CN116284184A - All-trans retinoic acid-gemcitabine prodrug and preparation and application thereof - Google Patents
All-trans retinoic acid-gemcitabine prodrug and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses an all-trans retinoic acid-gemcitabine prodrug, and preparation and application thereof. All-trans retinoic acid and gemcitabine are connected through ester bonds through chemical synthesis, the obtained all-trans retinoic acid-gemcitabine prodrug has the capability of self-assembly in water, and an iron death agonist RSL3 can be wrapped in nano particles in self-assembly engineering, so that the all-trans retinoic acid-gemcitabine/RSL 3 nano material is obtained. The all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial has the drug release performance of multi-drug delivery capability and esterase response, the stem cell differentiation treatment performance and the iron death and apoptosis induction performance, can be used for preparing a drug for treating breast cancer, and is used for the combined treatment of differentiation, chemotherapy and iron death of breast cancer tumor stem cells.
Description
Technical Field
The invention belongs to the technical field of preparation and application of an organic drug carrier, relates to a biomedical material and preparation and application thereof, and in particular relates to an all-trans retinoic acid-gemcitabine prodrug and preparation and application thereof.
Background
Tumor stem cells are a small subset of tumor cells, and are believed to have a strong ability to self-renew, and to differentiate into a variety of tumor cells. There is growing evidence that tumor stem cells play an important role in the development, metastasis and recurrence of tumors, and their high resistance renders traditional chemotherapy ineffective. Thus, therapeutic strategies directed against tumor stem cells would greatly aid in the treatment of tumors.
All-trans retinoic acid (ATRA) is a first-line drug approved by the food and drug administration for the treatment of leukemia and has also been demonstrated to have the ability to promote differentiation of tumor stem cells. Many studies report that ATRA can reduce the stem nature of cancer cells by converting tumor stem cells into normal cancer cells, which are more sensitive to currently available treatments.
Iron death is a newly discovered type of cell death, associated with iron, with morphological and physiological characteristics that differ from apoptosis. The study found that tumor stem cells highly resistant to chemotherapy remain susceptible to iron death. During iron-iron death, the Fenton reaction produces a large number of hydroxyl radicals, resulting in accumulation of Lipid Peroxidation (LPO), disrupting the redox balance of the cell, leading to cell death. The disparate death mechanism of apoptosis allows iron death to be used as a treatment for chemotherapy-resistant tumor cells. To enhance the activity of iron-bearing tumor cells, many studies aimed at modulating key proteins of the iron-bearing tumor signaling pathway, such as GPX4, protect cells from iron death by reducing LPO. Thus, intracellular LPO can be increased by delivering GPX4 inhibitors to tumor cells, promoting iron sagging. RSL3 is one of the GPX4 inhibitors, but its poor water solubility and severe systemic toxicity affect its use.
Disclosure of Invention
In order to solve the problems in the prior art, one of the objects of the present invention is to provide an all-trans retinoic acid-gemcitabine prodrug, which belongs to a small molecule compound, is obtained by esterification reaction, and comprises all-trans retinoic acid and gemcitabine 1:1 are linked by an ester linkage. The all-trans retinoic acid-gemcitabine prodrug is prepared from all-trans retinoic acid and gemcitabine serving as raw materials through esterification reaction. The carboxyl group of all-trans retinoic acid is covalently linked to the 5-hydroxy group of gemcitabine via an ester linkage. The structural formula of the all-trans retinoic acid-gemcitabine prodrug is as follows:
the synthesis of the all-trans retinoic acid-gemcitabine prodrug is obtained by the following steps:
(1) All-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate are dissolved in a sufficient amount of dry N, N-dimethylformamide. The molar equivalent of the benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate is 1 to 4 times that of all-trans retinoic acid, and the mixture is stirred at normal temperature for 0.5 to 2 hours.
(2) Gemcitabine and N, N-diisopropylethylamine are dissolved 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 the above step 2.1, and the molar equivalent of N, N-diisopropylethylamine is 4 to 8 times that of all-trans retinoic acid in the above step 2.1. And then the solution containing all-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate in the step 2.1 is dropwise added into the gemcitabine and N, N-diisopropylethylamine solution by using a constant pressure dropping funnel, and the mixture is stirred at normal temperature for 12 hours.
(3) Removing the N, N-dimethylformamide solvent in the step 2.2), adding enough methylene dichloride, washing with citric acid for 2 to 3 times, washing with saturated sodium bicarbonate for 2 to 3 times, washing with saturated salt water for 2 to 3 times, and separating with a silica gel column to obtain orange yellow solid, namely the all-trans retinoic acid-gemcitabine prodrug.
The specific reaction equation is as follows:
in the reaction formula, the structure 1 is gemcitabine, the structure 2 is all-trans retinoic acid, the structure 3 is all-trans retinoic acid-gemcitabine prodrug, pyBop is the abbreviation of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, and DIPEA is the abbreviation of N, N-diisopropylethylamine.
The second purpose of the invention is to provide the application of the all-trans retinoic acid-gemcitabine prodrug in preparing the all-trans retinoic acid-gemcitabine self-assembled nanomaterial, wherein the application is to obtain the self-assembled nanomaterial by taking the all-trans retinoic acid-gemcitabine prodrug as a raw material, and adding the drug dissolved in an organic phase into an aqueous phase through a nano precipitation method.
The preparation is realized by the following steps: dissolving all-trans retinoic acid-gemcitabine prodrug in dimethyl sulfoxide, slowly dripping the obtained solution into ultrapure water under ultrasound, wherein the volume of ultrapure water is 9-20 times that of the dimethyl sulfoxide, and obtaining the all-trans retinoic acid-gemcitabine self-assembled nanomaterial.
It is a further object of the present invention to provide the use of the all-trans retinoic acid-gemcitabine prodrug in the preparation of all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial.
The all-trans retinoic acid-gemcitabine/RSL 3 nano material is obtained by taking all-trans retinoic acid-gemcitabine prodrug and an iron death agonist RSL3 as raw materials, utilizing the self-assembly performance of the all-trans retinoic acid-gemcitabine prodrug, coating RSL3 into nano particles, and dripping the medicine dissolved in an organic phase into an aqueous phase through a nano precipitation method.
The preparation method comprises the following steps: dissolving all-trans retinoic acid-gemcitabine prodrug and an iron death agonist RSL3 in dimethyl sulfoxide, wherein the mass of the all-trans retinoic acid-gemcitabine prodrug is 1 to 10 times that of the iron death agonist RSL3, slowly dripping the obtained solution into ultrapure water under ultrasound, and obtaining the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial by the volume of ultrapure water being 9 to 20 times that of the dimethyl sulfoxide.
The invention aims at providing an application of the all-trans retinoic acid-gemcitabine/RSL 3 nano material in preparing a medicine for treating breast cancer. The all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial has the drug release performance of multi-drug delivery capability and esterase response, the stem cell differentiation treatment performance and the iron death and apoptosis causing performance, and can be used for the combined treatment of differentiation, chemotherapy and iron death of breast cancer tumor stem cells.
The invention has the following technical effects:
the all-trans retinoic acid-gemcitabine prodrug prepared by the method has amphipathy, can be self-assembled into a nano material, and can simultaneously encapsulate an iron death agonist RSL3 into nano particles. The invention simultaneously delivers all-trans retinoic acid, gemcitabine and RSL3, and can depolymerize and release medicines under the action of esterase. All-trans retinoic acid can promote the differentiation of tumor stem cells into common tumor cells, and gemcitabine and RSL3 can cause apoptosis and iron death of tumor cells, respectively. In summary, the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial of the invention is used as a drug carrier, has multifunction, multi-drug delivery capability and esterase response drug release performance, stem cell differentiation treatment performance and iron death and apoptosis causing performance, and can be used for combined treatment of malignant tumors.
Drawings
Fig. 1: nuclear magnetic resonance hydrogen spectrogram of all-trans retinoic acid-gemcitabine prodrug.
Fig. 2: high resolution mass spectrum of all-trans retinoic acid-gemcitabine prodrug.
Fig. 3: transmission electron microscopy of all-trans retinoic acid-gemcitabine self-assembled nanomaterial.
Fig. 4: transmission electron microscopy of all-trans retinoic acid-gemcitabine/RSL 3 nanomaterials.
Fig. 5: gemcitabine release profile for all-trans retinoic acid-gemcitabine self-assembled nanomaterial and all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial. Wherein 5-1 is the gemcitabine release profile of the all-trans retinoic acid-gemcitabine self-assembled nanomaterial, 5-2 is the gemcitabine release profile of the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial, 5-3 is the gemcitabine release profile of the all-trans retinoic acid-gemcitabine self-assembled nanomaterial under esterase stimulation, and 5-4 is the gemcitabine release profile of the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial under esterase stimulation.
Fig. 6: cell viability curve of the material after action on breast cancer 4T 1. Wherein 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/RSL 3 nanomaterial.
Fig. 7: western blot of the effect of material on breast cancer 4T1 cells on iron death-related protein GPX 4. Wherein 7-1 is a control group, 7-2 is total-reflection-type 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/RSL 3 nanomaterial. GAPDH is a cellular protein internal reference.
Fig. 8: flow cytometric analysis of the material for changes in stem cell ratio (aldehyde dehydrogenase ALDH positive) on breast cancer 4T1 cells. Wherein 8-1 is a control group, 8-2 is total-reflection-type retinoic acid, 8-3 is RSL3,8-4 is gemcitabine, 8-5 is all-trans-type retinoic acid-gemcitabine self-assembled nanomaterial, and 8-6 is all-trans-type retinoic acid-gemcitabine/RSL 3 nanomaterial.
Detailed Description
The invention will be further described with reference to the following examples and drawings, but is not limited to the disclosure of the examples.
Example 1
200mg of gemcitabine (equivalent 2 times of all-trans retinoic acid) and 196mg of N, N-diisopropylethylamine were dissolved in 5mL of dry N, N-dimethylformamide and filled into glass bottles. 114mg of all-trans retinoic acid and 296mg of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate were dissolved in 5mL of dried N, N-dimethylformamide and premixed at 25℃for 0.5 hours, and then a solution containing all-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate was dropped into the bottle and stirred at 25℃overnight. The N, N-dimethylformamide was removed with a vacuum pump, and the residue was dissolved in 50mL of methylene chloride and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated physiological saline (50 mL, twice). DCM was removed in vacuo and silica gel column dichloromethane: methanol (10:1) to give an orange-yellow oily substance (50.1 mg, 23.4% yield) as all-trans retinoic acid-gemcitabine prodrug.
Example 2
500mg of gemcitabine (equivalent 5 times of all-trans retinoic acid) and 196mg of N, N-diisopropylethylamine were dissolved in 5mL of dry N, N-dimethylformamide and filled into glass bottles. 114mg of all-trans retinoic acid and 296mg of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate were dissolved in 5mL of dried N, N-dimethylformamide and premixed at 25℃for 0.5 hours, and then a solution containing all-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate was dropped into the bottle and stirred at 25℃overnight. The N, N-dimethylformamide was removed with a vacuum pump, and the residue was dissolved in 50mL of methylene chloride and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated physiological saline (50 mL, twice). DCM was removed in vacuo and silica gel column dichloromethane: methanol (10:1) to give an orange-yellow oily substance (97.8 mg, yield 45.7%) as all-trans retinoic acid-gemcitabine prodrug.
Example 3
1000mg of gemcitabine (equivalent 10 times of all-trans retinoic acid) and 196mg of N, N-diisopropylethylamine were dissolved in 5mL of dry N, N-dimethylformamide and filled into glass bottles. 114mg of all-trans retinoic acid and 296mg of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate were dissolved in 5mL of dried N, N-dimethylformamide and premixed at 25℃for 0.5 hours, and then a solution containing all-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate was dropped into the bottle and stirred at 25℃overnight. The N, N-dimethylformamide was removed with a vacuum pump, and the residue was dissolved in 50mL of methylene chloride and washed with 5% citric acid (50 mL, twice), saturated sodium bicarbonate (50 mL, twice) and saturated physiological saline (50 mL, twice). DCM was removed in vacuo and silica gel column dichloromethane: methanol (10:1) to give an orange-yellow oily substance (103.1 mg, yield 48.2%) as all-trans retinoic acid-gemcitabine prodrug.
Characterization of the all-trans retinoic acid-gemcitabine prodrug obtained in example 2 using nuclear magnetic resonance hydrogen spectroscopy (fig. 1) and high resolution mass spectrometry (fig. 2) demonstrated that the all-trans retinoic acid-gemcitabine prodrug was structurally correct and successfully synthesized.
Example 4
(1) Dissolving 5mg of all-trans retinoic acid-gemcitabine prodrug in 100 mu L of dimethyl sulfoxide, and slowly dropwise adding into 900 mu L of ultrapure water under ultrasound to obtain the all-trans retinoic acid-gemcitabine self-assembled nanomaterial;
(2) Dissolving 5mg of all-trans retinoic acid-gemcitabine prodrug and 1mg of RSL3 in 100 mu L of dimethyl sulfoxide, slowly dropwise adding into 900 mu L of ultrapure water under ultrasound to obtain all-trans retinoic acid-gemcitabine/RSL3 nano material;
(3) Taking the prepared all-trans retinoic acid-gemcitabine self-assembled nanomaterial and the prepared all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial for representation of a transmission electron microscope image, wherein as shown in figure 3, the two nanomaterials are uniformly dispersed spheres with diameters of about 100 nanometers;
(4) Taking the prepared all-trans retinoic acid-gemcitabine self-assembled nano material and all-trans retinoic acid-gemcitabine/RSL 3 nano material, placing the nano materials into a dialysis bag with the molecular weight of 500, and adding no esterase or adding esterase. The dialysis bag was placed in phosphate buffer and placed in a 37 degree celsius shaker, and samples were taken at intervals to detect the rate of gemcitabine release. The results are shown in fig. 4, where both nanomaterials exhibited an increase in the rate of gemcitabine release in response to esterase.
Example 5
(1) 4T1 cells are paved on a flat bottom 96-hole cell plate, and after the cells are attached, all-trans retinoic acid, RSL3, gemcitabine and all-trans retinoic acid-gemcitabine self-assembly nano materials with concentration gradient are added, and the nano materials are all-trans retinoic acid-gemcitabine/RSL 3 nano materials. After 72 hours, the cell viability of each group was measured using CCK-8 reagent and plotted as a cell viability curve as shown in FIG. 6. The self-assembled nano material of all-trans retinoic acid-gemcitabine and the nano material of all-trans retinoic acid-gemcitabine/RSL 3 are strong in cytotoxicity of Yu Jixi of the gemcitabine on breast cancer 4T1 cells;
(2) 4T1 cells are paved on a flat-bottomed 6-hole cell plate, and after the cells are attached, all-trans retinoic acid, RSL3, gemcitabine, all-trans retinoic acid-gemcitabine self-assembled nano materials and all-trans retinoic acid-gemcitabine/RSL 3 nano materials are added in equal equivalent of 500nM RSL 3. After 48 hours, the cellular proteins were extracted, and western blot experiments with GPX4 were performed with GAPDH as an internal cellular protein control. The results are shown in fig. 7, where RSL3 and the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial cause GPX4 to decrease, indicating that iron death of the cells is caused;
(3) 4T1 cells are paved on a flat bottom 24-hole cell plate with ultralow adsorption, and after five days, all-trans-retinoic acid, RSL3, gemcitabine, all-trans-retinoic acid-gemcitabine self-assembled nano material and all-trans-retinoic acid-gemcitabine/RSL 3 nano material are added in an equivalent amount of 500nM all-trans-retinoic acid. The proportion of the acetaldehyde dehydrogenase (ALDH) positive of the cells is detected by using an ALDEFLUOR kit, namely the proportion of the tumor stem cells. As shown in fig. 8, the all-trans retinoic acid-gemcitabine self-assembled nanomaterial and the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial both have a greater capacity to reduce stem cell fraction on breast cancer 4T1 cells than all-trans retinoic acid.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as various modifications, equivalent arrangements, improvements, etc., which fall within the spirit and principles of the present invention.
Claims (8)
1. An all-trans tretinoin-gemcitabine prodrug characterized in that: the structural formula of the all-trans retinoic acid-gemcitabine prodrug is as follows:
2. a process for the preparation of an all-trans retinoic acid-gemcitabine prodrug as defined in claim 1, which is characterized by: the preparation method comprises the following steps of:
(1) Dissolving all-trans retinoic acid and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate in N, N-dimethylformamide, wherein the molar equivalent of the benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate is 1 to 4 times that of the all-trans retinoic acid, and stirring for 0.5 to 2 hours at normal temperature;
(2) Dissolving gemcitabine and N, N-diisopropylethylamine in N, N-dimethylformamide, wherein the molar equivalent of the gemcitabine is 2 to 10 times that of the all-trans retinoic acid in the step (1), the molar equivalent of the N, N-diisopropylethylamine is 4 to 8 times that of the all-trans retinoic acid in the step (1), and dropwise adding the solution containing the all-trans retinoic acid and the benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate in the step (1) into the gemcitabine and N, N-diisopropylethylamine solution by using a constant pressure dropping funnel, and stirring at normal temperature for 12 hours;
(3) Removing the N, N-dimethylformamide solvent in the step (2), adding dichloromethane, washing with citric acid for 2 to 3 times, washing with saturated sodium bicarbonate for 2 to 3 times, washing with saturated salt water for 2 to 3 times, and separating with a silica gel column to obtain orange yellow solid, namely the all-trans retinoic acid-gemcitabine prodrug.
3. An all-trans retinoic acid-gemcitabine self-assembled nanomaterial, characterized in that: an all-trans retinoic acid-gemcitabine self-assembled nanomaterial is formed by self-assembly using the all-trans retinoic acid-gemcitabine prodrug of claim 1 as a raw material.
4. A method for preparing an all-trans retinoic acid-gemcitabine self-assembled nanomaterial as claimed in claim 3, which is characterized in that: the preparation method comprises the following steps of:
dissolving all-trans retinoic acid-gemcitabine prodrug in the method of claim 1 in dimethyl sulfoxide, slowly dripping the obtained solution into ultrapure water under ultrasound, wherein the volume of ultrapure water is 9-20 times that of the dimethyl sulfoxide, and obtaining the all-trans retinoic acid-gemcitabine self-assembled nanomaterial.
5. An all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial, characterized in that: the all-trans retinoic acid-gemcitabine/RSL 3 nano material is prepared from the all-trans retinoic acid-gemcitabine prodrug in claim 1 and an iron death agonist RSL3 as raw materials by utilizing the self-assembly performance of the all-trans retinoic acid-gemcitabine prodrug in claim 1, and coating the RSL3 into nano particles at the same time, and is obtained by a nano precipitation method.
6. The method for preparing the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial of claim 5, wherein the method comprises the following steps: the preparation method comprises the following steps of:
dissolving all-trans retinoic acid-gemcitabine prodrug and an iron-death agonist RSL3 in dimethyl sulfoxide, wherein the mass of the all-trans retinoic acid-gemcitabine prodrug is 1-10 times that of the iron-death agonist RSL3, slowly dripping the obtained solution into ultrapure water under ultrasonic conditions, and obtaining the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial by using ultrapure water with the volume of 9-20 times that of the dimethyl sulfoxide.
7. The use of the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial of claim 6 in the preparation of a medicament for the treatment of breast cancer.
8. The use according to claim 7, wherein the all-trans retinoic acid-gemcitabine/RSL 3 nanomaterial is used as a drug carrier for the combined treatment of breast cancer tumors by multi-drug delivery capacity and esterase-responsive drug release properties, stem cell differentiation therapeutic properties, iron death and apoptosis properties of tumor cells.
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