An iridium(III) complex, a preparation method thereof, and applications thereof in head and neck cancer

Abstract

The present invention relates to an iridium(III) complex, a preparation method thereof, and an application thereof in head and neck cancer, belonging to the technical field of biomedicine. The novel iridium(III) complex provided by the present invention has cytotoxicity to head and neck cancer, and the cytotoxicity is better than that of cisplatin CDDP. When the iridium(III) complex of the present invention acts on head and neck cancer, it can inhibit the migration and proliferation of tumor cells, induce apoptosis of tumor cells, induce an increase in the expression level of CRT and its flipping to the cell surface, induce an increase in the expression level of HMGB1 and its transfer to the cytoplasm and release, induce tumor cells to release more ATP, activate the endoplasmic reticulum stress pathway, increase the intracellular Ca<supgt;2+< / supgt; level of tumor cells, reduce the mitochondrial membrane potential of tumor cells, and increase the level of reactive oxygen species; inhibit tumor growth, promote tumor apoptosis, induce tumor immunogenic cell death through endoplasmic reticulum stress, and induce a tumor immune response. It provides a new option for the preparation of drugs for treating head and neck cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to an iridium(III) complex, a preparation method thereof, and an 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 in the head and neck except the eyes, brain, ears, thyroid gland, and esophagus. More than 90% of head and neck tumors are squamous cell carcinomas (HNSCC). Smoking and alcohol consumption are the most common risk factors for head and neck cancer. Other risk factors include human papillomavirus (HPV) infection, as well as poor oral hygiene and nutritional deficiencies. Early diagnosis of head and neck cancer is crucial for prognosis. However, since its symptoms may not be obvious, many patients are already in the advanced stage at the time of diagnosis. The treatment of advanced head and neck cancer is more difficult and the prognosis is also poor. The treatment methods for head and neck cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. For advanced or metastatic head and neck cancer, chemotherapy is a commonly used and effective treatment method. However, these drugs have limitations such as multi-drug resistance, hepatotoxicity, nephrotoxicity, nerve damage, and immunosuppression. New anti-tumor metal drugs with lower toxicity are needed to improve the prognosis of patients. Therefore, researchers have begun to look for alternatives to platinum drugs, and have paid particular attention to iridium(III) metal complexes. These metal elements are similar to platinum in chemical properties, have rich multi-coordination characteristics and structural diversity, and can be flexibly regulated through ligands, so they have attracted much attention in drug research and development. Summary of the Invention

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

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] In the first aspect, the present invention provides an iridium(III) complex, and 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 respectively:

[0006]

[0007] The present invention prepares new iridium(III) complexes by reacting a new ligand IPM with precursors Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2, and Cis-[Ir(ppy)2Cl]2 respectively. The iridium(III) complexes have cytotoxicity against head and neck cancer and can be used to treat head and neck cancer. The molecular formula of [Ir(piq)2(IPM)]PF6 is: C 50 H 34 IrN6O; The molecular formula of [Ir(bzq)2(IPM)]PF6 is: C 46 H 30 IrN6O; The molecular formula of [Ir(ppy)2(IPM)]PF6 is: C 42 H 30 IrN6O.

[0008] In a second aspect, the present invention provides a ligand IPM for an iridium(III) complex, and its structural formula is:

[0009] Its molecular formula is: C 20 H 14 N4O.

[0010] In a third aspect, the present invention provides a preparation method for the ligand IPM of the iridium(III) complex. Dissolve 3-methylsalicylaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium acetate in glacial acetic acid, heat under reflux for sufficient reaction, cool to room temperature, adjust the pH to neutral, collect the precipitated solid and dry it to obtain the ligand IPM; the molar ratio of 3-methylsalicylaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium acetate is 1:1:(20 - 25). The present invention uses the reaction of 3-methylsalicylaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium acetate to prepare the new ligand IPM for the iridium(III) complex.

[0011] As a preferred embodiment of the preparation method for the ligand IPM of the iridium(III) complex of the present invention, the heating temperature is 130 °C and the reaction time is 2 h. As a preferred embodiment of the preparation method for the ligand IPM of the iridium(III) complex of the present invention, the molar ratio of 3-methylsalicylaldehyde, 1,10-phenanthroline-5,6-dione, and ammonium acetate is 1:1:20.

[0012] Fourthly, the present invention provides a preparation method of the iridium(III) complex. Mix Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2 or Cis-[Ir(ppy)2Cl]2 with the ligand IPM respectively, dissolve them in a mixed solution of dichloromethane and methanol, and heat ( ) under reflux in an inert gas environment for sufficient reaction; cool to room temperature, add NH4PF6, stir, collect the filtrate, dry the solution obtained after rotary evaporation of the filtrate to obtain a crude product; elute the crude product 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.

[0013] After the Cis-[Ir(piq)2Cl]2 of the present invention reacts fully with the ligand IPM, the filtrate collected is rotary evaporated to obtain a dark red solution, and its crude product is dark brown. After elution, the orange-yellow band is collected and concentrated to obtain [Ir(piq)2(IPM)]PF6; after the Cis-[Ir(bzq)2Cl]2 reacts fully with the ligand IPM, the filtrate collected is rotary evaporated to obtain a yellow solution, and its crude product is yellow. After elution, the bright yellow band is collected and concentrated to obtain [Ir(bzq)2(IPM)]PF6; after the Cis-[Ir(ppy)2Cl]2 reacts fully with the ligand IPM, the filtrate collected is rotary evaporated to obtain a yellow solution, and its crude product is yellow. After elution, the yellow band is collected and concentrated to obtain [Ir(ppy)2(IPM)]PF6.

[0014] As a preferred embodiment of the preparation method of 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 preparation method of the iridium(III) complex of the present invention, the heating temperature is 40°C and the reaction time is 6 h. As a preferred embodiment of the preparation method of 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 preparation method of the iridium(III) complex of the present invention, the inert gas is argon.

[0015] Fifth aspect, the present invention provides the use of the above iridium(III) complex in the preparation of a medicament for treating head and neck cancer.

[0016] The iridium(III) complexes [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6 of the present invention have cytotoxicity against 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 the cell uptake mechanism, inhibit the migration of tumor cells, induce cell cycle arrest in the G0 / G1 phase and then inhibit the proliferation of tumor cells, induce apoptosis of tumor cells, induce an increase in the expression level of CRT and its flipping to the cell surface, induce an increase in the expression level of HMGB1 and its transfer to the cytoplasm and release, induce tumor cells to release more ATP, activate the endoplasmic reticulum stress pathway, increase the Ca 2+ level in tumor cells, reduce the mitochondrial membrane potential of tumor cells, and increase the level of reactive oxygen species; inhibit tumor growth, promote tumor apoptosis, induce immunogenic cell death of tumors through endoplasmic reticulum stress, and induce a tumor immune response. The iridium(III) complexes of the present invention can be used to prepare a medicament for treating head and neck cancer. As a medicament, the iridium(III) complexes of the present invention will not cause damage to chronic organs within 40 mg / kg and have good safety.

[0017] As a preferred embodiment of the use of the present invention, the iridium(III) complex is [Ir(piq)2(IPM)]PF6 or [Ir(bzq)2(IPM)]PF6. The present invention has found through research that the iridium(III) complexes [Ir(piq)2(IPM)]PF6 or [Ir(bzq)2(IPM)]PF6 have better cytotoxicity against head and neck cancer. As a preferred embodiment of the use of 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 use of the present invention, the head and neck cancer includes squamous cell carcinoma or pharyngeal squamous cell carcinoma. The present invention has found through research that the iridium(III) complexes have better cytotoxicity against squamous cell carcinoma or pharyngeal squamous cell carcinoma.

[0018] Sixth aspect, the present invention provides the use of the above iridium(III) complex in activating the endoplasmic reticulum stress pathway.

[0019] Seventh aspect, the present invention provides a medicament for treating head and neck cancer, and the medicament includes the iridium(III) complex.

[0020] As a preferred embodiment of the medicament of the present invention, the medicament further includes a pharmaceutically acceptable carrier.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The novel iridium(III) complex provided by the present invention has cytotoxicity against various tumor cells of head and neck cancer, and the 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 apoptosis of tumor cells, induce an increase in the expression level of CRT and its flipping to the cell surface, induce an increase in the expression level of HMGB1 and its transfer to the cytoplasm and release, induce tumor cells to release more ATP, activate the endoplasmic reticulum stress pathway, increase the Ca 2+ level in tumor cells, reduce the mitochondrial membrane potential of tumor cells, and increase the level of reactive oxygen species; inhibit tumor growth, promote tumor apoptosis, induce immunogenic cell death of tumors through endoplasmic reticulum stress, and induce tumor immune response. It provides a new option for the preparation of drugs for treating head and neck cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 1H NMR spectrum of IPM in Example 1 of the present invention;

[0024] Figure 2 Synthesis route diagrams of ligand IPM and three iridium(III) metal complexes [Ir(piq)2(IPM)]PF6 (Ir1), [Ir(bzq)2(IPM)]PF6 (Ir2), [Ir(ppy)2(IPM)]PF6 (Ir3) in Example 1 of the present invention;

[0025] Figure 3 Ultraviolet absorption spectra (A) and fluorescence emission spectra (B) of iridium(III) metal complexes Ir1, Ir2, and Ir3 in Example 1 of the present invention;

[0026] Figure 4 Uptake of complexes Ir1-3 after acting on SCC7 cells (A) and FADU cells (B) for 24 h in Example 3 of the present invention; Figure 5 Wound healing of scratched SCC7 cells (A) and its quantitative analysis performance diagram (B), wound healing of scratched FADU cells (C) and its quantitative analysis performance diagram (D) after complexes Ir1-3 and CDDP act for 24 hours in Example 3 of the present invention;

[0027] Figure 6 Colony formation of SCC7 cells (A) and its quantitative analysis performance diagram (B), colony formation of FADU cells (C) and its quantitative analysis performance diagram (D) after complexes Ir1-3 and CDDP act for 24 h in Example 3 of the present invention;

[0028] Figure 7Apoptosis of SCC7 cells (A) and its quantitative analysis performance graph (B), apoptosis of FADU cells (C) and its quantitative analysis performance graph (D) after the complexes Ir1-3 and CDDP act for 24 or 48 hours in Example 3 of the present invention;

[0029] Figure 8 Cell cycle detection (A) of SCC7 cells and its quantitative analysis performance graph (C), cell cycle detection (B) of FADU cells and its quantitative analysis performance graph (D) after the complexes Ir1-3 and CDDP act for 24 hours in Example 3 of the present invention;

[0030] Figure 9 CRT expression of SCC7 cells (A) and FADU cells (B) after the complexes Ir1-3 and CDDP act for 24 hours in Example 4 of the present invention;

[0031] Figure 10 CRT expression of SCC7 cells (A) and its quantitative analysis performance graph (B), CRT expression of FADU cells (C) and its quantitative analysis performance graph (D) after the complexes Ir1-3 and CDDP act for 24 or 48 hours in Example 4 of the present invention;

[0032] Figure 11 HMGB1 expression of SCC7 cells (A) and FADU cells (B) after the complexes Ir1-3 and CDDP act for 24 hours in Example 4 of the present invention;

[0033] Figure 12 HMGB1 expression of SCC7 cells (A) and its quantitative analysis performance graph (B), HMGB1 expression of FADU cells (C) and its quantitative analysis performance graph (D) after the complexes Ir1-3 and CDDP act for 24 or 48 hours in Example 4 of the present invention;

[0034] Figure 13 ATP release in the supernatant of SCC7 cells (A) and FADU cells (B) after the complexes Ir1-3 and CDDP act for 24 or 48 hours in Example 4 of the present invention;

[0035] Figure 14 Results of co-localization experiments of SCC7 cells (A) and FADU cells (B) with the endoplasmic reticulum, and results of co-localization experiments of SCC7 cells (C) or FADU cells (D) with mitochondria after the complexes Ir1-3 and CDDP act for 24 hours in Example 5 of the present invention;

[0036] Figure 15Results of Western blotting detection of the expression of proteins related to the endoplasmic reticulum stress pathway in SCC7 and FADU cells by the complexes Ir1-3 and CDDP in Example 6 of the present invention;

[0037] Figure 16 Results of detecting the changes in intracellular calcium ion content in SCC7 cells (A) and FADU cells (B) by laser confocal microscopy and the results of detecting the changes in intracellular calcium ion content in SCC7 cells (C) and FADU cells (D) by flow cytometry after the complexes Ir1-3 and CDDP act for 24 hours in Example 7 of the present invention;

[0038] Figure 17 Results of detecting the changes in mitochondrial membrane potential in SCC7 cells (A) and FADU cells (B) by laser confocal microscopy and the results of detecting the changes in mitochondrial membrane potential in SCC7 cells (C) and FADU cells (D) by flow cytometry after the complexes Ir1-3 and CDDP act for 24 hours in Example 8 of the present invention;

[0039] Figure 18 Results of detecting the changes in reactive oxygen species in SCC7 cells (A) and FADU cells (B) by laser confocal microscopy and the results of detecting the changes in reactive oxygen species in SCC7 cells (C) and FADU cells (D) by flow cytometry after the complexes Ir1-3 and CDDP act for 24 hours in Example 9 of the present invention;

[0040] Figure 19 Survival curves (A) and body weight change curves (B) of mice under the action of different concentrations of complex Ir1 in Example 10 of the present invention, and the results of HE staining (C) and Masson staining (D) of mouse organs;

[0041] Figure 20 Digital pictures (A), quantitative analysis diagrams of weights (B), growth curves (C) of mouse xenograft tumor tissues and body weight change curves (D) of mice in Example 11 of the present invention;

[0042] Figure 21 Digital pictures (A), quantitative analysis diagrams of weights (B), growth curves (C) of mouse syngeneic tumor tissues and body weight change curves (D) of mice in Example 11 of the present invention;

[0043] Figure 22 TUNEL fluorescence staining pictures (A) of mouse syngeneic tumor tissues and their performance diagrams of quantitative analysis (B), TUNEL fluorescence staining pictures (C) of mouse xenograft tumor tissues and their performance diagrams of quantitative analysis (D) in Example 11 of the present invention; Figure 23 CD3, CD4, CD8 immunofluorescence staining pictures of mouse syngeneic tumor tissues in Example 11 of the present invention;

[0044] Figure 24 This is the Ki67 immunohistochemical staining map of mouse syngeneic tumor tissue (A) and xenogeneic tumor tissue (B), and the p-eIF2α immunohistochemical staining map of mouse syngeneic tumor tissue (C) and xenogeneic tumor tissue (D) in Example 11 of the present invention.

[0045] In the above-mentioned drawings, * P < 0.05, ** P < 0.01, *** P < 0.001; Detailed implementation manners

[0046] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions. The human pharyngeal squamous cell carcinoma cell line FADU and the mouse squamous cell carcinoma cell line SCC7 are derived from ATCC. Balb / c Nude mice, C3H mice and KM mice are all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0047] Example 1 Synthesis and characterization of ligands and complexes

[0048] 1. Synthesis and characterization of ligand IPM

[0049] Accurately weigh 0.208 g (1.5 mmol) of 3-methylsalicylaldehyde, 0.315 g (1.5 mmol) of 1,10-phenanthroline-5,6-dione, and 2.31 g (30 mmol) of ammonium acetate, dissolve them in 25 mL of glacial acetic acid, then place them in a three-necked round-bottom flask, and carry out a condensation reflux reaction at 130 °C in a water bath for 2 h. After the reaction is completed, cool to room temperature, neutralize with concentrated ammonia water until neutral, and a large amount of pink precipitate will precipitate. Filter by suction, collect the filter residue and dry it overnight in a vacuum drying oven to obtain the ligand IPM. Yield: 92%. C 20 H 14 N4O HRMS (CH3OH): m / z = 325.11 ([M-H] - ). The nuclear magnetic resonance hydrogen spectrum of IPM is as Figure 1 shown.

[0050] 2. Synthesis of precursors

[0051] (1) Synthesis of precursor Cis-[Ir(piq)2Cl]2

[0052] Accurately weigh 1.04 g (4 mmol) of iridium trichloride and 1.65 g (8 mmol) of phenylisoquinoline into a three-necked round-bottom flask. Add 60 mL of ethylene glycol ether and 20 mL of double-distilled water. Heat at 120°C under argon for 24 hours under reflux. After cooling to room temperature, filter and oven dry to obtain a dark red residue. Yield: 81%.

[0053] (2) Synthesis of the precursor Cis-[Ir(bzq)2Cl]2

[0054] Accurately weigh 1.04 g (4 mmol) of iridium trichloride and 1.43 g (8 mmol) of 7,8-benzoquinoline into a three-necked round-bottom flask. Add 60 mL of ethylene glycol ether and 20 mL of double-distilled water. Heat at 120°C under argon for 24 hours. After cooling to room temperature, filter and oven dry to obtain a bright yellow residue. Yield: 79%.

[0055] (3) Synthesis of the precursor Cis-[Ir(ppy)2Cl]2

[0056] Accurately weigh 1.04 g (4 mmol) of iridium trichloride and 1.24 g (8 mmol) of 2-phenylpyridine into a three-necked round-bottom flask. Add 60 mL of ethylene glycol ether and 20 mL of double-distilled water. Heat at 120°C under argon for 24 hours under reflux. After cooling to room temperature, filter and oven dry to obtain a bright yellow residue. Yield: 78%.

[0057] 3. Synthesis of complexes

[0058] (1) Synthesis of the complex [Ir(piq)2(IPM)]PF6(Ir1)

[0059] 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 at the same time, and heat at 40°C under argon protection, condense and reflux for 6h. 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 after vacuum drying, a dark brown crude product is obtained. 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 1H NMR (600 MHz, DMSO-d6): δ 9.07 (d, J = 8.7 Hz, 2H), 8.95 (s, 2H), 8.45 (d, J = 8.1 Hz, 2H), 8.14 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 7.9 Hz, 2H), 7.90 (dd, J = 14.2, 6.9 Hz, 7H), 7.83 (d, J = 5.0 Hz, 2H), 7.50 (d, J = 6.6 Hz, 2H), 7.44 (d, J = 6.5 Hz, 2H), 7.20 (t, J = 7.7 Hz, 2H), 7.11 (d, J = 7.2 Hz, 1H), 7.00 (t, J = 7.4 Hz, 2H), 6.80 (t, J = 6.7 Hz, 1H), 6.39 (d, J = 7.6 Hz, 2H), 2.27 (s, 3H). 13 13C NMR (151 MHz, 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.

[0060] (2) Synthesis of Complex [Ir(bzq)2(IPM)]PF6 (Ir2)

[0061] Accurately weigh 0.234 g (0.2 mmol) of Cis-[Ir(bzq)2Cl]2 and 0.13 g (0.4 mmol) of ligand IPM, place them in a three-necked round-bottom flask, and simultaneously add 28 mL of dichloromethane and 14 mL of methanol solution. Under argon protection, heat and reflux at 40 °C for 6 h. After the reaction solution is cooled to room temperature, add an excess of NH4PF6, continue to stir at room temperature for 2 h, carry out suction filtration, and collect the filtrate. After rotary evaporation of the yellow solution and vacuum drying, a yellow crude product is obtained. The crude product is eluted with dichloromethane and acetone (v / v 5:1) on a neutral alumina column chromatography, 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] + ). 11H NMR (600 MHz, DMSO-d6): δ 8.91 (d, J = 8.3 Hz, 2H), 8.53 (d, J = 8.0 Hz, 2H), 8.04–7.99 (m, 6H), 7.90 (d, J = 8.8 Hz, 2H), 7.74 (dd, J = 8.3, 5.1 Hz, 2H), 7.60 (d, J = 8.0 Hz, 3H), 7.49 (dd, J = 8.1, 5.5 Hz, 2H), 7.25 (t, J = 7.6 Hz, 2H), 7.08 (d, J = 7.2 Hz, 1H), 6.77 (t, J = 7.5 Hz, 1H), 6.40 (d, J = 7.1 Hz, 2H), 2.24 (s, 3H). 13 13C NMR (151 MHz, 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.

[0062] (3) Synthesis of Complex [Ir(ppy)2(IPM)]PF6 (Ir3)

[0063] Accurately weigh 0.215 g (0.2 mmol) of Cis-[Ir(ppy)2Cl]2 and 0.13 g (0.4 mmol) of ligand IPM, place them in a three-necked round-bottom flask, and simultaneously add 28 mL of dichloromethane and 14 mL of methanol solution. Under argon protection, heat and condense for reflux at 40 °C for 6 h. After the reaction solution is cooled to room temperature, add an excess of NH4PF6, continue to stir at room temperature for 2 h, carry out suction filtration, and collect the filtrate. After rotary evaporation of the yellow solution and vacuum drying, a yellow crude product is obtained. The crude product is eluted with dichloromethane and acetone (v / v 5:1) on a neutral alumina column chromatography, collect the yellow band, and concentrate to obtain a yellow powder. Yield: 75%, C 42 H 30 IrN6O HRMS (CH3CN): m / z = 828.1959 ([M - PF6] + ). 11H NMR(600 MHz, DMSO-d6): δ 11.08 (s, 1H), 9.39 (d, J = 8.2 Hz, 1H), 9.20 (d, J = 8.3 Hz, 1H), 8.39–8.30 (m, 5H), 8.22 (ddd, J = 13.1, 8.3, 5.1 Hz, 2H), 8.11 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.8 Hz, 2H), 7.95 (t, J = 7.9 Hz, 2H), 7.61 (d, J = 5.9 Hz, 2H), 7.50 (d, J = 7.3 Hz, 1H), 7.13 (t, J = 7.6 Hz, 2H), 7.11–7.05 (m, 3H), 7.03 (t, J = 7.5 Hz, 2H), 6.37 (d, J = 7.6 Hz, 2H), 2.33 (s, 3H). 13 13C NMR(151 MHz, DMSO-d6): 167.28, 167.26, 164.92, 156.23, 150.75, 150.71, 150.14, 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.

[0064] The synthetic route diagrams of the ligand IPM and three iridium(III) metal complexes [Ir(piq)2(IPM)]PF6 (Ir1), [Ir(bzq)2(IPM)]PF6 (Ir2), [Ir(ppy)2(IPM)]PF6 (Ir3) are as Figure 2 shown.

[0065] 4. UV and fluorescence spectra determination of the complexes

[0066] The iridium(III) metal complexes Ir1, Ir2, and Ir3 are dissolved in methanol as the solvent, and then the ultraviolet absorption spectra and fluorescence emission spectra are measured using an ultraviolet spectrophotometer and a fluorescence spectrophotometer, respectively. The ultraviolet absorption spectra of the iridium(III) metal complexes Ir1, Ir2, and Ir3 ( Figure 3 A in) and the fluorescence emission spectra ( Figure 3 B in) are as Figure 3As shown in [figure], the results show that the maximum peaks of Ir1, Ir2, and Ir3 in the ultraviolet absorption spectrum are at 278 nm, 279 nm, and 271 nm respectively, with good absorption characteristics. The fluorescence ranges of Ir1, Ir2, and Ir3 are between 550 and 700 nm, and their maximum peaks are at 628 nm, 610 nm, and 609 nm respectively.

[0067] Example 2 In vitro cytotoxicity test

[0068] To investigate the in vitro cytotoxic effects of iridium metal complexes on head and neck cancer cells, the CCK-8 method was used to detect the half-maximal inhibitory concentration (IC 50 .

[0069] The IC 50 values of different cells treated with complexes Ir1-3 and cisplatin (CDDP) are shown in Table 1 below. Table 1

[0070]

[0071] Table 1 results show that complexes Ir1-3 have better cytotoxic effects on six head and neck cancer cell lines compared to cisplatin CDDP, especially on SCC7 cells and FADU cells. For SCC7 cells, the IC values of complexes Ir1, Ir2, and Ir3 are 0.429 ± 0.074 μΜ, 0.529 ± 0.019 μΜ, and 0.806 ± 0.013 μΜ respectively; for FADU cells, the IC values of complexes Ir1, Ir2, and Ir3 are 0.144 ± 0.007 μΜ, 0.315 ± 0.027 μΜ, and 0.407 ± 0.014 μΜ respectively. Therefore, these two cell lines were selected for subsequent experiments.

[0072] Example 3 Tests on cell uptake, migration, proliferation, apoptosis, and cell cycle effects

[0073] 1. Effect of cell uptake of the complex

[0074] To further investigate the mechanism of action of iridium metal complexes Ir1-3 in cells, fluorescence staining was observed under a laser confocal microscope to determine whether cells took up the complexes. The uptake of complexes Ir1-3 in SCC7 cells ( Figure 4 A in [figure]) and FADU cells ( Figure 4 B in [figure]) after 24 hours of treatment with the complexes is shown in Figure 4As shown, complexes Ir1-3 are mainly distributed in the cytoplasm of SCC7 cells and FADU cells, indicating that complexes Ir1-3 can enter cells through the cellular uptake mechanism and achieve effective accumulation within cells, thereby exerting their effects.

[0075] 2. Effect of the complex on cell migration

[0076] The cell scratch assay was used to detect cell migration ability. As Figure 5 shown, after 24 h of treatment, compared with the blank control group, both complexes Ir1-3 and CDDP could significantly reduce the scratch wound healing rate of SCC7 cells, indicating that they both have the ability to inhibit the migration of SCC7 cells. The quantitative analysis results showed that the scratch wound healing rate of the untreated blank control group reached 49.91 ± 1.44%, while after treatment with complexes Ir1-3 and CDDP, the scratch wound healing rates decreased to 5.64 ± 1.82%, 8.98 ± 2.12%, 17.21 ± 2.80% and 23.12 ± 2.30%, respectively. It can be seen that complex Ir1-3 is more potent than CDDP in inhibiting the migration of SCC7 cells. After treatment with complexes Ir1-3 and CDDP, compared with the blank control group, the scratch width of FADU cells was significantly larger, which means that the degree of wound healing was significantly reduced, indicating that complex Ir1-3 can effectively inhibit the migration ability of FADU cells. The quantitative analysis results showed that the scratch wound healing rate of the blank control group was 28.04 ± 2.16%, while after treatment with complexes Ir1-3 and CDDP, the scratch wound healing rates decreased significantly, to 4.64 ± 0.78%, 6.97 ± 1.03%, 7.57 ± 0.43% and 13.30 ± 2.57%, respectively. It is shown that complex Ir1-3 is more prominent than CDDP in inhibiting the migration of FADU cells.

[0077] 3. Effect of the complex on cell proliferation

[0078] The colony formation assay was used to detect cell proliferation ability and the sensitivity of complex Ir1-3. The colony formation of SCC7 cells after treatment with complex Ir1-3 and CDDP for 24 h ( Figure 6 A in) and its quantitative analysis performance graph ( Figure 6 B in), the colony formation of FADU cells ( Figure 6 C in) and its quantitative analysis performance graph ( Figure 6 D in) are as Figure 6As shown; the results showed that after culturing for 7 days after 24 hours of action, compared with the blank control group, the number of SCC7 cell clones was significantly reduced, indicating that the complex Ir1-3 could effectively inhibit the proliferation of SCC7 cells. Compared with the dense cell colonies in the untreated blank control group, the number of FADU cell clones in the experimental group was significantly reduced and more sparsely distributed. It shows that the complex Ir1-3 can effectively inhibit the proliferation of FADU cells.

[0079] 4. Effect of the complex on cell apoptosis

[0080] The Annexin V-APC / PI double staining method was used to detect the effect of the complex Ir1-3 on cell apoptosis on a flow cytometer. The sum of early apoptosis and late apoptosis was calculated as the cell apoptosis rate. As Figure 7 shown, when treating SCC7 cells 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, which were 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 the control group of SCC7 cells increased to 2.18±0.07%. At the same time, the apoptosis rates of the cells treated with the complex Ir1-3 and CDDP were further increased, which were 16.25±1.41%, 15.37±1.56%, 14.93±0.72% and 10.52±0.34% in turn. 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 that of CDDP. At the same time, when treating FADU cells for 24 hours, the apoptosis rate of the control group of FADU cells was 0.91±0.09%, while the apoptosis rates after treatment with the complex Ir1-3 and CDDP were 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 rates of the complex 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 induction effect on the apoptosis of FADU cells, and the complexes Ir1 and Ir2 show stronger apoptosis induction efficiency than CDDP.

[0081] 5. Effect of the complex on cell cycle

[0082] The PI labeling method was adopted to detect the effect of the complex Ir1-3 on the cell cycle on a flow cytometer. As Figure 8As shown, during the normal cell cycle, the percentage of untreated blank control group SCC7 cells in the G0 / G1 phase was 54.0 ± 1.93%, while the percentage of SCC7 cells treated with complexes 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 complexes Ir1-3 was 47.67 ± 1.51%, 54.60 ± 1.32%, 66.47 ± 0.61%, and 62.13 ± 0.06%. The results showed that complexes Ir1-3 could effectively induce cell cycle arrest of SCC7 and FADU cells in the G0 / G1 phase, while CDDP induced cell cycle arrest in the G2 / M phase, thereby inhibiting cell proliferation.

[0083] Example 4 Test on the Influence of Complexes on Cell CRT Expression, Cell Membrane CRT Expression, HMGB1 Release, and ATP Release 1. Influence of Complexes on Cell CRT Expression

[0084] To test the influence of complex Ir1-3 on immunogenic cells, after adding complex Ir1-3 and acting for 24 h, the expression of CRT in cells was detected by immunofluorescence assay. As Figure 9 shown, the CRT expression of cells in the blank control group was less and distributed in the cytoplasm, while the CRT expression of cells in the complex Ir1-3 and CDDP groups was strong and flipped to the cell surface.

[0085] 2. Influence of Complexes on Cell Membrane CRT Expression

[0086] Flow cytometry was used to detect the expression level of CRT on the cell membrane. As Figure 10As shown, the percentage of CRT expression on the cell membrane of the untreated blank control group SCC7 was 2.76 ± 0.20%, while after treatment with complexes Ir1-3 and CDDP for 24 h, the expression of CRT on the cell membrane increased, rising 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 48 h, 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 on SCC7 cells, the percentages of CRT expression on the cell membrane of the blank control group, complexes Ir1-3 and CDDP groups of FADU cells after 24 h of treatment were 4.62 ± 0.48%, 23.75 ± 2.0%, 16.06 ± 0.53%, 16.53 ± 0.91%, 21.56 ± 0.08%, and the percentages after 48 h of treatment decreased to 1.16 ± 0.17%, 14.45 ± 0.81%, 4.01 ± 0.15%, 2.81 ± 0.13%, 8.95 ± 0.04%. It shows that complex Ir1-3 can effectively induce a significant increase in the expression level of CRT on the cell membrane surface of SCC7 and FADU at different time periods.

[0087] 3. Effect of the complex on the release of HMGB1

[0088] The immunofluorescence method was used to detect the expression of HMGB1 in cells. After the complexes Ir1-3 and CDDP acted for 24 hours, the expression of HMGB1 in SCC7 cells ( Figure 11 A in) and FADU cells ( Figure 11 B in) was as shown in Figure 11 The results showed that the expression of HMGB1 in the cells of the blank control group was less and distributed in the nucleus, while the expression of HMGB1 in the cells of the complexes Ir1-3 and CDDP groups was strong and partially transferred to the cytoplasm.

[0089] Flow cytometry was used to detect the expression level of HMGB1 in the cell nucleus. As shown in Figure 12As shown, after treating SCC7 cells for 24 h, the percentages of intracellular HMGB1 expression in the blank control group, complexes Ir1-3, and CDDP group were 0.79 ± 0.07%, 18.57 ± 0.58%, 4.39 ± 0.33%, 9.77 ± 0.33%, and 13.27 ± 0.58%. When extended to 48 h, the percentages of HMGB1 expression in the blank control group, complexes Ir1-3, and CDDP group 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 conducted on FADU cells. After 24 h of treatment, the percentages of HMGB1 expression in the blank control group, complexes Ir1-3, and CDDP group were 11.24 ± 1.05%, 21.44 ± 0.91%, 20.35 ± 1.18%, 19.35 ± 0.99%, and 12.05 ± 0.5%. After 48 h of treatment, the percentages 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 in the expression level of HMGB1 in SCC7 and FADU cells, thereby inducing the translocation of HMGB1 to the cytoplasm and its release.

[0090] The change in the concentration of ATP in the cell culture supernatant was detected by ELISA (enzyme-linked immunosorbent assay). As Figure 13 shown, after treating SCC7 cells for 24 h, the intracellular ATP concentration in the blank control group was 165.3 ± 11.30 nM, while the intracellular ATP concentrations in the cells treated with complexes Ir1-3 and CDDP increased significantly, reaching 464.22 ± 16.68 nM and 437.76

[0091] ±56.91 nM, 353.51 ± 55.17 nM, and 341.69 ± 23.67 nM. When the action time was extended to 48 h, the ATP concentration of SCC7 cells in the control group decreased to 140.22 ± 0.42 nM, while the ATP concentrations of cells treated with complexes Ir1-3 and CDDP were 286.0 ± 12.28 nM, 218.09 ± 28.92 nM, 145.0 ± 19.35 nM, and 234.05 ± 18.74 nM. The same experiment was carried out on FADU cells. The ATP concentrations of the blank control group, complexes Ir1-3, and CDDP group after 24 h of treatment were 120.58 ± 7.94 nM, 1227.03 ± 5.66 nM, 197.25 ± 5.90 nM, 187.0 ± 5.62 nM, 208.66 ± 3.42 nM, and the percentages after 48 h of treatment were 23.83 ± 5.65 nM, 236.16 ± 14.85 nM, 213.84 ± 4.78 nM, 209.47 ± 2.96 nM, 208.74 ± 9.43 nM. The results showed that complex Ir1-3 could effectively induce SCC7 and FADU cells to release more ATP.

[0092] Example 5 Endoplasmic reticulum and mitochondria co-localization experiment

[0093] ER Tracker Red and Mito Tracker Red were used to specifically fluorescently stain the endoplasmic reticulum and mitochondria of cells, respectively.

[0094] As Figure 14 shown, complex Ir1-3 accumulated in large amounts in the endoplasmic reticulum and mitochondria of cells. The images of complex Ir1-3 merged with ER Tracker Red or Mito Tracker Red showed a highly matching structural pattern, indicating that complex Ir1-3 tended to selectively localize in the endoplasmic reticulum and mitochondria of SCC7 and FADU cells to play a role.

[0095] Example 6 Effect of complex on endoplasmic reticulum stress in head and neck cancer cells

[0096] 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) in cells.

[0097] FADU and SCC7 cells in the logarithmic growth phase were cultured until the cell density reached 70-80%, and the final concentration of IC 50The complete medium of the complexes Ir1-3 was used to continue culturing for 24 h. The cells were digested and collected with trypsin digestion solution without EDTA, centrifuged after sufficient lysis to remove cell debris, and the supernatant was collected to obtain the protein sample to be measured. The protein concentration was determined by the BCA method. SDS-PAGE gel electrophoresis imaging was used for detection.

[0098] As Figure 15 shown, the complexes Ir1-3 increased the expression of PERK and p-PERK, further increasing the expression of downstream eIF2α, p-eIF2α, CHOP, and ATF4. It is indicated that the complexes Ir1-3 act on SCC7 and FADU cells to activate the endoplasmic reticulum stress pathway.

[0099] Example 7 Changes in intracellular calcium ion (Ca 2+ ) levels

[0100] Fluo-4 AM was used as the fluorescent probe for Ca 2+ . The free ligand of Fluo-4 is almost non-fluorescent. When it binds to intracellular calcium ions, strong fluorescence will be generated.

[0101] As Figure 16 shown, the results of laser confocal microscopy ( Figure 16 A, B in) showed that compared with the weak green fluorescence of the blank control group, the cells in the complexes Ir1-3 and CDDP groups emitted stronger green fluorescence, indicating that the complexes Ir1-3 and CDDP can cause an increase in intracellular calcium ion levels. The results of flow cytometry detection ( Figure 16In C and D), it shows that the fluorescence intensity of untreated blank control group SCC7 cells is 541.33 ± 19.04. After treatment with complexes Ir1-3 and CDDP for 24 h, the fluorescence intensities increase to 1792.0 ± 39.66, 1517.67 ± 45.28, 680.0 ± 33.0, and 655.67 ± 68.50 respectively. When the action time is extended to 48 h, the blank control group is 829.33 ± 29.54, while the fluorescence intensities after treatment with complexes Ir1-3 and CDDP increase to 2152.67 ± 57.29, 1799.67 ± 71.11, 963.67 ± 57.07, and 1469.67 ± 70.29. Similar to the experiment on SCC7 cells, within 24 h, the fluorescence intensity of the blank control group cells is 1272.0 ± 67.77, and the fluorescence intensities of FADU cells in the groups of complexes Ir1-3 and CDDP are 4167.0 ± 77.08, 4671.67 ± 85.78, 3530.0 ± 131.05, and 1405.33 ± 111.84. Within 48 h, the fluorescence intensity of the blank control group is 1837.33 ± 45.37, and the fluorescence intensities of the groups of complexes Ir1-3 and CDDP are 5979.33 ± 179.09, 3253.67 ± 108.26, 4843.33 ± 71.23, and 5117.33 ± 181.29. It shows that complex Ir1-3 can increase the intracellular Ca 2+ level.

[0102] Example 8 Effect of the complex on mitochondrial membrane potential

[0103] JC-1 was used as a fluorescent probe to detect the changes in mitochondrial membrane potential of cells.

[0104] As Figure 17 shown, the results of laser confocal microscopy ( Figure 17 in A and B) show that the blank control group presents strong red fluorescence and weak green fluorescence, indicating that the mitochondrial membrane potential of the control group is normal. While the groups of complexes Ir1-3 and CDDP emit strong green fluorescence and weak red fluorescence, indicating that complexes Ir1-3 and CDDP can cause a decrease in mitochondrial membrane potential. The detection results of flow cytometry ( Figure 17As shown in C and D), after 24 h of treatment with complexes Ir1-3 and CDDP, the percentage of JC-1 monomers in the untreated blank control group of SCC7 was 3.49±0.22%, while the expression of JC-1 monomers in the groups of complexes Ir1-3 and CDDP 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 h, the percentage of JC-1 monomers in the blank control group was 1.26±0.19%, while the percentages of JC-1 monomers after treatment with complexes Ir1-3 and CDDP were 13.54±0.15%, 2.81±0.20%, 1.90±0.11%, and 6.70±0.47% respectively. The experiments on FADU cells were similar to those on SCC7 cells. The percentage of JC-1 monomers in the blank control group after 24 h of treatment was 3.10±0.18%, while the percentages of JC-1 monomers in the groups of complexes Ir1-3 and CDDP were 19.93±0.58%, 16.32±0.38%, 14.39±0.46%, and 9.08±0.17% respectively. When the action time was extended to 48 h, this phenomenon became 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 groups of complexes Ir1-3 and CDDP rose to 95.36±0.22%, 99.43±0.08%, 86.10±1.03%, and 48.57±0.40% respectively. Generally speaking, complex Ir1-3 can effectively induce the decline of mitochondrial membrane potential in SCC7 and FADU cells at different time periods.

[0105] Example 9 Effect of Complexes on the Level of Reactive Oxygen Species (ROS)

[0106] DCFH-DA was used as a fluorescent probe to detect the generation of reactive oxygen species in cells.

[0107] As Figure 18 shown, the results of laser confocal microscopy ( Figure 18 in A and B) showed that the blank control group presented weak green fluorescence, while the groups of complexes Ir1-3 and CDDP emitted stronger green fluorescence, indicating that complex Ir1-3 could cause the generation of reactive oxygen species. The results of flow cytometry ( Figure 18As shown in C and D), after treatment with complexes Ir1-3 and CDDP for 24 h, the fluorescence intensity of untreated blank control group SCC7 cells was 463.0±8.19, while after treatment with complexes Ir1-3 and CDDP for 24 h, the fluorescence intensities increased to 5839.0±34.70, 4847.3±150.93, 3240.0±21.07, 1264.67±34.29, respectively. When the action time was extended to 48 h, the fluorescence intensity of the blank control group was 1862.0±34.22, while the fluorescence intensities after treatment with complexes Ir1-3 and CDDP increased to 5293.3±55.90, 4536.33±88.19, 3470.0±163.33, 5485.0±184.96. Similar to the experiment on SCC7 cells, within 24 h, the fluorescence intensity of the blank control group cells was 1563.67±15.04, and the fluorescence intensities of the FADU cells in the groups of complexes Ir1-3 and CDDP were 3765.33±95.69, 1767.67±8.39, 1906.0±26.46, 1661.67±29.30. Within 48 h, the fluorescence intensity of the blank control group was 1408.67±98.09, and the fluorescence intensities of the groups of complexes Ir1-3 and CDDP were 3151.33±35.64, 2342.0±22.61, 1345.33±15.63, 1198.67±20.60. In summary, complex Ir1-3 can increase the level of reactive oxygen species.

[0108] Example 10 In vivo safety analysis of the complex

[0109] The administration concentration of complex Ir1 was evaluated by acute toxicity experiment in mice. Five-week-old healthy female KM mice were randomly divided into 7 groups (0, 20, 30, 40, 60, 90, 120 mg / kg) according to the set concentration, with 6 mice in each group. The mice were raised in an SPF-level environment. They were fasted for 12 h before administration without water restriction and administered once by intraperitoneal injection. After administration, they were fed normally, and the status of the mice was continuously observed for 14 days and the body weight changes were recorded. After 14 days, the mice were sacrificed by cervical dislocation, and the hearts, livers, spleens, lungs and kidneys of the mice were weighed and fixed in 10% buffered neutral formalin solution. After fixation for 24 h, tissue paraffin embedding sections, HE staining and Masson staining were performed, and scanned, observed and photographed on a digital pathology analysis system. As Figure 19As shown, the body weight of mice in the 60, 90, and 120 mg / kg concentration groups decreased continuously for 14 days of observation and measurement. Among them, mice died in the 90 and 120 mg / kg groups, while the body weight of mice in the control group and the 20, 30, and 40 mg / kg concentration groups did not change significantly and no deaths occurred. 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 vacuolization, and fibrosis occurred in the liver. There were almost no significant differences in the heart, liver, spleen, lung, and kidney of the control group and the 20, 30, and 40 mg / kg concentration groups. Thus, it can be seen that the administration concentration of complex Ir1 within 40 mg / kg will not cause chronic organ damage.

[0110] Experimental Analysis of Transplanted Tumor Model in Example 11

[0111] (1) Experimental Study on Xenograft Tumor Model

[0112] A tumor model of xenograft human pharyngeal squamous cell carcinoma FADU cells was constructed. Healthy 5-week-old female Balb / c Nude mice were raised in an SPF-level environment. FADU cells in the logarithmic growth phase were collected, resuspended in PBS, and immediately subcutaneously inoculated into the right flank of the mice. Each mouse was given 5.0×10 6 cells, and the status of the mice and the tumor growth were observed daily. When the tumor volume of the mice reached 100 - 200 mm 3 , the mice were randomly divided into 4 groups (control group, 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, cisplatin group), with 6 mice in each group. Intraperitoneal injection was given every two days, the status of the mice was observed, and the changes in tumor volume and mouse body weight were recorded. After 15 days, the mice were sacrificed by cervical dislocation, the tumor tissues of the mice were taken for photographing and weighing, embedded in paraffin after fixation for 24 h, sectioned, and stained by Tunel and immunohistochemistry, and scanned, observed, and photographed on a digital pathology analysis system. As Figure 20 shown, compared with the control group, the xenograft tumors in the 2.5 mg / kg Ir1, 5 mg / kg Ir1, and CDDP groups were inhibited to a certain extent. Among them, the tumor inhibitory effect of the complex Ir1 group was better than that of the CDDP group, and there were no significant differences in the body weight of the mice in each group. On the 15th day of treatment, the average tumor weights of the control group, 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group were 0.5223 g, 0.2771 g, 0.2532 g, and 0.2880 g respectively, and the tumor inhibition rates of FADU reached 46.9%, 51.5%, and 44.9%. It shows that complex Ir1 can effectively inhibit the growth of FADU tumors.

[0113] (2) Experimental Study on Allograft Tumor Model

[0114] A tumor model of allogeneic transplanted mouse squamous cell carcinoma SCC7 cells was constructed. Healthy 5-week-old female C3H mice were raised in a SPF-level environment. SCC7 cells in the logarithmic growth phase were collected, resuspended in PBS, and immediately subcutaneously inoculated into the right flank of the shaved mice. Each mouse was given 5.0×10 5 cells, and the status of the mice and the tumor growth were observed daily. When the tumor volume of the mice reached 100-200 mm 3 , the mice were randomly divided into 4 groups (control group, 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, cisplatin group), with 6 mice in each group. Administration was performed by intraperitoneal injection every two days. The status of the mice was observed, and the changes in tumor volume and mouse body weight were recorded. After 15 days, the mice were sacrificed by cervical dislocation. The tumor tissues of the mice were taken, photographed, weighed, embedded in paraffin after fixation for 24 h, sectioned, subjected to Tunel staining, immunofluorescence staining, and immunohistochemical staining, and scanned, observed, and photographed on a digital pathology analysis system.

[0115] As Figure 21 shown, compared with the control group, the growth of transplanted tumors in the 2.5 mg / kg Ir1 group and 5 mg / kg Ir1 group was inhibited to varying degrees. In particular, the 5 mg / kg Ir1 group showed a better antitumor effect than the CDDP group, and there was no significant difference in the body weight of the mice in each group, indicating that these drugs did not cause obvious negative effects on the overall health of the mice while producing antitumor effects. At the end of the treatment cycle, the average tumor weight of the mice in the control group was 0.6629 g, while the average tumor weights of the 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group were 0.4054 g, 0.3215 g, and 0.3234 g, respectively. The antitumor rates of the 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group were 38.8%, 51.5%, and 51.2%, respectively. Thus, it can be seen that the complex Ir1 can effectively inhibit the growth of SCC7 tumors.

[0116] (3) To further test the effect of the complex Ir1 on tumor apoptosis, TUNEL fluorescence staining was performed on the tumor tissues of C3H and Balb / c Nude mice. ) As Figure 22 shown, compared with the weak fluorescence of the control group, the tissues of the mice in the 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group showed stronger fluorescence, especially in the 5 mg / kg Ir1 group. It is indicated that the complex Ir1 can effectively promote tumor apoptosis and is related to the drug dose.

[0117] (4) To further test the effect of 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.

[0118] As Figure 23 shown, compared with the control group showing weak or almost no fluorescence, the mouse tissues in the 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group all showed stronger fluorescence signals, and the fluorescence intensity in the 5 mg / kg Ir1 dose treatment group was more prominent and more obvious; it indicates that there is an active T cell response in the tumors of mice treated with complex Ir1, which can effectively induce tumor immune responses.

[0119] (5) To further explore the mechanism of complex Ir1-induced tumor death, the tumor tissues of C3H and Balb / c Nude mice were taken for immunohistochemical staining of the tumor proliferation marker Ki67 and the endoplasmic reticulum stress marker p-eIF2α.

[0120] As Figure 24 shown, compared with the control group, the expression of Ki67 in the mouse tissues of the 2.5 mg / kg Ir1 group, 5 mg / kg Ir1 group, and CDDP group decreased significantly, and the expression of p-eIF2α increased significantly, which was the same as the trend in in vivo animal experiments. Thus, it can be seen that complex Ir1 can induce immunogenic cell death of tumors through endoplasmic reticulum stress.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An iridium(III) complex, characterized in that, The iridium(III) complex is: [Ir(piq)2(IPM)]PF6, [Ir(bzq)2(IPM)]PF6 or [Ir(ppy)2(IPM)]PF6, and their structural formulas are respectively: 、 、 。 2. The preparation method of the iridium(III) complex according to claim 1, characterized in that, Cis-[Ir(piq)2Cl]2, Cis-[Ir(bzq)2Cl]2 or Cis-[Ir(ppy)2Cl]2 is respectively mixed with the ligand IPM, 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, the filtrate is collected, the solution obtained after rotary evaporation of the filtrate is dried to obtain a crude product; the crude product is eluted with dichloromethane and acetone by column chromatography on neutral alumina 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; The structural formula of the ligand IPM is: 。 3. Use of the iridium(III) complex according to claim 1 in the preparation of a drug for treating head and neck cancer.

4. The application according to claim 3, characterized in that, The head and neck cancer is tongue squamous cell carcinoma, squamous cell carcinoma or pharyngeal squamous cell carcinoma.

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

6. The drug according to claim 5, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

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