A docetaxel-fatty acid prodrug compound and preparation and application thereof

Abstract

The application belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and particularly relates to a docetaxel-fatty acid prodrug compound and construction of self-assembled nanoparticles thereof, and application of the compound in a drug delivery system. The compound is a compound shown in general formula (I) or a pharmaceutically acceptable salt thereof, wherein n=2 or 3; and R is oleic acid. The application designs and synthesizes docetaxel-fatty acid small molecule prodrugs containing different lengths of connecting chains and different configuration modifications, and the self-assembled nanoparticles of the docetaxel-fatty acid small molecule prodrugs can effectively improve the curative effect of docetaxel and reduce the toxic and side effects thereof; with the growth of the intermediate chemical connecting chain and the specific configuration, the docetaxel-fatty acid prodrug can not only specifically release the parent drug in the high reduction environment of tumor cells, but also achieve the effect of attenuation. The application provides a new strategy and selection for developing high-efficiency-low-toxicity chemotherapy preparations.

Description

Technical fields:

[0001] This invention belongs to the field of new excipients and dosage forms for pharmaceutical preparations, specifically relating to the construction of a docetaxel-fatty acid prodrug compound and its self-assembled nanoparticles, as well as its application in drug delivery systems. Background technology:

[0002] In recent years, the incidence of malignant tumors has been increasing, seriously threatening human health. Chemotherapy is one of the most effective strategies in cancer treatment. Docetaxel (DTX) belongs to the taxane class of antitumor drugs and has strong cytotoxic and antitumor effects. However, DTX has extremely low oral absorption efficiency, resulting in poor oral bioavailability. Therefore, DTX is formulated into an injectable dosage form (Tasocide). Doxorubicin has become the only option for its clinical application. However, large amounts of Tween 80 and ethanol are used to dissolve DTX in commercially available injectables, leading to serious excipient-related side effects. More importantly, Doxorubicin is metabolized rapidly after intravenous administration, resulting in less tumor accumulation and poorer antitumor activity. Therefore, designing and implementing an efficient and safe drug delivery system has always been a requirement for clinical chemotherapy.

[0003] Nanoparticle drug delivery systems can effectively prolong drug circulation time in vivo and enhance anti-tumor effects. Prodrug strategies are an effective method to improve the delivery efficiency of chemotherapy drugs. Structural modification of docetaxel using prodrug strategies can effectively improve problems such as poor solubility and high toxicity associated with docetaxel. Therefore, self-assembled nanoparticle drug delivery systems based on prodrug strategies combine the advantages of nanotechnology and prodrug strategies, and have advantages such as high drug loading capacity and no need for solubilizers, and have been widely studied in recent years.

[0004] Prodrug assemblies typically consist of three parts: a parent drug, a linker chain, and a side chain. The linker chain connects the parent drug and the side chain together. Aliphatic side chains can increase the structural flexibility of prodrug molecules, balance intermolecular forces, and promote prodrug self-assembly. Studies have found that different chemical linkages also affect the redox sensitivity of prodrug self-assembled nanoparticles, thereby affecting their antitumor activity. Changes in the chemical linker chain can impact both the safety and efficacy of the drug.

[0005] The tumor microenvironment differs significantly from that of normal tissue cells. Tumor cells generate large amounts of reactive oxygen species and glutathione, creating a redox imbalance in the tumor microenvironment. The efficacy and safety of prodrug nanocomposites are often mutually exclusive. If the linkage is too active, the prodrug will release the active drug in the systemic circulation, resulting in toxicity. If the linkage is too stable, tumor cells will have difficulty releasing the active drug, affecting the antitumor effect. Therefore, rationally controlling the sensitivity of the linkage is crucial for designing highly effective and low-toxicity drug combinations. Chinese patent CN2020106659037 describes a taxane-fatty acid prodrug with 2,2'-dithiodiacetic acid as the linker chain exhibiting excellent antitumor effects, but with slightly poor safety. Therefore, prodrugs that can balance efficacy and safety are currently a key research focus. Summary of the Invention:

[0006] The purpose of this invention is to design a docetaxel-fatty acid prodrug compound and construct its self-assembled nanoparticles, as well as its application in drug delivery systems.

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

[0008] A docetaxel-fatty acid prodrug compound, wherein the compound is a compound of general formula (I) or a pharmaceutically acceptable salt thereof.

[0009]

[0010] Where n = 2 or 3; R is oleic acid.

[0011] The compound is one of the following compounds or a pharmaceutically acceptable salt thereof.

[0012] When n = 2 in general formula (I), it is a docetaxel-oleic acid prodrug (β-DTX-SS-OA) with 3,3'-dithiodipropionic acid as the linking chain.

[0013]

[0014] When n = 3 in general formula (I), 4,4'-dithiodibutyric acid is used as the linking chain for docetaxel-oleic acid prodrug (γ-DTX-SS-OA).

[0015]

[0016] A method for synthesizing the aforementioned docetaxel-fatty acid prodrug compound:

[0017] Step 1: Fatty acids react with ethylene glycol under the catalysis of p-toluenesulfonic acid to obtain fatty acid-ethylene glycol esters;

[0018] Step 2: Fatty acid-ethylene glycol esters undergo ring-opening esterification with dicarboxylic acid anhydrides (3,3'-dithiodipropionic anhydride or 4,4'-dithiodibutyric anhydride) under the catalysis of DMAP to obtain intermediate products;

[0019] Step 3: The intermediate product undergoes esterification with docetaxel under the catalysis of HOBt, EDCI and DMAP, and is then separated and purified to obtain the compound shown in general formula (I).

[0020] In step one, the molar ratio of p-toluenesulfonic acid: fatty acid: ethylene glycol is 1:(1-3):(5-15).

[0021] In step two, the molar ratio is DMAP: fatty acid-glycol ester: dicarboxylic anhydride = 1:(1-10):(5-15).

[0022] In step three, the molar ratio of intermediate product:HOBt:EDCI:DMAP:docetaxel is 1:(1-10):(2-6):(0.2-5):(0.5-10).

[0023] Furthermore,

[0024] Step 1: Using p-toluenesulfonic acid as a catalyst, fatty acids are slowly added dropwise to an ethylene glycol solution and stirred at 110°C for 2-3 hours. The mixture is then separated by column chromatography to obtain fatty acid-ethylene glycol esters; wherein the fatty acid is oleic acid.

[0025] Step 2: Dissolve the diacid in acetic anhydride and stir for 2-4 hours to convert the diacid into anhydride. After the reaction is complete, add toluene and remove toluene and acetic anhydride by rotary evaporation to obtain the diacid anhydride. The diacid is 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, or 4,4'-dithiodibutyric acid.

[0026] The fatty acid-ethylene glycol ester, diacid anhydride and 4-dimethylaminopyridine (DMAP) obtained above were dissolved in dichloromethane and stirred at room temperature for 12-18 hours. The intermediate product was obtained by column chromatography.

[0027] Step 3: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP) together with the intermediate product in anhydrous dichloromethane. After stirring in an ice bath for 2-4 hours, add docetaxel and stir at room temperature for 24-48 hours. Then, purify the product by preparative liquid chromatography to obtain the final product. The entire reaction was carried out under nitrogen protection.

[0028] A docetaxel-fatty acid prodrug compound self-assembled nanoparticle:

[0029] The prodrug compound self-assembled nanoparticles shown in general formula (I) are non-PEGylated small molecule prodrug self-assembled nanoparticles, PEG-modified / actively targeting group-modified small molecule prodrug self-assembled nanoparticles, or small molecule prodrug self-assembled nanoparticles loaded with fluorescent substances / hydrophobic drugs.

[0030] The non-PEGylated small molecule prodrug self-assembled nanoparticles are:

[0031] (1) Dissolve the prodrug compound shown in general formula (I), and add the solution dropwise to water under stirring conditions. The docetaxel-fatty acid prodrug spontaneously forms uniform nanoparticles.

[0032] (2) The organic solvent in the formulation was removed by vacuum rotary evaporation to obtain non-PEGylated small molecule prodrug self-assembled nanoparticles.

[0033] The PEG-modified / actively targeting group-modified small molecule prodrug self-assembled nanoparticles:

[0034] (1) The modifier and the prodrug compound shown in general formula (I) are mixed and dissolved. Under stirring conditions, the solution is slowly added dropwise to water. Docetaxel-fatty acid small molecule prodrug spontaneously forms uniform nanoparticles. The modifier is a PEG modifier or an active targeting modifier. The mass ratio is: docetaxel-fatty acid small molecule prodrug: modifier = (20-1):1.

[0035] (2) The organic solvent in the formulation was removed by vacuum rotary evaporation to obtain PEG-modified / actively targeted group-modified small molecule prodrug self-assembled nanoparticles.

[0036] The small molecule prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances or drugs:

[0037] (1) Mix and dissolve the PEG modifier, hydrophobic fluorescent substance or drug with the prodrug compound shown in general formula (I), and slowly add the solution to water under stirring conditions. Docetaxel-fatty acid small molecule prodrug spontaneously forms uniform nanoparticles; by mass ratio, the prodrug compound shown in general formula (I):PEG modifier:hydrophobic fluorescent substance or drug = (20-1):1:(1-5);

[0038] (2) The organic solvent in the formulation is removed by vacuum rotary evaporation to obtain small molecule prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances or drugs.

[0039] The organic solvent used in each step (1) of the above preparation process is ethanol, tetrahydrofuran or acetone;

[0040] 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 a sugar residue, hormone, receptor or ligand, such as antibody, glycoprotein, lipoprotein, transferrin, polypeptide and folic acid.

[0041] An application of the docetaxel-fatty acid prodrug compound, the compound of general formula (I) or a pharmaceutically acceptable salt thereof, or the application of the docetaxel-fatty acid prodrug compound self-assembled nanoparticles in the preparation of antitumor drugs that combine efficacy and safety.

[0042] The application of the aforementioned docetaxel-fatty acid small molecule prodrug or self-assembled nanoparticles in the preparation of injection, oral or topical drug delivery systems.

[0043] The application of the aforementioned docetaxel-fatty acid small molecule prodrug or self-assembled nanoparticles in the preparation of drug delivery systems that improve efficacy and reduce toxicity.

[0044] The beneficial effects of this invention are:

[0045] (1) This invention designs and synthesizes docetaxel-fatty acid small molecule prodrugs containing linking chains of different lengths and different configuration modifications, and prepares docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles with small particle size and uniform particle size distribution. The synthesis and preparation methods are simple and easy to implement.

[0046] (2) This invention designs and synthesizes docetaxel-fatty acid small molecule prodrugs containing linker chains of different lengths and with different configuration modifications, as well as docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles, which can effectively improve the efficacy of docetaxel and reduce its toxic side effects. With the growth of the intermediate chemical linker chains and the specific configuration, the docetaxel-fatty acid prodrugs can not only specifically release the parent drug in the high-reducing environment of tumor cells, but also achieve a reduction in toxicity. This invention provides a new strategy and option for developing highly effective and low-toxicity chemotherapy agents. Attached Figure Description

[0047] Figure 1 To confirm the structure of the docetaxel-oleic acid prodrug (α-DTX-SS-OA) in the comparative examples of this invention, using 2,2'-dithiodiacetic acid as the linking chain; A: α-DTX-SS-OA 1 H-NMR spectrum, B: mass spectrum of α-DTX-SS-OA.

[0048] Figure 2To confirm the structure of 3,3'-dithiodipropionic acid as a linking chain docetaxel-oleic acid prodrug (β-DTX-SS-OA) in Example 1 of this invention; A: β-DTX-SS-OA 1 H-NMR spectrum, B: β-DTX-SS-OA mass spectrum.

[0049] Figure 3 To confirm the structure of 4,4'-dithiodibutyric acid as a linking chain docetaxel-oleic acid prodrug (γ-DTX-SS-OA) in Example 2 of this invention; A: γ-DTX-SS-OA 1 H-NMR spectrum, B: mass spectrum of γ-DTX-SS-OA.

[0050] Figure 4 The particle size distribution of the docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles prepared in the embodiments and comparative examples of the present invention is shown in Figure A: Particle size distribution of α-DTX-SS-OA prodrug self-assembled nanoparticles, Figure B: Particle size distribution of β-DTX-SS-OA prodrug self-assembled nanoparticles, and Figure C: Particle size distribution of γ-DTX-SS-OA prodrug self-assembled nanoparticles.

[0051] Figure 5 This is an FBS stability diagram of the docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles in an embodiment of the present invention.

[0052] Figure 6 This is an in vitro plasma chemical stability diagram of docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles in an embodiment of the present invention.

[0053] Figure 7 The following are in vivo antitumor experimental images of docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles in embodiments of the present invention: A: Effect of docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles on the growth of subcutaneous breast cancer tumors in Balb / C mice; B: Effect of docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles on the body weight of tumor-bearing mice; C: Effect of docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles on the tumor bearing rate in Balb / C mice; D: Tumor photographs of Balb / C tumor-bearing mice after treatment with docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0054] Figure 8 This is an in vivo pharmacokinetic diagram of the docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles in an embodiment of the present invention. Detailed implementation method:

[0055] The present invention will be further illustrated by way of embodiments below, but the invention is not limited to the scope of the embodiments described herein.

[0056] This invention synthesizes docetaxel-fatty acid small molecule prodrugs with chemically linked bonds at different positions, prepares self-assembled nanoparticle drug delivery systems based on these prodrugs, and describes their application in drug delivery. The stability, cytotoxicity, and pharmacodynamic effects of the resulting self-assembled prodrug nanoparticles provide new strategies and more options for developing intelligent, tumor microenvironment-responsive drug delivery systems, meeting the urgent clinical need for highly effective chemotherapy agents.

[0057] Example 1: Synthesis of 3,3'-dithiodipropionic acid as a linker chain docetaxel-oleic acid prodrug (β-DTX-SS-OA).

[0058] A suitable amount of ethylene glycol (0.1 mmol) was placed in a three-necked flask, and p-toluenesulfonic acid (0.3 mmol) was added to the flask. Under N2 protection, the mixture was heated to 110 °C. 5 mL of oleic acid solution dissolved in toluene (1 mmol) was slowly added dropwise to the reaction flask. The reaction was allowed to proceed for 2-3 hours, and TLC was used to monitor the reaction until it was complete. After the reaction was completed, the mixture was allowed to stand and separate into layers. The ethylene glycol layer was extracted with toluene until no product spot was observed on TLC. The toluene layers were combined and then washed with saturated NaHCO3 solution until neutral. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a reddish-brown oily liquid. The liquid was separated and purified by column chromatography using a cyclohexane-ethyl acetate elution system to obtain a colorless oily liquid as intermediate 1.

[0059] Dissolve an appropriate amount of 3,3'-dithiodipropionic acid (2 mmol) in 4 mL of acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, stir magnetically at 25 °C for 2 hours, then transfer to a 100 mL round-bottom flask. Add three times the amount of toluene, and remove toluene and acetic anhydride by vacuum distillation. Add an appropriate amount of dichloromethane to dissolve the resulting dithiodiacetic anhydride, then add intermediate 1 (1 mmol) dissolved in dichloromethane, and slowly add a solution of 4-dimethylaminopyridine (DMAP, 0.2 mmol) dissolved in dichloromethane. Stir magnetically at 25 °C for 12 hours. Intermediate product 2 was obtained and purified by column chromatography using a cyclohexane-acetone elution system. After obtaining the purified product from the previous step (1 mmol), 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, and the mixture was activated at 0°C in an ice bath for 2 hours. Then, docetaxel dissolved in dichloromethane (0.8 mmol) was added, and the mixture was stirred at 25°C for 36-48 hours. After the reaction was completed, the product was separated using a preparative solution to obtain 3,3'-dithiodipropionic acid as the linker chain docetaxel-oleic acid prodrug (β-DTX-SS-OA).

[0060] The structure of the prodrug in Example 2 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2As shown. The nuclear magnetic resonance spectral analysis results are as follows: β-DTX-SS-OA.

[0061] 1 H NMR(600MHz,Chloroform-d)δ8.11(d,J=7.5Hz,2H,Ar-H),7.61(t,J=7.4Hz,1H,Ar-H),7.50(t,J=7.8Hz,2H,Ar-H),7.39(t,J=7.7Hz,2H,Ar-H),7. 34–7.27(m,3H,Ar-H),6.23(s,1H,-OCO-NH-),5.69(d,J=7.1Hz,1H,2'-H) ,5.53-5.43(m,2H,3'-H,-OH),5.38(s,1H,-OH),5.37–5.31(m,2H,-CH2-C H =C H -CH2-), 5.23(s, 1H, 10- H ),4.96(dd,J=9.7,2.5Hz,1H,13-H),4.35–4.23(m,6H,20-C H 2a,2-H and-OC H2 C H2 O-), 4.19(d, J = 8.6 Hz, 1H, 20-C H 2b),3.93(d,J=7.2Hz,1H,5-H),2.89(t,J=7.1Hz,2H,-SC H2 ), 2.86–2.80 (m, 4H, -SC) H2 C H2 -), 2.79-2.70(m,3H,-SCH2C H2 -,-OH),2.62–2.55(m,1H,7-H),2.43(s,3H,-OCOC H3 ),2.36-2.27(m,3H,-COC H2 CH2-,14-Ha),2.19-2.11(m,1H,14-Hb),2.03–1.99(m,4H,-C H2 -CH=CH-C H2 -), 1.95(s, 3H, 18-C H3 ),1.90–1.81(m,2H,3-H,6-Ha),1.75(s,3H,19-C H3 ),1.68–1.59(m,3H,6-Hb,-OCOCH2C H2- ),1.39–1.24(m,29H,-C(CH3)3,-(CH2)10 ),1.23(s,3H,16-C H3 ),1.12(s,3H,17-C H3 ),0.88(t,J=7.0Hz,3H,-CH2C H3 ).

[0062] The mass spectrometry results are MS(ESI) m / z for C 69 H 97 NO 19 S2[M+Na] + =1330.6096.

[0063] Example 2: Synthesis of 4,4'-dithiodibutyric acid as a linker chain docetaxel-oleic acid prodrug (γ-DTX-SS-OA).

[0064] The synthesis of 4,4'-dithiodibutyric acid as a linking chain docetaxel-oleic acid prodrug (γ-DTX-SS-OA) was carried out using the preparation method of Example 1, by replacing 3,3'-dithiodipropionic acid with 4,4'-dithiodibutyric acid to obtain 4,4'-dithiodibutyric acid as a linking chain docetaxel-oleic acid prodrug (γ-DTX-SS-OA).

[0065] The structure of the prodrug in Example 3 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 3 As shown. The nuclear magnetic resonance spectral analysis results are as follows: γ-DTX-SS-OA.

[0066] 1 H NMR(600MHz,Chloroform-d)δ8.11(d,J=7.5Hz,2H,Ar-H),7.60(t,J=7.4Hz,1H,Ar-H),7.50(t,J=7.8Hz,2H,Ar-H),7.39(t,J=7.7 Hz,2H,Ar-H),7.35–7.27(m,3H,Ar-H),6.24(s,1H,-OCO-NH-),5.69(d,J=7.1Hz,1H,2'-H),5.52–5.31(m,5H,3'-H,2*-OH,-CH2-C H =C H -CH2-), 5.23(s, 1H, 10- H ),4.96(dd,J=9.6,2.5Hz,1H,13-H),4.32(d,J=8.5Hz,1H,20-C H 2a),4.30–4.24(m,5H,2-H and-OC H2 C H2O-), 4.19(d, J = 8.6 Hz, 1H, 20-C H 2b),3.93(d,J=7.1Hz,1H,5-H),2.84(s,1H,-OH),2.69(t,J=7.1Hz,2H,

[0067] -SC H2 -), 2.66–2.51(m,4H,-SC H2 CH2C H2 CO-),2.49-2.38(m 6H,7-H,-SCH2CH2C H2 CO-,-OCOC H3 ),2.36-2.27(m,3H,-COC H2 CH2-,14-Ha),2.21–2.13(m,1H,14-Hb),2.05–1.96(m,8H,-C H2 -CH=CH-C H2 -,2*-SCH2C H 2CH2CO-), 1.95 (s, 3H, 18-C) H3 ),1.92–1.80(m,2H,3-H,6-Ha),1.75(s,3H,19-C H3 ),1.67–1.58(m,3H,6-Hb,-OCOCH2C H2 -),1.35–1.25(m,29H,-C(CH3)3,-(CH2) 10 ),1.23(s,3H,16-C H3 ),1.12(s,3H,17-C H3 ),0.88(t,J=7.0Hz,3H,-CH2C H3 ).

[0068] The mass spectrometry results are MS(ESI) m / z for C 71 H 101 NO 19 S2[M+Na] + =1358.6306.

[0069] Comparative Example: Synthesis of 2,2'-dithiodiacetic acid as a linker chain docetaxel-oleic acid prodrug (α-DTX-SS-OA).

[0070] A suitable amount of ethylene glycol (0.1 mmol) was placed in a three-necked flask, and p-toluenesulfonic acid (0.3 mmol) was added to the flask. Under N2 protection, the mixture was heated to 110 °C. 5 mL of oleic acid solution dissolved in toluene (1 mmol) was slowly added dropwise to the reaction flask. The reaction was allowed to proceed for 2-3 hours, and TLC was used to monitor the reaction until it was complete. After the reaction was completed, the mixture was allowed to stand and separate into layers. The ethylene glycol layer was extracted with toluene until no product spot was observed on TLC. The toluene layers were combined and then washed with saturated NaHCO3 solution until neutral. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a reddish-brown oily liquid. The liquid was separated and purified by column chromatography using a cyclohexane-ethyl acetate elution system to obtain a colorless oily liquid as intermediate 1.

[0071] Dissolve an appropriate amount of 2,2'-dithiodiacetic acid (2 mmol) in 4 mL of acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, stir magnetically at 25 °C for 2 hours, then transfer to a 100 mL round-bottom flask. Add three times the amount of toluene, and remove toluene and acetic anhydride by vacuum distillation. Add an appropriate amount of dithiodiacetic anhydride dissolved in dichloromethane, then add intermediate 1 (1 mmol) dissolved in dichloromethane, and slowly add a solution of 4-dimethylaminopyridine (DMAP, 0.2 mmol) dissolved in dichloromethane. Stir magnetically at 25 °C for 12 hours. Intermediate product 2 was obtained and purified by column chromatography using a cyclohexane-acetone elution system. After obtaining the purified product from the previous step (1 mmol), 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, and the mixture was activated at 0°C in an ice bath for 2 hours. Then, docetaxel dissolved in dichloromethane (0.8 mmol) was added, and the mixture was stirred at 25°C for 36-48 hours. After the reaction was completed, the product was separated using a preparative solution to obtain 2,2'-dithiodiacetic acid as the linker chain docetaxel-oleic acid prodrug (α-DTX-SS-OA).

[0072] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown. The nuclear magnetic resonance spectral analysis results are as follows: α-DTX-SS-OA.

[0073] 1H NMR(600MHz,Chloroform-d)δ8.11(d,J=7.5Hz,2H,Ar-H),7.61(t,J=7.4Hz,1H,Ar-H),7.51(t,J=7.8Hz,2H,Ar-H),7.39(t,J=7.7Hz,2H,Ar-H),7.33(d,J=7.4Hz,2H,Ar-H),7.30(t,J=7.4Hz,1H,Ar-H),6.23(s,1H,-OCO-NH-),5.74-5.65(m,2H,2’-H,3’-H),5.48(s,1H,-OH),5.39-5.29(m,3H,-CH2-C H =C H -CH2-,-OH),5.23(s,1H,10- H ),4.96(dd,J=9.7,2.5Hz,1H,13-H),4.39–4.26(m,6H,20-C H 2a,2-H and-OC H2 C H2 O-),4.19(d,J=8.5Hz,1H,20-C H 2b),3.92(d,J=7.1Hz,1H,5-H),3.63–3.49(m,4H,-C H2 SSC H2- ),2.83(s,1H,-OH),2.61–2.54(m,1H,7-H),2.43(s,3H,-OCOC H3 ),2.35-2.27(m,3H,-COC H2 CH2-,14-Ha),2.19-2.11(m,1H,14-Hb),2.04-1.98(m,4H,-C H2 -CH=CH-C H2 -),1.94(s,3H,18-C H3 ),1.90–1.81(m,2H,3-H,6-Ha),1.75(s,3H,19-C H3 ),1.71-1.57(m,3H,6-Hb,-OCOCH2C H2- ),1.43–1.23(m,29H,-C(CH3)3,-(CH2) 10 ),1.22(s,3H,16-C H3 ),1.12(s,3H,17-C H3 ),0.88(t,J=7.0Hz,3H,-CH2C H3 )。

[0074] The mass spectrometry results are MS(ESI) m / z for C 67 H 93 NO 19 S2[M+Na] + =1302.5670.

[0075] Example 3: Preparation of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0076] PEGylated docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles: Accurately weigh DSPE-PEG 2k 2 mg of docetaxel-fatty acid small molecule prodrug prepared in Examples 1 and 2 and the comparative example were mixed and dissolved in 1 mL of ethanol. While stirring, the ethanol solution was slowly added dropwise to 4 mL of deionized water, spontaneously forming uniform nanoparticles. The ethanol was removed by vacuum distillation to obtain an ethanol-free nano-formulation, namely DSPE-PEG. 2k Modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles: specifically α-DTX-SS-OA nanoparticles, β-DTX-SS-OA nanoparticles, and γ-DTX-SS-OA nanoparticles (see [link to product description]). Figure 4 (and Table 1).

[0077] Table 1. Particle size and particle size distribution of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles

[0078]

[0079] The results are shown in Table 1. The particle size of the nanoparticles all meet the requirements, and the particle size distribution is less than 0.2.

[0080] Further evaluation was conducted using the above-mentioned examples of prodrug assemblies with different configurations:

[0081] 1) FBS stability experiment of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0082] The stability of the nanoparticles was investigated by incubating the self-assembled prodrug nanoparticles in PBS solution (containing 10% FBS, pH 7.4). Figure 5As shown, after incubation in PBS solution (containing 10% FBS, pH 7.4) for 24 hours, the particle size of α-DTX-SS-OA nanoparticles increased to some extent. In contrast, the particle size changes of β-DTX-SS-OA and γ-DTX-SS-OA nanoparticles were negligible. This suggests that α-DTX-SS-OA nanoparticles may be more sensitive to the surrounding environment and have poor stability, while β-DTX-SS-OA and γ-DTX-SS-OA nanoparticles have better assembly ability and stability.

[0083] 2) Plasma chemical stability test of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0084] The in vitro plasma chemical stability of PEG-modified small molecule prodrug self-assembled nanoparticles was investigated. Three types of prodrug nanoparticles were mixed with blank rat plasma (1:20, v / v) and incubated in a 37°C constant-temperature shaker (100 rpm). At 0, 2, 4, 6, 8, 12, and 24 h, 50 μL of the sample was collected, 150 μL of acetonitrile was added, the mixture was vortexed for 3 min, centrifuged at 13000 rpm for 10 min, and the supernatant was used to determine the plasma drug concentration.

[0085] The results are as follows Figure 6 As shown, β-DTX-SS-OA nanoparticles and γ-DTX-SS-OA nanoparticles exhibited good plasma chemical stability, with approximately 90% of the prodrug remaining undegraded after 24 hours of incubation with plasma. In contrast, α-DTX-SS-OA nanoparticles showed poorer chemical stability, with significantly higher docetaxel release after 24 hours of incubation compared to β-DTX-SS-OA and γ-DTX-SS-OA nanoparticles. This indicates that prodrugs bridged by β- and γ-disulfide bonds possess better plasma chemical stability, ensuring their pharmacokinetic behavior in vivo.

[0086] Structure-activity relationship analysis of FBS stability and plasma chemical stability studies revealed a correlation between the release rate of docetaxel and the carbon chain length between the sulfur atom and the ester bond. In α-DTX-SS-OA nanoparticles, the sulfur atom is located at the α-position of the ester bond, resulting in the shortest carbon chain and the fastest drug release. For γ-DTX-SS-OA nanoparticles, although the carbon chain between the disulfide bond and the ester bond is the longest, the thiol group at the γ-position can attack the carbon atom on the carbonyl group, causing the ester bond to break and forming a stable five-membered ring structure, thereby promoting rapid docetaxel release. Therefore, γ-DTX-SS-OA nanoparticles exhibit stronger responsive drug release than β-DTX-SS-OA nanoparticles, while β-DTX-SS-OA nanoparticles hold the potential for achieving long-acting sustained-release drugs.

[0087] 3) In vivo antitumor experiment of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0088] The antitumor activity of PEG-modified small molecule prodrug self-assembled nanoparticles against a 4T1 heterotopic tumor model was investigated. 4T1 cell suspensions (5 × 10⁻⁶ cells / year) were used. 6 4T1 ectopic tumor model was constructed by inoculating female Balb / C mice with cells / 100μL on the right back. The tumor volume was increased to 100mm². 3 In this study, tumor-bearing mice were randomly divided into groups of five, and each group was administered saline, Taxotere, or PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Examples 1-3, respectively. Administered every 3 days for 6 consecutive days, at a dose of 30 mg / kg (based on docetaxel dosage). After administration, the mice's survival status was observed daily, their body weight was measured, and tumor volume was determined. The day after the last administration, the tumor-bearing mice were sacrificed, and the tumors were harvested for further analysis and evaluation.

[0089] The results are as follows Figure 7 As shown in the endpoint tests, we found that the tumors in mice treated with saline grew rapidly, while all mice in the Taxotere group died after only three intravenous injections, demonstrating extremely high toxicity. Notably, α-DTX-SS-OA nanoparticles induced sustained tumor contraction, with an antitumor effect even superior to DTX. However, due to their redox-sensitive properties, the drug release was rapid, causing some weight loss in mice, raising concerns about its safety. This suggests that α-DTX-SS-OA nanoparticles may not be suitable for developing highly efficient and safe drug delivery systems. In contrast, β-DTX-SS-OA and γ-DTX-SS-OA nanoparticles possess both antitumor activity and safety, without causing significant non-specific toxicity to the body, making them promising formulations for cancer treatment.

[0090] 4) Pharmacokinetic experiments of PEG-modified docetaxel-fatty acid small molecule prodrug self-assembled nanoparticles.

[0091] SD rats were randomly divided into groups of six. Taxotere, α-DTX-SS-OA nanoparticles, β-DTX-SS-OA nanoparticles, and γ-DTX-SS-OA nanoparticles were administered via tail vein injection, respectively. The equivalent dose of Taxotere was 4 mg / kg. Following administration, blood samples were collected from the rats' orbital sinuses at predetermined time points. Plasma samples were obtained by centrifugation and promptly stored at -80°C. The plasma concentrations of the prodrugs (α-DTX-SS-OA, β-DTX-SS-OA, γ-DTX-SS-OA) and the parent drug docetaxel were determined using UPLC-MS-MS.

[0092] The results are as follows Figure 8As shown, docetaxel in Taxotere was rapidly cleared from the bloodstream after injection, while the prodrug self-assembled nanoparticles significantly prolonged the residence time of docetaxel in plasma. The area under the drug-time curve (AUC) of total DTX for α-DTX-SS-OA nanoparticles, β-DTX-SS-OA nanoparticles, and γ-DTX-SS-OA nanoparticles was 71.69, 223, and 143 times higher than that of docetaxel solution, respectively. In contrast, α-DTX-SS-OA nanoparticles exhibited the worst stability and high redox sensitivity, rapidly depolymerizing upon entering the bloodstream and releasing large amounts of docetaxel, thus affecting its pharmacokinetic behavior. γ-DTX-SS-OA nanoparticles showed slightly worse colloidal stability, but their AUC was significantly higher than that of docetaxel solution, and their AUC was twice that of α-DTX-SS-OA nanoparticles. It is noteworthy that the β-disulfide bond has the most significant impact on the pharmacokinetic behavior of the prodrug self-assembled nanoparticles. The circulation time of β-DTX-SS-OA nanoparticles in vivo is significantly prolonged, and the AUC is three times that of α-DTX-SS-OA nanoparticles.

[0093] In summary, the chemical linkages at different positions affect the antitumor effect of the prodrug. By extending the intermediate chemical linkages, the safety of the docetaxel-fatty acid prodrug is significantly improved. The above results once again demonstrate the advantages of the self-assembled nanoparticles of the docetaxel-fatty acid small molecule prodrug.

Claims

1. A docetaxel-fatty acid prodrug compound self-assembled nanoparticle, characterized in that: The docetaxel-fatty acid prodrug compound self-assembled nanoparticles are non-PEGylated small molecule prodrug self-assembled nanoparticles, PEG-modified / actively targeting group-modified small molecule prodrug self-assembled nanoparticles, or small molecule prodrug self-assembled nanoparticles encapsulating fluorescent substances / hydrophobic drugs. The non-PEGylated small molecule prodrug self-assembled nanoparticles are: (1) Dissolve the prodrug compound and add the solution dropwise to water under stirring conditions. The docetaxel-fatty acid prodrug spontaneously forms uniform nanoparticles. (2) The organic solvent in the formulation was removed by vacuum rotary evaporation to obtain non-PEGylated small molecule prodrug self-assembled nanoparticles. The PEG-modified / actively targeting group-modified small molecule prodrug self-assembled nanoparticles: (1) Mix and dissolve the modifier and prodrug compound, and slowly add the solution to water under stirring conditions. Docetaxel-fatty acid small molecule prodrug spontaneously forms uniform nanoparticles. The modifier is a PEG modifier or an active targeting modifier. The mass ratio is: docetaxel-fatty acid small molecule prodrug: modifier = (20-1):

1. (2) The organic solvent in the formulation was removed by vacuum rotary evaporation to obtain PEG-modified / actively targeted group-modified small molecule prodrug self-assembled nanoparticles. The small molecule prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substances or drugs: (1) Mix and dissolve the PEG modifier, hydrophobic fluorescent substance or drug with the prodrug compound, and slowly add the solution to water under stirring conditions. Docetaxel-fatty acid small molecule prodrug spontaneously forms uniform nanoparticles; by mass ratio, prodrug compound:PEG modifier:hydrophobic fluorescent substance or drug = (20-1):1:(1-5). (2) The organic solvent in the formulation is removed by vacuum rotary evaporation, and small molecule prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances or drugs are obtained. The prodrug compound is a compound of general formula (I) or a pharmaceutically acceptable salt thereof. General Formula (I) Where n = 2 or 3; R is oleic acid.

2. The docetaxel-fatty acid prodrug compound self-assembled nanoparticles according to claim 1, characterized in that: When n=2 in general formula (I), that is, the docetaxel-oleic acid prodrug β-DTX-SS-OA with 3,3'-dithiodipropionic acid as the linking chain; When n=3 in general formula (I), that is, the docetaxel-oleic acid prodrug γ-DTX-SS-OA with 4,4'-dithiodibutyric acid as the linking chain; 。 3. The application of the docetaxel-fatty acid prodrug compound self-assembled nanoparticles according to claim 1, characterized in that... The application of the docetaxel-fatty acid prodrug compound self-assembled nanoparticles of claim 1 in the preparation of antitumor drugs; The tumor is breast cancer.

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