Construction and application of endoperoxide-based traceable singlet oxygen carrier

Abstract

The invention discloses construction and application of endoperoxide-based traceable singlet oxygen carriers, and belongs to the technical field of biological medicines. The invention designs and synthesizes a drug molecule which can be used as a singlet oxygen carrier and has an imaging function, the fluorescence of the drug molecule is weakened or disappears when the drug molecule generates endoperoxide, and the fluorescence is recovered after the singlet oxygen is released. The invention not only develops a novel photodynamic therapy traceable peroxy singlet oxygen carrier with excellent performance, but also provides a brand new thought for the development and design of photodynamic diagnosis and treatment platform integration and multifunctional singlet oxygen carriers.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and particularly relates to the construction and application of a type of traceable singlet oxygen carrier based on endoperoxide. Background Art

[0002] As a singlet oxygen carrier, endoperoxides can generate singlet oxygen and effectively kill cancer cells without irradiating the surrounding area of ​​the tumor with a light source, and have great therapeutic potential. However, the three common types of endoperoxides currently encounter major bottlenecks in their application due to the inability to trace the delivery process of singlet oxygen, and there are limitations. Detecting the degree to which endoperoxide molecules deliver singlet oxygen is the key to their anti-cancer application. Currently, the commonly used methods mainly rely on singlet oxygen capture probes, but their operation process is complicated and is often affected by experimental conditions. Design a molecule that can deliver oxygen as a singlet oxygen carrier and has imaging function. When endoperoxides are generated, the fluorescence weakens or disappears, and after releasing singlet oxygen, the fluorescence can be restored. This concept of a tracer singlet oxygen carrier will undoubtedly promote the development of "integrated diagnosis and treatment" of endoperoxides. Summary of the invention

[0003] The purpose of the present invention is to develop an efficient singlet oxygen carrier with adjustable optical properties. Phenazine derivatives are used as new traceable endoperoxides for anti-cancer research by utilizing their good optical properties and excellent pharmacological activity. The endoperoxide has adjustable optical properties, which can not only effectively release singlet oxygen in a solution system, but also release a large amount of singlet oxygen in cells to induce tumor cell apoptosis, and can monitor the treatment process in real time through fluorescence enhancement. The singlet oxygen delivery process can be traced, showing good application prospects in photodynamic diagnosis and treatment.

[0004] The technical solution of the present invention is a singlet oxygen transport agent based on an endoperoxide structure, wherein the singlet oxygen transport agent has the structure of the following formula I or formula II:

[0005]

[0006] Wherein, 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 alkylamino, 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C5-C30 heteroaryl with or without substituents.

[0008] The substituents are each independently selected from hydrogen, C1-C30 alkyl, C1-C30 alkoxy, C1-C30 alkylamino, C1-C30 cycloalkyl, C5-C30 aryl, C3-C16 heterocyclic group, C2-C15 alkenyl, C2-C15 alkynyl, mesyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C15 ester, sulfonic acid, sulfinic acid, carboxyl,

[0009] X is an integer from 1 to 200.

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

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

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

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

[0014] In some specific singlet oxygen delivery agents, R3-R4 are each 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C15 aryl with or without substitution, and C2-C10 heteroaryl with or without substitution.

[0015] The heteroaryl group is selected from pyridyl, quinolyl, isoxazolyl, oxazolyl, pyrazolyl, furanyl, thiazolyl, imidazolyl, pyrrolyl, thiolyl, pyrimidine benzothiazolyl, benzoxazolyl, benzimidazolyl;

[0016] The substituents are each 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, methylsulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C10 ester, sulfonic acid, sulfinic acid, carboxyl,

[0017] X is an integer from 1 to 200.

[0018] In some specific singlet oxygen delivery agents, R3-R4 are each 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 alkylamino, C2-C6 alkoxycarbonyl, and C5-C15 aryl with or without substitution.

[0019] The substituents are each 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, methylsulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C5 ester, sulfonic acid, sulfinic acid, carboxyl,

[0020] X is an integer from 1 to 200.

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

[0022] The substituents are independently selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, carboxyl,

[0023]

[0024] X is an integer from 1 to 200.

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

[0026] The substituents are each independently selected from hydrogen and C1-C10 alkyl.

[0027] Specifically, the C1-C10 alkyl group includes a C1-C10 straight-chain alkyl group and a C1-C10 branched-chain alkyl group.

[0028] Specifically, the C1-C10 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl, n-hexyl, n-octyl, n-nonyl, and n-decyl.

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

[0030] The substituents are each independently selected from hydrogen and C1-C6 alkyl.

[0031] The present invention provides the use of the singlet oxygen carrier in preparing imaging functional materials for transporting singlet oxygen. The singlet oxygen carrier is used to prepare traceable drugs for transporting singlet oxygen to cells, tissues or organs.

[0032] The endoperoxide precursor is prepared by reacting a catechol compound (compound A) and an o-phenylenediamine compound (compound B). The endoperoxide is prepared by Diels-Alder reaction. The corresponding catechol compound and o-phenylenediamine compound can be obtained by conventional chemical synthesis methods, and the specific reaction formula is as follows:

[0033]

[0034] Wherein, 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C5-C30 heteroaryl with or without substituents.

[0035] The substituents are each independently selected from hydrogen, C1-C30 alkyl, C1-C30 alkoxy, C1-C30 alkylamino, C1-C30 cycloalkyl, C5-C30 aryl, C3-C16 heterocyclic group, C2-C15 alkenyl, C2-C15 alkynyl, mesyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C15 ester, sulfonic acid, sulfinic acid, carboxyl,

[0036] X is an integer from 1 to 200.

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

[0038] Beneficial effects of the present invention: The present invention designs phenazine endoperoxides as a new traceable endoperoxide singlet oxygen carrier, realizing that a simple structure of the endoperoxide matrix can simultaneously have the functions of photodynamic therapy and traceability, getting rid of the problem that the traditional endoperoxide matrix cannot achieve traceability during the treatment process or needs to connect additional fluorescent groups to achieve traceability, resulting in increased workload. Not only has a new type of photodynamic therapy traceable endoperoxide singlet oxygen carrier with excellent performance been developed, but it also provides a new idea for the development and design of an integrated photodynamic diagnosis and treatment platform and a multifunctional singlet oxygen carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the change in singlet oxygen release intensity of endoperoxide 8 over time.

[0040] Figure 2 UV-visible spectrum and fluorescence emission spectrum of endoperoxide 8 (200 μM) during decay at 37°C.

[0041] Figure 3 This is the H NMR spectrum of the aromatic region half-life of endoperoxide 8 in deuterated chloroform at 37°C.

[0042] Figure 4 This is the H NMR spectrum of the dimethyl half-life of endoperoxide 8 in deuterated chloroform at 37°C.

[0043] Figure 5 This is a diagram showing the effect of endoperoxide 8 on cancer cell growth under normoxic environment. Specific implementation methods

[0044] The present invention is specifically described below by way of 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.

[0045] The specific embodiments of the present invention are described in detail below in conjunction with the technical solution:

[0046]

[0047] Example 1

[0048] Step a: Preparation of compound 2

[0049] In a nitrogen environment at 0°C, compound 1 (1.2 g, 10 mmol) was added to a mixed solution of acetic anhydride (1 mL) and triethylamine (1.6 mL). 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 with 2 mol / L hydrochloric acid, sodium bicarbonate and brine in sequence, and dried over anhydrous sodium sulfate. A rotary evaporator was used for vacuum distillation, and the crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate = 1 / 1 as the eluent to obtain 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, 65% nitric acid (275 μL) 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 at room temperature overnight, then poured onto crushed ice and extracted with ethyl acetate. The combined extract was washed with sodium bicarbonate solution and brine, dried, and subjected to reduced pressure distillation using a rotary evaporator. The crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate = 3 / 1 as an eluent to obtain 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] A mixture of compound 3 (100 mg, 0.48 mmol) in 4 mol / L hydrochloride (3.7 mL) was heated to 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 obtain 50 mg of a light 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 replaced with hydrogen and stirred at room temperature overnight. After filtration, the filtrate was concentrated to obtain 40 mg of a brown-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 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 then the product was reacted with compound 5 (30 mg, 0.22 mmol) and acetic acid (130 μL) using dichloromethane (2.5 mL) as solvent. The reaction solution 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 a rotary evaporator was used for vacuum distillation. The crude product was purified by silica gel column chromatography using n-hexane as the eluent to obtain 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). 2-3 particles of methylene blue were added to make the solution appear distinctly blue-green. The reaction mixture was cooled to 0°C in an ice bath. While passing oxygen, red light (18 W, 630 nm) was used to irradiate the solution. 1 The progress of the reaction was monitored by H NMR. After 8 h of reaction, methylene blue was removed by 200-mesh activated carbon and the solvent was evaporated under reduced pressure to obtain 30 mg of a white solid with a calculated yield of 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 mixed solution of compound 9 (100 mg, 0.67 mmol) and water (1 mL), and then 30% hydrogen peroxide solution (88 μL) was added dropwise. The reaction was stirred at 30°C for 3 h, and a few drops of 10% hydrochloric acid were added to quench the reaction, and extracted with ethyl acetate. The ethyl acetate portion was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography using n-hexane / ethyl acetate = 5 / 1 as an eluent to obtain 55 mg of a white solid with a calculated yield of 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, the solvent was removed under reduced pressure, and the obtained product was reacted with compound 5 (30 mg, 0.22 mmol) and acetic acid (130 μL) using dichloromethane (2.5 mL) as solvent. The reaction solution 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 with anhydrous sodium sulfate, and a rotary evaporator was used for decompression operation to obtain a crude product, which was further purified by silica gel column chromatography, and the crude product was purified using n-hexane as an 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). 2-3 particles of methylene blue were added to make the solution appear distinctly blue. The reaction mixture was cooled to 0°C in an ice bath. While passing oxygen, red light (18 W, 630 nm) was used to irradiate the solution. 1 H NMR was used to monitor the progress of the reaction and the product was obtained as a white solid. HRMS-ESI(+) m / z calculated 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, compound 13 (1.1 g, 4 mmol), compound 14 (1.1 g, 8.8 mmol), potassium carbonate (2.2 g, 16 mmol), tetrakistriphenylphosphine palladium (231 mg, 0.2 mmol), 40 mL 1,4-dioxane and 40 mL water were added to a 250 mL three-necked flask. The reaction was stirred at 100 ° C for 24 h, and the completion of the reaction was monitored by TLC. Dichloromethane and water were added to the mixture, the organic layer was separated and concentrated in vacuo, and the crude product was purified by column chromatography using dichloromethane as an eluent to obtain 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, the solvent was removed under reduced pressure, and the obtained product was reacted with compound 15 (52 mg, 0.22 mmol) and acetic acid (130 μL) using dichloromethane (2.5 mL) as solvent. The reaction solution 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 with anhydrous sodium sulfate, and a rotary evaporator was used for decompression to obtain a crude product, which was further purified by silica gel column chromatography, and the crude product was purified using n-hexane as an 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). 2-3 particles of methylene blue were added to make the solution appear distinctly blue. The reaction mixture was cooled to 0°C in an ice bath. While passing oxygen, red light (18 W, 630 nm) was used to irradiate the solution. 1 H NMR was used to monitor the progress of the reaction and the product was obtained as a white solid. HRMS-ESI(+) m / z calculated 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(diphenylphosphino)ferrocene]palladium (73 mg, 0.1 mmol), potassium carbonate (414 mg, 3 mmol) and silver oxide (348 mg, 2.5 mmol) were stirred in tetrahydrofuran in a sealed tube at 80°C under an argon atmosphere.

[0079]

[0080] The suspension was added into 1% ethanol (75 mL). After 8 h, the mixture was cooled to room temperature, the dark solid was filtered, 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 product.

[0081] Step b: Preparation of compound 21

[0082] A mixed solution 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 cooled to room temperature. The mixture was diluted with methanol. The mixture was filtered through diatomaceous earth, rinsed thoroughly with methanol, filtered and concentrated. Saturated aqueous sodium bicarbonate solution (150 mL) and ethyl acetate (100 mL) were used for extraction, and the organic phase was separated and collected. The organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated in vacuo 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, the solvent was removed under reduced pressure, and the obtained product was reacted with compound 6 (24 mg, 0.22 mmol) and acetic acid (130 μL) using dichloromethane (2.5 mL) as solvent. The reaction solution 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 with anhydrous sodium sulfate, and a rotary evaporator was used for decompression to obtain a crude product, which was further purified by silica gel column chromatography, and the crude product was purified using n-hexane as an 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). 2-3 particles of methylene blue were added to make the solution appear distinctly blue. The reaction mixture was cooled to 0°C in an ice bath. While passing oxygen, red light (18 W, 630 nm) was used to irradiate the solution. 1 H NMR was used to monitor the progress of the reaction and the product was obtained as a white solid. HRMS-ESI(+) m / z calculated 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 was calculated for C 20 H 24 N2O2[M+H] + 325.18, found 325.25.

[0090] Example 6

[0091] Step a: Preparation of compound 30

[0092] At room temperature, sodium dimethyldithiocarbamate (740 mg, 1.2 mmol) was added to a 50 mL round-bottom flask equipped with a magnetic stirring bar, and a solution of compound 26 (246 mg, 1.0 mmol) in methanol (10 mL) was added thereto. The mixture was stirred at the same temperature for 5 h. After the reaction was quenched with a saturated aqueous sodium thiosulfate solution, ethyl acetate was added and extracted twice. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered with a cotton plug, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain an intermediate product. A solution of o-quinoline dimer (105 mg, 0.2 mmol) in toluene (2.0 mL) was refluxed in air by an organic synthesizer for 16 h. After the reaction, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain the 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, the solvent was removed under reduced pressure, and the obtained product was reacted with compound 26 (45 mg, 0.22 mmol) and acetic acid (130 μL) using dichloromethane (2.5 mL) as solvent. The reaction solution 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 with anhydrous sodium sulfate, and a rotary evaporator was used for decompression to obtain a crude product, which was further purified by silica gel column chromatography, and the crude product was purified using n-hexane as an 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). 2-3 particles of methylene blue were added to make the solution appear distinctly blue. The reaction mixture was cooled to 0°C in an ice bath. While passing oxygen, red light (18 W, 630 nm) was used to irradiate the solution. 1 H NMR was used to monitor the progress of the reaction and the product was obtained as a white solid. HRMS-ESI(+) m / z calculated for C 32 H 32 N2O2[M+H] + 479.25,found479.31

[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 calculated for C 36 H 40 N2O2[M+H] + 534.32, found 534.43.

[0100] Example 8

[0101] In vitro singlet oxygen release assay of endoperoxides.

[0102] Singlet oxygen is a molecular oxygen in an excited state. It is difficult to detect its existence directly because of its low lifetime and high activity. We used UV-visible spectrophotometry to select 1,3-diphenylbenzofuran

[0103]

[0104] (DPBF) is used to capture the singlet oxygen generated in the system, and studies have shown that the degradation rate of DPBF is proportional to the generation rate of singlet oxygen. The DPBF absorption change curve is obtained with the absorption wavelength as the horizontal axis and the absorbance as the vertical axis to reflect the release of singlet oxygen from endoperoxide 8 ( Figure 1 shown).

[0105] The specific test process is as follows: 1) Weigh 2 mg of endoperoxide 8 and dissolve it in 832 μL of N,N-dimethylformamide (DMF) solution to prepare the molecule to be tested into a 10 mM mother solution; 2) Accurately prepare a 200 μM DPBF DMF mother solution, and use a pipette to draw 1.8 mL of the above endoperoxide 8 mother solution before testing, add it to a quartz cuvette containing 1.2 mL of 200 μM DPBF mother solution, and adjust it to 3 mL. The test concentration of endoperoxide 8 obtained is 6 mM, the DPBF test concentration is 40 μM, and the UV-visible absorption spectrum scan range is 350 nm-550 nm; 3) The temperature of the heating module of the Agilent Cary 3500 UV-visible spectrometer is set to 37 ° C for preheating, and the cycle time is set to 20 min / time. When the temperature reaches 37 ° C, start monitoring the change of the absorption peak at 417 nm in the UV-visible absorption spectrum within 3 hours.

[0106] It can be clearly seen from the figure that within the first 60 minutes, endoperoxide 8 rapidly released a large amount of singlet oxygen, which caused the absorbance of DPBF in the solution to drop significantly. The rapid change of the curve at this stage shows the strong ability of endoperoxide 8 to release singlet oxygen. After that, its absorbance value dropped slowly, and after 120 minutes, it changed slightly and remained unchanged, indicating that endoperoxide 8 had stopped releasing singlet oxygen. This experiment strongly proves that endoperoxide 8 can be used as a good singlet oxygen carrier and has great potential in biological applications.

[0107] Example 9

[0108] The decay of endoperoxides at 37°C was determined by UV-visible and fluorescence emission spectra.

[0109] The specific test process is as follows: 1) Weigh 1 mg of compound 7 and 1 mg of endoperoxide 8, 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, and configure the molecules to be tested into a 640 μM mother solution; 2) Before the test, use a pipette to take 94 μL, 188 μL, 375 μL, 750 μL, and 1.5 mL of the mother solution, respectively, add it to a mixed solution containing N,N-dimethylformamide and acetonitrile (DMF / MeCN, v / v=1 / 2), make it dilute to 3 mL, and then transfer it to a quartz cuvette. The test concentrations of the obtained compounds were 20μM, 40μM, 80μM, 160μM, and 320μM, respectively; then, the UV-visible absorption spectrum and fluorescence emission spectrum were tested in sequence using Agilent Cary 3500 UV-visible spectrometer and Agilent Cary Eclipse fluorescence spectrophotometer, respectively. The excitation slit and emission slit widths were both set to 5nm, the fluorescence excitation wavelength of compound 7 was 363nm, and the fluorescence excitation wavelength of endoperoxide 8 was 318nm.

[0110] like Figure 2 As shown in the figure, with the increase of experimental time, the maximum absorption wavelength of the UV-visible absorption spectrum shifted from 318nm to 363nm, and the curve change of the fluorescence emission spectrum also showed that the maximum emission wavelength changed from 418nm to 478nm, which also corresponds to the maximum absorption and maximum emission wavelengths of compound 7 and endoperoxide 8. By monitoring the changes in the UV-visible absorption and fluorescence emission spectrum curves at 37°C over a certain period of time, it is further explained that endoperoxide 8 can spontaneously undergo a reverse cycloaddition reaction to convert into compound 7, the maximum absorption wavelength red-shifts, and the fluorescence is enhanced, which is conducive to the better realization of the fluorescence imaging function.

[0111] Example 10

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

[0113] The specific test process is as follows: 2-5 mg of endoperoxide 8 solid was weighed and dissolved in 0.5 mL of deuterated chloroform. At the start time (0 h), the endoperoxide was quickly taken out from the beaker filled with ice cubes, and care was taken to avoid the endoperoxide from undergoing a cyclic reaction and decomposition at room temperature. Then, the endoperoxide was tested by nuclear magnetic resonance (NMR) proton spectrum. After the test, it was immediately placed in a water bath at 37 °C and measured using a Bruker Avance II 400M NMR spectrometer at the determined intervals of 0 h, 19 h, 24 h, 42 h, 67 h, 144 h, 187 h, and 331 h. 1 H NMR test, and finally determine the proportion of endoperoxides by the peak area ratio of H at the same position (methyl H or aromatic H). Monitor and analyze the above time period 1 H NMR monitoring was stopped when the characteristic peak of endoperoxide disappeared.

[0114] like Figure 3 , Figure 4 The figure shows the change of the H NMR spectrum of endoperoxide 8 in deuterated chloroform at 37°C over time. 1 The H NMR characteristic peaks were integrated and normalized, and the material ratio between the endoperoxide 8 and the reduction product compound 7 can be inferred. Figure 3 As shown, the characteristic peak chemical shifts of the aromatic region of endoperoxide 8 are 7.99-8.04ppm, 7.65-7.70ppm and 6.76ppm, and the corresponding characteristic peak chemical shifts of the ring reduction product compound 7 are 8.18-8.23ppm, 7.71-7.76ppm and 7.46ppm respectively; Figure 4 As shown in the figure, the chemical shift of the dimethyl characteristic peak of endoperoxide 8 is 1.94 ppm, and the corresponding characteristic peak of the ring reduction product compound 7 is 2.81 ppm. Combining the characteristic peaks of the endoperoxide and the ring reduction product, it is calculated that the half-life of the release of singlet oxygen by endoperoxide 8 in deuterated chloroform is t 1 / 2 It is 88.8h.

[0115] Embodiment 11

[0116] Effects of endoperoxides on cancer cell growth under normoxic conditions.

[0117] A total of 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 explore the killing ability of endoperoxide 8 on tumor cells under normoxic environment.

[0118] The specific test process is as follows: After counting the cells, 5×10 3 The cells were cultured in a 96-well plate at a density of 100 μL complete medium per well, and incubated overnight at 37°C and 5% CO2 under normoxic conditions to allow the cells to completely adhere to the wall. After 24 hours, the original medium was removed and the drug was administered. The endoperoxide 8 solution with drug concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, and 100 μM was prepared using complete medium. After adding 100 μL to each well, the cells were incubated for 24 hours. 20 μL MTT (5 mg / mL in PBS) solution was added to each well under light-proof conditions, and the solution was removed after incubation for 4 hours. 200 μL DMSO solution was added to the wells to dissolve the formazan, and the absorbance at 570 nm was detected after shaking in a multifunctional microplate reader, and the cell survival rate was calculated.

[0119] The experimental results were analyzed, such as Figure 5 As shown, endoperoxide 8 exhibited strong cytotoxicity to the above cancer cell lines, and with the increase of the dosage concentration, the killing ability to multiple cancer cell lines also continued to increase. When the concentration of endoperoxide 8 reached 65μM, the killing ability to the above six cancer cell lines reached or even exceeded 50%, and human ovarian cancer cells (SKOV3) were almost all killed when the concentration of endoperoxide 8 exceeded 65μM. The above experiments show that endoperoxide 8 can still effectively release cytotoxic singlet oxygen in cells in vitro, and has a strong killing effect on different types of cancer cell lines.

Claims

1. A singlet oxygen delivery agent, characterized in that The singlet oxygen delivery agent has a structure of Formula I or Formula II: Wherein, 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C2-C30 heteroaryl with or without substituents; R1-R2, 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C30 aryl with or without substituents, and C5-C30 heteroaryl with or without substituents; The substituents are each independently selected from hydrogen, C1-C30 alkyl, C1-C30 alkoxy, C1-C30 alkylamino, C1-C30 cycloalkyl, C5-C30 aryl, C3-C16 heterocyclic group, C2-C15 alkenyl, C2-C15 alkynyl, mesyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C15 ester, sulfonic acid, sulfinic acid, carboxyl, X is an integer from 1 to 200; The heteroatoms on the heteroaryl group and the heterocyclic group are independently selected from at least one of O, N and S.

2. The singlet oxygen delivery agent according to claim 1, characterized in that R1-R2 and R5-R8 are each independently selected from hydrogen, hydroxyl, amino, C1-C20 alkyl, C2-C6 alkenyl, C1-C10 cycloalkyl, or C5-C10 aryl which may or may not be substituted.

3. The singlet oxygen delivery agent according to claim 2, characterized in that R1-R2, R5-R8 are each independently selected from hydrogen, C1-C10 alkyl, C1-C5 cycloalkyl, C2-C6 alkenyl.

4. The singlet oxygen delivery agent according to claim 3, characterized in that R1-R2, R5-R8 are each independently selected from hydrogen and C1-C10 alkyl.

5. The singlet oxygen delivery agent according to any one of claims 1 to 4, characterized in that R3-R4 are each 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 alkylamino, C2-C6 alkoxycarbonyl, C5-C15 aryl with or without substituents, and C2-C10 heteroaryl with or without substituents; The heteroaryl group is selected from pyridyl, quinolyl, isoxazolyl, oxazolyl, pyrazolyl, furanyl, thiazolyl, imidazolyl, pyrrolyl, thiolyl, pyrimidine benzothiazolyl, benzoxazolyl, benzimidazolyl; The substituents are each 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, methylsulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C10 ester, sulfonic acid, sulfinic acid, carboxyl, X is an integer from 1 to 200.

6. The singlet oxygen delivery agent according to claim 5, characterized in that R3-R4 are each 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 alkylamino, C2-C6 alkoxycarbonyl, or C5-C15 aryl with or without substitution; The substituents are each 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, methylsulfonyl, amino, nitro, hydroxyl, halogen, cyano, aldehyde, C2-C5 ester, sulfonic acid, sulfinic acid, carboxyl, X is an integer from 1 to 200.

7. The singlet oxygen delivery agent according to claim 6, characterized in that R3-R4 are each independently selected from hydrogen, C1-C15 alkyl, or C5-C10 aryl with or without substitution; The substituents are independently selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, carboxyl, X is an integer from 1 to 200.

8. The singlet oxygen delivery agent according to claim 7, characterized in that R1-R2, R5-R8 are each independently selected from a C1-C6 alkyl group; R3-R4 are each independently selected from hydrogen, C1-C10 alkyl, or C5-C10 aryl with or without substitution; The substituents are each independently selected from hydrogen and C1-C10 alkyl.

9. Use of the singlet oxygen carrier according to any one of claims 1 to 6 in preparing an imaging functional material for transporting singlet oxygen.

10. Use of the singlet oxygen carrier according to any one of claims 1 to 6 in the preparation of a traceable drug for transporting singlet oxygen to cells, tissues or organs.

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