An amphiphilic acid-sensitive docetaxel polymer prodrug based on acetal bond, its preparation method and its micelle preparation

Abstract

The present invention provides an amphiphilic acid-sensitive docetaxel polymer prodrug based on acetal bond and its preparation. The prodrug molecule provided by the present invention can be prepared by modular reaction, with simple, efficient, general and highly extensible methods. The prodrug molecule can be used to prepare docetaxel prodrug micelle preparations by self-assembly in aqueous solution. The obtained prodrug micelle preparations have the characteristics of simple preparation, good micelle stability and rapid acid-responsive activation. They are relatively stable under normal physiological pH conditions, while the acetal bond can be rapidly and responsively cleaved under acidic pH conditions, so as to realize the responsive and traceless release of docetaxel, improve the hydrophilicity of docetaxel and reduce its toxic and side effects while enhancing the anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to an amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond, a preparation method thereof, and a micelle preparation thereof. Background Art

[0002] Cancer is a major health threat to humanity, and shows a trend of younger patients and an increasing number of patients year by year. The treatment of cancer has always been a difficult problem and an important direction in scientific research. A variety of treatment methods have been developed and utilized. Among them, chemotherapy is a conventional and important cancer treatment method, and a variety of chemotherapy drugs have been discovered and applied. Taxane anti-tumor drugs, especially the second-generation taxane anti-tumor drug docetaxel, have good broad-spectrum anti-tumor activity and have been approved for the treatment of cancers such as breast cancer, advanced metastatic non-small cell lung cancer, advanced ovarian cancer, nasopharyngeal cancer, and advanced gastric cancer. However, due to its high toxicity, poor water solubility, low bioavailability and other characteristics, its clinical application has been greatly restricted.

[0003] In order to address the clinical application problems of chemotherapy drugs, various prodrug technologies have been developed and utilized. The prodrug technology is a way to modify drugs by chemical means. The drug is connected to the modification group through a chemical bond, which can improve the physicochemical properties such as the hydrophilicity and hydrophobicity of the drug, thereby improving the drug utilization rate and enhancing the curative effect. The key problem faced by the prodrug in exerting its effect is how to target and enrich and activate at the diseased site to achieve drug-responsive activation and release, so as to reduce the systemic toxicity while exerting the anti-tumor effect. To solve the problem of prodrug-responsive release, a variety of response activation technologies have been developed and utilized.

[0004] The acetal bond is an acid-sensitive chemical bond, and has been reported for use in prodrug construction in recent years. The prodrug based on the acetal bond has been confirmed to have high acid sensitivity, and can achieve efficient drug enrichment at diseased sites such as tumors without damaging normal tissues. The polymer prodrug can connect the drug to the polymer through a linker, and a prodrug nano-preparation can be obtained through in vitro self-assembly. While being convenient to use, it can achieve tumor-targeted enrichment relying on the EPR effect. To solve the problems faced in the clinical application of docetaxel, the development of docetaxel prodrugs, especially polymer-based acid-responsive prodrugs, has received increasing attention. Summary of the Invention

[0005] In view of this, the present invention aims to provide an amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond, a preparation method thereof, and a micelle preparation thereof. The docetaxel polymer prodrug can prepare a docetaxel prodrug micelle preparation by self-assembly in an aqueous solution. The obtained prodrug micelle preparation has good stability, sensitive acid responsiveness, high safety and anti-tumor activity.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows:

[0007] An amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond, the structure of which is shown in formula (I):

[0008]

[0009] Wherein, n = 10 - 100, m = 20 - 60.

[0010] Furthermore, the docetaxel polymer prodrug is an amphiphilic diblock polymer, with polyethylene glycol as the hydrophilic end, and the molecular weight is preferably 1000 - 3000 Daltons; and racemic polylactic acid as the hydrophobic end, and the molecular weight is preferably 1000 - 5000 Daltons.

[0011] The present invention also provides a preparation method of the docetaxel polymer prodrug as described in any one of the above, and the method includes the following steps:

[0012] 1) Docetaxel and formula (II) react under acid catalysis in an airtight condition to obtain a docetaxel acetal prodrug molecule containing a functionalized alkynyl group connected by an acetal bond as shown in formula (III);

[0013]

[0014]

[0015] The reaction formula is as follows:

[0016]

[0017] 2) The diblock polymer shown in formula (IV) and azidohexanoic acid carry out an esterification reaction under the action of a condensing agent and an organic base catalyst to obtain a diblock polymer containing an azide group as shown in formula (V);

[0018]

[0019] The reaction formula is as follows:

[0020]

[0021] Wherein, n = 10 - 100, m = 20 - 60;

[0022] 3) The azide-containing diblock polymer shown in formula (V) and the docetaxel acetonide prodrug molecule containing a functionalized alkynyl group shown in formula (III) undergo a click reaction under the catalysis of a copper catalyst and a ligand under an air-free condition to obtain the docetaxel polymer prodrug based on an acetonide bond shown in formula (I); after docetaxel is modified with acetonide, it is connected to the hydrophobic end of the racemic polylactic acid through a click reaction;

[0023] The reaction formula is as follows:

[0024]

[0025] wherein, n = 10 - 100, m = 20 - 60.

[0026] Furthermore, the specific steps of the step 1) are as follows:

[0027] Docetaxel and the molecule shown in (II) and a reaction solvent are added into a reaction flask. Under an air-free condition, an acid catalyst is added, and a nucleophilic addition reaction of docetaxel and the molecule shown in (II) occurs under acid catalysis. After the reaction is completed, the reaction is terminated with triethylamine to obtain the docetaxel acetonide prodrug molecule shown in formula (III).

[0028] Furthermore, the reaction solvent is any one of dichloromethane, tetrahydrofuran, toluene, benzene or acetone;

[0029] The acid catalyst is any one of p-toluenesulfonic acid, acetic acid, 1,2-dichloroethane or trifluoromethanesulfonic acid;

[0030] After the reaction in the step 1) is terminated with triethylamine, a purification step is further included. The specific method is as follows: after the reaction is completed, the solvent is removed by rotary evaporation under reduced pressure, and then the product is purified by silica gel column chromatography separation. The developing agent for silica gel column chromatography separation is petroleum ether or ethyl acetate; after concentration and drying, the docetaxel acetonide prodrug molecule shown in formula (III) can be obtained.

[0031] Furthermore, in the step 2), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide;

[0032] The organic base catalyst is any one of 4-dimethylaminopyridine or triethylamine;

[0033] The solvent is selected from tetrahydrofuran or N,N-dimethylformamide;

[0034] After the reaction in the step 2) is completed, a purification step is further included. The specific method is as follows: deionized water is added to terminate the reaction, then extraction is carried out with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, concentrated and then precipitated and purified with ether. The precipitate is dried under vacuum to obtain the azide-containing diblock polymer shown in formula (V).

[0035] Further, in the step 3), the copper catalyst is any one of copper sulfate, cuprous iodide or cuprous bromide;

[0036] The ligand is pentamethyldiethylenetriamine or triethylamine;

[0037] The reaction solvent is selected from N,N-dimethylformamide or tetrahydrofuran;

[0038] After the reaction in the step 3) ends, a purification step is further included. The specific method is as follows: Add water to terminate the reaction, then extract with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and then precipitated and purified with ether. After the obtained precipitate is dehydrated under vacuum, the docetaxel polymer prodrug shown in formula (I) can be obtained.

[0039] Further, in the step 2) and step 3), n = 20 - 60 and m = 30 - 50 are preferably selected.

[0040] The present invention also provides a micelle preparation of a docetaxel polymer prodrug. The micelle is prepared by a nanoprecipitation method from the amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond in an aqueous solution. The specific method is as follows:

[0041] 1) Dissolve the docetaxel polymer prodrug in a water-soluble organic solvent;

[0042] 2) Under stirring, slowly drop the organic solution obtained in step 1) into the aqueous solution, and continue to stir with an open mouth at room temperature. After the organic solvent volatilizes completely, sterilize it through a sterile filter membrane to obtain the micelle preparation of the prodrug.

[0043] Further, the organic solvent is any one of acetone, tetrahydrofuran, methanol or ethanol;

[0044] The aqueous solution is PBS or ultrapure water;

[0045] The volatilization time of the organic solvent is 1 - 24 h;

[0046] The ratio of the prodrug:organic solvent:aqueous solution is 10 - 100 mg:1 - 10 mL:10 - 100 mL;

[0047] The particle size of the micelle preparation is 5 - 40 nm.

[0048] Among them, the outer side of the micelle is a hydrophilic shell composed of methoxypolyethylene glycol, and the inner side is a hydrophobic core of docetaxel connected by a poly(lactic acid) through an acetal bond.

[0049] Compared with the prior art, the amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond of the present invention has the following advantages:

[0050] (1) The preparation method of the docetaxel polymer prodrug described in the present invention is mature and efficient, and the docetaxel acetone polymer prodrug is constructed through modular reactions. Specifically, through an efficient method for constructing acetone bonds, a docetaxel prodrug molecule with a reactive alkyne group is prepared; through a simple esterification reaction, the polymer is modified with azide groups, and finally, the prodrug module and the polymer module are connected through a click reaction to prepare the docetaxel acetone polymer prodrug. The design of the present invention is reasonable and ingenious, with strong innovation and practicality, and a variety of docetaxel polymer prodrugs based on acetone bonds can be derived and prepared through this method.

[0051] (2) The preparation of the prodrug micelle preparation described in the present invention is simple, and the prodrug micelle preparation can be obtained by the prodrug molecules through the nanoprecipitation method. The outer side of the micelle is a hydrophilic shell layer composed of methoxypolyethylene glycol, and the polyethylene glycol hydrophilic layer can extend the circulation time of the micelle preparation in the body, facilitating the targeting and enrichment of the micelle preparation to tumors through the EPR effect. The inner side of the prodrug micelle is docetaxel connected through an acetone bond. The acetone bond is relatively stable under normal physiological pH conditions, while it can be responsive and cleaved in the acidic environment of tumors, realizing the targeted and scarless release of docetaxel, so as to improve the hydrophilicity of docetaxel, reduce the toxic and side effects, and at the same time improve the anti-tumor effect. Description of the Drawings

[0052] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0053] Figure 1 1H NMR spectrum of the docetaxel polymer prodrug (I) prepared in Example 1 of the present invention;

[0054] Figure 2 1H NMR spectrum of the docetaxel polymer prodrug (I) prepared in Example 2 of the present invention;

[0055] Figure 3 1H NMR spectrum of the docetaxel polymer prodrug (I) prepared in Example 3 of the present invention;

[0056] Figure 4 Particle size distribution diagram measured by dynamic light scattering of the prodrug micelle prepared in Example 4;

[0057] Figure 5 In vitro stability investigation diagram of the prodrug micelle prepared in Example 4;

[0058] Figure 6 pH-responsive release performance test diagram of the docetaxel prodrug micelle in vitro;

[0059] Figure 7 It is a test chart for the killing performance of docetaxel prodrug micelles against tumor cells in vitro;

[0060] Figure 8 It is a chart for the antitumor efficacy of docetaxel prodrug micelles in vivo;

[0061] Figure 9 It is a chart for the safety evaluation of the antitumor effect of docetaxel prodrug micelles in vivo. Detailed implementation manners

[0062] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0063] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0064] Embodiment 1

[0065] A preparation method of an amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond, the method comprising the following steps:

[0066] 1) Preparation of prodrug formula (III):

[0067] Under nitrogen protection, add 1.44 g of the compound shown in formula (II), 0.82 g of docetaxel, and 5 mL of ultra-dry DCM to a 15 mL reaction flask. Add 3 mg of 1,2-dichloroacetic acid catalyst under stirring and react at room temperature. After the reaction is completed, add 0.3 mL of triethylamine to the system to terminate the reaction, concentrate, and use PE (petroleum ether) / EA (ethyl acetate) as the eluent, and separate by silica gel column chromatography to obtain the target prodrug molecule shown in formula (III).

[0068] 2) Preparation of a diblock polymer formula (V) containing an azide group:

[0069] Under nitrogen protection, add 1 g of 6-azidohexanoic acid, 6.3 g of mPEG 2000 -PDLLA 1750 , 50 mL of ultra-dry N,N-dimethylformamide, 1.3 g of 4-dimethylaminopyridine, and 1.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and react at room temperature for 24 h. After the reaction is completed, add 150 mL of deionized water to the system to terminate the reaction, extract the aqueous phase with dichloromethane, combine the organic phases, wash the organic phases successively with water, saturated brine, dry over anhydrous sodium sulfate, filter and desolventize, and then precipitate with ether, and vacuum dry to obtain the compound shown in formula (V).

[0070] 3) Preparation of prodrug formula (I):

[0071] Under nitrogen protection, 900 mg of the compound shown in formula (III) prepared in Example 1, 2.3 g of the polymer shown in formula (V) prepared in Example 2, 30 mL of ultra-dry N,N-dimethylformamide, and 68 mg of pentamethyldiethylenetriamine were added to a 100 mL reaction flask. The oxygen in the solvent was removed by the freeze-thaw method, repeated three times. 86 mg of copper(I) bromide was added to the system as a catalyst, and the reaction was carried out overnight at room temperature. After the reaction was completed, 100 mL of deionized water was added to the system to quench the reaction. The aqueous phase was extracted with dichloromethane. The organic phase was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, precipitated with diethyl ether, and the precipitate was dehydrated under vacuum to obtain the prodrug shown in formula (I).

[0072] The structure of the prodrug (I) was identified and characterized by nuclear magnetic resonance technology. Its hydrogen spectrum is shown as Figure 1 shown.

[0073] From Figure 1 the 1H NMR spectrum, the characteristic peaks of the benzene ring (6.0 - 8.0 ppm) in the docetaxel molecule and the characteristic peaks of the polymer (3.0 - 3.5 ppm and 5.0 - 5.5 ppm) can be observed, indicating that the target product was successfully obtained.

[0074] Example 2

[0075] A preparation method of an amphiphilic acid-sensitive docetaxel polymer prodrug based on acetone ketal bond, the method comprising the following steps:

[0076] 1) Preparation of the prodrug of formula (Ⅲ):

[0077] Under nitrogen protection, 1.44 g of the compound shown in formula (Ⅱ), 0.82 g of docetaxel, and 5 mL of ultra-dry DCM were added to a 15 mL reaction flask. 3 mg of 1,2-dichloroacetic acid was added under stirring, and the reaction was carried out at room temperature. After the reaction was completed, 0.3 mL of triethylamine was added to the system to terminate the reaction, concentrated, and the target prodrug molecule shown in formula (Ⅲ) was separated by silica gel column chromatography using PE (petroleum ether) / EA (ethyl acetate) as the eluent.

[0078] 2) Preparation of the double-block polymer of formula (V) containing azide groups:

[0079] Under nitrogen protection, 1 g of 6-azidohexanoic acid and 8.7 g of mPEG 2000 -PDLLA 3000, 50 mL of ultra-dry N,N-dimethylformamide, 1.3 g of 4-dimethylaminopyridine, 2.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and react at room temperature for 24 h. After the reaction is completed, add 150 mL of deionized water to the system to terminate the reaction. Extract the aqueous phase with dichloromethane, combine the organic phases, and wash the organic phases successively with water, saturated brine, dry over anhydrous sodium sulfate, filter and remove the solvent, and then precipitate with ether. After vacuum drying, the compound shown in formula (V) is obtained.

[0080] 3) Preparation of prodrug of formula (I):

[0081] Under nitrogen protection, add 900 mg of the compound shown in formula (III) prepared in the example, 2.9 g of the polymer shown in formula (V) prepared in Example 3, 30 mL of ultra-dry N,N-dimethylformamide, and 68 mg of pentamethyldiethylenetriamine to a 100 mL reaction flask. Remove the oxygen in the solvent by the freeze-thaw method, repeat three times, add 86 mg of copper bromide as a catalyst to the system, and react at room temperature overnight. After the reaction is completed, add 100 mL of deionized water to the system to quench the reaction, extract the aqueous phase with dichloromethane, wash the organic phase with water, saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, concentrate, precipitate with ether, and then remove water from the precipitate under vacuum to obtain the prodrug shown in formula (I).

[0082] The structure of prodrug (I) was identified and characterized by nuclear magnetic resonance technology, and the spectrum is as Figure 2 shown.

[0083] From Figure 2 the 1H NMR spectrum, the characteristic peaks of the benzene ring (6.0 - 8.0 ppm) in the docetaxel molecule and the characteristic peaks of the polymer (3.0 - 3.5 ppm and 5.0 - 5.5 ppm) can be observed, indicating that the target product was successfully obtained.

[0084] Example 3

[0085] A preparation method of an amphiphilic acid-sensitive docetaxel polymer prodrug based on acetone ketal bond, the method comprising the following steps:

[0086] 1) Preparation of prodrug of formula (Ⅲ):

[0087] Under nitrogen protection, add 1.44 g of the compound shown in formula (Ⅱ), 0.82 g of docetaxel, and 5 mL of ultra-dry DCM to a 15 mL reaction flask. Add 3 mg of 1,2-dichloroacetic acid under stirring and react at room temperature. After the reaction is completed, add 0.3 mL of triethylamine to the system to terminate the reaction, concentrate, and use PE (petroleum ether) / EA (ethyl acetate) as the eluent to separate the target prodrug molecule shown in formula (Ⅲ) by silica gel column chromatography.

[0088] 2) Preparation of the diblock polymer of formula (V) containing an azide group:

[0089] Under nitrogen protection, 1 g of 6-azidohexanoic acid, 10.1 g of mPEG 2000 -PDLLA 4000 , 50 mL of ultra-dry N,N-dimethylformamide, 1.3 g of 4-dimethylaminopyridine, and 2.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to a 250 mL reaction flask and reacted at room temperature for 24 h. After the reaction was completed, 150 mL of deionized water was added to the system to terminate the reaction. The aqueous phase was extracted with dichloromethane, and the organic phases were combined. The organic phase was successively washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and then precipitated with ether. After vacuum drying, the compound of formula (V) was obtained.

[0090] 3) Preparation of the prodrug of formula (I):

[0091] Under nitrogen protection, 900 mg of the compound of formula (III) prepared in Example 1, 3.6 g of the polymer of formula (V) prepared in Example 2, 30 mL of ultra-dry N,N-dimethylformamide, and 68 mg of pentamethyldiethylenetriamine were added to a 100 mL reaction flask. The oxygen in the solvent was removed by the freeze-thaw method three times. 86 mg of copper(I) bromide was added to the system as a catalyst, and the reaction was carried out overnight at room temperature. After the reaction was completed, 100 mL of deionized water was added to the system to quench the reaction. The aqueous phase was extracted with dichloromethane. The organic phase was washed with water, saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, precipitated with ether, and the precipitate was vacuum dehydrated to obtain the prodrug of formula (I).

[0092] The structure of the prodrug (I) was identified and characterized by nuclear magnetic resonance technology, as Figure 3 shown.

[0093] From Figure 3 the proton nuclear magnetic resonance spectrum, the characteristic peaks of the benzene ring (6.0 - 8.0 ppm) in the docetaxel molecule and the characteristic peaks of the polymer (3.0 - 3.5 ppm and 5.0 - 5.5 ppm) could be observed, indicating that the target product was successfully obtained.

[0094] Example 4 took Example 1 as an example for the preparation of the docetaxel prodrug micelle preparation:

[0095] 20 mg of the docetaxel prodrug prepared in Example 1 was weighed and dissolved in 100 μL of acetone, and slowly added dropwise to 1 mL of a PBS buffer solution with a concentration of 10 mM while continuously stirring with an open mouth at a rotation speed of 500 rpm. Stirring was continued at this rotation speed at room temperature for 2 h. After the acetone was completely evaporated, the above PBS solution was sterilized by passing through a 0.22 μm sterile water membrane to obtain the prodrug micelle preparation.

[0096] The particle size distribution of the obtained prodrug micelles was determined by DLS (dynamic light scattering) technology, and the particle size distribution is as Figure 4 shown.

[0097] As can be seen from Figure 4 it, the particle size of the preparation is about 20 nm. Nanoparticles with small particle size are more conducive to tumor penetration and play an anti-tumor effect.

[0098] Meanwhile, in order to illustrate the in vitro stability of the preparation, the particle size of the preparation was tracked and measured continuously for 20 days, and the results are as Figure 5 shown.

[0099] As can be seen from the figure, the particle size of the preparation hardly changed within 20 days, indicating that this preparation has good in vitro stability and is conducive to use and storage.

[0100] In addition, in addition to direct use, the obtained micelle preparation can also obtain prodrug powder through freeze-drying technology, and can be redissolved according to the required concentration during use.

[0101] Experiment 1: Acid-responsive release performance test of acid-sensitive docetaxel prodrug micelles:

[0102] The docetaxel prodrug preparation prepared in Example 4 was added to phosphate buffer solutions with pH values of 5.0 and 7.4 and a concentration of 30 mM, and the final docetaxel concentration was 40 μM. The hydrolysis system was incubated in a shaker (37 °C, 100 rpm). At a predetermined time point, 200 μL of the sample was taken, 200 μL of 200 mM phosphate buffer solution with pH 8.0 and 400 μL of acetonitrile were added to terminate the hydrolysis, and after vortexing, the docetaxel content was determined by high performance liquid chromatography (HPLC).

[0103] Chromatographic column: Agilent Poroshell 120 EC-C18, 4.6 mm × 100 mm, 2.7 μm; mobile phase: acetonitrile: ultrapure water (55:45, V / V); flow rate: 1.0 mL / min; ultraviolet detection wavelength: 230 nm; column temperature: 30 °C; injection volume: 20 μL.

[0104] The hydrolysis rate calculation formula is as follows:

[0105] Hydrolysis rate (%) = concentration of docetaxel in the sample tube / concentration of docetaxel after complete acidolysis of the prodrug nano-micelle preparation × 100%.

[0106] The acid responsiveness of docetaxel micelles is as Figure 6 shown.

[0107] As can be seen from the figure, under normal physiological conditions (pH 7.4), the hydrolysis rate of the prodrug is less than 8% within 24 hours, indicating that the prodrug can remain stable for a long time under normal physiological conditions. However, in the lysosomal environment (pH 5.0), the prodrug can undergo rapid responsive hydrolysis, and more than 95% of the prodrug has been hydrolyzed after about 12 hours.

[0108] Experiment 2 Killing ability of docetaxel prodrug micelle formulation against tumor cells

[0109] Culture conditions for mouse breast cancer 4T1 cells: RPMI-1640 medium, 10% FBS, 1% P / S. Collect 4T1-luc cells, and seed 100 μL of 4T1-luc cell suspension into 96-well plates at a density of 3×10 3 / well, and incubate in an incubator for 24 hours. Then, add docetaxel and the docetaxel prodrug micelle formulation prepared in Example 4 to the wells by changing the medium, with concentrations ranging from 1 nM to 1 μM in the medium, and incubate for 72 hours. Add 100 μL of medium containing CCK-8 to the wells by changing the medium, incubate for 1.5 hours, and measure the absorbance at 450 nm. Calculate the percentage of surviving cells and plot the graph to calculate the IC50 (half-inhibitory concentration).

[0110] The cytotoxicity results of docetaxel and the prodrug micelle formulation against mouse breast cancer 4T1 cells are as Figure 7 shown. After calculation, the IC50 values of docetaxel and the docetaxel prodrug micelle formulation prepared in Example 4 against 4T1 cells are 13.0 nM and 17.9 nM, respectively.

[0111] From the above data, it can be seen that compared with docetaxel, the prodrug formulation has comparable tumor cell killing efficiency, indicating that this prodrug formulation has good tumor cell killing ability.

[0112] Experiment 3 In vivo efficacy and safety evaluation of docetaxel prodrug micelle formulation

[0113] Purchase 6-week-old, 15-17 g female Balb / c mice from Vital River Laboratories. Seed 5×10 5 cells per mouse subcutaneously into the right chest of the mice to establish a subcutaneous tumor model. When the tumor volume is approximately 50 mm 3 , the mice are randomly divided into 3 groups (n = 8), and PBS, the docetaxel micelle formulation prepared in Example 4, and docetaxel injection (commercially available docetaxel formulation) are injected via the tail vein on days 0, 3, and 6, respectively, at a dosing dose of 10 mg / kg (docetaxel equivalent dose). Tumor volume calculation formula: V tumor =(a×b 2 ) / 2. a is the longest diameter of the tumor, and b is the widest diameter of the tumor.

[0114] The tumor inhibition results and mouse survival rate of tumor-bearing mice are as Figure 8 shown below.

[0115] As can be seen from the above figure, on the 12th day after treatment, the tumor volume of the commercially available control drug group was about 1.5 times that of the docetaxel micelle group. By the 12th day, the survival rate of the docetaxel micelle group was 100%, while that of the commercially available control drug group was 75%, and that of the PBS group was 62.5%. Through the above experiments, it can be shown that the docetaxel prodrug micelles prepared by the present invention have good safety and anti-tumor activity.

[0116] Through the above examples, it can be shown that the docetaxel prodrug based on the acetal bond and its micelle preparation prepared by the present invention have the characteristics of clear structure, simple and efficient preparation method and process, excellent acid responsiveness, high safety and high anti-tumor activity, and have high innovation and application prospects.

[0117] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An amphiphilic acid-sensitive docetaxel polymer prodrug based on acetal bond, characterized in that: The structure is as shown in formula (Ⅰ): Wherein, n = 10 - 100, m = 20 - 60.

2. The amphiphilic acid-sensitive docetaxel polymer prodrug based on acetal bond according to claim 1, wherein: The docetaxel polymer prodrug is an amphiphilic diblock polymer, with polyethylene glycol as the hydrophilic end and a molecular weight of 1000 - 3000 Daltons; racemic poly(lactic acid) as the hydrophobic end and a molecular weight of 1000 - 5000 Daltons.

3. A preparation method of an amphiphilic acid-sensitive docetaxel polymer prodrug based on an acetal bond as described in any one of claims 1-2, characterized in that: This method comprises the following steps: 1) Docetaxel and formula (Ⅱ) react under acid catalysis in an air-free condition to obtain a docetaxel acetal prodrug molecule with a functionalized alkyne group connected by an acetal bond as shown in formula (Ⅲ); The reaction formula is as follows: 2) The diblock polymer shown in formula (Ⅳ) and azidohexanoic acid carry out an esterification reaction under the action of a condensing agent and an organic base catalyst to obtain a diblock polymer containing an azide group as shown in formula (Ⅴ); The reaction formula is as follows: Wherein, n = 10 - 100, m = 20 - 60; 3) The diblock polymer containing an azide group shown in formula (Ⅴ) and the docetaxel acetal prodrug molecule containing a functionalized alkyne group shown in formula (Ⅲ) carry out a click reaction under the catalysis of a copper catalyst and a ligand in an air-free condition to obtain a docetaxel polymer prodrug based on an acetal bond as shown in formula (I); after docetaxel is modified with acetal, it is connected to the hydrophobic end of racemic poly(lactic acid) through a click reaction; The reaction formula is as follows: Wherein, n = 10 - 100, m = 20 - 60.

4. The preparation method of the docetaxel polymer prodrug according to claim 3, characterized in that: The specific steps of step 1) are as follows: Add docetaxel, the molecule shown in (Ⅱ) and a reaction solvent into a reaction flask. Under an air-free condition, add an acid catalyst, and carry out a nucleophilic addition reaction between docetaxel and the molecule shown in (Ⅱ) under acid catalysis. After the reaction is completed, terminate the reaction with triethylamine to obtain a docetaxel acetal prodrug molecule as shown in formula (Ⅲ).

5. The preparation method of the docetaxel polymer prodrug according to claim 4, wherein: The reaction solvent is any one of dichloromethane, tetrahydrofuran, toluene, benzene or acetone; The acid catalyst is any one of p-toluenesulfonic acid, acetic acid, 1,2-dichloroethane or trifluoromethanesulfonic acid; After step 1) terminates the reaction with triethylamine, it further includes a purification step. The specific method is: after the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, and then purify the product by silica gel column chromatography separation. The developing agent for silica gel column chromatography separation is petroleum ether / ethyl acetate; after concentration and drying, the docetaxel acetal prodrug molecule as shown in formula (III) can be obtained.

6. The preparation method of the docetaxel polymer prodrug according to claim 3, characterized in that: In step 2), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dicyclohexylcarbodiimide; The organic base catalyst is 4-dimethylaminopyridine or triethylamine; The solvent is selected from tetrahydrofuran or N,N-dimethylformamide; After the reaction in step 2) is completed, it further includes a purification step. The specific method is: add deionized water to terminate the reaction, then extract with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, concentrated and then precipitated and purified with ether. The precipitate is dried in vacuo to obtain a diblock polymer containing an azide group as shown in formula (V).

7. The preparation method of the docetaxel polymer prodrug according to claim 3, characterized in that: In step 3), the copper catalyst is any one of copper sulfate, cuprous iodide or cuprous bromide; The ligand is pentamethyldiethylenetriamine or triethylamine; The reaction solvent is selected from N,N-dimethylformamide or tetrahydrofuran; After the reaction in step 3) is completed, a purification step is further included. The specific method is as follows: Deionized water is added thereto to terminate the reaction, and then extracted with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, concentrated, and then precipitated and purified with ether. After the obtained precipitate is dehydrated under vacuum, the docetaxel polymer prodrug shown in formula (I) can be obtained.

8. The preparation method of the docetaxel polymer prodrug according to claim 3, characterized in that: In steps 2) and 3), n = 20 - 60 and m = 30 - 50.

9. A micellar preparation of a docetaxel polymer prodrug, characterized in that: This micelle preparation is obtained by the nanoprecipitation method from the acetal bond-based amphiphilic acid-sensitive docetaxel polymer prodrug described in any one of claims 1 - 2 in an aqueous solution. The specific method is as follows: 1) Dissolve the docetaxel polymer prodrug in a water-soluble organic solvent; 2) Under stirring, slowly drop the organic solution obtained in step 1) into the aqueous solution, and continue to stir with the opening at room temperature. After the organic solvent has completely volatilized, it is sterilized through a sterile filter membrane to obtain the micelle preparation of the prodrug.

10. The micellar preparation of the docetaxel polymer prodrug according to claim 9, characterized in that: The water-soluble organic solvent is any one of acetone, tetrahydrofuran, methanol, or ethanol; The aqueous solution is PBS buffer or ultrapure water; The volatilization time of the organic solvent is 1 - 24 h; The ratio of the prodrug:organic solvent:aqueous solution is 10 - 100 mg:1 - 10 mL:10 - 100 mL; The particle size of the micelle preparation is 5 - 40 nm.

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