Singlet oxygen-triplet oxygen synergistic delivery carrier and application thereof
By designing the inner peroxide-1,4-diazabicyclo[2.2.2]octane (DABCO) and inner peroxide-proline molecules, the coordinated delivery of singlet oxygen and triplet oxygen is achieved, solving the problems of tumor hypoxia and tumor cell killing, and improving the effect of tumor treatment.
Patent Information
- Application Number
- CN202510497013.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-08-19
AI Technical Summary
Existing oxygen carriers cannot achieve synergies between oxygen release and drug effects when relieving tumor hypoxia, and singlet oxygen is quickly quenched in biological media and cannot effectively kill tumor cells.
The inner peroxide-1,4-diazabicyclo[2.2.2]octane (DABCO) and inner peroxide-proline molecules are designed to release singlet oxygen through reverse cyclization reaction, and partially quenched into triplet oxygen using DABCO to achieve synergistic delivery of oxygen.
While alleviating tumor hypoxia, the killing effect of singlet oxygen is improved, and it shows good killing ability to non-small cell lung cancer cells, achieving the coordinated delivery of singlet oxygen and triplet oxygen.
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Figure CN120504677A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology and relates to a singlet oxygen-triplet oxygen coordinated delivery carrier and its application. Background Art
[0002] Hypoxia in tumor tissue can lead to a series of serious consequences, such as activating the expression of hypoxia-inducible factors (HIFs), which enhance the tumor's migration, invasion, and proliferation capabilities. It also enhances drug resistance through physiological and genetic pathways, reducing the effectiveness of radiotherapy and chemotherapy. Currently developed oxygen carriers, such as perfluorocarbon emulsions and hemoglobin oxygen carriers, combined with drugs, have alleviated tumor hypoxia to a certain extent. However, these systems often require a long time to release oxygen and fail to address the synergistic effect of oxygen release and drug action.
[0003] Singlet oxygen ( 1 O2) is a cytotoxic reactive oxygen species, but it is highly unstable and easily quenched to triplet oxygen. The relaxation half-life of singlet oxygen to triplet oxygen in aqueous solution is 4 microseconds. However, its half-life in biological media, particularly within cells, is much shorter because it is quenched by most biomolecules. This quenching occurs almost entirely chemically, meaning that singlet oxygen reacts with biological substrates and alters their structure without generating any triplet oxygen. If triplet oxygen is desired, it must be physically quenched before chemical reaction with the biological substrate. Summary of the Invention
[0004] The purpose of this invention is to develop a singlet oxygen-triplet oxygen synergistic carrier. The present invention designs endoperoxide-1,4-diazabicyclo[2.2.2]octane (DABCO) and endoperoxide-proline molecules. These molecules release toxic singlet oxygen through a reverse cyclization reaction of the endoperoxide structure within cells. Simultaneously, DABCO and L-proline act as physical singlet oxygen quenchers, quenching some of the singlet oxygen into triplet oxygen. This molecular carrier not only releases molecular oxygen to alleviate tumor hypoxia but also releases singlet oxygen to kill tumor cells.
[0005] The technical solution of the present invention: a class of compounds having a structure of formula I:
[0006] Where Q is 、
[0007] wherein R2 and R3 are each independently selected from hydrogen, hydroxyl, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R1 and R4 are each independently selected from hydrogen, hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R5 is selected from hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; Wherein, x is an integer from 1 to 2000.
[0008] Furthermore, R2 and R3 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R1 and R4 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R5 is selected from an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group.
[0009] Further, R2 and R3 are each independently selected from hydrogen and an alkyl group having 1 to 6 carbon atoms; R1 and R4 are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms; R5 is an alkyl group having 1 to 6 carbon atoms.
[0010] Furthermore, R1 and R4 are hydrogen, R2 and R3 are hydrogen, and R5 is methyl.
[0011] Use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of singlet oxygen and oxygen carrier materials.
[0012] Use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors.
[0013] Furthermore, the tumor is non-small cell lung cancer.
[0014] Furthermore, the compound or a pharmaceutically acceptable salt thereof transports singlet oxygen and triplet oxygen to cells, tissues or organs.
[0015] A pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.
[0016] Furthermore, the pharmaceutical composition is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsules, suppositories, pills, aerosols, sprays, powder inhalers, syrups, alcoholic preparations, tinctures, lotions, and films.
[0017] Beneficial effects of the present invention: The present invention combines endoperoxides with singlet oxygen physical quenching groups L-proline and DABCO to develop a singlet oxygen-triplet oxygen co-delivery carrier. Naphthalene endoperoxides are highly effective 1 An O2 storage and release unit, capable of releasing singlet oxygen at a certain temperature, exhibits advantages such as high singlet oxygen yield, moderate half-life, and excellent stability in water, making it a reliable singlet oxygen carrier. L-proline groups and DABCO are used to partially convert singlet oxygen released from endoperoxide molecules into triplet oxygen, alleviating tumor hypoxia while simultaneously exerting the killing effect of singlet oxygen. This endoperoxide carrier exhibits excellent singlet and triplet oxygen release capacity, as well as anti-tumor activity against human non-small cell lung cancer (A549) cells under hypoxic conditions, suggesting broad application prospects.
[0018] This vector is based on an endoperoxide structure, with L-proline and 1,4-diazabicyclo[2.2.2]octane (DABCO) as singlet oxygen physical quenching groups. At a certain temperature, the endoperoxide structure undergoes a reverse cyclization reaction to release toxic singlet oxygen. Simultaneously, the quenching group quenches some of the singlet oxygen to produce triplet oxygen, enabling the molecule to both alleviate tumor hypoxia and kill tumors. In vitro testing confirmed that the molecule provided by the present invention releases triplet oxygen simultaneously with singlet oxygen, achieving singlet-triplet oxygen co-delivery. This invention successfully establishes a singlet-triplet oxygen co-delivery vector that can be used to alleviate tumor hypoxia and enhance the killing effect of singlet oxygen, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a graph showing the change in singlet oxygen release intensity of endoperoxides over time.
[0020] Figure 2 The graph shows the change of singlet oxygen release intensity of endoperoxide at 531 nm as a function of time.
[0021] Figure 3 This is an imaging diagram of triplet oxygen released from cells by BTP detecting endoperoxides in a hypoxic environment.
[0022] Figure 4 This is an image of the release of singlet oxygen from endoperoxides within cells.
[0023] Figure 5 The toxicity of endoperoxides 10 and 15 to A549 cells under normoxia and hypoxia. DETAILED DESCRIPTION
[0024] The present invention will be described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention, but are not limited thereto, unless otherwise specified.
[0025] The compound or a pharmaceutically acceptable salt thereof is a singlet oxygen-triplet oxygen co-carrier and has the following structure: ; wherein R2 and R3 are each independently selected from hydrogen, hydroxyl, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R1 and R4 are each independently selected from hydrogen, hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R5 is selected from hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; Wherein, x is an integer from 1 to 2000.
[0026] In some specific singlet oxygen-triplet oxygen co-delivery vectors, R2 and R3 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R1 and R4 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R5 is selected from an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group.
[0027] In some specific singlet oxygen-triplet oxygen co-delivery vectors, R2 and R3 are independently selected from hydrogen and an alkyl group with 1-6 carbon atoms; R1 and R4 are independently selected from hydrogen and an alkyl group with 1-6 carbon atoms; and R5 is an alkyl group with 1-6 carbon atoms.
[0028] In some specific singlet oxygen-triplet oxygen co-delivery vectors, R1 and R4 are each independently hydrogen, R2 and R3 are hydrogen, and R5 is methyl.
[0029] Application of the singlet oxygen-triplet oxygen coordinated delivery carrier in the preparation of singlet oxygen and oxygen carrier materials.
[0030] The singlet oxygen-triplet oxygen coordinated delivery carrier is used in the preparation of a drug for delivering singlet oxygen and triplet oxygen to cells, tissues or organs.
[0031] Application of the singlet oxygen-triplet oxygen co-delivery carrier in the preparation of drugs for treating tumors.
[0032] The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, alcoholic preparations, tinctures, lotions, and membrane preparations.
[0033] A drug comprising at least one of the above-mentioned singlet oxygen-triplet oxygen co-delivery vectors; 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; The pharmaceutical excipients are selected from one or more of diluents, binders, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents.
[0034] The endoperoxide precursors of the present invention are prepared by reacting a 1-naphthylamine compound (Compound A) with Boc-L-proline (Compound B) and a 1-naphthylamine compound (Compound A) with a carboxyl compound linked to triethylenediamine (Compound C). The corresponding 1-naphthylamine compound and the carboxyl compound of triethylenediamine can be obtained by conventional chemical synthesis. The preparation steps and reaction formula are as follows:
[0035] Wherein, the definitions of R1-R5 are the same as those in the above-mentioned singlet oxygen-triplet oxygen co-delivery carrier structure.
[0036] The specific embodiments of the present invention are described in detail below in conjunction with the technical solutions: Example 1
[0037]
[0038] Step a: Dissolve compound 1 (2 g, 12.8 mmol) in carbon tetrachloride (30 mL). Add N-bromosuccinimide (2.28 g, 12.8 mmol) and azobisisobutyronitrile (105 mg, 0.64 mmol). Under nitrogen, reflux the reaction mixture in a 70°C oil bath for 4 h. After the reaction is complete, cool the reaction mixture, filter it, and wash it with carbon tetrachloride. The filtrate is concentrated under reduced pressure on a rotary evaporator and the crude product is purified by silica gel column chromatography using n-hexane as the eluent to obtain 1.5 g of a white solid in a calculated yield of 50%. 1 H NMR (400 MHz, Chloroform- d ) d 8.20 – 8.16 (m, 1H), 8.08 – 8.04 (m, 1H), 7.67 – 7.55 (m, 2H), 7.45 (d, J =7.1 Hz, 1H), 7.26 (d, J = 6.8 Hz, 1H), 4.97 (s, 2H), 2.70 (s, 3H). Step b: Diethyl malonate (480 mg, 3.0 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL), and sodium hydride (96 mg, 4 mmol) was slowly added. The mixture was refluxed in an oil bath at 66°C with stirring for 3 h. Compound 2 (235 mg, 1.0 mmol) was then dissolved in anhydrous tetrahydrofuran (5 mL) and slowly added dropwise to the reaction mixture. Reflux and stirring were continued at 66°C for 2 h. After the reaction was completed and the reaction mixture was cooled, water (1 mL) was added to quench the reaction. Hydrochloric acid was added to adjust the pH of the reaction mixture to 3, and the mixture was extracted with ethyl acetate. The combined extracts were washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure using a rotary evaporator. The crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate (50 / 1) as the eluent to obtain 475 mg of a colorless, transparent oil with a calculated yield of 84%. 1 H NMR (400 MHz, Chloroform- d ) d 8.09– 8.00 (m, 2H), 7.60 – 7.50 (m, 2H), 7.30 – 7.18 (m, 2H), 4.24 – 4.09 (m,4H), 3.83 (t, J = 7.5 Hz, 1H), 3.69 (d, J = 7.5 Hz, 2H), 2.66 (s, 3H), 1.20(t, J = 7.1 Hz, 6H). Step c: Dissolve compound 3 (250 mg, 0.8 mmol) in methanol (5 mL) and add sodium hydroxide solution (12.5%, 6 mmol). Heat under reflux at 90°C for 2 h. After the reaction, remove the solution using a rotary evaporator, adjust the pH to 3 with hydrochloric acid (2 M), and filter to obtain 200 mg of a white solid with a calculated yield of 97%. 1 H NMR (400 MHz, DMSO- d 6) d 8.12– 8.00 (m, 2H), 7.62 – 7.53 (m, 2H), 7.28 – 7.21 (m, 2H), 3.60 (t, J = 7.3Hz, 1H), 3.49 (d, J = 7.4 Hz, 2H), 2.61 (s, 3H). Step d: Compound 4 (200 mg, 0.77 mmol) was stirred at 150 °C for 24 h to obtain 150 mg of a light yellow solid with a calculated yield of 91%. 1 H NMR (400 MHz, DMSO- d 6) d 12.18 (s, 1H), 8.10 – 7.99 (m, 2H), 7.60 – 7.53 (m, 2H), 7.26 (s, 2H), 3.26 (t, J = 7.7 Hz, 2H), 2.65 –2.58 (m, 5H). Step e: Dissolve compound 5 (150 mg, 0.7 mmol) in thionyl chloride (3 mL, 41 mmol) and reflux at 80°C with stirring for 2 h. Remove the remaining thionyl chloride using a rotary evaporator to obtain a yellow solid. Dissolve the resulting solid in 1,4-dioxane (2 mL), and slowly add the mixture dropwise to 25% aqueous ammonia (2 mL). Stir the mixture at room temperature for 2 h, extract with ethyl acetate, wash the organic layer with saturated brine, dry over anhydrous sodium sulfate, and perform vacuum distillation using a rotary evaporator. Purify the crude product by silica gel column chromatography using dichloromethane / methanol = 50 / 1 as the eluent to obtain 94 mg of a yellow solid with a calculated yield of 63%. NMR data: 1 H NMR (400 MHz, Chloroform- d ) d 8.11 – 8.02(m, 2H), 7.59 – 7.52 (m, 2H), 7.30 – 7.19 (m, 2H), 5.98 (s, 1H), 5.57 (s,1H), 3.46 – 3.38 (m, 2H), 2.69 (s, 3H), 2.67 – 2.61 (m, 2H). Step f: Compound 6 (94 mg, 0.44 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL). Lithium aluminum hydride (0.35 mL, 2.5 M in THF) was added dropwise at 0°C under nitrogen protection. The mixture was stirred and refluxed at 75°C under nitrogen protection for 6 h. After the reaction was completed, the reaction solution was cooled and quenched with water (1 mL). 10% sodium hydroxide solution (1 mL) and water (3 mL) were added, respectively. The mixture was filtered through celite, and the filtrate was collected and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The crude product was purified by silica gel column chromatography using dichloromethane / methanol = 20 / 1 as the eluent to obtain 25 mg of a white solid with a calculated yield of 28%.1 H NMR (400 MHz, Methanol- d 4) d 8.12 – 8.02 (m, 2H), 7.60 – 7.50 (m, 2H), 7.26 (s, 2H), 3.17 (t, J = 7.7 Hz, 2H), 3.05 – 2.97 (m, 2H), 2.65 (s, 3H), 2.13 – 2.04 (m, 2H). 13 C NMR (101 MHz, Methanol- d 4) d 136.20,134.42, 134.12, 133.05, 127.23, 126.86, 126.68, 126.49, 125.87, 125.13,41.00, 30.88, 30.75, 19.49. Step g: Compound 7 (60 mg, 0.3 mmol) was dissolved in N,N-dimethylformamide (3 mL), and BOC-L-proline (64.6 mg, 0.3 mmol), benzotriazole-1-bis(trimethylamino)phosphine-hexafluorophosphate (132.7 mg, 0.3 mmol), and N,N-diisopropylethylamine (232.6 mg, 1.8 mmol) were added thereto, respectively. The reaction solution was stirred at room temperature for 44 h, quenched with saturated sodium bicarbonate solution (1 mL), and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated using a rotary evaporator. The crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate = 3 / 1 as the eluent to obtain 100 mg of a colorless, transparent oil with a calculated yield of 84%. 1 H NMR (400 MHz, Chloroform- d ) d 8.06 – 7.99 (m, J = 6.5, 3.4 Hz, 2H), 7.54 – 7.49 (m, 2H), 7.25 – 7.19 (m, 2H), 4.30 (d, J = 14.4 Hz, 1H), 3.38 (q, J = 8.9, 7.5 Hz,4H), 3.08 (t, J = 7.6 Hz, 2H), 2.67 (s, 3H), 2.25 – 1.77 (m, 6H), 1.45 (s, 9H). Step h: Compound 8 (100 mg, 0.25 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added thereto. The reaction solution was stirred at room temperature for 2 h. Excess trifluoroacetic acid was removed using a rotary evaporator, and the mixture was washed with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated using a rotary evaporator to obtain 67 mg of a yellow oil with a calculated yield of 90%. 1 H NMR (400 MHz, Chloroform- d ) d 8.05 – 7.99(m, 2H), 7.74 – 7.66 (m, 1H), 7.54 – 7.49 (m, 2H), 7.25 – 7.20 (m, 2H), 3.76 – 3.69 (m, 1H), 3.40 – 3.29 (m, 2H), 3.12 – 3.04 (m, 2H), 3.04 – 2.81 (m,2H), 2.66 (s, 3H), 2.19 – 2.07 (m, 1H), 2.00 – 1.83 (m, 3H), 1.72 – 1.63 (m,2H); 13 C NMR (101 MHz, Chloroform- d ) d 175.12, 135.77, 133.08, 132.78, 131.83,126.38, 125.84, 125.54, 125.39, 124.98, 124.25, 60.64, 47.31, 38.80, 30.84,30.83, 30.47, 26.23, 19.50. Step i: Compound 9 (30 mg, 0.1 mmol) was dissolved in deuterated chloroform (1 mL). A photosensitizer, methylene blue, was added, causing the solution to turn blue. The reaction mixture was stirred in an ice bath while irradiated with red light (18 W, 630 nm) and oxygen was introduced. Reaction progress was monitored by TLC. After 6 h, 200-mesh activated carbon was added to remove methylene blue. The reaction mixture was then filtered through a 0.45 μm organic filter membrane. The filtrate was concentrated on a rotary evaporator to remove the solvent, yielding 33 mg of the product as a yellow solid in a calculated yield of 99%. 1 H NMR (400 MHz, Chloroform- d ) d8.43 – 8.34 (m, 1H), 7.31 – 7.25 (m, 2H), 7.24 – 7.21 (m, 2H), 6.77 – 6.65 (m, 2H), 4.50 (s, 1H), 3.41 – 3.27 (m, 4H), 2.41 – 2.18 (m, 3H), 2.05 – 1.74 (m, 9H). Example 2
[0039]
[0040] Step a: Compound 11 (1 g, 11.61 mmol) was dissolved in toluene (30 mL). Triethylamine (3.24 mL) and ethyl 2,3-dibromopropionate (3 g, 11.61 mmol) were added, and the mixture was stirred in an 80°C oil bath for 12 h. After the reaction, the mixture was filtered and the solvent removed by rotary evaporation. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (40 / 1) as the eluent to obtain 1 g of a yellow oil with a calculated yield of 51%. 1 H NMR (400 MHz, Chloroform- d ) d 4.09 (q, J = 7.1 Hz, 2H), 3.35 – 3.29 (m, 1H), 2.99 – 2.86(m, 2H), 2.85 – 2.71 (m, 3H), 2.69 – 2.48 (m, 5H), 1.15 (t, J = 7.1 Hz, 3H). Step b: Compound 12 (100 mg, 0.54 mmol) was dissolved in methanol (2 mL), and sodium hydroxide solution (132 mg, 3.3 mmol) was added. The mixture was heated under reflux at 90 °C with stirring for 3 h. After the reaction, the solvent was removed by rotary evaporation. The pH was adjusted to 6 with hydrochloric acid, and the solvent was removed by rotary evaporation to obtain 77.6 mg of a yellow solid with a calculated yield of 92%. 1 H NMR (400 MHz, DMSO- d 6) d 3.58 (t, J = 8.8 Hz, 1H), 3.27 – 3.18 (m, 1H), 3.14 –3.08 (m, 2H), 3.08 – 3.01 (m, 2H), 2.95 – 2.84 (m, 5H). 13C NMR (101 MHz, DMSO- d 6) d 169.22, 56.58, 48.66, 46.02, 44.28, 43.77, 41.17. Step c: Compound 13 (60 mg, 0.38 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (171 mg, 0.45 mmol) and N,N-diisopropylethylamine (77.6 mg, 0.6 mmol) were dissolved in N,N-dimethylformamide (3 mL), and compound 7 (59 mg, 0.3 mmol) was dissolved in N,N-dimethylformamide (1 mL) and added to the mixture. The reaction solution was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography using dichloromethane / methanol = 50 / 1 as eluent to obtain 40 mg of a yellow oil with a calculated yield of 40%. 1 H NMR (400 MHz, Chloroform- d ) d 8.04 – 7.95 (m, 2H), 7.56 – 7.47 (m,2H), 7.22 – 7.14 (m, 2H), 5.45 (t, J = 5.7 Hz, 1H), 3.18 (q, J = 6.7 Hz, 2H),3.07 (t, J = 7.6 Hz, 2H), 2.80 (s, 10H), 2.63 (s, 3H), 2.09 – 1.99 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) d 161.54, 134.68, 133.30, 133.15, 131.73,126.42, 126.12, 126.00, 125.72, 125.07, 124.23, 56.02, 45.18, 30.50, 29.95,19.55, 18.59, 17.11. Step d: Compound 14 (30 mg, 0.09 mmol) was dissolved in deuterated chloroform (1 mL). A photosensitizer, methylene blue, was added, causing the solution to turn blue. The reaction mixture was stirred in an ice bath while irradiated with red light (18 W, 630 nm) and oxygen was introduced. Reaction progress was monitored by TLC. After 6 h, 200-mesh activated carbon was added to the reaction solution to remove methylene blue. The solution was then filtered through a 0.45 μm organic filter membrane. The filtrate was concentrated on a rotary evaporator to remove the solvent, yielding 33 mg of the product as a yellow oil with a calculated yield of 99%. 1 H NMR (400 MHz, Chloroform- d ) d 7.32– 7.28 (m, 2H), 7.26 – 7.20 (m, 2H), 6.81 (d, J = 8.1 Hz, 1H), 6.71 (d, J =8.1 Hz, 1H), 5.77 (t, J = 5.6 Hz, 1H), 3.36 – 3.29 (m, 2H), 2.93 (s, 10H), 2.51 – 2.41 (m, 1H), 2.34 – 2.24 (m, 1H), 2.11 – 1.94 (m, 2H), 1.84 (s, 3H). Example 3
[0041] Endoperoxide singlet oxygen release assay.
[0042] Using SOSG as a singlet oxygen capture probe, endoperoxides 10a, 10, and 15 (250 μM) were mixed with SOSG (10 μM). Endoperoxide 10a was an endoperoxide without a quenching group attached and served as a control endoperoxide. Singlet oxygen release was detected using excitation light at 504 nm. Figure 1 As shown, the horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.
[0043] By comparison, endoperoxide 10a can release a large amount of singlet oxygen in the absence of a singlet oxygen quenching group, while endoperoxides 10 and 15 release significantly less singlet oxygen than 10a, demonstrating that proline and DABCO groups can partially quench singlet oxygen. Example 4
[0044] In vitro imaging of triplet oxygen release from cells by endoperoxides.
[0045] Ir(btp)2(acac) (BTP) is an iridium complex that emits red phosphorescence. However, this phosphorescence is quenched by oxygen through energy transfer. Therefore, the lower the oxygen concentration, the stronger the phosphorescence intensity, and vice versa. To better characterize the intracellular oxygen content and the quenching effect of the quenching group on oxygen, this BTP probe was used for cell imaging.
[0046] A549 cells were incubated in a 37°C, 5% CO₂, 21% O₂ incubator for 24 hours under normoxia. The cells were then placed in a 37°C, 5% CO₂, 1% O₂ hypoxic incubator for an additional 24 hours. 5 mM stock solutions of endoperoxygen compounds 10a, 10, and 15, as well as BTP, were prepared and diluted to 50 μM in culture medium. The drug-containing medium was added to a 96-well plate and incubated under hypoxia. A blank control group remained untreated. After 2 hours, the medium was aspirated. BTP-containing medium, diluted to 50 μM in culture medium, was added to the 96-well plate containing the drug-treated and blank control groups. Incubation continued under hypoxia and in the dark. After 2 hours, the medium was aspirated, and the cells were washed three times with PBS. Finally, 100 μL of PBS was added to each well for cell imaging.
[0047] from Figure 3 As can be seen, due to the near-absence of oxygen in the environment, BTP in the blank control group emits distinct red phosphorescence. Furthermore, endoperoxide 10a, as an endoperoxide without a quenching group, releases a large amount of singlet oxygen, leading to distinct red phosphorescence in cells treated with 10a. However, the phosphorescence intensity of BTP is weakened in the images of endoperoxides 10 and 15, indicating that BTP phosphorescence is quenched by oxygen released by the endoperoxides. These results demonstrate that endoperoxides 10 and 15 can release oxygen, quenching the phosphorescence of the BTP probe. Example 5
[0048] Endoperoxide imaging of singlet oxygen release in cells in vitro.
[0049] To assess the intracellular release of singlet oxygen from endoperoxides, a commercial singlet oxygen fluorescent probe, DCFH-DA, was used for imaging. DCFH-DA itself is nonfluorescent. However, after entering the cell membrane, it is hydrolyzed to DCFH by intracellular esterases. Intracellular reactive oxygen species (ROS) oxidize DCFH to DCF, generating fluorescence. Therefore, monitoring the fluorescence of DCF provides an indicator of intracellular ROS levels. A549 cells were incubated in a 37°C, 5% CO₂, 21% O₂ incubator for 24 h. Stock solutions of endoperoxides 10a, 10, and 15 were prepared at 10 mM concentrations and diluted to 100 μM in serum-free culture medium (1% penicillin-streptomycin). The DCFH-DA probe was then added to the wells at a volume ratio of 1:1000 (DCFH-DA:culture medium) for a 10 μM probe concentration per well. A 96-well plate was incubated in a 37°C normoxic incubator for 30 min. The culture medium was then aspirated and washed with PBS. Cells were imaged using a high-content imaging system.
[0050] from Figure 4 As can be seen, the fluorescence intensity within A549 cells in the untreated blank control group is very weak, while the image of the control endoperoxide 10a, which releases singlet oxygen, exhibits distinct and intense green fluorescence, indicating that endoperoxide 10a can release a large amount of singlet oxygen. Compared with 10a, the fluorescence intensity of the other two groups of endoperoxides 10 and 15, which are attached to quenching groups, is greatly reduced, indicating that the amount of singlet oxygen released by endoperoxides 10 and 15 is lower than that of endoperoxide 10a. This is consistent with the results of the in vitro singlet oxygen release experiments in solution and the in vitro triplet oxygen release experiments in cells described above, further confirming that both endoperoxides 10 and 15 can quench some singlet oxygen into triplet oxygen. Example 6
[0051] Effects of endoperoxides on cell growth.
[0052] A549 cells were incubated in a 37°C, 5% CO₂, 21% O₂ incubator under normoxia for 24 hours. Cells in the hypoxia group were then incubated in a 37°C, 5% CO₂, 1% O₂ incubator for another 24 hours. Cells in the normoxia group were incubated in a 37°C, 5% CO₂, 21% O₂ incubator under normoxia for another 24 hours. Different concentrations of endoperoxide (10 and 15%) were added, and the cells were incubated under normoxia and hypoxia for another 24 hours. Cell survival was assessed using the MTT assay.
[0053] from Figure 5As can be seen, both endoperoxides exhibited greater cytotoxicity under hypoxic conditions than under normoxia. In normoxia, endoperoxide 10 began to show cytotoxicity at 100 μM and killed over 50% of tumor cells at 150 μM. In hypoxia, its cell killing activity exceeded 80% at 150 μM. Endoperoxide 15 had almost no killing effect on cells under normoxia, but in hypoxia, it achieved a cell killing activity of 40% at a concentration of 150 μM. Comparison of the cytotoxicity of each endoperoxide against A549 cells under normoxia and hypoxia revealed that the cytotoxicity of both endoperoxides against A549 cells increased with increasing concentration under hypoxia, and the killing effect was significantly greater than that under normoxia. This result suggests that the triplet oxygen released by both endoperoxides under hypoxia disrupts the hypoxic microenvironment of tumor cells. This effect, combined with the cytotoxicity of singlet oxygen released by the endoperoxides, enhances the cytotoxicity of the endoperoxides. In a normoxic environment, tumor cells are not in a hypoxic microenvironment, and the triplet oxygen released by endoperoxides has no effect on them. Tumor cells rely solely on singlet oxygen to kill, resulting in a reduced killing ability. This demonstrates that the synthesized endoperoxides have a certain effect on alleviating tumor hypoxia, and the singlet oxygen-triplet oxygen co-delivery vector designed in this system has great application potential.
[0054] The above embodiments are only used to illustrate the present invention. Any equivalent transformations and improvements based on the technical solution of the present invention should not be excluded from the protection scope of the present invention.
Claims
1. A compound characterized in that The compound has the structure of Formula I: ； Where Q is 、 ; wherein R2 and R3 are each independently selected from hydrogen, hydroxyl, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R1 and R4 are each independently selected from hydrogen, hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; R5 is selected from hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 5 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 14 carbon atoms, cycloalkyl having 3 to 10 carbon atoms, or ; Wherein, x is an integer from 1 to 2000.
2. The compound according to claim 1, characterized in that: R2 and R3 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R1 and R4 are each independently selected from hydrogen, an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group; R5 is selected from an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group.
3. The compound according to claim 1, characterized in that R2 and R3 are each independently selected from hydrogen and an alkyl group having 1 to 6 carbon atoms; R1 and R4 are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms; R5 is an alkyl group having 1 to 6 carbon atoms.
4. The compound according to claim 1, characterized in that R1 and R4 are hydrogen, R2 and R3 are hydrogen, and R5 is methyl.
5. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of singlet oxygen and oxygen carrier materials.
6. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.
7. The use according to claim 6, characterized in that: The tumor is non-small cell lung cancer.
8. The use according to claim 6, characterized in that: The compound or a pharmaceutically acceptable salt thereof transports singlet oxygen and triplet oxygen to cells, tissues or organs.
9. A pharmaceutical composition, characterized in that: The pharmaceutical combination comprises one of the compounds according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.