Iridium (III) complex as well as preparation method and application thereof in head and neck cancer

Abstract

The invention relates to an iridium (III) complex as well as a preparation method and application thereof in head and neck cancer, and belongs to the technical field of biological medicines. The novel iridium (III) complex provided by the invention has cytotoxicity to head and neck cancer, and the cytotoxicity is better than that of cis-platinum CDDP. The iridium (III) complex acts on head and neck cancer, and can inhibit migration and proliferation of tumor cells, induce apoptosis of the tumor cells, induce CRT expression quantity increase and overturn to cell surfaces, induce HMGB1 expression quantity increase and transfer to cytoplasm and release, induce tumor cells to release more ATP, activate endoplasmic reticulum stress pathways, improve Ca < 2 + > level in the tumor cells, inhibit tumor cell apoptosis, inhibit CRT expression quantity increase and overturn to cell surfaces, induce HMGB1 expression quantity increase and transfer to cytoplasm and release and inhibit tumor cell apoptosis. The mitochondrial membrane potential of tumor cells is reduced, and the level of active oxygen is improved; tumor growth is inhibited, tumor apoptosis is promoted, tumor immunogen cell death is induced through endoplasmic reticulum stress, and tumor immune response is induced. A new choice is provided for preparing the medicine for treating the head and neck cancer.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an iridium (III) complex and a preparation method thereof and application thereof in head and neck cancer. Background Art

[0002] Head and neck cancer (HNC), that is, head and neck tumors include tumors originating from any tissue or organ of the head and neck except the eyes, brain, ears, thyroid and esophagus. More than 90% of head and neck tumors are squamous cell carcinomas (HNSCC). Smoking and drinking are the most common risk factors for head and neck cancer. Other risk factors include infection with human papillomavirus (HPV), poor oral hygiene and nutritional deficiencies. Early diagnosis of head and neck cancer is crucial for prognosis. However, because its symptoms may not be obvious, many patients are already in the late stage when diagnosed. Advanced head and neck cancer is more difficult to treat and has a poorer prognosis. Treatments for head and neck cancer include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Chemotherapy is a common and effective treatment for advanced or metastatic head and neck cancer. However, these drugs have limitations such as multidrug resistance, hepatotoxicity, nephrotoxicity, nerve damage and immunosuppression. New anti-tumor metal drugs with lower toxicity are needed to improve patient prognosis. Therefore, researchers began to look for alternatives to platinum drugs, especially iridium (III) metal complexes. This type of metal element is similar to platinum in chemical properties, has rich multi-coordination characteristics and structural diversity, and can be flexibly regulated by ligands. Therefore, it has attracted much attention in drug development. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new iridium (III) complex and its preparation method and application in head and neck cancer.

[0004] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect, the present invention provides an iridium (III) complex, wherein the iridium (III) complex comprises: [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6, and the structural formulas thereof are:

[0005] The present invention prepares a new iridium (III) complex by using a new ligand IPM to react with precursors Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2, and Cis-[Ir(ppy)2Cl]2. The iridium (III) complex has cytotoxicity to head and neck cancer and has the effect of treating head and neck cancer. The molecular formula of [Ir(piq)2(IPM)]PF6 is: C50 H 34 IrN6O; [Ir(bzq)2(IPM)]PF6 molecular formula is: C 46 H 30 IrN6O; [Ir(ppy)2(IPM)]PF6 molecular formula is: C 42 H 30 IrN6O.

[0006] In a second aspect, the present invention provides a ligand IPM of an iridium (III) complex, the structural formula of which is: Its molecular formula is: C 20 H 14 N4O.

[0007] In the third aspect, the present invention provides a method for preparing the ligand IPM of the iridium (III) complex, wherein 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate are dissolved in glacial acetic acid, heated to reflux for sufficient reaction, cooled to room temperature, the pH is adjusted to neutral, the precipitate is collected and dried to obtain the ligand IPM; the molar ratio of the 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate is 1:1:(20-25). The present invention utilizes the reaction of 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate to prepare a new iridium (III) complex ligand IPM.

[0008] As a preferred embodiment of the method for preparing the ligand IPM of the iridium (III) complex of the present invention, the heating temperature is 130°C and the reaction time is 2 hours. As a preferred embodiment of the method for preparing the ligand IPM of the iridium (III) complex of the present invention, the molar ratio of 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate is 1:1:20.

[0009] In a fourth aspect, the present invention provides a method for preparing the iridium (III) complex, comprising mixing Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2 or Cis-[Ir(ppy)2Cl]2 with a ligand IPM, dissolving the mixture in a mixed solution of dichloromethane and methanol, heating () to reflux in an inert gas environment for a full reaction; cooling to room temperature, adding NH4PF6, stirring, collecting the filtrate, and drying the obtained solution after rotary evaporation of the filtrate to obtain a crude product; the crude product is subjected to neutral alumina column chromatography with dichloromethane and methanol for chromatographic separation; The product is eluted with alkane and acetone to obtain [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6 respectively; the molar ratio of Cis-[Ir(piq)2Cl]2 to the ligand IPM is (1-1.5):2; the molar ratio of Cis-[Ir(bzq)2Cl]2 to the ligand IPM is (1-1.5):2; the molar ratio of Cis-[Ir(ppy)2Cl]2 to the ligand IPM is (1-1.5):2.

[0010] According to the present invention, after Cis-[Ir(piq)2Cl]2 is fully reacted with the ligand IPM, the filtrate collected is subjected to rotary evaporation to obtain a dark red solution, and the crude product thereof is dark brown. After elution, an orange-yellow band is collected, and [Ir(piq)2(IPM)]PF6 is obtained by concentration. After Cis-[Ir(bzq)2Cl]2 is fully reacted with the ligand IPM, the filtrate collected is subjected to rotary evaporation to obtain a yellow solution, and the crude product thereof is yellow. After elution, a bright yellow band is collected, and [Ir(bzq)2(IPM)]PF6 is obtained by concentration. After Cis-[Ir(ppy)2Cl]2 is fully reacted with the ligand IPM, the filtrate collected is subjected to rotary evaporation to obtain a yellow solution, and the crude product thereof is yellow. After elution, a yellow band is collected, and [Ir(ppy)2(IPM)]PF6 is obtained by concentration.

[0011] As a preferred embodiment of the method for preparing the iridium (III) complex of the present invention, the molar ratio of Cis-[Ir(piq)2Cl]2 to the ligand IPM is 1:2; the molar ratio of Cis-[Ir(bzq)2Cl]2 to the ligand IPM is 1:2; the molar ratio of Cis-[Ir(ppy)2Cl]2 to the ligand IPM is 1:2. As a preferred embodiment of the method for preparing the iridium (III) complex of the present invention, the heating temperature is 40°C and the reaction time is 6h. As a preferred embodiment of the method for preparing the iridium (III) complex of the present invention, the volume ratio of dichloromethane to methanol is 2:1; the volume ratio of dichloromethane to acetone is 5:1. As a preferred embodiment of the method for preparing the iridium (III) complex of the present invention, the inert gas is argon.

[0012] In a fifth aspect, the present invention provides the use of the above-mentioned iridium (III) complex in the preparation of a drug for treating head and neck cancer.

[0013] The iridium (III) complexes [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6 of the present invention are cytotoxic to various tumor cells of head and neck cancer, and the cytotoxicity is better than that of cisplatin CDDP. The iridium (III) complexes of the present invention act on head and neck cancer, can enter cells through a cell uptake mechanism, inhibit the migration of tumor cells, induce cell cycle arrest at the G0 / G1 phase and thus inhibit the proliferation of tumor cells, induce tumor cell apoptosis, induce CRT expression to increase and flip to the cell surface, induce HMGB1 expression to increase and transfer to the cytoplasm and release, induce tumor cells to release more ATP, activate the endoplasmic reticulum stress pathway, and increase Ca in tumor cells. 2+ level, reduce the mitochondrial membrane potential of tumor cells, increase the level of reactive oxygen; inhibit tumor growth, promote tumor apoptosis, induce tumor immunogenic cell death through endoplasmic reticulum stress, and induce tumor immune response. The iridium (III) complex of the present invention can be used to prepare drugs for treating head and neck cancer. The iridium (III) complex of the present invention is used as a drug, and will not cause chronic organ damage within 40 mg / kg, and has good safety.

[0014] As a preferred embodiment of the application described in the present invention, the iridium (III) complex is [Ir(piq)2(IPM)]PF6 or [Ir(bzq)2(IPM)]PF6. The present invention has found that the iridium (III) complex [Ir(piq)2(IPM)]PF6 or [Ir(bzq)2(IPM)]PF6 has better cytotoxicity to head and neck cancer. As a preferred embodiment of the application described in the present invention, the head and neck cancer includes tongue squamous cell carcinoma, squamous cell carcinoma or pharyngeal squamous cell carcinoma. As a preferred embodiment of the application described in the present invention, the head and neck cancer includes squamous cell carcinoma or pharyngeal squamous cell carcinoma. The present invention has found that the iridium (III) complex has better cytotoxicity to squamous cell carcinoma or pharyngeal squamous cell carcinoma.

[0015] In a sixth aspect, the present invention provides the use of the above-mentioned iridium (III) complex in activating the endoplasmic reticulum stress pathway.

[0016] In a seventh aspect, the present invention provides a drug for treating head and neck cancer, the drug comprising the iridium (III) complex.

[0017] As a preferred embodiment of the drug of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The new iridium (III) complex provided by the present invention has cytotoxicity to various tumor cells of head and neck cancer, and its cytotoxicity is better than that of cisplatin CDDP. The iridium (III) complex of the present invention acts on head and neck cancer, can inhibit the migration and proliferation of tumor cells, induce tumor cell apoptosis, induce CRT expression to increase and flip to the cell surface, induce HMGB1 expression to increase and transfer to the cytoplasm and release, induce tumor cells to release more ATP, activate the endoplasmic reticulum stress pathway, and increase Ca in tumor cells. 2+ level, reduce the mitochondrial membrane potential of tumor cells, increase the level of reactive oxygen species; inhibit tumor growth, promote tumor apoptosis, induce tumor immunogenic cell death through endoplasmic reticulum stress, and induce tumor immune response. It provides a new option for the preparation of drugs for the treatment of head and neck cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the IPM in Example 1 of the present invention; Figure 2 The synthetic route of the ligand IPM and three iridium (III) metal complexes [Ir(piq)2(IPM)]PF6(Ir1), [Ir(bzq)2(IPM)]PF6(Ir2), and [Ir(ppy)2(IPM)]PF6(Ir3) in Example 1 of the present invention; Figure 3 The ultraviolet absorption spectra (A) and fluorescence emission spectra (B) of the iridium (III) metal complexes Ir1, Ir2 and Ir3 in Example 1 of the present invention are shown; Figure 4 The uptake of the complex Ir1-3 in Example 3 of the present invention by SCC7 cells (A) and FADU cells (B) after 24 hours of action; Figure 5 The wound healing conditions of the scratches of SCC7 cells after 24 hours of action of the complex Ir1-3 and CDDP in Example 3 of the present invention (A) and its quantitative analysis performance graph (B), and the wound healing conditions of the scratches of FADU cells (C) and its quantitative analysis performance graph (D); Figure 6 The clone formation of SCC7 cells (A) and its quantitative analysis performance diagram (B) after the complex Ir1-3 and CDDP in Example 3 of the present invention acted on for 24 hours, and the clone formation of FADU cells (C) and its quantitative analysis performance diagram (D); Figure 7 The apoptosis of SCC7 cells (A) and its quantitative analysis performance diagram (B) after the complex Ir1-3 and CDDP in Example 3 of the present invention act for 24 or 48 hours, and the apoptosis of FADU cells (C) and its quantitative analysis performance diagram (D); Figure 8The cycle detection (A) and quantitative analysis performance diagram (C) of SCC7 cells after 24 hours of the action of the complex Ir1-3 and CDDP in Example 3 of the present invention, and the cycle detection (B) and quantitative analysis performance diagram (D) of FADU cells; Fig. 9 The expression of CRT in SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP were treated for 24 hours in Example 4 of the present invention; Fig.10 The expression of CRT in SCC7 cells (A) and its quantitative analysis performance graph (B) after the complex Ir1-3 and CDDP in Example 4 of the present invention are shown, and the expression of CRT in FADU cells (C) and its quantitative analysis performance graph (D); Fig.11 The expression of HMGB1 in SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP were treated for 24 hours in Example 4 of the present invention; Fig.12 The expression of HMGB1 in SCC7 cells after 24 or 48 hours of action of the complex Ir1-3 and CDDP in Example 4 of the present invention (A) and its quantitative analysis performance graph (B), and the expression of HMGB1 in FADU cells (C) and its quantitative analysis performance graph (D); Fig.13 The ATP release in the supernatant of SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP in Example 4 of the present invention acted on the cells for 24 or 48 hours; Fig.14 The co-localization experimental results of the complex Ir1-3 and CDDP in Example 5 of the present invention after 24 hours of action between SCC7 cells (A) and FADU cells (B) and the endoplasmic reticulum, and the co-localization experimental results of SCC7 cells (C) or FADU cells (D) and mitochondria; Fig.15 The results of Western blotting of the complex Ir1-3 and CDDP in Example 6 of the present invention on the expression of proteins related to the endoplasmic reticulum stress pathway in SCC7 and FADU cells; Fig.16 The results of laser confocal microscopy detection of changes in intracellular calcium ion content in SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP in Example 7 of the present invention were treated for 24 hours, and the results of flow cytometry detection of changes in intracellular calcium ion content in SCC7 cells (C) and FADU cells (D) were obtained; Fig.17The results of laser confocal microscopy detection of changes in mitochondrial membrane potential of SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP in Example 8 of the present invention were treated for 24 hours, and the results of flow cytometry detection of changes in mitochondrial membrane potential of SCC7 cells (C) and FADU cells (D) were shown; Fig.18 The results of laser confocal microscopy detection of changes in active oxygen in SCC7 cells (A) and FADU cells (B) after the complex Ir1-3 and CDDP in Example 9 of the present invention were used for 24 hours, and the results of flow cytometry detection of changes in active oxygen in SCC7 cells (C) and FADU cells (D) were used; Fig.19 The survival curve (A) and body weight change curve (B) of mice under the action of the complex Ir1 at different concentrations in Example 10 of the present invention, and the results of HE staining (C) and Masson staining (D) of mouse organs; Fig. 20 The digital image (A) of the mouse xenogeneic tumor tissue, the quantitative analysis graph of the weight (B), the growth curve (C) and the weight change curve of the mouse (D) in Example 11 of the present invention; Fig.21 The digital image (A), the quantitative analysis graph of the weight (B), the growth curve (C) and the weight change curve (D) of the mouse homologous tumor tissue in Example 11 of the present invention; Fig. 22 The figures are the TUNEL fluorescence staining diagram of the mouse homologous tumor tissue (A) and the performance diagram of its quantitative analysis (B), and the TUNEL fluorescence staining diagram of the mouse heterologous tumor tissue (C) and the performance diagram of its quantitative analysis (D) in Example 11 of the present invention; Fig.23 This is an immunofluorescence staining image of CD3, CD4, and CD8 of the same tumor tissue of mice in Example 11 of the present invention; Fig.24 These are the Ki67 immunohistochemical staining images of the mouse homologous tumor tissue (A) and the heterologous tumor tissue (B), and the p-eIF2α immunohistochemical staining images of the mouse homologous tumor tissue (C) and the heterologous tumor tissue (D) in Example 11 of the present invention.

[0020] In the above-mentioned figures, * P<0.05, ** P<0.01, *** P < 0.001; DETAILED DESCRIPTION

[0021] To better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources. Human pharyngeal squamous cell carcinoma cell line FADU, mouse squamous cell carcinoma cell line SCC7, are from ATCC. Balb / c Nude mice, C3H mice and KM mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0022] Example 1 Synthesis and characterization of ligands and complexes 1. Synthesis and characterization of ligand IPM Accurately weigh 0.208g (1.5mmol) 3-methyl salicylaldehyde, 0.315g (1.5mmol) 1,10-phenanthroline-5,6-dione, and 2.31g (30mmol) ammonium acetate, dissolve in 25mL glacial acetic acid, and then place in a three-necked round-bottom flask. Heat in a water bath at 130°C and reflux for 2h. After the reaction is completed, cool to room temperature, neutralize with concentrated ammonia water to neutrality, and precipitate a large amount of pink precipitate. Filter by suction, collect the filter residue and dry it in a vacuum drying oven overnight to obtain the ligand IPM. Yield: 92%. C 20 H 14 N4O HRMS(CH3OH):m / z=325.11([MH] - ). The H NMR spectrum of IPM is shown in Figure 1 shown.

[0023] 2. Synthesis of Precursors (1) Synthesis of the precursor Cis-[Ir(piq)2Cl]2 Accurately weigh 1.04g (4mmol) of iridium trichloride and 1.65g (8mmol) of phenylisoquinoline, place in a three-necked round-bottom flask, and add ethylene glycol ether (60mL) and double distilled water (20mL). Under argon protection, heat at 120℃ and reflux for 24h. After the reaction solution is cooled to room temperature, filter and dry in an oven to obtain a dark red residue. Yield: 81%.

[0024] (2) Synthesis of the precursor Cis-[Ir(bzq)2Cl]2 Accurately weigh 1.04g (4mmol) of iridium trichloride and 1.43g (8mmol) of 7,8-benzoquinoline, place in a three-necked round-bottom flask, and add ethylene glycol ether (60mL) and double distilled water (20mL). Under argon protection, heat at 120℃ and reflux for 24h. After the reaction solution is cooled to room temperature, filter and dry in an oven to obtain a bright yellow residue. Yield: 79%.

[0025] (3) Synthesis of the precursor Cis-[Ir(ppy)2Cl]2 Accurately weigh 1.04g (4mmol) of iridium trichloride and 1.24g (8mmol) of 2-phenylpyridine, place in a three-necked round-bottom flask, and add ethylene glycol ether (60mL) and double distilled water (20mL). Under argon protection, heat at 120℃ and reflux for 24h. After the reaction solution is cooled to room temperature, filter and dry in an oven to obtain a bright yellow residue. Yield: 78%.

[0026] 3. Synthesis of complexes (1) Synthesis of the complex [Ir(piq)2(IPM)]PF6(Ir1) Accurately weigh Cis-[Ir(piq)2Cl]20.255g (0.2mmol) and ligand IPM 0.13g (0.4mmol), place in a three-necked round-bottom flask, add 28mL of dichloromethane and 14mL of methanol solution, and heat at 40°C for 6h under argon protection. After the reaction solution is cooled to room temperature, add excess NH4PF6, continue stirring at room temperature for 2h, filter and collect the filtrate. After rotary evaporation, a deep red solution is obtained, and a dark brown crude product is obtained after vacuum drying. The crude product is eluted with dichloromethane and acetone (v / v5:1) ​​on a neutral alumina column, the orange-yellow band is collected, and concentrated to obtain a red powder. Yield: 80%, C 50 H 34 IrN6OHRMS(CH3CN):m / z=927.2476([M-PF6] + ). 1 H NMR (600MHz, DMSO-d6): δ9.07(d,J=8.7Hz,2H),8.95(s,2H),8.45(d,J=8.1Hz,2H),8.14 (d,J=8.0Hz,1H),8.03(d,J=7.9Hz,2H),7.90(dd,J=14.2,6.9Hz,7H),7.83(d,J=5.0Hz, 2H),7.50(d,J=6.6Hz,2H),7.44(d,J=6.5Hz,2H),7.20(t,J=7.7Hz,2H),7.11(d,J=7.2H z,1H),7.00(t,J=7.4Hz,2H),6.80(t,J=6.7Hz,1H),6.39(d,J=7.6Hz,2H),2.27(s,3H). 13C NMR(151MHz,DMSO-d6):168.39,156.28,155.11,146.46,145.84,143.06,140.99,136.89,132.39,132.21,132.03, 131.03,130.92,130.44,129.77,128.13,126.87,126.21,126.08,126.03,124.94,124.70,122.61,118.21,16.54. (2) Synthesis of the complex [Ir(bzq)2(IPM)]PF6(Ir2) Accurately weigh Cis-[Ir(bzq)2Cl]20.234g (0.2mmol) and ligand IPM 0.13g (0.4mmol), place in a three-necked round-bottom flask, add 28mL of dichloromethane and 14mL of methanol solution, and heat at 40°C for 6h under argon protection. After the reaction solution is cooled to room temperature, add excess NH4PF6, continue stirring at room temperature for 2h, filter and collect the filtrate. After rotary evaporation, the yellow solution is vacuum dried to obtain a yellow crude product. The crude product is eluted with dichloromethane and acetone (v / v5:1) ​​on a neutral alumina column, collect the bright yellow band, and concentrate to obtain a yellow powder. Yield: 78%, C 46 H 30 IrN6O HRMS(CH3CN):m / z=875.2085([M-PF6] + ). 1 H NMR (600MHz, DMSO-d6): δ8.91(d,J=8.3Hz,2H),8.53(d,J=8.0Hz,2H),8.04–7.99(m,6H),7.90(d,J=8.8Hz,2H),7.74(dd,J=8.3,5.1Hz,2H),7.60(d ,J=8.0Hz,3H),7.49(dd,J=8.1,5.5Hz,2H),7.25(t,J=7.6Hz,2H),7.08(d ,J=7.2Hz,1H),6.77(t,J=7.5Hz,1H),6.40(d,J=7.1Hz,2H),2.24(s,3H). 13C NMR(151MHz,DMSO-d6):157.05,156.25,149.03,148.46,147.04,143.63,140.92,137.85,134.19,132 .04,130.15,129.94,129.04,127.14,126.20,125.93,124.96,124.65,123.19,120.63,118.19,16.50. (3) Synthesis of the complex [Ir(ppy)2(IPM)]PF6(Ir3) Accurately weigh Cis-[Ir(ppy)2Cl]20.215g (0.2mmol) and ligand IPM 0.13g (0.4mmol), place in a three-necked round-bottom flask, add 28mL of dichloromethane and 14mL of methanol solution, and heat at 40°C for 6h under argon protection. After the reaction solution is cooled to room temperature, add excess NH4PF6, continue stirring at room temperature for 2h, filter and collect the filtrate. After rotary evaporation, the yellow solution is vacuum dried to obtain a yellow crude product. The crude product is eluted with dichloromethane and acetone (v / v5:1) ​​on a neutral alumina column, the yellow band is collected, and concentrated to obtain a yellow powder. Yield: 75%, C 42 H 30 IrN6O HRMS(CH3CN):m / z=828.1959([M-PF6] + ). 1 H NMR (600MHz, DMSO-d6): δ11.08(s,1H),9.39(d,J=8.2Hz,1H),9.20(d,J=8.3Hz,1H),8.3 9–8.30(m,5H),8.22(ddd,J=13.1,8.3,5.1Hz,2H),8.11(d,J=7.9Hz,1H),8.03(d,J=7.8 Hz,2H),7.95(t,J=7.9Hz,2H),7.61(d,J=5.9Hz,2H),7.50(d,J=7.3Hz,1H),7.13(t,J=7 .6Hz,2H),7.11–7.05(m,3H),7.03(t,J=7.5Hz,2H),6.37(d,J=7.6Hz,2H),2.33(s,3H). 13C NMR(151MHz,DMSO-d6):167.28,167.26,164.92,156.23,150.75,150.71,150.1 4,150.12,149.87,149.84,145.72,145.35,144.50,144.48,143.04,139.29,135 .82,134.43,133.89,131.92,131.69,131.66,130.78,128.34,128.26,126.79,125.58,125.47,124.48,124.36,124.32,123.01,120.50,120.33,109.69,16.17. The synthetic routes of the ligand IPM and three iridium (III) metal complexes [Ir(piq)2(IPM)]PF6(Ir1), [Ir(bzq)2(IPM)]PF6(Ir2), and [Ir(ppy)2(IPM)]PF6(Ir3) are shown in the figure. Figure 2 shown.

[0027] 4. UV and fluorescence spectroscopic determination of the complex Iridium (III) metal complexes Ir1, Ir2, and Ir3 were dissolved in methanol and then measured for UV absorption and fluorescence emission spectra using UV spectrophotometer and fluorescence spectrophotometer, respectively. Figure 3 A) and fluorescence emission spectra ( Figure 3 B) Figure 3 As shown in the results, the maximum peaks of Ir1, Ir2 and Ir3 in the ultraviolet absorption spectrum are at 278nm, 279nm and 271nm respectively, with good absorption characteristics. The fluorescence range of Ir1, Ir2 and Ir3 is 550-700nm, and their maximum peaks are at 628nm, 610nm and 609nm respectively.

[0028] Example 2 In vitro cytotoxicity test In order to explore the in vitro cytotoxic effect of iridium metal complexes on head and neck cancer cells, the CCK-8 method was used to detect the half inhibition rate IC of complexes Ir1, Ir2, and Ir3 (Ir1-3) on 6 cell lines (all adherent cells): human tongue squamous cell carcinoma cell CAL-27, human tongue squamous cell carcinoma cell CAL-33, human tongue squamous cell carcinoma cell SAS, human tongue squamous cell carcinoma cell SCC25, mouse squamous cell carcinoma cell SCC7, and human pharyngeal squamous cell carcinoma cell FADU. 50 .

[0029] IC of different cells under the action of complex Ir1-3 and cisplatin (CDDP) 50 The values ​​are shown in Table 1 below. The results in Table 1 show that the complexes Ir1-3 have better cytotoxic effects on six head and neck cancer cell lines relative to cisplatin CDDP, especially SCC7 cells and FADU cells have more obvious toxic effects. For SCC7 cells, the complex Ir1 is 0.429±0.074μΜ, Ir2 is 0.529±0.019μΜ, and Ir3 is 0.806±0.013μΜ; for FADU cells, the complex Ir1 is 0.144±0.007μΜ, Ir2 is 0.315±0.027μΜ, and Ir3 is 0.407±0.014μΜ. Therefore, these two cell lines were selected to continue the subsequent experiments.

[0030] Example 3 Cell uptake, migration, proliferation, apoptosis, cell cycle effect test 1. Effect of Cellular Uptake of Complexes In order to further explore the mechanism of action of iridium metal complex Ir1-3 in cells, fluorescence staining was observed under a laser confocal microscope to determine whether cells took up the complex. Figure 4 A) and FADU cells ( Figure 4 B) Intake conditions such as Figure 4 As shown, the complex Ir1-3 is mainly distributed in the cytoplasm of SCC7 cells and FADU cells, indicating that the complex Ir1-3 can enter the cells through the cellular uptake mechanism and achieve effective accumulation in the cells, thereby exerting its effect.

[0031] 2. Effects of complexes on cell migration The cell migration ability was detected by cell scratch test. Figure 5As shown in the figure, after 24h of treatment, compared with the blank control group, the complexes Ir1-3 and CDDP can significantly reduce the wound healing rate of SCC7 cells, indicating that they have the ability to inhibit the migration of SCC7 cells. The quantitative analysis results show that the wound healing rate of the untreated blank control group reached 49.91±1.44%, while after treatment with the complexes Ir1-3 and CDDP, the wound healing rates dropped to 5.64±1.82%, 8.98±2.12%, 17.21±2.80% and 23.12±2.30%, respectively. It can be seen that the complex Ir1-3 is stronger than CDDP in inhibiting the migration of SCC7 cells. After treatment with the complexes Ir1-3 and CDDP, the scratch width of FADU cells was significantly larger than that of the blank control group, which means that the degree of wound healing was significantly reduced, indicating that the complex Ir1-3 can effectively inhibit the migration ability of FADU cells. The quantitative analysis results showed that the healing rate of scratch wounds in the blank control group was 28.04±2.16%, while the healing rates of scratch wounds treated with complexes Ir1-3 and CDDP were significantly reduced to 4.64±0.78%, 6.97±1.03%, 7.57±0.43% and 13.30±2.57%, respectively. This indicates that the performance of complex Ir1-3 in inhibiting FADU cell migration is more prominent than that of CDDP.

[0032] 3. Effect of complexes on cell proliferation The cell proliferation ability and sensitivity of the complex Ir1-3 were detected by clone formation assay. The clone formation of SCC7 cells after 24h of the complex Ir1-3 and CDDP treatment ( Figure 6 A) and its quantitative analysis performance diagram ( Figure 6 B) and the clone formation of FADU cells ( Figure 6 C) and its quantitative analysis performance diagram ( Figure 6 D) Figure 6 As shown; the results showed that after 24 hours of action and 7 days of culture, the number of SCC7 cell clones was significantly reduced compared with the blank control group, indicating that the complex Ir1-3 can effectively inhibit the proliferation of SCC7 cells. Compared with the untreated blank control group, which showed dense cell colonies, the number of FADU cell clones in the experimental group was significantly reduced and the distribution was more sparse. This shows that the complex Ir1-3 can effectively inhibit the proliferation of FADU cells.

[0033] 4. Effects of the complex on cell apoptosis Annexin V-APC / PI double staining method was used to detect the effect of complex Ir1-3 on cell apoptosis on flow cytometry. The sum of early apoptosis and late apoptosis was calculated as the cell apoptosis rate. Figure 7As shown in the figure, when SCC7 cells were treated for 24 hours, the apoptosis rate of the blank control group without any drug treatment was only 1.02±0.13%, while the apoptosis rates of the cells treated with the complex Ir1-3 and CDDP were significantly increased, 11.05±0.08%, 9.47±0.28%, 8.78±0.28% and 8.03±0.21%, respectively. When the treatment time was extended to 48 hours, the apoptosis rate of SCC7 cells in the control group increased to 2.18±0.07%. At the same time, the apoptosis rates of cells treated with the complex Ir1-3 and CDDP were further increased, 16.25±1.41%, 15.37±1.56%, 14.93±0.72% and 10.52±0.34%, respectively. These results show that the complex Ir1-3 has a significant induction effect on the apoptosis of SCC7 cells, and its ability to induce apoptosis is better than CDDP. At the same time, when FADU cells were treated for 24 hours, the apoptosis rate of FADU cells in the control group was 0.91±0.09%, while the apoptosis rate after treatment with complexes Ir1-3 and CDDP was greatly increased to 10.09±0.51%, 9.18±0.32%, 7.54±0.73% and 6.39±0.58%. When the action time was extended to 48 hours, the apoptosis rate of the control group was 2.11±0.29%, while the apoptosis rate of the complexes Ir1-3 and CDDP groups increased sharply to 36.58±0.68%, 25.63±0.56%, 11.97±0.81% and 12.40±0.87%. It can be seen that the complex Ir1-3 has a very significant effect on the apoptosis induction of FADU cells, and the complexes Ir1 and Ir2 show stronger apoptosis induction than CDDP.

[0034] 5. Effects of complexes on cell cycle The PI labeling method was used to detect the effect of the complex Ir1-3 on the cell cycle on a flow cytometer. Figure 8 As shown, in the normal cell cycle, the percentage of SCC7 cells in the G0 / G1 phase of the untreated blank control group was 54.0±1.93%, while the percentage of SCC7 cells treated with the complex Ir1-3 increased to 63.70±1.42%, 72.0±1.76%, and 65.53±2.47%. The percentage of FADU cells in the G0 / G1 phase of the blank control group and the complex Ir1-3 was 47.67±1.51%, 54.60±1.32%, 66.47±0.61%, and 62.13±0.06%. The results showed that the complex Ir1-3 can effectively induce SCC7 and FADU cell cycle arrest in the G0 / G1 phase, while CDDP induces cell cycle arrest in the G2 / M phase, thereby inhibiting cell proliferation.

[0035] Example 4 Cellular CRT expression, cell membrane CRT expression, HMGB1 release, ATP release effect test 1. Effect of complex on cellular CRT expression In order to test the effect of complex Ir1-3 on immunogenic cells, the expression of CRT in cells was detected by immunofluorescence after adding complex Ir1-3 for 24 hours. Fig. 9 As shown, the CRT expression in the blank control group cells was less and distributed in the cytoplasm, while the CRT expression in the complex Ir1-3 and CDDP group cells was strong and flipped to the cell surface.

[0036] 2. Effects of complexes on cell membrane CRT expression Flow cytometry was used to detect the expression level of CRT on the cell membrane. Fig.10 As shown, the percentage of SCC7 cell membrane CRT expression in the untreated blank control group was 2.76±0.20%, while after 24h of treatment with complexes Ir1-3 and CDDP, the cell membrane CRT expression increased to 7.40±0.28%, 4.49±0.25%, 5.07±0.12%, and 4.88±0.12%, respectively. When the action time was extended to 48h, the blank control group was 2.73±0.12%, while the corresponding data after treatment with complexes Ir1-3 and CDDP increased to 11.21±0.38%, 8.09±0.49%, 9.49±0.57%, and 3.78±0.08%. Similar to the experiment of SCC7 cells, the percentage of FADU cell membrane CRT expression in the blank control group, complex Ir1-3 and CDDP groups after 24h treatment was 4.62±0.48%, 23.75±2.0%, 16.06±0.53%, 16.53±0.91%, 21.56±0.08%, and the percentage after 48h treatment was reduced to 1.16±0.17%, 14.45±0.81%, 4.01±0.15%, 2.81±0.13%, 8.95±0.04%, indicating that complex Ir1-3 can effectively induce a significant increase in the expression of CRT on the surface of SCC7 and FADU cells in different time periods.

[0037] 3. Effects of complexes on HMGB1 release Immunofluorescence was used to detect the expression of HMGB1 in cells. After 24 hours of treatment with complex Ir1-3 and CDDP, SCC7 cells ( Fig.11 A) and FADU cells ( Fig.11 B) HMGB1 expression in Fig.11 As shown, the results showed that the HMGB1 expression in the blank control group cells was low and distributed in the nucleus, while the HMGB1 expression in the complex Ir1-3 and CDDP group cells was strong and partially transferred to the cytoplasm.

[0038] Flow cytometry was used to detect the expression level of HMGB1 in the cell nucleus. Fig.12 As shown in the figure, after 24h of treatment of SCC7 cells, the percentage of HMGB1 expression in the blank control group, complex Ir1-3 and CDDP groups was 0.79±0.07%, 18.57±0.58%, 4.39±0.33%, 9.77±0.33% and 13.27±0.58%. When extended to 48h, the percentage of HMGB1 expression in the blank control group, complex Ir1-3 and CDDP groups decreased to 0.06±0.02%, 15.77±0.13%, 3.58±0.15%, 2.43±0.16% and 7.67±0.11%. The same experiment was performed on FADU cells. The HMGB1 expression percentages of the blank control group, complex Ir1-3 and CDDP groups after 24h treatment were 11.24±1.05%, 21.44±0.91%, 20.35±1.18%, 19.35±0.99%, and 12.05±0.5%, and the percentages after 48h treatment were 11.94±1.42%, 23.16±1.51%, 16.25±2.43%, 12.63±0.54%, and 15.13±0.75%. In summary, complex Ir1-3 can effectively promote the increase of HMGB1 expression in SCC7 and FADU cells, thereby inducing the transfer of HMGB1 to the cytoplasm and its release.

[0039] The ELISA method (enzyme-linked immunosorbent assay) was used to detect the concentration changes of ATP in the cell culture supernatant. Fig.13 As shown in Figure 2, after 24h treatment of SCC7 cells, the intracellular ATP concentration of the blank control group was 165.3±11.30nΜ, while the intracellular ATP concentration of the cells treated with complexes Ir1-3 and CDDP increased significantly, reaching 464.22±16.68nΜ and 437.76 When the action time was extended to 48h, the ATP concentration of SCC7 cells in the control group decreased to 140.22±0.42nΜ, while the ATP concentration of cells treated with complexes Ir1-3 and CDDP was 286.0±12.28nΜ, 218.09±28.92nΜ, 145.0±19.35nΜ and 234.05±18.74nΜ. The same experiment was performed on FADU cells. The ATP concentrations of the blank control group, complex Ir1-3 and CDDP groups after 24h treatment were 120.58±7.94nΜ, 1227.03±5.66nΜ, 197.25±5.90nΜ, 187.0±5.62nΜ, 208.66±3.42nΜ, and the percentages after 48h treatment were 23.83±5.65nΜ, 236.16±14.85nΜ, 213.84±4.78nΜ, 209.47±2.96nΜ, 208.74±9.43nΜ. The results showed that complex Ir1-3 can effectively induce SCC7 and FADU cells to release more ATP.

[0040] Example 5 Endoplasmic reticulum mitochondria colocalization experiment ER Tracker Red and Mito Tracker Red were used to perform specific fluorescent staining of the endoplasmic reticulum and mitochondria of cells, respectively.

[0041] like Fig.14 As shown, complex Ir1-3 accumulates in the endoplasmic reticulum and mitochondria in the cells. The images of complex Ir1-3 merged with ER Tracker Red or Mito Tracker Red show highly matching structural patterns, indicating that complex Ir1-3 tends to selectively localize in the endoplasmic reticulum and mitochondria of SCC7 and FADU cells to exert its effects.

[0042] Example 6 Effect of the complex on endoplasmic reticulum stress in head and neck cancer cells Western blotting was used to detect the expression of proteins related to the endoplasmic reticulum stress pathway (PERK, p-PERK, eIF2α, p-eIF2α, CHOP and ATF4).

[0043] FADU and SCC7 cells in the logarithmic growth phase were cultured until the cell density reached 70-80%, and a final concentration of IC 50The complete medium of the complex Ir1-3 was added and cultured for 24 hours. The cells were digested and collected with EDTA-free trypsin digestion solution, centrifuged after full lysis, cell debris was removed, and the supernatant was collected to obtain the protein sample to be tested. The protein concentration was determined by BCA method. SDS-PAGE gel electrophoresis imaging detection was performed.

[0044] like Fig.15 As shown, the complex Ir1-3 increased the expression of PERK and p-PERK, and further increased the expression of downstream eIF2α, p-eIF2α, CHOP and ATF4. This shows that the complex Ir1-3 acts on SCC7 and FADU cells to activate the endoplasmic reticulum stress pathway.

[0045] Example 7 Complex induces intracellular calcium ions (Ca 2+ ) Level changes Fluo-4 AM was used as Ca 2+ Fluo-4 is a fluorescent probe. The free ligand of Fluo-4 is almost non-fluorescent. When it binds to intracellular calcium ions, it will produce strong fluorescence.

[0046] like Fig.16 As shown, the laser confocal microscopy results ( Fig.16 A and B in Figure 2 show that compared with the weaker green fluorescence of the blank control group, the cells in the complex Ir1-3 and CDDP groups emitted stronger green fluorescence, indicating that the complex Ir1-3 and CDDP can increase the intracellular calcium ion level. Flow cytometry test results ( Fig.16C and D) show that the fluorescence intensity of SCC7 cells in the untreated blank control group was 541.33±19.04, and after 24h of treatment with complexes Ir1-3 and CDDP, the fluorescence intensity increased to 1792.0±39.66, 1517.67±45.28, 680.0±33.0, and 655.67±68.50, respectively. When the action time was extended to 48h, the fluorescence intensity of the blank control group was 829.33±29.54, while the fluorescence intensity after treatment with complexes Ir1-3 and CDDP increased to 2152.67±57.29, 1799.67±71.11, 963.67±57.07, and 1469.67±70.29. Similar to the experiment of SCC7 cells, within 24 hours, the fluorescence intensity of the cells in the blank control group was 1272.0±67.77, and the fluorescence intensity of the FADU cells in the complex Ir1-3 and CDDP groups was 4167.0±77.08, 4671.67±85.78, 3530.0±131.05, and 1405.33±111.84. Within 48 hours, the fluorescence intensity of the blank control group was 1837.33±45.37, and the fluorescence intensity of the complex Ir1-3 and CDDP groups was 5979.33±179.09, 3253.67±108.26, 4843.33±71.23, and 5117.33±181.29. This indicates that the complex Ir1-3 can increase intracellular Ca 2+ level.

[0047] Example 8 Effect of the complex on mitochondrial membrane potential JC-1 was used as a fluorescent probe to detect changes in mitochondrial membrane potential.

[0048] like Fig.17 As shown, the laser confocal microscopy results ( Fig.17 A and B in the figure show that the blank control group exhibits strong red fluorescence and weak green fluorescence, indicating that the mitochondrial membrane potential of the control group is normal. The complex Ir1-3 and CDDP groups emit strong green fluorescence and weak red fluorescence, indicating that the complex Ir1-3 and CDDP can cause the mitochondrial membrane potential to decrease. Flow cytometry test results ( Fig.17C and D) show that after 24 hours of treatment with complexes Ir1-3 and CDDP, the percentage of JC-1 monomer in the untreated blank control group SCC7 was 3.49±0.22%, while the expression of JC-1 monomer in the complexes Ir1-3 and CDDP groups increased, and this value rose to 12.87±0.56%, 7.20±0.31%, 4.82±0.18%, and 10.76±0.62%, respectively. When the action time was extended to 48 hours, the percentage of JC-1 monomer in the blank control group was 1.26±0.19%, while the percentage of JC-1 monomer after treatment with complexes Ir1-3 and CDDP was 13.54±0.15%, 2.81±0.20%, 1.90±0.11%, and 6.70±0.47%. The experiments of FADU cells and SCC7 cells were similar. After 24h treatment, the percentage of JC-1 monomers in the blank control group was 3.10±0.18%, and the percentages of JC-1 monomers in the complex Ir1-3 and CDDP groups were 19.93±0.58%, 16.32±0.38%, 14.39±0.46%, and 9.08±0.17%. When the action time was extended to 48h, this phenomenon was more obvious. The percentage of JC-1 monomers in the blank control group was 8.58±0.68%, while the percentages of JC-1 monomers in the complex Ir1-3 and CDDP groups increased to 95.36±0.22%, 99.43±0.08%, 86.10±1.03%, and 48.57±0.40%. In summary, the complex Ir1-3 can effectively induce the decrease of mitochondrial membrane potential of SCC7 and FADU cells in different time periods.

[0049] Example 9 Effect of complexes on reactive oxygen species (ROS) levels DCFH-DA was used as a fluorescent probe to detect the production of cellular reactive oxygen species.

[0050] like Fig.18 As shown, the laser confocal microscopy results ( Fig.18 A and B in the figure show that the blank control group exhibits weak green fluorescence, while the complex Ir1-3 and CDDP groups emit stronger green fluorescence, indicating that the complex Ir1-3 can induce the production of reactive oxygen species. Flow cytometry test results ( Fig.18C and D) show that after 24 hours of treatment with complex Ir1-3 and CDDP, the fluorescence intensity of SCC7 cells in the untreated blank control group was 463.0±8.19, while after 24 hours of treatment with complex Ir1-3 and CDDP, the fluorescence intensity increased to 5839.0±34.70, 4847.3±150.93, 3240.0±21.07, and 1264.67±34.29, respectively. When the action time was extended to 48 hours, the fluorescence intensity of the blank control group was 1862.0±34.22, while the fluorescence intensity after treatment with complex Ir1-3 and CDDP increased to 5293.3.±55.90, 4536.33±88.19, 3470.0±163.33, and 5485.0±184.96. Similar to the experiment of SCC7 cells, the fluorescence intensity of the cells in the blank control group was 1563.67±15.04 within 24 hours, and the fluorescence intensity of the FADU cells in the complex Ir1-3 and CDDP groups was 3765.33±95.69, 1767.67±8.39, 1906.0±2646, 1661.67±29.30. The fluorescence intensity of the blank control group was 1408.67±98.09 within 48 hours, and the fluorescence intensity of the complex Ir1-3 and CDDP groups was 3151.33±35.64, 2342.0±22.61, 1345.33±15.63, 1198.67±20.60. In summary, the complex Ir1-3 can increase the level of reactive oxygen species.

[0051] Example 10: Safety Analysis of Compounds in vivo The administration concentration of the complex Ir1 was evaluated by an acute toxicity experiment on mice. Healthy 5-week-old female KM mice were randomly divided into 7 groups (0, 20, 30, 40, 60, 90, 120 mg / kg) according to the set concentrations, with 6 mice in each group. The mice were housed in an SPF environment. They were fasted but not watered for 12 hours before administration, and the drug was administered once by intraperitoneal injection. After administration, normal feeding was resumed, and the mice were observed and weight changes were recorded for 14 consecutive days. After 14 days, the mice were killed by cervical dislocation, and the hearts, livers, spleens, lungs, and kidneys of the mice were weighed and fixed in 10% buffered neutral formalin. After fixation for 24 hours, the tissues were paraffin-embedded, sectioned, HE-stained, and Masson-stained, and scanned, observed, and photographed on a digital pathology analysis system. Fig.19As shown, after continuous observation and measurement for 14 days, the weight of mice in the 60, 90, and 120 mg / kg concentration groups decreased, and mice died in the 90 and 120 mg / kg concentration groups, while the weight of mice in the control group and the 20, 30, and 40 mg / kg concentration groups did not change significantly and there was no death. The staining results showed that compared with the control group, the lung, liver, and kidney tissues of the 60, 90, and 120 mg / kg concentration treatment groups had congestion, edema, and cavitation, and the liver had fibrosis. There was almost no significant difference in the heart, liver, spleen, lung, and kidney of the control group and the 20, 30, and 40 mg / kg concentration groups. It can be seen that the administration concentration of the complex Ir1 within 40 mg / kg will not cause chronic organ damage.

[0052] Example 11 Experimental analysis of transplanted tumor model (1) Xenograft tumor model experiment A xenograft tumor model of human pharyngeal squamous cell carcinoma FADU cells was constructed. Healthy 5-week-old female Balb / c Nude mice were raised in an SPF environment. FADU cells in the logarithmic growth phase were collected, resuspended in PBS, and immediately inoculated subcutaneously in the right flank of the mice. Each mouse was given 5.0×10 6 The mice were then given a plate of 100-200 mm2 of tumor tissue. 3 At the same time, the mice were randomly divided into 4 groups (control group, 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, cisplatin group), 6 mice in each group. The drugs were intraperitoneally injected every two days, the mice were observed, and the changes in tumor volume and weight of the mice were recorded. After 15 days, the mice were killed by cervical dislocation, and the tumor tissues of the mice were taken, photographed and weighed. After fixing for 24 hours, the sections were embedded, Tunel stained and immunohistochemically stained, and scanned, observed and photographed on a digital pathology analysis system. Fig. 20 As shown, compared with the control group, the xenograft tumors in the 2.5mg / kg Ir1, 5mg / kg Ir1, and CDDP groups were inhibited to a certain extent, among which the complex Ir1 group had a better tumor inhibition effect than the CDDP group, and there was no significant difference in the weight of mice in each group. On the 15th day of treatment, the average tumor weights of the control group, 2.5mg / kg Ir1 group, 5mg / kg Ir1 group, and CDDP group were 0.5223g, 0.2771g, 0.2532g, and 0.2880g, respectively, and the FADU tumor inhibition rates reached 46.9%, 51.5%, and 44.9%, indicating that the complex Ir1 can effectively inhibit the growth of FADU tumors.

[0053] (2) Homograft tumor model experiment A tumor model of mouse squamous cell carcinoma SCC7 cells was constructed by homologous transplantation. Healthy 5-week-old female C3H mice were housed in an SPF environment. SCC7 cells in the logarithmic growth phase were collected, resuspended in PBS, and immediately inoculated subcutaneously in the right flank of shaved mice. Each mouse was given 5.0×10 5 The mice were then given a plate of 100-200 mm2 of tumor tissue. 3 At 14:00, the mice were randomly divided into 4 groups (control group, 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, cisplatin group), 6 mice in each group. The drug was intraperitoneally injected every two days, the mice were observed, and the changes in tumor volume and weight of the mice were recorded. After 15 days, the mice were killed by cervical dislocation, and the tumor tissues of the mice were taken and photographed and weighed. After 24 hours of fixation, the sections were embedded, Tunel stained, immunofluorescence stained and immunohistochemical stained, and scanned, observed and photographed on a digital pathology analysis system.

[0054] like Fig.21 As shown, compared with the control group, the growth of transplanted tumors in the 2.5mg / kg Ir1 group and the 5mg / kg Ir1 group was inhibited to varying degrees, especially the 5mg / kg Ir1 group showed a better tumor inhibition effect than the CDDP group, and there was no significant difference in the weight of mice in each group, indicating that these drugs did not have a significant negative impact on the overall health of mice while producing tumor inhibition effects. At the end of the treatment cycle, the average tumor weight of mice in the control group was 0.6629g, while the average tumor weights of the 2.5mg / kg Ir1 group, the 5mg / kg Ir1 group, and the CDDP group were 0.4054g, 0.3215g, and 0.3234g, respectively. The tumor inhibition rates of the 2.5mg / kg Ir1 group, the 5mg / kg Ir1 group, and the CDDP group were 38.8%, 51.5%, and 51.2%. It can be seen that the complex Ir1 can effectively inhibit the growth of SCC7 tumors.

[0055] (3) To further test the effect of the complex Ir1 on tumor apoptosis, tumor tissues from C3H and Balb / c Nude mice were subjected to TUNEL fluorescence staining. Fig. 22 As shown, compared with the weak fluorescence of the control group, the mouse tissues in the 2.5 mg / kg Ir1 group, the 5 mg / kg Ir1 group, and the CDDP group all showed strong fluorescence, especially in the 5 mg / kg Ir1 group. This shows that the complex Ir1 can effectively promote tumor apoptosis and is related to the drug dose.

[0056] (4) To further test the effect of the complex Ir1 on immunogenic cell death of head and neck cancer cells, the same tumor tissues of C3H mice were taken for immunofluorescence staining of CD3, CD4, and CD8, with the fluorescence being green, red, and yellow, respectively.

[0057] like Fig.23 As shown, compared with the weak or almost no fluorescence in the control group, the mouse tissues in the 2.5 mg / kg Ir1 group, the 5 mg / kg Ir1 group, and the CDDP group all showed strong fluorescence signals, and the fluorescence intensity of the 5 mg / kg Ir1 dose-treated group was more prominent and obvious, indicating that the mouse tumors after treatment with the complex Ir1 had active T cell responses and could effectively induce tumor immune responses.

[0058] (5) To further explore the mechanism of Ir1-induced tumor death, tumor tissues from C3H and Balb / c Nude mice were subjected to immunohistochemical staining for the tumor proliferation marker Ki67 and the endoplasmic reticulum stress marker p-eIF2α.

[0059] like Fig.24 As shown, compared with the control group, the expression of Ki67 in mouse tissues in the 2.5 mg / kg Ir1 group, the 5 mg / kg Ir1 group, and the CDDP group was significantly decreased, and the expression of p-eIF2α was significantly upregulated, which was consistent with the trend of in vivo animal experiments. This shows that the complex Ir1 can induce tumor immunogenic cell death through endoplasmic reticulum stress.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An iridium (III) complex, characterized in that The iridium (III) complex includes: [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6, and their structural formulas are:

2. A ligand IPM of an iridium (III) complex, characterized in that: Its structural formula is:

3. The method for preparing the ligand IPM of the iridium (III) complex according to claim 2, characterized in that: Dissolve 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate in glacial acetic acid, heat under reflux for a full reaction, cool to room temperature, adjust the pH to neutral, collect the precipitate, and dry to obtain the ligand IPM; The molar ratio of the 3-methyl salicylaldehyde, 1,10-phenanthroline-5,6-dione and ammonium acetate is 1:1:(20-25).

4. The method for preparing the iridium (III) complex according to claim 1, characterized in that: The ligand IPM of claim 2 is mixed with Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2 or Cis-[Ir(ppy)2Cl]2 respectively, dissolved in a mixed solution of dichloromethane and methanol, and heated under reflux in an inert gas environment for sufficient reaction; cooled to room temperature, NH4PF6 is added, stirred, and the filtrate is collected, and the filtrate is evaporated and the obtained solution is dried to obtain a crude product; the crude product is eluted with dichloromethane and acetone on a neutral alumina column to obtain [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6 respectively; The molar ratio of Cis-[Ir(piq)2Cl]2 to the ligand IPM is (1-1.5):2; the molar ratio of Cis-[Ir(bzq)2Cl]2 to the ligand IPM is (1-1.5):2; the molar ratio of Cis-[Ir(ppy)2Cl]2 to the ligand IPM is (1-1.5):

2.

5. Use of the iridium (III) complex according to claim 1 in the preparation of a drug for treating head and neck cancer.

6. The use according to claim 5, characterized in that The head and neck cancer includes tongue squamous cell carcinoma, squamous cell carcinoma or pharyngeal squamous cell carcinoma.

7. Use of the iridium (III) complex according to claim 1 in activating the endoplasmic reticulum stress pathway.

8. A drug for treating head and neck cancer, characterized in that: The drug comprises the iridium (III) complex according to claim 1.

9. The drug according to claim 8, characterized in that The drug also includes a pharmaceutically acceptable carrier.

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