All-trans retinoic acid-gemcitabine prodrug and its preparation and application

Abstract

The present invention discloses an all-trans retinoic acid-gemcitabine prodrug and its preparation and application. All-trans retinoic acid and gemcitabine are connected by ester bonds through chemical synthesis, and the obtained all-trans retinoic acid-gemcitabine prodrug has the ability to self-assemble in water, and the ferroptosis agonist RSL3 can be wrapped into nanoparticles in the self-assembly process to obtain all-trans retinoic acid-gemcitabine / RSL3 nanomaterials. The all-trans retinoic acid-gemcitabine / RSL3 nanomaterials of the present invention have multi-drug delivery capabilities and esterase-responsive drug release performance, stem cell differentiation treatment performance, ferroptosis and apoptosis-inducing performance, and can be used to prepare drugs for treating breast cancer, and are used for the combined treatment of differentiation, chemotherapy, and ferroptosis of breast cancer tumor stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation and application of organic drug carriers, relates to a biomedical material and its preparation and application, and specifically relates to an all-trans retinoic acid-gemcitabine prodrug and its preparation and application. Background Art

[0002] Cancer stem cells are a small subpopulation of tumor cells that are believed to have a strong ability to regenerate themselves and can differentiate into a variety of tumor cells. More and more evidence shows that cancer stem cells play an important role in the occurrence, metastasis and recurrence of tumors, and their high drug resistance makes traditional chemotherapy ineffective. Therefore, therapeutic strategies targeting cancer stem cells will be of great help in the treatment of tumors.

[0003] All-trans retinoic acid (ATRA), a first-line drug approved by the Food and Drug Administration for the treatment of leukemia, has also been shown to have the ability to promote the differentiation of cancer stem cells. Many studies have reported that ATRA can reduce the stemness of cancer cells by converting cancer stem cells into normal cancer cells, which are more sensitive to currently available treatments.

[0004] Ferroptosis is a newly discovered type of cell death that is iron-related and has different morphological and physiological characteristics from apoptosis. Studies have found that cancer stem cells that are highly resistant to chemotherapy are still sensitive to ferroptosis. During ferroptosis, the Fenton reaction produces a large number of hydroxyl radicals, leading to the accumulation of lipid peroxidation (LPO), which destroys the redox balance of the cell and causes cell death. The completely different death mechanism of apoptosis allows ferroptosis to be used as a treatment for chemotherapy-resistant tumor cells. In order to enhance the activity of ferroptotic cells, many studies aim to regulate key proteins in the ferroptotic signaling pathway, such as GPX4, to protect cells from ferroptosis by reducing LPO. Therefore, intracellular LPO can be increased by delivering GPX4 inhibitors to tumor cells to promote ferroptosis. RSL3 is one of the GPX4 inhibitors, but its poor water solubility and severe systemic toxicity affect its application. Summary of the invention

[0005] In order to solve the problems existing in the background technology, one of the purposes of the present invention is to provide an all-trans retinoic acid-gemcitabine prodrug, which is a small molecule compound obtained by an esterification reaction, and comprises all-trans retinoic acid and gemcitabine connected by an ester bond in a ratio of 1:1. The all-trans retinoic acid-gemcitabine prodrug is prepared by an esterification reaction using all-trans retinoic acid and gemcitabine as raw materials. The carboxyl group of all-trans retinoic acid is covalently linked to the 5-hydroxyl group of gemcitabine through an ester bond. The structural formula of the all-trans retinoic acid-gemcitabine prodrug is:

[0006]

[0007] The synthesis of the all-trans retinoic acid-gemcitabine prodrug is obtained by the following steps:

[0008] (1) All-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate are dissolved in a sufficient amount of dry N,N-dimethylformamide. The molar equivalent of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate is 1 to 4 times that of all-trans retinoic acid, and stirred at room temperature for 0.5 to 2 hours.

[0009] (2) Dissolve gemcitabine and N,N-diisopropylethylamine in a sufficient amount of dry N,N-dimethylformamide. The molar equivalent of gemcitabine is 2 to 10 times that of all-trans retinoic acid in step 2.1 above, and the molar equivalent of N,N-diisopropylethylamine is 4 to 8 times that of all-trans retinoic acid in step 2.1 above. Then, use a constant pressure dropping funnel to dropwise add the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate in step 2.1 into the gemcitabine and N,N-diisopropylethylamine solution, and stir at room temperature for 12 hours.

[0010] (3) The N,N-dimethylformamide solvent in the above step 2.2) is removed, and a sufficient amount of dichloromethane is added, and the mixture is washed with citric acid 2 to 3 times, washed with saturated sodium bicarbonate 2 to 3 times, and washed with saturated saline 2 to 3 times. The mixture is separated by a silica gel column to obtain an orange-yellow solid, which is the all-trans retinoic acid-gemcitabine prodrug.

[0011] The specific reaction equation is as follows:

[0012]

[0013] In the reaction formula, structure 1 is gemcitabine, structure 2 is all-trans retinoic acid, structure 3 is all-trans retinoic acid-gemcitabine prodrug, PyBop is the abbreviation of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, and DIPEA is the abbreviation of N,N-diisopropylethylamine.

[0014] The second purpose of the present invention is to provide an application of the all-trans retinoic acid-gemcitabine prodrug in the preparation of all-trans retinoic acid-gemcitabine self-assembled nanomaterials, wherein the application uses the all-trans retinoic acid-gemcitabine prodrug as a raw material, and through a nanoprecipitation method, the drug dissolved in the organic phase is added dropwise to the aqueous phase to obtain self-assembly to form the target nanomaterial.

[0015] The specific preparation is achieved by the following method: dissolving the all-trans retinoic acid-gemcitabine prodrug in dimethyl sulfoxide, and then slowly adding the obtained solution into ultrapure water under ultrasound, wherein the volume of the ultrapure water is 9-20 times that of the dimethyl sulfoxide, thereby obtaining the all-trans retinoic acid-gemcitabine self-assembled nanomaterial.

[0016] The third object of the present invention is to provide the use of the all-trans retinoic acid-gemcitabine prodrug in the preparation of all-trans retinoic acid-gemcitabine / RSL3 nanomaterials.

[0017] The all-trans retinoic acid-gemcitabine / RSL3 nanomaterial uses all-trans retinoic acid-gemcitabine prodrug and ferroptosis agonist RSL3 as raw materials, utilizes the self-assembly performance of all-trans retinoic acid-gemcitabine prodrug, simultaneously encapsulates RSL3 into nanoparticles, and obtains the material by dropping the drug dissolved in the organic phase into the aqueous phase through the nanoprecipitation method.

[0018] Specifically, it is obtained by the following preparation method: all-trans retinoic acid-gemcitabine prodrug and ferroptosis agonist RSL3 are dissolved in dimethyl sulfoxide, the mass of all-trans retinoic acid-gemcitabine prodrug is 1 to 10 times that of ferroptosis agonist RSL3, and then the obtained solution is slowly added dropwise into ultrapure water under ultrasound, the volume of ultrapure water is 9-20 times that of dimethyl sulfoxide, so as to obtain all-trans retinoic acid-gemcitabine / RSL3 nanomaterial.

[0019] The fourth purpose of the present invention is to provide the use of the all-trans retinoic acid-gemcitabine / RSL3 nanomaterial in the preparation of a drug for treating breast cancer. The all-trans retinoic acid-gemcitabine / RSL3 nanomaterial of the present invention has multi-drug delivery capability and esterase-responsive drug release performance, stem cell differentiation treatment performance, ferroptosis and apoptosis-inducing performance, and can be used for the combined treatment of differentiation, chemotherapy, and ferroptosis of breast cancer tumor stem cells.

[0020] The technical effects of the present invention are as follows:

[0021] The all-trans retinoic acid-gemcitabine prodrug prepared by the method of the present invention has amphiphilicity, can be self-assembled into a nanomaterial, and the ferroptosis agonist RSL3 is simultaneously loaded into the nanoparticles. The present invention delivers all-trans retinoic acid, gemcitabine and RSL3 simultaneously, and can depolymerize and release the drug under the action of esterase. All-trans retinoic acid can promote the differentiation of tumor stem cells into ordinary tumor cells, and gemcitabine and RSL3 can cause apoptosis and ferroptosis of tumor cells, respectively. In summary, the all-trans retinoic acid-gemcitabine / RSL3 nanomaterial of the present invention is used as a drug carrier, has multifunctionality, multi-drug delivery ability and esterase-responsive drug release performance, stem cell differentiation treatment performance, ferroptosis and apoptosis-inducing performance, and can be used for the combined treatment of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : H NMR spectrum of all-trans retinoic acid-gemcitabine prodrug.

[0023] Figure 2 : High-resolution mass spectrum of all-trans retinoic acid-gemcitabine prodrug.

[0024] Figure 3 : Transmission electron microscopy images of all-trans retinoic acid-gemcitabine self-assembled nanomaterials.

[0025] Figure 4 : Transmission electron microscopy image of all-trans retinoic acid-gemcitabine / RSL3 nanomaterials.

[0026] Figure 5 : Gemcitabine release curves of all-trans retinoic acid-gemcitabine self-assembled nanomaterials and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials. 5-1 is the gemcitabine release curve of all-trans retinoic acid-gemcitabine self-assembled nanomaterials, 5-2 is the gemcitabine release curve of all-trans retinoic acid-gemcitabine / RSL3 nanomaterials, 5-3 is the gemcitabine release curve of all-trans retinoic acid-gemcitabine self-assembled nanomaterials under esterase stimulation, and 5-4 is the gemcitabine release curve of all-trans retinoic acid-gemcitabine / RSL3 nanomaterials under esterase stimulation.

[0027] Figure 6 : Cell viability curve after the materials acted on breast cancer 4T1. Among them, 6-1 is all-trans retinoic acid, 6-2 is RSL3, 6-3 is gemcitabine, 6-4 is all-trans retinoic acid-gemcitabine self-assembled nanomaterial, and 6-5 is all-trans retinoic acid-gemcitabine / RSL3 nanomaterial.

[0028] Figure 7 : Western blot of the effect of the materials on ferroptosis-related protein GPX4 in breast cancer 4T1 cells. 7-1 is the control group, 7-2 is all-trans-retinoic acid, 7-3 is RSL3, 7-4 is gemcitabine, 7-5 is all-trans retinoic acid-gemcitabine self-assembled nanomaterial, and 7-6 is all-trans retinoic acid-gemcitabine / RSL3 nanomaterial. GAPDH is the cell protein internal reference.

[0029] Figure 8 : Flow cytometric analysis of the changes in the proportion of stem cells (ALDH positive) caused by the materials in breast cancer 4T1 cells. 8-1 is the control group, 8-2 is all-trans-retinoic acid, 8-3 is RSL3, 8-4 is gemcitabine, 8-5 is all-trans retinoic acid-gemcitabine self-assembled nanomaterials, and 8-6 is all-trans retinoic acid-gemcitabine / RSL3 nanomaterials. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with embodiments and the accompanying drawings, but is not limited to the contents disclosed in the embodiments.

[0031] Example 1

[0032] 200 mg of gemcitabine (equivalent to 2 times of all-trans retinoic acid) and 196 mg of N,N-diisopropylethylamine were dissolved in 5 mL of dry N,N-dimethylformamide and placed in a glass bottle. 114 mg of all-trans retinoic acid and 296 mg of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate were dissolved in 5 mL of dry N,N-dimethylformamide and premixed at 25°C for 0.5 hours, then the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate was dripped into the bottle and stirred overnight at 25°C. N,N-dimethylformamide was removed by vacuum pump, and the residue was dissolved in 50 mL of dichloromethane and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated saline (50 mL, twice). DCM was removed in vacuo and the product was purified by silica gel chromatography with dichloromethane:methanol (10:1) to obtain an orange-yellow oily substance (50.1 mg, yield 23.4%), which was all-trans retinoic acid-gemcitabine prodrug.

[0033] Example 2

[0034] 500 mg of gemcitabine (equivalent to 5 times that of all-trans retinoic acid) and 196 mg of N,N-diisopropylethylamine were dissolved in 5 mL of dry N,N-dimethylformamide and placed in a glass bottle. 114 mg of all-trans retinoic acid and 296 mg of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate were dissolved in 5 mL of dry N,N-dimethylformamide and premixed at 25°C for 0.5 hours, then the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate was dripped into the bottle and stirred overnight at 25°C. N,N-dimethylformamide was removed by a vacuum pump, and the residue was dissolved in 50 mL of dichloromethane and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated saline (50 mL, twice). DCM was removed in vacuo and purified by silica gel chromatography with dichloromethane:methanol (10:1) to obtain an orange-yellow oily substance (97.8 mg, yield 45.7%), which was all-trans retinoic acid-gemcitabine prodrug.

[0035] Example 3

[0036] 1000 mg of gemcitabine (equivalent to 10 times that of all-trans retinoic acid) and 196 mg of N,N-diisopropylethylamine were dissolved in 5 mL of dry N,N-dimethylformamide and placed in a glass bottle. 114 mg of all-trans retinoic acid and 296 mg of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate were dissolved in 5 mL of dry N,N-dimethylformamide and premixed at 25°C for 0.5 hours, then the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate was dripped into the bottle and stirred overnight at 25°C. N,N-dimethylformamide was removed by vacuum pump, and the residue was dissolved in 50 mL of dichloromethane and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated saline (50 mL, twice). DCM was removed in vacuo and purified by silica gel chromatography with dichloromethane:methanol (10:1) to obtain an orange-yellow oily substance (103.1 mg, yield 48.2%), which was all-trans retinoic acid-gemcitabine prodrug.

[0037] Using H NMR spectroscopy ( Figure 1 ) and high-resolution mass spectrometry ( Figure 2 ) Characterization The all-trans retinoic acid-gemcitabine prodrug obtained in Example 2 was characterized to prove that the all-trans retinoic acid-gemcitabine prodrug had a correct structure and was successfully synthesized.

[0038] Example 4

[0039] (1) 5 mg of all-trans retinoic acid-gemcitabine prodrug was dissolved in 100 μL of dimethyl sulfoxide, and slowly added dropwise to 900 μL of ultrapure water under ultrasound to obtain all-trans retinoic acid-gemcitabine self-assembled nanomaterials;

[0040] (2) 5 mg of all-trans retinoic acid-gemcitabine prodrug and 1 mg of RSL3 were dissolved in 100 μL of dimethyl sulfoxide, and slowly added dropwise to 900 μL of ultrapure water under ultrasound to obtain all-trans retinoic acid-gemcitabine / RSL3 nanomaterials;

[0041] (3) The all-trans retinoic acid-gemcitabine self-assembled nanomaterials and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials prepared above were characterized by transmission electron microscopy images. Figure 3 As shown, both nanomaterials are uniformly dispersed spheres with a diameter of about 100 nanometers;

[0042] (4) The above-prepared all-trans retinoic acid-gemcitabine self-assembled nanomaterials and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials were placed in a 500 molecular weight dialysis bag without or with esterase. The dialysis bag was placed in a phosphate buffer and placed in a shaker at 37 degrees Celsius. Samples were taken at regular intervals to detect the gemcitabine release rate. The results are as follows: Figure 4As shown, both nanomaterials exhibited an esterase-responsive enhancement of gemcitabine release rates.

[0043] Example 5

[0044] (1) 4T1 cells were plated on a flat-bottom 96-well cell plate. After the cells adhered to the plate, all-trans retinoic acid, RSL3, gemcitabine, all-trans retinoic acid-gemcitabine self-assembled nanomaterials, and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials were added in a concentration gradient. After 72 hours, the cell viability of each group was detected using CCK-8 reagent, and a cell viability curve was drawn as shown in the figure. Figure 6 As shown. It can be seen that all-trans retinoic acid-gemcitabine self-assembled nanomaterials and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials have stronger cytotoxicity than gemcitabine on breast cancer 4T1 cells;

[0045] (2) 4T1 cells were plated on a flat-bottomed 6-well cell plate. After the cells adhered to the plate, all-trans retinoic acid, RSL3, gemcitabine, all-trans retinoic acid-gemcitabine self-assembled nanomaterials, and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials were added in an equivalent amount of 500 nM RSL3. Cell proteins were extracted 48 hours later, and a Western blotting experiment of GPX4 was performed, with GAPDH as the internal reference of cell proteins. The results are shown in Figure 2. Figure 7 As shown, RSL3 and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials caused a decrease in GPX4, indicating that ferroptosis of cells was induced;

[0046] (3) 4T1 cells were plated on a flat-bottom 24-well cell plate with ultra-low adsorption. Five days later, all-trans retinoic acid, RSL3, gemcitabine, all-trans retinoic acid-gemcitabine self-assembled nanomaterials, and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials were added in an equivalent amount of 500 nM all-trans retinoic acid. The proportion of cells positive for acetaldehyde dehydrogenase (ALDH) was detected using the ALDEFLUOR kit, which is the proportion of tumor stem cells. The results are shown in Figure 2. Figure 8 As shown, all-trans retinoic acid-gemcitabine self-assembled nanomaterials and all-trans retinoic acid-gemcitabine / RSL3 nanomaterials have a stronger ability to reduce the proportion of stem cells in breast cancer 4T1 cells than all-trans retinoic acid.

[0047] The above description is only an example of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An all-trans retinoic acid-gemcitabine prodrug, characterized in that: The structural formula of the all-trans retinoic acid-gemcitabine prodrug is: Prepared by the following steps: (1) dissolving all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate in N,N-dimethylformamide, wherein the molar equivalent of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate is 1 to 4 times that of all-trans retinoic acid, and stirring at room temperature for 0.5 to 2 hours; (2) dissolving gemcitabine and N,N-diisopropylethylamine in N,N-dimethylformamide, wherein the molar equivalent of gemcitabine is 2 to 10 times that of all-trans retinoic acid in step (1), and the molar equivalent of N,N-diisopropylethylamine is 4 to 8 times that of all-trans retinoic acid in step (1), and then using a constant pressure dropping funnel to dropwise add the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate in step (1) into the gemcitabine and N,N-diisopropylethylamine solution, and stirring at room temperature for 12 hours; (3) Remove the N,N-dimethylformamide solvent in step (2), add dichloromethane, wash with citric acid 2 to 3 times, wash with saturated sodium bicarbonate 2 to 3 times, wash with saturated brine 2 to 3 times, and separate with silica gel column to obtain an orange-yellow solid, which is all-trans retinoic acid-gemcitabine prodrug.

2. An all-trans retinoic acid-gemcitabine self-assembled nanomaterial, characterized in that: Using the all-trans retinoic acid-gemcitabine prodrug in claim 1 as a raw material, forming an all-trans retinoic acid-gemcitabine self-assembled nanomaterial by self-assembly; Prepared by the following steps: The all-trans retinoic acid-gemcitabine prodrug of claim 1 is dissolved in dimethyl sulfoxide, and the obtained solution is slowly added dropwise into ultrapure water under ultrasound, wherein the volume of the ultrapure water is 9-20 times that of the dimethyl sulfoxide, thereby obtaining the all-trans retinoic acid-gemcitabine self-assembled nanomaterial.

3. An all-trans retinoic acid-gemcitabine / RSL3 nanomaterial, characterized in that: The all-trans retinoic acid-gemcitabine / RSL3 nanomaterial is obtained by using the all-trans retinoic acid-gemcitabine prodrug and the ferroptosis agonist RSL3 in claim 1 as raw materials, utilizing the self-assembly performance of the all-trans retinoic acid-gemcitabine prodrug in claim 1, and simultaneously encapsulating RSL3 into nanoparticles through a nanoprecipitation method; Prepared by the following steps: The all-trans retinoic acid-gemcitabine prodrug and the ferroptosis agonist RSL3 in claim 1 are dissolved in dimethyl sulfoxide, wherein the mass of the all-trans retinoic acid-gemcitabine prodrug is 1 to 10 times that of the ferroptosis agonist RSL3, and then the obtained solution is slowly added dropwise into ultrapure water under ultrasound, wherein the volume of the ultrapure water is 9 to 20 times that of the dimethyl sulfoxide, thereby obtaining the all-trans retinoic acid-gemcitabine / RSL3 nanomaterial.

4. Use of the all-trans retinoic acid-gemcitabine / RSL3 nanomaterial according to claim 3 in the preparation of a drug for treating breast cancer, wherein the all-trans retinoic acid-gemcitabine / RSL3 nanomaterial is used as a drug carrier to jointly treat breast cancer tumors through its multidrug delivery capability, esterase-responsive drug release performance, stem cell differentiation therapeutic performance, and ferroptosis and apoptosis performance of tumor cells.

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