Design and application of singlet oxygen-camptothecin derivative prodrug molecule

Abstract

The invention relates to design and application of singlet oxygen-camptothecin derivative prodrug molecules, and belongs to the technical field of biological medicine. The molecule does not have the capability of releasing singlet oxygen and inhibiting the activity of topoisomerase in a low-temperature environment, releases toxic singlet oxygen in cells through a temperature-controlled reverse cyclization reaction of an internal peroxide structure, and generates a camptothecin derivative with anticancer activity in situ. And singlet oxygen is generated to be combined with chemotherapeutic drugs for treatment. The DMC-ENDO test proves that DMC with topoisomerase inhibitory activity is generated in situ while the anti-cancer molecule provided by the invention releases singlet oxygen, so that singlet oxygen-CPT combined anti-cancer treatment is realized. The DPBF capture test proves that the anti-cancer molecule can normally release singlet oxygen, and the in-situ generation of DMC is successfully proved by utilizing UV-vis and fluorescence spectrum; the conclusion is also verified in cells. The invention successfully realizes the successful establishment of the singlet oxygen-CPT combined anticancer system, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the design and application of a class of singlet oxygen-camptothecin derivative prodrug molecules. Background Art

[0002] Camptothecin (CPT) is a pentacyclic quinoline alkaloid isolated from the stems and leaves of Camptotheca acuminata, and has good anti-tumor effects in animal models. Modern medicine has proven its anti-tumor mechanism as a broad-spectrum DNA topoisomerase I inhibitor, and it is clinically used in leukemia and various solid cancers (such as colon cancer, lung cancer, breast cancer, ovarian cancer and melanoma). As a chemotherapy drug that inhibits cell proliferation, CPT has no direct cytotoxicity, which is why it is easy to develop drug resistance in clinical practice. When it is used as a "scavenger" after photodynamic therapy (PDT), it can effectively cooperate with the reactive oxygen species (ROS) generated by PDT to kill cancer cells and achieve complete death of cancer cells.

[0003] Singlet oxygen ( 1 O2) is the main cytotoxic substance in PDT, so oxygen is one of the key elements for PDT. In photodynamic therapy, photosensitizers consume dissolved oxygen in cells within a few minutes to produce 1 O2, In addition, PDT-mediated tumor inhibition originates from different pathways, including the destruction of tumor blood vessels. During PDT, this damage to blood vessels may lead to a sharp drop in oxygen levels within the tumor tissue. Therefore, hypoxia reduces the efficiency of photodynamic therapy during treatment. However, this damage should not be regarded as an obstacle to be overcome, as it is one of the main ways of tumor destruction. In order to improve the effect of PDT, oxygen levels must be maintained as high as possible during treatment. Based on the above research, the researchers proposed: 1 O2 is not necessarily produced in the tumor by the photosensitizer. 1 O2 is “stored” and released to the required tissues for therapeutic purposes, and this 1 O2 transfer and release systems can be realized by using electron-rich aromatic structures.

[0004] 1,4-Dimethylnaphthalene, 9,10-diphenylanthracene and 2-pyridone derivatives are highly effective 1 O2 storage and release unit, this type of structure is similar to 1 O2 reacts quickly and can be stored for a long time at 193K; 1 O2 can be released under mild warming conditions.

[0005] As an emerging 1 O2 carrier, 5,8-dimethylquinoline is also a promising 1O2 storage carrier. On the one hand, the nitrogen atom on the quinoline ring can effectively prolong the half-life of the endoperoxide (>100h) after being substituted with an alkyl group to form a salt. Under certain conditions, after eliminating this substitution, the half-life of the endoperoxide will be restored to 1-2h, which can achieve 1 On the other hand, the quinoline ring is a common structure in natural products. By modifying the structure of natural products, it can be loaded with 1 The structure of O2, the release of natural product endoperoxides in tumor cells and tissues 1 After O2, precursor compounds with anticancer activity are generated in situ, achieving 1 O2 and natural products as synergistic agents for cancer therapy.

[0006] CPT is a natural product with quinoline structure. 1 The combined use of O2 has great prospects. 1 O2 is a powerful assistant to CPT in fighting cancer. It can be produced in various forms, including exogenous photosensitizers, sonosensitizers or endogenous induced production. 1 There are no reports on the combined use of O2 and CPT.

[0007] The planar structure of camptothecin derivatives is destroyed after the formation of endoperoxides, so camptothecin endoperoxides lose their topoisomerase inhibitory activity. When singlet oxygen is not released, camptothecin endoperoxides have no anticancer activity, but after the reverse cyclization reaction of the endoperoxide structure under temperature control releases toxic singlet oxygen, camptothecin derivatives with topoisomerase inhibitory activity are generated in situ. This prodrug design can also achieve controllable triggered release by structural modification of camptothecin or control of substituents. Considering the advantages of endoperoxides, it is very urgent to develop an endoperoxide containing CPT. Summary of the invention

[0008] Based on the anti-cancer properties of endoperoxides and camptothecin compounds, the present invention designs an endoperoxide-camptothecin combined therapeutic molecule: the molecule releases toxic singlet oxygen through the reverse cyclization reaction of the endoperoxide structure in the cell, and simultaneously generates a dimethylcamptothecin compound with anti-cancer activity in situ, thereby achieving a combined treatment of singlet oxygen and chemotherapeutic drugs.

[0009] The technical solution of the present invention is: a type of camptothecin derivative prodrug molecule with quinoline endoperoxide, having the following structure:

[0010]

[0011] 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

[0012] R1 and R4 are each independently selected from 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

[0013] Wherein, x is an integer from 1 to 2000.

[0014] In some specific prodrug molecules of camptothecin derivatives, R2 and R3 are independently selected from hydrogen, alkoxy with 1-2 carbon atoms, alkoxyalkyl with 2-6 carbon atoms, alkyl with 1-10 carbon atoms, and trifluoromethyl.

[0015] R1 and R4 are each independently selected from an alkoxy group having 1 to 2 carbon atoms, an alkoxyalkyl group having 2 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, and a trifluoromethyl group.

[0016] In some specific prodrug molecules of camptothecin derivatives, R2 and R3 are independently selected from hydrogen, alkoxy with 1-2 carbon atoms, alkyl with 1-6 carbon atoms, and trifluoromethyl.

[0017] R1 and R4 are each independently selected from an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group.

[0018] In some specific prodrug molecules of camptothecin derivatives, R2 and R3 are independently selected from hydrogen and an alkyl group having 1 to 6 carbon atoms; and R1 and R4 are independently selected from an alkyl group having 1 to 6 carbon atoms.

[0019] In some specific prodrug molecules of camptothecin derivatives, R1 and R4 are independently an alkyl group having 1 to 6 carbon atoms, and R2 and R3 are hydrogen.

[0020] In some specific prodrug molecules of camptothecin derivatives, R1 and R4 are independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl or n-hexyl, and R2 and R3 are hydrogen.

[0021] The camptothecin derivative is used in the preparation of singlet oxygen releasing materials.

[0022] The camptothecin derivative is used in preparing medicine for transporting singlet oxygen to cells, tissues or organs.

[0023] The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, lotions, and films.

[0024] A drug in the form of liposomes or micelles, wherein the drug in the form of liposomes or micelles comprises at least one of the above-mentioned camptothecin derivative prodrug molecules;

[0025] and / or carrier;

[0026] and / or pharmaceutical excipients;

[0027] 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.

[0028] The pharmaceutical excipients are selected from one or more of diluents, adhesives, disintegrants, lubricants, glidants, flavoring agents, coating agents, gelatin capsule shells, latent solvents, propellants, surfactants, preservatives, and freeze-drying protective agents.

[0029] A specific camptothecin derivative prodrug molecule has the following structure:

[0030]

[0031] The preparation steps and reaction formula of the camptothecin derivative prodrug molecule of the present invention are as follows:

[0032]

[0033] Under the protection of inert gas, compound 9, compound A and an inorganic base are heated in an organic solvent to react, and HBr is removed to obtain compound B.

[0034] Under the protection of inert gas, a mixture of compound B, potassium acetate and bis(triphenylphosphine) palladium acetate (II) was heated in an organic solvent for reaction, 25% NaOH was added to the reaction mixture, the organic phase was separated, the aqueous phase was extracted with a mixed solution of MeOH-CHCl3, and concentrated HCl was added dropwise to pH = 2, and organic matter was extracted with MeOH-CHCl3 (1:10, 2×50 mL). The organic layers were combined, dried, purified by column chromatography, and recrystallized (1,4-dioxane) to obtain compound C.

[0035] Compound C was dissolved in an organic solvent, cooled to 0°C in an ice bath, a catalytic amount of methylene blue was added, and the mixture was stirred under an oxygen atmosphere. During the reaction, 18W, 630nm red light was used to irradiate the reaction solution throughout the reaction to obtain the target compound.

[0036] Wherein, the definitions of R1-R4 are the same as those in the molecular structure of the above-mentioned camptothecin derivative prodrug.

[0037] The preparation process of compound A is as follows:

[0038]

[0039] Under an inert gas atmosphere, an aniline compound, TEA and Ac2O are reacted in an organic solvent at room temperature with stirring to obtain a benzamide compound.

[0040] The benzamide compound was added to a DMF solution containing POCl3 at room temperature and heated under reflux to obtain compound A-2.

[0041] At room temperature, compound A-1 is reduced by NaBH4 in an organic solvent to obtain compound A-2.

[0042] At 0°C, compound A-2 is reacted with PBr3 in an organic solvent with stirring to obtain compound A.

[0043] Wherein, the definitions of R1-R4 are the same as those in the molecular structure of the above-mentioned camptothecin derivative prodrug.

[0044] Beneficial effects of the present invention: The present invention jointly designs endoperoxides and camptothecin, and develops a camptothecin endoperoxy derivative prodrug molecule with a quinoline mother core. The camptothecin endoperoxy derivative prodrug molecule releases toxic singlet oxygen through the reverse cyclization reaction of the endoperoxy structure in the cell, and simultaneously generates a dimethyl camptothecin compound with anticancer activity in situ, thereby realizing the combined treatment of singlet oxygen and chemotherapeutic drugs. The present invention successfully establishes a singlet oxygen-CPT combined anticancer system, which has a wide range of application prospects. The A ring of camptothecin (CPT) is structurally modified by introducing substituents at positions 9 and 12. This allows the camptothecin derivative to be converted into endoperoxides (DMC-ENDO). After the endoperoxide is formed, singlet oxygen is released thermally, with a half-life of 1 hour at 37°C, and it is reduced to the original 9,12-dimethyl camptothecin (DMC). Compared with CPT and DMC, the cytotoxicity of the MDR cell line by endoperoxide modification is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the change in UV absorption after incubation of DPBF and DMC-ENDO.

[0046] Figure 2 This is the change in UV absorption after incubation of DPBF and DMC.

[0047] Figure 3 This is the UV absorption change of DMC-ENDO at 37°C over time.

[0048] Figure 4 This is a graph showing the fluorescence intensity change of DMC-ENDO at 37°C over time.

[0049] Figure 5 This is an imaging diagram of DMC-ENDO releasing DMC and ROS in cells.

[0050] Figure 6 The toxicity of different concentrations of DMC-ENDO, DMC and CPT to MCF-7 cells.

[0051] Figure 7 The toxicity of different concentrations of DMC-ENDO, DMC and CPT on MCF-7 / ADR cells.

[0052] Figure 8 The toxicity of different concentrations of DMC-ENDO, DMC and CPT on 3T3 cells.

[0053] Fig. 9 The toxicity of different concentrations of DMC-ENDO, DMC and CPT to LO2 cells. DETAILED DESCRIPTION

[0054] Preparation of DMC-ENDO:

[0055]

[0056] Step a: n-BuLi (20.0 mL, 2.5 M in n-Hex) was slowly added dropwise to a solution of 2,2,6,6-tetramethylpiperidine (6.0 mL, 35.6 mmol) in anhydrous THF (30 mL) at -78 °C under nitrogen. The mixture was stirred at this temperature for 30 min, and then a solution of 2-methoxynicotinic acid (1, 2.0 g, 13.1 mmol) in anhydrous THF (40 mL) was added. The mixture was stirred for another 30 min, and a solution of propionaldehyde (3.0 mL, 41.6 mmol) in anhydrous THF (20 mL) was added. The mixture was stirred for another 30 min at this temperature, then slowly warmed to room temperature, and the reaction was quenched with 1N HCl (50 mL). The mixture was extracted with EA (3×50 mL), and the obtained organic phase was dried over anhydrous Na 2 SO 4 and concentrated to give a crude product, which was further purified by silica gel chromatography (Hex:EA=3:1, v / v) to give a colorless oil 2 (yield 59.4%).

[0057] Step b: Under nitrogen at 0°C, a solution of compound 2 (3.0 g, 15.5 mmol) in anhydrous THF (30 mL) was added dropwise to a solution of LiAlH4 (12.0 mL, 2.5 M in THF). The reaction was returned to room temperature and continued to react for 3 h, then cooled to 0°C and carefully quenched with 1N NaOH (15 mL, slowly added dropwise over 15 min). The mixture was diluted with THF (30 mL), 1N NaOH was added until the precipitate completely disappeared, extracted with EA (3×50 mL), the combined organic layers were dried over anhydrous Na2SO4, and concentrated to give a crude product, which was further purified by silica gel chromatography (Hex:EA=1:1, v / v) to give a colorless oil 3 (80.0% yield).

[0058] Step c: At 0°C, tert-butyldimethylsilyl trifluoromethanesulfonate (0.5 mL, 2.2 mmol) was slowly added dropwise to a solution of anhydrous DCM (20 mL) containing 3 (440 mg, 2.2 mmol) and 2,6-lutidine (0.5 mL, 4.4 mmol) over a period of more than 2 min. After reacting at this temperature for 30 min, saturated NaHCO3 solution (50 mL) was added to quench the reaction and extracted with DCM (3×30 mL). The combined organic layer was washed with 1N HCl, saturated NaHCO3 solution and saturated brine, dried over anhydrous Na2SO4, and concentrated to give a crude product, which was further purified by silica gel chromatography (Hex:EA=3:1, v / v) to give a colorless oil 4 (43.1% yield).

[0059] Step d: Pyridinium chlorochromate (PCC, 660 mg, 3.0 mmol) was added to a solution of 4 (470 mg, 1.5 mmol) in DCM (20 mL), and the suspension was stirred at room temperature for 12 h. After quenching the reaction with MeOH (10 mL), the organic phase was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (Hex:EA=4:1, v / v) to give a colorless oil 5 (57.0% yield).

[0060] Step e: n-BuLi (3.0 mL, 2.5 M in n-Hex) was added to a suspension of methyltriphenylphosphonium bromide (2.1 g, 6.0 mmol) in toluene (30 mL) under nitrogen atmosphere. The mixture was stirred at room temperature for 30 min, followed by the addition of 2-5 (620 mg, 2.0 mmol), and the resulting reaction solution was refluxed for more than 12 h until the reaction was complete as detected by TLC. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel chromatography (Hex:EA=10:1, v / v) to obtain a colorless oil 6 (yield 63.1%).

[0061] Step f: (DHQD)2PHAL (70.9 mg, 0.09 mmol), K3Fe(CN)6 (4.5 g, 13.7 mmol), K2CO3 (1.9 g, 13.7 mmol), K2OsO4·2H2O (6.7 mg, 0.018 mmol) and MsNH2 (430 mg, 4.5 mmol) were dissolved in a mixed solution of H2O / t-BuOH (v / v=1:1, 50 mL). The mixture was stirred at room temperature for 30 min and then cooled to 0°C. 6 (1.4 g, 4.5 mmol) was added to the mixture and stirring was continued at 0°C for 40 h. After the reaction was completed, Na2SO3 (5.0 g) was added at this temperature to quench the reaction, and the mixture was then returned to room temperature and stirred for another 30 min. DCM (50 mL) and H2O (20 mL) were added, and the aqueous layer was further extracted with DCM (3×25 mL). The combined organic layers were dried (Na2SO4) and concentrated. The crude product was purified by silica gel column chromatography (Hex:EA=2:1, v / v) to afford 7 as a colorless oil (yield 96.5%).

[0062] Step g: To a mixture of 7 (1 g, 2.93 mmol), TEMPO (91.5 mg, 0.59 mmol) and PBS (20 mL, pH = 7.4) in MeCN (40 mL) was added NaClO2 solution (1.7 g in 12 mL H2O) and NaClO (10% aqueous solution, 4.0 mL) at 0°C. The reaction was monitored by TLC until the starting material was completely converted, and then the pH was adjusted to 6.0 with 1N HCl. The reaction was quenched with saturated Na2SO3 solution (20 mL), and the mixture was extracted with DCM (3×50 mL). The combined organic layers were dried (Na2SO4), concentrated under reduced pressure, and the residue was purified by silica gel chromatography (DCM:MeOH=50:1, v / v) to give 8 as a colorless oil (68.4% yield).

[0063] Step h: 8 (940 mg) was suspended in 3N HCl (12 mL) solution, the mixture was refluxed for 12 h, then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (EA: MeOH = 10: 1, v / v) to give a white solid 9 (54.9% yield).

[0064] Step i: Ac2O (2.0 mL, 21.3 mmol) was added to a solution of 2,5-dimethylaniline (10, 2.5 mL, 20.0 mmol) and TEA (3.1 mL, 22.5 mmol) in anhydrous DCM (20 mL) at 0°C under nitrogen atmosphere, and stirred overnight at room temperature. After TLC detection, the mixture was poured into ice and stirred vigorously for 30 min, and the organic layer was washed with 1N HCl, saturated NaHCO3 solution and saturated brine. The combined organic layer was dried over anhydrous Na2SO4 and concentrated to give 11 as a white solid (97.2% yield) without further purification.

[0065] Step j: POCl3 (14.0 mL, 140.0 mmol) was added dropwise to a DMF (4.0 mL, 50.0 mmol) solution over 30 min, and the reaction mixture was stirred for another 30 min at room temperature. 11 (3.3 g, 20 mmol) was subsequently added to the mixture over 10 min, and the reaction solution was refluxed for 10 h. After the reaction was complete according to TLC detection, the mixture was poured into ice (500 g) and stirred vigorously for 30 min. The pH was adjusted to 9.0 with Na2CO3, and the mixture was extracted with DCM (3×200 mL). The combined organic layers were dried (Na2SO4), concentrated under reduced pressure, and the residue was purified by a silica gel column (Hex:EA=4:1, v / v) to give 12 as a white solid (49.5% yield).

[0066] Step k: To a solution of 12 (435.2 mg, 2.0 mmol) in anhydrous methanol (20 mL) was added NaBH4 (150.0 mg, 4.0 mmol) at room temperature. After the reaction was complete according to TLC, the mixture was poured into water (100 mL) and extracted with EA (3×50 mL). The combined organic layers were dried (Na2SO4) and concentrated to give 13 as a white solid (98.7% yield) without further purification.

[0067] Step 1: PBr3 (1 mL) was slowly added to a solution of 13 (1.76 g, 7.9 mmol) in CHCl3 (50 mL) at 0°C. The mixture was stirred at this temperature for 1 hour and quenched with saturated NaHCO3 solution (100 mL). The organic matter was extracted with DCM (3×50 mL), and the combined organic phase was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to give a crude product. The residue was purified by silica gel chromatography (Hex:DCM=4:1, v / v) to give 14 as a white solid (yield 78.3%).

[0068] Step m: K2CO3 (276.4 mg, 2.0 mmol) was added to a solution of 9 (209.2 mg, 1.0 mmol) and 14 (316.5 mg, 1.1 mmol) in anhydrous DMF (5 mL). The mixture was then heated at 50 °C for 10 h under a nitrogen atmosphere. After the reaction was complete, the mixture was poured into a 1N HCl solution (25 mL) and extracted with DCM (3 × 20 mL). The combined organic phase was dried over anhydrous Na2SO4 and evaporated to dryness to give a crude product. The residue was purified by silica gel chromatography (DCM: MeOH = 50: 1, v / v) to give 15 as a white solid (yield 80.0%).

[0069] Step n: Anhydrous MeCN (10 mL) was added to a mixture of 15 (84.3 mg, 0.2 mmol), potassium acetate (60.1 mg, 0.6 mmol) and bis(triphenylphosphine) palladium (II) acetate (30.6 mg, 0.04 mmol) under a nitrogen atmosphere. The mixture was stirred at 100 ° C for 12 h. After the reaction was completed, 25% NaOH (20 mL) was added to the cooled mixture. The organic phase was separated and the aqueous layer was washed with a mixed solution of MeOH-CHCl3 (1:10, 2×20 mL). Concentrated HCl was added dropwise to the aqueous layer until pH=2, and organic matter was extracted with MeOH-CHCl3 (1:10, 2×50 mL). The combined organic layer was dried (Na2SO4) and concentrated, and the residue was purified by column chromatography (DCM:MeOH=50:1, v / v) to remove most of the impurities. The crude product was recrystallized (1,4-dioxane) to give yellow solid DMC (yield 32.5%).

[0070] Step o: Dissolve DMC (8.0 mg, 0.95 mmol) in CHCl3 (5 mL), and cool the reaction mixture to 0°C in an ice bath. Add methylene blue (catalytic amount) to the solution, and stir the mixture for 6 h under an oxygen atmosphere. During the reaction, 18 W, 630 nm red light was used to irradiate the reaction solution throughout the reaction. After the reaction was completed, activated carbon was added to the reaction solution to remove methylene blue, filtered, and the filtrate was concentrated by a rotary evaporator to obtain a light yellow solid DMC-ENDO with a yield of 98%. NMR data: 1 HNMR (400MHz, CDCl3) δ7.81(s,1H),7.45(s,1H),6.84–6.71(m,2H),5.71(d,J=16.3Hz,1H),5.28(d,J= 16.2Hz,1H),5.13(d,J=7.4Hz,2H),2.01(s,3H),1.95(s,3H),1.93–1.80(m,2H),1.03(t,J=7.0Hz,3H).

[0071] Refer to the above steps to The following four camptothecin endoperoxy derivative prodrug molecules can be prepared using four anilines as raw materials:

[0072]

[0073] Example 1

[0074] Experiments on the release of singlet oxygen from endoperoxide DMC-ENDO. 1,3-Diphenylisobenzofuran (DPBF) was used as 1 O2-specific probe, revealing the generation of singlet oxygen through the change of absorbance at 414nm. DMC-ENDO and DMC were prepared into 200μM solutions in anhydrous DMF, and then mixed with an equal volume of 100μM DPBF anhydrous DMF solution. The mixture was incubated at 37℃ in the dark, and the absorbance change of the solution system was detected by UV-vis spectrophotometer. Figure 1 The absorbance of the solution shown decreases, which is due to the 1 O2 has an irreversible reaction, indicating that the endoperoxide DMC-ENDO can be released normally. 1 O2. Figure 2 The absorbance of the DMC and DPBF co-incubation solution remained basically unchanged, indicating that endoperoxygenation is 1 A prerequisite for O2 production.

[0075] Example 2

[0076] Experiment on the generation of DMC from endoperoxide DMC-ENDO. DMC has specific ultraviolet absorption and fluorescence emission capabilities. After measurement, the maximum absorption wavelength of DMC is 366nm, and the maximum fluorescence wavelength is 418nm; while the ultraviolet absorption of endoperoxide DMC-ENDO shows an obvious blue shift, and the fluorescence intensity is weakened. The changes in absorbance and fluorescence intensity of the solution containing DMC-ENDO over time are detected by UV-vis photometer and fluorescence photometer, reflecting the release behavior of DMC. DMC-ENDO was prepared into a 100μM solution with DMF, incubated at 37℃ in the dark, and the changes in absorbance and fluorescence intensity were measured. From Figure 3 It can be seen that the absorption at 354nm gradually decreases, while the absorption at 366nm gradually increases, which indicates that the endoperoxide DMC-ENDO is constantly decaying and returning to the precursor compound DMC. At the end of the test, the absorbance of the solution no longer changes significantly, indicating the end of the release process. Figure 4It can be seen that during the decay of endoperoxides, the generated DMC gradually accumulates, the fluorescence intensity of the solution continues to rise, and the maximum emission wavelength also increases from 411nm to 418nm, and the fluorescence intensity no longer changes significantly in the later period. The above results all prove that DMC-ENDO releases 1 O2 can be restored to the precursor DMC, indicating the stability and reliability of the endoperoxide carrier. Combined with the above-mentioned Example 1, DMC-Endo releases 1 O2 and DMC achieved the original design intention of synchronous release, thus achieving a combined anti-cancer effect, which laid the foundation for subsequent biological tests.

[0077] Example 3

[0078] Experiment on the co-release of singlet oxygen and DMC by endoperoxide DMC-ENDO in cells. In order to evaluate the release of singlet oxygen by endoperoxide, a commercial singlet oxygen fluorescent probe DCFH-DA was used. Hela cells were seeded at 1×104 cells per well on Nest 96-well plates using DMEM medium containing 10% FBS and 1% penicillin. One day later, the cells were treated with medium containing DMC-ENDO (64μM) and DCFH-DA (10μM) for 24h, and then the cells were washed three times with PBS and then photographed using a high-content imager. Figure 5 As can be seen, DMC-ENDO is released in cells 1 O2, DMC was generated in situ. Cell imaging experiments fully proved that the compound DMC-ENDO achieved the simultaneous and isotopic release of two anticancer active substances in cells, and had the potential for anticancer activity.

[0079] Example 4

[0080] The MTT method was used to evaluate the cytotoxicity of endoperoxides. The cells were cultured in a 96-well plate at a density of 5×103 cells per well and placed under normoxic conditions at 37°C and 5% CO2 for 24 hours. The cells were then treated with culture medium containing different concentrations of endoperoxide DMC-ENDO, precursor DMC, and CPT (100 μL per well) for 24 hours, and then 10 μL MTT solution (5 mg / mL PBS solution) was added to each well. After incubation for 4 hours, the culture medium was removed, 150 μL DMSO was added to each well, and the plate was placed in a microplate reader. The plate was shaken at 37°C for 5 minutes and the absorbance at 570 nm was detected to calculate the cell survival rate. Figure 6 , Figure 7It can be seen that the cell survival rate in the CPT treatment group has no obvious concentration dependence. This is because CPT itself can only inhibit cell proliferation and cannot completely kill cells. This means that even if the concentration of CPT is increased, the cell survival rate will not decrease significantly. 1 After O2, DMC-ENDO showed effective cell killing ability. Secondly, DMC-ENDO showed better cell killing effect on MCF-7 / ADR cells, which is because DMC-ENDO released 1 O2 can effectively downregulate the expression of P-glycoprotein. P-glycoprotein can actively transport DMC out of cells, affecting the inhibitory effect of DMC on cancer cells. 1 While exerting its anti-cancer effect, O2 can effectively inhibit the transport of DMC by P-glycoprotein and enhance the inhibitory effect of DMC on cancer cells.

[0081] Example 5

[0082] The toxicity of endoperoxide DMC-ENDO and its precursor DMC to normal cells was detected by referring to the method of Example 4, revealing the safety of the compounds. Figure 8 , Fig. 9 It can be seen that DMC-ENDO has no obvious toxicity to normal cells, indicating that it has reliable safety.

Claims

1. A camptothecin derivative prodrug molecule having quinoline endoperoxide, characterized in that: It 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 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 Wherein, x is an integer from 1 to 2000.

2. The camptothecin derivative prodrug molecule 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 alkoxyalkyl group having 2 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, and a trifluoromethyl group; R1 and R4 are each independently selected from an alkoxy group having 1 to 2 carbon atoms, an alkoxyalkyl group having 2 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, and a trifluoromethyl group.

3. The camptothecin derivative prodrug molecule 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 an alkoxy group having 1 to 2 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a trifluoromethyl group.

4. The camptothecin derivative prodrug molecule 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 an alkyl group having 1 to 6 carbon atoms.

5. The camptothecin derivative prodrug molecule according to claim 1, characterized in that: R1 and R4 are each independently an alkyl group having 1 to 6 carbon atoms, and R2 and R3 are hydrogen.

6. The camptothecin derivative prodrug molecule according to claim 1, characterized in that: R1 and R4 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl or n-hexyl, and R2 and R3 are hydrogen.

7. Use of the camptothecin derivative prodrug molecule according to any one of claims 1 to 6 in the preparation of a singlet oxygen releasing material.

8. Use of the camptothecin derivative prodrug molecule according to any one of claims 1 to 6 in the preparation of a drug for transporting singlet oxygen to cells, tissues or organs.

9. The use according to claim 8, characterized in that: The medicine is one or more of tablets, capsules, granules, powders, oral preparations, injections, microcapsule preparations, suppositories, pills, aerosols, sprays, powder inhalers, syrups, wine preparations, tinctures, lotions, and films.

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

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