Design and application of a class of singlet oxygen- camptothecin derivative prodrug molecules

Abstract

The application relates to design and application of a class of singlet oxygen-camptothecin derivative pro-molecule, and belongs to the technical field of biological medicines. The molecule does not have the ability to release singlet oxygen and inhibit topoisomerase activity in a low-temperature environment, releases toxic singlet oxygen through a temperature-controlled internal peroxide structure reverse cyclization reaction in cells, generates camptothecin derivatives with anticancer activity in situ, and produces the effect of combined treatment of singlet oxygen and chemotherapy drugs. It is proved through DMC-ENDE that the anticancer molecule provided by the application releases singlet oxygen, generates DMC with topoisomerase inhibitory activity in situ, realizes combined anticancer treatment of singlet oxygen-CPT, and proves that the anticancer molecule can normally release singlet oxygen through DPBF capture test, and successfully proves the in-situ generation of DMC through UV-vis and fluorescence spectrum; the conclusion is also verified in cells. The application successfully realizes the successful establishment of a singlet oxygen-CPT combined anticancer system, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the design and application of a class of singlet oxygen-camptothecin derivative prodrug molecules. Background Technology

[0002] Camptothecin (CPT) is a pentacyclic quinoline alkaloid isolated from the stems and leaves of the camptotheca tree, and it exhibits good antitumor activity in animal models. Modern medicine has demonstrated its antitumor mechanism as a broad-spectrum DNA topoisomerase I inhibitor, and it is clinically used for leukemia and various solid cancers (such as colon cancer, lung cancer, breast cancer, ovarian cancer, and melanoma). As a chemotherapeutic drug that inhibits cell proliferation, CPT lacks direct cytotoxicity, which is why drug resistance easily develops clinically. However, when used as a "scavenger" after photodynamic therapy (PDT), it can effectively synergistically work with reactive oxygen species (ROS) generated by PDT to kill cancer cells, achieving complete cancer cell death.

[0003] Singlet oxygen ( 1 O2 is a major cytotoxic substance in photodynamic therapy (PDT), making oxygen a key element for PDT. In PDT, photosensitizers consume dissolved oxygen in cells within minutes to produce oxygen. 1 O2, in addition, PDT-mediated tumor suppression originates from various pathways, including the destruction of tumor-supplying blood vessels. During PDT, this damage to blood vessels can 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 considered an obstacle to overcome, as it is one of the main mechanisms of tumor destruction. To improve the effectiveness of PDT, the highest possible oxygen level must be maintained during treatment. Based on the above research, the researchers propose: 1 O2 is not necessarily produced by photosensitizers within the tumor. It will be produced outside the tumor. 1 O2 is "stored" and released into the tissues where it is needed for therapeutic purposes, and this 1 O2 delivery and release systems can be achieved by using electron-rich aromatic structures.

[0004] 1,4-Dimethylnaphthalene, 9,10-diphenylanthracene, and 2-pyridone derivatives are highly efficient 1 O2 storage and release units, this type of structure is similar to 1 O2 reacts quickly and can be stored at 193K for extended periods; simultaneously, it is stored in this type of structure. 1 O2 can also be released under conditions of gentle heating.

[0005] As an emerging 1 O2 carriers, such as 5,8-dimethylquinoline, are also promising. 1O2 storage carrier. On the one hand, the nitrogen atom on the quinoline ring can be substituted with an alkyl group to form a salt, which can effectively prolong the half-life of the internal peroxide (>100h). On the other hand, under certain conditions, eliminating this substitution will restore the half-life of the internal peroxide to 1-2h, thus achieving… 1 Controlled release of O2. On the other hand, quinoline rings are common structures in natural products, and structural modifications to natural products can yield carriers. 1 The structure of O2, and the release of endogenous peroxides from natural products into tumor cells and tissues. 1 Following O2, precursor compounds with anticancer activity are generated in situ, achieving... 1 Synergistic treatment of cancer with O2 and natural products.

[0006] CPT, as a natural product with a quinoline structure, and 1 The combined use of O2 shows great promise. 1 O2, as a powerful aid in CPT's anti-cancer action, is produced in various forms, including exogenous photosensitizers, acoustic sensitizers, or endogenous induction, with endogenous peroxides serving as a carrier. 1 There are no reports of the combined application of O2 and CPT.

[0007] The planar structure of the camptothecin derivative is disrupted after the formation of the internal peroxide, thus the camptothecin internal peroxide loses its topoisomerase inhibitory activity. Without the release of singlet oxygen, the camptothecin internal peroxide lacks anticancer activity. However, under temperature-controlled reverse cyclization of the internal peroxide structure, toxic singlet oxygen is released, leading to the in-situ formation of a camptothecin derivative with topoisomerase inhibitory activity. This prodrug design can also achieve controlled-trigger release through structural modification of camptothecin or control of substituents. Considering the advantages of internal peroxides, the development of an internal peroxide containing CPT is therefore urgently needed. Summary of the Invention

[0008] Based on the anticancer properties of endorphins and camptothecin compounds, this invention designs an endorphin-camptothecin combined therapeutic molecule: this molecule releases toxic singlet oxygen through a reverse cyclization reaction of the endorphin structure in cells, while simultaneously generating dimethylcamptothecin compounds with anticancer activity in situ, thus achieving combined treatment of singlet oxygen and chemotherapy drugs.

[0009] The technical solution of this invention: A class of camptothecin derivative prodrug molecules with quinoline intraperoxide, having the following structure:

[0010]

[0011] R2 and R3 are each independently selected from hydrogen, hydroxyl, trimethylsilyl, amino, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms, alkoxy with 1-5 carbon atoms, alkylamine with 1-6 carbon atoms, alkoxyalkyl with 2-6 carbon atoms, alkyl with 1-10 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl with 2-6 carbon atoms, aryl with 6-14 carbon atoms, cycloalkyl with 3-10 carbon atoms, or...

[0012] R1 and R4 are each independently selected from hydroxyl, trimethylsilyl, amino, alkenyl (2-6 carbon atoms), alkynyl (2-6 carbon atoms), alkoxy (1-5 carbon atoms), alkylamine (1-6 carbon atoms), alkoxyalkyl (2-6 carbon atoms), alkyl (1-10 carbon atoms), trifluoromethyl, halogen, alkoxycarbonyl (2-6 carbon atoms), aryl (6-14 carbon atoms), cycloalkyl (3-10 carbon atoms), or...

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

[0014] Some specific camptothecin derivative prodrug molecules, where R2 and R3 are each independently selected from hydrogen, alkoxy groups with 1-2 carbon atoms, alkoxyalkyl groups with 2-6 carbon atoms, alkyl groups with 1-10 carbon atoms, and trifluoromethyl groups.

[0015] R1 and R4 are each independently selected from alkoxy groups with 1-2 carbon atoms, alkoxyalkyl groups with 2-6 carbon atoms, alkyl groups with 1-10 carbon atoms, and trifluoromethyl groups.

[0016] Some specific camptothecin derivative prodrug molecules, where R2 and R3 are each independently selected from hydrogen, alkoxy groups with 1-2 carbon atoms, alkyl groups with 1-6 carbon atoms, and trifluoromethyl groups.

[0017] R1 and R4 are each independently selected from alkoxy groups with 1-2 carbon atoms, alkyl groups with 1-6 carbon atoms, and trifluoromethyl groups.

[0018] In some specific camptothecin derivative prodrug molecules, R2 and R3 are each independently selected from hydrogen and alkyl groups having 1-6 carbon atoms; R1 and R4 are each independently selected from alkyl groups having 1-6 carbon atoms.

[0019] Some specific camptothecin derivative prodrug molecules have R1 and R4 as alkyl groups with 1-6 carbon atoms, and R2 and R3 as hydrogen atoms.

[0020] Some specific camptothecin derivative prodrug molecules have R1 and R4 as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, or n-hexyl, and R2 and R3 as hydrogen.

[0021] The application of the camptothecin derivative in the preparation of materials that release singlet oxygen.

[0022] The application of the camptothecin derivative in the preparation of drugs that deliver singlet oxygen to cells, tissues or organs.

[0023] The drug is one or more of the following: tablets, capsules, granules, powders, oral preparations, injections, microcapsules, suppositories, pills, aerosols, sprays, powder inhalers, syrups, tinctures, lotions, and films.

[0024] A liposomal or micellar drug, wherein the liposomal or micellar drug 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 the following: metal nanocarriers, non-metal nanocarriers, micelles, liposomes, lactose, sucrose, gelatin, magnesium stearate, and stearic acid.

[0028] The pharmaceutical excipients are selected from one or more of the following: diluents, binders, disintegrants, lubricants, flow aids, flavoring agents, coating agents, gelatin capsule shells, cosolvents, propellants, surfactants, preservatives, and lyophilization protectants.

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

[0030]

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

[0032]

[0033] Under inert gas protection, compound 9, compound A, and an inorganic base reacted by heating in an organic solvent, and the HBr was removed to give compound B.

[0034] Under inert gas protection, a mixture of compound B, potassium acetate, and bis(triphenylphosphine)palladium(II) acetate was heated in an organic solvent. 25% NaOH was added to the reaction mixture, and the organic phase was separated. The aqueous phase was extracted with a mixed solution of MeOH-CHCl3, and concentrated HCl was added dropwise until pH = 2. The organic matter was then 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 give compound C.

[0035] Compound C was dissolved in an organic solvent and 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, the reaction solution was irradiated with 18W, 630nm red light throughout to obtain the target compound.

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

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

[0038]

[0039] A aniline compound, TEA, and Ac2O were reacted in an organic solvent at room temperature under an inert gas atmosphere to yield 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 in an organic solvent by NaBH4 to give compound A-2.

[0042] Compound A was obtained by reacting compound A-2 with PBr3 in an organic solvent at 0°C with stirring.

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

[0044] The beneficial effects of this invention: This invention combines endoperoxide and camptothecin to develop a camptothecin endoperoxide derivative prodrug molecule with a quinoline core. This camptothecin endoperoxide derivative prodrug molecule releases toxic singlet oxygen within cells through a reverse cyclization reaction of the endoperoxide structure, simultaneously generating a dimethylcamptothecin compound with anticancer activity in situ, achieving combined treatment with singlet oxygen and chemotherapeutic drugs. This invention successfully establishes a singlet oxygen-CPT combined anticancer system with broad application prospects. The A ring of camptothecin (CPT) was structurally modified by introducing substituents at positions 9 and 12. This allows the camptothecin derivative to be converted into endoperoxide (DMC-ENDO). After the formation of endoperoxide, singlet oxygen is thermally released, with a half-life of 1 hour at 37°C, reducing to the original 9,12-dimethylcamptothecin (DMC). Compared with CPT and DMC, the endoperoxide modification significantly enhances cytotoxicity against MDR cell lines. Attached Figure Description

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

[0046] Figure 2 This is a graph showing the changes in UV absorption after incubation of DPBF and DMC.

[0047] Figure 3 This is a graph showing the change in UV absorption of DMC-ENDO over time at 37℃.

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

[0049] Figure 5 This is an imaging image of DMC-ENDO releasing DMC and ROS within 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 to MCF-7 / ADR cells.

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

[0053] Figure 9 The toxicity of different concentrations of DMC-ENDO, DMC, and CPT to LO2 cells. Detailed Implementation

[0054] Preparation of DMC-ENDO:

[0055]

[0056] Step a: Under a nitrogen atmosphere at -78°C, 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). 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 then a solution of propionaldehyde (3.0 mL, 41.6 mmol) in anhydrous THF (20 mL) was added. The mixture was stirred at this temperature for another 30 min, then slowly heated to room temperature, and the reaction was quenched with 1 N HCl (50 mL). The mixture was extracted with EA (3 × 50 mL), the resulting organic phase was dried with anhydrous Na2SO4, concentrated to obtain crude product, and further purified by silica gel column chromatography (Hex:EA = 3:1, v / v) to obtain colorless oil 2 (yield 59.4%).

[0057] Step b: Under nitrogen atmosphere at 0°C, an anhydrous THF solution of compound 2 (3.0 g, 15.5 mmol) in 30 mL was added dropwise to a solution of LiAlH4 (12.0 mL, 2.5 M in THF). The reaction was brought back to room temperature and continued for 3 h, then cooled to 0°C and carefully quenched with 1 N NaOH (15 mL, slowly added dropwise over 15 min). The mixture was diluted with THF (30 mL), and 1 N NaOH was added until the precipitate completely disappeared. Extraction was performed with EA (3 × 50 mL), the combined organic layers were dried over anhydrous Na2SO4, and concentrated to obtain the crude product. Further purification was performed by silica gel column chromatography (Hex:EA = 1:1, v / v) to give 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 over a period of more than 2 min to an anhydrous DCM (20 mL) solution containing 3 (440 mg, 2.2 mmol) and 2,6-dimethylpyridine (0.5 mL, 4.4 mmol). After reacting at this temperature for 30 min, the reaction was quenched with saturated NaHCO3 solution (50 mL), and extracted with DCM (3 × 30 mL). The combined organic layers were washed with 1N HCl, saturated NaHCO3 solution, and saturated brine, dried over anhydrous Na2SO4, and concentrated to obtain the crude product. The crude product was further purified by silica gel column chromatography (Hex:EA = 3:1, v / v) to give a colorless oil 4 (43.1% yield).

[0059] Step d: Add pyridinium chlorochromate (PCC, 660 mg, 3.0 mmol) to a 20 mL solution of DCM (470 mg, 1.5 mmol) and stir the suspension at room temperature for 12 h. Quench the reaction with MeOH (10 mL), filter the organic phase, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography (Hex:EA = 4:1, v / v) to give colorless oil 5 (57.0% yield).

[0060] Step e: Under a nitrogen atmosphere, n-BuLi (3.0 mL, 2.5 M in n-Hex) was added to a suspension of methyltriphenylphosphine bromide (2.1 g, 6.0 mmol) in toluene (30 mL). 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 at least 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 column chromatography (Hex:EA = 10:1, v / v) to give a colorless oil 6 (yield 63.1%).

[0061] Step f: Dissolve (DHQD)₂PHAL (70.9 mg, 0.09 mmol), K₃Fe(CN)₆ (4.5 g, 13.7 mmol), K₂CO₃ (1.9 g, 13.7 mmol), K₂O₅SO₄·2H₂O (6.7 mg, 0.018 mmol), and MsNH₂ (430 mg, 4.5 mmol) in a mixed solution of H₂O / t-BuOH (v / v = 1:1, 50 mL). Stir the mixture at room temperature for 30 min, then cool to 0 °C. Add K₃Fe(CN)₆ (1.4 g, 4.5 mmol) to the mixture and continue stirring at 0 °C for 40 h. After the reaction is complete, add Na₂SO₃ (5.0 g) at this temperature to quench the reaction, then restore the mixture to room temperature and continue stirring for 30 min. Add DCM (50 mL) and H₂O (20 mL), and further extract the aqueous layer 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 give a colorless oil 7 (yield 96.5%).

[0062] Step g: At 0°C, 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 to a solution of NaClO2 (1.7 g, in 12 mL H2O) and NaClO (10% aqueous solution, 4.0 mL). The reaction was monitored by TLC until the starting material was completely converted, and then the pH was adjusted to 6.0 with 1 N 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 column chromatography (DCM:MeOH = 50:1, v / v) to give a colorless oil 8 (68.4% yield).

[0063] Step h: 8 (940 mg) was suspended in 12 mL of 3 N HCl 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 white solid 9 (54.9% yield).

[0064] Step i: Under a nitrogen atmosphere at 0 °C, Ac₂O (2.0 mL, 21.3 mmol) was added to an anhydrous DCM (20 mL) solution of 2,5-dimethylaniline (10 mL, 2.5 mL, 20.0 mmol) and TEA (3.1 mL, 22.5 mmol), and the mixture was stirred overnight at room temperature. After TLC analysis, the mixture was poured into ice and stirred vigorously for 30 min. The organic layer was washed with 1 N HCl, saturated NaHCO₃ solution, and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated to give a white solid 11 (97.2% yield), which required no 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. Then, 11 (3.3 g, 20 mmol) was added to the mixture over 10 min, and the reaction solution was refluxed for 10 h. After the reaction was confirmed to be complete by TLC, 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 silica gel column chromatography (Hex:EA = 4:1, v / v) to give a white solid 12 (49.5% yield).

[0066] Step k: Add NaBH4 (150.0 mg, 4.0 mmol) to a solution of 12 (435.2 mg, 2.0 mmol) in 20 mL of anhydrous methanol at room temperature. After the reaction was complete as detected by 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 a white solid 13 (98.7% yield), requiring no further purification.

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

[0068] Step m: K₂CO₃ (276.4 mg, 2.0 mmol) was added to 9 (209.2 mg, 1.0 mmol) and 14 (316.5 mg, 1.1 mmol) in 5 mL of anhydrous DMF. 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 25 mL of 1 N HCl solution and extracted with DCM (3 × 20 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and evaporated to dryness to give the crude product. The residue was purified by silica gel column chromatography (DCM:MeOH = 50:1, v / v) to give white solid 15 (yield 80.0%).

[0069] Step n: Under a nitrogen atmosphere, 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). The mixture was stirred at 100 °C for 12 h. After the reaction was complete, 25% NaOH (20 mL) was added to the cooled mixture. The organic phase was separated, and the aqueous layer was washed with a mixture of MeOH-CHCl3 (1:10, 2 × 20 mL). Concentrated HCl was added dropwise to the aqueous layer until pH = 2, and the organic matter was extracted with MeOH-CHCl3 (1:10, 2 × 50 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by column chromatography (DCM:MeOH = 50:1, v / v) to remove most of the impurities. The crude product (1,4-dioxane) was recrystallized to give a 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, the reaction solution is irradiated with 18 W, 630 nm red light throughout. After the reaction is complete, activated carbon is added to the reaction solution to remove methylene blue, the solution is filtered, and the filtrate is concentrated by rotary evaporator to give a pale yellow solid DMC-ENDO in 98% yield. NMR data: 1 H NMR (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=1 6.2Hz,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] Referring to the steps above, respectively using Four types of aniline can be used as raw materials to prepare the following four camptothecin endopeptide prodrug molecules:

[0072]

[0073] Example 1

[0074] Experiments on the release of singlet oxygen from the internal peroxide DMC-ENDO. 1,3-Diphenylisobenzofuran (DPBF) was used as... 1 An O2-specific probe was used to reveal the generation of singlet oxygen by measuring changes in absorbance at 414 nm. DMC-ENDO and DMC were each prepared into 200 μM solutions with anhydrous DMF, and then each solution was uniformly mixed with an equal volume of 100 μM DPBF anhydrous DMF solution. The mixtures were incubated in the dark at 37 °C, and the absorbance changes of the solution systems were detected using a UV-Vis spectrophotometer. Figure 1 The decrease in absorbance of the solution shown is due to the interaction between DPBF and... 1 The irreversible reaction of O2 indicates that the internal peroxide DMC-ENDO can be released normally. 1 O2. Figure 2 The absorbance of the DMC and DPBF co-incubation solution remained essentially unchanged, indicating that internal peroxide is... 1 The prerequisite for O2 production.

[0075] Example 2

[0076] Experiments were conducted to investigate the formation of DMC from the endogenous peroxide DMC-ENDO. DMC possesses specific UV absorption and fluorescence emission capabilities; measurements showed that the maximum absorption wavelength of DMC was 366 nm, and the maximum fluorescence wavelength was 418 nm. In contrast, the endogenous peroxide DMC-ENDO exhibited a significant blue shift in UV absorption and a decrease in fluorescence intensity. Changes in absorbance and fluorescence intensity of the DMC-ENDO-containing solution over time were detected using a UV-Vis spectrophotometer and a fluorophotometer to reflect the DMC release behavior. A 100 μM solution of DMC-ENDO was prepared with DMF and incubated at 37°C in the dark; changes in absorbance and fluorescence intensity were then measured. Figure 3 It can be seen that the absorption at 354 nm gradually decreases, while the absorption at 366 nm gradually increases. This indicates that the internal peroxide DMC-ENDO is continuously decaying and reverting to the precursor compound DMC. Finally, 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 internal peroxides, the generated DMC gradually accumulates, the fluorescence intensity of the solution continuously increases, and the maximum emission wavelength also increases from 411 nm to 418 nm, after which the fluorescence intensity no longer changes significantly. These results all demonstrate that DMC-ENDO releases... 1 The fact that it can revert to the precursor DMC after O2 decay demonstrates the stability and reliability of the internal peroxide carrier. In conjunction with Example 1, DMC-Endo releases [a substance / material] during its decay process. 1 O2 and DMC were released simultaneously, achieving the design goal of a combined anti-cancer effect, which laid the groundwork for subsequent biological testing.

[0077] Example 3

[0078] Experiments on the co-release of singlet oxygen by the endogenous peroxide DMC-ENDO in cells with DMC. To assess the release of singlet oxygen by endogenous peroxide, a commercially available fluorescent singlet oxygen probe, DCFH-DA, was used. HeLa cells were seeded at a density of 1 × 10⁴ cells per well in Nest 96-well plates using DMEM medium containing 10% FBS and 1% penicillin. After one day, cells were treated with medium containing DMC-ENDO (64 μM) and DCFH-DA (10 μM) for 24 h, followed by washing three times with PBS and imaging using a high-content imaging system. Figure 5 It can be seen that DMC-ENDO is released intracellularly. 1 Simultaneously with O2, DMC was generated in situ. Cell imaging experiments fully demonstrated that the compound DMC-ENDO achieved the simultaneous and isotopic release of two anticancer active substances within the cell, possessing the potential for anticancer activity.

[0079] Example 4

[0080] The MTT assay was used to assess the cytotoxicity of endogenous peroxides (DPCs). Cells were cultured at a density of 5 × 10³ cells per well in 96-well plates and incubated at 37°C under normoxic conditions (5% CO₂) for 24 h. Subsequently, cells were treated with 100 μL of medium containing different concentrations of DPCs (DMC-ENDO), precursor DMC, and CPT per well for 24 h each. Then, 10 μL of MTT solution (5 mg / mL PBS) was added to each well, and after incubation for 4 h, the medium was removed, and 150 μL of DMSO was added to each well. The plates were then placed in a microplate reader, and the absorbance at 570 nm was measured by shaking at 37°C for 5 min. Cell viability was calculated. Figure 6 , Figure 7It can be seen that the cell survival rate in the CPT-treated group did not show a significant concentration dependence. This is because CPT itself only has the ability to inhibit cell proliferation and cannot completely kill cells. Therefore, even if the CPT concentration is increased, the cell survival rate will not decrease significantly. In contrast, the combined treatment with CPT in the DMC-treated group showed a different result. 1 After O2 exposure, DMC-ENDO exhibited effective cell-killing ability. Secondly, DMC-ENDO showed even better cell-killing effects against MCF-7 / ADR cells, which is due to the release of [a specific chemical / particle] by DMC-ENDO. 1 O2 can effectively downregulate the expression of P-glycoprotein. P-glycoprotein can actively transport DMC out of the cell, affecting the inhibitory effect of DMC on cancer cells. Meanwhile, DMC-ENDO releases... 1 While exerting its anti-cancer effects, O2 can effectively inhibit the transport of DMC by P-glycoprotein, thereby enhancing the inhibitory effect of DMC on cancer cells.

[0081] Example 5

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

Claims

1. A class of camptothecin derivative prodrug molecules containing quinoline intraperoxide, characterized in that, It has the following structure: ； R2 and R3 are each independently selected from hydrogen and alkyl groups having 1-6 carbon atoms; R1 and R4 are each independently selected from alkoxy groups with 1-2 carbon atoms, alkyl groups with 1-6 carbon atoms, and trifluoromethyl groups.

2. The camptothecin derivative prodrug molecule according to claim 1, characterized in that, R2 and R3 are each independently selected from hydrogen and alkyl groups having 1-6 carbon atoms; R1 and R4 are each an alkyl group with 1-6 carbon atoms.

3. The camptothecin derivative prodrug molecule according to claim 1, characterized in that, R1 and R4 are each alkyl groups with 1-6 carbon atoms, while R2 and R3 are hydrogen atoms.

4. 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, while R2 and R3 are hydrogen.

5. The application of the camptothecin derivative prodrug molecule according to any one of claims 1-4 in the preparation of materials that release singlet oxygen.

6. The use of the camptothecin derivative prodrug molecule according to any one of claims 1-4 in the preparation of a drug for delivering singlet oxygen to cells, tissues or organs.

7. The application according to claim 6, characterized in that, The drug is one or more of the following: tablets, capsules, granules, powders, injections, microcapsules, suppositories, pills, aerosols, sprays, powder inhalers, syrups, tinctures, lotions, and films.

8. A drug in liposome or micelle form, characterized in that, The liposome or micellar form of the drug includes at least one of the camptothecin derivative prodrug molecules according to any one of claims 1-4; and / or carrier; The carrier is selected from one or more of micelles and liposomes.

9. A drug in liposome or micelle form, characterized in that, The liposome or micellar form of the drug includes at least one of the camptothecin derivative prodrug molecules according to any one of claims 1-4; And / or pharmaceutical excipients.

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