Preparation and application of radiotherapy sensitizer IR808-ATIPA

Abstract

The application belongs to the technical field of medicine, and specifically discloses a radiotherapy sensitizer IR808-ATIPA as well as a preparation method and application thereof. The compound is covalently connected by IR808 and ATIPA, has the functions of radiosensitization and CT imaging, and can prolong the retention time in tumors. IR808-ATIPA can enhance X-ray absorption and induce ferroptosis (down-regulate GPX4, up-regulate ACSL4 and ROS) to synergistically improve the effect of radiotherapy. The preparation method comprises the following steps: condensation of phenylhydrazine and 3-methyl-2-butanone to generate an intermediate 1, reaction of the intermediate 1 with 6-bromohexanoic acid to generate an intermediate 2, acyl chloride treatment of the intermediate 2, condensation of the intermediate 2 with triiodoisophthalic acid, and finally reaction and purification of the intermediate 2 with a cyclohexene derivative to obtain the target product. Experiments show that after intratumoral injection of IR808-ATIPA, the micro-CT can guide the radiotherapy target area in real time, and the combination of the micro-CT and low-dose X-ray (4 Gy) can significantly inhibit the growth of tumors and reduce the expression of Ki67. The compound is suitable for tumor treatment with integration of diagnosis and treatment, and has the potential of high biological safety and precise radiotherapy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the preparation and application of a radiosensitizer IR808-ATIPA. Background Technology

[0002] Radiation therapy is a primary treatment for many types of cancer in clinical practice. It utilizes high-energy X-rays to destroy cancer cells, and its mechanisms can be divided into direct and indirect effects. Direct effects are caused by the interaction between ionizing radiation and biomolecules; this effect does not involve any mediators in the biomolecular structure and primarily affects single-stranded and double-stranded DNA molecules. Indirect effects are caused by hydroxyl radicals and other reactive oxygen species (ROS) generated by the ionization of water molecules in tissues; these ROS can damage biomolecules and DNA. However, the dosage and efficacy of radiation therapy are limited by its toxicity to normal tissues. Excessive damage to normal tissues from high-energy rays can lead to serious side effects.

[0003] Therefore, radiosensitization is a process that increases the sensitivity of cancer cells to radiation damage. Radiosensitizers are substances that can store radiation energy and generate free radicals, thereby enhancing the radiosensitivity of tumor cells. These sensitizers can concentrate radiation energy within tumor tissue or reduce the tumor's resistance to X-rays, thus improving the efficacy of radiotherapy without increasing the radiation dose and potentially expanding the therapeutic window. Currently, radiosensitizers based on small molecule compounds or high atomic number (Z) materials have been used in cancer treatment. These materials possess characteristics such as chemical stability, slow metabolism, high selectivity, and significant effects at low doses. However, the biocompatibility and long metabolic cycle of metallic materials are significant drawbacks.

[0004] In clinical radiotherapy, localization via computed tomography (CT) imaging is an indispensable step. The real-time monitoring capabilities of imaging effectively prevent overtreatment and undertreatment. Due to its widespread availability, high efficiency, relatively low cost, and good penetration into deep tissues, CT is widely used clinically. Image-guided radiotherapy (IGRT) helps to more accurately manage patient radiotherapy, thereby significantly improving treatment outcomes and prognosis. Clinically, iodine is a commonly used contrast agent in CT imaging. Furthermore, iodine is a non-metallic high-Z material with good X-ray absorption and biocompatibility, showing promising potential in enhancing radiotherapy; however, its small molecular weight and rapid metabolism limit its application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes to covalently link IR808 with ATIPA to create a novel radiosensitizer (IR808-ATIPA). This sensitizer can accumulate at the tumor site, has a good retention time, and is beneficial for delineating the target area of ​​the lesion in radiotherapy, thereby enhancing the therapeutic effect.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] The method for preparing IR808-ATIPA of the present invention includes:

[0008] 2,3,3-trimethyl-3H-indol-1-ium (2,3,3-trimethylindolium ion):

[0009] Phenylated hydrazine (5.00 g, 46.24 mmol) was dissolved in 100 mL of acetic acid (AcOH) at room temperature. Then, 3-methyl-2-butanone (5.97 g, 69.31 mmol) was added and mixed thoroughly with stirring. The temperature was raised to 118°C and refluxed for 8 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure to obtain a yellow oily liquid. 50 mL of dichloromethane (CH2Cl2) was added, and the mixture was washed with saturated sodium bicarbonate solution (50 mL × 3). The organic phase was collected, dried overnight in sodium sulfate (Na2SO4), filtered, and concentrated to obtain a brownish-red oily liquid—Intermediate 1 (6.64 g, 90.21%).

[0010] 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium bromide:

[0011] Intermediate 1 (6.64 g, 41.62 mmol) and 6-bromohexanoic acid (16.21 g, 83.10 mmol) were added to a 100 mL round-bottom flask. 60 mL of acetonitrile (ACN) was added, and the mixture was refluxed (1 drop / second) for 72 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove the acetonitrile. 100 mL of ethyl acetate (EtOAc) was added, and the mixture was stirred to precipitate a pink solid. The precipitate was filtered off, the solid was washed with ethyl acetate, and dried in a vacuum oven to obtain a pink powder (8.54 g, 58.23%).

[0012] 1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-2,3,3-trimethyl-3H-indol-1-ium bromide (1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-2,3,3-trimethyl-3H-indol-1-ium bromide):

[0013] To a solution of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole bromide (0.50 g, 1.41 mmol) in dichloromethane (10 mL) and one drop of N,N-dimethylformamide (DMF), oxalyl chloride (0.89 g, 7.05 mmol) was added. The mixture was stirred overnight at room temperature in a well-ventilated fume hood. After the solvent had evaporated to dryness, 10 mL of N,N-dimethylacetamide (DMAc) was added, followed by dropwise addition of the oxalyl chloride solution to a solution of 5-amino-2,4,6-triiodoisophthalic acid (0.79 g, 1.41 mmol) and triethylamine (0.43 g, 4.23 mmol) dissolved in 10 mL of DMAc. The reaction mixture was heated to 80 °C for 5 hours under argon protection in the dark. The reaction mixture was quenched in 100 mL of ice water to obtain a solid precipitate. After filtration, the crude residue was purified by rapid column chromatography (dichloromethane / methanol = 10:1) to obtain the title compound as a white powder (0.15 g, 12.12%).

[0014] N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline hydrochloride(N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline hydrochloride):

[0015] Under ice-salt bath conditions, a 250 mL three-necked flask equipped with a thermometer was used. 20 mL of anhydrous DMF and 20 mL of anhydrous dichloromethane were added and stirred until homogeneous. When the temperature dropped to 0°C, 15 mL of phosphorus oxychloride and 18 mL of anhydrous dichloromethane were mixed and added to a constant-pressure dropping funnel equipped with a drying tube. The solution was added dropwise to the three-necked flask at a rate of 1 drop per second, ensuring that the temperature did not rise during the addition. After the addition was complete, the mixture was stirred for 30 minutes, and then cyclohexanone (5 g, 50.94 mmol) was added to the solution. After the addition of cyclohexanone, the ice-water bath was removed and the temperature was raised to reflux for 3 hours. After the reaction was complete, the reaction mixture was poured into crushed ice, allowed to stand, and then filtered to obtain a yellow solid (4.12 g, 23.94 mmol). This solid was dissolved in 50 mL of anhydrous ethanol, and aniline (6.69 g, 71.85 mmol) was added, followed by stirring for 1 hour. Pour the solution into 1 L of 10% hydrochloric acid solution and let it stand for 2 hours. Filter and collect the dark purple solid, dissolve it in methanol, and recrystallize it from methyl tert-butyl ether and n-hexane (v / v = 1:1) to give the purple solid product (4.32 g, 23.67%).

[0016] 2-((E)-2-((E)-2-chloro-3-(2-((E)-1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-ylidene) ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-3,3-dimethyl-3H-indol-1-ium Bromides:

[0017] In a 100 mL round-bottom flask, 1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-2,3,3-trimethyl-3H-indole-1-onium bromide (0.76 g, 0.8083 mmol), N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohexyl-2-en-1-yl)methyl)aniline hydrochloride (0.13 g, 0.38 mmol), anhydrous sodium acetate (0.02 g), and anhydrous ethanol (15 mL) were added. The mixture was refluxed under argon atmosphere in the dark for 8 hours (1 drop / second). The crude residue was then purified by rapid column chromatography (dichloromethane / methanol, 50:1) to give the title compound as a green powder (60 mg, 8.55%).

[0018] The chemical structure of the sensitizer disclosed in this invention is shown below:

[0019] .

[0020] The present invention also discloses the application of the above-mentioned IR808-ATIPA in the preparation of tumor radiotherapy drugs, wherein the drugs are administered by intratumoral injection and used in conjunction with X-ray irradiation.

[0021] The present invention also discloses the application of the above-mentioned IR808-ATIPA in CT imaging-guided radiotherapy. After intratumoral injection of IR808-ATIPA, the CT value changes are monitored by micro-CT to delineate the tumor target area and adjust the radiotherapy dose in real time.

[0022] The present invention also discloses the application of the above-mentioned IR808-ATIPA in the preparation of a drug that induces ferroptosis in tumor cells. The IR808-ATIPA enhances radiotherapy-induced ferroptosis by inhibiting GPX4 activity, increasing ACSL4 expression, and promoting lipid peroxidation.

[0023] The present invention also discloses a pharmaceutical composition of IR808-ATIPA described above, the composition comprising IR808-ATIPA and a pharmaceutically acceptable carrier.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0025] IR808-ATIPA is a compound formed by covalently linking IR808 and ATIPA. This compound not only directly enhances the efficacy of radiotherapy by increasing X-ray absorption but also exhibits a strong ability to induce ferroptosis. IR808-ATIPA increases post-radiotherapy ROS production, leading to GPX4 inactivation, which in turn exacerbates oxidative stress and promotes lipid peroxidation (LPO), thereby inducing and enhancing ferroptosis in cells, increasing the sensitivity of tumor cells to radiation, and achieving more significant growth inhibition. Furthermore, IR808-ATIPA has a molecular weight larger than iodine atoms. CT imaging results after intratumoral injection of IR808-ATIPA confirmed its long retention time within the tumor. Under CT imaging guidance, IR808-ATIPA can effectively administer radiotherapy. Therefore, IR808-ATIPA possesses the function of real-time guidance and monitoring of synergistic anticancer therapy and can be used as a small molecule drug for integrated tumor diagnosis and treatment. Attached Figure Description

[0026] Figure 1 The image shows compound IR808-ATIPA from Example 1. A represents the NMR mass spectrometry results of IR808-ATIPA; B is a flowchart of the synthesis of IR808-ATIPA.

[0027] Figure 2 The effects of compound IR808-ATIPA on cells are shown. A represents cytotoxicity assay; B represents cell colony formation assay; C represents apoptosis assay; and D represents cell DNA damage assay.

[0028] Figure 3 RNA sequencing was performed on HeLa cells treated with IR808-ATIPA. In the figures, A represents a comparison of the number of differentially expressed genes (DEGs) between groups; B represents a comparison of the number of DEGs between the IR808-ATIPA combined with radiotherapy group and the PBS group; CD represents the analysis of genes with high differential expression; E represents GO enrichment analysis; and FG represents KEGG database analysis.

[0029] Figure 4 The experiment involved IR808-ATIPA and ferroptosis in cells. A represents cell morphology and lipid droplet staining assays; B represents intracellular ROS detection; C represents protein immunoblotting assays; DF represents intracellular GSH, GSH-PX, and MDA measurements; and G represents cell transmission electron microscopy.

[0030] Figure 5 The images show the CT imaging results of the IR808-ATIPA. A represents external CT imaging; B represents internal CT imaging.

[0031] Figure 6 The effects of IR808-ATIPA on radiotherapy are shown. A represents the change in body weight in Hela model mice during treatment; B represents the change in tumor volume in Hela model mice during treatment; C represents the weight of the tumor in Hela model mice after treatment; and D represents HE staining of tumor sections in Hela model mice after treatment. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0033] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0034] Example 1 In this example, IR808-ATIPA is synthesized by the following method, which is as follows.

[0035] (1) At room temperature, phenylhydrazine (5.00 g, 46.24 mmol) was dissolved in 100 mL of acetic acid (AcOH) in a beaker. Then, 3-methyl-2-butanone (5.97 g, 69.31 mmol) was added and mixed thoroughly with stirring. The mixture was heated to 118 °C and refluxed (1 drop / second) for 8 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure to obtain a yellow oily liquid. 50 mL of dichloromethane (CH2Cl2) was added and the mixture was washed with saturated sodium bicarbonate solution (50 mL × 3). The organic phase was collected, dried overnight with sodium sulfate (Na2SO4), filtered, and concentrated to obtain a brownish-red oily liquid A (6.64 g, 90.21%).

[0036] (2) Add liquid A (6.64 g, 41.62 mmol) obtained in (1) and 6-bromohexanoic acid (16.21 g, 83.10 mmol) to a 100 mL round-bottom flask. Add 60 mL of acetonitrile (ACN) and raise the temperature to reflux (1 drop / second), reacting for 72 hours. After the reaction is complete, concentrate under reduced pressure to remove acetonitrile. Add 100 mL of ethyl acetate (EtOAc) and stir to precipitate a pink solid. Filter off the precipitate, wash the resulting solid with ethyl acetate, and dry in a vacuum drying oven to obtain pink powder B (8.54 g, 58.23%).

[0037] (3) To a solution of powder B (0.50 g, 1.41 mmol) obtained in (2) in dichloromethane (10 mL) and one drop of N,N-dimethylformamide (DMF), add oxaloyl chloride (0.89 g, 7.05 mmol). Stir overnight at room temperature in a well-ventilated fume hood. After the solvent has evaporated to dryness, add 10 mL of N,N-dimethylacetamide (DMAc), and then add the acyl chloride solution dropwise to a solution of 5-amino-2,4,6-triiodoisophthalic acid (0.79 g, 1.41 mmol) and triethylamine (0.43 g, 4.23 mmol) dissolved in 10 mL of DMAc. Heat to 80 °C for 5 hours under argon protection in the dark. Quench the reaction mixture in 100 mL of ice water to obtain a solid precipitate. After filtration, the crude residue was purified by rapid column chromatography (dichloromethane / methanol = 10:1) to obtain the compound of the present invention as white powder C (0.15 g, 12.12%).

[0038] (4) Under ice-salt bath conditions, take a 250 mL three-necked flask equipped with a thermometer, add 20 mL of anhydrous DMF and 20 mL of anhydrous dichloromethane, and stir until homogeneous. When the temperature drops to 0°C, mix 15 mL of phosphorus oxychloride and 18 mL of anhydrous dichloromethane, and add the mixture to a constant pressure dropping funnel equipped with a drying tube. Add the solution dropwise to the three-necked flask at a rate of 1 drop per second, ensuring that the temperature does not rise during the addition process. After the addition is complete, stir for 30 minutes, and then add cyclohexanone (5 g, 50.94 mmol) to the solution. After the addition of cyclohexanone, remove the ice-water bath and raise the temperature to reflux for 3 hours. After the reaction is complete, pour the reaction mixture into crushed ice, let it stand, and then filter to obtain a yellow solid (4.12 g, 23.94 mmol). Dissolve the solid in 50 mL of anhydrous ethanol, add aniline (6.69 g, 71.85 mmol), and then stir for 1 hour. Pour the solution into 1 L of 10% hydrochloric acid solution and let it stand for 2 hours. Filter and collect the dark purple solid, dissolve it in methanol, and recrystallize it with methyl tert-butyl ether and n-hexane (v / v = 1:1) to give the purple solid product D (4.32 g, 23.67%).

[0039] (5) Add the powder C (0.76 g, 0.8083 mmol) obtained in (3), the product D (0.13 g, 0.38 mmol) obtained in (4), anhydrous sodium acetate (0.02 g), and anhydrous ethanol (15 mL) to a 100 mL round-bottom flask. Recirculate under argon atmosphere in the dark for 8 hours (1 drop / second). Afterward, remove the solvent by vacuum distillation, and then perform silica gel column chromatography (dichloromethane:methanol = 50:1). v / v The crude residue was purified to obtain the compound of the present invention as a green powder (60 mg, 8.55%).

[0040] Example 2 The purpose of this example is to verify the basic damage to cells caused by IR808-ATIPA.

[0041] (1) The cytotoxicity of IR808-ATIPA under radiation-free and radiation-free conditions was investigated using the standard CCK-8 assay. HeLa cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated in DMEM medium (150 µL) at 37°C and 5% CO2 for 24 hours until cell adhesion. The medium was then replaced with 150 µL of freshly prepared medium containing different concentration gradients of IR808-ATIPA. After 4 hours, the cells were irradiated with X-rays (0 or 4 Gy). After 48 hours of incubation, the cells were washed with PBS, and each well was incubated with DMEM (90 µL) and CCK-8 solution (10 µL) for approximately 2 hours. The absorbance was measured at 450 nm using a microplate reader. The experiment demonstrated that... Figure 2 A) IR808-ATIPA has extremely low cytotoxicity; however, in the presence of radiation, cell survival gradually decreases with increasing IR808-ATIPA concentration.

[0042] (2) Stir HeLa cells at 10 per well 3 Cells were cultured at a density of [number] cells in 6-well plates with PBS or IR808-ATIPA added to the culture medium. The plates were incubated in a CO2 incubator for 4 hours and exposed to X-ray radiation (0 or 4 Gy). After approximately one week of incubation, the cells were fixed with paraformaldehyde (2.0 mL, 4 wt%) at room temperature for 30 minutes. After removing the fixative, each well was stained with 2 mL of crystal violet for 15 minutes. The 6-well plates were slowly washed with water, air-dried, and photographed. Untreated wells served as the control group. The experiment demonstrated [results]. Figure 2 B) The IR808-ATIPA+X-ray group showed more significant inhibition on the number and size of cell colonies.

[0043] (3) HeLa cells were cultured in T25 flasks and divided into various treatment groups (PBS group, IR808-ATIPA group, X-ray group, IR808-ATIPA+X-ray group). PBS or IR808-ATIPA was added to the culture medium and the cells were treated in a CO2 incubator for 4 hours. The culture medium was then replaced and the cells were irradiated with radiation (0 or 4 Gy) for another 48 hours. Adherent cells and cell supernatant were collected and resuspended in 1× Annexin Binding Buffer. The collected cells were mixed, and FITC-labeled Annexin V and phycoerythrin (PE) were added and gently mixed. The cells were incubated at room temperature in the dark for 30 minutes. Binding Buffer was then added to each tube, and the stained cells were analyzed by flow cytometry. Apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%. Compared with the control group, the IR808-ATIPA+X-ray group had higher early apoptosis rate, late apoptosis rate, and overall apoptosis rate than other groups. Figure 2 D).

[0044] Example 3 The purpose of this example is to verify the RNA sequencing characteristics of IR808-ATIPA-treated HeLa cells.

[0045] Cellular RNA sequencing analysis was performed on the PBS group, IR808-ATIPA group, PBS+X-ray group, and IR808-ATIPA+X-ray group. The number of differentially expressed genes (DEGs) differed significantly among the groups. Figure 3 A), the group receiving the combined treatment of IR808-ATIPA and radiotherapy showed the greatest change in the number of DEGs compared to the PBS group. Figure 3 B), analyzing related genes with large differences ( Figure 3 CD (Cellular Antioxidant) revealed that many genes are associated with antioxidant defense, ferroptosis, apoptosis, protein degradation, cell cycle regulation, fatty acid metabolism, and responses to DNA damage. Upregulated genes were associated with apoptosis, ferroptosis, and cellular antioxidant responses. GO enrichment analysis showed (…). Figure 3 DEGs expression in the IR808-ATIPA+X-ray group (E) is typically enriched in biological processes such as regulation of iron transport, cellular oxidative stress, and apoptosis.

[0046] Mapping differentially expressed gene transcripts to the KEGG database ( Figure 3 The results confirmed that the IR808-ATIPA+X-ray group was significantly enriched in the ferroptosis pathway, the apoptosis pathway, and the glutathione metabolism pathway, which is related to iron metabolism and oxidative stress.

[0047] Example 4 The purpose of this example is to verify the effect of IR808-ATIPA on ferroptosis-related damage in cells.

[0048] (1) HeLa cells were cultured in 96-well plates. PBS or IR808-ATIPA was added to the culture medium and the cells were incubated in a CO2 incubator for 4 hours, and exposed to X-ray radiation (0 or 4 Gy). After incubation for another 48 hours, the culture medium was changed, the cells were washed with PBS, and 4% paraformaldehyde fixative was added for fixation at room temperature for 10-15 minutes. Staining Solution was prepared (BODIPY 493 / 503 (1000X): Hoechst 33342 (1000X): Assay Buffer = 1:1:998). The paraformaldehyde fixative was removed, and the cells were washed 1-2 times with PBS. The PBS was removed, and 100 µL of Staining Solution was added to each well. The cells were incubated at room temperature in the dark for 10-20 minutes. The cells were washed twice with PBS, and the green fluorescence was observed using a fluorescence microscope. The morphology of HeLa cells changed significantly, and the number of intracellular lipid droplets increased significantly ( Figure 4 A).

[0049] (2) HeLa cells were cultured in 96-well plates, and PBS or IR808-ATIPA was added to the culture medium. The cells were then treated in a CO2 incubator for 4 hours and exposed to X-ray radiation (0 or 4 Gy). After another 48 hours of incubation, the culture medium was changed, the cells were washed with PBS, and HeLa cells were added to DCFH-DA and incubated in a 37°C cell culture incubator for 20 minutes. The cells were then washed twice with PBS, and green fluorescence was observed using a fluorescence microscope. Untreated cells served as the control group. Combined treatment with IR808-ATIPA and radiotherapy further increased intracellular ROS levels (…). Figure 4 B).

[0050] (3) HeLa cells cultured in T25 culture flasks were treated with PBS or IR808-ATIPA in a CO2 incubator for 4 hours, then the culture medium was changed and the cells were irradiated with radiation (0 or 4 Gy) for another 48 hours. The cells were washed with PBS, 100 µL of PBS was added to each flask, and the cell suspension was collected using a cell scraper. The cells were centrifuged at 4°C (3000 rpf, 5 min). After centrifugation, the supernatant was discarded, and a mixture of RIPA lysis buffer and PMSF (99:1) was added and the cells were mixed. The cell suspension was placed on ice and shaken for 30 minutes. The cells were centrifuged at 4°C (12000 rcf, 30 min), and the supernatant was retained. The protein concentration was quantified using a BCA protein assay kit. Each sample was loaded onto a PAGE precast gel, the target protein was separated by electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane, blocked with rapid blocking buffer for about 20 minutes, and incubated with primary antibody at 4°C overnight. The primary antibodies used here were rabbit anti-ACSL4, rabbit anti-GPX4, and rabbit anti-β-tubulin. Subsequently, the membrane was washed three times in TBST for 10 minutes each time and incubated with HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody at 25°C for 1 hour. Observation was performed using a chemiluminescence assay kit. In the IR808-ATIPA combined radiotherapy group, the reduction in GPX4 was more significant than in the radiotherapy-only group. Simultaneously, the combined treatment with radiation and IR808-ATIPA increased ACSL4 levels (…). Figure 4 C).

[0051] (4) HeLa cells cultured in T25 flasks were treated with PBS or IR808-ATIPA in a CO2 incubator for 4 hours, followed by culture medium replacement and irradiation (0 or 4 Gy) for another 48 hours. HeLa cells were washed with PBS and GSH, GSH-PX, and MDA were measured according to the manufacturer's instructions. GSH, GSH-PX, and MDA levels were normalized to quantitative total protein levels using a BCA protein assay kit. Untreated cells served as a control group. Radiation therapy decreased cellular GSH and GSH-PX levels while increasing MDA levels. Detection showed that an increase in IR808-ATIPA increased the intergroup differences (…). Figure 4 DF).

[0052] (5) HeLa cells cultured in T25 flasks were treated with PBS or IR808-ATIPA in a CO2 incubator for 4 hours, then the culture medium was changed and the cells were irradiated (0 or 4 Gy) for another 48 hours. HeLa cells were washed with PBS and digested with trypsin. After digestion with trypsin, culture medium was added to stop the digestion, and the cells were transferred to centrifuge tubes and centrifuged at 4°C (3000 rpf, 5 min). The supernatant was removed, and electron microscopy fixative was added. The cell clumps were dispersed and resuspended. The cells were fixed at room temperature in the dark for 30 minutes and then transferred to 4°C for storage and observation under an electron microscope. After 72 hours of irradiation, ferroptosis-like changes were observed in the mitochondria, such as reduced mitochondrial volume, rounding, increased HRTEM contrast, mitochondrial membrane rupture, increased outer membrane density, and a significant decrease in the number of mitochondrial cristae. Figure 4 G).

[0053] Example 5: The purpose of this example is to verify the CT imaging performance of the IR808-ATIPA.

[0054] CT values ​​of different concentrations of IR808-ATIPA were measured using a small animal micro-CT scanner (PerkinElmer, QuantumGX2). Gradient concentrations of IR808-ATIPA were dispensed into individual EP tubes, and scans were performed using the micro-CT scanner. CT values ​​were measured, and a drug concentration versus CT curve was calculated. With increasing IR808-ATIPA concentration, the CT value significantly increased, showing a clear linear relationship with iodine concentration. Figure 5 A).

[0055] After subcutaneous tumor formation in mice, 50 µL of IR808-ATIPA was injected intratumorally. CT imaging was performed before injection and at 3 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after injection, and regions of interest (ROIs) were delineated to measure and record changes in CT values ​​within the tumor. Immediately after drug injection, the CT value within the tumor increased unevenly. As time progressed, the drug gradually became more uniformly distributed, but the CT value of the tumor tissue remained significantly higher than before injection. The measurement results showed that the CT value reached its peak at 3 minutes after injection, then gradually decreased. Enhancement of the tumor was still observed at 4 hours, and the CT value was higher than that of the surrounding tissue. Figure 5 B).

[0056] Example 6: The purpose of this example is to verify the effect of IR808-ATIPA in radiotherapy.

[0057] When the tumor size reaches approximately 100 mm 3 Twenty-four BALB / c nude mice were randomly divided into four groups and treated on day 0: Group 1 received an intratumoral injection of 50 µL PBS; Group 2 received an intratumoral injection of 50 µL IR808-ATIPA; Group 3 received an intratumoral injection of 50 µL PBS and was irradiated with 4 Gy of X-rays; and Group 4 received an intratumoral injection of 50 µL IR808-ATIPA and was irradiated with 4 Gy of X-rays. The in vivo treatment experiment was terminated when the tumor size reached 15 mm in any dimension, and all mice were euthanized. During radiotherapy, almost no significant weight loss was observed in the nude mice. Figure 6 A) Tumors injected with IR808-ATIPA and exposed to low-dose X-rays were almost completely suppressed after 15 days. Figure 6 BD).

[0058] Tumor tissues and major organs were collected and fixed with paraformaldehyde (4 wt%). Hematoxylin and eosin (HE) staining and Ki67 and GPX4 immunohistochemical staining were performed on the tumor tissues. HE staining showed that, compared with other treatment groups, the tissue structure of the IR808-ATIPA-treated group exposed to X-rays was loose, and some staining intensities were reduced. Immunohistochemical staining results showed that Ki67 and GPX4 were downregulated (…). Figure 6 E).

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An inducer of ferroptosis IR808-ATIPA, characterized in that, The chemical structure of the ferroptosis inducer is as follows: ； The IR808 in the ferroptosis inducer is covalently connected to the triiodophenyl of ATIPA through an amide bond; the ferroptosis inducer IR808-ATIPA enhances radiotherapy-induced ferroptosis by inhibiting GPX4 activity, increasing ACSL4 expression, and promoting lipid peroxidation; the ferroptosis inducer IR808-ATIPA is used as a computed tomography (CT) imaging agent and a radiotherapy sensitizer, and realizes the functions of real-time monitoring and synergistic anticancer.

2. The method of preparing IR808-ATIPA of the ferroptosis inducer of claim 1, characterized by, The method comprises the following steps: (a) refluxing phenylhydrazine and 3-methyl-2-butanone in acetic acid to generate intermediate 1; (b) condensing intermediate 1 and 6-bromohexanoic acid in acetonitrile to generate 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole bromide; (c) reacting the product of step (b) with oxalyl chloride to generate an acyl chloride intermediate, and then condensing the acyl chloride intermediate with 5-amino-2,4,6-triiodoisophthalic acid to obtain a triiodophenyl-modified intermediate; and (d) refluxing the product of step (c) with N-((E)-(2-chloro-3-((phenylimino)methyl)cyclohexenyl)aniline hydrochloride in ethanol, and purifying to obtain IR808-ATIPA.

3. The method of claim 2, wherein: The reaction temperature of step (a) is 110-120 DEG C, and the refluxing time is 6-10 hours; the purification of step (d) uses an elution system with a volume ratio of dichloromethane to methanol of 50:

1.

4. The method of claim 2, wherein: The condensation reaction of step (c) is carried out at 80 DEG C under argon protection for 5 hours.

5. Use of the ferroptosis inducer IR808-ATIPA of claim 1 in the preparation of a tumor radiotherapy sensitization drug, wherein the drug is administered by intratumoral injection and is used in combination with X-ray irradiation.

6. Use of the ferroptosis inducer IR808-ATIPA of claim 1 in the preparation of a CT imaging guided radiotherapy drug, wherein the CT value change of the IR808-ATIPA after intratumoral injection is monitored by micro-CT, the tumor target region is real-time outlined, and the radiotherapy dose is adjusted.

7. Use of the IR808-ATIPA of claim 1 in the preparation of a drug for inducing ferroptosis of tumor cells, wherein the IR808-ATIPA enhances radiotherapy-induced ferroptosis by inhibiting GPX4 activity, increasing ACSL4 expression, and promoting lipid peroxidation.

8. A pharmaceutical composition comprising the ferroptosis inducer IR808-ATIPA of claim 1, wherein the composition comprises the ferroptosis inducer IR808-ATIPA and a pharmaceutically acceptable carrier.

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