Preparation and application of radiotherapy sensitizer IR808-ATIPA

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a radiotherapy sensitizer IR808-ATIPA as well as a preparation method and application thereof. According to the compound, IR808 and ATIPA are covalently connected, so that the compound has radiosensitization and CT imaging functions, and the residence time in tumors can be prolonged. The IR808-ATIPA synergistically improves the radiotherapy effect by enhancing X-ray absorption and inducing ferroptosis (down-regulating GPX4 and up-regulating ACSL4 and ROS). The preparation method comprises the following steps: condensing phenylhydrazine and 3-methyl-2-butanone to generate an intermediate 1, reacting with 6-bromohexanoic acid to obtain an intermediate 2, carrying out acylating chlorination, condensing with triiodo-isophthalic acid, and finally reacting with a cyclohexene derivative for purification to obtain a target product. Experiments show that after IR808-ATIPA is injected into a tumor, micro-CT can guide a radiotherapy target region in real time, and tumor growth is remarkably inhibited and Ki67 expression is reduced in combination with low-dose X-rays (4Gy). The compound is suitable for diagnosis and treatment integrated tumor treatment, and has high biological safety and precise radiotherapy potential.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to the preparation and application of a radiosensitizer IR808-ATIPA. Background Art

[0002] Radiotherapy is a major treatment modality for various types of cancer in clinical practice. It uses high-energy X-rays to destroy cancer cells, and its mechanism can be divided into direct action and indirect action. The direct action is caused by the interaction of ionizing radiation with biomolecules, which does not involve any intermediary in the biomolecular structure and mainly affects single-stranded and double-stranded DNA molecules. The indirect action is caused by the 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 dose and efficacy of radiotherapy are limited by the toxicity of normal tissues. Because excessive damage to normal tissues by high-energy rays may lead to serious side effects.

[0003] Therefore, radiosensitization is a process of increasing the sensitivity of cancer cells to radiation damage. Radiosensitizers are a class of substances that can store radiation energy and generate free radicals, thereby enhancing the radiosensitivity of tumor cells. These sensitizers can concentrate the radiation energy within the tumor tissue or reduce the resistance of the tumor to X-rays, so that the efficacy of radiotherapy can be improved 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 have characteristics such as chemical stability, slow metabolism, high selectivity, and significant effects at low doses. However, the biosafety of metal materials and their relatively long metabolic cycle are undeniable drawbacks.

[0004] In clinical radiotherapy, localization by computed tomography (CT) imaging is an essential step. The real-time monitoring ability of imaging examinations can effectively prevent over-treatment and under-treatment. Due to the wide availability, high efficiency, relatively low cost, and good deep tissue penetration of CT, it has been widely used in clinical practice. Image-guided radiotherapy (IGRT) helps to more accurately perform radiotherapy on patients, thereby significantly improving the treatment effect and prognosis of patients. Clinically, iodine-based agents are commonly used contrast agents for CT imaging. In addition, iodine is a non-metallic high-Z material with good X-ray absorption ability and biocompatibility, showing good prospects in enhancing radiotherapy, but its small molecular weight and fast metabolism make it difficult to apply. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention proposes covalently linking IR808 with ATIPA to create a new type of radiosensitizer (IR808-ATIPA), which can accumulate at the tumor site, has a good retention time, is conducive to target area delineation of the lesion during radiotherapy, and enhances the therapeutic effect.

[0006] To achieve the above invention purpose, the present invention provides the following technical solutions.

[0007] The preparation method of IR808-ATIPA of the present invention includes: 2,3,3-trimethyl-3H-indol-1-ium (2,3,3-trimethylindolium ion): At room temperature, dissolve phenylhydrazine (5.00 g, 46.24 mmol) in 100 mL of acetic acid (AcOH). Then, add 3-methyl-2-butanone (5.97 g, 69.31 mmol) and mix well under stirring. Raise the temperature to 118 °C and reflux for 8 hours. After the reaction is completed, cool the reaction mixture to room temperature, and then concentrate it under reduced pressure to obtain a yellow oily liquid. Add 50 mL of dichloromethane (CH 2 Cl 2 ), and wash it with saturated sodium bicarbonate solution (50 mL × 3). Collect the organic phase, add sodium sulfate (Na 2 SO 4 ) and dry it overnight, then filter and concentrate to obtain a brownish-red oily liquid - intermediate 1 (6.64 g, 90.21%); 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium bromide (1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole bromide): Add intermediate 1 (6.64 g, 41.62 mmol) 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 the reflux state (1 drop / second) and react for 72 hours. After the reaction is completed, concentrate it under reduced pressure to remove acetonitrile. Add 100 mL of ethyl acetate (EtOAc), stir to precipitate a pink solid. Filter off the precipitate, wash the obtained solid with ethyl acetate, and dry it in a vacuum drying oven to obtain a pink powder (8.54 g, 58.23%); 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): To a solution of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium bromide (0.50 g, 1.41 mmol) in dichloromethane (10 mL) and a 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 was evaporated to dryness, 10 mL of N,N-dimethylacetamide (DMAc) was added, and then the acyl chloride solution was added 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 and well mixed. The reaction was heated to 80 °C under argon protection and in the dark for 5 hours. The reaction mixture was poured into 100 mL of ice water to quench, and a solid precipitate was obtained. After filtration, the crude residue was purified by flash column chromatography (dichloromethane / methanol = 10:1) to give the title compound as a white powder (0.15 g, 12.12%); 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): 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 this mixture to a constant-pressure dropping funnel equipped with a drying tube. Add this 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. After addition, stir for 30 minutes, then add cyclohexanone (5 g, 50.94 mmol) to the solution. After adding 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 this 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 obtain a purple solid product (4.32 g, 23.67%); 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 bromide: Add 1-(6-((3,5-dicarboxy-2,4,6-triiodophenyl)amino)-6-oxohexyl)-2,3,3-trimethyl-3H-indol-1-ium bromide (0.76 g, 0.8083 mmol), N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline hydrochloride (0.13 g, 0.38 mmol), anhydrous sodium acetate (0.02 g), and anhydrous ethanol (15 mL) to a 100 mL round-bottom flask. Reflux the reaction under argon atmosphere in the dark for 8 hours (1 drop / second). Then, purify the crude residue by flash column chromatography (dichloromethane / methanol, 50:1) to obtain the title compound as a green powder (60 mg, 8.55%).

[0008] The chemical structure of the sensitizer disclosed by the present invention is as follows: .

[0009] The present invention also discloses an application of the above-mentioned IR808-ATIPA in the preparation of a tumor radiotherapy drug, and the drug is administered by intratumoral injection and used in combination with X-ray irradiation.

[0010] The present invention also discloses an application of the above-mentioned IR808-ATIPA in CT imaging-guided radiotherapy. After intratumoral injection of IR808-ATIPA, the change of its CT value is monitored by micro-CT, and the tumor target area is outlined in real time and the radiotherapy dose is adjusted.

[0011] The present invention also discloses an application of the above-mentioned IR808-ATIPA in the preparation of a drug for inducing ferroptosis of tumor cells. The IR808-ATIPA enhances the ferroptosis induced by radiotherapy by inhibiting the activity of GPX4, increasing the expression of ACSL4 and promoting lipid peroxidation.

[0012] The present invention also discloses a pharmaceutical composition of the above-mentioned IR808-ATIPA, and the composition contains IR808-ATIPA and a pharmaceutically acceptable carrier.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] IR808-ATIPA is formed by covalently connecting IR808 and ATIPA. This compound not only directly enhances the effect of radiotherapy by increasing the absorption of X-rays, but also exhibits a strong ferroptosis induction ability. IR808-ATIPA increases the production of ROS after radiotherapy, leading to the inactivation of GPX4, further exacerbating oxidative stress, promoting lipid peroxidation (LPO), thereby inducing and enhancing the ferroptosis of cells, enhancing the radiosensitivity of tumor cells, and achieving more significant growth inhibition. In addition, the molecular weight of IR808-ATIPA is greater than that of iodine atoms. After intratumoral injection of IR808-ATIPA, the CT imaging results verify its longer retention time in the tumor. Under the guidance of CT imaging, IR808-ATIPA can effectively implement radiotherapy. Therefore, IR808-ATIPA has the function of real-time guiding and monitoring of combined anti-cancer therapy and can be used as a small molecule drug for tumor diagnosis and treatment integration. Description of the Drawings

[0015] Figure 1 It is the compound IR808-ATIPA in Example 1. Wherein A is the nuclear magnetic mass spectrometry result of IR808-ATIPA; B is the synthesis flow chart of IR808-ATIPA.

[0016] Figure 2 Effects of compound IR808-ATIPA on cells. Among them, A is cytotoxicity detection; B is cell colony formation assay; C is cell apoptosis detection; D is cell DNA damage detection.

[0017] Figure 3 RNA sequencing of Hela cells treated with IR808-ATIPA. Among them, A is the comparison of the number of differentially expressed genes (DEGs) between groups; B is the comparison of the number of DEGs between the combined treatment group of IR808-ATIPA and radiotherapy and the PBS group; C-D are the analyses of related genes with large differences; E is GO enrichment analysis; F-G are KEGG database analysis.

[0018] Figure 4 IR808-ATIPA and ferroptosis of cells. Among them, A is the cell morphology and lipid droplet staining assay; B is the detection of intracellular ROS; C is the Western blot experiment; D-F are the measurements of intracellular GSH, GSH-PX and MDA; G is the transmission electron microscopy of cells.

[0019] Figure 5 CT imaging effect of IR808-ATIPA. Among them, A is in vitro CT imaging; B is in vivo CT imaging.

[0020] Figure 6 Effect of IR808-ATIPA in radiotherapy. Among them, A is the change in body weight of Hela model mice during the treatment course; B is the change in tumor volume of Hela model mice during the treatment course; C is the weight of the tumor after the treatment of Hela model mice; D is the HE section staining of the tumor after the treatment of Hela model mice. Specific implementation mode

[0021] The present invention will be further described in detail below with specific examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0022] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0023] Example 1 In this example, IR808-ATIPA was synthesized by the following method, and the method is as follows.

[0024] (1) At room temperature, dissolve phenylhydrazine (5.00 g, 46.24 mmol) in 100 mL of acetic acid (AcOH) in a beaker. Then, add 3-methyl-2-butanone (5.97 g, 69.31 mmol) and mix well with stirring. Heat to 118 °C and reflux (1 drop / second) for 8 hours. After the reaction is complete, cool the reaction mixture to room temperature and then concentrate it under reduced pressure to obtain a yellow oily liquid. Add 50 mL of dichloromethane (CH 2 Cl 2 ) and wash with saturated sodium bicarbonate solution (50 mL × 3). Collect the organic phase, add sodium sulfate (Na 2 SO 4 ) and dry overnight, then filter and concentrate to obtain a brownish-red oily liquid A (6.64 g, 90.21%).

[0025] (2) Add the 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) and react for 72 hours. After the reaction is complete, concentrate under reduced pressure to remove acetonitrile. Add 100 mL of ethyl acetate (EtOAc), stir to precipitate a pink solid. Filter off the precipitate, wash the obtained solid with ethyl acetate, and dry it in a vacuum drying oven to obtain a pink powder B (8.54 g, 58.23%).

[0026] (3) To a solution of the powder B (0.50 g, 1.41 mmol) obtained in (2) in dichloromethane (10 mL) and one drop of N,N-dimethylformamide (DMF), add oxalyl chloride (0.89 g, 7.05 mmol). Stir overnight at room temperature in a well-ventilated fume hood. After the solvent has evaporated, 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 and mixed well. Heat to 80 °C and react for 5 hours under argon protection in the dark. Pour the reaction mixture into 100 mL of ice water to quench, obtaining a solid precipitate. After filtration, purify the crude residue by flash column chromatography (dichloromethane / methanol = 10:1) to obtain the compound of the present invention as a white powder C (0.15 g, 12.12%).

[0027] (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 this mixture to a constant pressure dropping funnel equipped with a drying tube. Add this 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. After addition, stir for 30 minutes, then add cyclohexanone (5 g, 50.94 mmol) to the solution. After adding cyclohexanone, remove the ice-water bath and raise the temperature to the reflux state and react 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 this 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 obtain the purple solid product D (4.32 g, 23.67%).

[0028] (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. Under an argon atmosphere, reflux the reaction in the dark for 8 hours (1 drop / second). Then, distill off the solvent under reduced pressure, and purify the crude residue by silica gel column chromatography (dichloromethane:methanol = 50:1, v / v ) to obtain the compound of the present invention as a green powder (60 mg, 8.55%).

[0029] Example 2 The purpose of this example is to verify the basic damage of IR808-ATIPA to cells.

[0030] (1) Use the standard CCK-8 method to explore the cytotoxicity of IR808-ATIPA under conditions with and without radiation. Seed Hela cells into a 96-well plate at a density of 5×10 3 cells per well, and use DMEM medium (150 µL) at 37 °C, 5% CO 2Incubate for 24 hours until the cells adhere. Replace 150 µL of freshly prepared medium containing different concentration gradients of IR808-ATIPA. After 4 hours, perform X-ray irradiation (0 or 4 Gy). After incubating for 48 hours, wash the cells with PBS, add DMEM (90 µL) and CCK-8 solution (10 µL) to each well, and incubate for about 2 hours. Measure the absorbance at 450 nm using a microplate reader. The experiment demonstrated ( Figure 2 A) IR808-ATIPA has extremely low toxicity to cells; in the presence of radiation, as the concentration of IR808-ATIPA increases, the cell survival rate gradually decreases.

[0031] (2) Culture Hela cells in a 6-well plate at a density of 10 3 cells per well. Add PBS or IR808-ATIPA to the medium and treat for 4 hours in a CO 2 incubator, and expose to X-ray radiation (0 or 4 Gy). Incubate for about one week, fix the cells with paraformaldehyde (2.0 mL, 4 wt%) at room temperature for 30 minutes. After removing the fixative, stain the cells with 2 mL of crystal violet solution per well for 15 minutes. Slowly wash the 6-well plate with water, air dry, and take machine photos. The untreated wells serve as the control group. The experiment demonstrated ( Figure 2 B) The IR808-ATIPA + X-ray group showed more obvious inhibition of the number and size of cell colonies.

[0032] (3) Culture Hela cells in a T25 culture flask and divide them into various treatment groups (PBS group, IR808-ATIPA group, X-ray group, IR808-ATIPA + X-ray group). Add PBS or IR808-ATIPA to the medium and treat in a CO 2 incubator for 4 hours, then replace the medium and perform radiation irradiation (0 or 4 Gy), and continue to culture for 48 hours. Collect the adherent cells and cell supernatant, resuspend them in 1× Annexin binding buffer (1× Binding Buffer). Mix the collected cells well, add FITC-labeled Annexin V and phycoerythrin (PE), and gently mix. Incubate at room temperature in the dark for 30 minutes. Then add Binding Buffer to each tube and perform flow cytometry analysis on the stained cells. Apoptosis rate = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%. Compared with the control group, the early apoptosis rate, late apoptosis rate, and overall apoptosis rate of the IR808-ATIPA + X-ray group were higher than those of other groups ( Figure 2 D).

[0033] Example 3 The purpose of this example is to verify the RNA sequencing characteristics of IR808-ATIPA in treating Hela cells.

[0034] Cell RNA sequencing analysis was performed on the PBS group, IR808-ATIPA group, PBS+X-ray group, and IR808-ATIPA+X-ray group. There were significant differences in the comparison of the number of differentially expressed genes (DEGs) among the groups ( Figure 3 A), and the combined treatment group of IR808-ATIPA and radiotherapy had the largest change in the number of DEGs compared with the PBS group ( Figure 3 B). Analyzing the related genes with a large degree of difference ( Figure 3 C-D), it was found that many were related to genes such as antioxidant defense, ferroptosis, apoptosis, protein degradation, cell cycle regulation, fatty acid metabolism, and response to DNA damage. The upregulated genes were related to apoptosis, ferroptosis, and cellular antioxidant response. GO enrichment analysis showed ( Figure 3 E) that the DEGs expression in the IR808-ATIPA+X-ray group was typically enriched in biological processes such as "regulation of iron transport", "cellular oxidative stress", and "apoptosis".

[0035] The transcripts of the differential genes were mapped to the KEGG database ( Figure 3 F-G). The results confirmed that the IR808-ATIPA+X-ray group was significantly enriched in the "ferroptosis pathway", "apoptosis pathway", and the "glutathione metabolism pathway" related to iron metabolism and oxidative stress.

[0036] Example 4 The purpose of this example is to verify the ferroptosis-related damage performance of IR808-ATIPA on cells.

[0037] (1) Cultivate Hela cells in a 96-well plate, add PBS or IR808-ATIPA to the culture medium, and treat them in a CO 2 incubator for 4 hours, and expose them to X-ray radiation (0 or 4 Gy). After incubating for another 48 hours, change the culture medium, wash with PBS, and add 4% paraformaldehyde fixative to fix at room temperature for 10-15 minutes. Prepare Staining Solution (BODIPY 493 / 503(1000X): Hoechst 33342(1000X): Assay Buffer = 1:1:998). Remove the paraformaldehyde fixative and wash with PBS 1-2 times. Aspirate the PBS, and add 100 µL of Staining Solution to each well. Incubate in the dark at room temperature for 10-20 minutes. Wash with PBS twice and observe the green fluorescence using a fluorescence microscope. The morphology of Hela cells changed significantly, and the number of lipid droplets in the cells increased significantly (Figure 4 A).

[0038] (2) Cultivate Hela cells in a 96-well plate, add PBS or IR808-ATIPA to the culture medium, and incubate in a CO 2 incubator for 4 hours, and expose to X-ray radiation (0 or 4 Gy). After incubating for another 48 hours, change the culture medium, wash with PBS, add DCFH-DA to the Hela cells, and incubate in a 37 °C cell incubator for 20 minutes. Then wash twice with PBS and observe the green fluorescence using a fluorescence microscope. Use untreated cells as the control group. The combined treatment of IR808-ATIPA and radiotherapy can further increase the ROS level in cells ( Figure 4 B).

[0039] (3) Cultivate Hela cells in a T25 cell culture flask, add PBS or IR808-ATIPA to the culture medium, and incubate in a CO 2 incubator for 4 hours, then change the culture medium and perform radiotherapy (0 or 4 Gy), and continue to culture for 48 hours. Wash the cells with PBS, add 100 µL of PBS to each flask and use a cell scraper to collect the cell suspension, and centrifuge at 4 °C (3000 rpf, 5 min). After centrifugation, discard the supernatant, add a mixture of RIPA lysis buffer and PMSF (99:1), and mix the cells well. Place the cell suspension on ice and shake for 30 minutes. Centrifuge at 4 °C (12000 rcf, 30 min), and retain the supernatant after centrifugation. Quantify the protein concentration using a BCA protein assay kit. Load each sample onto a precast PAGE gel, electrophoretically separate the target protein, transfer the protein to a polyvinylidene fluoride membrane, block with a rapid blocking solution for about 20 minutes, and incubate with primary antibodies at 4 °C overnight. The primary antibodies used here are rabbit anti-ACSL4, rabbit anti-GPX4, and rabbit anti-β-tubulin. Subsequently, wash the membrane three times in TBST for 10 minutes each time, and incubate with an HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody at 25 °C for 1 hour. Observe using a chemiluminescence detection kit. In the combined treatment of IR808-ATIPA and radiotherapy, the decrease in GPX4 is more obvious than in the radiotherapy alone group. At the same time, the combined treatment of radiotherapy and IR808-ATIPA increases the level of ACSL4 ( Figure 4 C).

[0040] (4) Hela cells cultured in a T25 cell culture flask, add PBS or IR808-ATIPA to the culture medium, and incubate in a CO 2After treatment in an incubator for 4 hours, the culture medium was replaced and irradiation (0 or 4 Gy) was performed, and the culture was continued for 48 hours. Hela cells were washed with PBS and used for GSH, GSH-PX and MDA measurement according to the manufacturer's instructions. GSH, GSH-PX and MDA contents were normalized to the quantitative total protein amount using a BCA protein assay kit. Untreated cells were used as a control group. Radiation therapy can reduce the levels of cellular GSH and GSH-PX, while the level of MDA increases. It was found that the increase of IR808-ATIPA increased the difference between the groups ( Figure 4 DF).

[0041] (5) Hela cells cultured in T25 flasks were incubated with PBS or IR808-ATIPA in CO 2 Treat in the incubator for 4 hours, then replace the culture medium and perform radiation irradiation (0 or 4 Gy), and continue to culture for 48 hours. Wash Hela cells with PBS and add trypsin for digestion. After trypsin digestion, add culture medium to terminate digestion, transfer to a centrifuge tube, centrifuge at 4°C (3000 rpf, 5 min), remove the supernatant, add electron microscopy fixative, blow off the cell clusters and resuspend. Fix at room temperature in the dark for 30 minutes, transfer to 4°C for storage, and observe under an electron microscope. After 72 hours of irradiation, mitochondria in the cells showed ferroptosis-like changes, such as reduced mitochondrial volume, rounding, enhanced HRTEM contrast, mitochondrial membrane rupture, increased outer membrane density, and a significant decrease in the number of mitochondrial cristae ( Figure 4 G).

[0042] Example 5 The purpose of this example is to verify the CT imaging effect of IR808-ATIPA.

[0043] The CT values ​​of IR808-ATIPA at different concentrations were measured by small animal micro-CT (PerkinElmer, QuantumGX2). IR808-ATIPA with gradient concentrations was divided into EP tubes, scanned by micro-CT, the CT values ​​were measured, and the curve of drug concentration and CT was calculated. As the concentration of IR808-ATIPA increased, the CT value increased significantly and showed a clear linear relationship with the iodine concentration ( Figure 5 A).

[0044] After subcutaneous tumor formation in mice, 50 µL of IR808-ATIPA was injected into the tumor. CT imaging was performed before injection and at 3 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after injection, and the region of interest (ROI) was outlined to measure and record the change in CT value within the tumor. Immediately after drug injection, the CT value within the tumor increased unevenly. As time passed, the drug gradually distributed evenly, and the CT value of the tumor tissue was still significantly higher than that before injection. The measurement results showed that the CT value reached the highest at 3 minutes after injection and then gradually decreased. Enhancement of the tumor could still be observed 4 hours later, and the CT value was higher than that of the surrounding tissues ( Figure 5 B).

[0045] Example 6 The purpose of this example is to verify the effect of IR808-ATIPA in radiotherapy.

[0046] When the tumor size reached approximately 100 mm 3 ³, 24 BALB / C nude mice were randomly divided into 4 groups and treated on day 0: Group 1 was injected with 50 µL of PBS into the tumor; Group 2 was injected with 50 µL of IR808-ATIPA into the tumor; Group 3 was injected with 50 µL of PBS into the tumor and received 4 Gy of X-ray irradiation; Group 4 was injected with 50 µL of IR808-ATIPA into the tumor and received 4 Gy of X-ray irradiation. When the tumor size reached 15 mm in any dimension, the in vivo treatment experiment was terminated, and all mice were euthanized. During radiotherapy, it was observed that the body weight of the nude mice hardly decreased significantly ( Figure 6 A), and the tumors injected with IR808-ATIPA and exposed to low-dose X-rays were almost completely inhibited after 15 days ( Figure 6 B-D).

[0047] The tumor tissues and major organs were collected and fixed with paraformaldehyde (4 wt%). HE staining and immunohistochemical staining of Ki67 and GPX4 were performed on the tumor tissues. HE staining showed that compared with other treatment groups, the tissue structure of the group treated with IR808-ATIPA and exposed to X-rays was loose, and the staining intensity was partially reduced. The results of immunohistochemical staining showed that Ki67 and GPX4 were downregulated ( Figure 6 E).

[0048] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A radiotherapy sensitizer IR808-ATIPA, characterized in that: The chemical structure of the sensitizer is shown below: 。 2. The radiosensitizer IR808-ATIPA according to claim 1, characterized in that: In the sensitizer, IR808 is covalently linked to the triiodophenyl group of ATIPA via an amide bond.

3. A method for preparing IR808-ATIPA according to claim 1, characterized in that: The following steps are involved: (a) Phenylhydrazine and 3-methyl-2-butanone are refluxed in acetic acid to generate intermediate 1; (b) Intermediate 1 is condensed with 6-bromohexanoic acid in acetonitrile to generate 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole bromide; (c) The product of step (b) is reacted with oxalyl chloride to generate an acyl chloride intermediate, which is then condensed with 5-amino-2,4,6-triiodoisophthalic acid to obtain a triiodophenyl-modified intermediate; (d) The product of step (c) is reacted with N-((E)-(2-chloro-3-((phenylimino)methyl)cyclohexenyl)aniline hydrochloride in ethanol to reflux, and IR808-ATIPA is obtained after purification.

4. The preparation method according to claim 2, characterized in that: The reaction temperature of step (a) is 110-120° C., and the reflux time is 6-10 hours; the purification of step (d) adopts an elution system with a volume ratio of dichloromethane to methanol of 50:

1.

5. The preparation method according to claim 2, characterized in that: The condensation reaction in step (c) was carried out at 80°C under argon protection for 5 hours.

6. Use of the IR808-ATIPA according to claim 1 in the preparation of a drug for tumor radiotherapy, wherein the drug is administered by intratumoral injection and combined with X-ray irradiation.

7. A use of the IR808-ATIPA according to claim 1 in CT imaging-guided radiotherapy, wherein after the IR808-ATIPA is injected into the tumor, the CT value change is monitored by micro-CT, the tumor target area is delineated in real time, and the radiotherapy dose is adjusted.

8. Use of the IR808-ATIPA according to 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.

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

Images