Conjugated prodrug, nanoparticle, pharmaceutical composition, preparation method and application

Abstract

The present invention discloses a coupled prodrug, nanoparticles, pharmaceutical composition, preparation method and application based on the structure of taxane-artemisinin derivatives, wherein the coupled prodrug structure is as follows: P-L 1 ‑(CH 2 ) n ‑X‑(CH 2 ) m ‑L 2 ‑A easily achieves the spatiotemporal coordinated transport of different drugs through different connecting arms, and has controllable drug release behavior, which improves the tumor treatment effect. At the same time, it improves the drug solubility by self-assembly into nanoparticles, and solves the biocompatibility, preparation method complexity and safety problems caused by solvents and carrier materials. It can be prepared into drugs for multiple administration routes such as oral, inhalation, injection, ophthalmic, transdermal, mucosal, etc., and is used in the prevention and treatment of various tumors.

Description

Technical Field

[0001] The present invention relates to a coupled prodrug, nanoparticles, a pharmaceutical composition, a preparation method and an application thereof based on a taxane-artemisinin derivative structure, and in particular to a coupled prodrug, nanoparticles, a pharmaceutical composition, a preparation method and an application thereof with significantly improved solubility, biosafety and efficacy. Background Art

[0002] Taxanes are a broad-spectrum chemotherapy drug isolated from Taxus chinensis. They work by stimulating tubulin polymerization, leading to G1 or G2 / M cell cycle arrest and apoptosis. They are now widely used in the clinical treatment of various human cancers. Among them, the most representative paclitaxel (PTX) has good therapeutic properties, but due to its poor solubility in water, it is only 0.006 mg / mL. In order to improve the water solubility of paclitaxel, additional solubilization strategies are often required, including the use of solubilizers and carriers. Paclitaxel injection used in clinical practice The solubilizer Cremaphor EL (Cremaphor EL) associated with toxic effects is required for formulation and use; albumin-encapsulated PTX (Abraxane TM ), polymer micelles (NK105 and GenexolPM TM ) and liposomal preparations The use of carrier materials to encapsulate PTX has problems such as low drug loading, safety of solubilizers and carrier materials, and material complexity, which brings certain difficulties and challenges to the formulation development of taxane drugs.

[0003] Artemisinin drugs are sesquiterpene lactone drugs containing peroxide bridges extracted from the stems and leaves of the plant Artemisia annua. Artemisinin drugs are widely used to treat malaria. In recent years, artemisinin drugs have also shown strong and extensive anti-cancer properties and have anti-cancer effects on a variety of tumors. The main obstacles to the clinical application of artemisinin drugs are poor water solubility, short half-life and limited tissue distribution, which often require large doses of artemisinin compounds to exert greater anti-tumor effects.

[0004] In order to solve the shortcomings of the single application of the above drugs, Construction and anti-tumor activities of disulfide-linked docetaxel-dihydroartemisinin nanoconjugates (Colloids and Surfaces B: Biointerfaces, 191 (2020): 111018.) discloses a docetaxel-dihydroartemisinin prodrug coupled with a disulfide bond, specifically 3,3'-dithiodipropionic acid as a 6-carbon linker to couple docetaxel and dihydroartemisinin. The drug molecule is still highly hydrophobic, has low solubility in water, and has weak molecular self-assembly ability. It still needs to use up to 20% of vitamin E polyethylene glycol succinate (TPGS) as a solubilizer and stabilizer, which has potential biosafety risks. In addition, PEGylated-Paclitaxel and Dihydroartemisinin Nanoparticles for Simultaneously Delivering Paclitaxel and Dihydroartemisinin to Colorectal Cancer (Pharm Res, (2020) 37: 129.) also discloses a paclitaxel-dihydroartemisinin prodrug coupled with PEG, which uses PEG as a linker to couple carboxypaclitaxel and artesunate, and the synthesis process is complicated and cumbersome; PTX, DHA, lecithin and PTX-PEG-DHA are then prepared into nanoliposomes by O / W emulsion-solvent evaporation method, the prescription design and preparation process are complicated, the drug is not administered in the form of pure drug molecules, the drug loading is low (<15%), and there is still a biosafety risk. Therefore, although the existing prodrug molecules have overcome the defects of single drug use to a certain extent, their application is still limited by solubility, safety and preparability. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings of existing taxane-artemisinin derivative prodrugs, such as poor drug water solubility, low self-assembly ability, complex preparation process, and biosafety issues, the present invention aims to provide a taxane-artemisinin derivative prodrug, nanoparticle, pharmaceutical composition, preparation method and application that can significantly improve water solubility, biosafety and effectiveness and is convenient to prepare.

[0006] Technical solution: As the first aspect of the present invention, the conjugated prodrug of the present invention has the following structure PL 1 -(CH 2 ) n -X-(CH 2 ) m -L 2 -A,

[0007] in:

[0008] P is a structural fragment of a taxane compound with one hydroxyl group removed, wherein the taxane compound is selected from paclitaxel, docetaxel, cephalomannine or 10-deacetylpaclitaxel;

[0009] A is a structural fragment of an artemisinin compound with one hydroxyl group removed, wherein the artemisinin compound is selected from dihydroartemisinin and artesunate;

[0010] X is selected from -CC-, -S-, -Se-, -SS- or -Se-Se-;

[0011] m, n are selected from 0 or 1;

[0012] L 1 , L 2 It is an ester bond.

[0013] Preferably, in the above structure: the sum of n and m is selected from 0, 1 or 2, especially 0 or 2.

[0014] More preferably, it is any of the following compounds:

[0015]

[0016] Prodrugs, also known as precursor drugs, can change the key physical properties of drug molecules such as water solubility and stability based on structural modification of drug molecules, and can restore pharmacological activity after transformation of target tissues in vivo. Prodrug delivery strategies can achieve drug solubilization, efficacy enhancement and toxicity reduction without relying on carrier materials. At the same time, the prodrug design strategy derived from drug molecules as modification groups can further integrate the advantages of synergistic and synergistic combination drugs.

[0017] The present invention is based on taxane and artemisinin derivatives, and connects the two through different chemical linking arms. The synthesis process is simple and easy to industrialize. The prodrug molecules coupled by the target linking arms have good dispersibility in water and strong self-assembly ability. They can be self-assembled in the aqueous phase by nanoprecipitation to form stable nanomedicines. It is a "green" solution to the defects of poor water solubility of the two types of drugs, and no additional solubilization strategies are required, such as no need to use solubilizers or carriers, avoiding the biocompatibility, complexity, and safety issues caused by solvents and carrier materials. The prodrug nanoparticles have an appropriate nanometer size, good dispersion uniformity, and controllable drug release behavior, which can achieve the spatiotemporal coordinated transport of different drugs, and synergistically enhance the effect through multiple pharmacological mechanisms, and have excellent tumor treatment effects and clinical transformation prospects.

[0018] The preparation method of the above-mentioned conjugated prodrug is as follows:

[0019] Method 1:

[0020]

[0021] A-OH + P-O-CO-(CH 2 ) n -X-(CH 2 ) m -COOH → P-O-CO-(CH 2 ) n -X-(CH 2 ) m -CO-O-A (2)

[0022] Method 2:

[0023] P-OH + COCl-(CH 2 ) n -X-(CH 2 ) m -COCl → P-O-CO-(CH 2 ) n -X-(CH 2 ) m -COCI (1)

[0024] A-OH + P-O-CO-(CH 2 ) n -X-(CH 2 ) m -COCl → P-O-CO-(CH 2 ) n -X-(CH 2 ) m -CO-O-A (2)

[0025] Method 3:

[0026] P-OH + COOH-(CH 2 ) n -X-(CH 2 ) m -COOH → P-O-CO-(CH 2 ) n -X-(CH 2 ) m -COOH (1)

[0027] A-OH + P-O-CO-(CH 2 ) n -X-(CH 2 ) m -COOH → P-O-CO-(CH 2 ) n -X-(CH 2 ) m-CO-OA (2)

[0028] Method 4:

[0029] P-OH+COCl-(CH 2 ) n nX-(CH 2 ) m -COOH→PO-CO-(CH 2 ) n -X-(CH 2 ) m -COOH (1)

[0030] A-OH+PO-CO-(CH 2 ) n -X-(CH 2 ) m -COOH→PO-CO-(CH 2 ) n -X-(CH 2 ) m -O-CO-A (2)

[0031] A-OH+COCl-(CH 2 ) n -X-(CH 2 ) m -COOH→AO-CO-(CH 2 )-X-(CH 2 ) m -COOH (⑴)

[0032] or

[0033] P-OH+AO-CO-(CH 2 ) n -X-(CH 2 ) m -COOH→PO-CO-(CH 2 ) n -X-(CH 2 ) m -O-CO-A (2)

[0034] Wherein, P, A, X, m, and n are defined as above.

[0035] The present invention adopts TEA or DIPEA or pyridine as an acid-binding agent to catalyze the nucleophilic substitution reaction between anhydride, acyl chloride and hydroxyl to obtain an ester bond, or EDC and 4-dimethylaminopyridine (DMAP) as strong nucleophilic catalysts to attack the carboxyl group to generate a stable active intermediate ester, and then undergoes a condensation reaction with the hydroxyl group to obtain an ester bond, thereby achieving successful coupling between the connecting arm and the two types of small molecules.

[0036] The preparation method is as follows:

[0037] Method 1: Reaction using anhydride as linker

[0038] A certain amount of hydroxyl-containing taxane compound is dissolved in a reaction solvent, triethylamine is added as a catalyst, and then an excess of anhydride containing -CC-, -S-, -Se-, -SS- or -Se-Se- is added as a connecting arm, and the reaction is stirred for 12 hours. The single-end grafted intermediate product is obtained by column chromatography separation; a certain amount of the intermediate product is dissolved in a reaction solvent, and activators EDC and DMAP are added under an ice-water bath. After activation for a period of time, a certain amount of hydroxyl-containing artemisinin compound is added, and the reaction is stirred for 24 hours. The taxane-artemisinin prodrug molecule is obtained by column chromatography separation.

[0039] Method 2: Reaction with a linker with acyl chloride at both ends

[0040] A certain amount of hydroxyl-containing taxane compound is dissolved in a reaction solvent, triethylamine is added as an acid-binding agent, and then an excess of a linker containing -CC-, -S-, -Se-, -SS- or -Se-Se- and having acyl chlorides at both ends is added, and the reaction is stirred for 12 hours. The single-end grafted intermediate product is obtained by column chromatography separation; a certain amount of hydroxyl-containing artemisinin compound is dissolved in a reaction solvent, triethylamine is added as an acid-binding agent, and then a certain amount of the intermediate product is added, and the reaction is stirred for 12 hours. The taxane-artemisinin prodrug molecule is obtained by column chromatography separation.

[0041] Method 3: Reaction with a linker with carboxyl groups at both ends

[0042] An excess of a linker containing -CC-, -S-, -Se-, -SS- or -Se-Se- and having carboxyl groups at both ends is dissolved in a reaction solvent, and activators 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) are added under an ice-water bath. After activation for a period of time, a certain amount of a hydroxyl-containing taxane compound is added, stirred for reaction for 24 hours, and a single-end grafted intermediate product is obtained by column chromatography separation; an excess of an intermediate product containing a carboxyl group at one end is dissolved in a reaction solvent, and activators EDC and DMAP are added under an ice-water bath. After activation for a period of time, a certain amount of a hydroxyl-containing artemisinin compound is added, stirred for reaction for 24 hours. By column chromatography separation, a taxane-artemisinin prodrug molecule is obtained.

[0043] Method 4: Reaction with a linker with a carboxyl group at one end and an acyl chloride at the other end

[0044] A certain amount of hydroxyl-containing taxane compound is dissolved in a reaction solvent, triethylamine is added as an acid-binding agent, and then an excess of a linker containing -CC-, -S-, -Se-, -SS- or -Se-Se- and a reactive group at one end is a carboxyl group and a reactive group at the other end is an acyl chloride is added, and the reaction is stirred for 12 hours. The intermediate product is separated by column chromatography; an excess of the intermediate product containing a carboxyl group at one end is dissolved in a reaction solvent, and activators EDC and DMAP are added under an ice-water bath. After activation for a period of time, a certain amount of hydroxyl-containing artemisinin compound is added, and the reaction is stirred for 24 hours. Alternatively, a certain amount of hydroxyl-containing artemisinin compound is dissolved in a reaction solvent, triethylamine is added as an acid-binding agent, and then an excess of a linker containing -CC-, -S-, -Se-, -SS- or -Se-Se- and a reactive group at one end is a carboxyl group and a reactive group at the other end is an acyl chloride is added, and the reaction is stirred for 12 hours. The intermediate product is obtained by column chromatography separation; an excess of the intermediate product containing a carboxyl group at one end is dissolved in a reaction solvent, and activators EDC and DMAP are added under an ice-water bath. After activation for a period of time, a certain amount of a hydroxyl-containing taxane compound is added, and the reaction is stirred for 24 hours. The taxane-artemisinin prodrug molecule is obtained by column chromatography separation.

[0045] The reaction solvents used in the above preparation method are dichloromethane, acetone, methanol, ethanol, N,N-dimethylformamide and dimethyl sulfoxide solvents.

[0046] As a second aspect of the present invention, the nanoparticles self-assembled from the above-mentioned coupled prodrugs have a particle size of 10 to 900 nm, a polydispersity coefficient of less than 0.3, and good stability.

[0047] The above-mentioned coupled prodrug is dissolved and mixed with water, and the solvent is removed to obtain the nanoparticles.

[0048] The preparation method is as follows:

[0049] An appropriate amount of taxane-artemisinin prodrug molecules are weighed and dissolved in a good solvent to obtain a molecular solution, the solution is mixed with water, and self-assembled prodrug nanoparticles are prepared by conventional anti-solvent precipitation method, solvent evaporation method, microfluidics technology, or ultrasound method. The solvent is removed by dialysis, ultrafiltration or evaporation to obtain a highly efficient and stable self-assembled prodrug nanoparticle solution.

[0050] More specifically, the mixing time is 1-24h; the ultrasonic frequency is 80-600W, and the ultrasonic time is 1-1h; the homogenization is achieved by a high-pressure homogenizer or a microfluidizer, the homogenization pressure is 200-1200Pa, and the homogenization times are 1-30 times; the homogenization is achieved by a homogenizer, the speed is 1000-9000rpm, and the homogenization time is 30-300s; the medium grinding machine speed is 400-800rpm, and the grinding time is 1-10h. The pharmaceutically acceptable solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide, ethanol, acetone, methanol, tetrahydrofuran, dichloromethane, chloroform, and ethyl acetate.

[0051] As the third aspect of the present invention, the above-mentioned coupled prodrug or its pharmaceutically acceptable salt, isomer, solvate or isotope label and a pharmaceutically acceptable carrier form a pharmaceutical composition. The pharmaceutical composition can be in the form of injection, tablet, capsule, granule, powder, suppository, pill, cream, paste, gel, powder, oral solution, inhalant, suspension, dry suspension, patch, lotion.

[0052] As the fourth aspect of the present invention, the above-mentioned coupled drug is used for the preparation of drugs for preventing and / or treating tumors by oral, inhalation, injection, ophthalmic, transdermal or mucosal administration, especially for the preparation of drugs for preventing and / or treating breast cancer, liver cancer, ovarian cancer, colon cancer, lung cancer, melanoma, head and neck cancer, lymphoma, pancreatic cancer, brain tumor or blood cancer.

[0053] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0054] (1) Realize the spatiotemporal coordinated transport of different drugs and have controllable drug release behavior to improve the therapeutic effect of tumors;

[0055] (2) Improve drug solubility without the need for additional solubilization, solving the problems of biocompatibility, preparation method complexity, and safety caused by solvents and carrier materials;

[0056] (3) The preparation method is simple and easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The particle size distribution and dilution stability of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9 before and after freeze-drying;

[0058] Figure 2 The colloidal stability of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9 before and after freeze-drying;

[0059] Figure 3 The release behavior of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9 in blank medium and esterase-containing conditions;

[0060] Figure 4 The cytotoxicity of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9;

[0061] Figure 5 The tumor weight curve of the in vivo efficacy of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9;

[0062] Figure 6 The body weight changes of tumor-bearing mice in vivo treated with paclitaxel-dihydroartemisinin prodrug self-assembled nanoparticles (PTX-CC-DHA) prepared in Example 9. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below in conjunction with embodiments.

[0064] Example 1: Synthesis of Paclitaxel-Dihydroartemisinin Prodrug Molecule Linked to Succinic Anhydride

[0065] Accurately weigh an excess of succinic anhydride in a 50 mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 3 times the molar amount of TEA, and then add paclitaxel dissolved in dichloromethane dropwise, and stir to react at room temperature for 12 hours. The intermediate product is then separated by column chromatography. The intermediate product is dissolved in anhydrous dichloromethane, 1.5 times the molar amount of EDC and an equal amount of DMAP are added, and the mixture is stirred and activated in an ice bath for 1-2 hours. Then, dihydroartemisinin dissolved in dichloromethane is added dropwise, and the reaction is stirred for 24 hours. The target product, paclitaxel-dihydroartemisinin prodrug molecule obtained is separated and purified as above to obtain esterase-sensitive chemical bond-coupled paclitaxel-dihydroartemisinin prodrug molecule. The above reaction is performed throughout the N 2 Under protection.

[0066] The synthetic route is illustrated as follows:

[0067]

[0068] Since artesunate is also the product of the reaction between dihydroartemisinin and succinic anhydride, Example 1 can also be achieved by direct esterification of paclitaxel and artesunate.

[0069] Accurately weigh a certain amount of artesunate in a 50 mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir to activate in an ice bath for 1-2 hours. Then add paclitaxel dissolved in dichloromethane dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. That is, the esterase-sensitive chemical bond-coupled paclitaxel-dihydroartemisinin prodrug molecule is obtained. The above reaction is carried out in N 2 Under protection.

[0070] The synthetic route is illustrated as follows:

[0071]

[0072] Example 2: Synthesis of docetaxel-dihydroartemisinin prodrug molecules linked to succinyl chloride

[0073] Accurately weigh an appropriate amount of docetaxel in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve it, and add 3 times the molar amount of triethylamine. Then add an excess of succinyl chloride dissolved in anhydrous dichloromethane dropwise, and stir the reaction at room temperature for 24 hours. Then separate by column chromatography to obtain a single-end grafted intermediate product. Dissolve a certain amount of dihydroartemisinin obtained in anhydrous dichloromethane, and add 3 times the molar amount of triethylamine. Then add the intermediate dissolved in dichloromethane dropwise, and stir the reaction for 12 hours. The obtained target product, docetaxel-dihydroartemisinin prodrug molecule, is separated and purified as above to obtain an esterase-sensitive chemical bond-coupled docetaxel-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout the N 2 Under protection.

[0074] The synthetic route is illustrated as follows:

[0075]

[0076] Example 3: Synthesis of docetaxel-dihydroartemisinin prodrug molecules linked to thiodiacetic acid

[0077] Accurately weigh an excess of thiodiacetic acid in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add docetaxel dissolved in dichloromethane dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. Accurately weigh a certain amount of the intermediate product in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The obtained target product, docetaxel-dihydroartemisinin prodrug molecule, is separated and purified as above to obtain redox-sensitive chemical bond-coupled docetaxel-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout N2 Under protection.

[0078] The synthetic route is illustrated as follows:

[0079]

[0080] Example 4: Synthesis of Cephalomannine-Dihydroartemisinin Prodrug Molecule Linked with Thiodiacetic Acid

[0081] Accurately weigh an excess of thiodiacetic acid in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add the dichloromethane-dissolved cephalomannine dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. Accurately weigh a certain amount of the intermediate product in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add the dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The obtained target product, the cephalomannine-dihydroartemisinin prodrug molecule is separated and purified as above to obtain the redox-sensitive chemical bond-coupled cephalomannine-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout N 2 Under protection.

[0082] The synthetic route is illustrated as follows:

[0083]

[0084] Example 5: Synthesis of docetaxel-dihydroartemisinin prodrug molecules linked to selenodiacetic acid

[0085] Accurately weigh an excess of selenodiacetic acid in a 50 mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add docetaxel dissolved in dichloromethane dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. The intermediate product obtained is dissolved in anhydrous dichloromethane, 1.5 times the molar amount of EDC and an equal amount of DMAP are added, and stir and activate in an ice bath for 1-2 hours. Then add dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The target product, paclitaxel-dihydroartemisinin prodrug molecule obtained is separated and purified as above to obtain esterase-sensitive chemical bond-coupled paclitaxel-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout the N 2 Under protection.

[0086] The synthetic route is illustrated as follows:

[0087]

[0088] Example 6: Synthesis of selenodiacetic acid-linked cephalomannine-dihydroartemisinin prodrug molecules

[0089] Accurately weigh an excess of selenodiacetic acid in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add the dichloromethane-dissolved cephalomannine dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. Accurately weigh a certain amount of the intermediate product in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add the dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The obtained target product, the cephalomannine-dihydroartemisinin prodrug molecule is separated and purified as above, to obtain the redox-sensitive chemical bond-coupled cephalomannine-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout N 2 Under protection.

[0090] The synthetic route is illustrated as follows:

[0091]

[0092] Example 7: Synthesis of 2,2'-dithiodiacetic acid-linked docetaxel-dihydroartemisinin prodrug molecules

[0093] Accurately weigh an excess of 2,2'-dithiodiacetic acid in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add docetaxel dissolved in dichloromethane dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. Accurately weigh a certain amount of the intermediate product in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The obtained target product, docetaxel-dihydroartemisinin prodrug molecule, is separated and purified as above to obtain redox-sensitive chemical bond-coupled docetaxel-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout the N 2 Under protection.

[0094] The synthetic route is illustrated as follows:

[0095]

[0096] Example 8: Synthesis of docetaxel-dihydroartemisinin prodrug molecules linked to 2,2'-diselenodiacetic acid

[0097] Accurately weigh an excess of 2,2'-diselenodiacetic acid in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add docetaxel dissolved in dichloromethane dropwise, and stir and react at room temperature for 24 hours. Then separate the intermediate product by column chromatography. Accurately weigh a certain amount of the intermediate product in a 50mL round-bottom flask, add anhydrous dichloromethane to dissolve, add 1.5 times the molar amount of EDC and an equal amount of DMAP, and stir and activate in an ice bath for 1-2 hours. Then add dihydroartemisinin dissolved in dichloromethane dropwise, and stir and react for 24 hours. The obtained target product, docetaxel-dihydroartemisinin prodrug molecule, is separated and purified as above to obtain redox-sensitive chemical bond-coupled docetaxel-dihydroartemisinin prodrug molecule. The above reaction is carried out throughout the N 2 Under protection.

[0098] Example 9: Preparation of prodrug-coupled self-assembled nanoparticles

[0099] An appropriate amount of taxane-artemisinin prodrug molecules are weighed and dissolved in a good solvent to obtain a molecular solution, the solution is mixed with water, and self-assembled prodrug nanoparticles are prepared by conventional anti-solvent precipitation method, solvent evaporation method, microfluidics technology or ultrasound method. The solvent is removed by dialysis, ultrafiltration or evaporation to obtain a highly efficient and stable self-assembled prodrug nanoparticle solution.

[0100] Example 10: In vitro release test of paclitaxel-dihydroartemisinin small molecule prodrug self-assembled nanoparticles

[0101] Under a water bath thermostatic shaker, PBS buffer containing 0.1% Tween-80 at pH 7.4 was used as the release medium, and 1 mL of the paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) of Example 9 was transferred to a blank 20 mL release medium containing a certain amount of esterase for incubation. At certain time intervals (0, 0.5, 1, 2, 4, 6, 12, 24, 36, 48 h), 0.5 mL of the release medium was taken and fresh release medium was added to investigate the drug release behavior of the prodrug molecule self-assembled nanoparticles and determine the drug release amount.

[0102] The results are as follows Figure 3 As shown, the paclitaxel-dihydroartemisinin prodrug molecules are self-assembled nanoparticles formed by connecting the two ends of the connecting arm with ester bonds, which will not affect the release behavior of the two types of drugs. Based on the fact that the expression of esterases in the tumor microenvironment is higher than that in normal tissues, the active drugs can be freely released in the form of ester bond hydrolysis and cleavage, and will not be restricted by binding.

[0103] Example 11: Cytotoxicity experiment of paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles

[0104] The MTT method was used to investigate the effects of paclitaxel-dihydroartemisinin prodrug self-assembled nanoparticles (PTX-CC-DHA) and paclitaxel injection in Example 9. Abraxane TM ) and dihydroartemisinin (DHA) on 4T1 cells. 4T1 cells were cultured at 5×10 3 The cells were seeded at a density of 100 cells / well in a 96-well cell plate and the plate was placed at 37°C and 5% CO 2 Cultured overnight in an incubator. After the cells adhered, the culture medium in the wells was aspirated and washed three times with PBS. Prodrug self-assembled nanoparticle solutions diluted with serum-free culture medium to different concentrations were added respectively and placed in an incubator for further incubation for 48 hours. 20 μL of MTT solution with a concentration of 5 mg / mL was added to each well. After continuing to incubate for 4 hours, the liquid in the well was aspirated, 150 μL of DMSO was added to each well, and the wells were shaken thoroughly on a shaker to fully dissolve the crystals. The absorbance of each well was measured at 570 nm using an enzyme-linked immunosorbent assay. The cell survival rate was calculated.

[0105] The results are as follows Figure 4 As shown, compared with the commercially available paclitaxel injection Abraxane TM ) and dihydroartemisinin (DHA), the cytotoxicity of the prodrug molecular nanoparticles is in the order of Taxol>Abraxane>DHA>PTX-CC-DHA. The series of paclitaxel-dihydroartemisinin prodrug molecular self-assembled nanoparticles (PTX-CC-DHA) with high drug content and low toxicity and side effects prepared by the present invention have high efficiency in killing tumor cells and low cytotoxicity and side effects. The reason is that the nanoparticles are self-assembled using the nanoprecipitation method, which is a "green" solution to the problem of poor water solubility of the two types of drugs, and no additional solubilization strategy is required, such as the use of solubilizers or carriers, avoiding the biocompatibility, complexity, and safety issues caused by solvents and carrier materials; combined medication is used to combine the two types of small molecule drugs through chemical coupling to achieve spatiotemporal coordinated transport, and synergistic enhancement through multiple pharmacological mechanisms, with excellent tumor treatment effects and clinical transformation prospects.

[0106] Example 12: In vivo efficacy of paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles

[0107] Balb / c mice (18-20 g) were randomly divided into 6 groups (n=6) and inoculated with 4T1 cells orthotopically. When the tumor size reached 100 mm 3 Around 2:00 pm, normal saline and commercially available paclitaxel injection were intravenously injected. Albumin-bound paclitaxel Dihydroartemisinin (DHA) and paclitaxel-dihydroartemisinin prodrug molecule self-assembled nanoparticles (PTX-CC-DHA) in Example 9 were injected intravenously once every 3 days at a dose of 5 mg / kg for 4 times. The weight of mice and the width and length of tumors were measured during the treatment period. At the end of the experiment, mice were euthanized according to institutional guidelines, and the tumors were dissected and weighed.

[0108] The results are as follows Figure 5 As shown, the in vivo anti-tumor effect of the prodrug molecule self-assembled nanoparticles in Example 9 is consistent with the expected effect: PTX-CC-DHA>Taxol>Abraxane>DHA.

[0109] The results are as follows Figure 6 As shown, there was no significant change in the body weight of mice in each group, indicating that the paclitaxel-dihydroartemisinin prodrug nanoparticles proposed in the present invention achieved the effect of increasing efficacy and reducing toxicity; the drug loading capacity was high and the water solubility was good, avoiding the use of highly toxic solubilizing agents, which was beneficial to reducing the adverse reactions caused by excipients and biomaterials; while having obvious anti-tumor effects, it did not cause significant nonspecific toxicity to the body, and was a safe and effective anticancer drug delivery system with excellent tumor treatment effects and clinical transformation prospects.

Claims

1. A conjugated prodrug, characterized in that: The specific structure is as follows:

2. A nanoparticle, characterized in that: It is self-assembled by the coupled prodrug according to claim 1.

3. The nanoparticles according to claim 2, characterized in that The particle size is 10 to 900 nm, and the polydispersity coefficient is less than 0.

3.

4. A method for preparing nanoparticles according to claim 2, characterized in that: The coupled prodrug is dissolved and then mixed with water, and the solvent is removed to obtain the nanoparticles.

5. A pharmaceutical composition, characterized in that Comprising the coupled prodrug according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The preparation form is selected from injection, tablet, capsule, granule, powder, suppository, pill, cream, paste, gel, powder, oral solution, inhalant, suspension, dry suspension, patch and lotion.

7. Use of the coupled prodrug according to claim 1 in the preparation of a drug for preventing and / or treating tumors.

8. The use according to claim 7, characterized in that: The tumor is selected from breast cancer, liver cancer, ovarian cancer, colon cancer, lung cancer, melanoma, head and neck cancer, lymphoma, pancreatic cancer, brain tumor or blood cancer.

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