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

Abstract

The application provides a construction and application of a singlet oxygen-mechlorethamine synchronous delivery system, and belongs to the technical field of biological medicines.Based on the anticancer performance of singlet oxygen and mechlorethamine compounds and the overexpression of enzymes in a tumor microenvironment, an endoperoxide-mechlorethamine combined treatment molecule responding to azo reductase is designed and synthesized: the molecule plays a role in a specific tumor microenvironment, releases mechlorethamine and singlet oxygen, and produces specific damage to tumor cells.Further, the drug is wrapped in a liposome to improve the targeting and biological safety of the drug to tumor cells.Meanwhile, in vitro and tumor-bearing mouse experiments show that the molecule has great potential in cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to construction and application of a singlet oxygen-nitrogen mustard synchronous delivery system. BACKGROUND

[0002] Photodynamic therapy is a new type of minimally invasive tumor treatment method, which generates active oxygen (Reactive Oxygen Species, ROS) such as singlet oxygen 1 O2 by photosensitizer to achieve the purpose of cancer treatment. The method has the shortcomings of limited light penetration depth, great oxygen dependence and poor selectivity, which to a large extent limits its actual application effect. In order to break the limitations of traditional photodynamic therapy, endoperoxide therapy is developed on the basis of photodynamic therapy. 1 O2 is generated by cycloaddition reaction to generate endoperoxide, which is transported to a specific position, and then the endoperoxide ring-opening generates 1 O2, which kills tumors in situ.

[0003] Nitrogen mustard compounds are a kind of low-cost cancer chemotherapy drugs. In actual use, the drug indiscriminately damages cancer cells and normal cells, which can cause serious side effects; and tumor cells can develop resistance after multiple uses. In addition, under the action of 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 of great prospect to develop a new endoperoxide-chemotherapy combined small molecule which is not limited by traditional photodynamic therapy and chemotherapy. SUMMARY

[0005] Based on the anti-cancer performance of endoperoxide and nitrogen mustard compounds and the phenomenon of overexpression of enzymes under tumor microenvironment, the present application designs an endoperoxide-nitrogen mustard combined therapy molecule which is responsive to azoreductase: the molecule acts under a specific tumor microenvironment to produce specific damage to tumor cells. At the same time, in order to further improve its biological safety, the small molecule is wrapped in a liposome, and in vitro and tumor-bearing mouse experiments show its great potential in cancer treatment.

[0006] The technical scheme of the present application: a pyridone endoperoxide compound, the compound PRI-AZO-Cl-ENDO has the following structure:

[0007]

[0008] Among them, R 1, R 2 , R 3 each independently selected from the group consisting of hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, C1-C10 ester.

[0009] R 4 -R 7 each independently selected from the group consisting of hydrogen, deuterated C1-C5 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.

[0010] R each independently is C1-C5 alkyl.

[0011] Some specific pyridinone endoperoxide compounds, R 1 , R 2 , R 3 each independently selected from the group consisting of hydrogen, deuterated methyl, C1-C5 alkyl, trifluoromethyl, carboxyl.

[0012] R 4 -R 7 each independently selected from the group consisting of hydrogen, deuterated C1-C5 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.

[0013] R each independently is C1-C5 alkyl.

[0014] Some specific pyridinone endoperoxide derivatives, R 1 , R 2 , R 3 each independently selected from the group consisting of hydrogen, deuterated methyl, trifluoromethyl, methyl, carboxyl.

[0015] Some specific pyridinone endoperoxide derivatives, R 1 , R 3 each independently selected from the group consisting of hydrogen, methyl.

[0016] Some specific pyridinone endoperoxide derivatives, R 4 -R 7 each independently selected from the group consisting of 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.

[0017] R is independently C1-C5 alkyl.

[0018] Use of the pyridone endoperoxide derivative in the preparation of a singlet oxygen releasing material.

[0019] Use of the pyridone endoperoxide derivative in the preparation of a medicament for the delivery of singlet oxygen to a cell, tissue or organ.

[0020] The medicament is one or more of a tablet, a capsule, a granule, a powder, an oral preparation, an injection, a microcapsule preparation, a suppository, a pill, an aerosol, a spray, a powder inhalant, a syrup, a liquor, a tincture, a lotion, a film.

[0021] A liposome or micelle form medicament comprising at least one of the above-mentioned pyridone endoperoxide derivative;

[0022] and / or a carrier;

[0023] and / or a pharmaceutical excipient;

[0024] The carrier is selected from one or more of a metallic nanocarrier, a non-metallic nanocarrier, a micelle, a liposome, lactose, sucrose, gelatin, hard magnesium sulfate, stearic acid.

[0025] The pharmaceutical excipient is selected from one or more of a diluent, a binder, a disintegrant, a lubricant, a glidant, a flavoring agent, a coating agent, a gelatin capsule shell, a cosolvent, a propellant, a surfactant, a preservative, a lyophilization protecting agent.

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

[0027] The liposome, the loading amount of the pyridone endoperoxide derivative is 10%-17%.

[0028] The liposome, the loading amount of the pyridone endoperoxide derivative is 14%-17%.

[0029] The liposome, the pyridone endoperoxide derivative, soybean lecithin, DSPE-PEG2000, cholesterol are mixed in an organic solvent, and after removing the solvent, dispersed in a buffer, filtered or freeze-dried to obtain.

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

[0031] The organic solvent is selected from chloroform, n-hexane, dioxane, ethyl acetate, methanol and the like.

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

[0033] The application further provides a preparation method of the pyridone endoperoxide derivative, and a reaction formula is as follows:

[0034]

[0035] The preparation method of the pyridone endoperoxide derivative is all adopted in the prior art, the preparation of the phenylpyridone compound is referred to the patent document with the publication number CN116655654A, the phenylpyridone compound is connected with the azobenzene compound through a conventional ester condensation, and the obtained compound is further referred to the patent document CN116655654A to obtain the target endoperoxide derivative through dye light irradiation.

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

[0037]

[0038] The idea of the application is that the endoperoxide compound has anticancer potential, and is combined with the chemotherapeutic drug nitrogen mustard to form a prodrug, and is expected to develop an efficient anticancer strategy. The mechanism is shown in the following formula, the molecule is connected by the azo group from the left and right two functional parts, the left pyridone endoperoxide slowly releases singlet oxygen at 37 DEG C to kill cells, and the right nitrogen mustard group performs combined chemotherapy. In the presence of the azo group, the nitrogen mustard is "passivated" due to electron-withdrawing effect, showing low toxicity; and when the drug reaches the tumor tissue, the azo reductase overexpressed in the low-oxygen environment of the tumor tissue reduces the azo bond to break, and the generated aniline nitrogen mustard has enhanced toxicity. Therefore, the combination of the two can selectively kill cancer cells with high efficiency in the low-oxygen environment of the tumor.

[0039]

[0040] The application has the following beneficial effects:

[0041] (1) The endoperoxide-nitrogen mustard combined anticancer agent PRI-AZO-Cl-ENDO synthesized in the application has strong DNA damage and cancer cell killing effect, especially in the process of treating human cervical cancer cells (Hela cell line), the PRI-AZO-Cl-ENDO under the low-oxygen condition has a half lethal amount IC 50 45.19 muM under the normal-oxygen condition, which shows stronger anticancer effect;

[0042] (2) The azo group connected in the compound PRI-AZO-Cl-ENDO of the application responds to the overexpression of azo reductase by low oxygen, and can significantly reduce the toxic effect of normal cells under normoxia compared with traditional anticancer drugs; and the overall biological safety of the anticancer drug is further improved by the technical means of liposome wrapping.

[0043] (3) The whole 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 by using common and easily available raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 PRI-AZO-Cl-ENDO in deuterated chloroform: deuterated methanol = 1:5 mixed solvent over time (37℃);

[0045] Figure 2 PRI-AZO-Cl-ENDO and PRI-AZO-Cl of different concentrations in different cell lines under normoxia (Normoxia) and hypoxia (Hypoxia) environment cell toxicity;

[0046] Figure 3 DCFH-DA detects the imaging diagram of PRI-AZO-Cl-ENDO releasing singlet oxygen in Hela cells;

[0047] Figure 4 The bright field and fluorescence images of cells double-stained with calcein and iodopyridine;

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

[0049] Figure 6 The cell migration microscope photos of Hela cells after scratch and treatment with PRI-AZO-Cl-ENDO and PRI-AZO-Cl for 24h and 48h;

[0050] Figure 7 The microscope photos of the effect of internal peroxide compound (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 (Normoxia) or hypoxia (Hypoxia) environment;

[0052] Figure 9 The in vivo tumor inhibition effect of drug-loaded liposomes and the evaluation of mouse biological safety.

[0053] Figure 10 Structure of a pyridinone endoperoxide-based compound. DETAILED DESCRIPTION

[0054] The application is further illustrated by the following examples.

[0055] Preparation of compound 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, then N,N-dihydroxyethyl aniline (3.9 g, 21.5 mmol) was added slowly while maintaining 0 °C. After that, the mixture was heated to reflux at 110 °C for 1 h, then the mixture was cooled to room temperature and concentrated by rotary evaporation. The residue was dissolved in ethyl acetate and washed with water three times, saturated brine once. The organic layer was dried over anhydrous magnesium sulfate, the organic phase was collected and concentrated by rotary evaporation to give the crude product. Finally, column chromatography was performed with n-hexane: ethyl acetate (v / v) 10:1 as eluent to give 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 a solution of p-aminobenzyl alcohol (1 g, 8.1 mmol) in water containing concentrated hydrochloric acid (1.675 mL). After stirring at 0 °C for 20 min, the resulting solution was added to a solution of compound 1 (1.5 g, 6.8 mmol) in ethanol. After 2 h, the solution was diluted with dichloromethane (500 mL) and washed with water twice, saturated brine once. The organic layer was collected and dried over anhydrous sodium sulfate, purified by silica gel chromatography n-hexane: ethyl acetate (v / v) 5:1 to give compound 2 with a yield of 35%;

[0059] S3: To dry DMF containing 3-methylpyridin-2(lH)-one (1 g, 10 mmol) was added the corresponding aryl iodide (4.14 g, 15 mmol), to the resulting solution was added anhydrous potassium carbonate (2.76 g, 20 mmol) and copper iodide (190.45 mg, 1 mmol), then the reaction mixture was heated to 150 °C. After 6 h, the reaction mixture was cooled to room temperature, quenched with cold water, and extracted with ethyl acetate three times. 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. Extraction with ethyl acetate, followed by concentration under reduced pressure, yielded the product as a white solid with a yield of 85%;

[0061] S5: To a dry 50 mL two-necked round bottom flask, compound 4 (150 mg, 0.65 mmol), dichloromethane (3 mL) and DMF (2 μL) were added in sequence. 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 redissolved in dichloromethane (20 mL) and to the stirred solution was added DMAP (0.611 mg, 0.005 mmol) and compound 2 (345 mg, 0.98 mmol) 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, the combined organic layers were washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography with n-hexane: ethyl acetate (v / v) 2:1 to give a yellow solid with a yield of 60%; S6: Compound PRI-AZO-Cl (20 mg, 0.34 mmol) was dissolved in 3 mL 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 under an oxygen atmosphere for 2 h. During the reaction, 18 W, 630 nm red light was used for irradiation. After activated carbon was used to remove methylene blue, the pure product, compound PRI-AZO-Cl-ENDO, was obtained as a yellow solid with a yield of 97% after concentration under reduced pressure.

[0062] Example 1: Test of the half-life of the internal peroxide compound

[0063] As shown in Figure 1 , the test of the half-life of the internal peroxide compound was carried out by nuclear magnetic resonance hydrogen spectrum. 3-5 mg of PRI-AZO-Cl-ENDO solid was dissolved in 0.6 mL of deuterated reagent, and nuclear magnetic resonance hydrogen spectrum test was carried out at time points of 0 h, 19 h, 48 h, 72 h, 96 h, 120 h and 168 h, respectively. The nuclear magnetic tube was maintained in a 37 °C constant temperature water bath, the change of the peak area of the internal peroxide was observed, the proportion was determined by the ratio, and whether the decay was ended was judged. Plotting ln(A0 / A) against time t gave a straight line with a slope k of 0.0117, and the half-life t 1 / 2 = 59.23 h.

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

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

[0066] Due to the need of the experiment, a hypoxia (1% O2) cell model will be constructed for hypoxia experiment research. First, an appropriate amount of cells are cultured in a 96-well plate or a 6-well plate, and after the cells are stable in the cell state after overnight culture in a 37°C, 5% CO2 incubator, the cells are transferred to a low-oxygen incubator (incubator environment: 37°C, 5% CO2, 1% O2, 94% N2) for further culture. In order to make the intracellular environment in a hypoxic state, the culture time in the low-oxygen incubator is not less than 24h.

[0067] Other cell culture has no special standard and is in normoxic (21% O2) environment.

[0068] Example 3: MTT method to evaluate the cytotoxicity of endoperoxide

[0069] The MTT method is used to evaluate the cytotoxicity of endoperoxide. Cells are cultured in a 96-well plate at a density of 5x10 3 cells per well under normoxic conditions at 37°C, 5% CO2 overnight to allow the cells to adhere completely. Then, according to the experimental requirements, the cells are divided into normoxic and hypoxic groups and placed in two groups of different oxygen concentrations for 24h to make the cells in normoxic and hypoxic (1% O2) states. Then, gradient concentrations of endoperoxide PRI-AZO-Cl-ENDO and its precursor compound PRI-AZO-Cl (control) are added to the cells, respectively, and incubated for 24h. Then 20μL of MTT solution (5mg / mL PBS solution) is added to each well, incubated for 4h, the culture medium is removed, 150μL of DMSO is added to each well to dissolve the formazan, and the absorbance at 570nm is detected in an enzyme marker at 37°C for 5min. The cell survival rate is calculated according to the formula. As shown in Figure 2 endoperoxide exhibits excellent anti-cancer efficacy in MCF-7, Hela, SK-OV-3, 4T1 cells, and has a large gap between normoxic and hypoxic (1% O2) environments.

[0070] Example 4: In vitro release of singlet oxygen by endoperoxide in cells

[0071] To evaluate the release of singlet oxygen by endoperoxide, a fluorescent probe DCFH-DA was used to capture singlet oxygen. Hela cells were cultured in 96-well plates at a density of 9 x 10 3 cells per well and incubated overnight at 37 °C in a 5% CO2 incubator. After the cells were fully adhered, they were incubated in a hypoxic environment for 24 h. Then, 25 μM and 50 μM endoperoxide solutions were added to the medium, and after 3 h of incubation, the medium was removed and the cells were washed with phosphate buffered saline (PBS). Then, 100 μL of serum-free medium containing DCFH-DA was added to each well for staining, and the cells were incubated at 37 °C for 45 min. After removing the medium, the cells were washed once with PBS. Finally, 100 μL of fresh medium was added to each well, and the cells in each well were photographed under a high-content imaging instrument. As shown in FIG. 2, weak green fluorescence was observed in the Hela cells cultured with 25 μM PRI-AZO-Cl-ENDO, indicating that endoperoxide can release singlet oxygen, but the amount of singlet oxygen is small. When the concentration was increased to 50 μM, the green fluorescence intensity was significantly enhanced, indicating that the amount of singlet oxygen stored and released by endoperoxide also increased with increasing concentration, which is consistent with the expected results. Figure 3

[0072] Example 5: In vitro apoptosis experiment

[0073] To visually observe the process of singlet oxygen-induced apoptosis, an Annexin V-FITC / PI kit was used. Hela cells were cultured in 96-well plates at a density of 7 x 10 3 cells per well and incubated overnight at 37 °C in a 5% CO2 normoxic environment to allow the cells to fully adhere. Then, the cells were incubated in a hypoxic environment for 24 h. Then, 12.5 μM endoperoxide and its precursor solutions were added, respectively, and after the administration was completed, the cells were incubated in a hypoxic environment for 3-4 h. Immediately after incubation, the drug-containing medium was removed, and the cells were washed with PBS. Then, Annexin V-FITC / PI staining solution was added, and after incubating the cells at room temperature for 20 min in the dark, they were placed in an ice bath. Finally, the location of red and green fluorescence in the cells was observed under a high-content imaging instrument to determine the early and late stages of apoptosis. As shown in FIG. 3, the control group (0 μM) showed strong green fluorescence signals, clearly indicating that the number of normally surviving cells was large, thereby verifying that the toxicity to Hela cells was very low. In the second group, after adding the endoperoxide precursor (PRI-AZO-Cl), 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), the tumor cells began to die (red fluorescence). Figure 4 ​​

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

[0075] In vitro live / dead cell double staining assays were performed using the Calcein-AM / PI kit. HeLa cells were cultured at 7 × 10⁶ cells per well. 3 Cells were cultured at a density of [number] cells per well in 96-well plates overnight at 37°C and 5% CO2 under normoxic conditions to ensure complete cell adhesion. Subsequently, the cells were incubated under hypoxic conditions for 24 hours. Then, 35 μM of an endoperoxide compound and its precursor solution were added to the culture medium. After drug administration, the cells were incubated under hypoxic conditions for another 2-3 hours. Immediately after incubation, the drug-containing medium was removed, and the cells were washed with PBS. Serum-free medium containing Calcein-AM was then added, and the cells were incubated at 37°C in the dark for 20-25 minutes. The PI (pill-in-place) from the kit was added to the cells, and staining was performed at room temperature in the dark for 5 minutes. Figure 5 As shown, the precursor of endogenous peroxide (PRI-AZO-Cl) exhibited only weak fluorescence in HeLa cells under hypoxic (1% O2) conditions, with most cells showing no morphological changes, indicating that aniline mustard released only under hypoxia has a weak killing effect on tumor cells. However, under the same conditions, cells co-cultured with endogenous peroxide (PRI-AZO-Cl-ENDO) showed strong fluorescence and significant morphological changes. The exposed phosphatidylserine residues on the everted cell membrane specifically bound to Annexin V-FITC, emitting strong green fluorescence upon excitation at 488 nm, indicating early apoptosis. Simultaneously, cell nuclei exhibiting strong red fluorescence were also observed, indicating that PI had reacted with the nuclei, which had lost cell membrane integrity, leading to late apoptosis or necrosis. These results suggest that under hypoxic conditions, endogenous peroxide can react with overexpressed azoreductase, thereby releasing the chemotherapeutic drug aniline mustard, which synergistically fights cancer with the singlet oxygen released by endogenous peroxide.

[0076] Example 7: In vitro cell migration experiment

[0077] 1 cell scratch

[0078] HeLa cells in logarithmic growth phase were harvested at 7 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight at 37°C and 5% CO2 under normoxic conditions. After complete cell adhesion, the plates were placed in a hypoxic environment for another 24 hours. First, the culture medium was aspirated from the wells. Then, a uniform scratch was made on the plate using a pipette tip. Cells were washed with PBS 3 times per hour. The control group was replaced with fresh medium (containing 1% FBS), and the drug-treated group was replaced with drug-containing medium (containing 1% FBS). Immediately, images were taken under a microscope. Next, 10 μM solutions of PRI-AZO-Cl-ENDO and PRI-AZO-Cl were added, and incubation continued for 48 hours. Images were taken at 0h, 24h, and 48h to observe the healing of the scratch under drug treatment. Figure 6 As shown, the control group and cells treated with the endogenous peroxide precursor (PRI-AZO-Cl) exhibited the highest degree of scratch healing after 48 hours of culture, while the degree of scratch healing was significantly reduced after treatment with the endogenous peroxide compound (PRI-AZO-Cl-ENDO). The wound healing rates at 24 hours and 48 hours were 20% to 35.9%, 14% to 22.1%, and 9.3% to 13.6%, respectively, indicating that endogenous peroxides have a significant ability to inhibit tumor migration.

[0079] 2. Transwell cell migration

[0080] Prepare a culture medium containing 20% ​​FBS, and add 600 μL to the bottom of a 24-well plate in the lower chamber. Take HeLa cells in logarithmic growth phase and add 1 × 10⁻⁶ cells. 5 Cells were resuspended in the upper chamber at a density of cells / well, and the presence of air bubbles was checked. The upper chamber was gently added to the lower chamber using forceps, and the bottom was checked for air bubbles. The cells were then cultured in an incubator. After 4 hours, the cells were treated using the following different methods: Group 1: Incubated with DMEM containing 1% FBS at 37°C, 21% O2, and 1% O2. Group 2: Incubated with DMEM containing PRI-AZO-Cl (35 μM) and 1% FBS at 37°C, 21% O2, and 1% O2. Group 3: Incubated with DMEM containing PRI-AZO-Cl-ENDO (35 μM) and 1% FBS at 37°C, 21% O2, and 1% O2. After 24 hours of incubation, staining was performed. The culture medium in the chambers was removed, and the lower surface was fixed by immersing in 4% paraformaldehyde solution for 10 minutes. After fixation, the cells were rinsed again and stained with 10% crystal violet for 5-10 minutes. After staining, the cells were washed until no obvious purple color remained. Gently wipe the upper side of the chamber with a cotton swab, then place it on a glass slide for microscopic observation. Count the number of cells on the membrane surface and take the average value of the five regions (center and perimeter). Figure 7It can be seen that the number of cell migration is reduced and the cell survival rate is decreased when the cells are in the hypoxic environment. The migration rate of the cells treated with the endoperoxide at the 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 result, indicating that the molecule has a certain degree of ability to inhibit cell migration.

[0081] Example 9: Liposome-encapsulated drug delivery strategy

[0082] 1 Synthesis of coated liposomes

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

[0084] 2 Appearance and morphology

[0085] The sample was dropped on a 400 mesh copper mesh, and after natural evaporation and drying, it was dyed with 3% phosphotungstic acid solution. After 30 seconds of dyeing time, the excess dye was removed, and after drying, the sample was carefully observed and photographed using a transmission electron microscope (TEM). In addition, we also took pictures of the macroscopic state of the liposomes for further analysis. The liposomes appeared as a light yellow transparent state, uniformly dispersed without precipitation, indicating that the appearance state was excellent and the quality was excellent; after freeze-drying, it was a yellow powder solid.

[0086] 3 Calculation of encapsulation efficiency

[0087] The UV-visible spectrometer was used to calculate: 5 mmol / L of compound PRI-AZO-Cl-ENDO DMSO solution was diluted with DMSO step by step, and the absorbance at 415 nm was tested at different concentrations to establish a standard curve.

[0088] Accurately weigh 27.83 mg of the liposome powder prepared in Example 1, dissolve in 2 mL of DMSO to break the emulsion. Take out 500 μL and add to 3 mL of a cuvette, test its absorbance at 415 nm, and get the actual concentration corresponding to the standard curve. According to the tested concentration, the mass of PRI-AZO-Cl-ENDO encapsulated is obtained, and the total dosage of PRI-AZO-Cl-ENDO is calculated to get the encapsulation efficiency of 14.2%.

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

[0090] Cells were cultured according to the procedure in Example 2, "Cell Culture and Establishment of a Hypoxic (1% O2) Tumor Cell Model," with normoxic and hypoxic groups established. The cytotoxicity of the drug-loaded liposomes was assessed using the MTT assay. Figure 9 As shown, liposomes in HeLa cells IC50 50 The concentration was 652 μg / mL, demonstrating excellent anticancer efficacy, and showing a significant difference between normoxic and hypoxic environments.

[0091] Example 11: In vivo tumor-suppressing effect in a mouse model

[0092] Take 21 tumors, each approximately 50-80 mm in size. 3 4T1 tumor-bearing mice were randomly divided into four groups: a saline group (control group), a PRI-AZO-Cl@LP treatment group (500 mg / kg), a PRI-AZO-Cl-ENDO@LP treatment group (300 mg / kg), and a PRI-AZO-Cl-ENDO@LP treatment group (500 mg / kg). Mice in each experimental group were injected intravenously via the tail vein. The first administration was scheduled for day 1, and administration was repeated eight times on days 1, 3, 5, 7…15. The long diameter (L, mm) and short diameter (W, mm) of the tumor in each experimental group were measured and recorded daily using calipers, 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 administration. t The data, plotted with time (d) on the x-axis, V t A tumor growth curve was plotted with the vertical axis as the ordinate. The mice were observed for their condition, and their weight was recorded daily using a balance. After 15 days of drug administration (day 16), the tumor-bearing mice were euthanized by exsanguination of the eyeballs. Blood was collected, the tumor was carefully dissected, washed with physiological saline, excess water was absorbed with absorbent paper, and the weight was measured and photographed. Results are as follows: Figure 9The tumor volume of the control group, PRI-AZO-Cl@LP and PRI-AZO-Cl-ENDO@LP treatment groups showed a trend of gradual increase. The tumor growth of the control group was the fastest and the average tumor tissue weight was the largest. The PRI-AZO-Cl@LP (500 mg / kg) treatment group and the PRI-AZO-Cl-ENDO@LP (300 mg / kg) treatment group were the second fastest, and the PRI-AZO-Cl-ENDO@LP (500 mg / kg) treatment group was the slowest, showing a good effect of inhibiting tumor growth. In summary, the endoperoxide PRI-AZO-Cl-ENDO can effectively inhibit tumor growth in animals after being wrapped in liposomes.

[0093] Good biological safety is a prerequisite for clinical application. During the treatment of tumor-bearing mouse models, no significant changes in body weight were found by monitoring the changes in mouse body weight every two days, as shown in Table 5. This indicates that the physiological effects of the drug treatment by tail vein injection did not cause significant side effects in mice. Figure 10 Good biological safety is a prerequisite for clinical application. During the treatment of tumor-bearing mouse models, no significant changes in body weight were found by monitoring the changes in mouse body weight every two days, as shown in Table 5. This indicates that the physiological effects of the drug treatment by tail vein injection did not cause significant side effects in mice.

Claims

1. A class of pyridinone endoperoxide derivatives characterized in that, having the structure: ； wherein R 1 , R 2 , R 3 are each independently selected from the group consisting of hydrogen, deuterated methyl, C1-C10 alkyl, carboxyl, C1-C5 fluoroalkyl, nitro, C1-C10 ester; R 4 -R 7 each independently selected from the group consisting of hydrogen, C1-C5 deuterated alkyl, C1-C5 fluorinated alkyl, halogen, carboxyl, sulfonic acid group, aminosulfonic acid group, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether group; each R is independently a C1-C5 alkyl group.

2. The pyridinone endoperoxide derivative according to claim 1, characterized in that, R 1 , R 2 , R 3 each independently is selected from the group consisting of hydrogen, deuterated methyl, C1-C5 alkyl, trifluoromethyl, carboxyl; R 4 -R 7 each independently selected from the group consisting of hydrogen, C1-C5 deuterated alkyl, C1-C5 fluorinated alkyl, halogen, carboxyl, sulfonic acid group, aminosulfonic acid group, hydroxyl, amino, -COOR, -CONR, C1-C10 alkylamino, nitro, cyano, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 thioether group; each R is independently a C1-C5 alkyl group.

3. The pyridinone endoperoxide derivative according to claim 2, wherein R 1 , R 2 , R 3 each independently is selected from the group consisting of hydrogen, deuterated methyl, trifluoromethyl, methyl.

4. The pyridinone endoperoxide derivative according to claim 3, wherein R 1 , R 3 each independently is selected from the group consisting of hydrogen, methyl.

5. The pyridinone endoperoxide derivative according to claim 4, wherein R 4 -R 7 each independently selected from the group consisting of hydrogen, deuterated methyl, trifluoromethyl, halogen, carboxyl, sulfonic acid group, aminosulfonic acid group, hydroxyl, amino, -COOR, -CONR, C1-C5 alkylamino, nitro, cyano, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 thioether group; each R is independently a C1-C5 alkyl group.

6. Use of the pyridinone endoperoxide derivative according to any one of claims 1-5 for the preparation of a medicament for the delivery of singlet oxygen to cells, tissues or organs.

7. Use according to claim 6, characterized in that, The medicament is one or more of a tablet, a capsule, a granule, a powder, an oral preparation, an injection, a microcapsule preparation, a suppository, a pill, an aerosol, a spray, a powder inhalant, a syrup, a liquor, a tincture, a lotion, a film.

8. A drug in the form of a liposome or a micelle, characterized in that, The liposome or micelle form medicament comprises at least one of the pyridinone endoperoxide derivatives according to any one of claims 1-5; and / or a carrier; and / or a pharmaceutical adjuvant; The carrier is selected from one or more of a micelle, a liposome.

9. A liposome, characterized by, The pyridinone endoperoxide derivative according to any one of claims 1-5, soybean phosphatidylcholine, DSPE-PEG2000, cholesterol.

10. The liposome of claim 9, wherein, The pyridinone endoperoxide derivative, soybean phosphatidylcholine, DSPE-PEG2000, cholesterol are mixed in an organic solvent, and after removal of the solvent, dispersed in a buffer, filtered or freeze-dried to obtain the liposome. The mass ratio of soybean phosphatidylcholine, DSPE-PEG2000, cholesterol, the pyridinone endoperoxide derivative is 2-7:1:1:0.2.

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