Preparation and application of liposome prodrug overcoming doxorubicin resistance

Abstract

The application belongs to the technical field of medicine, and relates to preparation and application of a liposome prodrug overcoming adriamycin resistance. 2000 On the basis, the prodrug is loaded in a constructed targeted liposome (DSPE-PEG The preparation method is simple, the stability is good, high-efficiency drug loading and delivery are realized, and the liposome prodrug is stable, slow-released, safe, and the like. The liposome prodrug can target the folate receptor on the surface of a tumor, under the action of high-level GSH in a tumor cell, the disulfide bond is broken and BQR and DOX are released. BQR can induce tumor cell ferroptosis and hinder DNA repair, and can synergistically kill tumor cells with adriamycin while overcoming cell resistance to adriamycin. The liposome prodrug prepared in the application realizes targeted delivery of drugs, has good in-vitro anti-tumor activity, overcomes adriamycin resistance, and has a broad development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and pharmaceutical preparations, and mainly includes the synthesis of a prodrug compound (BQR-SS-DOX) that overcomes doxorubicin resistance and the synthesis of BQR-SS-DOX / DSPE-PEG 2000 -Construction of FA liposome nano-targeting system and its application in anti-tumor. Background Art

[0002] Doxorubicin (DOX) is a widely used chemotherapy drug in clinical practice. However, cancer patients are prone to developing drug resistance, and once this occurs, tumors rapidly progress, which has become a major obstacle to the effectiveness of doxorubicin in treating cancer. Therefore, there is an urgent need to identify new treatment options to overcome doxorubicin resistance. Buquina (BQR) blocks DNA repair in tumor cells, sensitizing them to the DNA damage effects of chemotherapy. It also induces ferroptosis, which can enhance the anticancer activity of chemotherapy drugs.

[0003] The present invention synthesizes and constructs a BQR-SS-DOX / DSPE-PEG with tumor targeting effect 2000 -FA reduction stimulus-responsive liposome nanodelivery system. DSPE-PEG 2000 -FA-coated prodrug nanoparticles can actively target the folate receptors highly expressed on the surface of tumor cells; the disulfide bonds can be selectively degraded and broken by the high concentration of GSH in tumor cells, releasing buquinol and doxorubicin; buquinol can promote ferroptosis of tumor cells, block DNA repair of tumor cells, and synergize with doxorubicin to kill tumor cells. Summary of the Invention

[0004] The purpose of the present invention is to synthesize a tumor-targeting prodrug compound and assemble it into a nanodrug, thereby obtaining a prodrug liposome with good stability, sustained release function and high safety, thereby improving anti-tumor activity and overcoming doxorubicin resistance.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] The prodrug compound of the present invention is a compound in which brequinar and doxorubicin are connected by a disulfide bond, and its structural formula is as follows:

[0007]

[0008] Wherein n is an integer from 1 to 6.

[0009] The prodrug compound synthesis method provided by the present invention comprises the following steps: 5-fluoroindigo red and 4-(2-fluorophenyl)propiophenone are used as starting materials to synthesize the drug brequinar (BQR); the hydroxyl group at one end of the 2-hydroxyethyl disulfide is connected to brequinar; the hydroxyl group at the other end reacts with p-nitrophenyl chloroformate having an acyl chloride structure and is finally connected to doxorubicin to obtain the final product prodrug BQR-SS-DOX.

[0010] Specifically, the present invention provides a method for synthesizing the prodrug compound BQR-SS-DOX:

[0011] (1) Synthesis of Buquinar: 5-fluoroindigo red was dissolved in an anhydrous ethanol / water mixture, a small amount of potassium hydroxide was added at room temperature, and 4-(2-fluorophenyl)propiophenone was added after stirring. The mixture was heated to reflux, cooled to room temperature, evaporated to dryness under reduced pressure, and water was added. The pH was adjusted to 2-3 with hydrochloric acid. The mixture was filtered and washed with ether to obtain a solid Buquinar.

[0012] (2) Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-(2-hydroxy-ethyldisulfide)-ethyl ester (2): Buquinar was dissolved in dichloromethane, EDCI and DMAP were added, and the mixture was stirred at room temperature. 2-Hydroxyethyl disulfide was then added. The mixture was reacted at room temperature for 12 hours under N2 protection. The mixture was washed with water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound (2) was isolated by silica gel column chromatography.

[0013] (3) Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-[2-(4-nitro-phenoxycarbonyloxy)-ethyldisulfide]-ethyl ester (3): Compound (2) was dissolved in dichloromethane, pyridine and p-nitrophenyl chloroformate were added under ice-cooling, and the mixture was reacted at room temperature for 12 hours. The mixture was washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound (3) was separated by silica gel column chromatography. The disulfide-bonded alkyl diol was one or more of dithiodimethyl, 2,2'-dithiodiethanol, 3,3'-dithiodipropanol, or 4,4'-dithiodibutanol.

[0014] (4) Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-(2-{3-hydroxy-2-methyl-6-[3,5,12-trihydroxy-3-(2-hydroxy-acetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-naphthalen-1-yloxy]-tetrahydro-pyran-4-ylcarbamoyloxy}-ethyldisulfanyl)-ethyl ester (4)

[0015] Compound (3) and doxorubicin hydrochloride were dissolved in DMF, DIPEA was added and reacted at room temperature, and then water was added to precipitate. The precipitate was filtered, dried, and dissolved in a solvent of methanol: dichloromethane = 1:20. The prodrug compound BQR-SS-DOX was separated by silica gel column chromatography.

[0016] The present invention also provides DSPE-PEG 2000 -FA encapsulated BQR-SS-DOX prodrug compound to form liposome nanomedicine.

[0017] BQR-SS-DOX / DSPE-PEG 2000 -FA liposome nano preparation method: The present invention adopts thin film dispersion method to prepare liposomes; egg yolk lecithin, cholesterol, DSPE-PEG 2000 , DSPE-PEG 2000 -FA and BQR-SS-DOX were placed in an eggplant-shaped flask and dissolved in an organic solvent. The organic solvent was removed under reduced pressure at 40°C, and a film formed on the flask wall. PBS solution was then added, hydrated at 50°C, sonicated in ice water, and filtered through microporous membranes (0.45μm, 0.22μm, and 0.1μm). The organic solvent included one or more of ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), or N,N-dimethylformamide (DMF).

[0018] The present invention synthesizes and constructs a BQR-SS-DOX / DSPE-PEG with tumor targeting effect 2000 -FA liposome nanodelivery system. DSPE-PEG 2000 -FA-coated prodrug nanoparticles actively target folate receptors on tumor surfaces. High concentrations of GSH in tumor cells selectively cleave disulfide bonds, releasing buquinol and doxorubicin. Buquinol promotes ferroptosis in tumor cells, blocks DNA repair, and synergizes with doxorubicin to kill tumor cells. This invention achieves a specific effect of chemotherapy drugs on tumor cells, significantly outperforming anticancer drugs used alone, and holds promising development prospects.

[0019] The DSPE-PEG of the present invention 2000 -FA liposomes encapsulating prodrug compounds composed of BQR-SS-DOX / DSPE-PEG 2000-FA liposome targeted drug delivery system has the following advantages: (1) good liposome stability, high drug encapsulation rate, sustained release effect, and reliable formulation process; (2) in the high GSH environment of tumor cells, the disulfide bond in the prodrug reacts with GSH to break, releasing the active drug; (3) it has tumor targeting, and the selective effect makes the drug more efficient in killing tumor cells, while at the same time avoiding the toxic side effects caused by anti-tumor drugs damaging normal cells to a certain extent, and the drug synergy significantly improves the anti-tumor effect.

[0020] The present invention has the following effects: (1) a BQR-SS-DOX prodrug compound is designed and synthesized, and the synthesis method is stable, convenient and feasible; (2) DSPE-PEG is prepared 2000 -FA liposomes encapsulating prodrug compounds composed of BQR-SS-DOX / DSPE-PEG 2000 -FA liposome targeted drug delivery system, the preparation method is simple and easy, and the encapsulation rate is high; (3) the synergistic application of the two drugs and their targeting effects are achieved, which significantly improves the anti-tumor effect and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Synthesis route of BQR-SS-DOX prodrug compound in Example 1

[0022] Figure 2 : BQR-SS-DOX prodrug compound in Example 1 1 H NMR spectrum

[0023] Figure 3 : BQR-SS-DOX prodrug compound in Example 1 13 C-NMR mass spectrum

[0024] Figure 4 : Mass spectrometry of the BQR-SS-DOX prodrug compound in Example 2 in GSH solution

[0025] Figure 5 :Schematic diagram of in vitro simulation of GSH-triggered disulfide bond cleavage and release

[0026] Figure 6 : BQR-SS-DOX / DSPE-PEG in Example 3 2000 -FA liposome nanoparticle size distribution map (A) DSPE-PEG 2000 -FA(B)BQR-SS-DOX / DSPE-PEG 2000 -FA

[0027] Figure 7 : BQR-SS-DOX / DSPE-PEG in Example 3 2000-FA liposome nano-TME electron microscopy map

[0028] Figure 8 : BQR-SS-DOX / DSPE-PEG in Example 5 2000 -FA liposome nanoparticle uptake diagram in Hela cells DOX (red), cell nucleus DAPI staining (blue)

[0029] Figure 9 : BQR-SS-DOX / DSPE-PEG in Example 6 2000 -FA induces apoptosis in Hela cells DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0031] Example 1: Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid (1)

[0032] 5-Fluoroidin (1.09 g, 6.6 mmol) was dissolved in 18 mL of anhydrous ethanol / water (2:1) mixture, and a small amount of potassium hydroxide (1.48 g, 26.4 mmol) was added at room temperature. After stirring for 30 min, 4-(2-fluorophenyl)propiophenone (1 g, 4.38 mmol) was added. The mixture was heated to reflux at 90°C for 12 h, cooled to room temperature, evaporated to dryness under reduced pressure, water was added, and the pH was adjusted to 2-3 with hydrochloric acid. The mixture was filtered and washed with ether. The resulting solid was compound (1), i.e., buquinar.

[0033] Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-(2-hydroxy-ethyldisulfide)-ethyl ester (2)

[0034] Buquinar (375 mg, 1 mmol) was dissolved in 20 mL of dichloromethane, and EDCI (479 mg, 2.5 mmol) and DMAP (30.5 mg, 0.25 mmol) were added. The mixture was stirred at room temperature for 30 min, and then 2-hydroxyethyl disulfide (509 mg, 3.3 mmol) was added. The mixture was reacted at room temperature for 12 h under N2 protection. The mixture was washed with water, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound (2) was obtained by separation using silica gel column chromatography (200-300 mesh) (methanol:dichloromethane = 1:20).

[0035] Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-[2-(4-nitro-phenoxycarbonyloxy)-ethyldisulfide]-ethyl ester (3)

[0036] Compound (2) (205 mg, 0.4 mmol) was dissolved in 15 mL of dichloromethane. Pyridine (0.5 mL, 6.3 mmol) and p-nitrophenyl chloroformate (120 mg, 0.6 mmol) were added under ice-cooling and reacted at room temperature for 12 h. The mixture was washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound (3) was obtained by separation using silica gel column chromatography (200-300 mesh) (pure dichloromethane).

[0037] Synthesis of 6-fluoro-2-(2'-fluoro-biphenyl-4-yl)-3-methylquinoline-4-carboxylic acid 2-(2-{3-hydroxy-2-methyl-6-[3,5,12-trihydroxy-3-(2-hydroxy-acetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-naphthalen-1-yloxy]-tetrahydro-pyran-4-ylcarbamoyloxy}-ethyldisulfanyl)-ethyl ester (4)

[0038] Compound (3) (135 mg, 0.2 mmol) and doxorubicin hydrochloride (98.6 mg, 0.17 mmol) were dissolved in 5 mL of DMF, and DIPEA (1 mL, 5.7 mmol) was added and reacted at room temperature for 12 h. Water was added to precipitate, and the precipitate was filtered, dried, and dissolved in a solvent of methanol:dichloromethane = 1:20. Compound (4), i.e., the small molecule prodrug BQR-SS-DOX, was obtained by separation using silica gel column chromatography (200-300 mesh) (methanol:dichloromethane = 1:20).

[0039] 1 H NMR (400MHz, CDCl3) δ13.92 (s, 1H), 13.14 (s, 1H), 8.15-8.11 (m, 1H), 7.96 (d, J = 4.0Hz, 1H), 7.75-7.62(m,5H),7.51-7.16(m,8H),5.47(s,1H),5.23-5.14(m,2H),4.74(brs,4H),4.54( s,1H),4.2(brs,2H),4.10-4.04(m,4H),3.81(brs,1H),3.56(s,1H),3.22-2.86(m,8H),2.6 1-2.48(m,1H),2.33-2.30(m,1H),2.16-2.12(m,1H),1.81-1.75(m,3H),1.25-1.22(m,3H).

[0040] 13C NMR (100MHz, CDCl3) δ213.88,186.95,186.55,161.01,156.18,155.54,155.20,143.43, 136.25,135.74,135.38,133.57,132.32,130.75,129.39,129.31,129.11,126.93,124. 52,119.80,118.45,116.35,116.12,111.51,107.99,107.76,100.77,69.69,69.36,67.31,65.52,63.65,62.58,56.63,47.05,37.74,37.27,35.60,33.91,30.09,18.29,16.82.

[0041] Example 2: Study on the cleavage mechanism of BQR-SS-DOX prodrug

[0042] Accurately measure 5 mg of BQR-SS-DOX prodrug and add it to 10 mM GSH PBS buffer solution (pH 7.4). Stir at 37°C for 1 h. The product is detected by LCMS to analyze the GSH cleavage mechanism of BQR-SS-DOX prodrug.

[0043] Example 3: BQR-SS-DOX / DSPE-PEG 2000 Preparation of FA-targeted liposomes

[0044] The present invention adopts the thin film dispersion method to prepare liposomes, and the preparation method is as follows: 56 mg of egg yolk lecithin, 8 mg of cholesterol, DSPE-PEG 2000 27mg, DSPE-PEG 2000 3 mg of α-FA and 5.6 mg of the drug BQR-SS-DOX were placed in an eggplant-shaped flask and dissolved in 10 mL of an organic solvent. The organic solvent was removed under reduced pressure at 40°C, and a film formed on the flask wall. 10 mL of PBS solution was then added and the mixture was hydrated at 50°C for 60 minutes. The mixture was then placed in ice water and ultrasonicated for 20 minutes. The mixture was then filtered through 0.45 μm, 0.22 μm, and 0.1 μm microporous membranes three times each.

[0045] Table 1 BQR-SS-DOX / DSPE-PEG 2000 -FA encapsulation efficiency and drug loading results

[0046]

[0047] Example 4: Detection of cell proliferation inhibition rate by CCK8 method

[0048] Hela cells in the logarithmic growth phase and doxorubicin-resistant Hela cells were seeded in 96-well plates (1.0×10 4 / well, 100 μL) and placed in an incubator for overnight culture. Different concentrations of BQR, DOX, BQR-SS-DOX, BQR-SS-DOX / DSPE-PEG were added. 2000 -FA was cultured for 72 h. After different incubation times, 10 μL of CCK-8 solution was added. The culture plate was incubated in the incubator for 1 h, and the absorbance A value (wavelength 450 nm) of the suspension in the well was measured using a microplate reader to calculate the IC 50 value.

[0049] Table 2 Cell proliferation inhibition rate detected by CCK8 method

[0050]

[0051]

[0052] Example 5: BQR-SS-DOX / DSPE-PEG 2000 In vitro cellular uptake experiment of -FA

[0053] HeLa cells were seeded in 6-well plates (3.0×10 5 / well, 2 mL), placed in a 37 ° C carbon dioxide incubator, cultured overnight, discarded the culture medium, and added DOX and BQR-SS-DOX / DSPE-PEG containing the same amount of DOX, respectively. 2000 Incubate the cells in fresh medium containing 1% paraformaldehyde (-FA) for 1 hour. Wash the cells three times with PBS and fix them with 4% paraformaldehyde for 10 minutes at room temperature. Wash them again with PBS and stain the cell nuclei with DAPI for 10 minutes. Protect the cells from light throughout the experiment. Observe the red fluorescence intensity of DOX in the cells under a laser confocal microscope and take photos.

[0054] The results showed that BQR-SS-DOX / DSPE-PEG 2000 The -FA group showed higher intracellular fluorescence intensity than the free DOX group, indicating increased cellular uptake and demonstrating the good cell targeting ability of the nanosystem.

[0055] Example 6: BQR-SS-DOX / DSPE-PEG 2000 Effects of -FA on apoptosis of Hela cells

[0056] HeLa cells were seeded in 6-well plates (3.0×10 5 After overnight culture, BQR-SS-DOX / DSPE-PEG was added to the wells at final concentrations of 50, 100, and 200 nM.2000 -FA, and continue to culture in the incubator for 72 hours. After collecting the cells, wash them twice with cold PBS solution, remove the supernatant, add appropriate amount of 1× Binding Buffer, and adjust the cell concentration to 1.0×10 6 Annexin V-FITC (5 μL) and PI (5 μL) were added, and the cells were incubated at room temperature in the dark for 15 min. The apoptosis rate was detected by flow cytometry.

Claims

1. A prodrug compound of brequinar BQR and doxorubicin DOX based on a disulfide bond, characterized in that: It has the following structural formula: Wherein n is an integer from 1 to 6.

2. The method for preparing the disulfide-linked prodrug compound of brequinar BQR and doxorubicin DOX according to claim 1, characterized in that: The steps include: (1) Using 5-fluoroindigo red and 4-(2-fluorophenyl)propiophenone as starting materials, the drug Buquina (BQR), i.e., compound (1), was synthesized; (2) The carboxyl group contained in the structure of (1) undergoes an esterification reaction with the hydroxyl group at one end of the alkyl diol containing a disulfide bond, thereby introducing a disulfide bond to obtain compound (2); (3) The hydroxyl group in compound (2) undergoes a substitution reaction with the acyl chloride structure in p-nitrophenyl chloroformate to obtain compound (3); (4) Compound (3) is connected to the amino group of doxorubicin to obtain the final product BQR-SS-DOX (4); the alkyl diol containing a disulfide bond is one or more of dithiodimethylol, 2,2'-dithiodiethanol, 3,3'-dithiodipropanol or 4,4'-dithiodibutanol.

3. A liposome prodrug nanoparticle, characterized in that Folic acid FA and distearate phosphatidylethanolamine-polyethylene glycol DSPE-PEG 2000 Constructed targeted liposome DSPE-PEG 2000 -FA encapsulates the nanoparticles formed by assembling the disulfide bond-linked prodrug compound of brequinar and doxorubicin according to claim 1.

4. The method for preparing liposome prodrug nanoparticles according to claim 3, wherein: The steps include: egg yolk lecithin, cholesterol, DSPE-PEG 2000 , DSPE-PEG 2000 -FA and drug BQR-SS-DOX were placed in an eggplant-shaped flask, dissolved in an organic solvent, and the organic solvent was removed under reduced pressure at 40 °C to form a film on the flask wall; PBS solution was then added, hydrated at 50 °C, ultrasonicated in ice water, and filtered through 0.45 μm, 0.22 μm, and 0.1 μm microporous membranes for three times each to prepare BQR-SS-DOX / DSPE-PEG. 2000 -FA targeting liposomes; the organic solvent includes one or more of ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).

5. Use of the disulfide-linked prodrug compound of brequinar and doxorubicin according to claim 1 or the liposome prodrug nanoparticles according to claim 3 in the preparation of anti-cervical cancer drugs.

6. Use of the disulfide-linked prodrug compound of brequinar and doxorubicin according to claim 1 or the liposome prodrug nanoparticle according to claim 3 in the preparation of an injection, oral administration or topical administration system.

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