Preparation and application of intelligent responsive doxorubicin monomeric prodrug bridged by trisulfide bond

Abstract

This invention discloses the preparation and application of intelligently responsive trisulfide-bridged doxorubicin monomer prodrugs, belonging to the field of pharmaceutical technology. This invention prepares doxorubicin-stearic acid prodrug (DOX-SSS-SA) with stearic acid as a side chain and doxorubicin-vitamin E prodrug (DOX-SSS-VE) with vitamin E as a side chain, which are used to prepare antitumor drugs. Utilizing the dual redox hypersensitivity of trisulfide bonds, intelligent responsive activation of the prodrugs within tumor cells can be achieved, ensuring the antitumor efficacy and safety of the prodrugs. The preparation method of this invention is simple and easy to implement, providing a new strategy and more options for developing intelligently responsive drug delivery systems, meeting the urgent clinical need for highly effective chemotherapy agents.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation and application of trisulfide-bridged doxorubicin monomer prodrugs, specifically the synthesis of trisulfide-bridged doxorubicin-vitamin E prodrug and trisulfide-bridged doxorubicin-stearyl prodrug, and their application in tumor treatment. Background Technology

[0002] Cancer is one of the major public health problems facing the world and the second leading cause of death. Conquering cancer remains a major medical challenge. Current cancer treatments mainly include traditional surgery, chemotherapy, radiotherapy, and emerging immunotherapy. Chemotherapy is a systemic treatment that uses chemotherapeutic drugs to kill tumor cells. Compared to local treatments like surgery and radiotherapy, chemotherapy is effective for potential metastatic lesions or tumors with a tendency to spread systemically, especially for inoperable or clinically metastatic advanced-stage tumors. There are many types of chemotherapeutic drugs with different mechanisms of action. Among them, the anthracycline drug doxorubicin is a broad-spectrum antitumor drug currently used clinically, effective against various tumors. Its mechanism of action involves embedding itself into the DNA double helix structure, affecting nucleic acid synthesis, thereby killing tumor cells; it is a cell cycle nonspecific drug. However, while treating the disease, doxorubicin can also cause cardiotoxicity, leading to serious adverse reactions such as arrhythmia, heart failure, and hypertrophic cardiomyopathy. It is worth noting that there is no absolutely safe dosage for doxorubicin in clinical practice. Genetic differences in patients' metabolism of anthracyclines mean that even low doses of doxorubicin can cause cardiotoxicity, which greatly limits its clinical use. Based on advancements in nanotechnology for drug delivery, doxorubicin hydrochloride liposome injection (trade name: Doxil) has effectively reduced its cardiotoxicity; however, up to 25% of patients experience severe skin toxicity. The toxic side effects of doxorubicin still limit its clinical application and affect patient prognosis. These issues highlight the urgent need to improve the selectivity of chemotherapeutic drugs, enhance their safety from the source, and construct a highly efficient and low-toxicity intelligent delivery system for antitumor drugs to overcome the limitations of clinical chemotherapy.

[0003] Prodrug technology involves modifying the chemical structure of a parent drug to obtain prodrug compounds, thereby improving the drug's adverse properties. Prodrugs themselves have little or no biological activity; after metabolism in vivo, they become active substances. This process helps increase drug bioavailability, enhance drug targeting and selectivity, and reduce toxic side effects. Unlike normal cells, tumor cells continuously proliferate and grow rapidly, exhibiting very high levels of reactive oxygen species (ROS). Simultaneously, to avoid damage caused by oxidative stress, the concentration of the reducing agent glutathione (GSH) within tumor cells is more than four times that of normal cells, maintaining a high level of redox homeostasis. The high redox level of tumor cells, unlike normal cells, provides a basis for designing intelligent prodrugs. Numerous studies have introduced redox-sensitive bonds, such as monosulfide and disulfide bonds, into prodrug design to achieve tumor-responsive drug release and selective killing of tumor cells. Trisulfide bonds are novel, higher-level redox-sensitive bonds with three redox reaction sites and a high redox potential, exhibiting higher GSH sensitivity than monosulfide and disulfide bonds. Furthermore, trisulfide bonds also exhibit oxidative sensitivity when linked to drugs via ester bonds. Therefore, prodrug molecules constructed using trisulfide bonds can respond hypersensitively to the tumor microenvironment, rapidly releasing active drugs to exert antitumor effects, thus improving the antitumor efficacy while reducing the toxic side effects on normal tissues. No research has yet been reported on doxorubicin monomeric prodrug molecules constructed using trisulfide bonds. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the preparation of a smart responsive trisulfide-bridged doxorubicin monomer prodrug and its application in tumor treatment, providing new strategies and more options for developing smart responsive drug delivery systems for the tumor microenvironment, and meeting the urgent clinical need for highly efficient chemotherapeutic agents.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a trisulfide-bridged prodrug, as shown in general formula (I):

[0007]

[0008] Wherein, R is stearic acid group, stearyl alcohol group, cholesterol, vitamin E, oleic acid-ethylene glycol ester, oleyl alcohol, linolenic acid alcohol, or linoleyl alcohol; the trisulfide bridging group is a dicarboxylic acid compound containing a trisulfide bridge, including 2,2'-trithiodiacetic acid, 3,3'-trithiodipropionic acid, and 4,4'-trithiodibutyric acid; Drug is an antitumor drug, wherein the antitumor drug is selected from taxanes, anthraquinones, nucleosides, camptothecins, platinum compounds, vincristine alkaloids, podosides, and artemisinin compounds.

[0009] Based on the above technical solution, R is further selected from stearic acid group and vitamin E; the trisulfide bridging group is 3,3'-trithiodipropionic acid; and the drug is doxorubicin.

[0010] Based on the above technical solution, the structure of the doxorubicin-stearic acid prodrug (DOX-SSS-SA) with trisulfide bonds bridging the stearic acid group as a side chain is as follows:

[0011]

[0012] Based on the above technical solution, the structure of the doxorubicin-vitamin E prodrug (DOX-SSS-VE) with vitamin E as the side chain and a trisulfide bond bridged is as follows:

[0013]

[0014] This invention provides a method for preparing the trisulfide-bridged prodrug, comprising the following steps:

[0015] (1) At 30-60℃, bromopropionic acid aqueous solution is slowly added dropwise to sodium thiosulfate pentahydrate aqueous solution under stirring, and the reaction is continued for 2-6 hours under inert gas protection. After cooling to room temperature, sodium sulfide nonahydrate aqueous solution is added dropwise, and the reaction is carried out at room temperature for 10-16 hours. 3,3'-trithiodipropionic acid is obtained by separation and purification.

[0016] (2) Dissolve the 3,3'-trithiodipropionic acid obtained in step (1) in acetic anhydride, stir at room temperature for 1 to 5 hours, add stearic acid / vitamin E and DMAP, stir at room temperature for 0.5 to 2 hours, and purify to obtain trisulfide-bridged stearic acid or trisulfide-bridged vitamin E intermediate.

[0017] (3) Dissolve the trisulfide-bridged stearic acid or the trisulfide-bridged vitamin E intermediate, HBTU and DIPEA obtained in step (2) in DMF, activate in an ice bath for 0.5-2 h, add DOX, react at room temperature in the dark for 20-50 h, and separate and purify to obtain the trisulfide-bridged prodrug.

[0018] Based on the above technical solution, further, in step (1), the concentration of the aqueous solution of sodium thiosulfate pentahydrate is 0.1-1 g / mL, the concentration of the aqueous solution of bromopropionic acid is 0.1-0.5 g / mL, the concentration of the aqueous solution of sodium sulfide nonahydrate is 0.05-0.2 g / mL, and the molar ratio of sodium thiosulfate pentahydrate, bromopropionic acid and sodium sulfide nonahydrate is 1:0.5-0.9:0.1-0.5.

[0019] Based on the above technical solution, further, the molar ratio of 3,3'-trithiodipropionic acid to acetic anhydride in step (2) is 1:2 to 6; the molar ratio of stearic acid / vitamin E, DMAP to 3,3'-trithiodipropionic acid is 1:0.1 to 0.3:1 to 3.

[0020] Based on the above technical solution, further, in step (3), the molar ratio of trisulfide-bridged stearic acid or trisulfide-bridged vitamin E intermediate, HBTU, DIPEA and DOX is 1:1 to 2:1 to 3:1.

[0021] Based on the above technical solution, furthermore, step (3) is carried out under the protection of inert gas throughout the entire process.

[0022] A pharmaceutical composition comprising the trisulfide-bridged prodrug and a pharmaceutically acceptable carrier and excipient.

[0023] The present invention also provides the use of the trisulfide-bridged prodrug or the above-described pharmaceutical composition in the preparation of antitumor drugs.

[0024] The advantages of this invention over the prior art are as follows:

[0025] (1) This invention designs and synthesizes a monomeric prodrug of doxorubicin containing trisulfide bonds bridging stearic acid or vitamin E as side chains. The long carbon chain structure of stearic acid can improve the flexibility of molecules, disrupt the tight packing between molecules, thereby improving the physicochemical properties of chemotherapeutic drugs and making them easier to cross cell membranes, thus having better pharmacokinetic behavior and antitumor effects. On the other hand, stearic acid has good biocompatibility, safety and high stability, which can improve the stability of drugs in the normal internal environment. Vitamin E is an important antioxidant in the body, which can scavenge free radicals in the body, reduce the level of lipid peroxides, and to a certain extent enhance the body's disease resistance. It can also work synergistically with antitumor drugs to inhibit the growth and metastasis of tumors. Moreover, the synthesis method is simple and easy to implement.

[0026] (2) This invention investigated the cytotoxicity of trisulfide-bridged doxorubicin monomer prodrugs. Based on the comprehensive experimental results, due to the ultrasensitive reduction response of trisulfide bonds, both trisulfide-bridged doxorubicin monomer prodrugs can intelligently respond to the abnormally high redox levels of tumor cells and have good anti-tumor cell proliferation effects. This invention provides new strategies and more options for the development of intelligent responsive drug delivery systems, meeting the urgent clinical demand for highly effective chemotherapy agents. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1For the structural verification of the trisulfide-bridged doxorubicin-stearic acid prodrug (DOX-SSS-SA) of Example 1 of the present invention, wherein A: mass spectrum of DOX-SSS-SA, and B: high performance liquid chromatography purity spectrum of DOX-SSS-SA.

[0029] Figure 2 For the structural verification of the trisulfide-bridged doxorubicin-vitamin E prodrug (DOX-SSS-VE) in Example 2 of the present invention, wherein A: mass spectrum of DOX-SSS-VE, and B: high performance liquid chromatography purity spectrum of DOX-SSS-VE.

[0030] Figure 3 The diagram shows the cytotoxicity of the trisulfide-bridged doxorubicin-stearic acid prodrug (DOX-SSS-SA) in Example 1 and the trisulfide-bridged doxorubicin-vitamin E prodrug (DOX-SSS-VE) in Example 2 to 4T1 cells. Detailed Implementation

[0031] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0032] Example 1: Synthesis of trisulfide-bridged doxorubicin-stearic acid prodrug (DOX-SSS-SA)

[0033] First, prepare 10 mL of an aqueous solution of sodium thiosulfate pentahydrate (concentration 0.5 g / mL). In an oil bath at 50 °C, dissolve 2.3 g of bromopropionic acid in 20 mL of pure water and slowly add it dropwise to the solution with stirring. Continue the reaction under nitrogen protection for 5 h. After the reaction is complete, allow the reaction solution to cool to room temperature. Dissolve 1.1 g of sodium thiosulfate nonahydrate in 10 mL of pure water and add it dropwise to the cooled reaction solution. React overnight at room temperature for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate. Combine the separated ethyl acetate layers and evaporate to dryness to obtain crude 3,3'-trithiodipropionic acid, which is then purified by preparative liquid chromatography.

[0034] Subsequently, 1.9 g of 3,3'-trithiodipropionic acid was added to a 50 mL round-bottom flask and dissolved in 3 mL of acetic anhydride. The mixture was stirred at room temperature for 2 hours, and the reaction process was monitored by thin-layer chromatography. Then, 20 mL of toluene was added to the system in three portions, and the mixture was dried by vacuum distillation. The resulting product was dissolved in 20 mL of dichloromethane, and 0.56 g of stearic acid and 0.025 g of DMAP were added. The mixture was stirred at room temperature for 1 hour, and the reaction process was monitored by thin-layer chromatography. The trisulfide-bridged stearic acid intermediate was purified by silica gel column chromatography.

[0035] Finally, 0.91 g of intermediate product, 1 g of HBTU and 0.32 mL of DIPEA (molar ratio of intermediate product, HBTU and DIPEA is 1:1.5:1) were dissolved in 50 mL of anhydrous N,N-dimethylformamide, and the mixture was placed in an ice bath for 1 h. Then, 1 g of doxorubicin was added, and the mixture was stirred at room temperature for another 24 h. The reaction process was monitored by thin-layer chromatography, and the target product was separated and purified by preparative liquid chromatography. The entire reaction was carried out under N2 protection.

[0036] The structure of DOX-SSS-SA prepared in Example 1 was verified by mass spectrometry, and the purity of DOX-SSS-SA prepared in Example 1 was verified by high performance liquid chromatography. The results are as follows: Figure 1 As shown. The mass spectrometry results are MS(ESI) m / z [M+Na]. + =1042 and [M+K] + =1058. The purity results show that the purity of DOX-SSS-SA is 97.395%, which meets the requirements for subsequent experiments.

[0037] Example 2: Synthesis of a trisulfide-bridged doxorubicin-vitamin E prodrug (DOX-SSS-VE)

[0038] First, prepare 10 mL of an aqueous solution of sodium thiosulfate pentahydrate (concentration 0.5 g / mL). In an oil bath at 50 °C, dissolve 2.3 g of bromopropionic acid in 20 mL of pure water and slowly add it dropwise to the solution with stirring. Continue the reaction under nitrogen protection for 5 h. After the reaction is complete, allow the reaction solution to cool to room temperature. Dissolve 1.1 g of sodium thiosulfate nonahydrate in 10 mL of pure water and add it dropwise to the cooled reaction solution. React overnight at room temperature for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate. Combine the separated ethyl acetate layers and evaporate to dryness to obtain crude 3,3'-trithiodipropionic acid, which is then purified by preparative liquid chromatography.

[0039] Subsequently, 1.9 g of the obtained 3,3'-trithiodipropionic acid was added to a 50 mL round-bottom flask and dissolved in 3 mL of acetic anhydride. The mixture was stirred at room temperature for 2 hours, and the reaction process was monitored by thin-layer chromatography. Then, 20 mL of toluene was added to the system in three portions, and the mixture was dried under reduced pressure by distillation. The resulting product was dissolved in 20 mL of dichloromethane, and 0.85 g of vitamin E and 0.025 g of DMAP were added. The mixture was stirred at room temperature for 1 hour, and the reaction process was monitored by thin-layer chromatography. The trisulfide-bridged vitamin E intermediate was purified by silica gel column chromatography.

[0040] Finally, 1.25 g of intermediate product, 1 g of HBTU and 0.32 mL of DIPEA (the molar ratio of intermediate product, HBTU and DIPEA was 1:1.5:1) were dissolved in 50 mL of anhydrous N,N-dimethylformamide, and the mixture was placed in an ice bath for 1 h. Then, 1 g of doxorubicin was added, and the mixture was stirred at room temperature for another 24 h. The reaction process was monitored by thin-layer chromatography, and the target product was separated and purified by preparative liquid chromatography. The entire reaction was carried out under N2 protection.

[0041] Mass spectrometry was used to verify the structure of DOX-SSS-VE prepared in Example 2, and high performance liquid chromatography was used to verify the purity of DOX-SSS-VE prepared in Example 2. The results are as follows: Figure 2 As shown. The mass spectrometry results are MS (ESI) m / z: [M+Na] + =1202 and [M+K] + =1234. The purity results show that the purity of DOX-SSS-SA is 96.838%, which meets the requirements for subsequent experiments.

[0042] Example 3: Cytotoxicity of trisulfide-bridged doxorubicin monomer prodrug

[0043] The MTT assay was used to investigate the toxicity of trisulfide-bridged doxorubicin monomer prodrug to mouse breast cancer (4T1) cells. First, morphologically sound cells were digested, diluted with culture medium to 10,000 cells / mL, and after homogenization, 200 μL of cell suspension was added to each well of a 96-well plate and incubated for 24 h to allow cell adhesion. After cell adhesion, DOX solution, and the trisulfide-bridged doxorubicin monomer prodrug solutions prepared in Examples 1 and 2 were added, respectively. All drug solutions used in this experiment were derived from the culture medium for the corresponding cells and were aseptically filtered through a 0.22 μm filter membrane. Add 200 μL of the test solution to each well, with three parallel wells for each concentration. 48 h after addition, remove the 96-well plate and add 20 μL of 5 mg / mL MTT solution to each well. Incubate for 4 h, then discard the culture medium. Invert the 96-well plate onto filter paper to thoroughly blot away any remaining liquid. Add 200 μL of DMSO to each well and shake for 10 min to dissolve the blue-purple crystals. Measure the absorbance of each well at 570 nm using a microplate reader after zeroing.

[0044] The results are as follows Figure 3 As shown, since the prodrugs require activation to exert their effects in cells, the cytotoxicity of both trisulfide-bridged doxorubicin monomer prodrugs is weaker than that of doxorubicin solution. However, due to the hypersensitive reduction response of trisulfide bonds, both trisulfide-bridged doxorubicin monomer prodrugs can intelligently respond to the abnormally high redox levels of tumor cells, exhibiting good anti-tumor cell proliferation effects. This will also be beneficial for reducing off-target toxicity and improving treatment safety in vivo.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A trisulfide-bridged prodrug, characterized in that, The structure of the trisulfide-bridged prodrug is as follows: or .

2. A process for the preparation of the trisulfide-bridged prodrug of claim 1, characterized in that, Includes the following steps: (1) At 30~60℃, bromopropionic acid aqueous solution was slowly added dropwise to sodium thiosulfate pentahydrate aqueous solution under stirring, and the reaction was continued for 2~6 h under inert gas protection. The temperature was lowered to room temperature, and then sodium sulfide nonahydrate aqueous solution was added dropwise. The reaction was carried out at room temperature for 10~16 h, and 3,3'-trithiodipropionic acid was obtained by separation and purification. (2) Dissolve the 3,3'-trithiodipropionic acid obtained in step (1) in acetic anhydride, stir at room temperature for 1-5 h, add stearic acid / vitamin E and DMAP, stir at room temperature for 0.5-2 h, and purify to obtain trisulfide-bridged stearic acid or trisulfide-bridged vitamin E intermediate. (3) Dissolve the trisulfide-bridged stearic acid or the trisulfide-bridged vitamin E intermediate, HBTU and DIPEA obtained in step (2) in DMF, activate in an ice bath for 0.5-2 h, add DOX, react at room temperature in the dark for 20-50 h, and separate and purify to obtain the trisulfide-bridged prodrug. The concentration of the aqueous solution of sodium thiosulfate pentahydrate in step (1) is 0.1~1 g / mL, the concentration of the aqueous solution of bromopropionic acid is 0.1~0.5 g / mL, the concentration of the aqueous solution of sodium sulfide nonahydrate is 0.05~0.2 g / mL, and the molar ratio of sodium thiosulfate pentahydrate, bromopropionic acid and sodium sulfide nonahydrate is 1:0.5~0.9:0.1~0.

5.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 3,3'-trithiodipropionic acid to acetic anhydride in step (2) is 1:2~6; the molar ratio of stearic acid / vitamin E, DMAP to 3,3'-trithiodipropionic acid is 1:0.1~0.3:1~3.

4. The production method according to claim 2, characterized by, The molar ratio of the trisulfide-bridged stearic acid or the trisulfide-bridged vitamin E intermediate, HBTU, DIPEA and DOX in step (3) is 1:1~2:1~3:

1.

5. The preparation method according to claim 2, characterized in that, Step (3) is carried out under the protection of inert gas throughout.

6. A pharmaceutical composition comprising a trisulfide-bridged prodrug as claimed in claim 1, and a pharmaceutically acceptable carrier and excipient.

7. The use of the trisulfide-bridged prodrug of claim 1 or the pharmaceutical composition of claim 6 in the preparation of an antitumor drug.

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