic acid or cyclopentanoic acid, 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI), 4-dimethylamino pyridine (DMAP) and taxol (PTX) in anhydrous dichloromethane, stirring at 15-30℃ for 10-24h (preferably 25℃ for 12h), and separating and purifying the obtained product by preparative liquid phase, wherein the whole reaction is carried out under N2 protection.

[0019] Further, the molar ratio of the 1,2-diselena-cyclopentane-4-carboxylic acid or 1,2-dithia-cyclopentane-4-carboxylic acid or cyclopentanoic acid to taxol is (1-5):1, preferably 1:1.

[0020] Further, the molar ratio of the 1,2-diselena-cyclopentane-4-carboxylic acid or 1,2-dithia-cyclopentane-4-carboxylic acid or cyclopentanoic acid to 1-ethyl-3(3-dimethylpropylamine) carbodiimide is 1:(0.2-4).

[0021] Further, the molar ratio of the 1,2-diselena-cyclopentane-4-carboxylic acid or 1,2-dithia-cyclopentane-4-carboxylic acid or cyclopentanoic acid to 4-dimethylamino pyridine is 1:(0.05-0.8).

[0022] The present application also provides a taxol prodrug self-assembled nanoparticle bridged by a cyclic structure, wherein the prodrug self-assembled nanoparticle is a non-PEGylated prodrug self-assembled nanoparticle, a PEG modified / active targeting modified prodrug self-assembled nanoparticle or a prodrug self-assembled nanoparticle loaded with a hydrophobic fluorescent substance / drug.

[0023] The present application also provides a preparation method of the taxol prodrug self-assembled nanoparticle bridged by a cyclic structure, comprising the following steps:

[0024] When the self-assembled nanoparticle is a non-PEGylated prodrug self-assembled nanoparticle, the preparation method comprises the following steps: dissolving a certain amount of prodrug in a proper amount of organic solvent (such as anhydrous ethanol, acetone and tetrahydrofuran, etc.), slowly adding the solution into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; removing the organic solvent in the preparation by a reduced pressure rotary evaporation method to obtain a nanoparticle colloidal solution without any organic solvent, i.e. a non-PEGylated prodrug self-assembled nanoparticle.

[0025] When the self-assembled nanoparticles are PEG-modified / actively targeted group-modified prodrug self-assembled nanoparticles, the preparation method is as follows: a certain amount of PEG modifier / actively targeted modifier and prodrug are dissolved in a proper amount of organic solvent, the solution is slowly added into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by reduced pressure rotary evaporation to obtain a nanoparticle colloidal solution without any organic solvent, namely the PEG-modified / actively targeted group-modified prodrug self-assembled nanoparticles, wherein the mass ratio of the prodrug with a cyclic structure bridged to the PEG modifier / actively targeted modifier is 1:(0.1-1), the PEG modifier is DSPE-PEG, TPGS, PLGA-PEG, PE-PEG or DSPE-PEG-FA, etc., which are amphiphilic polymers or targeting groups, wherein the molecular weight of PEG is 400-5000, and the actively targeted modifier is a substance capable of targeting specific tissues, such as an antibody, a sugar residue, a hormone, a receptor or a ligand.

[0026] When the self-assembled nanoparticles are prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances (such as coumarin-6 or DiR, etc.) / drugs, the preparation method is as follows: a certain amount of PEG modifier, hydrophobic fluorescent substance / drug and prodrug are dissolved in a proper amount of organic solvent, the solution is slowly added into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by reduced pressure rotary evaporation to obtain a nanoparticle colloidal solution without any organic solvent, namely the prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances / drugs, wherein the mass ratio of the prodrug with a cyclic structure bridged to the PEG modifier and the hydrophobic fluorescent substance / drug is 1:(0.1-1):(0.1-1).

[0027] The application further provides a pharmaceutical composition comprising the prodrug of paclitaxel with a cyclic structure bridged or the pharmaceutical composition.

[0028] The application further provides application of the prodrug of paclitaxel with a cyclic structure bridged or the prodrug self-assembled nanoparticles of paclitaxel or the pharmaceutical composition in preparation of an antitumor drug.

[0029] The application further provides application of the prodrug of paclitaxel with a cyclic structure bridged or the prodrug self-assembled nanoparticles of paclitaxel or the pharmaceutical composition in preparation of an injection administration system, an oral administration system or a topical administration system.

[0030] The application further provides application of the prodrug of paclitaxel with a cyclic structure bridged or the prodrug self-assembled nanoparticles of paclitaxel or the pharmaceutical composition in preparation of a drug delivery system, especially in a drug delivery system for improving curative effect and reducing toxicity.

[0031] The application has the following beneficial effects:

[0032] (1) The present application designs and synthesizes taxol prodrugs bridged by different cyclic structures, and the synthesis method is simple and easy to operate; and self-assembled nanoparticles of the taxol prodrugs bridged by cyclic structures with small particle size and uniform particle size distribution are prepared, and the preparation method is simple and easy to operate.

[0033] (2) The influence of the cyclic structure on the preparation property, in-vivo fate and anti-tumor activity of the prodrug self-assembled nanoparticles is investigated. The results show that the self-assembled nanoparticles of the taxol prodrugs bridged by cyclic structures can effectively improve the curative effect of taxol; different cyclic structures have significant influence on the preparation property, in-vivo fate and anti-tumor activity of the self-assembled nanoparticles of the taxol prodrugs; the self-assembled nanoparticles of the taxol prodrugs bridged by five-membered ring diselenium have the best colloid stability, cytotoxicity, oxidation responsiveness and anti-tumor effect; the self-assembled nanoparticles of the taxol prodrugs bridged by five-membered ring disulfide are superior to the self-assembled nanoparticles of the taxol prodrugs bridged by cyclopentanoic acid in colloid stability, cytotoxicity and reduction responsiveness. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The figure is the appearance diagram of the self-assembled nanoparticles of the non-PEG modified prodrug of Example 4 of the present application. 1 HNMR spectrum and mass spectrum of the five-membered ring diselenium bridged taxol prodrug (PTX-Cy-Se) of Example 1 of the present application.

[0035] Figure 2 The figure is the appearance diagram of the self-assembled nanoparticles of the non-PEG modified prodrug of Example 4 of the present application. 1 HNMR spectrum and mass spectrum of the five-membered ring disulfide bridged taxol prodrug (PTX-Cy-S) of Example 2 of the present application.

[0036] Figure 3 The figure is the appearance diagram of the self-assembled nanoparticles of the non-PEG modified prodrug of Example 4 of the present application. 1 HNMR spectrum and mass spectrum of the cyclopentanoic acid bridged taxol prodrug (PTX-Cy-C) of Example 3 of the present application.

[0037] Figure 4 The figure is the appearance diagram of the self-assembled nanoparticles of the non-PEG modified prodrug of Example 4 of the present application.

[0038] Figure 5 The figure is the transmission electron microscope diagram of the PEG modified prodrug self-assembled nanoparticles of Example 5 of the present application.

[0039] Figure 6 The figure is the colloid stability diagram of the PEG modified prodrug self-assembled nanoparticles of Example 5 of the present application in phosphate buffer solution containing 10% fetal bovine serum.

[0040] Figure 7 The figure is the oxidation-reduction activation curve diagram of the PEG modified prodrug self-assembled nanoparticles of Example 5 of the present application.

[0041] Figure 8Tumor growth curve of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-breast cancer experiment of tumor-bearing mice.

[0042] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0043] Figure 9 Body weight change graph of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-breast cancer experiment of tumor-bearing mice.

[0044] Figure 10 Tumor weight graph of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-breast cancer experiment of tumor-bearing mice.

[0045] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0046] Figure 11 Hematological analysis result graph of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-breast cancer experiment of tumor-bearing mice.

[0047] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0048] Figure 12 Tumor growth curve of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-lung cancer experiment of tumor-bearing mice.

[0049] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0050] Figure 13 Body weight change graph of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-lung cancer experiment of tumor-bearing mice.

[0051] Figure 14 Tumor weight graph of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application in the in vivo anti-lung cancer experiment of tumor-bearing mice.

[0052] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0053] Figure 15 Figure for the results of hematological analysis of tumor-bearing mice in the in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application.

[0054] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test)

[0055] Figure 16 Figure for the results of liver and kidney functions of tumor-bearing mice in the in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present application.

[0056] n.s.: P≥0.05 *: P<0.05 **: P<0.01 ***: P<0.001 ****: P<0.0001 (all two-tailed t-test) DETAILED DESCRIPTION

[0057] The present application will be further described in detail with reference to the following examples.

[0058] Example 1: Synthesis of a five-membered ring diseleno-bridged paclitaxel prodrug (PTX-Cy-Se)

[0059] Synthesis of 1,2-diselenapentane-4-carboxylic acid: Selenium powder (1.58 g, 20 mmol) and 30 mL of anhydrous ethanol were added into a 250 mL three-necked round-bottom flask, and 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 dropped into the three-necked flask, and 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, 3-bromo-2-bromomethylpropionic acid (4.92 g, 20 mmol) was added, and stirring was carried out at 25°C overnight. After the completion of the reaction, solid impurities were first removed by filtration, and the filtrate was diluted with a large amount of water and then subjected to liquid-liquid extraction with ethyl acetate to obtain the target compound. Finally, the ethyl acetate layer was dried over anhydrous magnesium sulfate, and after the removal of organic reagents by rotary evaporation, a dark red-brown solid was obtained.

[0060] Synthesis of the prodrug: 1,2-Diselenapentane-4-carboxylic acid and paclitaxel (molar ratio = 1:1) were dissolved in 20 mL of dichloromethane, and then 10 mL of 2.5 times the amount of 1-ethyl-3(3-dimethylpropylamine) carbodiimide and 0.2 times the amount of 4-dimethylaminopyridine dissolved in dichloromethane were added, and stirring was carried out at room temperature for 12 hours. The obtained product was purified by preparative liquid chromatography, and the above reaction was carried out under N2 protection throughout.

[0061] The structure of the prodrug was confirmed by nuclear magnetic resonance hydrogen spectrum and mass spectrometry, and the results were as follows: Figure 1The atlas analysis results are shown as follows:

[0062] 1 H NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 7.1 Hz, 2H, o-2O-ArH), 7.75 (d, J = 7.0 Hz, 2H, o-3’NH-ArH), 7.64 - 7.57 (m, 1H, p-2O-ArH), 7.55 - 7.31 (m, 10H, 3’-ArH, 2O-ArH, 3’NH-ArH), 6.87 (d, J = 9.2 Hz, 1H, 3’-NH-), 6.30 (s, 1H, 10-H), 6.25 (t, J = 9.0 Hz, 1H, 13-H), 6.02 (dd, J = 9.1, 3.5 Hz, 1H, 3’-H), 5.68 (d, J = 7.1 Hz, 1H, 2-H), 5.51 (d, J = 3.5 Hz, 1H, 2’-H), 4.97 (dd, J = 9.7, 2.3 Hz, 1H, 5-H), 4.44 (t, J = 8.6 Hz, 1H, 7-H), 4.32 (d, J = 8.5 Hz, 1H, 20-HH), 4.21 (d, J = 8.4 Hz, 1H, 20-HH), 3.96 (p, J = 5.8 Hz, 1H, OOCCHCH2), 3.81 (d, J = 7.0 Hz, 1H, 3-H), 3.61 (dd, J = 10.5, 5.9 Hz, 1H, OOCCHCH2), 3.51 (dd, J = 10.8, 6.1 Hz, 1H, OOCCHCH2), 3.37 (ddd, J = 15.9, 10.6, 5.9 Hz, 2H, OOCCHCH2), 2.56 - 2.53 (m, 1H, 6-HH), 2.47 (s, 4H, 7-OH, 4O-Ac-H), 2.35 (dd, J = 15.4, 9.4 Hz, 1H, 14-H), 2.23 (s, 3H, 10O-Ac-H), 2.12 (dd, J = 15.4, 8.8 Hz, 1H, 14-H), 1.94 (s, 3H, 18-CH3), 1.93 - 1.85 (m, 1H, 6-HH), 1.76 (s, 1H, 1-OH), 1.68 (s, 3H, 19-CH3), 1.23 (s, 3H, 17-CH3), 1.13 (s, 3H, 16-CH3).

[0063] MS (ESI) m / z for C51H56NO15Se2 [M+H] + : 1082.19949.

[0064] Example 2: Synthesis of a five-membered ring disulfide bridged paclitaxel prodrug (PTX-Cy-S)

[0065] 1,2-dithiolane-4-carboxylic acid and paclitaxel (molar ratio = 1:1) were dissolved in 20 mL of dichloromethane, followed by the addition of 10 mL of dichloromethane-dissolved 2.5 times the equivalent amount of 1-ethyl-3(3-dimethylpropylamine) carbodiimide and 0.2 times the equivalent amount of 4-dimethylamino pyridine, stirred at room temperature for 12 hours, and the resulting product was separated and purified by preparative liquid chromatography. The above reaction was carried out under N2protection throughout.

[0066] The structure of the prodrug was confirmed by nuclear magnetic resonance hydrogen spectrum and mass spectrum, and the results are shown in Figure 2 , and the spectrum analysis results are as follows:

[0067] 1 H NMR (400 MHz, Chloroform-d) δ 8.15 (d, J = 7.2 Hz, 2H, o-2O-ArH), 7.74 (d, J = 7.2 Hz, 2H, o-3’NH-ArH), 7.64-7.57 (m, 1H, p-2O-ArH), 7.57-7.30 (m, 10H, 3’-ArH and 2O-ArH and 3’NH-ArH), 6.87 (d, J = 9.3 Hz, 1H, 3’-NH-), 6.30 (s, 1H, 10-H), 6.25 (d, J = 9.3 Hz, 1H, 13-H), 6.02 (dd, J = 9.2, 3.2 Hz, 1H, 3’-H), 5.69 (d, J = 7.1 Hz, 1H, 2-H), 4.98 (d, J = 9.4 Hz, 1H, 5-H), 4.45 (t, J = 8.6 Hz, 1H, 7-H), 4.32 (d, J = 8.5 Hz, 1H, 20-HH), 4.21 (d, J = 8.5 Hz, 1H, 20-HH), 3.82 (d, J = 7.0 Hz, 1H, 3-H), 3.52-3.48 (m, J = 7.7, 5.0 Hz, 1H, OOCCHCH2), 3.41 (m, J = 11.7, 5.0 Hz, 1H, OOCCHCH2), 3.37-3.22 (m, 3H, OOCCHCH2, OOCCHCH2), 2.57-2.55 (m, 1H, 6-HH), 2.48 (s, 4H, 7-OH, 4O-Ac-H), 2.38 (dd, J = 15.4, 9.1 Hz, 1H, 14-H), 2.23 (s, 3H, 10O-Ac-H), 2.15 (dd, J = 15.4, 8.9 Hz, 1H, 14-H), 1.97-1.84 (m, 4H, 18-CH3, 6-HH), 1.71 (s, 1H, 1-OH), 1.69 (s, 3H, 19-CH3), 1.23 (s, 3H, 17-CH3), 1.14 (s, 3H, 16-CH3).

[0068] MS (ESI) m / z for C51H55NO15S2Na [M+Na] 1011.2883. + : 1008.29203.

[0069] Example 3: Synthesis of cyclopentanoic acid bridged paclitaxel prodrug (PTX-Cy-C)

[0070] Cyclopentanoic acid and paclitaxel (molar ratio = 1:1) were dissolved in 20 mL of dichloromethane, followed by the addition of 10 mL of 2.5 times the equivalent amount of 1-ethyl-3(3-dimethylpropylamine) carbodiimide and 0.2 times the equivalent amount of 4-dimethylamino pyridine dissolved in dichloromethane, stirred at room temperature for 12 hours, and the resulting product was separated and purified by preparative liquid chromatography. The above reaction was carried out under N2protection throughout.

[0071] The structure of the prodrug was confirmed by nuclear magnetic resonance hydrogen spectrum and mass spectrum, and the results are shown in Figure 3 , and the spectrum analysis results are as follows:

[0072] 1H NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 7.0 Hz, 2H, o-20-ArH), 7.73 (d, J = 7.0 Hz, 2H, o-3'NH-ArH), 7.64 - 7.58 (m, 1H, p-20-ArH), 7.55 - 7.31 (m, 10H, 3'-ArH and 20-ArH and 3'NH-ArH), 6.87 (d, J = 9.1 Hz, 1H, 3'-NH-), 6.30 (s, 1H, 10-H), 6.26 (t, J = 9.4 Hz, 1H, 13-H), 5.95 (dd, J = 9.1, 3.3 Hz, 1H, 3'-H), 5.68 (d, J = 7.1 Hz, 1H, 2-H), 5.50 (d, J = 3.3 Hz, 1H, 2'-H), 4.98 (dd, J = 9.9, 2.3 Hz, 1H, 5-H), 4.45 (dd, J = 10.9, 6.6 Hz, 1H, 7-H), 4.32 (d, J = 8.4 Hz, 1H, 20-HH), 4.21 (d, J = 8.4 Hz, 1H, 20-HH), 3.82 (d, J = 7.0 Hz, 1H, 3-H), 2.84 (m, 1H, OOCCHCH2), 2.61 - 2.52 (m, 1H, 6-HH), 2.47 (s, 4H, 7-OH, 40-Ac-H), 2.37 (dd, J = 15.3, 9.3 Hz, 1H, 14-H), 2.23 (s, 3H, 10O-Ac-H), 2.18 - 2.11 (m, 1H, 14-H), 1.94 (s, 3H, 18-CH3), 1.92 - 1.80 (m, 4H, 6-HH, OOCCHCH2CH2CH2CH2), 1.75 - 1.63 (m, 9H, OOCCHCH2CH2CH2CH2, 1-OH, 19-CH3), 1.23 (s, 3H, 17-CH3), 1.13 (s, 3H, 16-CH3).

[0073] MS (ESI) m / z for C53H59NO15Na [M + Na] + : 972.37978.

[0074] Example 4: Preparation and characterization of non-PEGylated prodrug self- assembled nanoparticles

[0075] An appropriate amount of prodrug (PTX-Cy-Se prepared in Example 1, PTX-Cy-S prepared in Example 2, or PTX-Cy-C prepared in Example 3) was dissolved in anhydrous ethanol. The drug-containing ethanol solution was added dropwise into deionized water (volume ratio of anhydrous ethanol and water was 1:5) under stirring to prepare 0.2 mg / mL (A) and 0.6 mg / mL (B) prodrug self-assembled nanoparticles. Figure 4 A) and 0.6 mg / mL (Figure 4 B) non-PEGylated nanoparticles. As shown in Figure 4 Figure 2, with the increase of drug concentration, both PTX-Cy-S and PTX-Cy-C prodrugs showed drug precipitation, while no drug precipitation was observed for PTX-Cy-Se prodrug. This indicated that the prodrug with cyclic diselenide bridge has the best self-assembly ability and stability.

[0076] Example 5: Preparation and characterization of PEGylated prodrug self- assembled nanoparticles

[0077] DSPE-PEG 2k 1 mg and prodrug (PTX-Cy-Se prepared in Example 1, PTX-Cy-S prepared in Example 2 or PTX-Cy-C prepared in Example 3) 4 mg were dissolved in 800 uL anhydrous ethanol, and the ethanol solution was added dropwise into 4 mL deionized water under stirring, and uniform nanoparticles (PTX-Cy-Se nanoparticles, PTX-Cy-S nanoparticles and PTX-Cy-C nanoparticles, respectively) were spontaneously formed. As shown in Table 1, the particle size of the nanoparticles was about 120-130 nm, the particle size distribution was less than 0.2, the surface charge was about -20 mV, and the drug loading was more than 60%. The particle size and morphology of the prodrug self-assembled nanoparticles were determined by transmission electron microscopy, and the results are shown in Figure 5 Figure 3. The transmission electron microscopy image showed that the prodrug nanoparticles were uniform spherical.

[0078] Table 1. Particle size, particle size distribution, surface charge and drug loading of PEG- modified prodrug self-assembled nanoparticles

[0079]

[0080] Example 6: Colloidal stability of prodrug self-assembled nanoparticles

[0081] The PEG-modified prodrug self-assembled nanoparticles prepared in Example 5 were uniformly mixed with phosphate buffer solution (PBS, pH 7.4) containing 10% FBS (1:10 = v:v), incubated at 37°C for 24 hours, and the particle size change was determined by dynamic light scattering method at the predetermined time points. As shown in Figure 6 Figure 4, PTX-Cy-Se nanoparticles had the best colloidal stability, and the particle size change was the smallest within 24 hours. In contrast, the colloidal stability of PTX-Cy-S nanoparticles and PTX-Cy-C nanoparticles was poor, and the particle size of the nanoparticles increased significantly as the incubation time prolonged.

[0082] Example 7: Redox activation of prodrug self-assembled nanoparticles

[0083] The release medium was phosphate buffer solution (PBS) containing 10% ethanol and 1% sodium dodecyl sulfate at pH 7.4. Hydrogen peroxide (H2O2) and dithiothreitol (DTT) were used to simulate the high expression of reactive oxygen species (ROS) and glutathione (GSH) at the tumor site, respectively. The activation behavior of the prodrug was investigated in the presence of different concentrations of hydrogen peroxide (0.1 mM, 1 mM and 2 mM, respectively) or dithiothreitol (1 mM, 2 mM and 5 mM, respectively). The prodrug nanoparticle equivalent to 67 nM paclitaxel was uniformly mixed with 10 mL of the release medium, incubated in a constant temperature shaker at 37°C, and sampled at 0, 1, 2, 4, 8 and 12 hours. The content of the prodrug in the release medium was determined by high performance liquid chromatography. The results are shown in Table 1. Figure 7 As shown in Table 1, in the presence of hydrogen peroxide, the oxidative activation sequence of the prodrug nanoparticle is PTX-Cy-Se nanoparticle > PTX-Cy-S nanoparticle > PTX-Cy-C nanoparticle. In the presence of dithiothreitol, the reduction activation sequence of the prodrug nanoparticle is PTX-Cy-S nanoparticle > PTX-Cy-Se nanoparticle > PTX-Cy-C nanoparticle. At the same time, with the increase of the concentration of H2O2 and DTT, the activation rate of the prodrug is accelerated.

[0084] Example 8: Cytotoxicity of the self-assembled prodrug nanoparticle

[0085] The cytotoxicity of the prodrug nanoparticle was evaluated by MTT method. Mouse breast cancer cells (4T1), mouse melanoma cells (B16-F10), human lung cancer cells (A549) and mouse fibroblasts (3T3) were seeded into 96-well plates at a density of 2000 cells per well and incubated overnight for adhesion. Then the cells were treated with a series of concentrations of taxol (5-500 nM) or the prodrug nanoparticle prepared in Example 5 (5-2000 nM). After 48 hours of treatment with the series of dilutions, the 96-well plates were removed, the drug-containing solution was discarded, and 200 μL of 0.5 mg / mL MTT solution was added to each well. After incubation in the incubator for 4 hours, the 96-well plates were removed, the residual liquid was thoroughly absorbed by placing the 96-well plates upside down on filter paper, and DMSO was added to the shaker for 10 minutes to dissolve the blue-purple crystalline material. Finally, the absorbance value was measured at 490 nm or 570 nm using a microplate reader.

[0086] The results are shown in Table 2. Compared with the paclitaxel solution (taxol), the cytotoxicity of the prodrug nanoparticle was reduced. This is because the release of paclitaxel from the prodrug requires a certain period of time. From the cytotoxicity results, it can be seen that different cyclic structures have a significant impact on the cytotoxicity of the prodrug nanoparticle. Compared with PTX-Cy-S nanoparticle and PTX-Cy-C nanoparticle, PTX-Cy-Se nanoparticle has the strongest cytotoxicity, which may be due to the fact that PTX-Cy-Se nanoparticle releases more paclitaxel, and the higher the drug release efficiency, the stronger the cytotoxicity.

[0087] Table 2. Half-inhibitory concentrations (IC50) of self-assembled nanoparticles of paclitaxel and three prodrugs on four cell types. 50 )

[0088]

[0089] Example 9: In vivo anti-breast cancer study of self-assembled nanoparticles of small molecule prodrugs

[0090] Mouse breast cancer cell suspension (4T1, 5×10) 6 Cells were subcutaneously inoculated into the right back of female BALB / c mice. The tumors were allowed to grow to 100-120 mm in size. 3 At that time, tumor-bearing mice were randomly divided into 6 groups of 5 mice each: saline group, paclitaxel group, paclitaxel (albumin-bound) nanoparticle group, PTX-Cy-Se nanoparticle group, PTX-Cy-S nanoparticle group, and PTX-Cy-C nanoparticle group. The dosage was 10 mg / kg (based on paclitaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5. Administration was once every other day for 5 consecutive days. After administration, the survival status of the mice was observed daily, and changes in body weight and tumor volume were measured. On day 11 after administration, the mice were sacrificed, and organs and tumor tissues were collected for further analysis and evaluation. The results are as follows: Figures 8 to 11 As shown. Figure 8 This indicates that the tumor volume in the saline group mice grew rapidly, reaching 800 mm on day 11. 3 Around 100%. In contrast, nanoparticles can inhibit tumor growth; Figure 9 This indicates that there were no significant changes in the body weight of mice in each formulation group; Figure 10 The results showed that, after the end of administration, the tumor weight of mice in the prodrug self-assembled nanoparticle group was smaller, with the PTX-Cy-Se nanoparticle group having the smallest tumor weight. Figure 11 The results showed that mice in both the paclitaxel group and the paclitaxel (albumin-bound) nanoparticle group exhibited a decrease in the number of blood cells (leukocytes, lymphocytes, monocytes, and neutrophils), indicating hematologic toxicity. However, the hematologic analysis of mice in the prodrug nanoparticle group showed no abnormalities, indicating that the prodrug nanoparticles have good in vivo safety. In summary, compared to paclitaxel solution (paclitaxel) and commercially available paclitaxel (albumin-bound) nanoparticles, cyclic diselenylene-bridged paclitaxel prodrug self-assembled nanoparticles exhibit better antitumor efficacy without causing significant systemic toxicity, making them a safe and efficient chemotherapeutic drug delivery method.

[0091] Example 10: In vivo anti-lung cancer study of self-assembled nanoparticles of small molecule prodrugs

[0092] A suspension of mouse lung cancer cells (5 × 10⁻⁶) was prepared. 6 Cells were subcutaneously inoculated on the right back of C57BL / 6j mice. The tumors were allowed to grow to 100-120 mm in size. 3 At that time, tumor-bearing mice were randomly divided into 6 groups of 5 mice each: saline group, paclitaxel group, paclitaxel (albumin-bound) nanoparticle group, PTX-Cy-Se nanoparticle group, PTX-Cy-S nanoparticle group, and PTX-Cy-C nanoparticle group. The dosage was 10 mg / kg (based on paclitaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5. Administration was once every other day for 5 consecutive days. After administration, the survival status and weight changes of the mice were monitored daily, and the tumor volume was measured. On the 10th day after administration, the mice were sacrificed, and their organs and tumor tissues were collected for further analysis and evaluation. The results are as follows: Figures 12 to 16 As shown. Figure 12 This indicates that the tumor volume in the saline group mice grew rapidly, reaching 800 mm on day 10. 3 Around 100%. In comparison, nanoparticles can inhibit tumor growth; Figure 13 This indicates that there were no significant changes in the body weight of mice in each formulation group; Figure 14 The results showed that the tumor weight of mice in the nanoparticle group was smaller after the administration was completed, with the PTX-Cy-Se nanoparticle group having the smallest tumor weight. Figure 15 and Figure 16 The results showed that mice in the Taxol group and the paclitaxel (albumin-bound) nanoparticle group exhibited decreased blood cell (leukocyte and lymphocyte) counts and increased aspartate aminotransferase (AST) levels, indicating bone marrow suppression and liver damage, respectively. However, the prodrug nanoparticle group showed no abnormalities in blood parameters and liver and kidney function indicators, suggesting that the prodrug nanoparticles have good in vivo safety. In conclusion, compared to paclitaxel solution (Taxol) and commercially available paclitaxel (albumin-bound) nanoparticles, cyclodiselement-bridged paclitaxel prodrug self-assembled nanoparticles exhibit better antitumor efficacy and safety.

Claims

1. Paclitaxel prodrugs with cyclic bridging as shown in general formula (I): in, X is Se, S, or CH2; R is Se, S, or CH2; n = 0-8; m = 0-8; the cyclic structure includes structures containing saturated or unsaturated bonds.

2. The cyclic-bridged paclitaxel prodrug as described in claim 1, characterized in that, Any one of the following specific structures:

3. The method for preparing the cyclically bridged paclitaxel prodrug according to claim 2, characterized in that, Includes the following steps: 1,2-diselenopentane-4-carboxylic acid or 1,2-dithiopentane-4-carboxylic acid or cyclopentanoic acid, 1-ethyl-3-(3-dimethylpropylamine)carbodiimide, 4-dimethylaminopyridine and paclitaxel were dissolved in anhydrous dichloromethane and stirred at 15-30°C. The resulting product was purified by preparative liquid phase separation. The entire reaction was carried out under N2 protection.

4. The self-assembled nanoparticles of the cyclically bridged paclitaxel prodrug as described in claim 1 or 2, characterized in that, This includes non-PEGylated self-assembled nanoparticles, PEG-modified / actively targeted modifier-modified self-assembled nanoparticles, or self-assembled nanoparticles loaded with hydrophobic fluorescent substances / drugs; wherein the PEG is TPGS, DSPE-PEG, PLGA-PEG, PE-PEG, or DSPE-PEG-AA.

5. The method for preparing self-assembled nanoparticles of cyclic-bridged paclitaxel prodrug according to claim 4, characterized in that, Includes the following steps: When the self-assembled nanoparticles are non-PEGylated self-assembled nanoparticles, the preparation method is as follows: the prodrug is 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 without any organic solvent, which is the non-PEGylated self-assembled nanoparticle. When the self-assembled nanoparticles are PEG-modified / actively targeted group-modified self-assembled nanoparticles, the preparation method is as follows: the PEG modifier / actively targeted modifier and the prodrug are dissolved in an organic solvent, and the solution is added dropwise to water under 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 targeted group-modified self-assembled nanoparticle. The mass ratio of the prodrug to the PEG modifier / actively targeted modifier is 1:(0.1-1). The PEG modifier is DSPE-PEG, TPGS, PLGA-PEG, PE-PEG or DSPE-PEG-FA, and the active targeting modifier is an antibody, sugar residue, hormone, receptor or ligand. When the self-assembled nanoparticles are self-assembled nanoparticles loaded with 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 self-assembled nanoparticle loaded with 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).

6. A pharmaceutical composition comprising a paclitaxel prodrug or a pharmaceutically acceptable excipient with a cyclic structure bridged according to any one of claims 1-2.

7. The use of the cyclic-bridged paclitaxel prodrug of any one of claims 1-2, or the self-assembled nanoparticles of the paclitaxel prodrug of claim 4, or the pharmaceutical composition of claim 6 in the preparation of a drug delivery system.

8. The use of the cyclic-bridged paclitaxel prodrug of any one of claims 1-2, or the self-assembled nanoparticles of the paclitaxel prodrug of claim 4, or the pharmaceutical composition of claim 6 in the preparation of an antitumor drug.

9. The use of the cyclic-bridged paclitaxel prodrug of any one of claims 1-2, or the self-assembled nanoparticles of the paclitaxel prodrug of claim 4, or the pharmaceutical composition of claim 6 in the preparation of an injectable, oral, or topical delivery system.

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