Cyclo-metallated ir(III) complexes, methods for their preparation and use

Abstract

The present application relates to the technical field of antitumor drugs, in particular to a kind of cyclometallated Ir (III) complex and its preparation method and application.The present application provides a kind of cyclometallated Ir (III) complex, the complex not only cell dark toxicity is significantly lower than the clinical commonly used chemotherapeutic drug cisplatin, simultaneously have excellent ultrasonic physical performance and significant sound catalytic activity.Under the excitation of ultrasound, the complex can efficiently generate I type and II type active oxygen, and show good lysosome targeting ability and synergistic enhancement of antitumor immunoregulation characteristics;When it is applied to antitumor therapeutic agent, not only the safety is greatly improved, but also excellent antitumor therapeutic effect can be realized, and wide application prospect is shown.

Description

Technical Field

[0001] This application relates to the field of antitumor drug technology, and in particular to a class of cyclic metallized Ir(III) complexes and their preparation methods and applications. Background Technology

[0002] Both photodynamic therapy (PDT) and sonodynamic therapy (SDT) utilize the dynamic effects of physical light or ultrasound to treat diseases. PDT has secured a place in tumor treatment due to its precise targeting advantage; while theoretically, unlike light which has limited tissue penetration, ultrasound has excellent tissue penetration, making it an effective clinical treatment for deep tumors such as pancreatic cancer and lung cancer. Their core mechanisms of action are consistent: after tumor tissue takes up a sensitizer (photosensitizer / soundsensitizer), the physical signal (light / ultrasound) acts on the target site. The sensitizer catalyzes the reaction of oxygen and substrate within the tumor through energy transfer or electron transfer, generating cytotoxic reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radicals, which then damage organelles and cellular structures and functions, inducing cell death and ultimately achieving tumor treatment.

[0003] Compared to traditional therapies, the aforementioned treatments offer significant advantages in terms of high efficiency, controllability, and safety. This technological direction aligns perfectly with the clinical needs of cancer treatment—taking oral squamous cell carcinoma (OSCC) as an example. OSCC is the most common oral malignancy, and the classic treatment method primarily involves surgery, supplemented by radiotherapy or chemotherapy. However, among current mainstream chemotherapy drugs, platinum-based complexes, represented by cisplatin, while widely used, are prone to severe systemic toxicity and can induce multidrug resistance in tumors, limiting their clinical application. Against this backdrop, iridium, a metal element homologue of platinum, exhibits a novel anti-tumor mechanism due to its excellent photoacoustic physical properties, intracellular catalytic activity, and unique subcellular organelle localization capabilities. It not only holds promise for development into a new type of highly efficient, low-toxicity, and highly targeted anti-tumor drug following platinum-based drugs, but also provides a high-quality material choice for the development of sensitizers in photo / acoustic dynamic therapy. The application of cyclic iridium(III) complexes in photodynamic therapy (PDT) has been reported. For example, a cyclic metallized Ir(III) complex disclosed in Chinese patent application CN117126205A exhibits excellent photodynamic efficacy, but suffers from excessively high dark toxicity: the dark toxicity of this complex to human oral mucosal precancerous cells (DOK) is approximately five times that of cisplatin, while its dark toxicity to human tongue squamous cell carcinoma (SCC1) is comparable to that of cisplatin. This excessively high dark toxicity allows it to spontaneously damage normal tissues and cells without photoactivation, significantly increasing the risk of non-targeted toxic side effects during treatment and drastically reducing the clinical safety window, creating serious therapeutic safety risks and greatly hindering its translation from laboratory research to clinical application.

[0004] Therefore, there is an urgent need to develop an iridium complex that combines low dark toxicity with excellent therapeutic effects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art in which cyclic iridium (III) complexes cannot simultaneously achieve low dark toxicity and excellent therapeutic effect, and to provide the application of bridging iridium precursors in the preparation of antitumor materials or antitumor drugs.

[0006] Another object of the present invention is to provide the use of organic ligands in the preparation of antitumor materials or antitumor drugs.

[0007] Another object of the present invention is to provide a class of cyclic metallized Ir(III) complexes.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned cyclometalated Ir(III) complex.

[0009] Another object of the present invention is to provide the use of the above-mentioned cyclic metallized Ir(III) complexes in the preparation of antitumor materials or antitumor drugs.

[0010] Another object of the present invention is to provide an antitumor therapeutic agent.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects the use of a bridged iridium precursor in the preparation of antitumor materials or antitumor drugs, the structural formula of which is shown below: .

[0012] When this bridging iridium precursor is used to prepare antitumor materials or drugs, it exhibits good biocompatibility and easily achieves a synergistic effect of enhanced efficacy and reduced toxic side effects.

[0013] Furthermore, the chemical formula of the bridging iridium precursor is [Ir(btp)2(μ-Cl)]2.

[0014] This invention protects the use of organic ligands in the preparation of antitumor materials or antitumor drugs, wherein the structural formula of the organic ligands is shown below: ; Wherein, R1 is hydrogen or C 1-6 The alkane, where R2 is hydrogen or a compound containing a five-membered heterocyclic structure, and the heteroatom of the five-membered heterocyclic structure is sulfur.

[0015] When this organic ligand is used to prepare antitumor materials or drugs, it can improve the biocompatibility of the materials / drugs, thereby easily achieving a synergistic effect of enhanced efficacy and reduced toxic side effects.

[0016] Furthermore, the structural formula of the organic ligand is shown below: ; Wherein, R1 is hydrogen or C 1-3 Alkanes, where R2 is hydrogen or a thiophene compound.

[0017] Furthermore, the organic ligand has any of the following structural formulas, which are respectively named organic ligands 1 to 5: .

[0018] In this invention, organic ligands 1 to 5 are merely formal names and have no technical limitation effect.

[0019] Preferably, the organic ligand has any of the following structural formulas: .

[0020] More preferably, the organic ligand has the following structural formula: .

[0021] This invention protects a class of cyclometalated Ir(III) complexes, the structural formula of which is shown below: ; Wherein, R1 is hydrogen or C 1-6 The alkane, where R2 is hydrogen or a compound containing a five-membered heterocyclic structure, and the heteroatom of the five-membered heterocyclic structure is sulfur.

[0022] The cyclic metallized Ir(III) complexes of this invention possess excellent acoustic physical properties and acoustic catalytic activity. Under ultrasound, they can efficiently generate type I and type II reactive oxygen species, exerting a sonodynamic effect. At the same time, the planar hydrophobic structure of these complexes enables them to target lysosomes, induce cell death, and thus synergistically enhance antitumor immunomodulatory properties. They are a novel antitumor sonodynamic therapeutic agent with good biosafety.

[0023] Furthermore, the structural formula of the cyclic metallized Ir(III) complex is shown below: ; Wherein, R1 is hydrogen or C 1-3 Alkanes, where R2 is hydrogen or a thiophene compound.

[0024] Furthermore, the cyclometalated Ir(III) complex has any of the following structural formulas: .

[0025] Preferably, the cyclometalated Ir(III) complex has any of the following structural formulas: .

[0026] More preferably, the cyclic metallized Ir(III) complex has the following structure: .

[0027] This invention protects a method for preparing the above-mentioned cyclic metallized Ir(III) complex, comprising the following steps: The bridging iridium precursor and the organic ligand are mixed and dispersed in an organic solvent, reacted completely, and purified to obtain the cyclometalated Ir(III) complex.

[0028] Furthermore, the preparation of the bridging iridium precursor includes the following steps: Under a protective gas atmosphere, iridium trichloride or its hydrate and 6-(benzo[b]thiophene-2-yl)phenanthridine are mixed and dispersed in a solvent, reacted completely, and then post-treated to obtain the bridged iridium precursor.

[0029] Furthermore, the protective gas includes one or more of nitrogen, helium, and argon.

[0030] Furthermore, the protective gas is nitrogen.

[0031] Furthermore, the hydrate is iridium trichloride trihydrate.

[0032] Furthermore, the molar ratio of iridium trichloride or its hydrate to 6-(benzo[b]thiophene-2-yl)phenanthridine is 1:(1~3).

[0033] Furthermore, the molar ratio of iridium trichloride or its hydrate to 6-(benzo[b]thiophene-2-yl)phenanthridine is 1:(1.1~2).

[0034] Furthermore, the solvent is a combination of ethylene glycol ether compounds and water.

[0035] Furthermore, the ethylene glycol ether compounds include one or more of 2-ethoxyethanol, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, and diethylene glycol monoethyl ether.

[0036] Preferably, the volume ratio of the ethylene glycol ether compound to water is (2~5):1.

[0037] Furthermore, the mass-to-volume ratio of the iridium trichloride or its hydrate to the solvent is 1 g: (40~60) mL.

[0038] Furthermore, in the preparation of the bridging iridium precursor, the temperature for the complete reaction is 60-90 °C.

[0039] Furthermore, in the preparation of the bridging iridium precursor, the sufficient reaction time is 20-30 h.

[0040] Furthermore, the post-processing includes cooling, filtration, and washing.

[0041] Furthermore, the washing process involves sequentially washing the filtered solid product with methanol and diethyl ether.

[0042] Furthermore, the organic ligands can be obtained commercially or in-house.

[0043] Preferably, the preparation of the organic ligand 1 includes the following steps: Formaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium salt were mixed thoroughly in an acidic reagent, allowed to react completely, and purified to obtain organic ligand 1.

[0044] Furthermore, the molar ratio of formaldehyde to 1,10-phenanthroline-5,6-dione is 1:(0.8~2).

[0045] Furthermore, the ammonium salt includes one or more of ammonium acetate, ammonium formate, ammonium bicarbonate, and ammonium carbonate.

[0046] Furthermore, the molar ratio of formaldehyde to ammonium salt is 1:(15~20).

[0047] Furthermore, the acidic reagent includes one or more of acetic acid, formic acid, and propionic acid.

[0048] Furthermore, the acidic reagent is acetic acid.

[0049] Furthermore, the mass-to-volume ratio of formaldehyde to acidic reagent is 1 g: (400~800) mL.

[0050] Furthermore, the mass-to-volume ratio of formaldehyde to acidic reagent is 1 g: (500~600) mL.

[0051] Furthermore, in the preparation of organic ligand 1, the temperature for the complete reaction is 80~100 °C.

[0052] Furthermore, in the preparation of organic ligand 1, the reaction time is 5-10 h.

[0053] Furthermore, in the preparation of organic ligand 1, the purification includes the following steps: cooling the system after the reaction, adjusting the pH to 7-8, filtering, washing, and drying.

[0054] Preferably, the solvent for adjusting the pH is an alkaline reagent.

[0055] More preferably, the alkaline reagent is ammonia.

[0056] Furthermore, the concentration of the ammonia water is 5%~10% (mass fraction, w / w).

[0057] Preferably, the organic ligand 2 and organic ligand 5 are prepared by the following method: S1. Mix 2-thiophenecarboxaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium salt in an acidic reagent until homogeneous, react thoroughly, and purify to obtain organic ligand 2; S2. Under alkaline conditions, the organic ligand 2 obtained in step S1 and the methylating agent are mixed in an organic solvent, reacted completely, and purified to obtain organic ligand 5.

[0058] Furthermore, the molar ratio of 2-thiophenecarboxaldehyde and 1,10-phenanthroline-5,6-dione is 1:(0.8~2).

[0059] Furthermore, the molar ratio of 2-thiophenecarboxaldehyde to ammonium salt is 1:(15~20).

[0060] Furthermore, the mass-to-volume ratio of the 2-thiophene formaldehyde to the acidic reagent is 1 g: (100~200) mL.

[0061] Furthermore, in step S1, the temperature for the complete reaction is 80~100 °C.

[0062] Furthermore, in step S1, the time for the full reaction is 5 to 10 hours.

[0063] Further, in step S1, the purification includes cooling, adjusting the cooled reaction mixture to pH 7-8, filtering, washing, and drying.

[0064] Preferably, the solvents used for washing are water and diethyl ether, in sequence.

[0065] Preferably, the washing is of the solid product after washing and filtration.

[0066] The drying process refers to drying the product after washing.

[0067] Further, in step S2, the organic solvent includes one or more of formonitrile, acetonitrile, and propionitrile.

[0068] Furthermore, in step S2, the organic solvent is formonitrile.

[0069] Furthermore, the mass-to-volume ratio of the organic ligand 2 to the organic solvent is 1 g: (100~150) mL.

[0070] Furthermore, in step S2, the alkaline conditions are provided by a metal hydride.

[0071] Furthermore, the metal hydride includes one or more of sodium hydride, potassium hydride, and calcium hydride.

[0072] Furthermore, the metal hydride is sodium hydride.

[0073] Furthermore, when the alkaline conditions are provided by the metal hydride, the molar ratio of the organic ligand 2 to the metal hydride is 1:(1~3).

[0074] Furthermore, the methylating agent includes halomethanes and / or methyl esters.

[0075] Furthermore, the halomethane includes potassium iodide and / or potassium bromide.

[0076] Furthermore, the methyl ester compounds include one or more of dimethyl sulfate, methyl toluenesulfonate, and dimethyl carbonate.

[0077] Preferably, the methylating agent is potassium iodide.

[0078] Furthermore, the molar ratio of the organic ligand 2 to the methylating agent is 1:(1~3).

[0079] Furthermore, in step S2, the temperature at which the reaction is fully completed is 80~100 °C.

[0080] Furthermore, in step S2, the time for the complete reaction is 8-10 hours.

[0081] Further, in step S2, the purification involves removing the solvent, washing, and drying.

[0082] Preferably, the solvent removal is performed by rotary evaporation under reduced pressure.

[0083] Preferably, the washing involves washing the product after removing the solvent with water.

[0084] Preferably, the drying is the drying of the washed product.

[0085] Preferably, the preparation of the organic ligand 3 includes the following steps: 2,2-Bithiophene-5-carboxaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium salt were mixed evenly in an acidic reagent, allowed to react completely, and purified to obtain organic ligand 3.

[0086] Furthermore, the molar ratio of 2,2-bithiophene-5-carboxaldehyde and 1,10-phenanthroline-5,6-dione is 1:(0.8~2).

[0087] Furthermore, the molar ratio of the 2,2-bithiophene-5-carboxaldehyde to the ammonium salt is 1:(15~20).

[0088] Furthermore, the mass-to-volume ratio of the 2,2-bithiophene-5-carboxaldehyde to the acidic reagent is 1 g: (60~120) mL.

[0089] Furthermore, the temperature for the complete reaction is 80~100 °C.

[0090] Furthermore, the time for the complete reaction is 5 to 10 hours.

[0091] Furthermore, the purification includes cooling, adjusting the cooled reaction mixture to pH 7-8, filtering, washing, and drying.

[0092] Preferably, the preparation of the organic ligand 4 includes the following steps: 2,2':5',2''-trithiophene-5-carboxaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium salt were mixed thoroughly in an acidic reagent, allowed to react completely, and purified to obtain organic ligand 4.

[0093] Furthermore, the molar ratio of 2,2':5',2''-trithiophene-5-carboxaldehyde and 1,10-phenanthroline-5,6-dione is 1:(0.8~2).

[0094] Furthermore, the molar ratio of 2,2':5',2''-trithiophene-5-carboxaldehyde to ammonium salt is 1:(15~20).

[0095] Furthermore, the mass-to-volume ratio of the 2,2':5',2''-trithiophene-5-carboxaldehyde and the acidic reagent is 1 g:(50~80) mL.

[0096] Furthermore, the temperature for the complete reaction is 80~100 °C.

[0097] Furthermore, the time for the complete reaction is 5 to 10 hours.

[0098] Furthermore, the purification includes cooling, adjusting the cooled reaction mixture to pH 7-8, filtering, washing, and drying.

[0099] Furthermore, the molar ratio of the bridging iridium precursor to the organic ligand is 1:(1.8~2.5).

[0100] Preferably, the molar ratio of the bridging iridium precursor to the organic ligand is 1:(1.8~2.2).

[0101] Furthermore, the organic solvent is a chloromethane reagent or an alcohol reagent.

[0102] Furthermore, the chloromethane reagents include dichloromethane and / or trichloromethane.

[0103] Preferably, the chloromethane reagent is dichloromethane.

[0104] Furthermore, the alcohol reagent is one or more of methanol, ethanol, and isopropanol.

[0105] Preferably, the alcohol reagent is methanol.

[0106] Preferably, the volume ratio of the chloromethane reagent to the alcohol reagent is (1~3):1.

[0107] Furthermore, in the preparation of the cyclometalated Ir(III) complex, the temperature for the complete reaction is 50~70°C.

[0108] Furthermore, in the preparation of the cyclometalated Ir(III) complex, the reaction time is 5-10 h.

[0109] Furthermore, the purification includes cooling, solvent removal, and column chromatography.

[0110] Preferably, the solvent removal is performed by vacuum evaporation to obtain a crude solid sample.

[0111] Preferably, the chromatographic column used for column chromatography is a silica flash column chromatography column.

[0112] Preferably, the eluent for the column chromatography is dichloromethane or a combination of dichloromethane and methanol.

[0113] Preferably, the volume ratio of dichloromethane to methanol is 1:(0.01~1).

[0114] More preferably, the volume ratio of dichloromethane to methanol is 1:(0.02~0.5).

[0115] This invention protects the use of the above-mentioned cyclic metallized Ir(III) complexes in the preparation of antitumor materials or antitumor drugs.

[0116] Furthermore, the antitumor material is an anti-oral squamous cell carcinoma material, and the antitumor drug is an anti-oral squamous cell carcinoma drug.

[0117] This invention protects an antitumor therapeutic agent comprising the above-mentioned cyclic metallized Ir(III) complex.

[0118] Furthermore, the antitumor therapeutic agent includes an antitumor photodynamic therapy agent and / or an antitumor sonodynamic therapy agent. These complexes not only possess sonodynamic properties, but also generate a certain amount of reactive oxygen species under laser irradiation, thereby exhibiting photodynamic therapy effects.

[0119] Furthermore, the antitumor therapeutic agent is an antitumor sonodynamic therapeutic agent.

[0120] Compared with the prior art, the present invention has the following beneficial effects: The cyclic metallized Ir(III) complex of the present invention has significantly lower dark toxicity than cisplatin, high sonotoxicity index, good biosafety, and can be selectively taken up by tumor cells, further targeting tumor cell lysosomes, causing lysosomal damage and inducing inflammatory cell death; moreover, it can generate a large amount of ROS under sonication, exert a strong SDT effect, and effectively kill SCC1, HN6 and DOK cells.

[0121] The cyclic metallized Ir(III) complex of the present invention can also regulate the tumor immune microenvironment under ultrasound conditions, induce immunogenic death (ICD) in tumor cells, activate the immune response through damage-associated molecular patterns (DAMPs), such as the expression of calreticulin, the secretion of high-mobility group box 1 (HMGB1) and the release of ATP molecules, and can synergistically block PD-L1 immune checkpoint, thereby inhibiting tumor growth. It is an anti-tumor drug with outstanding potential for sonodynamic immunotherapy. Attached Figure Description

[0122] Figure 1 The diagram shows the synthetic routes of the cyclic metallized Ir(III) complexes in Examples 3-7.

[0123] Figure 2 The above are the HPF fluorescence spectra of the ·OH-specific fluorescent probes of the cyclic metallized Ir(III) complexes in Examples 3-7.

[0124] Figure 3 O2 of the cyclometalated Ir(III) complexes in Examples 3-7 ·- Fluorescence spectrum of the specific fluorescent probe DHR 123.

[0125] Figure 4 The cyclometalated Ir(III) complexes in Examples 3-7 1 Absorbance spectrum of O2-specific probe ABDA.

[0126] Figure 5 This is a drug uptake diagram of the cyclic metallized Ir(III) complexes in Examples 3-7.

[0127] Figure 6 The diagram shows the colocalization of the cyclic metallized Ir(III) complex with lysosomes in Example 3 (A) and the damage analysis of the cyclic metallized Ir(III) complex to lysosomes under ultrasound in Example 3 (B).

[0128] Figure 7The images show the volcano diagram (A) and the GSEA pathway enrichment analysis diagram (B) of the RNA sequencing results of the cyclic metallized Ir(III) complex in SCC1 cells in Example 3.

[0129] Figure 8 The figures show the changes in intracellular ROS levels after sonodynamic therapy with different concentrations of cyclic metallized Ir(III) complexes in Example 3, and statistical data on the changes in ROS levels (A) and GSH levels (B).

[0130] Figure 9 This is a diagram showing the cell death of tumor cells after treatment with the cyclic metallized Ir(III) complex in Example 3, analyzed by live-dead staining.

[0131] Figure 10 The expression of Cleaved caspase-3, Fl-GSDME, and N-GSDME proteins in cells after sonodynamic therapy treatment with cyclic metallized Ir(III) complexes in Example 3 was analyzed by Western blot (A), and statistical data based on changes in protein expression levels were plotted (B).

[0132] Figure 11 The cell morphology diagram is obtained by transmission electron microscopy analysis after sonodynamic therapy treatment of the cyclic metallized Ir(III) complex in Example 3.

[0133] Figure 12 The graph shows the ATP content in the cell supernatant after sonodynamic therapy treatment with the cyclic metallized Ir(III) complex in Example 3, analyzed by chemiluminescence.

[0134] Figure 13 The graphs show the changes in intracellular HMGB1 expression and statistical data based on the changes in HMGB1 expression after sonodynamic therapy with the cyclic metallized Ir(III) complex in Example 3, as well as the changes in CRT expression on the cell membrane and statistical data based on the changes in CRT expression. (A)

[0135] Figure 14 The images show the gene expression level of PD-L1 in cells after treatment with cyclic metallized Ir(III) complexes via RT-qPCR (A), the protein electrophoresis characterization of PD-L1 in cells after treatment with cyclic metallized Ir(III) complexes via Western blot (B), and the statistical data of protein expression level changes based on the electrophoresis characterization (C).

[0136] Figure 15The following diagrams illustrate the therapeutic effect of sonodynamic therapy on oral tumors using cyclic metallized Ir(III) complexes in Example 3 of the CDX model study, and its synergistic anti-tumor effect when combined with PD-L1 inhibitors. (A) shows the tumor volume growth curve during treatment; (B) shows the gross image of the tumor tissue after treatment; (C) shows the statistical data of tumor tissue weight analysis; (D) shows the experimental results of H&E pathological examination and IHC staining of Ki67 and PD-L1 expression levels after treatment; (E) shows the H&E staining of organ tissues after treatment; and (F) shows the mouse body weight change curve during treatment.

[0137] Figure 16 The image shows the therapeutic effect of sonodynamic therapy on oral tumors using cyclic metallized Ir(III) complexes in Example 3 of the CDX model study, and its synergistic effect with PD-L1 inhibitors in enhancing the anti-tumor immune response. (A) shows the expression map of PD-L1 on the surface of tumor dendritic cells and macrophages after treatment, along with statistical data based on the corresponding expression maps; (B) shows the expression map of CD8 infiltrating tumor cells after treatment. + T cell content graph and statistical data based on this content graph; (C) shows the in situ infiltration of CD4 cells at the end of treatment. + T and CD8 + T-surface PD-1 expression map and statistical data based on the corresponding expression map.

[0138] Figure 17 The image shows the therapeutic effect of sonodynamic therapy on oral tumors treated with cyclic metallized Ir(III) complexes in Example 3 of the CDX model study, and the synergistic enhancement of anti-tumor cytokine expression levels in combination with PD-L1 inhibitors. Among them, (A) is a statistical graph of tumor necrosis factor α (TNF-α) expression level in situ after treatment; (B) is a statistical graph of tumor interferon γ (IFN-γ) expression level in situ after treatment; and (C) is a statistical graph of tumor interleukin 6 (IL-6) expression level in situ after treatment. Detailed Implementation

[0139] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0140] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0141] Figure 6 A is Figure 6 Figure A in the text, Figure 6 B indicates Figure 6 The order of the other figures follows the same pattern, starting with Figure B.

[0142] Example 1 Organic ligands 1-5 and their preparation methods The structural formula of organic ligand 1 is: The structural formula of organic ligand 2 is: The structural formula of organic ligand 3 is: The structural formula of organic ligand 4 is The structural formula of organic ligand 5 is: It is prepared by the following method: (1) Preparation of organic ligand 1: Formaldehyde (0.036 g, 1.2 mmol), 1,10-phenanthroline-5,6-dione (1 mmol), and ammonium acetate (20 mmol) were mixed in acetic acid (20 mL) and heated to 100 mL. o After stirring for 5 hours, the mixture was cooled to room temperature and the pH of the mixture was adjusted to 7-8 with ammonia (5%, w / w). The mixture was then filtered to obtain a crude product, which was washed successively with water and diethyl ether and dried to obtain organic ligand 1. (2) Preparation of organic ligands 2 and 5: 2-Thiophenecaraldehyde (0.15 g, 1.2 mmol), 1,10-phenanthroline-5,6-dione (1 mmol), and ammonium acetate (20 mmol) were mixed and stirred until homogeneous, and then heated in acetic acid (20 mL) at 100 °C. o Stir at 80°C for 5 h. After cooling to room temperature, adjust the pH of the mixture to 7-8 with ammonia (5%, w / w), filter to obtain a solid sample, wash successively with water and diethyl ether, and dry to obtain organic ligand 2; then mix organic ligand 2 (0.16 g, 0.5 mmol), sodium hydride (0.5 mmol), and methyl iodoformate (0.75 mmol) in formonitrile (19.2 mL) at 80°C. o Heating at C for 8 h. After the reaction was complete, the solvent was removed under reduced pressure, the residue was washed with water and dried to obtain organic ligand 5; (3) Preparation of organic ligand 3: 2,2-Bithiophene-5-carboxaldehyde (0.24 g, 1.2 mmol), 1,10-phenanthroline-5,6-dione (1 mmol), and ammonium acetate (20 mmol) were mixed in acetic acid (20 mL), stirred at 100 °C for 5 h, cooled to room temperature, and the pH of the mixture was adjusted to 7-8 with ammonia (5%, w / w). The mixture was filtered, and the solid was washed with water and diethyl ether in sequence and dried to obtain organic ligand 3. (4) Preparation of organic ligand 4: 2,2':5',2''-trithiophene-5-carboxaldehyde (0.31 g, 1.2 mmol), 1,10-phenanthroline-5,6-dione (1 mmol), and ammonium acetate (20 mmol) were mixed in acetic acid (20 mL), stirred at 100 °C for 5 h, cooled to room temperature, and the pH of the mixture was adjusted to 7-8 with ammonia (5%, w / w). The mixture was filtered, and the solid was washed with water and diethyl ether in sequence and dried to obtain organic ligand 4.

[0143] Example 2: Bridged Iridium Precursor and its Preparation Method The structural formula of the bridging iridium precursor ([Ir(btp)2(μ-Cl)]2) is as follows: It is prepared by the following method: Iridium trichloride trihydrate (0.35 g, 1 mmol) and 6-(benzo[b]thiophene-2-yl)phenanthridine (1.2 mmol) were mixed and dispersed in 20 mL of organic solvent (2-ethoxyethanol and water in a volume ratio of 3:1). The mixture was refluxed at 80 °C for 24 h under nitrogen protection, cooled and filtered, and the resulting red solid product was washed with methanol and diethyl ether, which was the bridged iridium precursor.

[0144] Example 3 Cyclic metallized Ir(III) complex Ir5 and its preparation method A cyclometalated Ir(III) complex, Ir5, has the following structural formula: ; The above-mentioned cyclometalated Ir(III) complex Ir5 can be prepared by the following methods (e.g. Figure 1 (as shown) The bridged iridium precursor (400.0 mg, 0.236 mmol) and organic ligand 5 (149.3 mg, 0.472 mmol) were mixed and dispersed in 40 mL of organic solvent (dichloromethane and methanol in a volume ratio of 3:1). The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the solvent was evaporated under vacuum to obtain a crude solid. The solid was purified by silica gel flash column chromatography (eluted first with dichloromethane, then with a mixed solvent of dichloromethane and methanol in a volume ratio of 30:1) to obtain the cyclometalated Ir(III) complex Ir5.

[0145] Example 4 Cyclic metallized Ir(III) complex Ir1 and its preparation method A cyclometalated Ir(III) complex, Ir1, has the following structural formula: ; The above-mentioned cyclometalated Ir(III) complex Ir1 can be prepared by the following methods (e.g. Figure 1(as shown) The bridged iridium precursor (400.0 mg, 0.236 mmol) and organic ligand 1 (103.8 mg, 0.472 mmol) were mixed and dispersed in 40 mL of organic solvent (dichloromethane and methanol in a volume ratio of 3:1). The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the solvent was evaporated under vacuum to obtain a crude solid. The solid was purified by silica gel flash column chromatography (eluted first with dichloromethane, then with a mixed solvent of dichloromethane and methanol in a volume ratio of 30:1) to obtain the cyclometalated Ir(III) complex Ir1.

[0146] Example 5 Cyclic metallized Ir(III) complex Ir2 and its preparation method A cyclometalated Ir(III) complex, Ir2, has the following structural formula: ; The above-mentioned cyclometalated Ir(III) complex Ir2 can be prepared by the following methods (e.g. Figure 1 (as shown) The bridged iridium precursor (400.0 mg, 0.236 mmol) and organic ligand 2 (142.5 mg, 0.472 mmol) were mixed and dispersed in 40 mL of organic solvent (dichloromethane and methanol in a volume ratio of 3:1). The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the solvent was evaporated under vacuum to obtain a crude solid. The solid was purified by silica gel flash column chromatography (eluted first with dichloromethane, then with a mixed solvent of dichloromethane and methanol in a volume ratio of 30:1) to obtain the cyclometalated Ir(III) complex Ir2.

[0147] Example 6 Cyclic metallized Ir(III) complex Ir3 and its preparation method A cyclometalated Ir(III) complex, Ir3, has the following structural formula: ; The above-mentioned cyclometalated Ir(III) complex Ir3 can be prepared by the following methods (e.g. Figure 1 (as shown) The bridged iridium precursor (400.0 mg, 0.236 mmol) and organic ligand 3 (181.5 mg, 0.472 mmol) were mixed and dispersed in 40 mL of organic solvent (dichloromethane and methanol in a volume ratio of 3:1). The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the solvent was evaporated under vacuum to obtain a crude solid. The solid was purified by silica gel flash column chromatography (eluted first with dichloromethane, then with a mixed solvent of dichloromethane and methanol in a volume ratio of 30:1) to obtain the cyclometalated Ir(III) complex Ir3.

[0148] Example 7 Cyclic metallized Ir(III) complex Ir4 and its preparation method A cyclometalated Ir(III) complex, Ir4, has the following structural formula: ; The above-mentioned cyclometalated Ir(III) complex Ir4 can be prepared by the following methods (e.g. Figure 1 (as shown) The bridged iridium precursor (400.0 mg, 0.236 mmol) and organic ligand 4 (220.2 mg, 0.472 mmol) were mixed and dispersed in 40 mL of organic solvent (dichloromethane and methanol in a volume ratio of 3:1). The reaction was carried out at 50 °C for 5 h. After the reaction was completed, the solvent was evaporated under vacuum to obtain a crude solid. The solid was purified by silica gel flash column chromatography (eluted first with dichloromethane, then with a mixed solvent of dichloromethane and methanol in a volume ratio of 30:1) to obtain the cyclometalated Ir(III) complex Ir4.

[0149] Experimental Example 1: Acoustodynamic Effect Test of Cyclic Metallized Ir(III) Complexes 1. Experimental Methods To detect the generation of reactive oxygen species in cyclic metallized Ir(III) complexes, the ·OH-specific fluorescent probes HPF and O2 were used. ·- Specific fluorescent probe DHR 123 (both correspond to type I ROS) and 1 The O2-specific probe ABDA (corresponding to type II ROS) was used in the experiment. The specific method is as follows: Ir5 from Example 3, Ir1-2 from Examples 4-5, and Ir3-4 from Examples 6-7 (all 20 μM) were used as test samples. Each sample was separately mixed with HPF (10 μM), DHR 123 (10 μM), and ABDA (100 μM) in PBS buffer to construct three independent mixing systems. Each system was sonicated (1 MHz, 2 W cm⁻¹). -2 (50% duty cycle) Three treatments, 10 minutes each, followed immediately by characterization, with •OH and O2 measured sequentially. ·- and 1 The generation of O2.

[0150] 2. Experimental Results according to Figures 2-4 It can be seen that, with the extension of ultrasonic time, the cyclic metallized Ir(III) complexes in Examples 3-7 gradually increased the fluorescence intensity of HPF and DHR 123. Figures 2-3 ), and caused the absorbance of ABDA to gradually decrease ( Figure 4 This indicates that as the ultrasonic time increases, Ir can generate a large amount of •OH and O2.·- as well as 1 O2 indicates that these cyclic metallized Ir(III) complexes are acoustic sensitizers that can generate type I / II ROS.

[0151] Experimental Example 2: Cellular Uptake of Cyclic Metallized Ir(III) Complexes 1. Experimental Methods Cell uptake efficiency of Ir1–Ir5 cells was assessed by flow cytometry (LSRFortessa, BD Biosciences, USA). SCC1 cells were cultured at 3 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells / well in six-well plates and cultured under normoxic conditions for 12 hours until cell adhesion. The culture medium was then changed: blank medium was added to the control group, and medium containing Ir1–Ir5 (4.0 μM) was added to the experimental groups. The cells were then incubated at 37 °C in a 5% CO2 incubator for another 12 hours. After incubation, cells from each group were collected, washed three times with PBS, and resuspended in 300 μL of PBS solution for flow cytometry analysis. Detection parameters: excitation wavelength (λ… ex The wavelength range is 488 nm, the long-pass filter is 685 nm, and the band-pass filter is 695 / 40 nm. Experimental data were statistically analyzed using FlowJo v10.0 software (BD Life Sciences, USA).

[0152] 2. Experimental Results according to Figure 5 It can be seen that the cyclometalated Ir(III) complexes Ir1-2 and Ir5 in Examples 3-5 all possess good cellular uptake capabilities. Among them, Ir5 and Ir1 in Examples 3-4 exhibit excellent uptake effects, while Ir2 in Example 5, although slightly less efficient, still meets basic requirements. Although the cyclometalated Ir(III) complexes Ir3-4 in Examples 6-7 did not reach optimal cellular uptake levels, their outstanding extracellular ROS generation capacity indicates significant development potential.

[0153] Experimental Example 3: Cytotoxicity Evaluation of Cyclic Metallized Ir(III) Complexes 1. Experimental Methods The cytotoxic effects of sonodynamic therapy on oral squamous cell carcinoma were investigated using the CCK8 assay. Human squamous cell carcinoma cell line (SCC1), human tongue squamous cell carcinoma cell line (HN6), and human oral mucosal precancerous lesion cells (DOK) were seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 3Cells were cultured normally in an incubator (37 ℃, 5% CO2) until they adhered to the culture vessel. The original culture medium was then aspirated, and different concentrations of Ir1-2, Ir5 (products of Examples 3-5), and Ir3-Ir4 (products of Examples 6-7) were added to each well (100 μL per well, 3 parallel wells). Blank culture medium and untreated cells were also included as control wells. After 24 h of culture, the cells were sonicated (1 MHz, 2 W cm⁻¹). -2 The cells were treated with a 50% duty cycle for 15 min and incubated for another 24 h. 100 μl of CCK8 solution was added to each well and incubated at 37 ℃ for 1 h. The absorbance (OD) of each well was measured at 450 nm using a multi-functional microplate reader. Cell viability was calculated as follows: Cell viability % = (OD value of experimental group – OD value of blank group) / (OD value of control group – OD value of blank group).

[0154] 2. Experimental Results Table 1. Cytotoxicity test results (unit: μM)

[0155] Note: No ultrasound condition (NS); Ultrasonic condition (US); Acoustic toxicity coefficient (SI): the ratio of IC50 values ​​obtained under non-ultrasonic and ultrasonic conditions.

[0156] As shown in Table 1, Ir1-Ir5 exhibit low dark cytotoxicity and good therapeutic potential against SCC1, HN6, and DOK cells, specifically: the dark cytotoxicity of this series of complexes against SCC1, HN6, and DOK cells is lower than that of cisplatin, meaning that their spontaneous toxicity is lower under ultrasound-free conditions, significantly reducing the risk of non-targeted damage to normal tissues and cells, and resulting in better therapeutic safety. Under ultrasound, Ir1-Ir2 and Ir5 show excellent tumor cell killing effects, while Ir3-4 in Examples 6-7 have slightly lower sonotoxicity coefficients due to insufficient cellular uptake. Among the three cell lines mentioned above, the SI of Ir1 is 21.15-28.50, the SI of Ir2 is 18.79-21.87, and the SI of Ir5 is as high as 49.53-77.08, all three showing significantly better performance than Ir3-4 and cisplatin. In summary, the cyclic metallized Ir(III) complexes of Examples 3-5 not only exhibit low dark toxicity under ultrasound, but also possess significant antitumor activity. Among them, the Ir5 complex of Example 3 showed the best therapeutic activity, and therefore it was selected as the preferred material for further characterization studies.

[0157] Example 4: Characterization of Cyclic Metallized Ir(III) Complexes 1. Experimental Methods The cyclic metallized Ir(III) complex Ir5 from Example 3 was analyzed by nuclear magnetic resonance spectroscopy using a Bruker Advance III 400 MHz spectrometer (Bruker, Germany) and by mass spectrometry using a Thermo Scientific LTQ linear ion trap mass spectrometer (Thermo Scientific, USA).

[0158] 2. Experimental Results The mass spectrometry analysis results are as follows: 1 HNMR (400 MHz, DMSO- d 6) δ 9.45 (d, J = 8.3 Hz, 2H), 9.07 (d, J = 8.7 Hz, 1H), 9.04 (d, J = 5.2 Hz, 2H), 8.79 (t, J = 7.7 Hz, 2H), 8.41 (d, J = 8.5 Hz, 2H), 8.17 (td, J = 8.4, 2.8 Hz, 2H), 8.10 (dd, J = 8.2, 3.5Hz, 4H), 8.01 (ddd, J = 12.2, 8.4, 5.3 Hz, 2H), 7.81 (d, J = 5.1 Hz, 1H), 7.63(d, J = 3.8 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 7.22(dt, J = 9.3, 4.9 Hz, 4H), 7.17 (d, J = 7.5 Hz, 1H), 6.79 – 6.70 (m, 6H), 4.22(s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ167.71, 162.30, 159.34, 158.66, 149.32, 147.23, 146.42, 145.37, 143.44, 143.36, 143.31, 143.27, 143.03, 142.87, 138.84, 138.73, 135.11, 134.08, 133.03, 132.87, 130.29, 130.22, 129.80, 129.50, 128.46, 128.34, 127.64, 127.58, 127.20, 126.85, 126.70, 126.34, 126.20, 126.13, 125.02, 124.39, 124.28, 124.23, 123.58, 123.34, 123.24, 122.70, 121.92, 121.01, 29.00. Electrospray ionization mass spectrometry m / z (Methanol): Theoretical value is [M–Cl] + [C] 60 H 36 IrN6S3] + :1129.18; Measured value:1129.36. Purity:97.1% (determined by high performance liquid chromatography).

[0159] Experimental Example 5: Cellular Sublocalization Analysis and Organelle Damage Analysis of Cyclic Metallized Ir(III) Complexes 1. Cellular sublocalization analysis of Ir(III) complexes (1) Experimental methods The colocalization of the cyclic metallized Ir(III) complex (Ir5) with lysosomes in Example 3 was determined. SCC1 cells were incubated with Ir5 (8 μM) for 6 h, followed by incubation with the lysosomal deep red probe (LTDR, 1 μM) for 15 min. The cells were then photographed using a confocal laser scanning microscope (FV3000, Olympus, Japan), and the results were analyzed using the Oly FV3000 software platform. Scale bar: 10 μm.

[0160] (2) Experimental results according to Figure 6 Experimental results for A: After co-incubation with cells, Ir5's localization highly overlapped with the fluorescence localization of LTDR in the cells, with a Pearson correlation coefficient (PCC) of 0.71, indicating that it can effectively target lysosomes.

[0161] 2. Analysis of lysosomal damage in cells using acridine orange (AO) dye. (1) Experimental methods SCC1 cells were seeded in confocal culture dishes and, after normal culture and adhesion, were incubated with culture medium and Ir5 (product of Example 3, concentration 4.0 μM) for 24 hours, followed by sonication (1 MHz, 2 W cm⁻¹). -2 After 50% duty cycle, the solution was incubated for 15 min and then 6 h incubated. The solution was then stained with 5 μM AO working solution at 37 °C in the dark for 15 min. After rinsing with PBS to remove excess dye, the solution was photographed using a confocal laser scanning microscope (FV3000, Olympus, Japan). The results were analyzed using the Oly FV3000 software platform. Scale bar: 20 μm.

[0162] (2) Experimental results Figure 6 The experimental results of B showed that in the control group, the ultrasound-only group, and the Ir5-only group, the AO dye emitted red dotted fluorescence, indicating good lysosomal integrity. This confirmed the low dark toxicity of Ir5 and also confirmed the safety of ultrasound treatment. Furthermore, the red dotted fluorescence of Ir5 (4.0 μM) disappeared after ultrasound treatment, indicating that this ring-metallized Ir(III) complex can significantly induce lysosomal membrane rupture and severe lysosomal damage under ultrasound, thereby killing tumor cells.

[0163] Experiment 6: RNA Sequencing SCC1 cells were treated with the drug (Ir5, a cyclic metallized Ir(III) complex from Example 3, at a concentration of 4.0 μM) and cultured at 37°C for 24 h. Total RNA was extracted using an RNA rapid purification kit and then sequenced.

[0164] 1. Experimental Methods Total RNA was subjected to quality checks and quantitative analysis using a Qubit quantitative PCR instrument and a Qsep400 high-throughput biological fragment analyzer, respectively, to assess RNA integrity and concentration. Only samples with an RNA integrity score of 7 or higher were used for subsequent sequencing steps. RNA sequencing was performed by Wuhan Maiwei Metabolism Biotechnology Co., Ltd. After constructing mRNA libraries, different libraries were sequenced using the Illumina HiSeq™ sequencing platform.

[0165] Gene expression levels were quantified using the featureCounts method, and alignment statistics were calculated. Next, based on gene length, the fragment-per-kilobase (FPKM) and transcript-per-million (TPM) values ​​were calculated for each gene. For data analysis, genes with an absolute Log2 (fold change) value ≥1 and a false discovery rate (FDR) significance score <0.05 were defined as differentially expressed genes (DEGs) for subsequent analysis. Finally, gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package.

[0166] 2. Experimental Results like Figure 7 As shown in the gene volcano plot of A, RNA sequencing (RNA-seq) analysis revealed 4483 significantly differentially expressed genes (DEGs) compared to the control group, of which 3532 genes were upregulated and 951 genes were downregulated. Further gene set enrichment analysis (GSEA) yielded the following results: Figure 7 As shown in B, after Ir5 treatment in Example 3, the genes responsible for cellular redox activity were significantly downregulated ( Figure 7 (Left figure B), while genes related to cellular pathways associated with inflammatory responses were significantly upregulated ( Figure 7 (Figure B, right). This shows that Ir5-mediated SDT therapy significantly disrupts cellular redox reactions, while simultaneously initiating cellular inflammatory responses and promoting tumor cell death.

[0167] Experimental Example 7: Analysis of Antitumor Effect 1. Oxidative stress analysis The ability of S1Ir5 cells to produce ROS (1) Experimental methods The ability of the cyclic metallized Ir(III) complex Ir5 in Example 3 to generate ROS was determined by flow cytometry. Specifically, well-grown SCC1 cells were collected, routinely washed, digested, and counted, at a concentration of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured normally in an incubator (37 ℃, 5% CO2) until cell adhesion. Ir5 (product of Example 3, concentrations of 1.0 μM, 2.0 μM, and 4.0 μM) was added, with drug-free cell culture medium as a control. Each group had 3 replicates, and the cells were incubated in a 37 ℃ incubator in the dark for 24 h. Sonication (1 MHz, 2 W cm⁻¹) was then performed. -2The cells were incubated for 15 min at 50% duty cycle. After 4 h of incubation, the cells were washed with PBS, and 10 μM DCFH-DA dye was added. The cells were then incubated at 37 °C in the dark for 30 min. The cells were washed with PBS, digested with trypsin for 2 min, and digestion was terminated by adding complete culture medium. The cell suspension was collected in flow cytometry tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in PBS, centrifuged again, the supernatant was discarded, and 300 mL of PBS was added to each tube to resuspend the cells before flow cytometry analysis.

[0168] (2) Experimental results according to Figure 8 The experimental results of A showed that in cells treated with Ir5-mediated sonodynamic therapy (Ir5-SDT), the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) increased in a concentration-dependent manner. At an Ir5 concentration of 4.0 μM and under sonication treatment, the intracellular ROS level was significantly increased compared to the control group (0 μM), approximately 4.8 times that of the control group. Figure 8 (Statistical chart of data A), the test results show that it can effectively generate reactive oxygen species in tumor cells, thereby achieving anti-tumor effects.

[0169] S2. Analyze intracellular oxidative stress levels by detecting glutathione (GSH) levels. (1) Experimental methods SCC1 cells were cultured in 12-well plates, and Ir5 (the product of Example 3, with concentrations of 1.0 μM, 2.0 μM, and 4.0 μM) was added. Drug-free cell culture medium was used as a control. Each group had three replicates, and the cells were incubated at 37 ℃ in the dark for 24 h. Sonication (1 MHz, 2 W cm⁻¹) was then performed. -2 Cells were incubated at 50% duty cycle for 15 min. After 4 h of incubation, cells were washed with PBS, centrifuged, and counted. Next, cells were rapidly frozen and thawed twice, centrifuged at 8000 g for 10 min each time. Finally, the supernatant was placed in a GSH quantification kit for total GSH determination.

[0170] (2) Experimental results like Figure 8 As shown in Figure B, GSH is a key antioxidant that protects cells from oxidative stress. In SCC1 cells treated with Ir5 sonication, the GSH levels were significantly reduced. These results indicate that Ir5 sonication treatment can effectively induce intracellular oxidative stress responses, thereby exerting a potent anti-tumor effect.

[0171] 2. Analysis of cell death and its modes S1. Cell viability staining analysis (1) Experimental methods SCC1 cells were seeded in confocal culture dishes and, after normal culture and adhesion, were incubated with culture medium and Ir5 (product of Example 3, 4.0 μM) for 24 hours, followed by sonication (1 MHz, 2 W cm⁻¹). -2 After 50% duty cycle treatment for 15 min and 6 h incubation, the cells were stained with Calcein / PI cell live / dead dye working solution at 37 °C in the dark for 15 min. After rinsing with PBS to remove excess dye, the cells were photographed using a confocal laser scanning microscope (FV3000, Olympus, Japan). The results were analyzed using the Oly FV3000 software platform. Scale bar: 50 μm.

[0172] (2) Experimental results like Figure 9 As shown, in the control group, the ultrasound-only group, and the Ir5-only group, cells emitted green fluorescence, and almost no red fluorescent cells were observed, indicating that live cells constituted the vast majority. However, when Ir5 (4.0 μM) was treated with ultrasound, a large number of cells emitted red fluorescence, indicating that this cyclic metallized Ir(III) complex could significantly kill tumor cells under ultrasound.

[0173] S2. Analysis of cell death-related protein expression levels using Western blotting. (1) Experimental methods SCC1 cells were cultured with Ir5 (the product of Example 3, at concentrations of 0, 1.0, 2.0, 4.0, and 6.0 μM) in a 37°C incubator for 24 h, followed by sonication (1 MHz, 2 W cm⁻¹). -2 Cells were treated with a 50% duty cycle for 15 min, and collected 6 h later. The collected cells were lysed on ice for 20 min with RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor. Protein concentration was quantified using BCA, and proteins were separated by SDS-PAGE gel electrophoresis. The separated proteins were transferred from the gel to a PVDF membrane and incubated overnight with primary antibody at 4 °C. After washing, the membrane was incubated with secondary antibody. Chemiluminescent substrate was added, and the membrane was developed under a gel imaging system. Images were acquired, protein expression was detected, and analysis was performed using ImageJ software.

[0174] (2) Experimental results like Figure 10As shown in Figure A, Ir5 (concentrations of 1.0, 2.0, 4.0, and 6.0 μM) induced inflammatory cell death via SDT, and the expression levels of related proteins, caspase-3 splice variant and N-terminal GSDME (N-GSDME), were significantly upregulated with increasing Ir5 concentration. Specifically, compared to the control group (Ir5 concentration of 0 μM), cleaved caspase-3 protein was significantly upregulated as the Ir5 concentration increased from 1.0 μM to 6.0 μM. Figure 10 Statistical analysis of the data (B) showed that this change was significantly different when the Ir5 concentration reached 4.0 μM and 6.0 μM. P <0.0001). Furthermore, the full-length GSDME (FL-GSDME) protein gradually cleaved to produce the functional protein N-GSDME. These results indicate that Ir5 sonication can induce inflammatory cell death via a GSEME-dependent pathway, a cell death mechanism that effectively induces an immune response.

[0175] S3. Observe the changes in cell morphology after Ir5-SDT treatment using transmission electron microscopy.

[0176] (1) Experimental methods SCC1 cells in good growth condition were seeded into six-well plates and cultured at 37 ℃ to allow cell adhesion. SCC1 cells were then cultured with Ir5 (product of Example 3, concentrations of 0 and 4.0 μM) at 37 ℃ for 24 h, followed by sonication (1 MHz, 2 W cm⁻¹). -2 After incubation for 15 min at 50% duty cycle for 6 h, the culture medium was discarded, and the cells were digested with trypsin, centrifuged, and collected. The collected cells were washed three times with pre-cooled PBS, centrifuged at 1000 rpm for 5 min, and the resulting cell clusters were fixed overnight at 4 °C with glutaraldehyde. After staining with osmium tetroxide, the cell morphology changes were observed using a transmission electron microscope (TEM, Hitachi HT7800, Japan). Scale bars: 5 µm and 2 µm.

[0177] (2) Experimental results according to Figure 11 Experimental results: Compared with the control group ( Figure 11 A) Compared to cells with intact organelles and plasma membranes, cells treated with Ir5-SDT, the product of Example 3 (A) Figure 11 B) Exhibits typical ultrastructural features of cell damage. Specifically, the treated cells are significantly swollen, producing large vesicles, and cell membrane rupture and leakage of cytoplasmic contents are also visible. This indicates that the structural integrity of tumor cells has been disrupted, ultimately leading to tumor cell death.

[0178] 3. Immunogenicity and death effect analysis During typical immunogenic cell death, tumor cells express damage-associated molecular patterns (DAMPs), characterized by ATP release, high-mobility group box 1 (HMGB1) release, and calreticulin (CRT) exposure. DAMPs promote antigen-presenting cell activation and T cell activation, thereby enabling anti-tumor immunotherapy. Meanwhile, programmed death ligand (PD-L1), a protein expressed on the surface of both tumor and immune cells, inhibits T cell immune activity upon binding to programmed death protein 1 (PD-1). Insufficient PD-L1 expression on tumor cell surface is one of the causes of immunotherapy resistance, while increased PD-L1 expression on tumor cell surface is beneficial for improving the efficacy of immunotherapeutic drugs.

[0179] Effects of S1.Ir5 sonication on ATP release levels (1) Experimental methods Take SCC1 cells in good growth condition and use 6×10 3 Cells were seeded at a density of 10 cells / well in 24-well plates and incubated at 37 °C until adherence was achieved. Ir5 (product of Example 3, concentrations of 0, 1.0, 2.0, and 4.0 μM) was added, and the plates were incubated at 37 °C in the dark for 24 h. The cells were then sonicated (1 MHz, 2 W cm⁻¹). -2 After incubation for 15 min at 50% duty cycle, the supernatant was collected after 6 h of incubation. The supernatant was then transferred to a 96-well plate with a black wall and a transparent bottom, ATP assay reagent was added, and ATP levels were detected using chemiluminescence immunoassay on a microplate reader.

[0180] (2) Experimental results Chemiluminescence detection such as Figure 12 The results showed that Ir5 sonication significantly increased ATP release from cell supernatants, with the increase increasing with increasing drug concentration. Specifically, compared with the untreated control group (0 μM), the ATP content in cell culture supernatants treated with 1.0 μM, 2.0 μM, and 4.0 μM Ir5-SDT was upregulated. The P-value for upregulation of ATP in cell culture supernatants after sonication treatment with 2.0 μM Ir5 was 0.031, and the P-value for upregulation after sonication treatment with 4.0 μM Ir5 was 0.0136, indicating that this upregulation was statistically significant. This confirms that Ir5 sonication treatment can effectively promote ATP release from cells.

[0181] Effects of S2.Ir5 sonication on intracellular HMGB1 and cell surface CRT expression (1) Experimental methods Take SCC1 cells in good growth condition, at 2×10⁻⁶ 5Cells were seeded at a density of 10 cells / well in 6-well plates and incubated at 37 °C to allow cell adhesion. After washing with PBS, Ir5 (the product of Example 3, at concentrations of 0, 1.0, 2.0, and 4.0 μM) was added, and the cells were sonicated (1 MHz, 2 W cm⁻¹). -2 After treatment with 50% duty cycle for 15 min, cells were incubated for 6 h, then collected, washed three times with PBS, and fixed with 4% PFA at room temperature for 15 min. To detect the expression of high-mobility group box 1 (HMGB1) in cells, cells were permeabilized with 0.1% Triton X-100 at room temperature for 15 min, then incubated with PE anti-HMGB1 Antibody at 4 °C for 30 min, and analyzed by flow cytometry. To detect extracellular calreticulin (CRT), fixed cells (without Triton X-100 permeabilization) were incubated with Calreticulin (D3E6) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) at 4 °C for 30 min, and analyzed by flow cytometry.

[0182] (2) Experimental results according to Figure 13 Flow cytometry results showed that the intranuclear HMGB1 content decreased significantly with increasing Ir5 treatment concentration. At 1.0 μM Ir5 sonication, the HMGB1 content already decreased significantly (P < 0.0001). With further increases in Ir5 concentration to 2.0 μM and 4.0 μM, HMGB1 levels decreased further (P < 0.0001 in both cases). Figure 13 (Data statistics chart for A). Meanwhile... Figure 13 Experiment B showed that Ir5 sonication significantly increased cell surface calreticulin (CRT), and the increase was more pronounced with increasing concentration. Specifically, compared with the untreated control group (0 μM), cell surface CRT exposure was significantly increased at Ir5-SDT concentrations of 1.0 μg / mL, 2.0 μg / mL, and 4.0 μg / mL, and this increase was statistically significant. Figure 13 (Data statistics for B). These results indicate that Ir5 sonication effectively downregulates the level of HMGB1 in the cell nucleus while promoting CRT exposure on the cell surface.

[0183] Analysis of PD-L1 expression levels in tumor cells after S3.Ir5 ultrasound treatment (1) Experimental methods RT-qPCR analysis of PD-L1 gene expression: SCC1 cells were cultured with Ir5 (product of Example 3, concentrations of 1.0, 2.0, and 4.0 μM) in a 37 ℃ incubator for 24 h, followed by sonication (1 MHz, 2 W cm⁻¹). -2 After 15 min of incubation at 50% duty cycle, and 6 h of incubation, total RNA was extracted from the sample according to the kit instructions. An RT-qPCR reverse transcription system was prepared and reverse transcribed using a reverse transcriptase instrument. An RT-qPCR reaction system was then prepared and placed in a LightCycler 96 real-time quantitative PCR instrument to begin the reaction. The target gene and internal reference gene were mixed at a ratio of 2... -ΔΔCt The method calculates relative gene expression levels and normalizes them using GAPDH gene expression levels.

[0184] Western blot detection of PD-L1 protein expression: SCC1 cells were cultured with Ir5 (1.0, 2.0, 4.0, 6.0 μM) in a 37 ℃ incubator for 24 h, and then sonicated (1 MHz, 2 W cm⁻¹). -2 After treatment with a 50% duty cycle for 15 min, cells were collected after 6 h of incubation. The collected cells were lysed on ice for 20 min with RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor. Protein concentration was quantified using BCA, and proteins were separated by SDS-PAGE gel electrophoresis. The separated proteins were transferred from the gel to a PVDF membrane and incubated overnight with primary antibody at 4 °C. After washing, the membrane was incubated with secondary antibody. Chemiluminescent substrate was added, and the membrane was developed under a gel imaging system. Images were acquired, protein expression was detected, and analysis was performed using ImageJ software.

[0185] (2) Experimental results according to Figure 14 The experimental results of A showed that Ir5 (concentrations of 1.0, 2.0, and 4.0 μM) induced upregulation of programmed death-ligand (PD-L1) at the gene level via SDT. Specifically, compared with the control group (0 μM), treatment with 4.0 Mm Ir5 significantly upregulated the intracellular PD-L1 gene expression level (P = 0.0002), which was statistically significant. Figure 14 The experimental results of B showed that, compared with the control group, SCC1 tumor cells treated with 6.0 μM Ir5 ultrasound showed significantly upregulated PD-L1 protein expression (P=0.0474), which was statistically significant. Figure 14 C). These results indicate that Ir5 ultrasound can effectively upregulate PD-L1 expression levels in a concentration-dependent manner.

[0186] In summary, the above test results further demonstrate that Ir5 ultrasound treatment can effectively induce immunogenic death in tumor cells and significantly upregulate PD-L1 expression, thereby exerting an immunomodulatory effect.

[0187] 4. Using a CDX infection model, evaluate the in vivo antitumor effect and immunomodulatory capacity of Ir5 ultrasound treatment. (1) Experimental methods Four-week-old female C3H-HeN mice were housed in the SPF-grade barrier system of South China Agricultural University. Their food and water were sterilized and provided to them freely in the animal room.

[0188] 100 μL (1×10⁻⁶) was subcutaneously injected into the right axilla of C3H-HeN mice. 6 Live cell suspensions of SCC7 (squamous cell carcinoma line) were used to establish a tumor model, combined with sonodynamic therapy. When the tumor volume was approximately 150 mm²... 3 The tumors were randomly divided into four groups of five each: saline (50 μL); PD-L1 inhibitor (BMS1, 50 μL, 2.5 mg / kg); Ir5 (50 μL, 5 mg / kg) + ultrasound; and Ir5 + ultrasound + PD-L1 inhibitor. Ir5 was injected intratumorally every two days for a total of five times. Ultrasound (1 MHz, 2 Wcm²) was used after each injection. -2 Sonodynamic therapy was administered for 15 minutes (50% duty cycle). PD-L1 inhibitors were injected intraperitoneally every 3 days for a total of 5 times. Tumor volume and body weight were recorded every other day from the date of administration. Mice were sacrificed 16 days after treatment, tumors were removed, and weighed. A portion of the tumor tissue was fixed in 10% buffered formalin, embedded in paraffin, and stained with immunohistochemical staining (IHC) for H&E, Ki67, and PD-L1, with scale bars of 50 μm (H&E) and 20 μm (IHC), respectively. Flow cytometry and cytokine detection were performed on a portion of the tumor tissue. Major organs such as the heart, liver, spleen, lungs, and kidneys were taken from mice, embedded in paraffin, and subjected to histopathological examination, with a scale bar of 100 μm.

[0189] (2) Experimental results Based on the tumor volume growth curve ( Figure 15 A) Gross image of tumor tissue ( Figure 15 B) and tumor tissue weight bar chart ( Figure 15 As shown in C), both Ir5 ultrasound treatment and PD-L1 inhibitors significantly inhibited tumor growth, and their combined effect further significantly promoted tumor regression; these anti-tumor effects were statistically significant. According to the H&E results (… Figure 15(D) In ​​the group treated with the combination of Ir5-SDT and PD-L1 inhibitors, tumor tissue showed severe necrosis, significant vacuolation, extensive nucleus loss, and marked nuclear pyknosis, indicating that this treatment regimen has an effective anti-tumor killing effect. Immunohistochemical staining results showed ( Figure 15 (D) The efficacy of the Ir5 ultrasound treatment group was comparable to that of PD-L1 inhibitors alone, and the Ki67 positivity rate was further reduced in the combination therapy group with PD-L1 inhibitors, indicating that this treatment regimen has a significant inhibitory effect on tumor cell proliferation. Furthermore, in the Ir5 ultrasound treatment group, the PD-L1 positivity rate was upregulated, while in the Ir5 ultrasound treatment combined with PD-L1 inhibitors group, PD-L1 expression was significantly downregulated, with levels lower than those treated with PD-L1 inhibitors alone. This suggests that Ir5 ultrasound treatment-induced PD-L1 upregulation enhances the efficacy of the immunotherapy drug PD-L1 inhibitors.

[0190] Meanwhile, no obvious toxic side effects were found in the H&E sections of the major organs of mice. Figure 15 E), and during the treatment, the body weight of mice in all groups did not change significantly, and there was no sharp decrease or increase in body weight in a short period of time. Figure 15 F) indicates that the medication is safe.

[0191] Flow cytometry results showed that in the group treated with Ir5 ultrasound and combined with PD-L1 inhibitors, PD-L1 levels on the surface of local dendritic cells and macrophages in the tumor were significantly downregulated. Figure 16 A), tumor-specific CD8 + T cell infiltration levels are upregulated ( Figure 16 B), and the combination of Ir5 ultrasound treatment and PD-L1 inhibitor therapy significantly reduced CD4. + T and CD8 + PD-1 on the T surface ( Figure 16 (C) This indicates that the treatment regimen can reduce the inhibition of dendritic cells and macrophages, increase the infiltration of T cells in the tumor site, alleviate T cell inhibition, restore the killing function of T cells, and has a synergistic effect in enhancing anti-tumor immunity.

[0192] Cytokine ELISA Detection Results Figure 17 The results showed that, compared with the control group, Ir5 ultrasound treatment and the application of PD-L1 inhibitors alone were equally effective, both upregulating tumor necrosis factor α (TNF-α) in tumor tissue. Figure 17 A) Interferon-γ (IFN-γ) Figure 17 B) Interleukin-6 (IL-6) Figure 17 C). In particular, the combined use of Ir5 ultrasound treatment and PD-L1 inhibitors further upregulated the levels of cytokines in tumor tissues, confirming that Ir5 ultrasound treatment combined with PD-L1 inhibitors can significantly enhance the anti-tumor immune response.

[0193] The above test results confirm that the combination therapy of cyclic metallized Ir(III) complex Ir5 with ultrasound treatment and PD-L1 inhibitor has a strong tumor-suppressive effect and can alleviate the immunosuppressive tumor microenvironment. It synergistically enhances anti-tumor adaptive immunity, which is feasible and has high biosafety.

[0194] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A class of cyclometalated Ir(III) complexes, characterized in that, The cyclometalated Ir(III) complex has any of the following structural formulas: 。 2. The method for preparing the cyclic metallized Ir(III) complex according to claim 1, characterized in that, Includes the following steps: The bridged iridium precursor and the organic ligand were mixed and dispersed in an organic solvent, reacted fully, and purified to obtain the cyclometalated Ir(III) complex. The structural formula of the bridging iridium precursor is shown below: ； The organic ligand has any of the following structural formulas: 。 3. The preparation method according to claim 2, characterized in that, The preparation of the bridging iridium precursor includes the following steps: Under a protective gas atmosphere, iridium trichloride or its hydrate and 6-(benzo[b]thiophene-2-yl)phenanthridine are mixed and dispersed in a solvent, reacted completely, and then post-treated to obtain the bridged iridium precursor.

4. The use of the cyclic metallized Ir(III) complex of claim 1 in the preparation of anti-oral squamous cell carcinoma materials or anti-oral squamous cell carcinoma drugs.

5. A sonodynamic therapeutic agent for oral squamous cell carcinoma, characterized in that, The sonodynamic therapeutic agent against oral squamous cell carcinoma comprises the cyclic metallized Ir(III) complex of claim 1.

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