Light-operated switch ring metal iridium (III) complex as well as synthesis method and application thereof

Abstract

The invention discloses a light-operated switch cyclometalated iridium (III) complex as well as a synthesis method and application thereof, the complex is characterized in that a diarylethene derivative is connected with cyclometalated iridium to obtain a photosensitizer with active oxygen'switch 'control capability, high active oxygen generation capability, high anti-cancer activity and low toxic and side effects; according to the complex disclosed by the invention, a closed-loop drug is converted into an open-loop complex in cells through near-infrared light irradiation, the complex has good cell uptake capability and capability of targeting mitochondria and cell nucleuses in tumor cells, and active oxygen generated after irradiation causes damage to the mitochondria and the cell nucleuses; according to the present invention, with the application of the polypeptide, the membrane potential is reduced, the intracellular ATP level and the intracellular GSH level are reduced, and the GSSG level is increased, the DNA double strand is broken, such that the cell cycle arrest is caused, the tumor cell apoptosis, the tumor cell necrosis and the autophagy arrest are induced, the triple negative breast cancer cell death is further induced, the good antitumor activity is achieved, and the wide application prospects are provided.

Description

Technical Field

[0001] The invention relates to a light-controlled switch cyclometal iridium (III) complex, and also relates to a synthesis method and application of the complex. Background Art

[0002] More than 50 years ago, Rosenberg discovered the anticancer drug cisplatin, which is activated by hydrolysis when it enters cells. Since then, the importance of metal complexes in medical development has steadily increased. For example, other platinum-based drugs such as carboplatin, oxaliplatin, nedaplatin and other metal-based drugs have been developed as anticancer therapeutic compounds and have been approved for marketing or entered clinical trials. However, these drugs act in a non-specific manner and often lead to severe side effects. To circumvent this problem, light can be used as an external stimulus to control the spatial and temporal activation of the drug.

[0003] Photodynamic therapy (PDT) is a well-established anticancer approach that converts oxygen into various ROS when photosensitizers (PS) are irradiated, including 1 O2、Superoxide (O2 - ) and hydroxyl radicals (·OH), killing tumor cells. Although PDT has the advantages of non-invasiveness and high spatiotemporal resolution, it still has shortcomings that severely limit its clinical application. In addition to the limited light penetration depth and hypoxic microenvironment in solid tumors, the inevitable dark toxicity of traditional PS in the "always on" mode can cause unnecessary damage to normal tissues and serious side effects. Therefore, the development of controllable metal-based PS as an advanced alternative material is crucial to improve the overall efficacy, precision therapy, and the development of inorganic pharmaceutical chemistry.

[0004] In the prior art, 1,2-bis(2'-methyl-5'-bromothiophene-3'-yl)perfluorocyclopentene (DTE derivative) has good photostability, fatigue resistance and thermal irreversibility, and reversibly converts between non-conjugated open ring and π-conjugated closed ring in the ultraviolet and visible light spectrum ranges, respectively. This change in the π-conjugated chain can be effectively used to control the donor-acceptor interaction, making the switch control of reactive oxygen possible. In addition, the addition of cyclometal iridium makes the complex as a whole have good solubility, which is conducive to the development of single-molecule light-controlled switch photosensitizers. The photosensitization of PS can be reversibly adjusted by DTE derivatives, realizing PDT with high precision, low side effects and high spatiotemporal resolution. However, DTE derivatives have poor water solubility and unstable structure when used as light-controlled switches. The poor control ability of cyclometal iridium as a photosensitizer for reactive oxygen will cause side effects in practical applications.

[0005] In recent years, Tang et al. have successfully developed new photoactivated photosensitizers related to DTE. Most of these reported photosensitizers are non-covalently linked systems without good stability, so the reversible control of singlet oxygen is not mature. This may be because the two components are separated under certain physiological conditions, losing their stable structure and reversible control of singlet oxygen. In addition, most of these photosensitizers are encapsulated in nanoparticles due to solubility reasons, and there are relatively few reports on single-molecule light-controlled switch photosensitizers. Therefore, it is urgent to develop a single-molecule light-activated switch photosensitizer with excellent stable structure. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a light-controlled switchable cyclometal iridium (III) complex with high reactive oxygen generation ability, high anticancer activity and low toxic side effects, and also provide a method for synthesizing the above-mentioned complex and its use in the preparation of photosensitizers or anti-tumor drugs.

[0007] Technical solution: The present invention discloses a light-controlled switch cyclometal iridium (III) complex, the structure of which is shown in Formula I:

[0008]

[0009] The complex is irradiated with near-infrared light to obtain an open-ring complex, the structure of which is shown in Formula II, and the open-ring complex is irradiated with ultraviolet light to obtain a light-controlled switchable cyclometal iridium (III) complex shown in Formula I:

[0010]

[0011] The method for synthesizing the above-mentioned light-controlled switchable cyclometalated iridium (III) complex comprises the following steps:

[0012] (1) Under an inert atmosphere, a bipyridine ligand modified with a diarylethene (DTE) derivative and a cyclometallated iridium dimer are heated to reflux in a mixed solution of dichloromethane and methanol, and the solvent is removed by distillation under reduced pressure;

[0013] (2) obtaining a crude product after centrifugation, and separating and purifying the crude product to obtain a light-controlled switchable cyclometalated iridium (III) complex;

[0014]

[0015] Among them, the preparation method of cyclometalated iridium dimer is: under an inert atmosphere, phenylpyridine and iridium chloride hydrate are dissolved in a mixed solution of ethylene glycol ethyl ether and water, the mixed material is reacted at reflux temperature to obtain a crude product, and the crude product is purified by filtering and washing with water; the molar ratio of the phenylpyridine and iridium chloride hydrate is 2 to 2.5:1.

[0016] The preparation method of the DTE derivative modified bipyridine ligand is as follows: under an inert atmosphere, the DTE derivative and the bridging ligand 5-ethynyl-2,2'-bipyridine are dissolved in anhydrous triethylamine, catalyzed by Pd(PPh3)2Cl2 and CuI, the mixture reacts at reflux temperature to obtain a crude product, and the crude product is separated and purified by column chromatography; the molar ratio of the DTE derivative to 5-ethynyl-2,2'-bipyridine is 1:2 to 2.5;

[0017]

[0018] Wherein, in step (1), the reaction molar ratio of the bipyridine ligand modified by the DTE derivative to the cyclometallated iridium dimer is 2:1-1.2, the volume ratio of dichloromethane to methanol in the mixed solution is 1:1-1.5, the reflux reaction time is 12-13 hours, and the temperature is 40-45°C.

[0019] Wherein, in step (2), the separation and purification of the crude product is specifically separation and purification by column chromatography, and the developing solvent is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 20:1.

[0020] Wherein, the inert atmosphere uses nitrogen or argon as protective gas.

[0021] The above-mentioned light-controlled switchable cyclometalated iridium (III) complex can also be used in the preparation of photosensitizers.

[0022] The above-mentioned light-controlled switchable cyclometallated iridium (III) complex can also be used in the preparation of anti-tumor drugs.

[0023] Wherein, the tumor is human triple-negative breast cancer cells.

[0024] Principle of the invention: The light-controlled switch cyclometal iridium (III) complex of the present invention is obtained by connecting a diarylethene derivative with cyclometal iridium, and by introducing a cyclometal iridium (III) part to adjust the lipid solubility of the complex, enhance cell uptake, and provide a donor part for generating reactive oxygen; the DTE derivative part can provide a receptor for FRET by changing the structure to achieve energy transfer; the light-controlled opening ring is directly connected to the metal center in a triple-bond manner, which enhances the stability of the complex structure, enhances the ability to control reactive oxygen, and improves the anti-cancer effect; a photosensitizer with reactive oxygen "switch" control ability, high reactive oxygen generation ability, high anti-cancer activity and low toxic side effects is obtained.

[0025] The complex of the present invention has good cellular uptake ability and the ability to target mitochondria and nuclei in tumor cells. It can convert closed-loop drugs into open-loop drugs in cells through near-infrared light irradiation. The reactive oxygen species generated after 420nm irradiation causes damage to mitochondria and nuclei, which is specifically manifested as a decrease in membrane potential, a decrease in intracellular ATP level, a decrease in GSH level and an increase in GSSG level. At the same time, the DNA double strand breaks and causes cell cycle arrest, inducing tumor cell apoptosis, necrosis and autophagy blockade, and further inducing triple-negative breast cancer cell death, achieving good anti-tumor activity and having broad application scenarios.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The light-controlled switchable cyclometal iridium (III) complex of the present invention has good lipid solubility and anti-tumor cell proliferation ability, generates ROS through intracellular ring opening and illumination, and effectively inhibits tumor cell proliferation and induces tumor cell apoptosis, necrosis and autophagy blockade through intracellular redox imbalance caused by mitochondrial and nuclear damage; (2) The preparation method of the complex is simple, the conditions are mild, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The localization map and cell uptake quantitative map of the complex of Example 1 in MDA-MB-231 cells; wherein a is the localization map and b is the cell uptake quantitative map

[0028] Figure 2 The laser confocal fluorescence imaging and flow cytometric images of the complex of Example 1 in MDA-MB-231 cells generating reactive oxygen species under light irradiation are shown; wherein a is the confocal fluorescence imaging image, and b is the cell flow cytometric image;

[0029] Figure 3 This is a graph showing the decrease in cellular ATP content and the imbalance in GSH and GSSG content caused by illumination of the complex in Example 1 in MDA-MB-231 cells; wherein a is a graph showing ATP content, and b is a graph showing the imbalance in GSH and GSSG content;

[0030] Figure 4 The light-induced mitochondrial damage in MDA-MB-231 cells by the complex of Example 1; wherein a is a confocal fluorescence imaging image, and b is a cell flow cytometry image;

[0031] Figure 5 The light-induced nuclear damage of the complex in Example 1 in MDA-MB-231 cells; wherein a is a cell flow cytometry graph, and b is a protein immunoblot graph;

[0032] Figure 6The figure is a picture of apoptosis and necrosis of MDA-MB-231 cells induced by light of the complex of Example 1; wherein a is a cell flow cytometry picture, and b is a protein immunoblot picture;

[0033] Figure 7 This is a diagram of light-induced autophagy in MDA-MB-231 cells by the complex of Example 1;

[0034] Figure 8 The results of light-induced cell death of the complex of Example 1 in MDA-MB-231 cells are shown in Figure 1; wherein a is a microscopic image, and b is a confocal image of live-dead staining. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0036] Example 1

[0037] The synthesis formula of the optically controlled switchable cyclometallated iridium (III) complex (M-Ir-DTE) of the present invention is as follows:

[0038]

[0039] Py-N was prepared by the following method: under an argon atmosphere, 5-bromo-2,2-bipyridine (1175 mg, 5 mmol), trimethylethynylsilane (1.2 mL, 6 mmol), triphenylphosphine palladium dichloride (105 mg, 0.15 mmol) and cuprous iodide (29 mg, 0.15 mmol) were dissolved in triethylamine, and stirred at reflux temperature overnight; after the reaction was completed, the solvent was removed by distillation under reduced pressure, and the crude product was further purified by column chromatography to obtain a yellow oil, which was then placed in methanol with potassium carbonate and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by distillation under reduced pressure; the crude product was further purified by column chromatography to obtain a white solid Py-N with a yield of 68%; 1 H NMR(400MHz,Chloroform-d)δ8.77(s,1H),8.69(d,J=4.9Hz,1H),8.40(dd,J=8.2,4.6Hz,2H),7. 90(dd,J=8.3,1.9Hz,1H),7.83(td,J=7.8,1.7Hz,1H),7.33(dd,J=7.4,5.0Hz,1H),3.29(s,1H).

[0040] B-bpy-DTE-C was prepared by the following method: under an argon atmosphere, Py-N (82 mg, 0.456 mmol) and 1,2-bis(2'-methyl-5'-bromothiophene-3'-yl)perfluorocyclopentene (100 mg, 0.19 mmol) were dissolved in triethylamine, catalyzed by triphenylphosphine palladium dichloride (8 mg, 0.0114 mmol) and cuprous iodide (2.2 mg, 0.0114 mmol), and stirred at reflux temperature overnight; after the reaction was completed, the solvent was removed by distillation under reduced pressure, and the crude product was further purified by column chromatography to obtain a blue solid B-bpy-DTE-C with a yield of 8%; 1 H NMR(400MHz,Chloroform-d)δ8.81-8.77(m,2H),8.71-8.68(m,2H),8.42(dt,J=8.1, 1.1Hz,4H),7.92(dd,J=8.3,2.2Hz,2H),7.86-7.81(m,2H),7.34(s,4H),1.98(s,6H).

[0041] The cyclometalated iridium dimer is prepared by the following method: under an inert atmosphere, phenylpyridine and iridium chloride hydrate are dissolved in a mixed solution of ethylene glycol ethyl ether and water, the mixed material is reacted at reflux temperature to obtain a crude product, and the crude product is purified by filtering and washing with water.

[0042] The synthesis method of the light-controlled switchable cyclometallated iridium (III) complex (M-Ir-DTE) of the present invention specifically comprises the following steps:

[0043] (1) Under an argon atmosphere, the DTE derivative-modified bipyridine ligand B-bpy-DTE-C (closed ring) (30 mg, 0.0414 mmol) and the cyclometallated iridium dimer (22 mg, 0.0207 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (the volume ratio of anhydrous dichloromethane to anhydrous methanol in the mixed solution was 1:1), and heated under reflux and stirred at 40°C for 12 h; after the reaction was completed, the solvent was removed by distillation under reduced pressure;

[0044] (2) After centrifugation, a crude product is obtained, which is further purified by column chromatography to obtain a dark blue solid, namely, the light-controlled switchable cyclometallic iridium (III) complex of the present invention, denoted as M-Ir-DTE-C, with a yield of 20%. 1H NMR(400MHz,Chloroform-d)δ9.77(d,J=8.7Hz,1H),9.68(d,J=8.2Hz,1H),8.77(s,1H),8.70(d,J=4.6Hz,1H),8.45(t,J=9.4 Hz,2H),8.31(d,J=8.8Hz,1H),8.26(t,J=8.1Hz,1H),7.93(q,J=9.1,8.4Hz,5H),7.84(t,J=8.3Hz,1H),7.79(d,J=5.7Hz,2H) ,7.70(dd,J=15.0,7.8Hz,2H),7.51(d,J=5.9Hz,1H),7.45(d,J=5.6Hz,1H),7.42-7.38(m,1H),7.35(d,J=5.2Hz,1H),7.05(d t,J=12.7,6.5Hz,4H),6.94(dt,J=14.7,7.5Hz,2H),6.51(d,J=21.2Hz,2H),6.28(dd,J=13.6,7.7Hz,2H),2.32-2.11(m,6H).

[0045] The complex was irradiated with M-Ir-DTE-C at 630 nm red light to obtain its ring-opening complex, which was recorded as M-Ir-DTE-O; 1 H NMR(400MHz,Chloroform-d)δ9.70(d,J=8.6Hz,1H),9.62(d,J=8.1Hz,1H),8.78(d,J=2.1Hz,1H),8.72-8.66(m,1H),8.42(d,J=8.2H z,2H),8.32(d,J=8.4Hz,1H),8.25(t,J=7.8Hz,1H),7.97-7.89(m,4H),7.88(d,J=5.3Hz,1H),7.86-7.81(m,1H),7.78(q,J=7.2Hz,2 H),7.70(dd,J=13.7,7.7Hz,2H),7.52(d,J=5.8Hz,1H),7.46(d,J=5.8Hz,1H),7.38(t,J=6.6Hz,1H),7.33(t,J=6.3Hz,1H),7.28(d, J=7.4Hz,2H),7.03(td,J=12.0,11.3,6.7Hz,4H),6.93(dt,J=14.6,7.5Hz,2H),6.28(dd,J=15.5,7.5Hz,2H),1.96(d,J=10.3Hz,6H).

[0046]

[0047] Comparative Example 1

[0048] The structural formula of the complex B-Ir-DTE in which the cyclometalated iridium (III) is connected via a DTE derivative is:

[0049]

[0050] The complex B-Ir-DTE-C was prepared by the following method:

[0051] (1) Under an argon atmosphere, the DTE derivative-modified bipyridine ligand B-bpy-DTE-C (closed ring) (30 mg, 0.0414 mmol) and the cyclometallated iridium dimer (44 mg, 0.0414 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (the volume ratio of anhydrous dichloromethane to anhydrous methanol in the mixed solution was 1:1), and heated under reflux at 40°C with stirring for 12 h; after the reaction was completed, the solvent was removed by distillation under reduced pressure;

[0052] (2) The crude product was further purified by column chromatography to obtain a dark blue solid B-Ir-DTE-C with a yield of 25%. 1 H NMR(400MHz,Chloroform-d)δ9.67(d,J=43.2Hz,4H),8.29(d,J=25.7Hz,4H),8 .05–7.86(m,8H),7.79(s,4H),7.70(dd,J=13.8,7.7Hz,4H),7.51(s,2H),7.43 (d,J=11.4Hz,3H),7.34(d,J=2.5Hz,1H),7.05(dt,J=13.1,7.2Hz,8H),6.93(d t,J=14.0,7.4Hz,4H),6.47(s,2H),6.27(dd,J=13.5,7.5Hz,4H),2.12(s,6H).

[0053] The complex B-Ir-DTE-O can be converted from B-Ir-DTE-C under irradiation of red light at 630 nm; 1H NMR(400MHz,Chloroform-d)δ9.59(d,J=47.0Hz,4H),8.28(d,J=36.5Hz,4H),7.97–7.84(m,8H),7.77(d,J=8.2Hz,4H),7.68( d,J=8.2Hz,3H),7.55–7.31(m,8H),7.13(s,2H),7.09–6.95(m,8H),6.91(t,J=7.0Hz,4H),6.26(d,J=7.4Hz,3H),2.02(s,6H).

[0054] The complex M-Ir-DTE-C / O prepared in Example 1 and the complex B-Ir-DTE-C / O prepared in Comparative Example 1 were subjected to the following experiment:

[0055] Cytotoxicity to human triple-negative breast cancer MDA-MB-231, human breast cancer cells MCF-7 and human mammary epithelial cells MCF-10A:

[0056] MTT colorimetry was used to analyze the antiproliferative effects of DTE derivative-modified cyclometallated iridium (III) complexes M-Ir-DTE-C / O, B-Ir-DTE-C / O and cisplatin CDDP. MTT (thiazolyl blue) is a tetrazolium salt that can be reduced by succinate dehydrogenase in the mitochondria of living cells to generate a blue-purple product, formazan (the product is soluble in DMSO), and the product has an absorption peak at 490nm, so it can be used to analyze the antiproliferative effects of cisplatin CDDP. 490 nm To analyze cell proliferation.

[0057] The specific experimental steps are as follows:

[0058] (1) First, thaw a tube of tumor cells, culture them with fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use them after three passages;

[0059] (2) When the cells reached the logarithmic growth phase, they were seeded into a 96-well plate at a density of 5,000 cells / well (100 μL of culture medium per well), and then placed in an incubator (37°C, 5% CO2) for culture;

[0060] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing different concentrations of compound M-Ir-DTE-C / O, B-Ir-DTE-C / O, and cisplatin CDDP was added to each well, and then placed in a constant temperature box for further incubation. After 4 hours of incubation, the light-exposed group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) irradiate for 40 minutes and continue incubation;

[0061] (4) After incubation for 24 hours, 20 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 hours in a 37°C incubator. 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 the IC 50 The MTT test results of compounds M-Ir-DTE-C / O, B-Ir-DTE-C / O and cisplatin CDDP are shown in Table 1.

[0062] Table 1. IC values ​​of compounds M-Ir-DTE-C / O, B-Ir-DTE-C / O and cisplatin CDDP 50 Value (μM)

[0063]

[0064] The results showed that M-Ir-DTE-O could effectively kill breast cancer cells, especially MDA-MB-231 cells, under irradiation. In addition, compared with B-Ir-DTE, M-Ir-DTE showed significantly better toxicity control ability, and the toxicity was negligible under dark conditions. The cytotoxicity of directly incubated M-Ir-DTE-O after irradiation and the cytotoxicity of M-Ir-DTE-C converted to M-Ir-DTE-O under 365nm light irradiation were studied. The results showed that after intracellular conversion, M-Ir-DTE-O still maintained a strong toxicity similar to that of directly incubated M-Ir-DTE-O, thus verifying the feasibility of intracellular drug conversion.

[0065] Example 2

[0066] Application of the light-controlled switchable metal iridium (III) complex M-Ir-DTE-O / C prepared in Example 1 to the localization of intracellular subcellular organelles (mitochondria).

[0067] Methods: The uptake and distribution of M-Ir-DTE-O and M-Ir-DTE-C by MDA-MB-231 cells were determined by measuring the iridium content. Briefly, cells were seeded and incubated overnight under standard growth conditions. They were then incubated with M-Ir-DTE-O (10 μM) and M-Ir-DTE-C (10 μM) complexes for 6 h, washed with PBS, collected with trypsin, stored in tubes, and resuspended in PBS. Mitochondria and nuclei were isolated from the pellet using a cell mitochondrial isolation kit (KeyGen, China). The buffer was then digested with concentrated nitric acid (100 μM) at 95 °C for 2 h, hydrogen peroxide (30%, 50 μM) at 95 °C for 1.5 h, and concentrated hydrochloric acid (50 μM) at 95 °C for 1.5 h to obtain a fully homogenized buffer. The remaining solution was diluted to 2 ml with double distilled water, and the iridium content in the sample was determined by ICP-MS (X Series 2, Thermo Fisher, USA). The average of the data from three parallel experiments was taken as the final result.

[0068] The subcellular distribution of the synthesized light-controlled switchable cyclometallated iridium (III) complexes M-Ir-DTE-O and M-Ir-DTE-C is shown in Example 1. Figure 1 As shown, the results showed that the photoswitchable cyclometallated iridium (III) complexes M-Ir-DTE-O and M-Ir-DTE-C were mainly distributed in the mitochondria and nucleus after being taken up by MDA-MB-231 cells, indicating that M-Ir-DTE-O and M-Ir-DTE-C have excellent ability to target mitochondria and nuclei.

[0069] Example 3

[0070] Application of the cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 to generate reactive oxygen species in cells upon light excitation:

[0071] Method 1: Confocal microscopy was used to detect ROS in cancer cells. MDA-MB-231 cells were inoculated in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, different concentrations of M-Ir-DTE-O and M-Ir-DTE-C were added for 4 h. The illumination group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 min, and then the cells were stained with serum-free medium containing 10 μM H2DCFH-DA at 37°C in the dark for 30 min, washed twice with PBS, and then immediately observed under a confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 530±30 nm.

[0072] Method 2: Flow cytometry was used to detect ROS in tumor cells. MDA-MB-231 cells were seeded in 6-well plates and grown overnight. Different concentrations of M-Ir-DTE-O and M-Ir-DTE-C were added for 4 h. The illumination group was illuminated with a 420 nm laser (3.5 mW cm -2 ) After irradiation for 40 minutes, the cells were stained with serum-free medium containing 10 μM H2DCFH-DA at 37°C in the dark for 30 minutes, centrifuged and discarded the supernatant, and washed three times with serum-free medium to remove H2DCFH-DA that did not enter the cells; the green fluorescence intensity was measured by flow cytometry within half an hour after the cells were collected; the excitation wavelength was 488 nm and the emission wavelength was 530 ± 20 nm. The average fluorescence intensity of green light was analyzed using FlowJo 7.6 (Tree Star, OR, USA) software.

[0073] The results of the generation of active oxygen by the cyclometallated iridium (III) complexes M-Ir-DTE-O and M-Ir-DTE-C under light irradiation are shown in Figure 2 The results showed that compared with the control group and M-Ir-DTE-C, the green fluorescence was significantly enhanced after treatment with M-Ir-DTE-O by flow cytometry and confocal microscopy, indicating that the complex M-Ir-DTE-O can produce a large amount of reactive oxygen species in MDA-MB-231 cells when stimulated by light.

[0074] Example 4

[0075] The light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 induces an imbalance of ATP and GSH\GSSG in cells:

[0076] Methods: MDA-MB-231 cells were seeded at a density of 10 5 The cells were cultured in a 6-well culture dish with a concentration of 100 μg / mL until the cells grew to 70%. The medium was then replaced with the medium containing M-Ir-DTE-O and M-Ir-DTE-C. ATP concentration was measured after 24 hours of incubation. After 4 hours of incubation, the cells in the illumination group were illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes and culture for 20 hours. Wash three times with PBS, then add 100 μM lysis reagent. Centrifuge the cell lysate at 12000g, 4°C for 5 minutes. Culture the cells in a 96-well white plate as instructed. Add the supernatant of the cell lysate to the ATP working solution. The luminescence of each well is immediately detected by a multifunctional microplate reader.

[0077] Methods: MDA-MB-231 cells were seeded at a density of 10 5The cells were cultured in 6-well culture dishes with a concentration of 10 μM / mL until the cells grew to 70%. Cell culture solutions containing different concentrations of M-Ir-DTE-O and 10 μM M-Ir-DTE-C were added to 6-well plates inoculated with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 h, the light group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, and then incubated in the dark for 20 hours. The drug-treated MDA-MB-231 cells were collected and homogenized. The total glutathione level was determined by glutathione reductase and 5,5-dithiobis(2-nitrobenzoic acid). The thiol group of GSH reacts with DTNB to generate yellow 5-thio-2-nitrobenzoic acid (TNB) with an absorbance of 405-414nm. TNB (A405) can be detected by enzyme labeling, and the reduced GSH level is calculated by subtracting the GSSG level from the total GSH (GSH = total GSH-2×GSSG).

[0078] The results of the cyclometallated iridium (III) complex M-Ir-DTE-O on the changes of intracellular ATP and GSH / GSSG are shown in Figure 3 The results showed that compared with the control group and M-Ir-DTE-C, after treatment with compound M-Ir-DTE-O, it was observed that the ATP content and GSH content decreased, and the GSSG content increased, indicating that compound M-Ir-DTE-O effectively induced mitochondrial dysfunction.

[0079] Example 5

[0080] The light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 induces mitochondrial damage in cells:

[0081] Method 1: Confocal microscopy was used to detect changes in mitochondrial membrane potential in tumor cells. MDA-MB-231 cells were inoculated in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, different concentrations of M-Ir-DTE-O and 10 μM M-Ir-DTE-C were added for 4 h. The illumination group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 min, and then continue to incubate in the dark for 20 h. Then, the cells were stained with the pre-prepared JC-1 working solution at 37°C for 30 min in the dark, and then immediately observed with a confocal microscope.

[0082] Method 2: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. Cell culture medium containing different concentrations of M-Ir-DTE-O and 10 μM M-Ir-DTE-C was added to 6-well plates inoculated with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 h, the illumination group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, and then continue to incubate in the dark for 20 hours, collect the cells, wash with PBS, and then add the prepared JC-1 working solution to stain for 30 minutes; wash the cells with 1× binding buffer and resuspend them, and immediately use BD C6 flow cytometer to detect the samples, and use FlowJo7.6 software to process and analyze the results. The detection fluorescence channel is λ ex =488nm,λ em =530±30nm;λ ex =488nm,λ em =590±30nm.

[0083] The results of the cyclometallated iridium (III) complex M-Ir-DTE-O on the changes in mitochondrial membrane potential in cells are as follows Figure 4 The results showed that compared with the control group and M-Ir-DTE-C, after treatment with compound M-Ir-DTE-O, it was observed that the red fluorescence in the cells was weakened and the green fluorescence was significantly enhanced, indicating that compound M-Ir-DTE-O effectively induced a decrease in mitochondrial membrane potential. At the same time, the results of flow cytometry also showed similar conclusions.

[0084] Example 6

[0085] The light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 induces nuclear damage in cells:

[0086] Method 1: Flow cytometry was used to detect changes in cell cycle arrest in tumor cells. Cell culture medium containing different concentrations of M-Ir-DTE-O and 10 μM M-Ir-DTE-C was added to 6-well plates inoculated with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 h, the illumination group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, and then continue to incubate in the dark for 20 hours, collect the cells, wash with PBS, and then add the prepared propidium iodide (PI) working solution to stain for 30 minutes; wash the cells with 1× binding buffer and resuspend them, and immediately use BD C6 flow cytometer to detect the samples, and use FlowJo 7.6 software to process and analyze the results. The detection fluorescence channel is λ ex =488nm,λ em=530±30nm;λ ex =488nm,λ em =590±30nm.

[0087] Method 2: Western blotting (WB) was used to detect the changes in γ-H2AX protein content. The pre-prepared cell culture medium containing the complexes M-Ir-DTE-O (5, 10 μM) and M-Ir-DTE-C (10 μM) was added to the 100 mm culture dish of MDA-MB-231 cells that had grown on the wall. After 4 h of drug treatment, the light group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, continue to incubate in the dark for 20 hours, collect cells by centrifugation, wash twice with PBS, add RIPA strong lysis solution containing PMSF, and perform whole cell lysis for 25 minutes at a low temperature of 4°C. After the end, centrifuge at 13400rpm for 20 minutes at 4°C, and the supernatant obtained by centrifugation is the cell whole protein sample required for the experiment; use the BCA protein content detection kit to determine the protein concentration in the above protein sample; use SDS-PAGE gel electrophoresis to detect the expression content of different proteins in the sample. After the gel is prepared, add the same volume of protein sample to each well for gel electrophoresis experiment, and stop electrophoresis immediately after appropriate separation; use the wet method to transfer the target protein to the PVDF membrane, and after the end, place the membrane in 5% skim milk powder for blocking for 2 hours. Dilute the primary antibody with skim milk powder according to the corresponding ratio according to the instructions for use of the antibody, and place the blocked membrane in the primary antibody incubation solution at 4°C overnight to make it specifically bind to the target protein. After the end, wash with PBST (5×6min / time). The washed membrane was placed in the pre-prepared secondary antibody incubation solution for 2 hours to allow it to bind to the primary antibody, and then washed with PBST. An equal volume ratio of ECL developer was prepared and covered on the PVDF membrane. After 2 minutes of treatment, the membrane was photographed using a chemiluminescence imaging system.

[0088] The experimental results of the light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O on inducing nuclear damage in MDA-MB-231 cells are as follows Figure 5 The results showed that compared with the control group or M-Ir-DTE-C group without drug treatment, the cells were blocked in the G2 / M phase after being treated with the complex M-Ir-DTE-O, indicating that the complex M-Ir-DTE-O can induce nuclear damage in MDA-MB-231 cells. At the same time, the protein immunoblot also confirmed the damage of the drug to the cell nucleus.

[0089] Example 7

[0090] The cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 induces apoptosis and necrosis in MDA-MB-231 cells:

[0091] Method 1: Flow cytometry was used to detect apoptosis in tumor cells. Cell culture medium containing different concentrations of M-Ir-DTE-O and 10 μM M-Ir-DTE-C was added to 6-well plates inoculated with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 h, the illumination group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, and then continue to incubate in the dark for 20 hours. Collect the cells, wash with PBS, resuspend in binding buffer, then add 5μM FITC working solution to stain in the dark for 5 minutes, then add 5μM PI to stain in the dark for 5-10 minutes, and immediately use BD C6 flow cytometer to detect the samples, and use FlowJo7.6 software to process and analyze the results. The detection fluorescence channel is λ ex =488nm,λ em =530±30nm;λ ex =561nm,λ em =600±30nm.

[0092] Method 2: Western blotting (WB) was used to detect changes in apoptotic protein content. Pre-prepared cell culture medium containing M-Ir-DTE-O (5, 10 μM) and M-Ir-DTE-C (10 μM) was added to the 100 mm culture dish of MDA-MB-231 cells that had grown on the wall. After 4 h of drug treatment, the light group was illuminated with a 420 nm laser (3.5 mW·cm -2) for 40 minutes, continue to incubate in the dark for 20 hours, collect cells by centrifugation, wash twice with PBS, add RIPA strong lysis solution containing PMSF, and perform whole cell lysis for 25 minutes at a low temperature of 4°C. After the end, centrifuge at 13400rpm for 20 minutes at 4°C, and the supernatant obtained by centrifugation is the cell whole protein sample required for the experiment; use the BCA protein content detection kit to determine the protein concentration in the above protein sample; use SDS-PAGE gel electrophoresis to detect the expression content of different proteins in the sample. After the gel is prepared, add the same volume of protein sample to each well for gel electrophoresis experiment, and stop electrophoresis immediately after appropriate separation; use the wet method to transfer the target protein to the PVDF membrane, and after the end, place the membrane in 5% skim milk powder for blocking for 2 hours. Dilute the primary antibody with skim milk powder according to the corresponding ratio according to the instructions for use of the antibody, and place the blocked membrane in the primary antibody incubation solution at 4°C overnight to make it specifically bind to the target protein. After the end, wash with PBST (5×6min / time). The washed membrane was placed in the pre-prepared secondary antibody incubation solution for 2 hours to allow it to bind to the primary antibody, and then washed with PBST. An equal volume ratio of ECL developer was prepared and covered on the PVDF membrane. After 2 minutes of treatment, the membrane was photographed using a chemiluminescence imaging system.

[0093] The experimental results of the cyclometallated iridium (III) complex M-Ir-DTE-O on inducing apoptosis of MDA-MB-231 cells are as follows Figure 6 The results showed that compared with the control group or M-Ir-DTE-C group without drug treatment, the proportion of apoptotic cells increased significantly after the cells were treated with the complex M-Ir-DTE-O, and the pro-apoptotic protein Caspase-9 was cleaved and Cleaved Caspase-9 was upregulated, indicating that the complex M-Ir-DTE-O can induce apoptosis and necrosis of A549 cells.

[0094] Example 8

[0095] Application of the light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 to inducing autophagy inhibition in MDA-MB-231 cells:

[0096] Methods: Western blotting (WB) was used to detect changes in autophagy protein content. The primary antibodies used in this application included autophagy marker proteins PINK 1, LC3, and p62.

[0097] The experimental results of the light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O on the expression of autophagy-related proteins in MDA-MB-231 cells are shown in Figure 7The results showed that compared with the control group and M-Ir-DTE-C without drug treatment, the expression of mitochondrial autophagy-related protein PINK1 was significantly upregulated after cells were treated with the complex M-Ir-DTE-O, and the levels of autophagy marker proteins LC3-II and p62 increased, indicating that the complex M-Ir-DTE-O can induce autophagy blockade in MDA-MB-231 cells.

[0098] Example 9

[0099] Application of the cyclometallated iridium (III) complex M-Ir-DTE-O prepared in Example 1 to inhibit the growth of MDA-MB-231 3D cell spheres:

[0100] Methods: MDA-MB-231 cells were divided into prepared and ultra-low adhesion 96-well round-bottom microplates (Corning) with 2500 cells per well. MDA-MB-231 MCTSs with a diameter of 500 μM were formed after 24 h. For 3D models, MDA-MB-231 MCTSs were divided into different groups and cultured in medium containing M-Ir-DTE-O (40 μM) and M-Ir-DTE-C (40 μM), respectively. The 3D models were treated with (420 nm, 3.5 mW·cm -2 ,40min) laser irradiation of MCTSs was performed and incubated for 4h. The culture medium was changed every two days. Three rounds of irradiation were performed in this experiment. Spheroid growth was monitored using a live cell phase contrast microscope (Axio Observer, Zeiss). Spheroids were then washed twice with PBS, stained with calcein AM / PI according to the manufacturer's instructions (Beyotime, China), and fixed in 4% paraformaldehyde. Spheroids were placed in glass-bottom dishes and imaged using a confocal scanning microscope system (Calcein AM:λ ex =488nm,λ em Range 500-550nm, PI:λ ex =561nm,λ em range 570-620nm) were imaged at different depths (z-stacking).

[0101] Application of photoswitchable cyclometallated iridium(III) complex M-Ir-DTE-O to inhibit the growth of MDA-MB-231 3D cell spheroids Figure 8 The results showed that compared with the control group and M-Ir-DTE-C without drug treatment, the cell spheres died after being treated with the complex M-Ir-DTE-O.

[0102] Therefore, the photoswitchable cyclometal iridium (III) complex of the present invention has good cellular uptake ability and the ability to target mitochondria and nuclei in tumor cells. The closed-loop drug is opened in the cell by near-infrared light irradiation, and ROS is generated by light to cause damage to the mitochondria and nucleus, thereby decreasing the membrane potential, the intracellular ATP level, the GSH level, and the GSSG level. At the same time, the DNA double strand breaks and causes cell cycle arrest, inducing tumor cell apoptosis, necrosis, and autophagy blockade, and further inducing triple-negative breast cancer cell death, achieving good anti-tumor activity, and having a wide range of application scenarios.

Claims

1. A light-controlled switchable cyclometalated iridium (III) complex, characterized in that: The structure of the complex is shown in Formula I:

2. The light-controlled switchable cyclometalated iridium (III) complex according to claim 1, characterized in that: The complex is irradiated with near-infrared light to obtain an open-ring complex, the structure of which is shown in Formula II. The open-ring complex is irradiated with ultraviolet light to obtain a light-controlled switchable cyclometal iridium (III) complex shown in Formula I:

3. A method for synthesizing the light-controlled switchable cyclometalated iridium (III) complex according to claim 1, characterized in that: The following steps are involved: (1) Under an inert atmosphere, a bipyridine ligand modified with a diarylethene (DTE) derivative and a cyclometallated iridium dimer are heated to reflux in a mixed solution of dichloromethane and methanol, and the solvent is removed by distillation under reduced pressure; (2) obtaining a crude product after centrifugation, and separating and purifying the crude product to obtain a light-controlled switchable cyclometalated iridium (III) complex; 4. The synthesis method according to claim 3, characterized in that The preparation method of the DTE derivative modified bipyridine ligand is as follows: under an inert atmosphere, the DTE derivative and the bridging ligand 5-ethynyl-2,2'-bipyridine are dissolved in anhydrous triethylamine, catalyzed by Pd(PPh3)2Cl2 and CuI, the mixture reacts at reflux temperature to obtain a crude product, and the crude product is separated and purified by column chromatography to obtain the crude product; 5. The synthesis method according to claim 4, characterized in that The molar ratio of the DTE derivative to 5-ethynyl-2,2'-bipyridine is 1:2-2.

5.

6. The synthesis method according to claim 3, characterized in that In step (1), the reaction molar ratio of the bipyridine ligand modified by the DTE derivative to the cyclometallated iridium dimer is 2:1-1.2, the volume ratio of dichloromethane to methanol in the mixed solution is 1:1-1.5, the reflux reaction time is 12-13 hours, and the temperature is 40-45°C.

7. The synthesis method according to claim 3, characterized in that In step (2), the crude product is separated and purified by column chromatography, and the developing solvent is a mixture of petroleum ether and ethyl acetate.

8. The synthesis method according to claim 3 or 4, characterized in that: The inert atmosphere uses nitrogen or argon as a protective gas.

9. Use of the photoswitchable cyclometallated iridium (III) complex according to claim 1 in a photosensitizer.

10. Use of the photoswitchable cyclometallated iridium (III) complex according to claim 1 in the preparation of anti-tumor drugs.

Images