An icd inducer ru(ii) complex with the ability of photodynamic DNA cleavage, and a preparation method and application thereof

Abstract

The application discloses an ICD inducer Ru(II) complex with a light-driven DNA breaking ability, a preparation method and application thereof, relates to the field of antitumor drugs, and the structural formula of the Ru(II) complex is [Ru(dip)2(L)](PF6)2. The Ru(II) complex can enter a cell nucleus to break DNA and activate immunogenic death mediated by the pyroptosis and cGAS-STING pathways by using photodynamic therapy, so that the antitumor immune effect of the body is improved. In the in-vivo experiment, the immune cells in the tumor microenvironment of the mice in the vaccine group are good, and the antitumor immune effect of the three injections of the vaccine is better. The results of H&E staining show that the vaccine does not cause obvious pathological damage to the main organs of the mice, and the vaccine has good biological safety.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, and particularly relates to an ICD inducer Ru(II) complex with photodynamic DNA breaking ability, its preparation method and application. Background Technology

[0002] Photodynamic therapy (PDT), a non-invasive cancer treatment, works by activating photosensitizers with light energy. Through the synergistic effect of light energy, oxygen, and photosensitizers (PSs), a large amount of reactive oxygen species (ROS) are generated within tumor cells. These ROS interact with intracellular proteins and other biomolecules, targeting multiple key sites such as mitochondria, endoplasmic reticulum, and the nucleus, disrupting the normal function and organelle structure of tumor cells, thereby inducing tumor cell death. Compared to traditional cancer treatments such as surgery, chemotherapy, and radiotherapy, PDT offers significant advantages, including less physiological trauma, lower toxicity, and greater spatiotemporal precision. These advantages make PDT play a positive role in improving treatment efficacy and reducing adverse reactions, and it has received increasing attention in recent years, gradually becoming a cutting-edge field in cancer research.

[0003] Pyroptosis is a novel form of cell death that relies on the formation of pores in the cell membrane by the Gasdermin protein family, simultaneously stimulating the formation of inflammasomes. In the tumor cell environment, pyroptosis can alter the tumor microenvironment by releasing specific inflammatory mediators and cellular contents, making it easier for the immune system to recognize and eliminate these altered cancer cells. PDT can induce pyroptosis in HeLa cells, thereby enhancing their sensitivity to the immune system. This process enhances the inflammatory response by leading to the release of immunostimulatory factors and damage-associated molecular patterns (DAMPs) through endoplasmic reticulum stress, while also facilitating the activation of innate immune responses and enhancing immunogenic death (ICD).

[0004] ICD (Internal Catheterization Discharge) refers to the release of specific molecules and DAMPs during the cancer cell death process induced by certain chemotherapy drugs, physiochemotherapy, photodynamic therapy, and radiotherapy. One important molecule is calreticulin (CRT), which is normally located in the endoplasmic reticulum but migrates to the cell membrane surface during ICD. This change is considered an important "signaling" process; its extracellular exposure helps attract antigen-presenting cells. High-mobility group box 1 (HMGB1) is released extracellularly as a "danger signal" to trigger an immune cell response. ATP is also released extracellularly as a chemokine to attract immune cells and activate inflammasomes. These molecules are considered "danger signals" that can promote tumor-specific immune responses, thus directly participating in the elimination of cancer cells and enhancing the long-term effects of anti-cancer therapy by activating the immune system.

[0005] The genetic information and signal transduction factors carried by the cell nucleus play a crucial role in cell growth and proliferation, and are considered the primary targets of many anti-tumor drugs. Through photodynamic therapy, photosensitizers can enhance the induction of tumor cell death, thereby improving the body's ability to fight tumors.

[0006] Ru(II) complexes are considered as candidates for next-generation photosensitizers due to their excellent photochemical properties, tunable cellular uptake capacity, and low dark toxicity. However, existing ruthenium complexes still have the following limitations: (1) most remain at the stage of inducing apoptosis / necrosis; (2) lack clear nuclear-targeted DNA breakage ability; (3) the activation mechanism of immune pathways such as cGAS-STING is unclear; and (4) the in vivo anti-tumor immune effect lacks systematic verification. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes an ICD inducer Ru(II) complex with photodynamic DNA-breaking ability, its preparation method, and its applications. This Ru(II) complex exhibits antitumor immunomodulatory activity. The antitumor activity of this Ru(II) complex was studied, revealing that it possesses good antitumor activity against tumor cells, significant phototoxicity against HeLa cells, and the ability to induce pyroptosis in HeLa cells and enhance their immunogenic death through the cGAS-STING pathway.

[0008] To achieve the above objectives, the present invention provides an ICD inducer Ru(II) complex with photodynamic DNA fragmentation capability, having the following structure:

[0009] .

[0010] The present invention also provides a method for preparing the above-mentioned ICD inducer Ru(II) complex with photodynamic DNA fragmentation ability, comprising the following steps:

[0011] Under argon protection, Ru(dip)2Cl2 and ligand L were refluxed in an organic solvent to obtain Ru(II) complex;

[0012] The ligand L is 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide; dip is 4,7-diphenyl-1,10-phenanthroline.

[0013] Furthermore, the organic solvent is anhydrous ethanol.

[0014] Furthermore, the reflux reaction temperature is 70-80℃, and the reaction time is 1-3 hours.

[0015] Furthermore, after the reflux reaction is completed, the process further includes: adding an excess of saturated NH4PF6 solution, filtering and drying the precipitate, and purifying it.

[0016] The present invention also provides an antitumor pharmaceutical composition comprising the above-mentioned ICD inducer Ru(II) complex with photodynamic DNA-breaking ability and a pharmaceutically acceptable carrier.

[0017] Furthermore, the antitumor drug composition is a photodynamic therapy agent, and the antitumor drug is used to induce immunogenic death of tumor cells.

[0018] The present invention also provides the application of the above-mentioned ICD inducer Ru(II) complex with photodynamic DNA breaking ability in the preparation of antitumor drugs.

[0019] Furthermore, the antitumor drug is used to induce immunogenic death of tumor cells; the tumor is an in situ solid tumor.

[0020] Furthermore, the tumor cells are murine breast cancer cells or human cervical cancer cells.

[0021] This invention also provides the application of the above-mentioned ICD inducer Ru(II) complex with photodynamic DNA-breaking ability in the preparation of an antitumor vaccine, wherein the antitumor vaccine is prepared by the following steps:

[0022] (a) Co-incubating tumor cells with the ICD inducer Ru(II) complex;

[0023] (b) Irradiate with a light source with a wavelength of 400-500 nm;

[0024] (c) Wash and resuspend the cells to obtain an anti-tumor vaccine;

[0025] The tumor cells are murine breast cancer cells or human cervical cancer cells.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] This invention provides a ruthenium(II) complex with an o-phenanthroline derivative as the main ligand, exhibiting excellent antitumor immunomodulatory activity. Due to the presence of a metal ion, it carries an inherent charge, enhancing its in vivo penetration and retention effects compared to traditional small organic molecules. Furthermore, the metal complex possesses a multi-coordination configuration, allowing for modification with different ligands to achieve its superior biological activity.

[0028] The ruthenium(II) complex provided by this invention is not only a metallic ruthenium(II) complex with good antitumor activity, but also, under photodynamic therapy, ruthenium(II) complex Ru1 induces mitochondrial damage (such as decreased membrane potential and ROS burst) and endoplasmic reticulum stress (calcium ion disorder) through a cascade delivery mechanism from mitochondria and endoplasmic reticulum to the nucleus. Ultimately, it enters the nucleus, breaks DNA, and activates pyroptosis and cGAS-STING pathway-mediated immunogenic death, thereby enhancing the body's antitumor immune response. In in vivo experiments, the vaccine group showed good efficacy against immune cells in the tumor microenvironment of mice, and the three-dose vaccine showed even better antitumor immune efficacy. H&E staining results showed that the vaccine did not cause significant pathological damage in the major organs of mice, confirming its good biocompatibility. Attached Figure Description

[0029] Figure 1 This is a diagram illustrating the effect of complex Ru1 entering the HeLa cell nucleus in Example 3.

[0030] Figure 2 The image shows the effect of complex Ru1 photolyzing DNA in Example 4; where (A) is a DNA gel electrophoresis image of Ru1 under dark conditions; and (B) is a DNA gel electrophoresis image of Ru1 under light conditions.

[0031] Figure 3 The diagram shows the effect of complex Ru1 on the cell cycle of HeLa cells in Example 5; (A) flow cytometry analysis results of complex Ru1 on the cell cycle distribution of HeLa cells; (B) bar chart of flow cytometry data processing; (C) Western blotting analysis of complex Ru1 on HeLa cell cycle-related proteins.

[0032] Figure 4The image shows the effect of complex Ru1 in inducing γ-H2AX expression in HeLa cells in Example 6; where (A) is a confocal image of Ru1 without the addition of antioxidant NAC; (B) is a confocal image of Ru1 with the addition of antioxidant NAC.

[0033] Figure 5 This is a diagram showing the effect of complex Ru1 in Example 7 on the expression of cGAS-STING pathway-related proteins in HeLa cells under light irradiation;

[0034] Figure 6 This image shows the immunogenicity of subcutaneously implanted tumors in female BALB / c mice after inoculation with 4T1 cells treated with the complex Ru1 in Example 8. (A) Statistical graph of mouse body weight change; (B) Statistical graph of mouse tumor volume change; (C) Solid image of mouse tumor size on day 30 after 4T1 cell injection; (D) H&E staining of sections of major mouse organs. Note: Cisplatin was used as a control drug. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0037] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0038] Example 1: Preparation of Ru1, an ICD inducer Ru(II) complex with photodynamic DNA fragmentation ability.

[0039] Under argon protection, Ru(dip)₂Cl₂·₂H₂O (0.33 g, 0.4 mmol) and ligand L (0.22 g, 0.4 mmol) were placed in anhydrous ethanol and refluxed at 78 °C for 2 hours. After the reaction was completed, most of the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography using dichloromethane and methanol in a ratio of 10:1, v / v. The yellow main band was collected, dried, and 0.24 g of yellow powder Ru₁ was obtained, with a yield of 36.9%.

[0040] The reaction equation is as follows:

[0041]

[0042] The structural formula of Ru(II) complex Ru1 is as follows:

[0043]

[0044] The above complex can be simply referred to as [Ru(dip)2(L)](PF6)2, where the ligand L = 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide, and the dip structure = 4,7-diphenyl-1,10-phenanthroline.

[0045] Elemental analysis C 81 H 64 ClF 12 N9O2P2Ru (molecular weight 1621.32), theoretical values: C, 59.98%; H, 3.98%; N, 7.77%; experimental values: C, 59.89%; H, 3.88%; N, 7.89%.

[0046] ESI-MS: Theoretical value: [M-2PF6] - ] 2+ m / z = 665.7, experimental value: [M-2PF6] - ] 2+ m / z = 666.0.

[0047] Example 2: In vitro antitumor activity experiment of Ru(II) in combination with other substances

[0048] The MTT assay was used to evaluate the cytotoxic effects of ruthenium(II) complexes on cells.

[0049] (1) Cell culture: Human cervical cancer cells (HeLa) and mouse breast cancer cells (4T1) were seeded in 96-well plates (density 5×10⁻⁶). 3 / well), incubate at 37℃ and 5% CO2 for 24 hours.

[0050] (2) Drug administration:

[0051] Dark toxicity group: 100 μL of culture medium containing different concentrations of Ru1 (0.001–10 μM) was added and cultured in the dark for 48 hours.

[0052] Phototoxicity group: Ru1 was added and the mixture was incubated for 4 hours, followed by exposure to a 450 nm LED light source (40 mW / cm²). 2 Irradiation for 10 minutes (total light energy 36 J / cm²) 2 Continue training for 44 hours.

[0053] (3) Detection: Add MTT solution (10 μL, 5 mg / mL) to each well, incubate for 4 hours, add 150 μL of DMSO to dissolve formazan, and measure the absorbance (OD) at 595 nm using an ELISA reader.

[0054] The phototoxicity index (PI) reflects the median lethal concentration (IC50) of a drug under both dark and light conditions. 50 The ratio of ruthenium(II) to ruthenium(II) complex was used as the reference group. The cell viability of the control group without ruthenium(II) complex was set at 100%, and the cell viability was calculated according to the following formula.

[0055] Cell viability % = (mean OD of the drug-treated group) / (mean OD of the control group) × 100%

[0056] The test results are shown in Table 1 below:

[0057] Table 1 IC of Ru(II) complexes 50 value a (μM)

[0058]

[0059] Note: IC 50 a This refers to the concentration of the complex required to kill 50% of tumor cells. The duration of drug action is 48 hours, and this data was obtained through at least three repeated experiments and is expressed as mean ± standard deviation.

[0060] Example 3: Nuclear localization of Ru1 (confocal imaging)

[0061] (1) HeLa cells were seeded in confocal culture dishes (density 3×10⁻⁶). 4 / dish), after adhering to the wall, add Ru1 (0.5 μM).

[0062] (2) Illumination treatment: Illuminate with 450 nm LED for 10 minutes and incubate in the dark for 3 hours.

[0063] (3) Staining: Wash 3 times with PBS, then stain with DAPI (5 μg / mL) for 15 minutes in the dark.

[0064] (4) Imaging: Observation using laser confocal microscopy (Ru1:λ) ex =448 nm, λ em =530±20 nm; DAPI: λ ex =405 nm, λ em =460±20 nm).

[0065] like Figure 1As shown, laser confocal imaging indicates that the blue fluorescence representing DAPI highly overlaps with the red fluorescence produced by Ru1, and that the complex Ru1 can enter the HeLa cell nucleus and accumulate in the nucleolus after illumination.

[0066] Example 4: Photodynamic DNA fragmentation of Ru1 (gel electrophoresis)

[0067] (1) Mix pBR322 DNA (0.1 μg) with different concentrations of Ru1 (0–0.5 μM) and in the dark or under 450 nm light (40 mW / cm²). 2 Processing time: 10 minutes.

[0068] (2) Loading: Add the DNA-loading buffer mixture to the wells of a 1% agarose gel.

[0069] (3) Electrophoresis: TBE buffer, 50 V voltage, 45 minutes.

[0070] (4) Staining: The gel was stained with GoldView and then imaged with ultraviolet light.

[0071] like Figure 2 As shown, the experiment revealed that Ru1 can cause DNA breaks (Form II loose circular, Form III linear) under light conditions, and the degree of breakage increases with increasing Ru1 concentration (the dark treatment group had almost no breakage).

[0072] Example 5: Effect of Ru1 on the cell cycle (flow cytometry + Western blotting)

[0073] (1) HeLa cells were seeded in 60 mm culture dishes (Corning). When the cells adhered to the wall and grew to 70%, Ru1 (0-0.5 μM) was added and the cells were treated with light and cultured for 24 hours.

[0074] (2) Cell fixation: Cells were collected by digestion with trypsin. Cells were washed three times with PBS, resuspended in 500 μL of PBS, and fixed overnight with 70% cold ethanol at 4°C.

[0075] (3) Staining: RNase (50 μg / mL) and propidium iodide PI (50 μg / mL) were used for staining for 30 minutes in the dark.

[0076] (4) Detection: Flow cytometry analysis of cycle distribution (ModFit software).

[0077] (5) Western blot: Extract total cell protein and detect Cyclin D1 / CDK4 expression.

[0078] like Figure 3 As shown, Figure 3Specific analysis of A-3B showed that the proportion of cells in the G0 / G1 phase significantly increased from 62.3% in the blank control group to 79.1%. It should be noted that the proportion of cells in the G2 / M phase decreased from 31.6% in the blank control group to 14.8%. Meanwhile, the proportion of cells in the S phase did not change significantly. The inhibitory effect of the drug on the cell cycle after light exposure is directly related to the drug concentration; this observation is consistent with Western blotting analysis of cell cycle-related proteins. Figure 3 The results shown in C) are consistent. Based on cell cycle distribution analysis, it can be inferred that light exposure causes DNA damage, thereby reducing the number of S-phase cells. Furthermore, the number of G2 / M-phase cells, which are involved in DNA repair, shows a decreasing trend under different drug concentrations. This indicates that Ru1 interferes with the repair process of damaged DNA.

[0079] Example 6 Immunofluorescence staining detection of Ru1-induced γ-H2AX expression in HeLa cells

[0080] The cell density was 3×10 4 HeLa cells were seeded in confocal culture dishes. After cell attachment, different concentrations of Ru1 were added and the cells were exposed to light for 10 minutes, followed by incubation for 24 hours. Following the instructions for the γ-H2AX antibody, rabbit anti-IgG (H+L) antibody was added, and the cells were incubated at room temperature in the dark for 1 hour. The cells were washed (three times with immunostaining wash buffer), followed by nuclear staining with DAPI in the dark for 15 minutes. The cells were washed three times with washing buffer and observed under a laser confocal microscope. (DAPI: λ) ex = 405 nm, λ em = 460 ± 20 nm; Rabbit anti-IgG (H+L): λ ex = 488 nm, λ em = 520 ± 20 nm)

[0081] The formation of γ-H2AX is one of the earliest reactions following DNA damage, and this protein is often used as an important marker for diagnosing DNA damage. In HeLa cells treated with different concentrations of Ru1 light, the intensity of the green fluorescence of γ-H2AX correlated with the administered concentration, indicating intracellular DNA damage. Importantly, the green fluorescence of γ-H2AX did not completely disappear in cells treated with the antioxidant NAC.

[0082] like Figure 4 As shown, the number of γ-H2AX foci increases with increasing Ru1 concentration, confirming DNA double-strand breaks. Furthermore, the experimental results reveal that the main mechanism of Ru1-induced DNA damage may not solely stem from the generation of large amounts of ROS.

[0083] Example 7: Western blot analysis of cGAS-STING pathway-related proteins

[0084] HeLa cells were seeded in six-well plates. When the cell density reached 70%, different concentrations of Ru1 were added and the cells were cultured in the dark for 4 hours. Then, a 450 nm wavelength LED light source (40 mW·cm⁻¹) was used. -2 Irradiate for 10 minutes (36 J·cm⁻¹). -2 After treating cells with Ru1 complex, perform the following steps in sequence according to the product manual: extract total protein, prepare gel and load sample, perform electrophoresis, transfer membrane, block, incubate with primary antibody, incubate with secondary antibody, and develop.

[0085] The classic signaling pathway of pyroptosis is based on the activation of Caspase-1. When tumor cells are stimulated by damage-associated molecular patterns (DAMPs), the inflammasome is activated and recruits Caspase-1 to cleave GSDMD, generating an active N-terminal fragment (GSDMD-NT) and a C-terminal fragment. In the classic Caspase-1-dependent pyroptosis mechanism, cells exhibit swelling, membrane rupture, and release of contents. Compared to traditional cell death mechanisms, the structure of the cell nucleus is often preserved during pyroptosis, while the process is accompanied by DNA damage. The cGAS-STING pathway plays a crucial role in mobilizing anti-tumor immune responses. In the field of tumor immunotherapy, cyclic guanylate synthase (cGAS) has shown great potential in effectively promoting STING expression. In this process, STING activates nuclear factor κB (NF-κB) through ubiquitination, thereby further enhancing the body's anti-tumor immune response.

[0086] like Figure 5 As shown, the expression levels of cell-related proteins were analyzed by Western blot. Caspase-1 was activated, and the expression level of N-GSDMD also increased. With the gradual increase of Ru1 concentration, the expression level of cGAS also increased, leading to an increase in p-STING level, which ultimately promoted the enhanced expression of p-NF-κB. Therefore, it can be concluded that Ru1 in HeLa cells not only induces Caspase-1-mediated pyroptosis but also activates the cGAS-STING signaling pathway, enhancing the anti-tumor immune response.

[0087] Example 8: In vivo experimental evaluation of tumor immune effect in mice

[0088] (1) Preparation of tumor cell vaccine: Mouse breast cancer 4T1 cells in logarithmic growth phase were seeded into cell culture dishes and the cell concentration was adjusted to 1×10⁻⁶. 6 / plate, placed in a cell culture incubator and cultured overnight. Different drugs were administered according to different groups: 1) 4T1 cells were co-cultured with CDDP (20 μM) for 36 hours; 4T1 (mouse breast cancer cells) were added to Ru1 and treated with light, followed by incubation for another 36 hours. After drug treatment, the cell suspension was collected into centrifuge tubes, washed three times with PBS, centrifuged to collect the cell pellet, and resuspended in PBS to obtain a single-cell suspension, which is the tumor cell vaccine.

[0089] (2) Evaluation of tumor immune effect in mice: Forty healthy 4- to 5-week-old SPF-grade female BALB / c mice were selected. 4T1 cell suspension was injected subcutaneously into the right hind limb of the mice to promote subcutaneous tumor formation, thereby evaluating whether the prepared vaccine could effectively stimulate an anti-tumor immune response in vivo. The experimental mice were randomly divided into four groups: a control group, a CDDP-treated group, a single Ru1 vaccine injection group, and a three-Ru1 vaccine injection group. The specific methods of the animal experiment were as follows: The midpoint between the right groin and the dorsal side of the thigh of the mice was disinfected, and a syringe was used to inject subcutaneously into this area: Control group: 100 μL PBS was injected only; CDDP group: cell suspension treated with CDDP was injected; Ru1 single-dose vaccine group: cell suspension treated with Ru1 was injected; the Ru1 three-dose vaccine group consisted of three repetitions of the single-dose injection process. Seven days later, the area from the right groin to the midpoint of the dorsal thigh of 30 mice was disinfected, and viable 4T1 cells were subcutaneously injected into this region. Tumor size and body weight in female BALB / c mice were monitored and recorded every two days. The formula for calculating tumor volume (V) is V = W. 2 × L / 2, where W is the width of the tumor and L is the length of the tumor. In the final in vivo mouse experiments, the major organs of the remaining mice were stained with H&E to observe any potential histological changes.

[0090] like Figure 6 As shown, throughout the animal experiment, the mice's body weight gradually increased without significant fluctuations. Figure 6 A). The experiment revealed that the growth rate of tumors in the control group and mice treated with cisplatin (CDDP) was significantly higher than that in mice that had been vaccinated. Tumor samples were collected from each group of mice on day 30 after tumor inoculation and observed. Figure 6 B-6C). Analysis by H&E staining ( Figure 6(D) No significant pathological changes were observed in the major organs of mice (including the heart, spleen, liver, lungs, and kidneys), indicating that the vaccine did not cause significant tissue damage or pathological changes in the major organs of mice, confirming its good biosafety. Therefore, this study provides crucial support for the biosafety and efficacy of vaccines.

[0091] The Ru(II) complex provided by this invention can induce pyroptosis in HeLa cells by damaging DNA with a low dose of Ru1 photosensitizer and enhance immunogenic death through the cGAS-STING pathway.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An ICD-inducing agent Ru(II) complex having a DNA-photodynamic cleavage ability, characterized by, A compound having the structure shown below: 。 2. A method for producing the ICD inducer Ru(II) complex having a DNA photodynamic cleavage ability according to claim 1, characterized by, The method comprises the following steps: Under the protection of argon, Ru(dip)2Cl2 and ligand L are refluxed in an organic solvent to obtain a Ru(II) complex; The ligand L is 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide; and dip is 4,7-diphenyl-1,10-phenanthroline.

3. The production method according to claim 2, characterized by, The organic solvent is anhydrous ethanol.

4. The production method according to claim 2, characterized by, The reflux reaction temperature is 70-80°C, and the reaction time is 1-3 hours.

5. The preparation method according to claim 2, characterized in that, After the reflux reaction, the following steps are further included: adding an excess of saturated NH4PF6 solution, filtering and drying the precipitate, and purifying.

6. An antitumor pharmaceutical composition, characterized by comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2. The ICD inducer Ru(II) complex having the ability to photodynamically break DNA according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the ICD inducer Ru(II) complex having the ability to photodynamically break DNA according to claim 1 in the preparation of an antitumor drug.

8. Use according to claim 7, characterized in that, The antitumor drug is used to induce immunogenic death of tumor cells; the tumor cells are murine breast cancer cells or human cervical cancer cells.

9. Use of the ICD inducer Ru(II) complex having a DNA light-activated cleavage ability according to claim 1 in the preparation of an antitumor vaccine, characterized by, The antitumor vaccine is prepared by the following steps: (a) co-incubating tumor cells with the ICD inducer Ru(II) complex; (b) irradiating with a light source with a wavelength of 400-500 nm; (c) washing and resuspending the cells to obtain an antitumor vaccine; The tumor cells are murine breast cancer cells or human cervical cancer cells.

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