Ring metal iridium (III) complex for hypoxia photodynamic therapy as well as synthesis method and application of ring metal iridium (III) complex

Abstract

The invention discloses a cyclometalated iridium (III) complex for hypoxia photodynamic therapy and a synthesis method and application thereof.The cyclometalated iridium (III) complex has good tumor cell proliferation resistance, I-type active oxygen is generated through illumination under the hypoxia condition, NADH balance is disturbed, and the tumor cell proliferation resistance is improved. Pyroptosis and immunogen cell death are activated by destroying lysosome to release cathepsin; the preparation method is simple, mild in condition and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to a cyclometalated iridium (III) complex for hypoxia photodynamic therapy, and also relates to a synthesis method and application of the complex. Background Art

[0002] Photodynamic therapy (PDT) is a treatment method that uses photosensitizers (PS) and excitation light to kill tumor cells. Compared with traditional therapies, PDT has the advantage of providing precise and effective treatment with minimal side effects. Most organic PSs that have been discovered mainly produce single linear oxygen ( 1 O2), which induces oxidative stress in tumor cells and causes tumor cell death. However, many solid tumors exhibit hypoxic characteristics, which limits the efficacy of photodynamic therapy. During PDT, the consumption of oxygen in tumor cells will further aggravate the hypoxic condition of the tumor, thereby promoting tumor proliferation, metastasis and invasion, leading to poor treatment prognosis. 3 PS * The electron transfer process between adjacent substrates and molecular oxygen produces superoxide anions (O2 -· ) and hydroxyl radicals (·OH), which disrupt the NADH balance in tumor cells and trigger tumor cell death. Type I PS is less dependent on oxygen, making it an effective option for photodynamic therapy of hypoxic tumors.

[0003] Cyclometallated Ir(III) complexes, as common photosensitizers, have been widely used in CO2 photoreduction research. However, due to their short absorption wavelength (maximum ~280 nm) and weak penetrating ability (light of this wavelength has a poor ability to penetrate cells), they cause significant photodamage to the skin, making them unsuitable for cancer PDT. To red-shift the absorption wavelength to the near-infrared (NIR) region (>600 nm), existing technologies have successfully achieved the maximum absorption wavelength of PS (BDP-Ir-ppy) by conjugating a 4,4-difluoro-4-boron-3a,4a-diaza-s-indane (BODIPY) core and a cyclometallated Ir(III) fragment. This has successfully pushed the maximum absorption wavelength to 630 nm and induced photodynamic immunotherapy under normoxic conditions. However, this compound exhibits low toxicity in the hypoxic environment of tumors, making it unsuitable for photodynamic therapy in hypoxic environments. Summary of the Invention

[0004] Invention purposes: The application aims to provide a fluorine boron dipyrrin modified cyclometalated iridium (III) complex for hypoxic photodynamic therapy, which can effectively solve the inhibition of traditional type II photodynamic effect by tumor hypoxic microenvironment, and can induce photodynamic immunotherapy in normoxic and hypoxic environments; the application also provides a synthesis method of the cyclometalated iridium (III) complex and application of the cyclometalated iridium (III) complex as a photosensitizer in hypoxic photodynamic immunotherapy.

[0005] Technical solutions: The cyclometalated iridium (III) complex for hypoxic photodynamic therapy has the following structural formula:

[0006] The synthesis method of the cyclometalated iridium (III) complex comprises the following steps:

[0007] (1) In an inert atmosphere, fluorine boron dipyrrin (BODIPY) modified bipyridine ligand and cyclometalated iridium dimer are heated and refluxed in a mixed solvent of dichloromethane and methanol, and the solvent is removed by reduced pressure distillation;

[0008] (2) The obtained crude product is replaced with NH4PF6, and the fluorine boron dipyrrin modified cyclometalated iridium (III) complex is obtained by column chromatography separation and purification of the crude product;

[0009] The structure of the fluorine boron dipyrrin modified bipyridine ligand is as follows:

[0010]

[0011] The structure of the cyclometalated iridium dimer is as follows:

[0012]

[0013] The preparation method of the cyclometalated iridium dimer is as follows: in an inert atmosphere, 2-phenylbenzothiazole and iridium chloride hydrate are dissolved in a mixed solution of ethylene glycol ethyl ether and water, and the mixture is reacted at reflux temperature to obtain a crude product, which is purified by filtration and water washing; the molar ratio of the 2-phenylbenzothiazole and the iridium chloride hydrate is 2-2.5:1.

[0014] The fluorine boron dipyrrin modified bipyridine ligand is prepared by the following method, and the specific steps are as follows:

[0015] (1.1) Fluorine boron dipyrrin and N-iodosuccinimide are dissolved in an organic solvent and reacted at room temperature; after the reaction is completed, the solvent is removed by reduced pressure distillation, and the crude product is purified by column chromatography to obtain iodinated fluorine boron dipyrrin;

[0016] (1.2) iodofluorobodipyrrin, 2-thiophene carboxaldehyde, piperidine and p-toluenesulfonic acid are dissolved in an organic solvent to react at a reflux temperature; after the reaction is completed, the solvent is removed by distillation under reduced pressure, and the crude product is purified by column chromatography to obtain an iodofluorobodipyrrin derivative;

[0017] (1.3) iodofluorobodipyrrin derivative, 5-ethynyl-2,2'-bipyridine, Pd(PPh3)2Cl2 (bis-triphenylphosphine palladium dichloride), PPh3 (triphenylphosphine oxide) and CuI are dissolved in anhydrous triethylamine, and the mixture is reacted at a reflux temperature to obtain a crude product, which is separated and purified by column chromatography.

[0018] In step (1.3), the molar ratio of the iodofluorobodipyrrin derivative to 5-ethynyl-2,2'-bipyridine is 1:1-1.5.

[0019] In step (1), the molar ratio of the fluorobodipyrrin-modified bipyridine ligand to the cyclometalated iridium dimer is 2:1-1.2; the organic solvent is a mixed solvent of dichloromethane and methanol; in the mixed solvent, the volume ratio of dichloromethane to methanol is 2:1-1.5; the reflux reaction is carried out for 12-13 h at a temperature of 45-48℃.

[0020] In step (2), the crude product is obtained by replacing NH4PF6, specifically, a saturated NH4PF6 methanol solution is added and stirred for 2-3 h.

[0021] Application of the above cyclometalated iridium (III) complex as a photosensitizer in hypoxic photodynamic immunotherapy

[0022] Invention principle: The cyclometalated iridium (III) complex for hypoxic photodynamic therapy in the present application adjusts the liposolubility of the complex by introducing a cyclometalated iridium (III) moiety, thereby enhancing cell uptake; the fluorobodipyrrin skeleton is modified by a thiophene group to red-shift the spectrum to the near-infrared light region, which can effectively enhance the penetration of the light into tissues and reduce the light damage to the skin; the introduction of the C^N heterocycle of the cyclometalated iridium (III) moiety can adjust the triplet excited state energy of the complex in a hypoxic environment, change the active oxygen production pathway, and mainly produce type I active oxygen in a hypoxic environment, thereby greatly improving the hypoxic tumor treatment effect of the complex as a photosensitizer.

[0023] Advantages: Compared with the prior art, the present application has the following obvious advantages: (1) the cyclometalated iridium (III) complex of the present application has good anti-tumor cell proliferation ability, generates type I active oxygen under light irradiation in a hypoxic condition, disturbs the NADH balance, and releases cathepsin to activate pyroptosis and immunogenic cell death by destroying lysosomes; (2) the preparation method of the complex of the present application is simple, the conditions are mild, and it is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Graph of theoretical calculation results for the complex of Example 1;

[0025] Figure 2 Graph of localization of the complex of Example 1 in A549 cells;

[0026] Figure 3 Laser confocal fluorescence imaging graph and flow cytometry graph of the complex of Example 1 in A549 cells under normoxic conditions (21% O2) for the generation of reactive oxygen species under light irradiation;

[0027] Figure 4 Laser confocal fluorescence imaging graph and flow cytometry graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the generation of reactive oxygen species under light irradiation;

[0028] Figure 5 Graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the consumption of NADH and the generation of H2O2 under light irradiation;

[0029] Figure 6 Cell flow cytometry graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the induction of changes in mitochondrial membrane potential after light irradiation;

[0030] Figure 7 Confocal fluorescence imaging graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the induction of lysosome membrane permeabilization under light irradiation;

[0031] Figure 8 Laser confocal fluorescence imaging graph and immunoblotting protein graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the induction of pyroptosis after light irradiation;

[0032] Figure 9 Graph of the complex of Example 1 in A549 cells under hypoxic conditions (1% O2) for the induction of immunogenic cell death after light irradiation;

[0033] Figure 10 Graph of the complex of Example 1 for the implementation of the immunoprophylaxis mouse experiment; wherein a is an experimental schematic diagram; b is an image after tumor peeling; c, d are graphs of changes in tumor volume and weight, respectively; e is a graph of changes in mouse body weight; f, g are expressions of mature dendritic cells in distal tumors; h, i are expressions of related inflammatory factors. DETAILED DESCRIPTION

[0034] Example 1

[0035] The preparation method of the cyclometalated iridium (III) complex (referred to as BDP-Ir-bpt) of the application comprises the following steps:

[0036]

[0037] (1) Dissolve fluorobodopyrrole (BDP) (780 mg, 2.4 mmol) and N-iodosuccinimide (540 mg, 2.4 mmol) in dichloromethane, stir at room temperature for 3 hours; after the reaction is completed, remove the solvent by distillation under reduced pressure, and further purify the crude product by column chromatography to obtain a red solid (iodinated fluorobodopyrrole) with a yield of 83%;

[0038] (2) Dissolve iodinated BDP (450 mg, 1 mmol), 2-thiophene carboxaldehyde (0.28 ml, 3 mmol), piperidine (1 ml) and p-toluenesulfonic acid (5 mg) in toluene, connect a water separator and a spherical condenser, and heat to reflux for 8 hours; after the reaction is completed, remove the solvent by distillation under reduced pressure, and further purify the crude product by column chromatography to obtain a purple solid (iodinated fluorobodopyrrole derivative BDP-I) with a yield of 24%; 1 H NMR (400 MHz, CD2Cl2) δ (ppm) 8.24 (d, J = 6.4 Hz, 1H), 7.56-7.52 (m, 3H), 7.48 (d, J = 7.5 Hz, 2H), 7.44 (d, J = 4.7 Hz, 2H), 7.39 (d, J = 5.1 Hz, 1H), 7.34-7.31 (m, 4H), 7.15-7.06 (m, 2H), 6.70 (s, 1H), 1.45 (s, 6H). 13 C NMR (101 MHz, CD2Cl2) δ 154.90, 145.17, 143.07, 142.29, 135.29, 131.49, 130.55, 129.81, 129.70, 129.66, 128.82, 128.79, 128.69, 128.52, 127.20, 119.41, 119.39, 118.68, 118.07, 17.26, 15.05.

[0039] (3) Dissolve BDP-I (100 mg, 0.16 mmol), 5-ethynyl-2,2'-bipyridine (34 mg, 0.19 mmol), Pd(PPh3)2Cl2 (7 mg), PPh3 (2 mg) and CuI (2 mg) in anhydrous triethylamine under an argon protective atmosphere, heat to reflux overnight; after the reaction is completed, remove the solvent by distillation under reduced pressure, and further purify the crude product by column chromatography to obtain a brown solid (fluorobodopyrrole modified bipyridine ligand

[0040] BDP-Ir-bpy, yield 45%; 1 H NMR (400 MHz, CD2Cl2) δ (ppm) 8.78 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.53 - 8.49 (m, 1H), 8.46 - 8.43 (m, 2H), 7.92 - 7.90 (m, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.60 (s, 1H), 7.56 - 7.55 (m, 3H), 7.50 (d, J = 6.1 Hz, 2H), 7.46 - 7.42 (m, 2H), 7.39 - 7.37 (m, 2H), 7.33 (s, 2H), 7.13 - 7.12 (m, 2H), 6.71 (s, 1H), 1.60 (s, 3H), 1.49 (s, 3H).

[0041] (4) Preparation of cyclometalated iridium dimer: under argon atmosphere, 2-phenylbenzothiazole (148 mg) and IrCl3·3H2O (100 mg) were dissolved in a mixed solution of ethylene glycol ethyl ether and water (v:v = 3:1), the mixture was reacted at reflux temperature overnight, after the reaction was completed, the solid was separated by centrifugation, and the crude product was washed with water twice to obtain a yellow solid (cyclometalated iridium dimer coordinated with 2-phenylbenzothiazole), with a yield of 69%;

[0042] (5) Preparation of complex BDP-Ir-bpt: under argon protection atmosphere, fluorine boron dipyrrin modified bipyridine ligand BDP-bpy (64 mg, 0.08 mmol) and cyclometalated iridium dimer (50 mg, 0.04 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (the volume ratio of anhydrous dichloromethane and anhydrous methanol in the mixed solvent was 2:1), heated and stirred at 45°C under reflux for 12 h; after the reaction was completed, the solvent was removed by reduced pressure distillation, a saturated NH4PF6 methanol solution was added, and the solid was collected by centrifugation after stirring for 2 h, and the crude product was further purified by column chromatography to obtain dark green solid BDP-Ir-bpt, with a yield of 23%. 1H NMR (400 MHz, CD2Cl2) δ 8.43 - 8.40 (m, 2H), 8.16 - 8.10 (m, 3H), 8.08 (d, J = 2.2 Hz, 2H), 7.92 (t, J = 7.6 Hz, 2H), 7.86 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.60 - 7.50 (m, 5H), 7.49 - 7.31 (m, 9H), 7.23 - 7.17 (m, 1H), 7.15 - 7.07 (m, 4H), 7.06 - 7.00 (m, 1H), 6.95 - 6.86 (m, 2H), 6.77 - 6.68 (m, 2H), 6.36 (t, J = 8.2 Hz, 2H), 6.28 (d, J = 8.4 Hz, 1H), 6.13 (d, J = 8.5 Hz, 1H), 1.49 (s, 3H), 1.38 (s, 3H). HR-MS (positive mode, m / z): Calcd. 1303.2279, found 1303.2274 for [BDP-Ir-bpt-PF6 - ] + .

[0043] Comparative Example 1 is BDP-Ir-ppy, which has the following structural formula:

[0044]

[0045] The complex BDP-Ir-bpt prepared in Example 1 and the comparative example 1 BDP-Ir-ppy were subjected to the following experiments:

[0046] Cytotoxicity on human lung cancer cells A549, mouse lung cancer cells LLC and human lung fibroblast-like cells HLF:

[0047] The fluorine boron dipyrrin modified cyclometalated iridium (III) complex BDP-Ir-bpt, BDP-Ir-ppy and cisplatin CDDP were analyzed for their anti-proliferation effects by MTT colorimetric method. MTT (thiazolyl blue) is a kind of tetrazolium salt, which can be reduced by succinate dehydrogenase in the mitochondria of living cells to form a blue-violet product formazan (the product is soluble in DMSO), and the product has an absorption peak at 490 nm, so A 490nm can be used to analyze cell proliferation.

[0048] The specific experimental steps are as follows:

[0049] (1) First, resuscitate a tube of tumor cells, and culture with fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use after 3 passages;

[0050] (2) When the cells reached the logarithmic growth phase, they were seeded into 96-well plates at a density of 5000 cells / well (100 μL of culture medium per well) and cultured in a normoxic (21% O2) or hypoxic (1% O2) incubator (37°C, 5% CO2).

[0051] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing different concentration gradients of BDP-Ir-bpt, BDP-Ir-ppy and cisplatin CDDP was added to each well, and then placed in a constant temperature box for further incubation. After incubation for 6 hours, the light group was illuminated with a 630 nm laser (0.6 W / cm 2 ) Irradiate for 5 minutes and continue incubation;

[0052] (4) After incubation for 48 hours, 20 μL of MTT (5 mg / mL) was added to each well and incubated in a 37°C incubator for another 4 hours. The supernatant was removed and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. A was detected using an enzyme-linked immunosorbent assay (ELISA). 490nm , calculate the cell proliferation inhibition rate and find IC 50 The MTT test results of compounds BDP-Ir-bpt, BDP-Ir-ppy and cisplatin CDDP are shown in Table 1.

[0053] Table 1 IC values ​​of compounds BDP-Ir-bpt, BDP-Ir-ppy and cisplatin CDDP 50 Value (μM)

[0054]

[0055]

[0056] The results showed that the phototoxicity of the complex BDP-Ir-bpt to A549 and LLC cells under hypoxic conditions was higher than that of the compounds BDP-Ir-ppy and CDDP, while its dark toxicity was very low and it had low toxicity to normal cells, indicating that the complex BDP-Ir-bpt modified with C^N ligand had higher anti-tumor activity under hypoxic environment.

[0057] Example 2

[0058] Application of theoretical calculations for the cyclometallated iridium (III) complexes BDP-Ir-bpt and BDP-Ir-ppy prepared in Example 1:

[0059] Methods: The ground state geometries of BDP-Ir-ppy and BDP-Ir-bpt were optimized using time-dependent density functional theory (TDDFT) and B3LYP functional, where LANL2DZ basis set was used for Ir and 6-31G(d,p) basis set for the rest of the atoms. The ground state energies were calculated based on the geometry optimization. Within the TDDFT framework, the lowest singlet and triplet excited state geometries were optimized based on the analytical gradient method to obtain the emission energies. All the above calculations were performed using Gaussian 09.

[0060] The theoretical calculation results of the cyclometalated iridium(III) complexes BDP-Ir-bpt and BDP-Ir-ppy prepared in Example 1 are shown in Table 1. Figure 1 As shown in Table 1, the results show that the energy difference (ΔE T1-S0 ) of BDP-Ir-ppy from the first triplet excited state (T1) to the ground state (S0) is calculated to be 1.674 eV, which can overcome the energy barrier between O2 3 O2 1 O2 1 under irradiation. BDP-Ir-bpt can minimize ΔE T1-S0 to 1.425 eV. Under irradiation, the energy gap between the first singlet excited state (S1) and the T1 state of BDP-Ir-bpt is 0.149 eV, which supports high intersystem crossing (ISC) and subsequently induces efficient electron transfer in the case of poor energy transfer, while activating the transformation of O2to O2 -· .

[0061] Example 3

[0062] Application of the cyclometalated iridium(III) complex BDP-Ir-bpt prepared in Example 1 to the localization of subcellular organelles in cells:

[0063] Methods: A549 cells were seeded in a 35mm Corning laser confocal culture dish, and when the cell density grew to 70%, 1 μM of BDP-Ir-bpt was added for 6h treatment, then the culture medium was aspirated, and PBS was used for washing twice, 500 μL of the prepared commercial fluorescent probes Hoechst 33342, ER-Tracker Green, Lyso-Tracker Green, Golgi-Tracker Green and Mito-Tracker Green were added, and after incubation in a 37℃ incubator for 30min, the probes were aspirated, and PBS was used for washing twice, then fresh preheated serum-free medium was replaced, and then immediately observed by confocal microscopy. Compound BDP-Ir-bpt: λ ex = 630nm, λ em = 670±20nm; commercial probes: λex = 365-488 nm. The colocalization coefficient was analyzed by ImageJ software.

[0064] The intracellular localization of the synthesized cyclometalated iridium(III) complex BDP-Ir-bpt after co-incubation with commercial probes is shown in Figure 1. Figure 2 The results show that the cyclometalated iridium(III) complex BDP-Ir-bpt is mainly distributed in the lysosomes after being taken up by A549 cells, with a colocalization coefficient of 0.80, indicating that BDP-Ir-bpt has excellent lysosome-targeting ability.

[0065] Example 4

[0066] Application of the cyclometalated iridium(III) complex BDP-Ir-bpt prepared in Example 1 to generate reactive oxygen species in cells under light excitation under normoxic and hypoxic conditions:

[0067] Method 1: Confocal microscopy detection of ROS in cancer cells. A549 cells were seeded in 35 mm Corning laser confocal culture dishes and incubated in a normoxic or hypoxic incubator, respectively. When the cell density reached 70%, different concentrations of BDP-Ir-bpt and BDP-Ir-ppy were added for 6 h. The light irradiation group was irradiated with a 630 nm laser (0.6 W / cm 2 ) for 5 min, and then the cells were stained with 10 μM H2DCFH-DA in serum-free culture medium at 37°C for 30 min in the dark. After washing twice with PBS, the cells were immediately observed under a confocal microscope, with an excitation wavelength of 488 nm and an emission wavelength of 530 ± 20 nm.

[0068] Method 2: Flow cytometry detection of ROS in tumor cells. A549 cells were seeded in 6-well plates and incubated in a normoxic or hypoxic incubator, respectively. Different concentrations of BDP-Ir-bpt and BDP-Ir-ppy were added for 6 h. After irradiation with a 630 nm laser (0.6 W / cm 2 ) for 5 min, the cells were stained with 10 μM H2DCFH-DA in serum-free culture medium at 37°C for 30 min in the dark. After centrifugation, the supernatant was discarded, and the cells were washed three times with serum-free medium to remove the H2DCFH-DA that did not enter the cells. The green fluorescence intensity was measured within half an hour of collecting the cells using a flow cytometer. The excitation wavelength was 488 nm, and the emission wavelength was 530 ± 20 nm. The average green fluorescence intensity was analyzed using FlowJo 7.6 (Tree Star, OR, USA) software.

[0069] The results of the generation of reactive oxygen species by the cyclometalated iridium(III) complex BDP-Ir-bpt under light irradiation are shown in Figures 2 and Figure 3 ​4 The results show that bright green fluorescence was observed for both BDP-Ir-ppy and BDP-Ir-bpt treated A549 cells after irradiation under normoxic conditions. In addition, green fluorescence was also observed for BDP-Ir-bpt treated A549 cells after irradiation under hypoxic conditions, indicating that ROS generation is independent of oxygen. In contrast, ROS generation for BDP-Ir-ppy treated A549 cells after irradiation is oxygen dependent, as no green fluorescence was observed under hypoxic conditions. Flow cytometry analysis further indicates that the ROS generation ability of BDP-Ir-bpt is comparable under normoxic and hypoxic conditions.

[0070] Example 5

[0071] Application of cyclometalated iridium(III) complex BDP-Ir-bpt prepared in Example 1 to affect intracellular NADH and H2O2 levels:

[0072] Method: 1 x 105A549 cells per well were incubated in 6-well plates under hypoxic atmosphere at 37°C for 24 hours. The cells were then incubated with BDP-Ir-ppy (6 μΜ) and BDP-Ir-ppy (2, 4, 6 μΜ) for 6 hours, followed by irradiation with light (630 nm, 0.6 W / cm2) for 5 minutes. The NADH and H2O2 concentrations of the cells were then measured using NADH / NAD+ Assay Kit and H2O2 Assay Kit, respectively, according to the manufacturer's suggested protocol. 5 2 +

[0073] Results of cyclometalated iridium(III) complex BDP-Ir-bpt to affect intracellular NADH and H2O2 levels are shown in Figure 6. The results show that BDP-Ir-bpt induced a dramatic decrease in intracellular NADH under hypoxic conditions upon light irradiation (-35% at 6 μΜ). The concomitant increase in intracellular H2O2 content further indicates cellular oxidative stress. In contrast, BDP-Ir-ppy had no significant effect on intracellular NADH and H2O2 at a concentration of 6 μΜ. Figure 5

[0074] Example 6

[0075] Application of cyclometalated iridium(III) complex BDP-Ir-bpt prepared in Example 1 to induce changes in mitochondrial membrane potential in cells:

[0076] Method: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. 1 x 105A549 cells per well were incubated in 6-well plates under hypoxic atmosphere at 37°C for 24 hours. The cells were then incubated with BDP-Ir-ppy (6 μΜ) and BDP-Ir-ppy (2, 4, 6 μΜ) for 6 hours, followed by irradiation with light (630 nm, 0.6 W / cm2) for 5 minutes. The changes in mitochondrial membrane potential of the cells were then measured using JC-1 Assay Kit according to the manufacturer's suggested protocol. 5 ​​​​A549 cells were seeded in 6-well plates and incubated at 37°C under anoxic atmosphere for 24 hours. Then the cells were incubated with BDP-Ir-ppy (6 μM) and BDP-Ir-ppy (2, 4, 6 μM) for 6 hours, then irradiated with light (630 nm, 0.6 W / cm 2 ) for 5 minutes, and incubated in the dark for 18 h. The cells were harvested, washed with PBS, and then stained with prepared JC-1 working solution for 20 min. The cells were washed with 1x binding buffer and resuspended, and immediately detected using a BD C6 flow cytometer. The results were processed and analyzed using FlowJo 7.6 software. The detection fluorescence channels were λ ex = 488 nm, λ em = 530 ± 30 nm; λ ex = 488 nm, λ em = 590 ± 30 nm.

[0077] The results of the effect of the cyclometalated iridium (III) complex BDP-Ir-bpt on inducing changes in mitochondrial membrane potential in cells are shown in Figure 6 The results show that, compared with the control group and the BDP-Ir-ppy treatment group, the red fluorescence in the cells is weakened and the green fluorescence is significantly enhanced after treatment with the compound BDP-Ir-bpt, indicating that the compound BDP-Ir-bpt effectively induces a decrease in mitochondrial membrane potential under anoxic conditions, while BDP-Ir-ppy has no effect.

[0078] Example 7

[0079] Application of the cyclometalated iridium (III) complex BDP-Ir-bpt prepared in Example 1 to induce lysosome membrane permeabilization in cells:

[0080] Method: Confocal microscopy was used to detect changes in lysosome integrity in tumor cells. A549 cells were seeded in a 35 mm Corning laser confocal culture dish and incubated at 37°C under anoxic atmosphere for 24 hours. When the cell density reached 70%, different concentrations of BDP-Ir-bpt and BDP-Ir-ppy at a concentration of 6 μM were added for 6 h. The light irradiation group was irradiated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, and then incubated in the dark for 1 h. The cells were then stained with 5 μM AO working solution at 37°C in the dark for 30 min, and then washed twice with PBS and immediately observed using confocal microscopy. The excitation wavelength of the green light channel was 488 nm, and the excitation wavelength of the red light channel was 561 nm.

[0081] The results of the effect of the cyclometalated iridium (III) complex BDP-Ir-bpt on inducing lysosome membrane permeabilization in cells are shown in Figure 7The results show that compared with the control group and the BDP-Ir-ppy treatment group, the lysosome red fluorescence is obviously weakened after the complex BDP-Ir-bpt is treated by light, indicating that the complex BDP-Ir-bpt can effectively induce lysosome membrane permeabilization under the light excitation of hypoxia.

[0082] Example 8

[0083] Application of the cyclometalated iridium (III) complex BDP-Ir-bpt prepared in Example 1 to induce pyroptosis of A549 cells:

[0084] Method 1: Observation of cell morphology by confocal microscope. A549 cells were seeded in a 35mm Corning laser confocal culture dish and incubated in hypoxia overnight in the confocal culture dish. Then the cells were treated with BDP-Ir-bpt (6μM) for 6 hours. A549 cells were exposed to 5 minutes of irradiation (630nm, 0.6W cm -2 ). After 1 hour of incubation, the cells were washed with PBS and stained with Dil-Red for 30 minutes. After PBS washing, the cells were observed under a confocal microscope. The detection fluorescence channel was λ ex =561nm, λ em =600±30nm.

[0085] Method 2: Detection of pyroptosis protein content change by Western Blot (WB). A549 cells were seeded in a 100mm culture dish and incubated in hypoxia overnight. Add cell culture medium containing complex BDP-Ir-bpt (2, 4, 6μM) and BDP-Ir-ppy (6μM), after 6h of drug treatment, the light group uses 630nm laser (0.6W / cm 2) irradiation for 5 min, and then incubated in the dark for 18 h. The cells were collected by centrifugation, washed twice with PBS, and lysed in RIPA lysis buffer containing PMSF for 25 min at 4 °C. After centrifugation at 13,400 rpm for 20 min at 4 °C, the supernatant was collected as the whole-cell protein sample. The protein concentration of the sample was determined using a BCA protein assay kit. The expression levels of different proteins in the sample were detected by SDS-PAGE gel electrophoresis. After the gel was prepared, the same volume of protein sample was added to each well for gel electrophoresis. After appropriate separation, the electrophoresis was stopped immediately. The target protein was transferred to a PVDF membrane using a wet method. After blocking for 2 h in 5% skim milk, the membrane was incubated with the primary antibody diluted in skim milk according to the manufacturer's instructions. After incubation overnight at 4 °C, the membrane was washed with PBST (5x6 min / time). The washed membrane was incubated with the secondary antibody for 2 h, and then washed with PBST. Equal volumes of ECL developing solution were prepared and covered on the PVDF membrane. After 2 min of treatment, the chemical luminescence imaging system was used for imaging.

[0086] The results of the related experiments of the cyclometalated iridium(III) complex BDP-Ir-bpt inducing pyroptosis in A549 cells are shown in FIG. 6. Figure 8 As shown in FIG. 6, after the cells were treated with the complex BDP-Ir-bpt under light irradiation, bubbles appeared on the cell membrane, which is a typical feature of pyroptosis. GSDMD was cleaved into GSDMD-N, and the cleaved Caspase-1 was up-regulated, indicating that the complex BDP-Ir-bpt can induce pyroptosis in A549 cells. There was no obvious change in the bands of the dark control group and the BDP-Ir-ppy treatment group, indicating that BDP-Ir-ppy cannot induce pyroptosis under hypoxic conditions.

[0087] Example 9

[0088] Application of the cyclometalated iridium(III) complex BDP-Ir-bpt prepared in Example 1 to induce immunogenic death in A549 cells under hypoxic conditions:

[0089] Method 1: Confocal microscopy was used to detect the immunofluorescence of CRT and HMGB1. A549 cells were seeded in a 35 mm Corning laser confocal culture dish and incubated under hypoxia overnight. When the cell density reached 70%, different concentrations of BDP-Ir-bpt were added for 6 h. The light irradiation group used a 630 nm laser (0.6 W / cm 2) irradiation for 5 min, and then incubated in dark for 18 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 10-15 min, washed with PBS, permeabilized with 0.2% Triton X-100 for 30 min, washed with PBS, and incubated with the primary antibody diluted with skim milk according to the proportion in the instruction manual of the antibody at 4°C overnight. After washing twice with PBS, the cells were incubated with the diluted FITC-labeled secondary antibody at room temperature for 1 h, washed twice with PBS, and then stained with 0.3 μΜ DAPI for 10 min. After washing twice with PBS, the cells were observed immediately by a confocal microscope.

[0090] Method 2: Detection of immunofluorescence of CRT and HMGB1 by flow cytometry. 1 × 10 5 A549 cells were inoculated in a 6-well plate and cultured at 37°C under an anaerobic atmosphere for 24 h. The cell culture solution containing the complex BDP-Ir-bpt (4, 6 μΜ) and BDP-Ir-ppy (6 μΜ) was added, and after drug treatment for 6 h, the light irradiation group was irradiated with a 630 nm laser (0.6 W / cm 2 ) for 5 min, and then incubated in dark for 18 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 10-15 min, washed with PBS, permeabilized with 0.2% Triton X-100 for 30 min, washed with PBS, and incubated with the primary antibody diluted with skim milk according to the proportion in the instruction manual of the antibody at 4°C overnight. After washing twice with PBS, the cells were incubated with the diluted FITC-labeled secondary antibody at room temperature for 1 h, washed twice with PBS, and then stained with 0.3 μΜ DAPI for 10 min. After washing twice with PBS, the cells were observed immediately by a confocal microscope. ex em = 488 nm and λ em = 530 ± 30 nm.

[0091] Method 3: Detection of change in ATP content in cell culture solution by a multifunctional enzyme label meter. 1 × 10 5 A549 cells were inoculated in a 6-well plate and cultured at 37°C under an anaerobic atmosphere for 24 h. The cell culture solution containing the complex BDP-Ir-bpt (4, 6 μΜ) and BDP-Ir-ppy (6 μΜ) was added, and after drug treatment for 6 h, the light irradiation group was irradiated with a 630 nm laser (0.6 W / cm 2 ) for 5 min, and then incubated in dark for 18 h. The supernatant was collected by centrifugation and stored at low temperature, and the ATP content was detected by using an ATP detection kit.

[0092] The results of the complex BDP-Ir-bpt in inducing immunogenic death of A549 cells are shown in Fig. 2. Figure 9The results show that immunofluorescence shows that the green fluorescence of the BDP-Ir-bpt treatment group is significantly enhanced compared with the control group and the BDP-Ir-ppy treatment group, indicating the up-regulation of calreticulin CRT and the release of high mobility group protein B1 HMGB1, as well as ATP efflux, which together indicate that the phototreatment of the complex BDP-Ir-bpt under hypoxia induces immunogenic death of A549 cells.

[0093] Example 10

[0094] Anti-tumor and immune-activating applications of the cyclometalated iridium (III) complex BDP-Ir-bpt prepared in Example 1:

[0095] Methods: C57BL / 6J female mice (6-8 weeks old) were randomly divided into four groups of 6, and pre-treated with PBS, PBS+Light, BDP-Ir-bpt (5 mM) and BDP-Ir-bpt+Light (5 mM) LLC cells were injected subcutaneously 3 times at -7 days, -5 days and -1 day. Then on day 0, the mice were injected subcutaneously with untreated LLC cells (1 x 10 6 2 The tumor volume was calculated as follows: tumor volume (V) = a x b2 / 2, where a is the major axis of the tumor and b is the minor axis. All mice were euthanized at the end of the experiment and the tumors were harvested. The maturation of dendritic cells (DCs) and different types of T cells and their biomarkers in the tumor were also determined. The tumor was prepared into a single cell suspension, and the collected lymphocytes were incubated with anti-CD-86-PE, anti-CD-80-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE and analyzed by flow cytometry. Serum was isolated from the mice and analyzed. The secretion of TNF-a and IL-10 was detected using an ELISA kit.

[0096] The experimental results are shown in Figure 10 The results show that the tumor was significantly inhibited in the mice immunized with BDP-Ir-bpt+light treated LLC cells compared with the control group. Based on the tumor weight, the tumor inhibition rate of the BDP-Ir-bpt+light group was 73.6%, which was significantly higher than that of the control group. No significant weight loss was observed in any of the mice during the experiment. In addition, hematoxylin-eosin (H&E) staining showed no obvious organ damage, indicating that the complex exhibited good biocompatibility. At the same time, the expression of CD86 and CD80 in the tumor increased from 29.05% to 60.73%, which indicates that a large number of mature dendritic cells are distributed in the tumor to exert immune function. In addition, flow cytometry data shows that the percentage of CD4 + ​and CD8 + The expression level of T cells was significantly up-regulated, indicating that helper and effector T cells were formed in the tumor at the same time. At the same time, the serum anti-inflammatory cytokine secretion concentration of the immunized group treated with BDP-Ir-bpt was the highest, indicating that PDT treatment with BDP-Ir-bpt can stimulate the strongest systemic anti-tumor immune response.

[0097] The method can obtain a photosensitizer for generating type I active oxygen in a hypoxic environment (1% O2); the complex can participate in intracellular NADH circulation under near-infrared light irradiation at 630 nm, generate hydroxyl radicals and superoxide anions, cause lysosome membrane permeability, release cathepsin B, activate the Caspase-1 / GSDMD pathway to induce pyroptosis, further induce immunogenic cell death, achieve good hypoxic anti-tumor activity, and have a wide application scenario.

Claims

1. A cyclometalated iridium (III) complex for use in hypoxic photodynamic therapy, characterized in that, The structural formula is:

2. The method of synthesis of cyclometalated iridium (III) complexes according to claim 1, characterized in that, The method comprises the following steps: (1) under an inert atmosphere, heating and refluxing fluorine boron dipyrrin modified bipyridine ligand and cyclometalated iridium dimer in an organic solvent, and removing the solvent by reduced pressure distillation; (2) replacing the obtained crude product with NH4PF6, and separating and purifying the crude product by column chromatography to obtain the fluorine boron dipyrrin modified cyclometalated iridium (III) complex; The structural formula of the fluorine boron dipyrrin modified bipyridine ligand is as follows: The structural formula of the cyclometalated iridium dimer is as follows:

3. The method of synthesis of claim 2, wherein: The cyclometalated iridium dimer is prepared by the following method: under an inert atmosphere, dissolving 2-phenylbenzothiazole and iridium chloride hydrate in a mixed solution of ethylene glycol ethyl ether and water, and obtaining a crude product after reacting the mixture at a reflux temperature, and purifying the crude product by filtration and water washing.

4. The method of synthesis of claim 3, wherein: The molar ratio of the 2-phenylbenzothiazole and the iridium chloride hydrate is 2-2.5:

1.

5. The method of synthesis of claim 2, wherein: The fluorine boron dipyrrin modified bipyridine ligand is prepared by the following method, and the specific steps are as follows: (1.1) dissolving fluorine boron dipyrrin and N-iodosuccinimide in an organic solvent and reacting at room temperature; after the reaction is completed, the solvent is removed by reduced pressure distillation, and the crude product is purified by column chromatography to obtain iodinated fluorine boron dipyrrin; (1.2) dissolving iodinated fluorine boron dipyrrin, 2-thiophene formaldehyde, piperidine and p-toluenesulfonic acid in an organic solvent and reacting at a reflux temperature; after the reaction is completed, the solvent is removed by reduced pressure distillation, and the crude product is purified by column chromatography to obtain iodinated fluorine boron dipyrrin derivative; (1.3) under an inert atmosphere, dissolving iodinated fluorine boron dipyrrin derivative, 5-ethynyl-2,2'-bipyridine, Pd (PPh3) 2Cl2, PPh3 and CuI in anhydrous triethylamine, and reacting the mixture at a reflux temperature to obtain a crude product, and the crude product is separated and purified by column chromatography.

6. The method of synthesis of claim 5, wherein: In step (1.3), the molar ratio of the iodinated fluorine boron dipyrrin derivative and the 5-ethynyl-2,2'-bipyridine is 1:1-1.

5.

7. The method of synthesis of claim 2, wherein, In step (1), the reaction molar ratio of the fluorine boron dipyrrin modified bipyridine ligand and the cyclometalated iridium dimer is 2:1-1.

2.

8. The method of synthesis of claim 2, wherein, In step (1), the organic solvent is a mixed solvent of dichloromethane and methanol; in the mixed solvent, the volume ratio of dichloromethane to methanol is 2:1-1.

5.

9. The method of synthesis of claim 2, wherein, In step (1), the reflux reaction time is 12-13 h, and the temperature is 45-48℃.

10. The cyclometalated iridium (III) complex of claim 1 as a photosensitizer in the application of hypoxic photodynamic immunotherapy.

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