Construction and application of a class of traceable singlet oxygen carrier based on endoperoxide

Abstract

The application belongs to the technical field of biological medicine, and relates to construction and application of a class of traceable singlet oxygen carriers based on endoperoxides. The application designs and synthesizes a drug molecule with imaging function which can be used as a singlet oxygen carrier. When endoperoxides are generated, the fluorescence of the drug molecule is weakened or disappears, and after the singlet oxygen is released, the fluorescence will recover. The application not only develops a new type of endoperoxide singlet oxygen carrier with excellent performance for photodynamic therapy, but also provides a new idea for the development and design of a photodynamic diagnosis and treatment platform integration and a multifunctional singlet oxygen carrier.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the construction and application of a class of traceable singlet oxygen carriers based on internal peroxides. Background Technology

[0002] Internal peroxides, as singlet oxygen carriers, can generate singlet oxygen and effectively kill cancer cells without the need for light source irradiation around the tumor, demonstrating significant therapeutic potential. However, the current three common types of internal peroxides face significant limitations due to the inability to trace the singlet oxygen delivery process. Detecting the extent to which internal peroxide molecules deliver singlet oxygen is crucial for their anti-cancer applications. Current methods mainly rely on singlet oxygen capture probes, but these procedures are complex and often affected by experimental conditions. Designing a molecule that can act as a singlet oxygen carrier to deliver oxygen while also possessing imaging capabilities, exhibiting reduced or absent fluorescence during internal peroxide generation and restored fluorescence after singlet oxygen release, would undoubtedly promote the "therapeutic integration" of internal peroxides. Summary of the Invention

[0003] The purpose of this invention is to develop a highly efficient singlet oxygen carrier with adjustable optical properties. Utilizing the excellent optical properties and pharmacological activity of phenazine derivatives, they are used as novel traceable internal peroxides for anticancer research. This internal peroxide possesses adjustable optical properties, effectively releasing singlet oxygen not only in solution systems but also releasing large amounts of singlet oxygen intracellularly to induce tumor cell apoptosis. Furthermore, the treatment progress can be monitored in real time through fluorescence enhancement, and the singlet oxygen delivery process can be traced, demonstrating promising applications in photodynamic therapy.

[0004] The technical solution of this invention: A type of singlet oxygen delivery agent based on an internal peroxy structure, wherein the singlet oxygen delivery agent has the structure of Formula I or Formula II as follows:

[0005]

[0006] R3-R4 are each independently selected from hydrogen, hydroxyl, amino, trifluoromethyl, halogen, trimethylsilyl, C1-C30 alkyl, C1-C10 cycloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 amino-substituted alkyl, C2-C6 alkoxyalkyl, C1-C6 alkylamine, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C2-C30 heteroaryl with or without substituents.

[0007] R1-R2 and R5-R8 are each independently selected from hydrogen, hydroxyl, amino, trifluoromethyl, halogen, trimethylsilyl, C1-C30 alkyl, C1-C10 cycloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 amino-substituted alkyl, C2-C6 alkoxyalkyl, C1-C6 alkylamine, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C5-C30 heteroaryl with or without substituents.

[0008] Each of the substituents is independently selected from hydrogen, C1-C30 alkyl, C1-C30 alkoxy, C1-C30 alkylamino, C1-C30 cycloalkyl, C5-C30 aryl, C3-C16 heterocyclic, C2-C15 alkenyl, C2-C15 alkynyl, methanesulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C15 ester, sulfonic acid, sulfinic acid, carboxyl, etc.

[0009] X is an integer between 1 and 200.

[0010] The heteroatoms on the heteroaryl and heterocyclic groups are each independently selected from at least one of O, N, and S.

[0011] Some specific singlet oxygen delivery agents, R1-R2 and R5-R8, are each independently selected from hydrogen, hydroxyl, amino, C1-C20 alkyl, C2-C6 alkenyl, C1-C10 cycloalkyl, and C5-C10 aryl with or without substituents.

[0012] Some specific singlet oxygen delivery agents, R1-R2 and R5-R8, are each independently selected from hydrogen, C1-C10 alkyl, C1-C5 cycloalkyl, and C2-C6 alkenyl.

[0013] Some specific singlet oxygen delivery agents, R1-R2 and R5-R8, are each independently selected from hydrogen and C1-C10 alkyl groups.

[0014] Some specific singlet oxygen delivery agents, R3-R4, are independently selected from hydrogen, hydroxyl, amino, trifluoromethyl, halogen, trimethylsilyl, C1-C15 alkyl, C1-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C10 amino-substituted alkyl, C2-C6 alkoxyalkyl, C1-C6 alkylamine, C2-C6 alkoxycarbonyl, C5-C15 aryl with or without substituents, and C2-C10 heteroaryl with or without substituents.

[0015] The heteroaryl group is selected from pyridyl, quinolinyl, isoxazolyl, oxazolyl, pyrazolyl, furanyl, thiazolyl, imidazolyl, pyrroleyl, thiazolyl, pyrimidinebenzothiazolyl, benzoxazolyl, and benzimidazolyl.

[0016] Each of the substituents is independently selected from hydrogen, C1-C15 alkyl, C1-C15 alkoxy, C1-C15 alkylamino, C1-C10 cycloalkyl, C5-C10 aryl, C2-C10 alkenyl, C2-C10 alkynyl, methanesulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C10 ester, sulfonic acid, sulfinic acid, carboxyl, etc.

[0017] X is an integer between 1 and 200.

[0018] Some specific singlet oxygen delivery agents, R3-R4, are independently selected from hydrogen, hydroxyl, amino, trifluoromethyl, halogen, trimethylsilyl, C1-C15 alkyl, C1-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C5 amino-substituted alkyl, C2-C6 alkoxyalkyl, C1-C6 alkylamine, C2-C6 alkoxycarbonyl, and C5-C15 aryl with or without substituents.

[0019] Each of the substituents is independently selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C0 alkylamino, C1-C10 cycloalkyl, C5-C10 aryl, C2-C5 alkenyl, C2-C5 alkynyl, methanesulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C5 ester, sulfonic acid, sulfinic acid, carboxyl, etc.

[0020] X is an integer between 1 and 200.

[0021] Some specific singlet oxygen delivery agents, R3-R4 are each independently selected from hydrogen, C1-C15 alkyl groups, and C5-C10 aryl groups with or without substituents.

[0022] Each of the substituents is independently selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, carboxyl,

[0023]

[0024] X is an integer between 1 and 200.

[0025] Some specific singlet oxygen delivery agents, R1-R2 and R5-R8 are each independently selected from C1-C10 alkyl groups; R3-R4 are each independently selected from hydrogen, C1-C10 alkyl groups, and C5-C10 aryl groups with or without substituents.

[0026] Each of the substituents is independently selected from hydrogen and C1-C10 alkyl groups.

[0027] Specifically, the C1-C10 alkyl groups include C1-C10 straight-chain alkyl groups and C1-C10 branched-chain alkyl groups.

[0028] Specifically, the C1-C10 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl, n-hexyl, n-germyl, n-octyl, n-nonyl, and n-decane.

[0029] Some specific singlet oxygen delivery agents, R1-R2 and R5-R8 are each independently selected from C1-C6 alkyl groups; R3-R4 are each independently selected from hydrogen, C1-C6 alkyl groups, and C5-C10 aryl groups with or without substituents.

[0030] Each of the substituents is independently selected from hydrogen and C1-C6 alkyl groups.

[0031] This invention provides the application of the singlet oxygen carrier in the preparation of imaging functional materials that deliver singlet oxygen. The singlet oxygen carrier is used to prepare traceable drugs that deliver singlet oxygen to cells, tissues or organs.

[0032] The internal peroxide precursor is prepared by reacting a catechol compound (compound A) with an o-phenylenediamine compound (compound B). The internal peroxide is prepared by the Diels-Alder reaction. The corresponding catechol and o-phenylenediamine compounds can be obtained using conventional chemical synthesis methods, as shown in the following reaction formulas:

[0033]

[0034] R is independently selected from hydrogen, hydroxyl, amino, trifluoromethyl, halogen, trimethylsilyl, C1-C30 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 amino-substituted alkyl, C2-C6 alkoxyalkyl, C1-C6 alkylamine, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C5-C30 heteroaryl with or without substituents.

[0035] Each of the substituents is independently selected from hydrogen, C1-C30 alkyl, C1-C30 alkoxy, C1-C30 alkylamino, C1-C30 cycloalkyl, C5-C30 aryl, C3-C16 heterocyclic, C2-C15 alkenyl, C2-C15 alkynyl, methanesulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C15 ester, sulfonic acid, sulfinic acid, carboxyl, etc.

[0036] X is an integer between 1 and 200.

[0037] The heteroatoms on the heteroaryl and heterocyclic groups are each independently selected from at least one of O, N, and S.

[0038] The beneficial effects of this invention are as follows: By designing phenazine-based internal peroxides as novel traceable internal peroxide singlet oxygen carriers, this invention enables a simple internal peroxide matrix to simultaneously possess photodynamic therapy and traceability functions. This overcomes the problems of traditional internal peroxide matrices failing to achieve traceability during treatment or requiring additional fluorescent groups to achieve traceability, thus increasing workload. This invention not only develops a novel, high-performance, traceable internal peroxide singlet oxygen carrier for photodynamic therapy but also provides a completely new approach for the development and design of integrated and multifunctional singlet oxygen carriers for photodynamic diagnostic and therapeutic platforms. Attached Figure Description

[0039] Figure 1 The graph shows the change in singlet oxygen release intensity of internal peroxide 8 over time.

[0040] Figure 2 The UV-Vis and fluorescence emission spectra of internal peroxide 8 (200 μM) during its decay process at 37 °C are shown.

[0041] Figure 3 The NMR spectrum of the aromatic region of deuterated chloroform inner peroxide 8 at 37 °C is shown as a 1H NMR spectrum.

[0042] Figure 4 The NMR spectrum of the dimethyl region of deuterated chloroform, specifically the 1H NMR spectrum of internal peroxide 8, at 37 °C.

[0043] Figure 5 This figure shows the effect of internal peroxide 8 on cancer cell growth under normoxic conditions. Specific implementation methods

[0044] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention, but are not limited thereto, unless otherwise stated.

[0045] The specific embodiments of the present invention are described in detail below with reference to the technical solutions:

[0046]

[0047] Example 1

[0048] Step a: Preparation of compound 2

[0049] Compound 1 (1.2 g, 10 mmol) was added to a mixed solution of acetic anhydride (1 mL) and triethylamine (1.6 mL) under nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 2 h, then poured into ice and stirred vigorously for 30 min. The organic layer was then washed successively with 2 mol / L hydrochloric acid, sodium bicarbonate, and brine, and dried over anhydrous sodium sulfate. The crude product was purified by vacuum distillation using a rotary evaporator, followed by silica gel column chromatography with hexane / ethyl acetate (1 / 1) as eluent, yielding 1.5 g of a white solid, with a calculated yield of 93%. 1 H NMR (400MHz, Methanol-d4) δ7.15–7.06 (m, 2H), 6.94 (d, J = 7.6Hz, 1H), 2.27 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H).

[0050] Step b: Preparation of compound 3

[0051] At 0 °C, 275 μL of 65% nitric acid was added dropwise to a mixed solution of compound 2 (500 mg, 3.06 mmol) in acetic acid (1.5 mL) and acetic anhydride (1.5 mL). The mixture was stirred overnight at room temperature, then poured onto crushed ice and extracted with ethyl acetate. The combined extracts were washed with sodium bicarbonate solution and brine, dried, and purified by vacuum distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate = 3 / 1 as eluent to give 180 mg of a white solid, with a calculated yield of 28%. 1 H NMR (400MHz, Methanol-d4) δ7.35(d,J=7.9Hz,1H),7.23(d,J=7.9Hz,1H),2.27(s,3H),2.23(s,3H),2.09(s,3H).

[0052] Step c: Preparation of compound 4

[0053] The mixture of compound 3 (100 mg, 0.48 mmol) in 4 mol / L hydrochloride (3.7 mL) was heated under reflux for 4 h. After cooling to room temperature, the mixture was neutralized with sodium bicarbonate solution and extracted with ethyl acetate. The extract was washed with brine, dried, and concentrated to give 50 mg of a pale yellow solid, with a calculated yield of 63%. 1 H NMR (400MHz, Methanol-d4) δ7.02 (d, J = 7.5Hz, 1H), 6.44 (d, J = 7.5Hz, 1H), 2.28 (s, 3H), 2.13 (s, 3H).

[0054] Step d: Preparation of compound 5

[0055] 8 mg of 10% Pd / C was added to a mixed solution of compound 4 (90 mg, 0.54 mmol), ethyl acetate (0.7 mL), and methanol (88 μL). The mixture was purged with hydrogen and stirred overnight at room temperature. After filtration, the filtrate was concentrated to give 40 mg of a brownish-yellow solid, with a calculated yield of 56%. 1 H NMR (400MHz, Methanol-d4) δ6.39 (s, 2H), 2.08 (s, 6H).

[0056] Step e: Preparation of compound 7

[0057] Compound 6 (60 mg, 0.54 mmol) was dissolved in dichloromethane (8.62 mL), and this solution was added dropwise to a stirred solution of potassium dichromate (253 mg, 0.86 mmol) in 1 mol / L sulfuric acid (4.3 mL). After stirring at room temperature for 10 min, the organic layer was collected, the solvent was removed under reduced pressure, and the product was then reacted with compound 5 (30 mg, 0.22 mmol) and acetic acid (130 μL) in dichloromethane (2.5 mL). The reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Saturated sodium bicarbonate solution was added and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography using a rotary evaporator under reduced pressure and hexane as eluent, yielding 21 mg of a yellow solid, with a calculated yield of 46%. 1 H NMR (400MHz, Chloroform-d) δ8.20 (d, J = 10.1Hz, 2H), 7.73 (d, J = 10.1Hz, 2H), 7.46 (s, 2H), 2.81 (s, 6H); 13 C NMR (101MHz, Chloroform-d) δ142.2,141.1,134.3,128.8,128.7,128.5,16.6.

[0058] Step f: Preparation of compound 8

[0059] Compound 7 (30 mg, 0.14 mmol) was dissolved in deuterated chloroform (1 mL). Two to three grains of methylene blue were added, resulting in a distinct blue-green color. The reaction mixture was cooled to 0°C in an ice bath. While oxygen was being introduced, the mixture was irradiated with red light (18 W, 630 nm). 1 The reaction progress was monitored by ¹H NMR. After 8 hours of reaction, the methylene blue was removed by passing it through 200-mesh activated carbon and the solvent was evaporated under reduced pressure to obtain 30 mg of white solid. The yield was calculated to be 88%. 1H NMR(400MHz,Chloroform-d)δ8.00(d,J=9.7Hz,2H),7.67(d,J=9.7Hz,2H),6.76(s,2H),1.94(s,6H); 13 C NMR(101MHz,Chloroform-d)δ152.1,139.3,138.5,128.7,128.2,80.1,13.7.

[0060] Example 2

[0061]

[0062] Step a: Preparation of compound 10

[0063] Under nitrogen atmosphere, sodium hydroxide (27 mg, 0.67 mmol) was added to a stirred mixture of compound 9 (100 mg, 0.67 mmol) and water (1 mL), followed by dropwise addition of 30% hydrogen peroxide solution (88 μL). The reaction was stirred at 30 °C for 3 h, and then rapidly cooled with a few drops of 10% hydrochloric acid. The mixture was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using n-hexane / ethyl acetate = 5 / 1 as eluent, yielding 55 mg of a white solid. The yield was calculated to be 60%. 1 H NMR (400MHz, Methanol-d4) δ6.43(s,2H),2.10(s,6H).

[0064] Step b: Preparation of compound 11

[0065] Compound 10 (60 mg, 0.43 mmol) was dissolved in dichloromethane (8.62 mL) and added dropwise to a stirred solution of potassium dichromate (253 mg, 0.86 mmol) in 1 mol / L sulfuric acid (4.3 mL). After stirring at room temperature for 10 min, the organic layer was collected, and the solvent was removed under reduced pressure. The resulting product was reacted with compound 5 (30 mg, 0.22 mmol) and acetic acid (130 μL) in dichloromethane (2.5 mL). The reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Saturated sodium bicarbonate solution was added and extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulfate. The crude product was obtained by rotary evaporation under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane as the eluent to obtain 14 mg of a yellow solid, with a calculated yield of 14%. 1 H NMR (400MHz, Chloroform-d) δ7.43 (s, 4H), 2.80 (s, 12H).

[0066] Step c: Preparation of compound 12

[0067] Compound 11 (20 mg, 0.07 mmol) was dissolved in deuterated chloroform (1 mL). Two to three grains of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. The mixture was then irradiated with red light (18 W, 630 nm) while oxygen was being introduced. 1 The reaction progress was monitored by 1H NMR, and the product was a white solid. HRMS-ESI(+) m / z calcd for C 16 H 16 N₂O₂[M+H] + 301.11, found 301.12.

[0068] Example 3

[0069]

[0070] Step a: Preparation of compound 15

[0071] Under nitrogen atmosphere, compound 13 (1.1 g, 4 mmol), compound 14 (1.1 g, 8.8 mmol), potassium carbonate (2.2 g, 16 mmol), tetrakis(triphenylphosphine)palladium (231 mg, 0.2 mmol), 40 mL of 1,4-dioxane, and 40 mL of water were added to a 250 mL three-necked flask. The reaction was stirred at 100 °C for 24 h, and the reaction progress was monitored by TLC. Dichloromethane and water were added to the mixture, the organic layer was separated and concentrated under vacuum, and the crude product was purified by column chromatography using dichloromethane as eluent to give a white solid.

[0072] Step b: Preparation of compound 16

[0073] Compound 10 (60 mg, 0.43 mmol) was dissolved in dichloromethane (8.62 mL) and added dropwise to a stirred solution of potassium dichromate (253 mg, 0.86 mmol) in 1 mol / L sulfuric acid (4.3 mL). After stirring at room temperature for 10 min, the organic layer was collected, and the solvent was removed under reduced pressure. The resulting product was reacted with compound 15 (52 mg, 0.22 mmol) and acetic acid (130 μL) in dichloromethane (2.5 mL). The reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Saturated sodium bicarbonate solution was added and extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulfate. The crude product was obtained by rotary evaporation under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane as the eluent to obtain a yellow solid.

[0074] Step c: Preparation of compound 17

[0075] Compound 16 (26 mg, 0.1 mmol) was dissolved in deuterated chloroform (1 mL). Two to three grains of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. The mixture was then irradiated with red light (18 W, 630 nm) while oxygen was being introduced. 1 The reaction progress was monitored by 1H NMR, and the product was a white solid. HRMS-ESI(+) m / z calcd for C 26 H 22 N₂O₂[M+H] + 395.17, found 395.21.

[0076] Example 4

[0077] Step a: Preparation of compound 20

[0078] Compound 19 (298 mg, 1 mmol), compound 18 (390 mg, 3 mmol), dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium (73 mg, 0.1 mmol), potassium carbonate (414 mg, 3 mmol), and silver oxide (348 mg, 2.5 mmol) were stirred in a sealed tube under argon atmosphere at 80 °C.

[0079]

[0080] The suspension was obtained in 75 mL of uranium. After 8 h, the mixture was cooled to room temperature, the dark solid was filtered off, water was added to the solution, and the solution was extracted with chloroform. The crude product after evaporation was purified by column chromatography to obtain the final product.

[0081] Step b: Preparation of compound 21

[0082] A mixture of compound 20 (1.6 g, 5.2 mmol), zinc powder (1.7 g, 26 mmol), and acetic acid (20 mL) was refluxed under stirring for 15 min. The reaction mixture was stirred for 1 h, and the mixture was allowed to cool to room temperature. The mixture was diluted with methanol. The mixture was filtered through diatomaceous earth, thoroughly washed with methanol, filtered, and concentrated. The organic phase was separated and collected by extraction with saturated sodium bicarbonate aqueous solution (150 mL) and ethyl acetate (100 mL). This organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated under vacuum to obtain the product.

[0083] Step c: Preparation of compound 22

[0084] Compound 21 (119 mg, 0.43 mmol) was dissolved in dichloromethane (9 mL) and added dropwise to a stirred solution of potassium dichromate (253 mg, 0.86 mmol) in 1 mol / L sulfuric acid (4.3 mL). After stirring at room temperature for 10 min, the organic layer was collected, and the solvent was removed under reduced pressure. The resulting product was reacted with compound 6 (24 mg, 0.22 mmol) and acetic acid (130 μL) in dichloromethane (2.5 mL). The reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Saturated sodium bicarbonate solution was added and extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulfate. The crude product was obtained by rotary evaporation under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane as the eluent to obtain a yellow solid.

[0085] Step d: Preparation of compound 23

[0086] Compound 22 (35 mg, 0.1 mmol) was dissolved in deuterated chloroform (1 mL). Two to three grains of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. The mixture was then irradiated with red light (18 W, 630 nm) while oxygen was being introduced. 1 The reaction progress was monitored by 1H NMR, and the product was a white solid. HRMS-ESI(+) m / z calcd for C 24 H 32 N₂O₂[M+H] + 381.25, found 381.31.

[0087] Example 5

[0088]

[0089] The synthesis method was as described in Example 4. The structure of compound 28 was verified by mass spectrometry, HRMS-ESI(+) m / z calcd for C 20 H 24 N₂O₂[M+H] + 325.18, found 325.25.

[0090] Example 6

[0091] Step a: Preparation of compound 30

[0092] Sodium dimethyl dithiocarbamate (740 mg, 1.2 mmol) was added to a 50 mL round-bottom flask equipped with a magnetic stir bar at room temperature, followed by a methanol (10 mL) solution of compound 26 (246 mg, 1.0 mmol). The mixture was stirred for 5 h at the same temperature. After quenching the reaction with saturated sodium thiosulfate aqueous solution, the mixture was extracted twice with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution and brine, dried over anhydrous sodium sulfate, filtered through a cotton plug, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain an intermediate product. The intermediate product was then heated under reflux in air for 16 h using an organic synthesizer to a solution of o-quinoline dimer (105 mg, 0.2 mmol) in toluene (2.0 mL). After the reaction, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain the final product.

[0093] Step b: Preparation of compound 31

[0094]

[0095] Compound 30 (155 mg, 0.43 mmol) was dissolved in dichloromethane (10 mL) and added dropwise to a stirred solution of potassium dichromate (253 mg, 0.86 mmol) in 1 mol / L sulfuric acid (4.3 mL). After stirring at room temperature for 10 min, the organic layer was collected, and the solvent was removed under reduced pressure. The resulting product was reacted with compound 26 (45 mg, 0.22 mmol) and acetic acid (130 μL) in dichloromethane (2.5 mL). The reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Saturated sodium bicarbonate solution was added and extracted with dichloromethane. The organic layer was collected and dried over anhydrous sodium sulfate. The crude product was obtained by rotary evaporation under reduced pressure. Further purification was performed by silica gel column chromatography using n-hexane as the eluent to obtain a yellow solid.

[0096] Step c: Preparation of compound 32

[0097] Compound 31 (44 mg, 0.1 mmol) was dissolved in deuterated chloroform (1 mL). Two to three grains of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. The mixture was then irradiated with red light (18 W, 630 nm) while oxygen was being introduced. 1 The reaction progress was monitored by 1H NMR, and the product was a white solid. HRMS-ESI(+) m / z calcd for C 32 H 32 N₂O₂[M+H] + 479.25, found 479.31

[0098] Example 7

[0099] The synthesis method was as described in Example 6. The structure of compound 34 was verified by mass spectrometry, HRMS-ESI(+) m / z calcd for C 36 H 40 N₂O₂[M+H] + 534.32, found 534.43.

[0100] Example 8

[0101] In vitro singlet oxygen release experiment of internal peroxides.

[0102] Singlet oxygen is a type of molecular oxygen in an excited state. Due to its short lifetime and high reactivity, it is difficult to detect its presence directly. We used ultraviolet-visible spectrophotometry, selecting 1,3-diphenylbenzofuran.

[0103]

[0104] DPBF (dihydropyridine dioxide) was used to capture singlet oxygen generated in the system, and studies showed that the degradation rate of DPBF was proportional to the generation rate of singlet oxygen. A DPBF absorption curve was obtained by plotting absorption wavelength on the x-axis and absorbance on the y-axis to reflect the release of singlet oxygen from internal peroxide 8. Figure 1 (As shown).

[0105] The specific testing procedure is as follows: 1) Weigh 2 mg of internal peroxide 8 and dissolve it in 832 μL of N,N-dimethylformamide (DMF) solution to prepare a 10 mM stock solution for the analyte; 2) Accurately prepare a 200 μM DPBF stock solution. Before testing, pipette 1.8 mL of the above internal peroxide 8 stock solution and add it to a quartz cuvette containing 1.2 mL of the 200 μM DPBF stock solution, and dilute to 3 mL. The resulting test concentration of internal peroxide 8 is 6 mM, the test concentration of DPBF is 40 μM, and the UV-Vis absorption spectrum scanning range is 350 nm-550 nm; 3) Set the temperature of the Agilent Cary 3500 UV-Vis spectrometer heating module to 37 °C for preheating, and set the cycle time to 20 min / cycle. When the temperature reaches 37 °C, start monitoring the change of the absorption peak at 417 nm in the UV-Vis absorption spectrum within 3 hours.

[0106] As clearly shown in the figure, within the first 60 minutes, internal peroxide 8 rapidly releases a large amount of singlet oxygen, leading to a significant decrease in the absorbance of DPBF in the solution. This rapid change in the curve during this phase demonstrates the strong ability of internal peroxide 8 to release singlet oxygen. After this, the absorbance decreases more slowly, and after 120 minutes, the change becomes minimal and eventually remains constant, indicating that internal peroxide 8 has ceased releasing singlet oxygen. This experiment strongly demonstrates that internal peroxide 8 can serve as a good singlet oxygen carrier and has great potential for biological applications.

[0107] Example 9

[0108] UV-Vis and fluorescence emission spectra of internal peroxides during their decay process at 37 °C.

[0109] The specific test procedure is as follows: 1) Weigh 1 mg of compound 7 and 1 mg of internal peroxide 8, and dissolve them in 7.5 mL and 6.5 mL of a mixed solution of N,N-dimethylformamide and acetonitrile (DMF / MeCN, v / v = 1 / 2), respectively, to prepare a 640 μM stock solution for the analyte; 2) Before the test, pipette 94 μL, 188 μL, 375 μL, 750 μL, and 1.5 mL of the stock solution, respectively, and add them to the mixed solution of N,N-dimethylformamide and acetonitrile (DMF / MeCN, v / v = 1 / 2), and dilute to 3 mL before transferring to a quartz cuvette. The obtained compound concentrations were 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM, respectively. Subsequently, UV-Vis absorption and fluorescence emission spectra were measured using an Agilent Cary 3500 UV-Vis spectrometer and an Agilent Cary Eclipse fluorescence spectrophotometer, respectively. The excitation and emission slit widths were both set to 5 nm. The fluorescence excitation wavelength for compound 7 was 363 nm, and the fluorescence excitation wavelength for the internal peroxide 8 was 318 nm.

[0110] like Figure 2 As shown, with increasing experimental time, the maximum absorption wavelength of the UV-Vis absorption spectrum shifted from 318 nm to 363 nm, and the change in the fluorescence emission spectrum curve also indicated that the maximum emission wavelength changed from 418 nm to 478 nm. This corresponds one-to-one with the maximum absorption and maximum emission wavelengths of compound 7 and internal peroxide 8. By monitoring the changes in the UV-Vis absorption and fluorescence emission spectrum curves at 37℃ over a certain period of time, it was further demonstrated that internal peroxide 8 can spontaneously undergo a reverse cycloaddition reaction to transform into compound 7, resulting in a red shift of the maximum absorption wavelength and enhanced fluorescence, which is beneficial for better realization of fluorescence imaging function.

[0111] Example 10

[0112] Calculation of the half-life of singlet oxygen released by internal peroxides.

[0113] The specific testing procedure is as follows: Weigh 2-5 mg of internal peroxide 8 solid and dissolve it in 0.5 mL of deuterated chloroform. At the initial time (0 h), quickly remove the internal peroxide from the beaker filled with ice, taking care to avoid the internal peroxide decomposing due to a cyclic reaction at room temperature. Then, perform 1H NMR spectroscopy. After the test, immediately place it in a water bath at 37°C and perform NMR spectroscopy at predetermined intervals of 0 h, 19 h, 24 h, 42 h, 67 h, 144 h, 187 h, and 331 h using a Bruker Avance II 400M NMR spectrometer. 1 ¹H NMR analysis was performed, and the proportion of internal peroxides was ultimately determined by the peak area ratio of H atoms at the same position (methyl H or aromatic H). Monitoring and analysis were conducted within the aforementioned time period. 1 H NMR, monitoring was stopped when the characteristic peak of internal peroxide disappeared.

[0114] like Figure 3 , Figure 4 The image shows the 1H NMR spectrum of internal peroxide 8 in deuterated chloroform at 37°C over time. First, internal peroxides were selected... 1 The characteristic peaks of H NMR, after integration and normalization, allow for the deduction of the material ratio between internal peroxide 8 and reduction product compound 7. For example... Figure 3 As shown, the characteristic peak chemical shifts of the aromatic region of the internal peroxide 8 are 7.99-8.04 ppm, 7.65-7.70 ppm, and 6.76 ppm, respectively, while the corresponding characteristic peak chemical shifts of the cycloreduction product compound 7 are 8.18-8.23 ppm, 7.71-7.76 ppm, and 7.46 ppm; Figure 4 As shown, the chemical shift of the dimethyl characteristic peak of internal peroxide 8 is 1.94 ppm, while the corresponding chemical shift of the characteristic peak of the cycloreduction product compound 7 is 2.81 ppm. Based on the characteristic peaks of the internal peroxide and the cycloreduction product, the half-life t of the singlet oxygen released by internal peroxide 8 in deuterated chloroform was calculated. 1 / 2 It is 88.8h.

[0115] Example 11

[0116] The effect of internal peroxides on cancer cell growth under normoxic conditions.

[0117] Six cancer cell lines, including human cervical cancer cells (HeLa), human breast cancer cells (MCF-7), human liver cancer cells (HepG2), mouse breast cancer cells (4T1), human non-small cell lung cancer cells (A549), and human ovarian cancer cells (SKOV3), were selected to further investigate the killing ability of internal peroxide 8 on tumor cells under normoxic conditions.

[0118] The specific testing process is as follows: After counting the cells, they were divided into groups of 5 × 10⁶ cells per well. 3 Cells were cultured at a density of [number] cells per well in 96-well plates. 100 μL of complete culture medium was added to each well, and the plates were incubated overnight at 37°C under normoxic conditions (5% CO2) to ensure complete cell adhesion. After 24 hours, the original culture medium was aspirated, and the cells were drug-treated. Intraperoxide 8 solutions with drug concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, and 100 μM were prepared using complete culture medium. 100 μL of each solution was added to each well, and the cells were incubated for another 24 hours. Then, 20 μL of MTT (in 5 mg / mL PBS) was added to each well under dark conditions, and the cells were incubated for 4 hours. After incubation, the MTT solution was aspirated, and 200 μL of DMSO solution was added to each well to dissolve the formazan. The cells were then agitated using a multi-sensor microplate reader, and the absorbance at 570 nm was measured. Cell viability was calculated.

[0119] The experimental results were analyzed, such as Figure 5 As shown, internal peroxide 8 exhibited strong cytotoxicity against all the aforementioned cancer cell lines, and its killing ability against various cancer cell lines increased with increasing concentration. When the concentration of internal peroxide 8 reached 65 μM, the killing effect on all six cancer cell lines reached or exceeded 50%, and human ovarian cancer cells (SKOV3) were almost completely killed when the concentration of internal peroxide 8 exceeded 65 μM. These experiments demonstrate that internal peroxide 8 can effectively release cytotoxic singlet oxygen in vitro and has a strong killing effect on different types of cancer cell lines.

Claims

1. A class of singlet oxygen delivery agents, characterized in that, This singlet oxygen delivery agent has the structure of formula I or formula II: ； Among them, R1-R2 and R5-R8 are each independently selected from C1-C6 alkyl groups; R3-R4 are each independently selected from hydrogen, C1-C10 alkyl groups, and C5-C10 aryl groups with or without substituents; Each of the substituents is independently selected from hydrogen and C1-C10 alkyl groups.

2. The application of the singlet oxygen delivery agent according to claim 1 in the preparation of imaging functional materials that deliver singlet oxygen.

3. The use of the singlet oxygen delivery agent according to claim 1 in the preparation of traceable drugs for delivering singlet oxygen to cells, tissues or organs.

Images