A light-controlled switchable cyclometallated iridium (III) complex and its synthesis method and application

Abstract

The present invention discloses a light-controlled switchable cyclometal iridium (III) complex, a synthesis method thereof, and an application thereof. The complex is formed by connecting a diarylethene derivative with a cyclometal iridium to obtain a photosensitizer with reactive oxygen "switch" control capability, high reactive oxygen generation capability, high anti-cancer activity, and low toxic side effects. The complex of the present invention converts a closed-loop drug into an open-loop complex intracellularly upon irradiation with near-infrared light, has good cellular uptake capability and the ability to target mitochondria and nuclei in tumor cells. The reactive oxygen species generated after irradiation damage the mitochondria and nucleus, causing a decrease in membrane potential, a decrease in intracellular ATP level and GSH level, and an increase in GSSG level. Simultaneously, DNA double strands are broken and cell cycle arrest occurs, inducing apoptosis, necrosis, and autophagy block in tumor cells, further inducing death of triple-negative breast cancer cells, achieving good anti-tumor activity, and having broad application scenarios.

Description

Technical Field

[0001] The present 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] Over 50 years ago, Rosenberg discovered the anticancer drug cisplatin, which is activated by hydrolysis upon entering 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 nonspecific manner, often leading to serious 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 being non-invasive and having 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 improving overall efficacy, precision therapy, and the development of inorganic pharmaceutical chemistry.

[0004] Prior art indicates that 1,2-bis(2'-methyl-5'-bromothiophen-3'-yl)perfluorocyclopentene (DTE derivatives) exhibit excellent photostability, fatigue resistance, and thermal irreversibility, reversibly converting between a non-conjugated open ring and a π-conjugated closed ring within the ultraviolet and visible light spectra, respectively. This π-conjugated chain variation can effectively control donor-acceptor interactions, enabling on-off control of reactive oxygen species. Furthermore, the addition of cyclometallated iridium improves the overall solubility of the complex, facilitating the development of single-molecule photoswitchable photosensitizers. This allows for reversible regulation of PS photosensitization using DTE derivatives, enabling high-precision, low-side effect, and high-spatiotemporal resolution PDT. However, DTE derivatives suffer from poor water solubility and structural instability when used as photoswitches. Furthermore, the poor control of reactive oxygen species by cyclometallated iridium as a photosensitizer can lead to side effects in practical applications.

[0005] In recent years, Tang and his research group have successfully developed novel photoactivated photosensitizers related to DTE. However, most of these reported photosensitizers are non-covalently linked systems, lacking good stability and, therefore, immature in their ability to reversibly control singlet oxygen. This is likely due to the separation of the two components under certain physiological conditions, resulting in a loss of their stable structure and reversible control of singlet oxygen. Furthermore, most of these photosensitizers are encapsulated in nanoparticles due to solubility concerns, and reports on single-molecule photoswitchable photosensitizers are relatively rare. Therefore, the development of single-molecule photoswitchable photosensitizers with excellent stability is urgently needed. Summary of the Invention

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

[0007] Technical solution: The present invention discloses a light-controlled switchable cyclometallated 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. 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 synthesis method of 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 photoswitchable cyclometalated iridium (III) complex;

[0014]

[0015] The preparation method of the cyclometalated iridium dimer is as follows: under an inert atmosphere, phenylpyridine and iridium chloride hydrate are dissolved in a mixed solution of ethylene glycol ethyl ether and water, the mixed materials are 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 DTE derivative-modified bipyridine ligand 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 carried out by 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 utilizes nitrogen or argon as a 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 switchable cyclometallated iridium (III) complex of the present invention is obtained by connecting a diarylethene derivative with a cyclometallated iridium, and adjusting the lipid solubility of the complex by introducing the cyclometallated iridium (III) part, thereby enhancing cellular uptake and providing a donor part for generating reactive oxygen species; the DTE derivative part can provide a receptor for FRET by changing the structure to achieve energy transfer; the light-controlled ring opening is directly connected to the metal center in a triple bond manner, thereby enhancing the stability of the complex structure, enhancing the ability to control reactive oxygen species, and improving the anti-cancer effect; thus, 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 cell nuclei in tumor cells. It can convert closed-ring drugs into open-ring drugs in cells through near-infrared light irradiation. The reactive oxygen species generated after 420nm irradiation cause damage to mitochondria and cell nuclei, which is specifically manifested as a decrease in membrane potential, a decrease in intracellular ATP level and GSH level, and an increase in GSSG level. At the same time, DNA double strands are broken and cell cycle arrest occurs, 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 light irradiation, and effectively inhibits tumor cell proliferation and induces tumor cell apoptosis, necrosis and autophagy inhibition through intracellular redox imbalance caused by mitochondrial and cell 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 cellular uptake quantitative map of the complex of Example 1 in MDA-MB-231 cells; wherein a is the localization map and b is the cellular uptake quantitative map

[0028] Figure 2 These are laser confocal fluorescence imaging and flow cytometry images of the complex of Example 1 generating reactive oxygen species in MDA-MB-231 cells under illumination; wherein a is the confocal fluorescence imaging image and b is the flow cytometry image;

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

[0030] Figure 4 This is 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 This is the light-induced nuclear damage of the complex in MDA-MB-231 cells in Example 1; wherein a is a flow cytometry image, and b is a protein immunoblot image;

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

[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 Graphs showing the results of light-induced cell death in MDA-MB-231 cells by the complex of Example 1; wherein, a is a micrograph, and b is a confocal image of live-dead staining. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below with reference to 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 light-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 overnight. After the reaction, the solvent was removed by distillation under reduced pressure, and the crude product was further purified by column chromatography to obtain a yellow oil. The oil was then placed in methanol with potassium carbonate and stirred at room temperature overnight. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was further purified by column chromatography to obtain Py-N as a white solid in 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'-bromothiophen-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 overnight; after completion of the reaction, the solvent was removed by distillation under reduced pressure, and the crude product was further purified by column chromatography to obtain B-bpy-DTE-C as a blue solid in an 8% yield; 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 mixture 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 argon protection 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 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 was obtained, which was further purified by column chromatography to obtain a dark blue solid, namely, the photoswitchable cyclometalated 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 cyclometalated iridium (III) is connected via a DTE derivative is:

[0049]

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

[0051] (1) Under argon protection 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 following experiments were conducted on the complex M-Ir-DTE-C / O prepared in Example 1 and the complex B-Ir-DTE-C / O prepared in Comparative Example 1:

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

[0056] The MTT colorimetric method 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 produce a blue-purple product, formazan (the product is soluble in DMSO), and the product has an absorption peak at 490 nm, so it can be used as an A 490 nm To analyze cell proliferation.

[0057] The specific experimental steps are as follows:

[0058] (1) First, thaw a tube of tumor cells and culture them in fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin). Passage them three times before use.

[0059] (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 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 concentration gradients of compound M-Ir-DTE-C / O, different concentration gradients of B-Ir-DTE-C / O, and different concentration gradients of cisplatin CDDP was added to each well, and then placed in a constant temperature box for further incubation. After incubation for 4 hours, the light-irradiated 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 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 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, under irradiation, M-Ir-DTE-O effectively killed breast cancer cells, particularly MDA-MB-231 cells. Furthermore, compared with B-Ir-DTE, M-Ir-DTE exhibited significantly better toxicity control, with negligible toxicity 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 365 nm light irradiation were investigated. The results showed that after intracellular conversion, M-Ir-DTE-O retained a potent toxicity similar to that of directly incubated M-Ir-DTE-O, thus validating 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 was determined by measuring iridium content. Briefly, cells were seeded and incubated overnight under standard growth conditions. Cells were then incubated with M-Ir-DTE-O (10 μM) and M-Ir-DTE-C (10 μM) complexes for 6 h, washed with PBS, harvested 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 of the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) (X Series 2, Thermo Fisher, USA). The average value of the three parallel experimental data was taken as the final result.

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

[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 seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 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, cells were stained with serum-free medium containing 10 μM H2DCFH-DA at 37°C in the dark for 30 minutes. The supernatant was discarded by centrifugation and the cells were washed three times with serum-free medium to remove H2DCFH-DA that had not entered the cells. Within half an hour of harvesting the cells, green fluorescence intensity was measured by flow cytometry using an excitation wavelength of 488 nm and an emission wavelength of 530 ± 20 nm. The mean green fluorescence intensity was analyzed using FlowJo 7.6 software (Tree Star, OR, USA).

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

[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 Cells were cultured in 6-well culture dishes at a concentration of 100 μg / mL until the cells reached 70% growth. 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 light group were illuminated using a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes and incubate for 20 hours. Wash three times with PBS, then add 100 μM lysis reagent. Centrifuge the cell lysate at 12,000 g at 4°C for 5 minutes. Culture the cells in a 96-well white plate as directed. Add the supernatant of the cell lysate to the ATP working solution. Luminescence from each well is immediately detected using a multi-function microplate reader.

[0077] Methods: MDA-MB-231 cells were seeded at a density of 10 5Cells were cultured in 6-well culture dishes with a concentration of 10 μM M-Ir-DTE-O and 10 μM M-Ir-DTE-C 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 seeded 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 incubated in the dark for an additional 20 hours. Drug-treated MDA-MB-231 cells were then harvested and homogenized. Total glutathione levels were measured using glutathione reductase and 5,5-dithiobis(2-nitrobenzoic acid). The thiol group of GSH reacts with DTNB to form yellow 5-thio-2-nitrobenzoic acid (TNB), with an absorbance of 405-414 nm. TNB (A405) can be detected by enzyme labeling, and reduced GSH levels are calculated by subtracting GSSG levels from total GSH (GSH = total GSH - 2 × GSSG).

[0078] The results of the cyclometallated iridium (III) complex M-Ir-DTE-O on the changes of ATP and GSH / GSSG in cells are as follows: 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 seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 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 minutes, and then continue to incubate in the dark for 20 hours. Then, the cells were stained with the pre-prepared JC-1 working solution at 37°C for 30 minutes 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 seeded with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 hours, the light group was illuminated using a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, then continue to incubate in the dark for 20 hours, harvest 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 a BD C6 flow cytometer to analyze 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, the red fluorescence in the cells was weakened and the green fluorescence was significantly enhanced after treatment with the compound M-Ir-DTE-O, indicating that the compound M-Ir-DTE-O effectively induced a decrease in mitochondrial membrane potential. The results of flow cytometry also showed a similar conclusion.

[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 seeded with MDA-MB-231 cells with good morphology and normal growth. After drug treatment for 4 hours, the light group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) for 40 minutes, then continue to incubate in the dark for 20 hours, harvest 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 a BD C6 flow cytometer to analyze 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 changes in γ-H2AX protein content. 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 a 100 mm culture dish of MDA-MB-231 cells that had grown on the wall. After 4 hours of drug treatment, the light group was illuminated with a 420 nm laser (3.5 mW·cm -2 ) irradiated for 40 minutes, continued to incubate in the dark for 20 hours, centrifuged to collect cells, washed twice with PBS, added RIPA strong lysis buffer containing PMSF, and lysed the whole cells for 25 minutes at a low temperature of 4°C. After the end, centrifuged at 13400rpm for 20 minutes at 4°C, and the supernatant obtained by centrifugation was the cell total protein sample required for the experiment; the protein concentration in the above protein sample was determined using the BCA protein content detection 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 experiment, and the electrophoresis was stopped immediately after appropriate separation; the target protein was transferred to the PVDF membrane using a wet method. After the end, the membrane was placed in 5% skim milk powder for blocking for 2 hours. According to the instructions for use of the antibody, the primary antibody was diluted with skim milk powder in the corresponding ratio, and the blocked membrane was placed in the primary antibody incubation solution at 4°C overnight to allow it to specifically bind to the target protein. After the end, it was washed with PBST (5×6min / time). The washed membrane was incubated in pre-prepared secondary antibody incubation solution for 2 hours to allow binding with the primary antibody, and then washed with PBST. An equal volume of ECL developer solution was prepared and overlaid on a PVDF membrane. After 2 minutes of treatment, the membrane was imaged using a chemiluminescence imaging system.

[0088] The experimental results of the light-controlled switchable cyclometallated iridium (III) complex M-Ir-DTE-O on the induction of nuclear damage in MDA-MB-231 cells are as follows: Figure 5 The results showed that, compared with the untreated control group or the M-Ir-DTE-C group, cells treated with the M-Ir-DTE-O complex were arrested in the G2 / M phase after light exposure, indicating that the M-Ir-DTE-O complex can induce nuclear damage in MDA-MB-231 cells. Western blotting also confirmed the drug's nuclear damage.

[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 seeded 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, then continue to incubate in the dark for 20 hours, harvest the cells, wash with PBS, resuspend in binding buffer, then add 5μM FITC working solution and stain in the dark for 5 minutes, then add 5μM PI and stain in the dark for 5-10 minutes, 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 levels. Pre-prepared cell culture medium containing M-Ir-DTE-O (5, 10 μM) and M-Ir-DTE-C (10 μM) was added to a 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) irradiated for 40 minutes, continued to incubate in the dark for 20 hours, centrifuged to collect cells, washed twice with PBS, added RIPA strong lysis buffer containing PMSF, and lysed the whole cells for 25 minutes at a low temperature of 4°C. After the end, centrifuged at 13400rpm for 20 minutes at 4°C, and the supernatant obtained by centrifugation was the cell total protein sample required for the experiment; the protein concentration in the above protein sample was determined using the BCA protein content detection 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 experiment, and the electrophoresis was stopped immediately after appropriate separation; the target protein was transferred to the PVDF membrane using a wet method. After the end, the membrane was placed in 5% skim milk powder for blocking for 2 hours. According to the instructions for use of the antibody, the primary antibody was diluted with skim milk powder in the corresponding ratio, and the blocked membrane was placed in the primary antibody incubation solution at 4°C overnight to allow it to specifically bind to the target protein. After the end, it was washed with PBST (5×6min / time). The washed membrane was incubated in pre-prepared secondary antibody incubation solution for 2 hours to allow binding with the primary antibody, and then washed with PBST. An equal volume of ECL developer solution was prepared and overlaid on a PVDF membrane. After 2 minutes of treatment, the membrane was imaged using a chemiluminescence imaging system.

[0093] The experimental results of the cyclometallated iridium (III) complex M-Ir-DTE-O on inducing apoptosis in MDA-MB-231 cells are as follows: Figure 6 The results showed that compared with the untreated control group or M-Ir-DTE-C group, the proportion of apoptotic cells increased significantly after cells were treated with the complex M-Ir-DTE-O. At the same time, 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 in 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 levels. Primary antibodies used in this application included autophagy marker proteins PINK1, 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 as follows: Figure 7The results showed that compared with the untreated control group and M-Ir-DTE-C, 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 inhibition 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 spheroids:

[0100] Methods: MDA-MB-231 cells were plated in prepared and ultra-low-adhesion 96-well round-bottom microplates (Corning) at 2500 cells per well. MDA-MB-231 MCTSs with a diameter of 500 μM were formed after 24 h. For 3D modeling, 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 cells were illuminated every two days with a 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). The 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. The spheroids were placed in a glass-bottom dish 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 570-620 nm) were imaged at different depths (z-stacking).

[0101] Application of photoswitchable cyclometallated iridium(III) complex M-Ir-DTE-O in inhibiting the growth of MDA-MB-231 3D cell spheroids Figure 8 The results showed that compared with the untreated control group and M-Ir-DTE-C, the cells treated with the complex M-Ir-DTE-O showed cell spheroid death.

[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. Through near-infrared light irradiation, the closed-loop drug is opened in the cell and light generates ROS, causing damage to the mitochondria and nucleus, resulting in 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.

Claims

1. A photoswitchable cyclometallated iridium (III) complex, characterized in that: The structure of the complex is shown in Formula I: 。 2. A method for synthesizing the photoswitchable cyclometallated 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 under reflux in a mixed solution of dichloromethane and methanol, and the solvent is removed by distillation under reduced pressure; (2) After centrifugation, the crude product is obtained, and the photoswitchable cyclometalated iridium (III) complex is obtained by separation and purification; 。 3. The synthesis method according to claim 2, characterized in that The preparation method of the bipyridine ligand modified with a diarylethene DTE derivative is as follows: under an inert atmosphere, a DTE derivative DTE-C and a bridging ligand 5-ethynyl-2,2'-bipyridine are dissolved in anhydrous triethylamine, catalyzed by Pd(PPh3)2Cl2 and CuI, and the mixture reacts at reflux temperature to obtain a crude product, which is then separated and purified by column chromatography. 。 4. The synthesis method according to claim 3, characterized in that The molar ratio of the DTE derivative DTE-C to 5-ethynyl-2,2'-bipyridine is 1:2-2.

5.

5. The synthesis method according to claim 2, characterized in that In step (1), the reaction molar ratio of the bipyridine ligand modified with the diarylethylene 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.

6. The synthesis method according to claim 2, 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.

7. The synthesis method according to claim 2 or 3, characterized in that The inert atmosphere utilizes nitrogen or argon as a protective gas.

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

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

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