A docetaxel prodrug antitumor preparation

Abstract

The present invention discloses a docetaxel prodrug anti-tumor preparation, which belongs to the field of new excipients and new dosage forms for pharmaceutical preparations. A docetaxel-fatty alcohol prodrug containing different fatty alcohol side chains and different connecting chains as shown in the following general formula (I) was specifically designed and synthesized, and self-assembled nanoparticles were prepared. The results show that docetaxel-fatty alcohol prodrug self-assembled nanoparticles can effectively improve the efficacy of docetaxel and reduce its toxic side effects; the length of the branched fatty alcohol side chain, the structure of the fatty alcohol side chain (branched or straight chain), and the length of the connecting chain have a significant impact on the pharmaceutical properties, in vivo fate, anti-tumor activity and safety of the docetaxel prodrug self-assembled nanoparticles. Docetaxel-branched fatty alcohol prodrug self-assembled nanoparticles have higher anti-tumor activity and lower toxicity than docetaxel-straight chain fatty alcohol prodrug self-assembled nanoparticles.

Description

Technical field:

[0001] The present invention belongs to the field of new excipients and new dosage forms for pharmaceutical preparations, and relates to a docetaxel prodrug anti-tumor preparation, and specifically to the construction of a docetaxel-branched fatty alcohol prodrug and self-assembled nanoparticles thereof, as well as their application in a drug delivery system. Background technology:

[0002] In recent years, the incidence of malignant tumors has been increasing, posing a serious threat to human health. Chemotherapy is one of the most effective strategies for cancer treatment. Docetaxel (DTX) is a taxane anti-tumor drug with strong cytotoxicity and anti-tumor effects. However, the use of docetaxel can cause many side effects, including strong bone marrow suppression and immunosuppression. In addition, docetaxel has extremely poor solubility in water, and the commercially available docetaxel solution is Tween 80 and ethanol are essential for solubilization. However, even with the aid of solubilizers, docetaxel solutions are unstable, prone to precipitation upon dilution, and exhibit poor pharmacokinetic properties. These shortcomings limit the clinical application of docetaxel.

[0003] Prodrug strategies are an effective method for improving the delivery efficiency of chemotherapy drugs. Structural modification of docetaxel through prodrug strategies can effectively improve docetaxel's poor solubility and significant toxic side effects. Nanodrug delivery systems can effectively prolong drug circulation in the body and enhance anti-tumor effects. Therefore, self-assembled nanodrug delivery systems based on prodrug strategies combine the advantages of nanotechnology and prodrug strategies, offering advantages such as high drug loading and the absence of solubilizers, and have been widely studied in recent years.

[0004] Prodrugs typically consist of a parent drug, a linker, and a side chain. The linker connects the parent drug and the side chain. To construct prodrugs with self-assembly capabilities, existing docetaxel prodrugs mostly use linear fatty acids or fatty alcohols as side chains. Aliphatic side chains can increase the structural flexibility of prodrug molecules, balance intermolecular forces, and promote prodrug self-assembly. We hypothesize that branched fatty alcohols can effectively disrupt the tight packing of prodrug molecules, potentially further enhancing their self-assembly ability. Furthermore, the carbon chain length of the branched fatty alcohol may affect the pharmaceutical properties, in vivo fate, and antitumor efficacy of prodrug self-assembled nanoparticles. Currently, no studies have compared the effects of branched fatty alcohol carbon chain length on prodrug self-assembled nanoparticles, nor have any studies reported the effects of branched and linear fatty alcohols as side chains on prodrug self-assembled nanoparticles.

[0005] The tumor microenvironment differs significantly from that of normal tissue cells. Tumor cells produce large amounts of reactive oxygen species and glutathione, creating a redox-unbalanced tumor microenvironment. Disulfide bonds possess dual redox sensitivity, enabling intelligent response to high redox states within tumor cells and drug release. Furthermore, the length of the linker chain can influence the redox sensitivity of prodrug self-assembled nanoparticles, and thus their antitumor activity. Summary of the invention:

[0006] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a docetaxel prodrug anti-tumor preparation, specifically a docetaxel-branched fatty alcohol small molecule prodrug and its self-assembled nanoparticles, as well as its preparation and application. The present invention specifically relates to the synthesis of a disulfide-bridged docetaxel-branched fatty alcohol small molecule prodrug, the construction of self-assembled nanoparticles containing the prodrug, and the application of the prodrug in drug delivery. A disulfide-bridged docetaxel-branched fatty alcohol small molecule prodrug and the preparation of prodrug self-assembled nanoparticles have the advantages of small and uniform particle size distribution, high drug loading capacity, good stability, good anti-tumor effect and good safety.

[0007] The purpose of the present invention is to design and synthesize docetaxel-branched fatty alcohol small molecule prodrugs with disulfide bonds as connecting chains, prepare prodrug self-assembled nanoparticles, explore the effects of branched fatty alcohols and the length of the connecting chain on the prodrug self-assembled nanoparticles, and examine the effects of branched fatty alcohols and straight-chain fatty alcohols with the same carbon chain length on the pharmacokinetics and pharmacodynamics of the prodrug self-assembled nanoparticles, thereby providing new strategies and more options for the development of tumor microenvironment intelligent responsive drug delivery systems to meet the urgent demand for highly effective chemotherapy preparations in clinical practice.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Docetaxel-branched fatty alcohol small molecule prodrug or a pharmaceutically acceptable salt thereof, wherein the docetaxel-branched fatty alcohol small molecule prodrug structure is shown in the following general formula (I):

[0010]

[0011] Wherein, n=1 to 3;

[0012] R is a saturated or unsaturated C3-C 30 Hydrocarbon group, wherein R is a hydrocarbon group containing a branched structure, wherein the branched chain is C1-C 18 Alkyl, C2-C 18 Alkenyl or C2-C 18 one or more of alkynyl groups;

[0013] Preferably, R is a saturated or unsaturated C3-C 24Hydrocarbon group, wherein R is a hydrocarbon group containing a branched structure, wherein the branched chain is a straight chain C6-C 10 Alkyl, C6-C 10 Alkenyl or C6-C 10 One or more of alkynyl groups.

[0014] Preferably, R is a saturated or unsaturated C 10 -C 24 Hydrocarbon group, wherein R is a hydrocarbon group containing a branched structure, wherein the branched chain is a straight chain C6-C 10 Alkyl, C6-C 10 Alkenyl or C6-C 10 One or more of alkynyl groups.

[0015] Preferably, R is C 10 -C 24 Alkyl, wherein R is an alkyl containing a branched structure, wherein the branched chain is a straight chain C6-C 10 alkyl.

[0016] Preferably, R is a saturated or unsaturated C 16 -C 24 Hydrocarbon group, wherein R is a hydrocarbon group containing a branched structure, wherein the branched chain is a straight chain C6-C 10 alkyl.

[0017] Preferably, R is C 16 -C 24 Alkyl, wherein R is an alkyl containing a branched structure, wherein the branched chain is a straight chain C6-C 10 alkyl.

[0018] When R is an unsaturated hydrocarbon group, the number of alkenyl groups, alkynyl groups, or the sum of alkenyl groups and alkynyl groups contained in the unsaturated hydrocarbon group is 1-5.

[0019] The branched-chain fatty alcohols are 2-hexyl-octanol, 1-heptyl-octanol, 2-hexyl-decanol, 1-butyl-dodecanol, 1-heptyl-nonanol, 1-octyl-nonanol, 2-octyl-decanol, 2-heptyl-undecanol, 1-nonyl-decanol, 2-octyl-dodecanol, 2-decyl-tetradecanol or 2-dodecyl-tetradecanol.

[0020] Preferably, the branched fatty alcohol is 2-hexyl-decanol, 2-heptyl-undecanol, 2-octyl-dodecanol, or 2-decyl-tetradecanol.

[0021] In the docetaxel-branched fatty alcohol small molecule prodrug, docetaxel and the branched fatty alcohol are connected via a dibasic acid as a connecting chain, and the dibasic acid is 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid.

[0022] The docetaxel-branched fatty alcohol small molecule prodrug uses 2,2'-dithiodiacetic acid or 4,4'-dithiodibutyric acid as a connecting chain, specifically:

[0023] (1) When 2,2'-dithiodiacetic acid is used as the connecting chain, the branched fatty alcohol small molecule prodrug is docetaxel-2-hexyl-decanol prodrug, docetaxel-2-heptyl-undecanol prodrug, docetaxel-2-octyl-dodecanol prodrug or docetaxel-2-decyl-tetradecanol prodrug; the corresponding prodrugs are named DTX-SS-HD, DTX-SS-HUA, DTX-SS-OD, and DTX-SS-DTD, respectively; the structural formulas are shown in II, III, IV and V below;

[0024] (2) When 4,4'-dithiodibutyric acid is used as the connecting chain, the branched fatty alcohol small molecule prodrug is docetaxel-2-octyl-dodecanol prodrug, and the corresponding prodrug is named γ-DTX-SS-OD, with the structural formula shown in Figure VI below.

[0025] The present invention further provides two docetaxel-straight-chain fatty alcohol small molecule prodrugs, specifically docetaxel-stearyl alcohol prodrugs, with thiodiacetic acid or 2,2'-dithiodiacetic acid as the connecting chain. The corresponding prodrugs are named DTX-S-SA and DTX-SS-SA, respectively; their structural formulas are shown in VII and VIII below.

[0026]

[0027] (II) docetaxel-2-hexyl-decanol prodrug (DTX-SS-HD) with 2,2'-dithiodiacetic acid as the linker;

[0028]

[0029] (III) docetaxel-2-heptyl-undecanol prodrug (DTX-SS-HUA) with 2,2'-dithiodiacetic acid as the linker;

[0030]

[0031] (IV) docetaxel-2-octyl-dodecanol prodrug (DTX-SS-OD) with 2,2'-dithiodiacetic acid as the linker;

[0032]

[0033] (V) Docetaxel-2-decyl-tetradecanol prodrug (DTX-SS-DTD) with 2,2'-dithiodiacetic acid as the linker.

[0034]

[0035] (VII) docetaxel-stearyl alcohol prodrug (DTX-S-SA) with thiodiacetic acid as the linker;

[0036]

[0037] (VIII) docetaxel-stearyl alcohol prodrug (DTX-SS-SA) with 2,2'-dithiodiacetic acid as the linker;

[0038]

[0039] (VI) docetaxel-2-octyl-dodecanol prodrug (γ-DTX-SS-OD) with 4,4'-dithiodibutyric acid as the linker;

[0040] A method for synthesizing docetaxel-branched fatty alcohol small molecule prodrug comprises the following steps:

[0041] Step 1: converting a dibasic acid into an anhydride and then esterifying it with a branched fatty alcohol to obtain an intermediate product, a branched fatty alcohol-dibasic acid monoester, wherein the molar ratio of the branched fatty alcohol to the dibasic acid anhydride is 1:(1.5-5);

[0042] Step 2: Branched fatty alcohol-diacid unilateral ester reacts with docetaxel to form an ester, thereby obtaining the final product, docetaxel-branched fatty alcohol small molecule prodrug, wherein the molar ratio of branched fatty alcohol-diacid unilateral ester to docetaxel is 1:(0.5-10), and the reaction equation is as follows:

[0043]

[0044] Wherein, n=1 to 3;

[0045] R is a saturated or unsaturated C3-C 30 Hydrocarbon group, wherein R is a hydrocarbon group containing a branched structure, wherein the branched chain is C1-C 18 Alkyl, C2-C 18 Alkenyl or C2-C 18 One or more of alkynyl groups.

[0046] The synthesis method of docetaxel-branched fatty alcohol small molecule prodrug, the specific process is:

[0047] (1) dissolving a dibasic acid in acetic anhydride and stirring for 2-4 hours to convert the dibasic acid into anhydride; after the reaction is complete, adding toluene, and removing the toluene and acetic anhydride by rotary evaporation to obtain the dibasic acid anhydride;

[0048] (2) taking a branched fatty alcohol and 4-dimethylaminopyridine (DMAP), dissolving them together with the dibasic acid anhydride obtained in step (1) in dichloromethane, stirring at room temperature for 12-18 hours, and separating by column chromatography to obtain an intermediate product, a branched fatty alcohol-dibasic acid monoester;

[0049] (3) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP) were dissolved in anhydrous dichloromethane together with a branched fatty alcohol-diacid monoester. After stirring in an ice bath for 2-4 hours, docetaxel was added. The mixture was stirred at room temperature for 24-48 hours, and then purified by preparative liquid phase separation to obtain the final product. The above reaction was carried out under nitrogen protection throughout the process.

[0050] In the step (1), the dibasic acid is 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, or 4,4'-dithiodibutyric acid.

[0051] In the step (2), the branched fatty alcohol is C3-C 30 Saturated or unsaturated fatty alcohol, the side chain is C1-C 18 Alkyl, C2-C 18 Alkenyl or C2-C 18 One or more of alkynyl groups.

[0052] In the step (1), the ratio of dibasic acid to acetic anhydride is 1:(1-10), and the unit is mmol:ml.

[0053] In the step (1), preferably, the ratio of dibasic acid to acetic anhydride is 1:(1-2).

[0054] In the step (2), the molar ratio of 4-dimethylaminopyridine (DMAP): branched fatty alcohol: dibasic acid anhydride is 1: (1-10): (5-15).

[0055] In the step (2), preferably, the molar ratio of 4-dimethylaminopyridine (DMAP):branched fatty alcohol:dibasic acid anhydride is 1:(2-5):(10-15).

[0056] In the step (3), the molar ratio of the intermediate product branched fatty alcohol-diacid monoester: 1-hydroxybenzotriazole (HOBt): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI): 4-dimethylaminopyridine (DMAP): docetaxel is 1: (1-10): (2-6): (0.2-5): (0.5-10).

[0057] In the step (3), preferably, in molar ratio, the intermediate product branched fatty alcohol-diacid monoester: 1-hydroxybenzotriazole (HOBt): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI): 4-dimethylaminopyridine (DMAP): docetaxel = 1: (1-2): (2-4): (0.5-2): (0.8-2).

[0058] The self-assembled nanoparticles of docetaxel-branched fatty alcohol small molecule prodrug are non-PEGylated small molecule prodrug self-assembled nanoparticles, PEG-modified / active targeting group-modified small molecule prodrug self-assembled nanoparticles, or small molecule prodrug self-assembled nanoparticles loaded with fluorescent substances / hydrophobic drugs.

[0059] The preparation method of the docetaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticles comprises the following steps:

[0060] When the small molecule prodrug self-assembled nanoparticles are non-PEGylated, the preparation steps are as follows:

[0061] (1) A certain amount of docetaxel-branched fatty alcohol small molecule prodrug is dissolved in an appropriate amount of ethanol, tetrahydrofuran or acetone, and the solution is slowly added dropwise to water under stirring, and the docetaxel-branched fatty alcohol small molecule prodrug spontaneously forms uniform nanoparticles;

[0062] (2) The organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nanocolloid solution without any organic solvent, namely, non-PEGylated small molecule prodrug self-assembled nanoparticles.

[0063] When the small molecule prodrug self-assembled nanoparticles are modified with PEG or active targeting groups, the preparation method is as follows:

[0064] (1) A certain amount of a modifier and docetaxel-branched fatty alcohol small molecule prodrug are dissolved in an appropriate amount of ethanol, tetrahydrofuran or acetone, and the solution is slowly added dropwise to water under stirring conditions, and the docetaxel-branched fatty alcohol small molecule prodrug spontaneously forms uniform nanoparticles, wherein the modifier is a PEG modifier or an active targeting modifier, the PEG modifier is an amphiphilic polymer or targeting group such as 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; in terms of mass ratio, docetaxel-branched fatty alcohol prodrug:modifier=(20-1):1;

[0065] (2) 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 a PEG-modified / active targeting group-modified small molecule prodrug self-assembled nanoparticle.

[0066] When the small molecule prodrug self-assembled nanoparticles encapsulate hydrophobic fluorescent substances or drugs, the preparation method is as follows:

[0067] (1) A certain amount of PEG modifier, hydrophobic fluorescent substance or drug and docetaxel-branched fatty alcohol small molecule prodrug are dissolved in an appropriate amount of ethanol, tetrahydrofuran or acetone, and the solution is slowly added dropwise to water under stirring, and the docetaxel-branched fatty alcohol small molecule prodrug spontaneously forms uniform nanoparticles; the mass ratio of docetaxel-branched fatty alcohol small molecule prodrug: PEG modifier: hydrophobic fluorescent substance or drug = (20-1):1:(1-5);

[0068] (2) The organic solvent in the preparation is removed by a reduced pressure rotary evaporation method to obtain a nanocolloid solution free of organic solvent, which is a small molecule prodrug self-assembled nanoparticle encapsulating a hydrophobic fluorescent substance or a drug.

[0069] Application of the docetaxel-branched fatty alcohol small molecule prodrug or self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0070] The docetaxel-branched fatty alcohol small molecule prodrug or self-assembled nanoparticles are used in preparing injection, oral or local drug delivery systems.

[0071] The docetaxel-branched fatty alcohol small molecule prodrug or self-assembled nanoparticles are used in preparing a drug delivery system with improved efficacy and reduced toxicity.

[0072] Beneficial effects of the present invention:

[0073] (1) We designed and synthesized docetaxel-branched fatty alcohol small molecule prodrugs containing different fatty alcohol side chains and different connecting chain modifications, and prepared docetaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticles with small particle size and uniform particle size distribution. The synthesis and preparation methods are simple and easy.

[0074] (2) The effects of four lengths of branched fatty alcohol side chains, the structure of fatty alcohol side chains (branched or linear), and connecting chains of different lengths on the pharmaceutical properties, antitumor activity, and safety of prodrug self-assembled nanoparticles were investigated.

[0075] Results indicate that docetaxel-fatty alcohol small molecule prodrug self-assembled nanoparticles can effectively enhance the efficacy of docetaxel and reduce its toxic side effects. Side chains and linkers of varying structures and lengths significantly influence the pharmaceutical properties, in vivo fate, antitumor activity, and safety of the docetaxel prodrug self-assembled nanoparticles. 2-Octyl-dodecanol side chains exhibited the best safety profile. Docetaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticles exhibited superior antitumor efficacy and safety compared to docetaxel-linear fatty alcohol small molecule prodrug self-assembled nanoparticles containing the same carbon number. This invention provides new strategies and options for developing highly effective and low-toxic chemotherapy formulations. Description of the drawings:

[0076] Figure 1 This is a blood drug concentration-time curve of the PEG-modified prodrug self-assembled nanoparticles of Example 8 of the present invention (prodrug);

[0077] Figure 2 This is a blood drug concentration-time curve of the PEG-modified prodrug self-assembled nanoparticles of Example 8 of the present invention (mother drug);

[0078] Figure 3 This is a blood drug concentration-time curve (sum) of the PEG-modified prodrug self-assembled nanoparticles of Example 8 of the present invention;

[0079] Figure 4 This is a diagram showing changes in tumor volume during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 9 of the present invention;

[0080] ns: no significant difference (two-tailed t-test) **: P < 0.01 (two-tailed t-test) ***: P < 0.001 (two-tailed t-test)

[0081] Figure 5 This is a graph showing changes in mouse body weight during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 9 of the present invention;

[0082] ****: P < 0.0001 (two-tailed t-test)

[0083] Figure 6 This is a diagram of tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 9 of the present invention;

[0084] ns: no significant difference (two-tailed t-test) **: P < 0.01 (two-tailed t-test)

[0085] ***: P < 0.001 (two-tailed t-test) ****: P < 0.0001 (two-tailed t-test)

[0086] Figure 7This is a graph showing changes in tumor volume during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 10 of the present invention;

[0087] *: P < 0.05 (two-tailed t-test) ***: P < 0.001 (two-tailed t-test)

[0088] Figure 8 This is a graph showing changes in mouse body weight during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 10 of the present invention;

[0089] ***: P < 0.001 (two-tailed t-test)

[0090] Figure 9 This is a graph showing the tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 10 of the present invention;

[0091] ns: no significant difference (two-tailed t-test) ***: P < 0.001 (two-tailed t-test)

[0092] Figure 10 This is a graph showing routine blood indicators in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 10 of the present invention;

[0093] ns: no significant difference (two-tailed t-test) *: P < 0.05 (two-tailed t-test)

[0094] **: P < 0.01 (two-tailed t-test)

[0095] Figure 11 This is a graph showing the alanine aminotransferase index in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 10 of the present invention;

[0096] ns: no significant difference (two-tailed t test)

[0097] Figure 12 This is a graph showing the aspartate aminotransferase index in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 10 of the present invention;

[0098] ns: no significant difference (two-tailed t-test) *: P < 0.05 (two-tailed t-test)

[0099] **: P < 0.01 (two-tailed t-test)

[0100] Figure 13 This is a graph showing changes in tumor volume during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 11 of the present invention;

[0101] ns: no significant difference (two-tailed t-test) *: P < 0.05 (two-tailed t-test)

[0102] Figure 14 This is a graph showing changes in mouse body weight during an in vivo anti-tumor experiment using PEG-modified small molecule prodrug self-assembled nanoparticles according to Example 11 of the present invention;

[0103] ***: P < 0.001 (two-tailed t-test)

[0104] Figure 15 This is a graph showing the tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 11 of the present invention;

[0105] ns: no significant difference (two-tailed t-test) **: P < 0.01 (two-tailed t-test)

[0106] ***: P < 0.001 (two-tailed t-test)

[0107] Figure 16 This is a graph showing changes in mouse body weight during a long-term toxicity experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 13 of the present invention;

[0108] ****: P < 0.0001 (two-tailed t-test)

[0109] Figure 17 This is a mouse survival curve in a long-term toxicity experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 13 of the present invention. Specific implementation method:

[0110] The present invention will be further described in detail below with reference to the embodiments.

[0111] Example 1: Synthesis of docetaxel-2-hexyl-decanol prodrug (DTX-SS-HD) with 2,2'-dithiodiacetic acid as the linker

[0112] An appropriate amount of 2,2'-dithiodiacetic acid (2 mmol) was dissolved in 4 mL of acetic anhydride and placed in a 25 mL eggplant-shaped flask. After complete dissolution, the mixture was magnetically stirred at 25°C for 2 hours and then transferred to a 100 mL eggplant-shaped flask. Three times the amount of toluene was added. The toluene and acetic anhydride were removed by distillation under reduced pressure. An appropriate amount of dichloromethane was added to dissolve the formed dithiodiacetic anhydride. Then, 2-hexyl-decanol (1 mmol) dissolved in dichloromethane was added, and a solution of 4-dimethylaminopyridine (DMAP, 0.2 mmol) dissolved in dichloromethane was slowly added dropwise. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate product. The product, 2-hexyl-decanol-dithiodiacetic acid monoester, was isolated and purified by column chromatography using a cyclohexane-acetone elution system. After obtaining the purified product from the previous step (1 mmol), a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2 mmol), 1-hydroxybenzotriazole (HOBt, 1 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) was added. The mixture was activated in an ice bath at 0°C for 2 h. Docetaxel (0.8 mmol) dissolved in dichloromethane was then added, and the mixture was stirred at 25°C for 48 h. After the reaction, the product was separated using preparative phase chromatography to obtain docetaxel-2-hexyl-decanol prodrug (DTX-SS-HD) with 2,2'-dithiodiacetic acid as the linker.

[0113] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy. The spectral analysis results are as follows:

[0114] 1 H NMR (600MHz): δ8.12(d,2H,Ar-H),7.62(t,1H,Ar-H),7.51(t,2H,Ar-H),7.40(m,4H,Ar-H),7.35(m,1H,A r-H),5.68(d,1H,2-H),5.37(d,1H,2'-H),4.95~4.95(d,1H,5-H),4.33(dd,1H,7-H),4.05~4.20(m,2H,C H 2OCO),3.63(d,1H,3-H),3.54~3.61(m,4H,C H 2SSC H 2),2.59(m,2H,14-H),2.44(s,3H,COC H 3),1.83(s,1H,(CH2)6C H (CH2)8),1.75(s,3H,18-CH3),1.58(s,3H,19-CH3),1.47(s,9H,C(C H 3)3),1.23~1.33(m,28H,CH3(C H2)6CH,CH3(C H 2)8CH),1.12(s,6H,16-CH3,17-CH3),0.88(t,6H,C H 3-(CH2)8-,C H 3-(CH2)6-).

[0115] MS (ESI) m / z for C 63 H 88 NO 17 S2[MH] - =1193.7553.

[0116] Example 2: Synthesis of docetaxel-2-heptyl-undecanol prodrug (DTX-SS-HUA) with 2,2'-dithiodiacetic acid as the linker

[0117] An appropriate amount of 2,2'-dithiodiacetic acid (2 mmol) was dissolved in 4 mL of acetic anhydride and placed in a 25 mL eggplant flask. After complete dissolution, the mixture was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL eggplant flask. Three times the amount of toluene was added. The toluene and acetic anhydride were removed by distillation under reduced pressure. An appropriate amount of dichloromethane was added to dissolve the formed dithiodiacetic anhydride. Then, 2-heptyl-undecanol (1 mmol) dissolved in dichloromethane was added, and 4-dimethylaminopyridine (DMA) dissolved in dichloromethane was slowly added dropwise. A solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2 mmol), 1-hydroxybenzotriazole (HOBt, 1 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) in dichloromethane was added to the purified product (1 mmol) and activated in an ice bath at 0°C for 2 h. Docetaxel (0.8 mmol) dissolved in dichloromethane was then added and stirred at 25°C for 48 h. After the reaction, the product was separated using preparative phase chromatography to obtain docetaxel-2-heptyl-undecanol prodrug (DTX-SS-HUA) with 2,2'-dithiodiacetic acid as the linker.

[0118] The structure of the prodrug in Example 2 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy. The spectral analysis results are as follows:

[0119] 1H NMR (600MHz): δ8.11(d,2H,Ar-H),7.62(t,1H,Ar-H),7.53(t,2H,Ar-H),7.51(m,4H,Ar-H),7.35(m,1 H,Ar-H),5.70(d,1H,2-H),5.21(d,1H,2'-H),4.97(d,1H,5-H),4.33(dd,1H,7-H),4.05~4.20(m,2H,C H 2OCO),3.63(d,1H,3-H),3.54~3.61(m,4H,C H 2SSC H 2),2.59(m,2H,14-H),2.44(s,3H,COC H 3),1.83(s,1H,(CH2)5C H (CH2)7),1.75(s,3H,18-CH3),1.58(s,3H,19-CH3),1.47(s,9H,C(C H 3)3),1.23~1.33(m,24H,CH3(C H 2)5CH,CH3(C H 2)7CH),1.12(s,6H,16-CH3,17-CH3),0.87(t,6H,C H 3-(CH2)5-,C H 3-(CH2)7-).

[0120] MS (ESI) m / z for C 65 H 93 NO 17 S2Cl[M+Cl] - =1258.5546.

[0121] Example 3: Synthesis of docetaxel-2-octyl-dodecanol prodrug (DTX-SS-OD) with 2,2'-dithiodiacetic acid as the linker

[0122] An appropriate amount of 2,2'-dithiodiacetic acid (2 mmol) was dissolved in 4 ml of acetic anhydride and placed in a 25 mL eggplant flask. After magnetic stirring at 25 ° C for 2 hours, it was transferred to a 100 mL eggplant flask, and three times the amount of toluene was added. The toluene and acetic anhydride were removed by distillation under reduced pressure. An appropriate amount of dichloromethane was added to dissolve the formed dithiodiacetic anhydride. Then, 2-octyl-dodecanol (1 mmol) dissolved in dichloromethane was added, and a dichloromethane solution of 4-dimethylaminopyridine (DMAP, 0.2 mmol) was slowly added dropwise. The mixture was magnetically stirred at 25 ° C for 12 hours to obtain the intermediate product 2- Octyl-dodecanol-dithiodiacetic acid monoester was isolated and purified by column chromatography using a cyclohexane-acetone elution system. The purified product from the previous step (1 mmol) was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2 mmol), 1-hydroxybenzotriazole (HOBt, 1 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol). The mixture was activated in an ice bath at 0°C for 2 h. Docetaxel (0.8 mmol) dissolved in dichloromethane was then added, and the mixture was stirred at 25°C for 48 h. After the reaction, the product was separated using preparative phase chromatography to obtain docetaxel-2-octyl-dodecanol prodrug (DTX-SS-OD) with 2,2'-dithiodiacetic acid as the linker.

[0123] The structure of the prodrug in Example 3 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy. The spectral analysis results are as follows:

[0124] 1 H NMR (600MHz): δ8.12(d,2H,Ar-H),7.62(t,1H,Ar-H),7.52(t,2H,Ar-H),7.39(m,4H,Ar-H),7.35(m,1 H,Ar-H),5.70(d,1H,2-H),5.21(d,1H,2'-H),4.97(d,1H,5-H),4.33(dd,1H,7-H),4.05~4.20(m,2H,C H 2OCO),3.63(d,1H,3-H),3.54~3.61(m,4H,C H 2SSC H 2),2.59(m,2H,14-H),2.44(s,3H,COC H 3),1.83(s,1H,(CH2)7C H (CH2)9),1.73(s,3H,18-CH3),1.66(s,3H,19-CH3),1.58(s,9H,-C(C H 3)3),1.23~1.33(m,32H,CH3(C H2)7CH,CH3(C H 2)9CH),1.12(s,6H,16-CH3,17-CH3),0.88(t,6H,C H 3-(CH2)7-,C H 3-(CH2)9-).

[0125] MS (ESI) m / z for C 67 H 97 NO 17 S2K[M+K] + =1290.4748.

[0126] Example 4: Synthesis of docetaxel-2-decyl-tetradecanol prodrug (DTX-SS-DTD) with 2,2'-dithiodiacetic acid as the linker

[0127] An appropriate amount of 2,2'-dithiodiacetic acid (2 mmol) was dissolved in 4 mL of acetic anhydride and placed in a 25 mL eggplant-shaped flask. After complete dissolution, the mixture was magnetically stirred at 25°C for 2 hours and then transferred to a 100 mL eggplant-shaped flask. Three times the amount of toluene was added. The toluene and acetic anhydride were removed by distillation under reduced pressure. An appropriate amount of dichloromethane was added to dissolve the formed dithiodiacetic anhydride. Then, 2-decyl-tetradecanol (1 mmol) dissolved in dichloromethane was added, and a solution of 4-dimethylaminopyridine (DMAP, 0.2 mmol) dissolved in dichloromethane was slowly added dropwise. The mixture was magnetically stirred at 25°C for 12 hours. The intermediate product, 2-decyl-tetradecanol-dithiodiacetic acid monoester, was isolated and purified by column chromatography using a cyclohexane-acetone elution system. A dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2 mmol), 1-hydroxybenzotriazole (HOBt, 1 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) was added to the purified product (1 mmol). The mixture was activated in an ice bath at 0°C for 2 hours. Docetaxel dissolved in dichloromethane was then added and stirred at 25°C for 48 hours. After the reaction, the product was separated using preparative phase chromatography to obtain docetaxel-2-decyl-tetradecanol prodrug (DTX-SS-DTD) with 2,2'-dithiodiacetic acid as the linker.

[0128] The structure of the prodrug in Example 4 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy. The spectral analysis results are as follows:

[0129] 1H NMR (600MHz): δ8.12(d,2H,Ar-H),7.62(t,1H,Ar-H),7.52(t,2H,Ar-H),7.39(m,4H,Ar-H),7.35(m,1 H,Ar-H),5.77(d,1H,2-H),5.21(d,1H,2'-H),4.97(d,1H,5-H),4.33(dd,1H,7-H),4.05~4.20(m,2H,C H 2OCO),3.63(d,1H,3-H),3.54~3.61(m,4H,C H 2SSC H 2),2.59(m,2H,14-H),2.44(s,3H,COC H 3),1.83(s,1H,(CH2)9C H (CH2) 11 ),1.73(s,3H,18-CH3),1.66(s,3H,19-CH3),1.58(s,9H,-C(C H 3)3),1.23~1.33(m,40H,CH3(C H 2)9CH,CH3(C H 2) 11 CH),1.12(s,6H,16-CH3,17-CH3),0.87(t,6H,C H 3-(CH2)9-,C H 3-(CH2) 11 -).

[0130] MS (ESI) m / z for C 71 H 106 NO 17 S2[M+H] + =1307.8946.

[0131] Example 5: Synthesis of docetaxel-2-octyl-dodecanol prodrug (γ-DTX-SS-OD) with 4,4'-dithiodibutyric acid as the linker

[0132] A suitable amount of 4,4'-dithiodibutyric acid (2mmol) was dissolved in 4mL of acetic anhydride and placed in a 25mL eggplant-shaped flask. After complete dissolution, the mixture was magnetically stirred at 25℃ for 2 hours and then transferred to a 100mL eggplant-shaped flask. Three times the amount of toluene was added. The toluene and acetic anhydride were removed by vacuum distillation. A suitable amount of dichloromethane was added to dissolve the formed dithiodibutyric anhydride. Then, 2-octyl-dodecanol (1mmol) dissolved in dichloromethane was added. A solution of 4-dimethylaminopyridine (DMAP, 0.2mmol) dissolved in dichloromethane was slowly added dropwise. The mixture was magnetically stirred at 25℃ for 12 hours to obtain the intermediate. The product, 2-octyl-dodecanol-dithiodibutyric acid monoester, was isolated and purified by column chromatography using a cyclohexane-acetone elution system. The purified product from the previous step (1 mmol) was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2 mmol), 1-hydroxybenzotriazole (HOBt, 1 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol). The mixture was activated in an ice bath at 0°C for 2 hours. Docetaxel (0.8 mmol) dissolved in dichloromethane was then added, and the mixture was stirred at 25°C for 48 hours. After the reaction, the product was separated using preparative phase chromatography to obtain docetaxel-2-octyl-dodecanol prodrug (γ-DTX-SS-OD) with 4,4'-dithiodibutyric acid as the linker.

[0133] The structure of the prodrug in Example 5 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy. The spectral analysis results are as follows:

[0134] 1 H NMR (600MHz): δ8.12(d,2H,Ar-H),7.62(t,1H,Ar-H),7.52(t,2H,Ar-H),7.39(m,4H,Ar-H),7.35(m,1 H,Ar-H),5.70(d,1H,2-H),5.21(d,1H,2'-H),4.97(d,1H,5-H),4.33(dd,1H,7-H),4.05~4.20(m,2H,C H 2OCO),3.63(d,1H,3-H),3.54~3.61(m,4H,C H 2SSC H 2),2.59(m,2H,14-H),2.451~2.518(m,4H,C H2 CH2CH2SSCH2CH2C H 2),2.44(s,3H,COC H 3),1.925(m,4H,CH2C H 2CH2SSCH2C H 2CH2), 1.83(s,1H,(CH2)7CH (CH2)9),1.73(s,3H,18-CH3),1.66(s,3H,19-CH3),1.58(s,9H,-C(C H 3)3),1.23~1.33(m,32H,CH3(C H 2)7CH,CH3(C H 2)9CH),1.12(s,6H,16-CH3,17-CH3),0.88(t,6H,C H 3-(CH2)7-,C H 3-(CH2)9-).

[0135] MS (ESI) m / z for C 71 H 106 NO 17 S2[M+Na] + =1331.5622.

[0136] Example 6: DSPE-PEG 2k Preparation of modified docetaxel-fatty alcohol small molecule prodrug self-assembled nanoparticles

[0137] Accurately weigh DSPE-PEG 2k 2 mg of docetaxel-branched fatty alcohol small molecule prodrugs prepared in Examples 1-5 and 8 mg of two docetaxel-linear fatty alcohol small molecule prodrugs prepared using existing technology were dissolved in 1 mL of ethanol. The ethanol solution was slowly added dropwise to 4 mL of deionized water under stirring to spontaneously form uniform nanoparticles. The ethanol was removed by vacuum distillation to obtain an ethanol-free nanoparticle preparation, namely DSPE-PEG. 2k Modified docetaxel-fatty alcohol small molecule prodrug self-assembled nanoparticles: specifically DTX-SS-HD nanoparticles, DTX-SS-HUA nanoparticles, DTX-SS-OD nanoparticles, DTX-SS-DTD nanoparticles, γ-DTX-SS-OD nanoparticles, DTX-S-SA nanoparticles, and DTX-SS-SA nanoparticles.

[0138] As shown in Table 1, the particle size of the prodrug nanoparticles is about 75 nm, the particle size distribution PDI is <0.2, the surface charge is about -20 mV, and the drug loading is about 50%.

[0139] Table 1. Particle size, size distribution, surface charge, and drug loading of PEG-modified prodrug self-assembled nanoparticles

[0140]

[0141] Example 7: Cytotoxicity of PEG-modified small molecule prodrug self-assembled nanoparticles

[0142] The MTT assay was used to investigate the cytotoxicity of PEG-modified small molecule prodrug self-assembled nanoparticles against mouse breast cancer (4T1) cells, human alveolar adenocarcinoma basal epithelial cells (A549), and human liver (L02) cells. Healthy cells were digested and diluted with culture medium to a cell density of 1000 cells / mL. After agitation, 200 μL of the cell suspension was added to each well of a 96-well plate and incubated in an incubator for 24 hours to allow adherence. After cell adherence, docetaxel and the DTX-S-SA, DTX-SS-SA, and DTX-SS-HUA prodrug nanoparticles prepared in Example 6 were added.

[0143] For experiments with mouse breast cancer (4T1) cells and human liver (L02) cells, drug solutions and nanoparticle formulations were prepared and diluted in 1640 culture medium. For experiments with human alveolar adenocarcinoma basal epithelial cells (A549), drug solutions and nanoparticle formulations were prepared and diluted in DMEM culture medium and sterile-filtered through a 0.22 μm filter. 200 μL of the test solution was added to each well, with three replicates per concentration.

[0144] In the control group, no test solution was added, and 200 μL of culture medium was added alone, and the cells were incubated in an incubator. 48 hours after drug addition, the 96-well plate was removed, and 35 μL of 5 mg / mL MTT solution was added to each well. The plate was incubated in an incubator for 4 hours, then shaken. The 96-well plate was inverted on filter paper to fully absorb the residual liquid. 200 μL of DMSO was added to each well and shaken on a shaker for 10 minutes to dissolve the blue-purple crystals. Well A1 (containing only 200 μL DMSO) was set as the zero well. The absorbance value of each well after zeroing was measured at 490 nm using a microplate reader.

[0145] Because the release of docetaxel from prodrug nanoparticles takes time, the efficacy of docetaxel is limited. Therefore, the cytotoxicity of prodrug nanoparticles is lower than that of the docetaxel group. The order of cytotoxicity of prodrug nanoparticles is DTX-SS-SA nanoparticles > DTX-SS-HUA nanoparticles > DTX-S-SA nanoparticles. The selectivity of docetaxel solution and prodrug nanoparticles for normal and tumor cells was investigated. As shown in Table 2, the toxicity of prodrug nanoparticles towards L02 cells was significantly reduced compared to the docetaxel solution. A selectivity index (SI) greater than 1 indicates that the drug is more toxic to tumor cells than to normal cells. A larger value indicates a more pronounced toxicity difference. Prodrug nanoparticles can distinguish between tumor and normal cells, selectively releasing the active parent drug within tumor cells, significantly reducing the toxicity of docetaxel. Among them, DTX-SS-HUA nanoparticles have the highest selectivity index and the best cell selectivity.

[0146] Table 2. Half-inhibitory concentration (IC50) of docetaxel and prodrug nanoparticles on three cell lines50 nmol / L) and selectivity index (SI)

[0147]

[0148] Example 8: Pharmacokinetic Study of PEG-Modified Small Molecule Prodrug Self-Assembled Nanoparticles

[0149] Twenty healthy male rats weighing 180-220 g were randomly divided into four groups and fasted for 12 hours before dosing, with free access to water. Each of the four groups received a tail vein injection of docetaxel solution and PEGylated DTX-S-SA, DTX-SS-SA, and DTX-SS-HUA prodrug self-assembled nanoparticles prepared in Example 6. The dose was 4 mg / kg (calculated as docetaxel). Orbital blood was collected at the specified time points, and plasma was obtained by centrifugation. Drug concentrations in plasma were determined by liquid chromatography-mass spectrometry.

[0150] The experimental results are as follows Figures 1 to 3 shown. Figure 1 、 Figure 2 、 Figure 3 The blood concentration curves for each prodrug, the released parent drug docetaxel, and their sum are shown. The figure shows that the docetaxel solution has a very short circulation time and is rapidly metabolized and cleared from the body after administration. In contrast, the PEGylated small molecule prodrug self-assembled nanoparticles have a significantly longer circulation time, significantly improved bioavailability, and significantly enhanced pharmacokinetic parameters. This shows that different side chains have a significant impact on the pharmacokinetic behavior of prodrug nanoparticles. Compared with DTX-SS-SA nanoparticles and DTX-S-SA nanoparticles, DTX-SS-HUA nanoparticles have a greater area under the bloodstream (AUC) and a longer circulation time in vivo, which promotes nanoparticle accumulation at the tumor site. Furthermore, DTX-SS-HUA nanoparticles are the most stable in the blood circulation, releasing only a small amount of docetaxel.

[0151] Example 9: In vivo anti-tumor experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0152] Mouse breast cancer cell suspension (4T1, 5x10 6 cells / 100 μL) were inoculated subcutaneously on the dorsal flank of female Balb / c mice. 3Around 24 hours, tumor-bearing mice were randomly divided into 8 groups, 5 mice in each group: blank control group (Saline), docetaxel solution 2.5 mg / kg group, docetaxel solution 10 mg / kg group, docetaxel solution 20 mg / kg group, DTX-S-SA nanoparticles 2.5 mg / kg group (calculated as docetaxel), DTX-SS-SA nanoparticles 2.5 mg / kg group (calculated as docetaxel), DTX-SS-HUA nanoparticles 2.5 mg / kg group, DTX-SS-HUA nanoparticles 10 mg / kg group (calculated as docetaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 6. 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. One day after the last administration, the mice were sacrificed, and the organs and tumors were obtained for further analysis and evaluation.

[0153] The experimental results are as follows Figures 4 to 6 As shown. Figure 4 The tumor volume change curve showed that compared with the saline group, the docetaxel solution and prodrug nanoparticle groups showed stronger anti-tumor activity and significantly reduced tumor volume. Figure 6 As shown in the results, the prodrug self-assembled nanoparticles group with a dose of 2.5 mg / kg (calculated as docetaxel) showed stronger antitumor activity than the docetaxel solution group with the same dose. The order of tumor-bearing rate was docetaxel solution group > DTX-S-SA nanoparticles group > DTX-SS-SA nanoparticles group > DTX-SS-HUA nanoparticles group. Among them, the DTX-SS-HUA nanoparticles group had the best effect and the lowest tumor-bearing rate. When the dose was increased to 10 mg / kg, the antitumor activity of the DTX-SS-HUA nanoparticles group was also significantly stronger than that of the solution group with the same dose, and the tumor-bearing rate ( Figure 6 ) is lower. In addition, Figure 5 As shown, the prodrug nanoparticle group also had a very obvious attenuating effect on docetaxel. Due to the high toxicity of the docetaxel solution group, the body weight of the medium dose (10 mg / kg) and high dose groups (20 mg / kg) decreased severely after administration, while the body weight of the two dose groups of DTX-SS-HUA prodrug nanoparticles remained almost unchanged after 5 doses, showing good safety.

[0154] Example 10: In vivo anti-tumor experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0155] Mouse breast cancer cell suspension (4T1, 5x10 6 cells / 100 μL) were inoculated subcutaneously on the dorsal flank of female Balb / c mice. 3Around 24 hours after administration, the tumor-bearing mice were randomly divided into 7 groups, with 5 mice in each group: blank control group (Saline), DTX-SS-SA nanoparticles 10 mg / kg group, 20 mg / kg group, 30 mg / kg group (calculated as docetaxel); DTX-SS-HUA nanoparticles 10 mg / kg group, 20 mg / kg group, 30 mg / kg group (calculated as docetaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 6. 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. The mice were killed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation.

[0156] The experimental results are as follows Figures 7 to 12 As shown, from Figure 7 Tumor volume curve and Figure 9 In terms of tumor loading rate, compared with the saline group, the prodrug nanoparticle group showed stronger anti-tumor activity, with smaller tumor volume and lower tumor loading rate. However, at the same dose, there was no significant difference in tumor volume and tumor loading rate between the two nanoparticle groups, and Figure 8 As shown in Figure 2, body weight loss occurred at a dose of 30 mg / kg. Figure 10 As shown in the blood routine evaluation, at a dose of 30 mg / kg, the white blood cell count in the DTX-SS-SA group decreased more significantly, indicating a certain degree of immunosuppression, and the liver function test results ( Figure 11 , Figure 12 ) also showed that its alanine transaminase and aspartate transaminase were more abnormal than those in the DTX-SS-HUA group, exceeding or reaching the critical value of the normal range, and obvious liver damage occurred, indicating that DTX-SS-HUA prodrug self-assembled nanoparticles have better safety.

[0157] Example 11: In vivo anti-tumor experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0158] Mouse breast cancer cell suspension (4T1, 5x10 6 cells / 100 μL) were inoculated subcutaneously on the dorsal flank of female Balb / c mice. 3Around 24 hours after administration, tumor-bearing mice were randomly divided into 6 groups, with 5 mice in each group: blank control group (Saline), docetaxel solution 2.5 mg / kg group, DTX-SS-HD nanoparticles 2.5 mg / kg group (calculated as docetaxel), DTX-SS-HUA nanoparticles 2.5 mg / kg group (calculated as docetaxel), DTX-SS-OD nanoparticles 2.5 mg / kg group (calculated as docetaxel), DTX-SS-DTD nanoparticles 2.5 mg / kg group (calculated as docetaxel). The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 6. 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. The mice were sacrificed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation.

[0159] The experimental results are as follows Figures 13 to 15 As shown in Figure 3, compared with the saline group, both the docetaxel solution and prodrug nanoparticle groups showed stronger antitumor activity. Figure 13 As shown in the table, there was no significant difference in tumor volume among the groups at the same dose, but all were smaller than those in the saline group. Figure 15 ), the prodrug self-assembled nanoparticles group had a lower tumor-bearing rate than the docetaxel solution group at the same dose, but there was no significant difference between the different nanoformulation groups. In addition, the prodrug nanoparticles group also had a very obvious attenuation effect on docetaxel, such as Figure 14 As shown, the docetaxel solution group had a severe weight loss after administration due to its greater toxicity, while the body weight of each group of the prodrug nanoparticle group remained almost unchanged after 5 doses, showing better safety.

[0160] Example 12: Acute toxicity experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0161] Twenty-four healthy female Balb / c mice were randomly divided into eight groups, each containing three mice: a docetaxel solution group (50 mg / kg), a docetaxel solution group (60 mg / kg), a DTX-SS-SA nanoparticle group, a DTX-SS-HD nanoparticle group, a DTX-SS-HUA nanoparticle group, a DTX-SS-OD nanoparticle group, a DTX-SS-DTD nanoparticle group, and a γ-DTX-SS-OD nanoparticle group. The nanoparticles used for administration were the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 6. The solution group was administered in a single dose, while the nanoparticle group was administered at 100 mg / kg (based on docetaxel) every 15 minutes. When the cumulative dose increased to 500 mg / kg (based on docetaxel), the dosing interval was extended to 4 hours. Mouse mortality was recorded after administration.

[0162] The results are shown in Table 3. All nanoformulations demonstrated significantly improved safety compared to the solution formulation. Specifically, the DTX-SS-HUA nanoparticle group demonstrated superior safety compared to the DTX-SS-SA group, assuming the same carbon chain length. This suggests that branched-chain fatty alcohols are safer than linear fatty alcohols. When comparing branched fatty alcohols with different carbon numbers, the longer-chain DTX-SS-OD, DTX-SS-DTD, and γ-DTX-SS-OD nanoparticle groups demonstrated superior safety compared to the DTX-SS-HD and DTX-SS-HUA nanoparticle groups, demonstrating higher tolerable and lethal doses.

[0163] Table 3. Statistics of acute toxicity test results for each preparation group

[0164]

[0165] Example 13: Long-term toxicity experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0166] Thirty-six healthy female Balb / c mice were randomly divided into six groups, with six mice in each group: docetaxel solution group (30 mg / kg), docetaxel solution group (45 mg / kg), DTX-SS-OD nanoparticle group (30 mg / kg, calculated as docetaxel), DTX-SS-OD nanoparticle group (45 mg / kg, calculated as docetaxel), DTX-SS-DTD nanoparticle group (30 mg / kg, calculated as docetaxel), and DTX-SS-DTD nanoparticle group (45 mg / kg, calculated as docetaxel). The nanoparticles used for administration were the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 6. The mice were administered once every four days for three consecutive doses, and the weight changes and mortality of the mice were recorded every day.

[0167] The experimental results are as follows Figures 16-17 As shown. All the patients in the docetaxel solution group administered with 30 mg / kg and 45 mg / kg died on the 9th and 11th day respectively. Figure 17 When the dosage was 30 mg / kg, there was no death in the DTX-SS-OD nanoparticle group and the body weight was normal. Although there was no death in the DTX-SS-DTD nanoparticle group, there was a significant weight loss ( Figure 16 When the dose was 45 mg / kg, there was still no death in the DTX-SS-OD nanoparticle group, while 4 mice in the DTX-SS-DTD nanoparticle group had died on the 11th day of administration ( Figure 17Thus, although the DTX-SS-DTD nanoparticle group experienced significant weight loss at a dose of 30 mg / kg, and four animals died on day 11 in the DTX-SS-DTD nanoparticle group at a dose of 45 mg / kg, their safety profile was superior to that of the docetaxel solution group at the same dose. In summary, among the prodrug nanoparticles described above, the DTX-SS-OD prodrug nanoparticles possessed a superior safety profile.

Claims

1. Self-assembled nanoparticles of docetaxel-branched fatty alcohol prodrug, characterized in that: The self-assembled nanoparticles are PEG-modified small molecule prodrug self-assembled nanoparticles, and the docetaxel-branched fatty alcohol prodrug is selected from the following structural formula: Docetaxel-2-hexyl-decanol prodrug DTX-SS-HD with 2,2'-dithiodiacetic acid as the linker; Docetaxel-2-heptyl-undecanol prodrug DTX-SS-HUA with 2,2'-dithiodiacetic acid as the linker; Docetaxel-2-octyl-dodecanol prodrug DTX-SS-OD with 2,2'-dithiodiacetic acid as the linker; Docetaxel-2-decyl-tetradecanol prodrug DTX-SS-DTD with 2,2'-dithiodiacetic acid as the linker; Docetaxel-2-octyl-dodecanol prodrug γ-DTX-SS-OD with 4,4'-dithiodibutyric acid as the linker; The preparation method of the PEG-modified small molecule prodrug self-assembled nanoparticles comprises the following steps: Step 1: dissolving a PEG modifier and docetaxel-branched fatty alcohol prodrug in ethanol, tetrahydrofuran, or acetone to form a solution, and slowly adding the solution dropwise into water under stirring, so that the docetaxel-branched fatty alcohol prodrug spontaneously forms uniform nanoparticles; the PEG modifier is DSPE-PEG; and the mass ratio of docetaxel-branched fatty alcohol prodrug to PEG modifier is (20-1):1; Step 2: The organic solvent in the preparation is removed by rotary evaporation under reduced pressure to obtain a nanocolloid solution free of any organic solvent, namely, the PEG-modified small molecule prodrug self-assembled nanoparticles.

2. A method for preparing the self-assembled nanoparticles of docetaxel-branched fatty alcohol prodrug according to claim 1, characterized in that: The self-assembled nanoparticles are PEG-modified small molecule prodrug self-assembled nanoparticles, comprising the following steps: Step 1: dissolving a PEG modifier and docetaxel-branched fatty alcohol prodrug in ethanol, tetrahydrofuran, or acetone to form a solution, and slowly adding the solution dropwise into water under stirring, so that the docetaxel-branched fatty alcohol prodrug spontaneously forms uniform nanoparticles; the PEG modifier is DSPE-PEG; and the mass ratio of docetaxel-branched fatty alcohol prodrug to PEG modifier is (20-1):1; Step 2: The organic solvent in the preparation is removed by rotary evaporation under reduced pressure to obtain a nanocolloid solution free of any organic solvent, namely, the PEG-modified small molecule prodrug self-assembled nanoparticles.

3. Use of the self-assembled nanoparticles of docetaxel-branched fatty alcohol prodrug according to claim 1 in the preparation of anti-tumor drugs.

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