Construction and application of singlet oxygen-nitrogen mustard synchronous delivery system

Abstract

The invention provides construction and application of a singlet oxygen-nitrogen mustard synchronous delivery system, and belongs to the technical field of biological medicine. Based on the anti-cancer performance of singlet oxygen and nitrogen mustard compounds and the phenomenon of enzyme overexpression in a tumor microenvironment, an endoperoxide-nitrogen mustard combined treatment molecule for azo reductase response is designed and synthesized, the molecule plays a role in the specific tumor microenvironment, nitrogen mustard and singlet oxygen are released, and the treatment effect is improved. The specific damage to tumor cells is generated. And the drug is further wrapped in the liposome so as to improve the targeting property and the biological safety of the drug to tumor cells. Meanwhile, in-vitro and tumor-bearing mouse experiments show that the polypeptide has huge potential in cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the construction and application of a singlet oxygen-nitrogen mustard synchronous delivery system. Background Art

[0002] Photodynamic therapy is a novel, minimally invasive tumor treatment method that uses photosensitizers to generate cytotoxic reactive oxygen species (ROS), such as singlet oxygen 1 O2 is used to treat cancer. However, the method has the disadvantages of limited light penetration depth, high oxygen dependence and poor selectivity, which greatly limit its practical application effect. In order to break the limitations of traditional photodynamic therapy, endoperoxide therapy was developed based on it. 1 O2 undergoes a cycloaddition reaction to generate an endoperoxide. After being transported to a specific location, the endoperoxide is ring-opened to produce 1 O2, kills tumor in situ.

[0003] Nitrogen mustard compounds are a low-cost cancer chemotherapy drug. In actual use, the drug indiscriminately damages cancer cells and normal cells, which can lead to serious side effects; and after repeated use, tumor cells can develop a certain degree of drug resistance. In addition, under the influence of the tumor hypoxic microenvironment, the expression levels of enzymes such as azoreductase (AzoR), nitroreductase (NTR), and quinone oxidoreductase (QR) are significantly enhanced.

[0004] Therefore, it is very promising to develop new endoperoxide-chemotherapeutic small molecules that are not limited by traditional photodynamic therapy and chemotherapy. Summary of the invention

[0005] Based on the anticancer properties of endoperoxides and nitrogen mustard compounds and the phenomenon of enzyme overexpression in the tumor microenvironment, the present invention designs an endoperoxide-nitrogen mustard combined therapeutic molecule that responds to azoreductase: the molecule acts in a specific tumor microenvironment and causes specific damage to tumor cells. At the same time, in order to further improve its biosafety, the small molecule is encapsulated in liposomes, and in vitro and tumor-bearing mouse experiments show its great potential in cancer treatment.

[0006] The technical solution of the present invention is a pyridone endoperoxide compound, wherein the compound PRI-AZO-Cl-ENDO has the following structure:

[0007]

[0008] Among them, R 1, R 2 , R 3 Each is independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, and C1-C10 ester.

[0009] R 4 -R 7 Each is independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, and C1-C10 thioether.

[0010] R is independently a C1-C5 alkyl group.

[0011] Some specific pyridone endoperoxides, R 1 , R 2 , R 3 Each is independently selected from hydrogen, deuterated methyl, C1-C5 alkyl, trifluoromethyl, and carboxyl.

[0012] R 4 -R 7 Each is independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, and C1-C10 thioether.

[0013] R is independently a C1-C5 alkyl group.

[0014] Some specific pyridone endoperoxy derivatives, R 1 , R 2 , R 3 Each is independently selected from hydrogen, deuterated methyl, trifluoromethyl, methyl, and carboxyl.

[0015] Some specific pyridone endoperoxy derivatives, R 1 , R 3 Each is independently selected from hydrogen and methyl.

[0016] Some specific pyridone endoperoxy derivatives, R 4 -R 7 Each is independently selected from hydrogen, deuterated methyl, trifluoromethyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C5 alkylamino, nitro, cyano, C1-C5 alkyl, C1-C5 alkoxy, and C1-C5 thioether.

[0017] R is independently a C1-C5 alkyl group.

[0018] The application of the pyridone endoperoxy derivative in the preparation of singlet oxygen releasing materials.

[0019] The invention relates to the use of the pyridone endoperoxy derivative in preparing a medicine for transporting singlet oxygen to cells, tissues or organs.

[0020] The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, lotions, and films.

[0021] A drug in the form of liposomes or micelles, wherein the drug in the form of liposomes or micelles comprises at least one of the above-mentioned pyridone endoperoxy derivatives;

[0022] and / or carrier;

[0023] and / or pharmaceutical excipients;

[0024] The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, micelles, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid.

[0025] The pharmaceutical excipients are selected from one or more of diluents, adhesives, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents.

[0026] The present invention provides a specific liposome, comprising the above-mentioned pyridone endoperoxy derivative, soybean lecithin, DSPE-PEG2000 and cholesterol.

[0027] The liposome has a loading amount of 10%-17% of the pyridone endoperoxy derivative.

[0028] The liposome has a loading amount of 14%-17% of the pyridone endoperoxy derivative.

[0029] The liposome is obtained by mixing the pyridone endoperoxy derivative, soybean lecithin, DSPE-PEG2000 and cholesterol in an organic solvent, removing the solvent, dispersing the mixture in a buffer solution, filtering or filtering and freeze-drying.

[0030] The mass ratio of soybean lecithin, DSPE-PEG2000, cholesterol and the pyridone endoperoxy derivative is 2-7:1:1:0.2.

[0031] The organic solvent can be selected from solvents that can dissolve the four substances of pyridone endoperoxy derivatives, soybean lecithin, DSPE-PEG2000 and cholesterol.

[0032] The organic solvent is selected from chloroform, n-hexane, dioxane, ethyl acetate, methanol and the like.

[0033] The present invention also provides a method for preparing the pyridone endoperoxy derivative, and the reaction formula is as follows:

[0034]

[0035] The preparation methods of the pyridone endoperoxy derivatives of the present invention all adopt existing technologies. The preparation of the phenylpyridone compound refers to the patent document with publication number CN116655654A. The phenylpyridone compound is connected with the azobenzene compound through conventional ester condensation. The obtained compound is further obtained by dye illumination with reference to the patent document CN116655654A to obtain the target endoperoxy derivative.

[0036] A specific pyridone endoperoxy derivative has the following structure:

[0037]

[0038] The idea of ​​the present invention is that endoperoxide compounds have anti-cancer potential, and combined with the chemotherapy drug nitrogen mustard to make prodrugs, it is expected to develop an efficient anti-cancer strategy. The mechanism is shown in the following formula. The molecule is composed of two functional parts on the left and right connected by an azo group. The pyridone endoperoxide on the left will slowly release singlet oxygen to kill cells at 37°C, and the nitrogen mustard group on the right performs combined chemotherapy. When the azo group exists, the nitrogen mustard is "passivated" due to the electron-withdrawing effect, showing low toxicity; and when the drug reaches the tumor tissue, the azoreductase overexpressed in the hypoxic environment of the tumor tissue will reduce the azo bond to break it, and the generated aniline nitrogen mustard will have enhanced toxicity. Therefore, the combination of the two can selectively kill cancer cells efficiently in the hypoxic environment of the tumor.

[0039]

[0040] The beneficial effects of the present invention are:

[0041] (1) The endoperoxide-nitrogen mustard combined anticancer agent PRI-AZO-Cl-ENDO synthesized in the present invention has strong DNA damage and cancer cell killing effects, especially in the treatment of human cervical cancer cells (Hela cell line), PRI-AZO-Cl-ENDO has a median lethal dose of 14.76 μM under hypoxic conditions. 50 Compared with 45.19 μM under normoxic conditions, it showed a stronger anticancer effect;

[0042] (2) The azo group connected in the compound PRI-AZO-Cl-ENDO of the present invention responds to the azoreductase overexpressed in hypoxia, which can significantly reduce the toxic effects on normal cells under normoxia compared with traditional anticancer drugs; and the overall biosafety of anticancer drugs is further improved through liposome encapsulation technology.

[0043] (3) The entire synthetic route is simple and convenient, the reaction conditions are mild and controllable, and the target product PRI-AZO-Cl-ENDO can be efficiently synthesized using common and readily available raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The NMR changes of PRI-AZO-Cl-ENDO in a mixed solvent of deuterated chloroform: deuterated methanol = 1:5 over time (37°C);

[0045] Figure 2 The cytotoxicity of different concentrations of PRI-AZO-Cl-ENDO and PRI-AZO-Cl in normoxia and hypoxia environments of different cell lines;

[0046] Figure 3 This is an imaging image of DCFH-DA detecting the release of singlet oxygen by PRI-AZO-Cl-ENDO in Hela cells;

[0047] Figure 4 These are bright field and fluorescent images of cells double-stained with calcein-iodide;

[0048] Figure 5 Annexin V-FITC / PI imaging of PRI-AZO-Cl-ENDO-induced cell apoptosis under hypoxic conditions;

[0049] Figure 6 Microscopic photos of cell migration after Hela cells were scratched and treated with PRI-AZO-Cl-ENDO and PRI-AZO-Cl for 24h and 48h;

[0050] Figure 7 Microscope photos of the effects of endoperoxide (PRI-AZO-Cl-ENDO) and its precursor (PRI-AZO-Cl) on Hela cell migration;

[0051] Figure 8 The cytotoxicity of liposome PRI-AZO-Cl-ENDO@LP under normoxia or hypoxia;

[0052] Fig. 9 To evaluate the in vivo tumor inhibition effect and biosafety of drug-loaded liposomes in mice.

[0053] Fig.10 A structural diagram of a pyridone-based peroxide compound. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with embodiments.

[0055] Preparation steps of compound PRI-AZO-Cl-ENDO:

[0056]

[0057] S1: Phosphorus oxychloride (5 mL, 53.8 mmol) was added to a round-bottom flask and cooled to 0°C, and then N,N-dihydroxyethylaniline (3.9 g, 21.5 mmol) was slowly added while maintaining 0°C. After that, the mixture was heated to reflux at 110°C for 1 h, and then the mixture was cooled to room temperature and concentrated by rotary evaporation. The residue was dissolved in ethyl acetate, washed three times with water and once with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and the organic phase was collected and concentrated by rotary evaporation to obtain a crude product. Finally, column chromatography was performed using n-hexane: ethyl acetate (v / v) 10:1 as the eluent to obtain compound 1 as a light yellow viscous liquid with a yield of 90%;

[0058] S2: A solution of sodium nitrite (555 mg, 8 mmol) in water (30 mL) was added to an aqueous solution of p-aminobenzyl alcohol (1 g, 8.1 mmol) containing concentrated hydrochloric acid (1.675 mL). After stirring at 0°C for 20 min, the obtained solution was added to an ethanol solution of compound 1 (1.5 g, 6.8 mmol). After 2 h, the solution was diluted with dichloromethane (500 mL) and washed twice with water and once with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate and purified by silica gel chromatography with n-hexane: ethyl acetate (v / v) 5:1 to obtain compound 2 with a yield of 35%;

[0059] S3: To dry DMF containing 3-methylpyridin-2(1H)-one (1g, 10mmol) was added the corresponding aryl iodide (4.14g, 15mmol), and anhydrous potassium carbonate (2.76g, 20mmol) and cuprous iodide (190.45mg, 1mmol) were added to the resulting solution, and the reaction mixture was heated to 150°C. After 6h, the reaction mixture was cooled to room temperature, quenched with cold water, and extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by column chromatography to give compound 3 with a yield of 66%;

[0060] S4: A solution of compound 3 (900 mg, 3.5 mmol) in ethanol (135 mL) was added to a solution of sodium hydroxide (684 mg, 17.1 mmol) in water (45 mL). The solution was stirred at 83 °C for 5 h. The product was extracted with ethyl acetate and concentrated under reduced pressure to a white solid with a yield of 85%;

[0061] S5: Compound 4 (150 mg, 0.65 mmol), dichloromethane (3 mL) and DMF (2 μL) were added to a dry 50 mL double-necked round-bottom flask. Then, thionyl chloride (57 μL, 0.785 mmol) was added dropwise. The resulting mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was dissolved in dichloromethane (20 mL) again, and DMAP (0.611 mg, 0.005 mmol) and compound 2 (345 mg, 0.98 mmol) were added to the stirred solution at room temperature. Then, triethylamine (272 μL, 1.96 mmol) was added dropwise at 0 ° C and stirred at room temperature. After 2 h, the solution was diluted with ethyl acetate (20 mL), extracted with water, and the combined organic layer was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with n-hexane: ethyl acetate (v / v) 2:1 to obtain a yellow solid with a yield of 60%; S6: Compound PRI-AZO-Cl (20 mg, 0.34 mmol) was dissolved in 3 mL of deuterated chloroform. After the reaction mixture was cooled to 0°C in an ice bath, methylene blue hydrate (10 mg, 0.0296 mmol) was added to the solution, and the mixture was stirred for 2 h under an oxygen atmosphere. During the reaction, 18W, 630 nm red light was used for irradiation. After the methylene blue was removed by activated carbon, the pure product was obtained after reduced pressure concentration. The compound PRI-AZO-Cl-ENDO was a yellow solid with a yield of 97%.

[0062] Example 1: Testing of the half-life of endoperoxides

[0063] like Figure 1 As shown, the half-life of endoperoxide compounds is tested by nuclear magnetic resonance hydrogen spectrum. 3-5 mg of PRI-AZO-Cl-ENDO solid is dissolved in 0.6 mL of deuterated reagent. When the nuclear magnetic resonance hydrogen spectrum test is performed at 0 h, 19 h, 48 h, 72 h, 96 h, 120 h, and 168 h, the nuclear magnetic resonance tube needs to be kept in a constant temperature water bath at 37 ° C. The change in the characteristic peak area of ​​the endoperoxide is observed, and the proportion is determined by the ratio to determine whether the decay is completed. Plotting ln(A0 / A) against time t, a straight line with a slope k of 0.0117 is obtained, and the half-life t 1 / 2 =59.23h.

[0064] Example 2: Establishment of hypoxic (1% O2) cell model

[0065] The present invention uses a variety of cancer cells for in vitro medical biological research and anti-tumor effect evaluation. The cells are placed in an incubator at 37°C and 5% CO2, and the cells are cultured to the logarithmic growth phase using a complete culture medium containing 89% basal culture medium DMEM, 10% fetal bovine serum and 1% penicillin / streptomycin. The cell suspension prepared after 0.25% trypsin digestion can be used in subsequent different experimental schemes.

[0066] Due to experimental requirements, a hypoxic (1% O2) cell model was constructed for hypoxic experiments. First, an appropriate amount of cells were cultured in a 96-well plate or a 6-well plate, and cultured overnight in a 37°C, 5% CO2 incubator until the cell state was stable, and then the cells were transferred to a hypoxic incubator (incubator environment: 37°C, 5% CO2, 1% O2, 94% N2) for continued culture. In order to keep the intracellular environment in a hypoxic state, the culture time in the hypoxic incubator was not less than 24 hours.

[0067] Other cell cultures were kept in a normoxic (21% O2) environment without any special standards.

[0068] Example 3: MTT assay to assess cytotoxicity of endoperoxides

[0069] The MTT assay was used to assess the cytotoxicity of endoperoxides. 3 The cells were cultured at a density of 100 μL in a 96-well plate and incubated overnight at 37°C and 5% CO2 under normoxic conditions to allow the cells to completely adhere to the wall. Subsequently, according to the experimental requirements, the cells were divided into normoxic and hypoxic groups, and placed in two groups with different oxygen concentrations for 24 hours, respectively, so that the cells were in normoxia and hypoxia (1% O2) states. After that, gradient concentrations of the endoperoxide PRI-AZO-Cl-ENDO and its precursor compound PRI-AZO-Cl (control) were added to the cells, and the cells were incubated for 24 hours. Then, 20 μL of MTT solution (5 mg / mL in PBS solution) was added to each well. After incubation for 4 hours, the culture medium was removed, and 150 μL of DMSO was added to each well to dissolve the formazan. The cells were placed in a microplate reader and shaken at 37°C for 5 minutes to detect the absorbance at 570 nm, and the cell survival rate was calculated according to the formula. Figure 2 As shown, endoperoxides exhibited excellent anticancer efficacy in MCF-7, Hela, SK-OV-3, and 4T1 cells, and there was a large difference between normoxic and hypoxic (1% O2) environments.

[0070] Example 4: Release of singlet oxygen from intracellular peroxides in vitro

[0071] To evaluate the release of singlet oxygen from endoperoxides, a fluorescent probe DCFH-DA that captures singlet oxygen was used. HeLa cells were plated at 9 × 10 3 The cells were cultured at a density of 100 cells in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. After they were completely attached to the wall, they were placed in a low-oxygen environment and incubated for 24 hours. Then, 25 μM and 50 μM culture medium solutions of endoperoxides were added. After incubation for 3 hours, the culture medium was discarded and the cells were washed with phosphate buffered saline (PBS). Then, 100 μL of serum-free culture medium containing DCFH-DA was added to each well for staining, and incubated at 37°C for 45 minutes. After removing the culture medium, the cells were washed once with PBS. Finally, 100 μL of fresh culture medium was added to each well, and the cells in each well were photographed using a high-content imager. Figure 3 As shown in the figure, very weak green fluorescence can be observed in Hela cells cultured with 25μM PRI-AZO-Cl-ENDO, indicating that endoperoxides can release singlet oxygen, but the amount of singlet oxygen is small. When the concentration is increased to 50μM, the green fluorescence intensity is significantly enhanced, indicating that with the increase in concentration, the amount of singlet oxygen stored and released by endoperoxides is also increasing, which is consistent with the expected results.

[0072] Example 5: In vitro cell apoptosis experiment

[0073] In order to directly observe the process of singlet oxygen-induced cell apoptosis, the Annexin V-FITC / PI kit was used. Hela cells were plated at 7×10 3 The cells were cultured at a density of 100 cells in a 96-well plate and cultured overnight at 37°C and 5% CO2 under normoxic conditions to allow the cells to completely adhere to the wall. Subsequently, the cells were placed in a hypoxic environment and incubated for 24 hours. Then, 12.5 μM of the culture medium solution of endoperoxides and their precursors were added respectively, and the cells were incubated in a hypoxic environment for 3-4 hours after administration. After the incubation was completed, the drug-containing culture medium was immediately removed and the cells were washed with PBS. Annexin V-FITC / PI staining solution was then added, and the cells were incubated in the dark at room temperature for 20 minutes, then placed in an ice bath, and finally the position of the red and green fluorescence in the cells was observed under a high-content imager to determine the early and late stages of cell apoptosis. Figure 4 As shown, a strong green fluorescence signal was shown in the control group (0 μM), which clearly indicated that there were a large number of normal surviving cells, thus verifying that the toxicity to Hela cells was extremely low. After the addition of endoperoxide precursor (PRI-AZO-Cl) in the second group, a large number of cells still survived. In addition, in the third group, due to the release of singlet oxygen by endoperoxide (PRI-AZO-Cl-ENDO), tumor cells began to die (red fluorescence).

[0074] Example 6: In vitro live cell / dead cell double staining experiment

[0075] In vitro live cell / dead cell double staining experiments were performed using the Calcein-AM / PI kit. Hela cells were plated at 7×10 3 The cells were cultured at a density of 100 cells in a 96-well plate and cultured overnight at 37°C and 5% CO2 under normoxic conditions to allow the cells to completely adhere to the wall. Subsequently, the cells were placed in a hypoxic environment and incubated for 24 hours. Then, 35 μM of the culture medium solution of endoperoxide compounds and their precursors were added thereto respectively. After administration, the cells were incubated in a hypoxic environment for 2-3 hours. After the incubation was completed, the drug-containing culture medium was immediately removed and the cells were washed with PBS. Serum-free culture medium containing Calcein-AM was added and incubated at 37°C in the dark for 20 minutes to 25 minutes. The PI that comes with the kit was added to the above-mentioned stained cells and stained at room temperature in the dark for 5 minutes. Figure 5 As shown, the endoperoxide precursor (PRI-AZO-Cl) under hypoxic (1% O2) conditions has only weak fluorescence in Hela cells, and most of the cell morphology has not changed, indicating that the aniline mustard released only under hypoxia has a weak killing ability on tumor cells. Under the same conditions, strong fluorescence can be observed in cells co-cultured with endoperoxide compounds (PRI-AZO-Cl-ENDO), and the cell morphology is also significantly changed, resulting in the exposed phosphatidylserine of the everted cell membrane specifically binding to Annexin V-FITC, emitting strong green fluorescence under 488nm excitation, indicating that the cells have undergone early apoptosis; at the same time, cell nuclei with strong red fluorescence also appear, indicating that PI has reacted with the cell nucleus that has lost the integrity of the cell membrane, resulting in late apoptosis or necrosis of the cells. The above results show that under hypoxic conditions, endoperoxides can react with overexpressed azoreductase, thereby releasing the chemotherapy drug aniline mustard, and synergistically fight cancer with the singlet oxygen released by endoperoxides.

[0076] Example 7: In vitro cell migration assay

[0077] 1 Cell scratch

[0078] Hela cells in the logarithmic growth phase were taken at 7×10 5The cells were seeded at a density of 100 cells / well in a 6-well culture plate and cultured overnight under normoxic conditions at 37°C and 5% CO2. After the cells were completely attached, they were placed in a hypoxic environment and cultured for 24 hours. First, the culture medium in the well was aspirated and then a scratch was made evenly on the well plate with a gun tip. The cells were then washed with PBS for 3 hours. The control group was replaced with fresh culture medium (containing 1% FBS), and the drug-treated group was replaced with drug-containing culture medium (containing 1% FBS), and pictures were taken immediately under a microscope. Next, 10 μM culture medium solutions of PRI-AZO-Cl-ENDO and PRI-AZO-Cl were added respectively, and the incubation continued for 48 hours. Pictures were taken at 0h, 24h and 48h to observe the healing of the scratches under the action of the drug. Figure 6 As shown, the control group and cells treated with endoperoxide precursor (PRI-AZO-Cl) had the highest degree of wound healing after 48 hours of culture, while the degree of wound healing after treatment with endoperoxide compound (PRI-AZO-Cl-ENDO) was significantly reduced. The wound healing rates of each group at 24h and 48h were 20% to 35.9%, 14% to 22.1%, and 9.3% to 13.6%, respectively, indicating that endoperoxides have the ability to significantly inhibit tumor migration.

[0079] 2 Transwell cell migration

[0080] Prepare a medium containing 20% ​​FBS, take 600 μL and add it to the bottom of the 24-well plate in the lower chamber. Take Hela cells in the logarithmic growth phase and plate them at a concentration of 1×10 5 Resuspend the cells in the upper chamber at a density of 100 μM / well, check for bubbles, and gently add the upper chamber to the lower chamber with tweezers. After completion, check for bubbles at the bottom and culture in an incubator. After 4 hours, treat the cells with the following different methods: Group 1, incubate with DMEM containing 1% FBS at 37°C, 21% O2, and 1% O2. Group 2, incubate with DMEM containing PRI-AZO-Cl (35 μM) and 1% FBS at 37°C, 21% O2, and 1% O2. Group 3, incubate with DMEM containing PRI-AZO-Cl-ENDO (35 μM) and 1% FBS at 37°C, 21% O2, and 1% O2. After incubation for 24 hours, staining was performed, the culture medium in the chamber was removed, and the lower surface was immersed in 4% paraformaldehyde solution for 10 minutes. After fixation, rinse again, stain with 10% crystal violet for 5-10 minutes, and wash after staining until there is no obvious purple. Gently wipe the upper side of the chamber with a cotton swab and finally place it on a slide for observation under a microscope. Count the number of cells on the membrane surface and take the average of the middle and surrounding 5 areas. Figure 7It can be seen that when cells are in a hypoxic environment, the number of cell migration is decreasing and the cell survival rate is decreasing. In addition, the migration rate of cells treated with endoperoxide at a concentration of 35 μM (PRI-AZO-Cl-ENDO) is lower than that of the precursor compound (PRI-AZO-Cl), which is consistent with our expected results, indicating that the molecule has a certain degree of ability to inhibit cell migration.

[0081] Example 9: Liposome encapsulation drug delivery strategy

[0082] 1 Synthesis of coated liposomes

[0083] Soy lecithin (70 mg), DSPE-PEG2000 (10 mg), cholesterol (10 mg), PRI-AZO-Cl-ENDO (2 mg), and PRI-AZO-Cl (2 mg) were completely dissolved in chloroform. The solvent in the mixture was removed by decompression, and the residual solvent was blown dry with nitrogen, dispersed in 20 mL of phosphate buffer (pH 7.4), and hydrated at 40°C for 1 h. Then, it was quickly filtered with a 220 nm polycarbonate filter, pre-frozen at minus 80°C for 12 h, and finally the obtained liposome material was freeze-dried.

[0084] 2 Appearance and shape

[0085] The sample was dropped onto a 400-mesh copper mesh, and after it evaporated and dried naturally, it was dyed with a 3% phosphotungstic acid solution. After 30 seconds of dyeing, the excess dye was sucked off, and after it was dried, the sample was carefully observed using a transmission electron microscope (TEM), and its morphology was recorded. In addition, we also took photos of the macroscopic state of the liposomes for further analysis. The liposomes were light yellow and transparent, evenly dispersed, and had no signs of precipitation, indicating that their appearance was excellent and of high quality; after freeze-drying, they were yellow powder solids.

[0086] 3 Encapsulation efficiency calculation

[0087] Calculation by UV-Vis spectrometer: Take 5 mmol / L DMSO solution of compound PRI-AZO-Cl-ENDO and dilute it step by step with DMSO, test the absorbance at 415 nm at different concentrations, and establish a standard curve.

[0088] 27.83 mg of the liposome powder prepared in Example 1 was accurately weighed and dissolved in 2 mL of DMSO to break the emulsion. 500 μL was taken out and added to a 3 mL cuvette, and its absorbance at 415 nm was tested, and the actual concentration was obtained by comparing with the standard curve. The mass of the PRI-AZO-Cl-ENDO encapsulated was obtained according to the tested concentration, and the quotient was calculated by comparing with the total dosage of PRI-AZO-Cl-ENDO to obtain an encapsulation efficiency of 14.2%.

[0089] Example 10: Study on the toxicity of drug-loaded liposomes to cancer cells

[0090] The cells were cultured according to the "Cell Culture and Establishment of Hypoxic (1% O2) Tumor Cell Model" in Example 2 and two groups, normoxic and hypoxic, were set up. The cytotoxicity of the drug-loaded liposomes was evaluated using the MTT method. Fig. 9 As shown, liposomes were expressed in HeLa cells. 50 It is 652μg / mL, showing excellent anti-cancer efficacy, and there is a large gap between normoxic and hypoxic environments.

[0091] Example 11: In vivo tumor inhibition effect in mouse models

[0092] The tumor volume of 21 mice was about 50-80 mm. 3 The 4T1 tumor-bearing mice were randomly divided into 4 groups, namely, normal saline group (control group), PRI-AZO-Cl@LP treatment group (500 mg / kg), PRI-AZO-Cl-ENDO@LP (300 mg / kg) treatment group and PRI-AZO-Cl-ENDO@LP (500 mg / kg) treatment group. The mice in each experimental group were injected with the above-mentioned experimental group preparations through the tail vein. The first administration time was set on the first day, and the drug was administered 8 times on the 1st, 3rd, 5th, 7th...15th days respectively. The long diameter (L, mm) and short diameter (W, mm) of the tumor of each experimental group were measured and recorded with a vernier caliper every day, and the tumor volume (V) was calculated according to the following formula t , mm 3 ):V t =L×W 2 / 2, record V within 15 days of medication t The data is plotted with time (d) as the horizontal axis, V t The tumor growth curve was drawn with y as the ordinate. The mice were observed and their weights were weighed and recorded daily. After 15 days of administration (day 16), the tumor-bearing mice were killed by bleeding from their eyes, blood was collected, the tumor was carefully peeled off, and washed with saline. Excess water was absorbed with absorbent paper, weighed on a scale, and photographed with a camera. The results are shown in Figure 2. Fig. 9The results showed that the tumor volume of the control group, PRI-AZO-Cl@LP and PRI-AZO-Cl-ENDO@LP treatment groups all showed a trend of gradual increase, among which the tumor of the control group mice grew the fastest and the average mass of tumor tissue was the largest, followed by the PRI-AZO-Cl@LP (500 mg / kg) treatment group and the PRI-AZO-Cl-ENDO@LP (300 mg / kg) treatment group, and the slowest was the PRI-AZO-Cl-ENDO@LP (500 mg / kg) treatment group, showing a good tumor growth inhibition effect. In summary, the endoperoxide compound PRI-AZO-Cl-ENDO can effectively inhibit tumor growth in animals after being encapsulated in liposomes.

[0093] Good biosafety is a prerequisite for clinical application. Fig.10 As shown, no significant weight changes were found by monitoring the changes in the weight of mice every two days, indicating that the tail vein injection of drugs did not cause obvious side effects on the mice's physiology.

Claims

1. A pyridone endoperoxy derivative characterized in that: It has the following structure: Among them, R 1 , R 2 , R 3 Each independently selected from hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, C1-C10 ester; R 4 -R 7 Each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether; R is independently a C1-C5 alkyl group.

2. The pyridone endoperoxy derivative according to claim 1, characterized in that R 1 , R 2 , R 3 Each independently selected from hydrogen, deuterated methyl, C1-C5 alkyl, trifluoromethyl, and carboxyl; R 4 -R 7 Each independently selected from hydrogen, C1-C5 deuterated alkyl, C1-C5 fluoroalkyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether; R is independently a C1-C5 alkyl group.

3. The pyridone endoperoxy derivative according to claim 2, characterized in that R 1 , R 2 , R 3 Each is independently selected from hydrogen, deuterated methyl, trifluoromethyl, and methyl. Preferably, R 1 , R 3 Each is independently selected from hydrogen and methyl.

4. The pyridone endoperoxy derivative according to claim 3, characterized in that R 4 -R 7 Each is independently selected from hydrogen, deuterated methyl, trifluoromethyl, halogen, carboxyl, sulfonic acid, aminosulfonic acid, hydroxyl, amino, -COOR, -CONR, C1-C5 alkylamino, nitro, cyano, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 thioether; R is independently a C1-C5 alkyl group.

5. Use of the pyridone endoperoxy derivative according to any one of claims 1 to 4 in the preparation of a material releasing singlet oxygen.

6. Use of the pyridone endoperoxy derivative according to any one of claims 1 to 4 in the preparation of a medicament for transporting singlet oxygen to cells, tissues or organs.

7. The use according to claim 6, characterized in that: The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, dew preparations, and membrane preparations.

8. A drug in the form of liposomes or micelles, characterized in that: The liposome or micelle form of the drug comprises at least one of the pyridone endoperoxy derivatives according to any one of claims 1 to 5; and / or carrier; and / or pharmaceutical excipients; The carrier is selected from one or more of metal nanocarriers, non-metal nanocarriers, micelles, liposomes, lactose, sucrose, gelatin, hard magnesium sulfate, and stearic acid.

9. A liposome, characterized in that: The invention comprises the pyridone endoperoxy derivative according to any one of claims 1 to 4, soy lecithin, DSPE-PEG2000 and cholesterol.

10. The liposome according to claim 9, characterized in that The pyridone endoperoxy derivative, soybean lecithin, DSPE-PEG2000 and cholesterol are mixed in an organic solvent, dispersed in a buffer solution after removing the solvent, and filtered or filtered and freeze-dried to obtain the liposome; The mass ratio of soybean lecithin, DSPE-PEG2000, cholesterol and the pyridone endoperoxy derivative is 2-7:1:1:0.2.

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