Cyclic diselenide / disulfide / carbon bond bridged paclitaxel prodrug as well as preparation and application of self-assembled nanoparticles of cyclic diselenide / disulfide / carbon bond bridged paclitaxel prodrug

Abstract

The invention belongs to the technical field of medicines, and relates to a paclitaxel prodrug anti-tumor preparation, in particular to a paclitaxel prodrug bridged by an annular structure, construction of self-assembled nanoparticles of the paclitaxel prodrug and application of the paclitaxel prodrug in a drug delivery system. According to the invention, different ring structures are designed and synthesized (wherein X is Se, S or CH2; r is Se, S or CH2; n is equal to 0-8; m = 0-8; the paclitaxel prodrug is bridged by a cyclic structure (the cyclic structure comprises a structure containing a saturated or unsaturated bond), and the self-assembled nanoparticles are prepared. The paclitaxel prodrug self-assembled nanoparticles bridged by the annular structure can effectively improve the curative effect of paclitaxel and reduce the toxic and side effects of paclitaxel. The ring structure can significantly influence the pharmaceutical properties, in-vivo fate and antitumor activity of the paclitaxel prodrug self-assembled nanoparticles. The prodrug or the pharmaceutical composition thereof or the micromolecular prodrug self-assembled nanoparticles thereof can be used for preparing a tumor microenvironment intelligent response type drug delivery system, so that the anti-tumor effect of the drug is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a paclitaxel prodrug anti-tumor preparation bridged by a ring structure, and specifically to the preparation of a paclitaxel prodrug bridged by a cyclic diselenide / disulfide / carbon bond and its self-assembled nanoparticles, and the application of the prodrug in a drug delivery system. Background Art

[0002] The incidence and mortality of cancer are rising year by year, posing a serious threat to human health. Chemotherapy is one of the most commonly used and effective strategies in cancer treatment. However, most chemotherapy drugs have disadvantages such as 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 (<0.3μg / ml), and its commercially available injection Taxol requires the use of polyoxyethylene castor oil and ethanol as solubilizers, which in turn causes serious toxic and side effects, greatly limiting its clinical application. Therefore, it is urgent to explore more efficient drug delivery strategies to improve the adverse physical and chemical properties of drugs and reduce toxic side effects.

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

[0004] Smart responsive prodrugs usually consist of three parts: a parent drug, a responsive linker, and a side chain. The parent drug and the side chain are connected together by a responsive linker. Among them, the responsive linker and the side chain play an important role in the assembly process of nanoparticles. Currently, the commonly used responsive linkers are linear monosulfide bonds, disulfide bonds, monoselenium bonds, and diselenium bonds. There are no reports on self-assembled nanoparticles of prodrugs bridged by cyclic diselenium bonds and cyclic disulfide bonds. For the first time, we brought the responsive linker and side chain of paclitaxel prodrug into a ring structure, which significantly reduced the structural complexity of the prodrug. We speculate that the ring structure can disrupt the close stacking between prodrug molecules and is expected to enhance the self-assembly ability of the prodrug. In addition, previous studies have shown that linear disulfide bonds and diselenium bonds have redox-sensitive properties, which can intelligently respond to the high redox state in tumor cells and release drugs. Different linkers have different elemental compositions and redox sensitivities. Therefore, paclitaxel prodrugs modified with different linkers have different pharmaceutical properties, in vivo fates, and anti-tumor effects. Therefore, we compared the differences in redox sensitivity of paclitaxel prodrug self-assembled nanoparticles bridged by cyclic diselenide bonds and cyclic disulfide bonds and investigated the in vivo fate of different prodrug nanoparticles. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a paclitaxel prodrug anti-tumor preparation bridged by a ring structure, specifically the preparation and application of a paclitaxel small molecule prodrug bridged by a ring structure and its nanoparticles. The nanoparticles are self-assembled nanoparticles and 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 invention 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 diselenide 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 invention provides more options for developing new prodrug self-assembled nano delivery systems.

[0007] The present invention achieves the above object through the following technical solutions:

[0008] The present invention provides a paclitaxel prodrug or a pharmaceutically acceptable salt thereof bridged by a ring structure represented by general formula (I), the structure of which 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 a saturated or unsaturated bond.

[0011] The paclitaxel in the paclitaxel prodrug bridged by the ring structure is replaced by anti-tumor drugs, anti-metabolites, anti-inflammatory drugs or other poorly soluble drugs, wherein the anti-tumor drugs are selected from taxanes, anthraquinones, nucleosides, camptothecins, platinums, vinblastines, pocosides, and artemisinin compounds; the anti-metabolites are selected from pyrimidines, purines, and tadalafil; the anti-inflammatory drugs or other poorly soluble drugs are selected from halofantrine, griseofulvin, cyclosporine A, dexamethasone and their derivatives.

[0012] Specifically, the present invention provides paclitaxel prodrugs bridged by a ring structure, the prodrugs are named PTX-Cy-Se, PTX-Cy-S and PTX-Cy-C, and their structural formulas are as follows:

[0013]

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

[0015] The present invention provides a method for synthesizing 1,2-diselenopentane-4-carboxylic acid, comprising the following steps: firstly, stirring a first portion of selenium powder and sodium borohydride in an ice bath for reaction for 0.5-1.5 hours, and then reacting with a second portion of selenium powder at 60-100° C. for 0.5-2 hours to obtain an intermediate product. The intermediate product reacts with 3-bromo-2-bromomethylpropionic acid at 20-40° C. for 10-24 hours (preferably at 25° C. for 10 hours) to obtain 1,2-diselenopentane-4-carboxylic acid.

[0016] Furthermore, 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] Furthermore, the reaction solvent in the synthesis of 1,2-diselenopentane-4-carboxylic acid is ethanol.

[0018] Furthermore, the present invention provides a method for synthesizing the paclitaxel prodrug with a ring structure bridge, comprising the following steps: dissolving 1,2-diselenolane-4-carboxylic acid or 1,2-dithiolane-4-carboxylic acid or cyclopentanoic acid, 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and paclitaxel (PTX) in anhydrous dichloromethane, stirring at 15-30° C. for 10-24 h (preferably reacting at 25° C. for 12 hours), and separating and purifying the obtained product by preparative liquid phase separation, and the above reaction is carried out under N2 protection throughout the process.

[0019] Furthermore, the molar ratio of the 1,2-diselenolane-4-carboxylic acid or 1,2-dithiolane-4-carboxylic acid or cyclopentanoic acid to paclitaxel is (1-5):1, preferably 1:1.

[0020] Furthermore, the molar ratio of the 1,2-diselenolane-4-carboxylic acid or 1,2-dithiolane-4-carboxylic acid or cyclopentanoic acid to 1-ethyl-3 (3-dimethylpropylamine) carbodiimide is 1: (0.2-4).

[0021] Furthermore, the molar ratio of the 1,2-diselenolane-4-carboxylic acid or 1,2-dithiolane-4-carboxylic acid or cyclopentanoic acid to 4-dimethylaminopyridine is 1:(0.05-0.8).

[0022] The present invention also provides paclitaxel prodrug self-assembled nanoparticles bridged by annular structures, wherein the prodrug self-assembled nanoparticles are non-PEGylated prodrug self-assembled nanoparticles, PEG-modified / actively targeted modified prodrug self-assembled nanoparticles, or prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances / drugs.

[0023] The present invention also provides a method for preparing the paclitaxel prodrug self-assembled nanoparticles bridged by the ring structure, comprising the following steps:

[0024] When the self-assembled nanoparticles are non-PEGylated prodrug self-assembled nanoparticles, the preparation method is as follows: a certain amount of prodrug is dissolved in an appropriate amount of organic solvent (such as anhydrous ethanol, acetone and tetrahydrofuran, etc.), and the solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nanocolloid solution that does not contain any organic solvent, that is, the non-PEGylated prodrug self-assembled nanoparticles.

[0025] When the self-assembled nanoparticles are PEG-modified / active targeting group-modified prodrug self-assembled nanoparticles, the preparation method is as follows: a certain amount of PEG modifier / active targeting modifier and prodrug are dissolved in an appropriate amount of organic solvent, and the solution is slowly added dropwise into water under stirring, so that the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nanocolloid solution without any organic solvent, which is the PEG-modified / active targeting group-modified prodrug self-assembled nanoparticles, wherein the mass ratio of the paclitaxel prodrug bridged by a ring structure to the PEG modifier / active targeting modifier is 1:(0.1-1), the PEG modifier is an amphiphilic polymer or targeting group such as DSPE-PEG, TPGS, PLGA-PEG, PE-PEG or DSPE-PEG-FA, wherein the molecular weight of PEG is 400-5000, and the active targeting modifier is a substance that can target specific tissues such as antibodies, sugar residues, hormones, receptors or ligands.

[0026] When the self-assembled nanoparticles are prodrug self-assembled nanoparticles encapsulating 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 substances / drugs and prodrugs are dissolved in an appropriate amount of organic solvent, and the solution is slowly added dropwise into water under stirring, and the prodrugs spontaneously form uniform nanoparticles; the organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nano-colloidal solution without organic solvent, which is the prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances / drugs, wherein the mass ratio of the paclitaxel prodrug bridged by a ring structure to the PEG modifier and the hydrophobic fluorescent substance / drug is 1:(0.1-1):(0.1-1).

[0027] The present invention also provides a pharmaceutical composition comprising the paclitaxel prodrug bridged by the ring structure or a pharmaceutically acceptable excipient.

[0028] The present invention also provides the use of the paclitaxel prodrug bridged by a ring structure, the paclitaxel prodrug self-assembled nanoparticles, or the pharmaceutical composition in the preparation of anti-tumor drugs.

[0029] The present invention also provides the use of the paclitaxel prodrug bridged by the ring structure, the paclitaxel prodrug self-assembled nanoparticles or the pharmaceutical composition in the preparation of an injection, oral administration or local administration system.

[0030] The present invention also provides the use of the paclitaxel prodrug bridged by the ring structure, the paclitaxel prodrug self-assembled nanoparticles or the pharmaceutical composition in preparing a drug delivery system, especially in a drug delivery system for improving efficacy and reducing toxicity.

[0031] Beneficial effects of the present invention:

[0032] (1) The present invention designs and synthesizes paclitaxel prodrugs bridged by different cyclic structures, and the synthesis method is simple and easy; and prepares paclitaxel prodrug self-assembled nanoparticles bridged by cyclic structures with small particle size and uniform particle size distribution, and the preparation method is simple and easy.

[0033] (2) The effects of the ring structure on the pharmaceutical properties, in vivo fate and antitumor activity of prodrug self-assembled nanoparticles were investigated. The results showed that the paclitaxel prodrug self-assembled nanoparticles bridged by the ring structure can effectively improve the efficacy of paclitaxel. Different ring structures have a significant effect on the pharmaceutical properties, in vivo fate and antitumor activity of paclitaxel prodrug self-assembled nanoparticles; the colloidal stability, cytotoxicity, oxidation responsiveness and antitumor effect of paclitaxel prodrug self-assembled nanoparticles bridged by five-membered ring diselenide are the best; the colloidal stability, cytotoxicity and reduction responsiveness of paclitaxel prodrug self-assembled nanoparticles bridged by five-membered ring disulfide are better than those of paclitaxel prodrug self-assembled nanoparticles bridged by cyclopentanoic acid. Description of the drawings:

[0034] Figure 1 The five-membered ring diselenide-bridged paclitaxel prodrug (PTX-Cy-Se) of Example 1 of the present invention 1 H NMR spectra and mass spectra.

[0035] Figure 2 The five-membered ring disulfide-bridged paclitaxel prodrug (PTX-Cy-S) of Example 2 of the present invention 1 HNMR spectra and mass spectra.

[0036] Figure 3 The cyclopentanoic acid bridged paclitaxel prodrug (PTX-Cy-C) of Example 3 of the present invention 1 HNMR spectra and mass spectra.

[0037] Figure 4 This is an appearance diagram of the non-PEG-modified prodrug self-assembled nanoparticles of Example 4 of the present invention.

[0038] Figure 5 This is a transmission electron microscopy image of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0039] Figure 6 This is a graph showing the colloidal stability of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention in a phosphate buffer solution containing 10% fetal bovine serum.

[0040] Figure 7 This is a redox activation curve of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0041] Figure 8This is a graph showing the tumor growth curve of tumor-bearing mice in an in vivo anti-breast cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0042] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0043] Fig. 9 This is a graph showing the weight changes of tumor-bearing mice in an in vivo anti-breast cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0044] Fig.10 This is a graph showing the tumor weight of tumor-bearing mice in an in vivo anti-breast cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0045] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0046] Fig.11 This is a graph showing the results of hematological analysis of tumor-bearing mice in an in vivo anti-breast cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0047] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0048] Fig.12 This is a graph showing the tumor growth curve of tumor-bearing mice in an in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0049] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0050] Fig.13 This is a graph showing the weight changes of tumor-bearing mice in an in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0051] Fig.14 This is a graph showing the tumor weight of tumor-bearing mice in an in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0052] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0053] Fig.15 This is a graph showing the results of hematological analysis of tumor-bearing mice in an in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0054] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests)

[0055] Fig.16 This is a graph showing the liver and kidney function results of tumor-bearing mice in an in vivo anti-lung cancer experiment of the PEG-modified prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0056] ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (both two-sided t-tests) Specific implementation method:

[0057] The present invention is further described in detail below in conjunction with embodiments.

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

[0059] Synthesis of 1,2-diselenol-4-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, 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 dripped into the three-necked flask. After stirring in an ice bath for 0.5 hours, the second portion of selenium powder (1.97 g, 25 mmol) was added and reacted at 80 ° C for 0.5 hours. Subsequently, 3-bromo-2-bromomethylpropionic acid (4.92 g, 20 mmol) was added and stirred at 25 ° C overnight. After the reaction was completed, solid impurities were first filtered to remove the solid impurities, and the filtrate was diluted with a large amount of water and then liquid-liquid extracted with ethyl acetate to obtain the target compound. Finally, the ethyl acetate layer was dried over anhydrous magnesium sulfate, and the organic reagent was removed by rotary evaporation to obtain a dark red-brown solid.

[0060] Synthesis of prodrug: 1,2-diselenopentane-4-carboxylic acid and paclitaxel (molar ratio = 1:1) were dissolved in 20 mL of dichloromethane, followed by the addition of 2.5 equivalents of 1-ethyl-3(3-dimethylpropylamine)carbodiimide and 0.2 equivalents of 4-dimethylaminopyridine dissolved in 10 mL of dichloromethane, and the mixture was stirred at room temperature for 12 hours. The obtained product was separated and purified by preparative liquid phase separation. The above reaction was carried out under N2 protection throughout the process.

[0061] The prodrug structure was confirmed by H NMR and mass spectrometry. Figure 1As shown, the results of the spectrum analysis are as follows:

[0062] 1 H NMR(400MHz,Chloroform-d)δ8.14(d,J=7.1Hz,2H,o-2O-ArH),7.75(d,J=7.0Hz,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=9.2Hz,1H,3'-NH-),6.30(s,1H,10-H),6.25(t ,J=9.0Hz,1H,13-H),6.02(dd,J=9.1,3.5Hz,1H,3'-H),5.68(d,J=7.1Hz,1H,2-H),5.51(d,J=3.5Hz,1H,2'-H),4.97 (dd,J=9.7,2.3Hz,1H,5-H),4.44(t,J=8.6Hz,1H,7-H),4.32(d,J=8.5Hz,1H,20-HH),4.21(d,J=8.4Hz,1H,20-HH),3 .96(p,J=5.8Hz,1H,OOCCHCH2),3.81(d,J=7.0Hz,1H,3-H),3.61(dd,J=10.5,5.9Hz,1H,OOCCHCH2),3.51(dd,J=10.8 ,6.1Hz,1H,OOCCHCH2),3.37(ddd,J=15.9,10.6,5.9Hz,2H,OOCCHCH2),2.56-2.53(m,1H,6-HH),2.47(s,4H,7-OH,4O -Ac-H),2.35(dd,J=15.4,9.4Hz,1H,14-H),2.23(s,3H,10O-Ac-H),2.12(dd,J=15.4,8.8Hz,1H,14-H),1.94(s,3H,1 8-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 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 2.5 equivalents of 1-ethyl-3(3-dimethylpropylamine)carbodiimide and 0.2 equivalents of 4-dimethylaminopyridine dissolved in 10 mL of dichloromethane. The mixture was stirred at room temperature for 12 hours, and the obtained product was purified by preparative liquid separation. The above reaction was carried out under N2 protection throughout the process.

[0066] The prodrug structure was confirmed by H NMR and mass spectrometry. Figure 2 As shown, the results of the spectrum analysis are as follows:

[0067] 1 H NMR(400MHz,Chloroform-d)δ8.15(d,J=7.2Hz,2H,o-2O-ArH),7.74(d,J=7.2Hz,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 and3'NH-ArH),6.87(d,J=9.3Hz,1H,3'-NH-),6.30(s,1H,10-H),6.25(d,J=9.3Hz,1H ,13-H),6.02(dd,J=9.2,3.2Hz,1H,3'-H),5.69(d,J=7.1Hz,1H,2-H),4.98(d,J=9.4H z,1H,5-H),4.45(t,J=8.6Hz,1H,7-H),4.32(d,J=8.5Hz,1H,20-HH),4.21(d,J=8.5Hz ,1H,20-HH),3.82(d,J=7.0Hz,1H,3-H),3.52-3.48(m,J=7.7,5.0Hz,1H,OOCCHCH2),3 .41(m,J=11.7,5.0Hz,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=15.4,9.1Hz,1H,14-H),2.2 3(s,3H,10O-Ac-H),2.15(dd,J=15.4,8.9Hz,1H,14-H),1.97-1.84(m,4H,18-CH3,6-H H),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] + :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 2.5 equivalents of 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 0.2 equivalents of 4-dimethylaminopyridine dissolved in 10 mL of dichloromethane, and the mixture was stirred at room temperature for 12 hours. The obtained product was separated and purified by preparative liquid phase separation. The above reaction was carried out under N2 protection throughout the process.

[0071] The prodrug structure was confirmed by H NMR and mass spectrometry. Figure 3 As shown, the results of the spectrum analysis are as follows:

[0072] 1H NMR(400MHz,Chloroform-d)δ8.14(d,J=7.0Hz,2H,o-2O-ArH),7.73(d,J=7.0Hz,2H,o-3'NH-ArH),7.64-7.58(m,1H,p-2O-ArH),7.55-7.31(m,10H,3'-ArH and 2O-ArH and3'NH-ArH),6.87(d,J=9.1Hz,1H,3'-NH-),6.30(s,1H,10-H),6.26(t,J=9.4Hz, 1H,13-H),5.95(dd,J=9.1,3.3Hz,1H,3'-H),5.68(d,J=7.1Hz,1H,2-H),5.50(d,J= 3.3Hz,1H,2'-H),4.98(dd,J=9.9,2.3Hz,1H,5-H),4.45(dd,J=10.9,6.6Hz,1H,7-H ),4.32(d,J=8.4Hz,1H,20-HH),4.21(d,J=8.4Hz,1H,20-HH),3.82(d,J=7.0Hz,1H, 3-H),2.84(m,1H,OOCCHCH2),2.61-2.52(m,1H,6-HH),2.47(s,4H,7-OH,4O-Ac-H), 2.37(dd,J=15.3,9.3Hz,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] Weigh 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) and dissolve it in anhydrous ethanol. Under stirring, add the drug-containing ethanol solution dropwise into deionized water (the volume ratio of anhydrous ethanol to water is 1:5) to prepare 0.2 mg / mL ( Figure 4 A) and 0.6 mg / mL ( Figure 4 B) non-PEGylated nanoparticles. Figure 4 As shown in the figure, with the increase of drug concentration, both PTX-Cy-S and PTX-Cy-C prodrugs precipitated, while no drug aggregation was observed for PTX-Cy-Se prodrug, indicating that the cyclodiselenide-bridged paclitaxel prodrug has the best self-assembly ability and stability.

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

[0077] Weigh DSPE-PEG 2k 1mg and 4mg of the prodrug (PTX-Cy-Se prepared in Example 1, PTX-Cy-S prepared in Example 2, or PTX-Cy-C prepared in Example 3) were dissolved in 800uL of anhydrous ethanol. Under stirring, the ethanol solution was added dropwise to 4mL of deionized water to spontaneously form uniform nanoparticles (PTX-Cy-Se nanoparticles, PTX-Cy-S nanoparticles, and PTX-Cy-C nanoparticles, respectively). As shown in Table 1, the particle size of the nanoparticles is about 120-130nm, the particle size distribution is less than 0.2, the surface charge is about -20mV, and the drug loading is more than 60%. The particle size and morphology of the prodrug self-assembled nanoparticles were measured by transmission electron microscopy, and the results are as follows: Figure 5 As shown, the transmission electron microscopy image shows that the prodrug nanoparticles are 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 buffered saline (PBS, pH 7.4) containing 10% FBS (1:10 = v:v), incubated at 37°C for 24 hours, and the particle size change was measured by dynamic light scattering at predetermined time points. Figure 6 As shown in the figure, PTX-Cy-Se nanoparticles have the best colloidal stability and the smallest change in particle size within 24 hours. In contrast, the colloidal stability of PTX-Cy-S nanoparticles and PTX-Cy-C nanoparticles is poor, and the particle size of the nanoparticles increases significantly with the extension of incubation time.

[0082] Example 7: Redox Activation of Prodrug Self-Assembled Nanoparticles

[0083] Phosphate buffer (PBS) at pH 7.4 containing 10% ethanol and 1% sodium dodecyl sulfate was used as the release medium, and hydrogen peroxide (H2O2) and dithiothreitol (DTT) were used to simulate reactive oxygen species (ROS) and glutathione (GSH) highly expressed in tumor sites, respectively. The activation behavior of the prodrug in the presence of different concentrations of hydrogen peroxide (0.1mM, 1mM and 2mM, respectively) or dithiothreitol (1mM, 2mM and 5mM, respectively) was investigated. The prodrug nanoparticles equivalent to 67nM paclitaxel were evenly mixed with 10mL of the release medium, incubated in a constant temperature oscillator at 37°C, and samples were taken at 0, 1, 2, 4, 8, and 12 hours, and the content of the prodrug in the release medium was determined by high performance liquid chromatography. The results are shown in Figure 2. Figure 7 As shown in the figure, when hydrogen peroxide is present, the order of oxidative activation of prodrug nanoparticles is PTX-Cy-Se nanoparticles>PTX-Cy-S nanoparticles>PTX-Cy-C nanoparticles. When dithiothreitol is present, the order of reductive activation of prodrug nanoparticles is PTX-Cy-S nanoparticles>PTX-Cy-Se nanoparticles>PTX-Cy-C nanoparticles. At the same time, with the increase of H2O2 and DTT concentrations, the activation rate of prodrugs is accelerated.

[0084] Example 8: Cytotoxicity of Prodrug Self-Assembled Nanoparticles

[0085] The cytotoxicity of prodrug nanoparticles was evaluated by the MTT method. Mouse breast cancer cells (4T1), mouse melanoma cells (B16-F10), human lung cancer cells (A549) and mouse fibroblasts (3T3) were inoculated into 96-well plates at a density of 2000 cells per well and incubated overnight to allow them to adhere. The cells were then treated with a series of concentrations of Taxol (5-500nM) or the prodrug nanoparticles (5-2000nM) prepared in Example 5. After the cells were treated with the serial dilutions for 48 hours, the 96-well plates were taken out, the drug-containing solution was discarded, 200μL of 0.5mg / mL MTT solution was added to each well, and the plates were shaken after incubation in an incubator for 4 hours. The 96-well plates were turned upside down on filter paper to fully absorb the residual liquid, and DMSO was added to the oscillator for 10 minutes to dissolve the blue-purple crystals. Finally, the absorbance value was measured at 490nm or 570nm using an ELISA reader.

[0086] The results are shown in Table 2. Compared with paclitaxel solution (Taxol), the cytotoxicity of the prodrug nanoparticles is reduced. This is because the release of paclitaxel from the prodrug takes a certain amount of time. From the cytotoxicity results, it can be seen that different ring structures have a significant effect on the cytotoxicity of the prodrug nanoparticles. Compared with PTX-Cy-S nanoparticles and PTX-Cy-C nanoparticles, PTX-Cy-Se nanoparticles have the strongest cytotoxicity, which may be caused by the fact that PTX-Cy-Se nanoparticles can release more paclitaxel. The higher the drug release efficiency, the stronger the cytotoxicity.

[0087] Table 2. The half inhibitory concentration (IC) of taxol and three prodrug self-assembled nanoparticles on four types of cells 50 )

[0088]

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

[0090] Mouse breast cancer cell suspension (4T1, 5×10 6 The cells were inoculated subcutaneously on the right side of the back of female BALB / c mice. 3 At the same time, tumor-bearing mice were randomly divided into 6 groups, 5 mice in each group, namely normal saline group (Saline), taxol 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 (in terms of paclitaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5. The drug was administered once every other day for 5 consecutive times. After administration, the survival status of the mice was observed every day, and the weight changes and tumor volume were measured. On the 11th day after administration, the mice were killed, and the organs and tumor tissues were collected for further analysis and evaluation. The results are as follows Figures 8 to 11 shown. Figure 8 The results showed that the tumor volume of mice in the saline group increased rapidly, reaching 800 mm on the 11th day. 3 In contrast, the nanoparticles all inhibited tumor growth; Fig. 9 It showed that there was no significant change in the body weight of mice in each preparation group; Fig.10 It showed that after the end of drug administration, the tumor weight of mice in the prodrug self-assembled nanoparticle group was smaller, among which the tumor weight of the PTX-Cy-Se nanoparticle group was the smallest. Fig.11 The results showed that the number of blood cells (leukocytes, lymphocytes, monocytes and neutrophils) decreased in both the Taxol group and the Paclitaxel (albumin-bound) nanoparticle group, indicating that it has blood toxicity. The hematological analysis results of the mice in the prodrug nanoparticle group were normal, indicating that the prodrug nanoparticles have good in vivo safety. In summary, compared with paclitaxel solution (Taxol) and commercially available paclitaxel (albumin-bound) nanoparticles, the cyclodiselenide-bridged paclitaxel prodrug self-assembled nanoparticles have better anti-tumor effects and do not cause obvious systemic toxicity, and are a safe and efficient way to deliver chemotherapy drugs.

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

[0092] Mouse lung cancer cell suspension (Lewis lung cells, 5×10 6 Cells) were inoculated subcutaneously on the right side of the back of C57BL / 6j mice. When the tumor volume grew to 100-120mm 3 The tumor-bearing mice were randomly divided into 6 groups, 5 mice in each group, namely the normal saline group (Saline), the taxol group, the paclitaxel (albumin-bound) nanoparticle group, the PTX-Cy-Se nanoparticle group, the PTX-Cy-S nanoparticle group and the PTX-Cy-C nanoparticle group. The dosage was 10 mg / kg (in terms of paclitaxel). The nanoparticles used for administration were the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5. The drug was administered once every other day for 5 consecutive times. After administration, the survival status and weight changes of the mice were detected every day, and the tumor volume was measured. On the 10th day after administration, the mice were killed, and the mouse organs and tumor tissues were collected for further analysis and evaluation. The results are as follows Figures 12 to 16 shown. Fig.12 The results showed that the tumor volume of mice in the saline group increased rapidly, reaching 800 mm on the 10th day. 3 In contrast, the nanoparticles all inhibited tumor growth; Fig.13 It showed that there was no significant change in the body weight of mice in each preparation group; Fig.14 It showed that after the end of drug administration, the tumor weight of mice in the nanoparticle group was smaller, among which the tumor weight of the PTX-Cy-Se nanoparticle group was the smallest. Fig.15 and Fig.16 The results showed that the number of blood cells (leukocytes and lymphocytes) decreased and the level of aspartate aminotransferase (AST) increased in the Taxol group and the Paclitaxel (albumin-bound) nanoparticle group, indicating that the mice in the Taxol group and the Paclitaxel (albumin-bound) nanoparticle group had bone marrow suppression and liver damage, respectively. However, the blood indicators and liver and kidney function indicators of the mice in the prodrug nanoparticle group were normal, indicating that the prodrug nanoparticles had good in vivo safety. In summary, compared with paclitaxel solution (Taxol) and commercially available paclitaxel (albumin-bound) nanoparticles, cyclodiselenide-bridged paclitaxel prodrug self-assembled nanoparticles have better anti-tumor effects and safety.

Claims

1. Paclitaxel prodrugs bridged by a ring structure represented by 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 a structure containing a saturated or unsaturated bond.

2. The paclitaxel prodrug with a ring structure bridged by the general formula (I) according to claim 1, characterized in that: The paclitaxel is replaced by anti-tumor drugs, antimetabolites, anti-inflammatory drugs or other poorly soluble 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, and ribavirin; the anti-inflammatory drugs or other poorly soluble drugs are selected from halofantrine, griseofulvin, cyclosporine A, dexamethasone and their derivatives.

3. The paclitaxel prodrug with a cyclic structure bridged as claimed in claim 1 or 2, characterized in that: The specific structure is any of the following:

4. The method for preparing the paclitaxel prodrug with a ring structure bridge according to claim 2, characterized in that: The steps include: 1,2-diselenopentane-4-carboxylic acid or 1,2-dithiolane-4-carboxylic acid or cyclopentanoic acid, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 4-dimethylaminopyridine and paclitaxel are dissolved in anhydrous dichloromethane, stirred at 15-30° C., and the obtained product is separated and purified by preparative liquid phase. The above reaction is carried out under N2 protection throughout the process.

5. The ring-structure-bridged paclitaxel prodrug self-assembled nanoparticles according to claim 1 or 2, characterized in that: The invention comprises non-PEGylated prodrug self-assembled nanoparticles, PEG-modified / active targeting modifier-modified prodrug self-assembled nanoparticles or prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances / drugs; the PEG is TPGS, DSPE-PEG, PLGA-PEG, PE-PEG or DSPE-PEG-AA.

6. The method for preparing the ring-structure-bridged paclitaxel prodrug self-assembled nanoparticles according to claim 5, characterized in that: The steps include: When the self-assembled nanoparticles are non-PEGylated prodrug self-assembled nanoparticles, the preparation method is as follows: the prodrug is dissolved in an organic solvent, and the solution is added dropwise into water under stirring, so that the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nanocolloid solution that does not contain any organic solvent, namely, the non-PEGylated prodrug self-assembled nanoparticles. When the self-assembled nanoparticles are PEG-modified / active targeting group-modified prodrug self-assembled nanoparticles, the preparation method is as follows: dissolving a PEG modifier / active targeting modifier and a prodrug in an organic solvent, adding the solution dropwise 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 nanocolloid solution free of any organic solvent, namely, the PEG-modified / active targeting group-modified prodrug self-assembled nanoparticles, wherein the mass ratio of the prodrug to the PEG modifier / active targeting 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, a sugar residue, a hormone, a receptor or a ligand. When the self-assembled nanoparticles are prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance / drug, the preparation method is as follows: dissolving a PEG modifier, a hydrophobic fluorescent substance / drug and a prodrug in an organic solvent, dropping 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 nano-colloidal solution free of an organic solvent, namely, the prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance / drug, wherein the mass ratio of the prodrug to the PEG modifier and the hydrophobic fluorescent substance / drug is 1:(0.1-1):(0.1-1).

7. A pharmaceutical composition comprising the cyclic structure-bridged paclitaxel prodrug according to any one of claims 1 to 3 or a pharmaceutically acceptable excipient.

8. Use of the paclitaxel prodrug with a ring structure bridged as claimed in any one of claims 1 to 3, the paclitaxel prodrug self-assembled nanoparticles as claimed in claim 5, or the pharmaceutical composition as claimed in claim 7 in the preparation of a drug delivery system.

9. Use of the paclitaxel prodrug with a ring structure bridged as claimed in any one of claims 1 to 3, the paclitaxel prodrug self-assembled nanoparticles as claimed in claim 5, or the pharmaceutical composition as claimed in claim 7 in the preparation of anti-tumor drugs.

10. Use of the paclitaxel prodrug with a ring structure bridged by any one of claims 1 to 3, the paclitaxel 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.

Images