A ruthenium complex with dual activation ability of near-infrared light and X-rays, a preparation method thereof and application thereof in anti-tumor drugs

Abstract

This invention belongs to the fields of pharmaceutical chemistry and radiomedicine, specifically relating to a ruthenium complex with dual activation capabilities for near-infrared light and X-rays, its preparation method, and its application in antitumor drugs. The complex is characterized by the chemical structural formula [Ru(NO2-tpy)(Pyr-phen)Cl](PF6). The preparation method involves weighing the ligand p-Pyr-phen, the ruthenium precursor Ru(NO2-tpy)Cl3, and lithium chloride LiCl in an ethanol / water mixed solvent, and carrying out a reflux reaction in the presence of triethylamine. After the reaction is complete, the mixture is cooled to room temperature, and then an aqueous solution of ammonium hexafluorophosphate is added. After stirring, a precipitate is formed, collected, and washed sequentially with water and ethanol to remove soluble inorganic salts and polar impurities. The ruthenium complex is obtained after washing and alumina column chromatography. The advantages include a narrower bandgap, stronger near-infrared absorption, and direct X-ray activation capability, enabling dose-dependent generation of singlet oxygen.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical chemistry and radiation medicine, specifically relating to a ruthenium complex with dual activation capabilities for near-infrared light and X-rays, its preparation method, and its application in antitumor drugs. Background Technology

[0002] Photodynamic therapy (PDT) has attracted much attention in cancer treatment due to its high spatiotemporal selectivity. However, traditional photosensitizers mainly rely on visible light excitation, and their limited tissue penetration depth (usually <5 mm) severely restricts their application in deep or solid tumors. To overcome this bottleneck, extending the absorption spectrum of photosensitizers to the near-infrared (NIR) window (especially 650-900 nm) has become a key research direction.

[0003] Existing research indicates that modifying the ligands of ruthenium(II) polypyridine complexes using a donor-acceptor (DA) engineering strategy is an effective way to regulate their photophysical properties and achieve near-infrared absorption. Typical DA-type ruthenium complexes usually contain an electron donor and an electron acceptor, bridged by a metal center. For example, in the [Ru(acceptor-bipyridine)(donator-phenanthroline)Cl]... + In complexes with a basic framework, the near-infrared absorption performance mainly originates from intramolecular charge transfer between the strongly electron-donating groups on the donor ligand and the strongly electron-withdrawing groups on the acceptor ligand. When the donor is a moderately strong electron-donating group (such as methoxy, OCH3), although the HOMOLUMO band gap can be reduced to some extent, resulting in a redshift in the absorption spectrum, the degree of band gap compression and the efficiency of NIR light capture still need to be significantly improved.

[0004] More importantly, existing DA-type ruthenium complexes, regardless of their near-infrared absorption performance optimization, lack the ability to directly respond to highly penetrating X-rays. This prevents them from being directly integrated with widely used clinical radiotherapy equipment (X-rays), thus hindering the effective treatment of deep tumors. Therefore, developing a single-molecule system that can directly and efficiently respond to X-rays while possessing excellent near-infrared photoactivity to achieve true dual-mode treatment of deep tumors has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a ruthenium(II) complex with a narrower bandgap, stronger near-infrared absorption and X-ray direct activation capability, capable of dose-dependently generating singlet oxygen, and having dual activation capabilities of near-infrared light and X-rays, as well as its preparation method and its application in anti-tumor drugs. When used in combination with X-rays, the complex can synergistically increase the intracellular ROS level and significantly induce tumor cell apoptosis, resulting in significant synergistic effects.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: One objective of this invention is to provide a ruthenium complex with dual activation capabilities for near-infrared light and X-rays, the structural formula of which is shown below: .

[0007] The second objective of this invention is to provide a method for preparing ruthenium complexes with dual activation capabilities for near-infrared light and X-rays, comprising the following steps: Ligand p-Pyr-phen, ruthenium precursor Ru(NO2-tpy)Cl3, and lithium chloride LiCl were weighed and placed in an ethanol / water mixed solvent. The reaction was carried out under reflux in the presence of triethylamine. After the reaction was completed, the mixture was cooled to room temperature, and then an aqueous solution of ammonium hexafluorophosphate (NH4PF6) was added. After stirring, a precipitate formed. The precipitate was collected and washed successively with water and ethanol to remove soluble inorganic salts and polar impurities. A solution of acetonitrile and toluene in a 1:1 volume ratio was used as the eluent. After washing and alumina column chromatography, a ruthenium complex with dual activation capabilities for near-infrared and X-ray light was obtained, the structure of which is shown below: Pyr-phen is 4-(1-pyrrolidinyl)-1,10-phenanthroline, and NO2-tpy is 4'-(4-nitrophenyl)-2,2':6',2''-terpyridine.

[0008] Further, the molar ratio of Pyr-phen, Ru(NO2-tpy)Cl3, lithium chloride, and ammonium hexafluorophosphate is 0.4-0.6:0.4-0.6:1.8-2.2:1.2-1.4. Preferably, the molar ratio of Pyr-phen, Ru(NO2-tpy)Cl3, lithium chloride, and ammonium hexafluorophosphate is 0.5:0.5:2:1.3. Further, the volume ratio of ethanol to water in the alcohol / water mixed solvent is 2-4:1. Preferably, the volume ratio of ethanol to water in the alcohol / water mixed solvent is 3:1. The present invention also provides the use of the above-mentioned ruthenium complex in the preparation of antitumor drugs, wherein the tumor includes, but is not limited to, colorectal cancer.

[0009] The present invention also provides the application of the above-mentioned ruthenium complex in the preparation of X-ray sensitizers.

[0010] The present invention also provides the application of the above-mentioned ruthenium complex in the preparation of photodynamic therapy agents, wherein the application is to activate the complex by irradiation with light of wavelength above 660 nm.

[0011] The present invention also provides the use of the above-mentioned ruthenium complex in the preparation of radiodynamic therapy sensitizers or radiotherapy sensitizers.

[0012] Furthermore, the radiodynamic therapy or radiotherapy is performed in conjunction with X-rays. Under X-ray irradiation, singlet oxygen can be generated in a dose-dependent manner.

[0013] The present invention also provides an antitumor pharmaceutical composition comprising an effective amount of the ruthenium complex of claim 1 and a pharmaceutically acceptable carrier.

[0014] Compared with the prior art, the advantages of the present invention are as follows: I. Superior molecular design, possessing highly efficient dual-mode activation properties 1. Narrowing of band gap and enhanced absorption: Through strong donor-acceptor engineering, the HOMO-LUMO band gap of the complex is reduced to 2.28 eV, resulting in a redshift of about 25 nm in the metal-to-ligand charge transfer (MLCT) absorption band, which significantly improves its light-harvesting ability in the near-infrared (NIR) region.

[0015] 2. High intersystem crossing efficiency: This molecular structure promotes charge separation and intersystem crossing, facilitating the efficient generation of singlet oxygen (…). 1 O2) laid the foundation.

[0016] 3. Unique Dual-Mode Activation Capabilities: 1) NIR Photoactivation: Activated by NIR light with weak penetration, suitable for precise superficial treatment (such as skin and mucous membrane lesions). 2) Direct X-ray Activation: Activated by highly penetrating clinical X-rays, breaking through the limitation of traditional photosensitizers being only applicable to superficial areas, and achieving effective treatment of deep tumors. This represents a paradigm shift from "single photoactivation" to "photo / X-ray dual activation," broadening the scope of therapeutic applications.

[0017] II. Dual Mechanism of Action, Synergistically Enhancing Therapeutic Effect 1. Dual mechanisms for generating reactive oxygen species (ROS): Type I mechanism: Efficiently catalyzes the oxidation of biomolecules (such as NADH), exerting a catalytic oxidative damage effect. Type II mechanism: Efficiently generates singlet oxygen (…). 1 O2) directly kills tumor cells.

[0018] The dual mechanisms work synergistically to improve ROS generation efficiency and anti-tumor reliability.

[0019] 2. Clear radiosensitizing effect: Under X-ray irradiation, the complex can generate singlet oxygen in a dose-dependent manner, exhibiting a significant radiodynamic effect. Cellular and molecular level experiments have confirmed that it can effectively enhance the effect of radiotherapy and possesses excellent radiosensitizing properties.

[0020] III. Significant in vitro antitumor effects 1. It has significant toxicity to colorectal cancer cells.

[0021] 2. Synergistic effect when used in combination with X-rays: It can significantly increase intracellular ROS levels. It increases the apoptosis rate from about 11% with radiotherapy or drug administration alone to more than 30%, demonstrating a significant synergistic anti-tumor effect.

[0022] IV. Flexible Treatment Strategies and Clinical Application Potential 1. Combined use with radiotherapy (primary direction): During conventional tumor radiotherapy, this complex preparation is administered via intravenous or local injection. While X-rays irradiate the tumor, they simultaneously activate the complex enriched within the tumor, producing additional... 1 O2 kills tumor cells. Achieving synergy between radiotherapy and radiodynamic therapy is expected to reduce the required radiotherapy dose, overcome radiotherapy resistance, and improve treatment efficiency and safety.

[0023] 2. Combined with phototherapy: For skin cancer, oral cancer, or digestive / respiratory tract tumors that can be accessed endoscopically, NIR laser / LED irradiation can be used after local application of the compound to perform precise and minimally invasive photodynamic therapy.

[0024] 3. Flexible dosage form, easy to apply: It can be prepared into injection solutions and perfusion solutions (such as mixing with physiological saline / PBS) for the treatment of solid tumors or intracavitary tumors. It can also be further prepared into nano-formulations or liposomes to achieve targeted drug delivery, increase local tumor concentration and reduce systemic side effects.

[0025] V. Simple preparation and easy to transform and promote. The synthesis method of the complex provided by this invention is simple, reproducible, and easy to scale up for production, and has good prospects for industrialization and clinical application.

[0026] In summary, this invention presents a ruthenium complex with dual activation capabilities for near-infrared light and X-rays, its preparation method, and its application in antitumor drugs. Through innovative molecular design, this ruthenium complex achieves dual activation by NIR light and X-rays, and synergistic effects of both type I and type II mechanisms. Experiments have demonstrated its significant radiosensitizing effect and synergistic antitumor efficacy. Its dual-mode activation characteristics overcome the limitation of insufficient penetration depth in traditional photodynamic therapy, providing a novel integrated "photosensitizer / radiosensitizer" solution for deep tumor treatment, and possessing significant clinical translational value. Attached Figure Description

[0027] Figure 1 Methods for synthesizing coordination compounds; Figure 2 The X-ray diffraction (XRD) structure identification diagrams of the complexes are shown, where A is a wireframe structure, B is a single crystal structure, C is a single crystal structure without toluene (solvent), and D is a filled crystal structure. Figure 3The diagram shows the HOMO and LUMO orbital distributions and energy levels of the complex; Figure 4 A comparison of the UV-Vis absorption spectra of the complexes; Figure 5 The change in absorbance of the Ru(II)-NADH system under near-infrared light irradiation; Figure 6 Fluorescence changes of the Ru(II)-DPBF system under near-infrared light irradiation. Figure 7 Fluorescence changes of the Ru(II)-DPBF system under X-ray irradiation. Figure 8 IC for coordination compounds 50 Value curve graph; Figure 9 Representative images of HCT116 cells under different treatment conditions are compared: control (Ctrl), irradiation (4 Gy), 10 µM complex (complex), and combined treatment (complex+4 Gy). Figure 10 DCFH-DA staining and flow cytometry were used to detect intracellular ROS levels under different treatment conditions. Figure 11 Annexin V / 7-AAD staining and flow cytometry analysis were performed to detect apoptosis under different treatment conditions. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0029] Example 1: Synthesis method of the complex [Ru(NO2-tpy)(Pyr-phen)Cl] (PF6), as follows: Figure 1 The steps are as follows: Ligand p-Pyr-phen (0.5 mmol, Pyr-phen being 1-pyrrolidinyl)-1,10-phenanthroline), ruthenium precursor Ru(NO2-tpy)Cl3 (0.5 mmol, NO2-tpy being 4'-(4-nitrophenyl)-2,2':6',2''-terpyridine), and lithium chloride LiCl (2.0 mmol) were weighed and placed in an ethanol / water mixture (50 mL, v / v 3:1). Three drops of triethylamine were added, and the mixture was refluxed for 12 h. After cooling to room temperature, an aqueous solution of NH4PF6 (1.3 mmol) was added, and the mixture was stirred for 10 min, resulting in precipitation. The precipitate was filtered, collected, and washed successively with water and ethanol to remove soluble inorganic salts and polar impurities. The precipitate was purified by alumina column chromatography (eluent composed of acetonitrile and toluene in a 1:1 v / v ratio) to obtain the target complex, the structure of which is shown below: .

[0030] The proton and mass spectrometry analyses of the complex are as follows: 1 H-NMR (500 MHz, CD3CN): δ 9.79 (d, J = 6.5Hz, 1H, Ar-H), 9.21 (s, 2H, Ar-H), 8.63 (d, J = 8.1 Hz, 2H, Ar-H), 8.45 (d, J= 9.7 Hz, 1H, Ar-H), 8.22 (d, J = 9.8 Hz, 1H, Ar-H), 7.93 (t, J = 7.9 Hz, 2H,Ar-H), 7.73 (d, J = 5.6 Hz, 1H, Ar-H), 7.35 (t, J = 6.1 Hz, 2H, Ar-H), 7.35(d, J = 6.5 Hz, 1H, Ar-H), 6.58 (d, J = 6.6 Hz, 1H, Ar-H), 6.12 (d, J = 6.7Hz, 1H, Ar-H), 4.05 (t, J = 6.1 Hz, 4H, 4 × -CH2), 3.64 (t, J = 6.4 Hz, 4H,4 × -CH2) and 2.53 (pentet, J = 6.9 Hz, 8H, 4 × -CH2) ppm. 13 C-NMR (125 MHz, CD3CN): δ 160.85, 158.78, 152.85, 152.19, 151.52, 150.31, 149.47, 149.20,148.28, 147.03, 137.23, 128.32, 124.42, 121.87, 121.53, 121.31, 121.21,116.12, 107.78, 106.80, 53.14, 52.78, 26.35 and 25.99 ppm. MS (MALDI-TOF) m / z: [M-PF6]+ calcd for C 35 H 22 ClN8O2Ru, 733.14; found, 733.11. Among them, Figure 2 The X-ray diffraction (XRD) structures of the complexes are shown, where A is the wireframe structure, B is the single crystal structure, C is the single crystal structure without toluene (solvent), and D is the filled crystal structure.

[0031] Example 2: Theoretical calculations and photophysical properties of the coordination compound.

[0032] 1. Theoretical calculations for coordination compounds: (1) Software and Model: The molecular model of the coordination compound was constructed using the Gaussian16 quantum chemistry calculation software package.

[0033] (2) Calculation method: Ground state calculation: Density functional theory (DFT) was used, employing the B3LYP hybrid functional.

[0034] Basis set selection: For C, H, O, and N atoms, the 6-31G(d) basis set is used; for the heavy metal Ru atom, the LANL2DZ basis set is used, taking into account the relativistically effective nuclear potential.

[0035] Solvent effect: The aqueous environment was simulated using a polarized continuum model (PCM).

[0036] Excited state calculation: Time-dependent density functional theory (TD-DFT) is used to calculate the electronic excitation process.

[0037] (3) Analysis content: Calculate the energy and spatial distribution (plot) of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), as well as the energy and intensity of the excited state.

[0038] (4) Experimental measurement method: Accurately weigh the solid powder of the complex and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 10.0 μM solution. The test was performed using a UV-Vis spectrophotometer. Scan range: 300 nm to 800 nm. Interval: Record one data point every 2 nm. Background correction: Use pure DMSO solvent as a blank for background subtraction.

[0039] (5) Results and Data Analysis: like Figure 3 As shown, density functional theory (DFT) calculations indicate that the HOMO (-5.54 eV) of this complex is mainly distributed in the pyrrolidine-modified phenanthroline ligand and the RuCl axis region, while the LUMO (-3.26 eV) is highly concentrated in the NO2 terpyridine ligand, exhibiting significant donor-acceptor spatial separation. Its HOMO-LUMO band gap is 2.28 eV. This narrow band gap facilitates electron transitions and charge transfer in the visible and near-infrared regions, forming a crucial structural basis for the complex's efficient light-harvesting capability. This significant band gap compression is directly attributed to the electronic structure optimization resulting from the introduction of a strong electron-donating pyrrolidine group.

[0040] 2. Photophysical properties like Figure 4As shown, the UV-Vis absorption spectrum of this complex reveals a sharp, strong absorption peak in the high-energy region of 250–350 nm, attributed to the π→π* transition (LC absorption band) of the ligand itself. In the critical low-to-medium energy region of 400–700 nm, a broad and strong metal-to-ligand charge transfer (MLCT) absorption band is observed.

[0041] The MLCT absorption band exhibits a significant redshift, effectively extending into the near-infrared (NIR) window (>660 nm). This redshift directly confirms the narrowing of its HOMO-LUMO band gap. Simultaneously, the significantly enhanced absorption intensity in this region indicates higher electron transfer efficiency from the Ru center and donor ligand (HOMO region) to the acceptor ligand (LUMO region). These spectral characteristics suggest that this complex can more effectively absorb photons with longer wavelengths and lower energies. Since near-infrared light has stronger penetrating power into human tissues, this characteristic provides an important photophysical basis for its efficient photodynamic therapy in deep tissues.

[0042] In summary, theoretical calculations and experimental spectra corroborate each other. The ruthenium(II) complex provided in this invention was designed using a donor-acceptor (DA) engineering strategy. By introducing a strong electron-donating pyrrolidinyl group into the complex, its HOMO energy level was successfully raised further, and it was spatially separated from the LUMO, thereby narrowing the optical band gap. This directly leads to a redshift and enhancement of its absorption spectrum, making it an excellent near-infrared light trap, thus enabling photodynamic therapy in deep tissues.

[0043] Example 3: Photocatalytic and X-ray activation properties of the complex.

[0044] Quantitative assessment of the complex's ability to produce two key active substances under irradiation from two different energy sources (near-infrared light and X-rays): 1) exhibiting type I activity through the oxidation of the biomolecule nicotinamide adenine dinucleotide (NADH); 2) through the generation of singlet oxygen (… 1 O2) showed type II activity.

[0045] 1. Near-infrared (NIR) driven experiment: (1) Type I activity (NADH photo-oxidation experiment): Reaction system: Mix 10.0 μM of the complex and 240 μM of NADH in phosphate-buffered saline (PBS).

[0046] Irradiation conditions: A 680nm laser was used as the light source, with an irradiation power density of 3mW / cm². 2 .

[0047] Monitoring method: Using a UV-Vis spectrophotometer, the absorbance of the reaction solution at 339 nm was measured at regular intervals. 339 nm is the characteristic absorption peak of NADH, and its decrease indicates that NADH has been oxidized and consumed.

[0048] Data processing: Calculate the turnover frequency (TOF, unit: h) based on the initial rate of absorbance decrease. -1 This refers to how many substrate molecules each catalyst molecule can convert per hour.

[0049] like Figure 5 As shown, under 680 nm illumination, this complex effectively catalyzes the oxidation of NADH, as evidenced by a decrease in absorbance at 339 nm, and its time-of-flight (TOF) is calculated to be 8.0 h⁻¹. -1 It exhibits highly efficient type I photocatalytic activity.

[0050] (2) Type II reactivity: singlet oxygen ( 1 O2 generation experiment: Reaction system: In DMSO, a mixture of 10.0 μM of the complex and 10.0 μM of 1,3-diphenylisobenzofuran (DPBF) was prepared. DPBF reacts with... 1 The specific reaction with O2 causes its fluorescence or color to fade.

[0051] Irradiation conditions: 680nm light source, power density of 5mW / cm² 2 Irradiate for 10 minutes.

[0052] Detection method: The decay of DPBF fluorescence intensity at 458 nm was monitored using a fluorescence spectrometer, and the changes in its absorption peak were monitored using ultraviolet-visible spectroscopy.

[0053] Data processing: Plot the irradiation time on the x-axis and the ratio of fluorescence intensity or absorbance (I0 / I) on the y-axis. The slope of the curve reflects... 1 The rate of O2 generation.

[0054] like Figure 6 As shown, the complex can generate singlet oxygen via a type II mechanism ( 1 O2) causes DPBF fluorescence quenching. Its quenching rate constant is as high as 18.0 × 10⁻⁶. -2 .

[0055] In summary, this complex exhibits excellent photocatalytic activity. Its narrower band gap and optimized charge separation capability enable it to efficiently drive type I and type II photochemical reactions under NIR light.

[0056] 2. X-ray driven experiment: Irradiation source: Use of medical or research X-ray radiation sources.

[0057] Experimental design: A series of different radiation dose groups were set up: 0, 3, 6, 9, 12 Gy (Gy, the unit of absorbed radiation dose). Test Procedure: Solutions containing the complex and either NADH or DPBF were irradiated with different doses of X-rays as described above. Immediately after irradiation, the remaining amount of NADH or the consumption of DPBF was measured using the same method as in the NIR experiment. The specific experimental procedure was the same as in the near-infrared (NIR) driven experiment.

[0058] X-ray activation results analysis: X-ray irradiation could not effectively drive the oxidation of NADH in the complex, and there was no significant difference between the dose groups and the 0 Gy group. This indicates that the energy mode of X-ray photons does not match the electronic transitions required for the redox reaction catalyzed by the complex. However, as... Figure 7 As shown, X-ray irradiation can dose-dependently activate complex 2 to produce 1 O2: As the X-ray dose increased from 0 Gy to 12 Gy, the consumption of DPBF increased significantly and continuously. This indicates that the complex possesses a unique X-ray sensitization ability not found in other DA-type complexes. Although it cannot be used to catalyze redox reactions, its electronic structure can effectively absorb X-ray energy and transfer it to surrounding oxygen molecules, generating cytotoxic... 1 O2. This reveals a completely new activation mode—radiodynamic therapy (RDT).

[0059] In summary, this complex is a dual-activated photosensitizer. Under near-infrared light (680 nm) irradiation, it is a highly efficient "photocatalyst" and "photodynamic agent," capable of efficiently catalyzing the oxidation of NADH with a turnover rate of up to 8.0 h. -1 It can efficiently generate singlet oxygen. Under X-ray irradiation, it transforms into a highly efficient "radiosensitizer," and its complexes also exhibit dose-dependent singlet oxygen generation capabilities, indicating that it possesses a radiodynamic effect directly activated by X-rays. This characteristic expands its application from traditional superficial phototherapy to combined radiodynamic therapy for treating deep-seated tumors.

[0060] Example 4: In vitro antitumor activity of the complex In the human colorectal cancer HCT116 cell line, the antitumor effects of the complex alone and in combination with X-rays were verified, and the possible mechanisms of its cell-killing effect were explored. Details are as follows: Cell line: Human colorectal cancer cells HCT116.

[0061] Culture medium: High glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) was used.

[0062] Culture conditions: Cultured in a constant temperature and humidity incubator at 37℃ and 5% CO2.

[0063] 1. Cytotoxicity assay (CCK-8 assay): Plate seeding: HCT116 cells were seeded into 96-well plates at a density of 5000 cells per well and cultured overnight to allow the cells to adhere.

[0064] Drug administration: Prepare a series of complex solutions (from 0.5 μM to 100 μM) with fresh culture medium, add them to cell wells, and return the cells to the incubator for 24 hours. Each concentration has 5 replicate wells.

[0065] Detection: Add 100 μL of Cell Counting Kit-8 (CCK-8; Proteintech, Wuhan, China) reagent to each well and continue incubation for a specified time (according to the instructions). The CCK-8 reagent will be reduced by enzymes in living cells to produce orange-yellow formazan dye.

[0066] Readings: The absorbance of each well was measured at 450 nm using an iMark microplate reader (Bio-Rad). The absorbance value is directly proportional to the number of live cells.

[0067] Calculations: Data were processed using GraphPad Prism 10 software to plot cell viability-drug concentration curves and calculate the half-inhibitory concentration (IC50). 50 The optimal concentration of a drug is the concentration required to kill 50% of the cells.

[0068] Cytotoxicity test results as follows Figure 8 As shown, the complex, when acting alone, can kill HCT116 cells in a dose-dependent manner, and its IC50 value was calculated. 50 The value was 13.38 μM (i.e., the half-maximal inhibitory concentration of the drug was 13.38 µM).

[0069] 2. Combined treatment trial: Seeding: HCT116 cells were seeded into 6-well plates at a density of 500,000 per well and cultured overnight until the cells adhered.

[0070] Grouping: Four experimental groups were set up: ① blank control; ② X-ray irradiation only (4 Gy); ③ drug treatment only (10 μM complex, 6 hours); ④ combined group (10 μM complex treatment for 6 hours, followed by 4 Gy irradiation). After cells adhered for 24 hours, the above group treatments were performed.

[0071] Irradiation conditions: A linear accelerator (Siemens Healthineers AG, Erlangen, Germany) was used to generate a 6MV photon beam with a source-to-skin distance (SSD) of 100cm, delivering a single irradiation dose of 4Gy.

[0072] Cell morphology observation and recording: After treatment, the morphological changes of each group of cells were observed and photographed using a SOPTOPICX41 inverted microscope (10x objective).

[0073] The results are as follows Figure 9 As shown, the morphological effects of the combined treatment are as follows: Under the microscope, the number of cells in the combined treatment group decreased sharply, and the remaining cells became round, shrunken, and floated, showing strong signs of growth inhibition and death.

[0074] 3. Detection of intracellular reactive oxygen species (ROS): Staining: Collect cells from the above groups, discard the old culture medium, and add fresh serum-free culture medium containing 10 μM of the fluorescent probe 2',7'-dichlorodihydrofluorescein (DCFH-DA, Beyotime, Shanghai, China) for flow cytometry. Incubate at 37°C in the dark for 30 minutes. DCFH-DA itself is non-fluorescent; after entering the cells, it is hydrolyzed by esterases and oxidized by ROS to generate DCF with strong green fluorescence.

[0075] Detection: Cells were collected by trypsin digestion, and the fluorescence intensity of each cell was detected using a CytoFLEXS flow cytometer (Beckman Coulter). The stronger the fluorescence, the higher the intracellular ROS level.

[0076] Flow cytometry results as follows Figure 10 As shown, the combined treatment group had the highest intracellular fluorescence signal (representing ROS), which was significantly higher than that of the drug-only group or the radiotherapy-only group, indicating that the combined treatment produced strong oxidative stress.

[0077] 4. Apoptosis detection: Staining: Cells from each group were collected and stained at room temperature for 15 min using the Annexin V-PE / 7-AAD apoptosis detection kit (Cat; 62700-80, BioGems, CA, USA). Annexin V-PE binds to phosphatidylserine residues that are everted in apoptotic cells. 7-AAD is a nucleic acid dye that cannot enter live cells and early apoptotic cells, but can enter dead cells and late apoptotic cells.

[0078] Detection and Analysis: Cells were detected using a CytoFLEXS flow cytometer. Data were analyzed using FlowJo 10.8 software, and cells were divided into: viable cells (double-negative) and early apoptotic cells (Annexin V). + / 7-AAD - ), late apoptosis / necrosis (Annexin V) + / 7-AAD + The total apoptosis rate was the sum of the early and late apoptosis rates.

[0079] likeFigure 11 As shown, the baseline apoptosis rate was observed in the blank control group and the X-ray-only (4 Gy) group.

[0080] In the single complex 2 (10 μM) group, the apoptosis rate was 11.64%. In the combination therapy group (drug + X-ray), the apoptosis rate increased sharply to 31.7%. Conclusion: The apoptosis rate induced by the combination therapy was much higher than the sum of the effects of the two single therapies, showing a significant synergistic enhancement effect.

[0081] In summary, CCK-8 assay showed that the complex had an effect on the IC50 of HCT116 cells. 50 The concentration was 13.38 μM. When used in combination with X-rays (4 Gy), cell viability was significantly reduced, intracellular ROS levels were significantly increased, and the apoptosis rate increased to 31.7%, which was significantly higher than that of the drug-only or radiotherapy-only groups.

[0082] At the cellular level, the complex provided by this invention is not only cytotoxic itself but also a potent radiosensitizer. When used in conjunction with clinical X-rays, it synergistically induces a burst of intracellular ROS accumulation, thereby synergistically inducing massive apoptosis in cancer cells. This provides solid in vitro experimental evidence for developing the complex into a novel tumor radiosensitizer.

[0083] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A ruthenium complex with dual activation capabilities for near-infrared light and X-rays, characterized in that: The structural formula of the ruthenium complex is shown below: 。 2. A method for preparing the ruthenium complex with dual activation capabilities for near-infrared light and X-rays as described in claim 1, characterized in that... Includes the following steps: Ligand p-Pyr-phen, ruthenium precursor Ru(NO2-tpy)Cl3, and lithium chloride LiCl were weighed and placed in an ethanol / water mixed solvent. The reaction was carried out under reflux in the presence of triethylamine. After the reaction was completed, the mixture was cooled to room temperature, and then an aqueous solution of ammonium hexafluorophosphate was added. After stirring, a precipitate formed. The precipitate was collected and washed successively with water and ethanol to remove soluble inorganic salts and polar impurities. A solution of acetonitrile and toluene in a 1:1 volume ratio was used as the eluent. After washing and alumina column chromatography, a ruthenium complex with dual activation capabilities for near-infrared and X-ray light was obtained, the structure of which is shown below: Pyr-phen is 4-(1-pyrrolidinyl)-1,10-phenanthroline, and NO2-tpy is 4'-(4-nitrophenyl)-2,2':6',2''-terpyridine.

3. The preparation method according to claim 2, characterized in that: The molar ratio of Pyr-phen, Ru(NO2-tpy)Cl3, lithium chloride, and ammonium hexafluorophosphate is 0.4-0.6:0.4-0.6:1.8-2.2:1.2-1.

4.

4. The preparation method according to claim 2, characterized in that: The volume ratio of ethanol to water in the alcohol / water mixed solvent is 2-4:

1.

5. The use of the ruthenium complex according to claim 1 in the preparation of antitumor drugs.

6. The use of the ruthenium complex according to claim 1 in the preparation of photodynamic therapy agents, characterized in that: The application involves activating the complex by irradiating it with light of wavelengths above 660 nm.

7. The use of the ruthenium complex according to claim 1 in the preparation of X-ray sensitizers.

8. The use of the ruthenium complex according to claim 1 in the preparation of radiodynamic therapy sensitizers or radiotherapy sensitizers.

9. The application according to claim 8, characterized in that: The radiodynamic therapy or radiotherapy is performed in combination with X-rays.

10. An antitumor drug composition, characterized in that... The mixture comprises an effective amount of the ruthenium complex of claim 1 and a pharmaceutically acceptable carrier.

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