A fluoroboron dipyrrole ring metal iridium (III) complex and its synthesis method and application

Abstract

The present invention discloses a fluoroboron dipyrrole cyclometallated iridium (III) complex, a synthesis method thereof, and applications thereof. The complex is obtained by connecting fluoroboron dipyrrole with cyclometallated iridium to obtain a photosensitizer with high reactive oxygen species generation capacity, high anticancer activity, and low toxic side effects. The complex has good cellular uptake capacity and the ability to target lysosomes in tumor cells. It can generate reactive oxygen species through near-infrared light irradiation, causing lysosomal membrane permeability, the release of acidic hydrolases leading to tumor cell acidification, and calcium ion homeostasis imbalance causing mitochondrial damage, which is specifically manifested as a decrease in membrane potential and intracellular ATP level, inducing tumor cell apoptosis and autophagy blockade, and further inducing immunogenic cell death, thereby achieving good anti-tumor activity and having broad application scenarios.

Description

Technical Field

[0001] The present invention relates to a fluoroboron dipyrrole ring metal iridium (III) complex, and also relates to a synthesis method and application of the complex. Background Art

[0002] Cancer is a difficult disease to cure and has a high mortality rate. Chemotherapy remains the primary clinical treatment for cancer, but while it kills cancer cells, it can also damage normal cells and tissues, including hematopoiesis. Therefore, the development of novel cancer treatments is becoming increasingly important.

[0003] Photodynamic therapy (PDT) has been widely used in cancer treatment due to its unique advantages, including minimal side effects, non-invasiveness, and resistance to drug resistance. Non-toxic photosensitizers (PSs) can be precisely delivered to tumor sites and absorb light to generate reactive oxygen species (ROS), effectively killing cancer cells. Therefore, the development of photosensitizers is considered a key factor in improving the efficacy of phototherapy. To improve tissue penetration and reduce photodamage, PSs should have a low photobleaching rate and maintain structural stability under laser irradiation. Furthermore, PSs should absorb wavelengths in the near-infrared (NIR) region and have a high molar absorption coefficient to ensure efficient photon absorption. Typically, upon irradiation, PSs undergo photoexcitation, transitioning from the ground state (S0) to the excited singlet state (S1), then to the excited triplet state (T1) via intersystem crossing (ISC). These ROS then react with biomolecules or oxygen via energy or electron transfer, generating cytotoxic ROS. ROS not only directly kill tumor cells but also activate innate and adaptive immune responses. Therefore, the rational design of the chemical structure of PSs has become a key issue in the application of PDT.

[0004] In the prior art, 4,4-difluoro-4-boron-3a,4a-diaza-s-indene (BODIPY) derivatives have high molar absorption coefficients and excellent photochemical stability. In addition, the BODIPY core also has good reactivity and can be doped with various functional groups to adjust the absorption and emission bands. However, due to its high fluorescence quantum yield, the possibility of inactivating the S1 state through ISC is very small, resulting in unsatisfactory ROS yield. On the other hand, cyclometallated iridium (III) complexes exhibit a heavy atom effect and can effectively generate T1 states, thereby generating ROS. However, the absorption wavelength of cyclometallated I iridium (III) complexes is usually in the ultraviolet (UV) region, which limits their application in phototherapy and requires the development of chemical structures more suitable for PDT applications. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a fluoroboron dipyrrole-modified cyclometallated iridium (III) photosensitizer. On the one hand, the complex can solve the problems of poor water solubility and low ROS yield when fluoroboron dipyrrole is used as a photosensitizer. On the other hand, near-infrared light excitation can reduce photodamage and enhance tissue penetration. Also provided are a method for synthesizing the above-mentioned fluoroboron dipyrrole-modified cyclometallated iridium (III) complex and its application in the preparation of anti-tumor drugs or anti-tumor drug components.

[0006] Technical solution: The present invention discloses a fluoroboron dipyrrole ring metal iridium (III) complex, the structural formula of which is shown below:

[0007]

[0008] The synthesis method of the above-mentioned complex comprises the following steps:

[0009] (1) Under an inert atmosphere, a bipyridine ligand modified with boron dipyrrole (BODIPY) 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;

[0010] (2) replacing the crude product with NH4PF6, and separating and purifying the crude product by column chromatography to obtain a fluoroboron dipyrrole-modified cyclometalated iridium (III) complex;

[0011] The structural formula of the fluoroborane dipyrrole-modified bipyridine ligand is as follows:

[0012]

[0013] The structural formula of the cyclometallated iridium dimer is shown below:

[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] Among them, the preparation method of the fluoroborane dipyrrole-modified bipyridine ligand is as follows: under an inert atmosphere, iodinated fluoroborane dipyrrole and the 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; the molar ratio of the fluoroborane dipyrrole and 5-ethynyl-2,2'-bipyridine is 1:1 to 1.5.

[0017] Wherein, the inert atmosphere utilizes nitrogen or argon as a protective gas.

[0018] Wherein, in step (1), the reaction molar ratio of the fluoroboron dipyrrole-modified bipyridine ligand to the cyclometallated iridium dimer is 2:1 to 1.2.

[0019] Wherein, in step (1), the volume ratio of dichloromethane to methanol in the mixed solution is 2:1 to 1.5, more preferably 2:1.

[0020] Wherein, in step (1), the reflux reaction time is 12 to 13 hours at a temperature of 45 to 48°C, and more preferably, the reflux time is 12 hours at a temperature of 45°C.

[0021] Wherein, in step (2), NH4PF6 is used for replacement to obtain a crude product, specifically, a saturated NH4PF6 methanol solution is added and stirred for 2 to 3 hours.

[0022] The present invention also discloses the application of the fluoroboron dipyrrole-modified cyclometallated iridium (III) complex in the preparation of antitumor drugs or antitumor drug components.

[0023] Wherein, the tumor is human lung cancer cell.

[0024] Principle of the invention: The fluoroboron dipyrrole-modified cyclometallated iridium (III) complex of the present invention regulates the lipid solubility of the complex by introducing a cyclometallated iridium (III) moiety, thereby enhancing cellular uptake; the fluoroboron dipyrrole skeleton is modified with a thiophene group to red-shift the spectrum to the near-infrared region, thereby enhancing tissue penetration and reducing photodamage; the fluorophore is directly connected to the metal center in a conjugated manner, which greatly enhances the intersystem crossing effect, increases the ROS yield, and improves the anti-cancer effect.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The fluoroboron dipyrrole-modified cyclometallated iridium (III) complex of the present invention has good lipid solubility and anti-tumor cell proliferation ability, generates ROS under light, effectively inhibits tumor cell proliferation, induces tumor cell apoptosis and autophagy blockade through calcium ion imbalance caused by lysosomal damage, and can also activate immunogenic cell death; (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

[0026] Figure 1 This is the localization map of the complex of Example 1 in A549 cells;

[0027] Figure 2These are laser confocal fluorescence imaging and flow cytometry images of the complex of Example 1 generating reactive oxygen species in A549 cells under illumination; wherein a is the confocal fluorescence imaging image and b is the flow cytometry image;

[0028] Figure 3 This is a confocal fluorescence imaging image of the light-induced lysosomal membrane permeabilization of the complex in Example 1 in A549 cells;

[0029] Figure 4 These are laser confocal fluorescence images of the complex of Example 1 in A549 cells induced by light-induced cell acidification; wherein a is a confocal fluorescence imaging image, and b is a fluorescence intensity quantitative image;

[0030] Figure 5 This is a laser confocal fluorescence imaging image of calcium ion overload in the cytoplasm and mitochondria induced by light in A549 cells by the complex of Example 1;

[0031] Figure 6 The figures are flow cytometry and confocal fluorescence imaging of the changes in mitochondrial membrane potential induced by the complex in Example 1 in A549 cells after illumination; wherein a is the confocal fluorescence imaging figure; b is the flow cytometry figure at different concentrations;

[0032] Figure 7 The flow cytometry and immunoblot images of apoptosis induced by the complex of Example 1 in A549 cells after illumination; wherein, a is the flow cytometry image at different concentrations; b is the immunoblot image;

[0033] Figure 8 This is an immunoblot protein image showing that the complex of Example 1 induces autophagy inhibition in A549 cells after illumination; wherein a is an immunoblot protein image, and b is a quantitative image of protein expression;

[0034] Figure 9 The results of the immunogenic cell death induced by the complex of Example 1 in A549 cells after illumination are shown in Figures a and b; Figures c and c are immunofluorescence confocal images; Figure c is a flow cytometry graph at different concentrations; and Figure d is a quantitative bar graph of extracellular ATP content.

[0035] Figure 10 These are in vivo experimental diagrams of mice subjected to photodynamic therapy using the complex of Example 1; wherein a is a schematic diagram of the experiment; b is an image after tumor peeling; c and d are diagrams of changes in proximal and distal tumor volumes, respectively; e is a diagram of changes in mouse weight; f to h are the expression of mature dendritic cells in the distal tumor; and i to k are the expression of related anti-inflammatory factors. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] The preparation method of the fluoroboron dipyrrole-modified cyclometallated iridium (III) complex (abbreviated as Ir-1) of the present invention comprises the following steps:

[0039]

[0040] (1) Benzoyl chloride (2.44 mL, 21 mmol) and 2,4-dimethylpyrrole (4.12 mL, 40 mmol) were added dropwise to dichloromethane under an argon atmosphere and stirred at room temperature overnight. Triethylamine was added dropwise in an ice bath, and after 15 minutes, boron trifluoride etherate was added dropwise, and stirred at room temperature 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 an orange-yellow solid with a yield of 24%. 1 H NMR (400MHz, CDCl3) δ (ppm) 7.51–7.45 (m, 3H), 7.30–7.24 (m, 2H), 5.98 (s, 2H), 2.55 (s, 6H), 1.37 (s, 6H).

[0041] (2) 1 (780 mg, 2.4 mmol) and iodosuccinimide (540 mg, 2.4 mmol) were dissolved in dichloromethane and stirred at room temperature for 3 hours. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was further purified by column chromatography to obtain a red solid with a yield of 83%.

[0042] (3) 2 (450 mg, 1 mmol), 2-thiophenealdehyde (0.28 ml, 3 mmol), piperidine (1 ml), and p-toluenesulfonic acid (5 mg) were dissolved in toluene, connected to a water separator and a spherical condenser, and heated under reflux for 8 hours. 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 purple solid with a yield of 24%. 1 H NMR(400MHz, CD2Cl2)δ(ppm)8.24(d,J=6.4Hz,1H),7.56–7.52(m,3H),7.48(d,J=7.5Hz,2H),7.44(d,J=4.7Hz,2H),7.39(d,J=5.1Hz,1H),7.34 -7.31(m,4H),7.15–7.06(m,2H),6.70(s,1H),1.45(s,6H). 13C NMR(101MHz,CD2Cl2)δ154.90,145.17,143.07,142.29,135.29,131.49,130.55,129.81,129.70, 129.66,128.82,128.79,128.69,128.52,127.20,119.41,119.39,118.68,118.07,17.26,15.05.

[0043] (4) Under an argon atmosphere, 3 (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) were dissolved in anhydrous triethylamine and heated under 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 brown solid with a yield of 45%. 1 H NMR (400MHz, CD2Cl2) δ (ppm) 8.78 (s, 1H), 8.67 (d, J = 4.5Hz, 1H), 8.53–8.49 (m, 1H ),8.46–8.43(m,2H),7.92–7.90(m,1H),7.84(t,J=7.7Hz,1H),7.60(s,1H),7.56 -7.55(m,3H),7.50(d,J=6.1Hz,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).

[0044] (5) 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, and the mixture is reacted at reflux temperature to obtain a crude product, which is then purified by filtering and washing with water.

[0045] Complex Ir-1 was prepared by the following method:

[0046] Under argon protection atmosphere, fluoroborane dipyrrole-modified bipyridine ligand 4 (42.0 mg, 0.08 mmol) and cyclometallated iridium dimer (42.8 mg, 0.04 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 2:1), and heated under reflux at 45°C with stirring for 12 h. After the reaction, the solvent was removed by distillation under reduced pressure, and a saturated methanol solution of NH4PF6 was added. After stirring for 2 h, the solid was collected by centrifugation, and the crude product was further purified by column chromatography to obtain a dark green solid Ir-1 with a yield of 20%. 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.89 (d, J = 8.0Hz, 1H), 8.39 (d, J = 8.4Hz, 1H), 8.26 (dd, J = 19.0, 8.3Hz, 1H), 8.11 (d, J = 1 6.2Hz,1H),7.99–7.92(m,1H),7.87(d,J=6.2Hz,1H),7.81–7.79(m,1H),7.75–7.66(m,1H),7.65–7.58(m,1H),7.45(s ,1H),7.33(s,1H),7.30–7.24(m,1H),7.23–7.13(m,2H),7.02(t,J=7.5Hz,1H),6.90(t,J=7.4Hz,1H),6.79(t,J=7.5H z,1H),6.62(t,J=7.4Hz,1H),6.16(d,J=7.3Hz,1H),6.11(d,J=7.4Hz,1H),1.45(s,1H),1.31(s,1H).HR-MS(positive mode,m / z):Calcd.1191.2837,found1191.2836for[Ir-1-PF6 - ] + .

[0047] Comparative Example 1

[0048] The structural formula of the complex Ir-2, in which the cyclometalated iridium (III) is connected via the meso position of the BODIPY core, is:

[0049]

[0050] Complex Ir-2 was prepared by the following method:

[0051] Under an argon atmosphere, the bipyridine ligand (70 mg, 0.1 mmol) and the cyclometallated iridium dimer (49 mg, 0.05 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 2:1), and the mixture was heated under reflux and stirred at 45°C for 12 h. After the reaction, the solvent was removed by distillation under reduced pressure, and a saturated methanol solution of NH4PF6 was added. The mixture was stirred for 2 h and then centrifuged to collect the solid. The crude product was further purified by column chromatography to obtain a dark green solid Ir-2 with a yield of 25%. 1 H NMR(400MHz, CD2Cl2)δ8.54(t,J=8.0Hz,1H),8.26–8.24(m,1H),8.20–8.10(m,1H),8.03–7.96(m ,1H),7.86–7.73(m,1H),7.65(d,J=8.1Hz,1H),7.58(d,J=5.5Hz,1H),7.45(d,J=5.8Hz,1H),7.4 3–7.37(m,1H),7.28(d,J=2.8Hz,1H),7.10–7.08(m,1H),7.04(d,J=5.9Hz,1H),7.00–6.93(m,1H ),6.67–6.65(m,1H),6.34(d,J=7.4Hz,1H),6.29(d,J=7.3Hz,1H),1.46(s,1H).HR-MS(positive mode,m / z):Calcd.1191.2837,found 1191.2837for[Ir-2-PF6 - ] + .

[0052] The complex Ir-1 prepared in Example 1 and Ir-2 prepared in Comparative Example 1 were subjected to the following experiments:

[0053] Cytotoxicity against human lung cancer cells A549, mouse lung cancer cells LLC and human lung fibroblast-like cells HLF:

[0054] The MTT colorimetric method was used to analyze the antiproliferative effects of fluoroboron dipyrrole-modified iridium (III) complexes Ir-1 and Ir-2, commercial photosensitizer CE6, 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 490nm, so it can be used as an A 490 nm To analyze cell proliferation.

[0055] The specific experimental steps are as follows:

[0056] (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.

[0057] (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;

[0058] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing different concentration gradients of compound Ir-1, Ir-2, CE6 and 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 group was illuminated with a 630 nm laser (0.6 W / cm 2 ) Irradiate for 5 minutes and continue incubation;

[0059] (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 compound CE6, Ir-1, Ir-2 and cisplatin CDDP are shown in Table 1.

[0060] Table 1. IC values ​​of compounds CE6, Ir-1, Ir-2, and cisplatin CDDP 50 Value (μM)

[0061]

[0062] The results showed that the phototoxicity of complex Ir-1 to A549 and LLC cells was higher than that of commercial photosensitizer CE6, compound Ir-2 and CDDP, while its dark toxicity was very low and it had low toxicity to normal cells, indicating that the anti-tumor activity of complex Ir-1 modified at position 2 was higher.

[0063] Example 2

[0064] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to the localization of intracellular subcellular organelles (lysosomes).

[0065] Methods: A549 cells were seeded in 35mm Corning laser confocal culture dishes. When the cell density reached 70%, 1μM Ir-1 was added for 6 hours. The culture medium was aspirated and washed twice with PBS. 500μL of the commercially prepared lysosomal green fluorescent probe Lyso-Tracker was added and incubated in a 37°C incubator for 30 minutes. The probe was aspirated and washed twice with PBS. Fresh preheated serum-free culture medium was replaced and the cells were immediately observed using a confocal microscope. Compound Ir-1: λ ex =630nm,λ em =670±20nm; commercial probe Lyso-Tracker: λ ex =443nm,λ em = 505 nm. Colocalization coefficient analysis was performed using ImageJ software.

[0066] The intracellular localization of the cyclometallated iridium (III) complex Ir-1 synthesized in Example 1 after co-incubation with the lysosomal probe is shown in FIG. Figure 1 As shown, the results showed that the cyclometallated iridium (III) complex Ir-1 was mainly distributed in the mitochondria after being taken up by A549 cells, with a colocalization coefficient of 0.88, indicating that Ir-1 has excellent ability to target lysosomes.

[0067] Example 3

[0068] Application of the cyclometalated iridium (III) complex Ir-1 prepared in Example 1 to generate reactive oxygen species in cells upon light excitation:

[0069] Method 1: Confocal microscopy was used to detect ROS in cancer cells. A549 cells were seeded in a 35 mm Corning laser confocal culture dish. When the cell density reached 70%, different concentrations of Ir-1 and Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 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±20 nm.

[0070] Method 2: Flow cytometry was used to detect ROS in tumor cells. A549 cells were seeded in 6-well plates and grown overnight. Different concentrations of Ir-1 and Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2After 5 minutes of irradiation, 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 after 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).

[0071] The results of the generation of active oxygen by the cyclometalated iridium (III) complex Ir-1 under light irradiation are as follows Figure 2 The results showed that compared with the control group, the green fluorescence was significantly enhanced after Ir-1 treatment by flow cytometry and confocal microscopy, indicating that the complex Ir-1 can produce a large amount of reactive oxygen species in A549 cells when stimulated by light.

[0072] Example 4

[0073] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to induce intracellular lysosomal membrane permeabilization:

[0074] Methods: Confocal microscopy was used to examine the changes in lysosomal integrity in tumor cells. A549 cells were seeded in 35 mm Corning laser confocal microplates. When the cell density reached 70%, different concentrations of Ir-1 and 2 μM Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, followed by incubation in the dark for 1 hour. Cells were then stained with 5 μM AO working solution at 37°C for 30 minutes in the dark, washed twice with PBS, and immediately observed using a confocal microscope. The excitation wavelength for the green channel was 488 nm, and the excitation wavelength for the red channel was 561 nm.

[0075] The results of the cyclometallated iridium (III) complex Ir-1 on inducing intracellular lysosomal membrane permeabilization are as follows Figure 3 The results showed that compared with the control group, the red fluorescence of lysosomes was significantly weakened after treatment with the complex Ir-1, indicating that the complex Ir-1 can effectively induce lysosomal membrane permeabilization under light excitation.

[0076] Example 5

[0077] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to induce a decrease in the intracellular pH of A549 cells:

[0078] Methods: Confocal microscopy was used to detect changes in pH within tumor cells. A549 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, 1 μM Ir-1 was added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes. The cells were then stained with 5 μM BCECF working solution at 37°C for 30 minutes in the dark, washed twice with PBS, and immediately observed under a confocal microscope. A549 cells in the control group were cultured in media with pH values ​​of 6.5, 7.4, and 8.6, respectively. The excitation wavelength was 488 nm, and the emission wavelength was 530 ± 20 nm.

[0079] The results of the cyclometallated iridium (III) complex Ir-1 on inducing a decrease in the intracellular pH of A549 cells are shown in Figure 2. Figure 4 The results showed that compared with the control group, the green fluorescence was weakened after the complex Ir-1 was treated with light, indicating that the complex Ir-1 could effectively damage the lysosomes under light excitation, release acidic hydrolases, and reduce the intracellular pH.

[0080] Example 6

[0081] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to affecting the intracellular calcium ion level in A549 cells:

[0082] Method 1: Confocal microscopy was used to detect changes in intracellular calcium levels in tumor cells. A549 cells were seeded in 35 mm Corning laser confocal microplates. When the cell density reached 70%, different concentrations of Ir-1 and Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, then cells were stained with 2 μM Fluo-4 AM working solution at 37°C in the dark for 30 minutes, washed twice with PBS, and immediately observed using a confocal microscope. The excitation wavelength was 488 nm, and the emission wavelength was 530 ± 20 nm.

[0083] Method 2: Confocal microscopy was used to detect changes in mitochondrial calcium levels in tumor cells. A549 cells were seeded in 35 mm Corning laser confocal microplates. When the cell density reached 70%, different concentrations of Ir-1 and Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, then cells were stained with 4 μM Rhod-2 AM working solution at 37°C for 30 minutes in the dark, washed twice with PBS, and immediately observed using a confocal microscope. The excitation wavelength was 561 nm, and the emission wavelength was 600 ± 20 nm.

[0084] The results of the effect of cyclometallated iridium (III) complex Ir-1 on the intracellular calcium level of A549 cells are as follows Figure 5 The results showed that compared with the control group, the green fluorescence in the cells and the red fluorescence in the mitochondria were significantly enhanced after treatment with the complex Ir-1, indicating that a large amount of calcium ions were released after lysosomal membrane permeabilization, resulting in an imbalance of intracellular calcium ion homeostasis and inducing calcium ion overload in the mitochondria.

[0085] Example 7

[0086] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to induce changes in intracellular mitochondrial membrane potential:

[0087] Method 1: Confocal microscopy was used to detect changes in mitochondrial membrane potential in tumor cells. A549 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, different concentrations of Ir-1 and 2 μM Ir-2 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes and continued to incubate in the dark for 18 hours. Then, the cells were stained with the pre-prepared JC-1 working solution at 37°C for 20 minutes in the dark and immediately observed with a confocal microscope.

[0088] Method 2: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. Cell culture medium containing different concentrations of Ir-1 and 2 μM Ir-2 was added to 6-well plates seeded with A549 cells with good morphology and normal growth. After 6 hours of drug treatment, the light group was illuminated using a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, then continue to incubate in the dark for 18 hours, harvest the cells, wash with PBS, and then add the prepared JC-1 working solution to stain for 20 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 the obtained results and analyze them. The detection fluorescence channel is λ ex =488nm, λ em =530±30nm;λ ex =488nm, λ em =590±30nm.

[0089] The results of the cyclometallated iridium (III) complex Ir-1 on the changes in mitochondrial membrane potential in cells are as follows Figure 6The results showed that compared with the control group, after treatment with compound Ir-1, the red fluorescence in the cells was weakened and the green fluorescence was significantly enhanced, indicating that compound Ir-1 effectively induced a decrease in mitochondrial membrane potential. At the same time, the results of flow cytometry also showed a similar conclusion.

[0090] Example 8

[0091] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to inducing apoptosis in A549 cells:

[0092] Method 1: Flow cytometry was used to detect apoptosis in tumor cells. Cell culture medium containing different concentrations of Ir-1 and 2 μM Ir-2 was added to 6-well plates seeded with A549 cells with good morphology and normal growth. After 6 hours of drug treatment, the light group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, then continue to incubate in the dark for 18 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 FlowJo 7.6 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.

[0093] Method 2: Western blotting (WB) was used to detect changes in apoptotic protein levels. Pre-prepared cell culture medium containing complexes Ir-1 (0.5, 1, 2 μM) and Ir-2 (2 μM) was added to a 100 mm culture dish of A549 cells that had grown on the wall. After 6 hours of drug treatment, the light group was illuminated with a 630 nm laser (0.6 W / cm 2) irradiated for 5 minutes, continued to incubate in the dark for 18 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 whole 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.

[0094] The experimental results of the cyclometallated iridium (III) complex Ir-1 on inducing apoptosis of A549 cells are as follows Figure 7 The results showed that compared with the control group or Ir-2 group without drug treatment, the proportion of apoptotic cells increased significantly after cells were treated with the complex Ir-1. At the same time, the pro-apoptotic protein bid was cleaved into tbid, Bax was upregulated, the anti-apoptotic protein bcl-2 was downregulated, and Cytochrome C was significantly upregulated, indicating that the complex Ir-1 can induce apoptosis in A549 cells.

[0095] Example 9

[0096] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to inducing autophagy inhibition in A549 cells:

[0097] Methods: Western blotting (WB) was used to detect changes in autophagy protein levels. Primary antibodies used in this application included autophagy marker proteins PINK1, Parkin, LC3, and p62.

[0098] The experimental results of the cyclometallated iridium (III) complex Ir-1 on the expression of autophagy-related proteins in A549 cells are as follows Figure 8The results showed that compared with the untreated control group, the expression of mitochondrial autophagy-related proteins PINK1 and Parkin was significantly upregulated after cells were treated with the complex Ir-1, and the levels of autophagy marker proteins LC3-II and p62 increased, indicating that the complex Ir-1 can induce autophagy inhibition in A549 cells.

[0099] Example 10

[0100] Application of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1 to inducing immunogenic death of A549 cells:

[0101] Method 1: Immunofluorescence detection of CRT and HMGB1 by confocal microscopy. A549 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, different concentrations of Ir-1 were added for 6 h. The illumination group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes and incubate in the dark for another 18 hours. After washing with PBS, fix with 4% paraformaldehyde fixative for 10-15 minutes, wash with PBS, add 0.2% Triton X-100 to promote permeation for 30 minutes, wash with PBS, dilute the primary antibody with skim milk powder according to the antibody instructions, incubate in primary antibody incubation solution at 4°C overnight, wash twice with PBS, add diluted FITC secondary antibody and incubate at room temperature for 1 hour, wash twice with PBS, add 0.3μM DAPI to stain the nuclei for 10 minutes, wash twice with PBS, and then immediately observe with a confocal microscope.

[0102] Method 2: Immunofluorescence detection of CRT and HMGB1 by flow cytometry. Cell culture medium containing different concentrations of Ir-1 was added to 6-well plates seeded with A549 cells with good morphology and normal growth. After 6 hours of drug treatment, the light group was illuminated with a 630 nm laser (0.6 W / cm 2 ) for 5 minutes, and then continue to incubate in the dark for 18 hours. The cells were harvested, washed with PBS, and incubated at room temperature for 1 hour with the appropriate concentration of primary antibody incubation solution. After washing with PBS, the corresponding FITC secondary antibody incubation solution was added and incubated at room temperature for 1 hour. After washing with PBS, the samples were immediately detected using a BD C6 flow cytometer, and the results were processed and analyzed using FlowJo 7.6 software. The fluorescence channel for detection was λ ex =488nm, λ em =530±30nm.

[0103] Method 3: Multifunctional microplate reader was used to detect the changes in ATP content in cell culture medium. Cell culture medium containing different concentrations of Ir-1 was added to 6-well plates seeded with A549 cells with good morphology and normal growth. After 6 hours of drug treatment, the light group was illuminated with a 630nm laser (0.6W / cm 2 ) for 5 minutes, and then incubate in the dark for 18 hours. The supernatant was collected by centrifugation and stored at low temperature. The ATP content was detected using an ATP detection kit.

[0104] The results of the cyclometallated iridium (III) complex Ir-1 on inducing immunogenic death of A549 cells are shown in Figure 2. Figure 9 The results showed that compared with the control group, the green fluorescence was significantly enhanced by immunofluorescence, indicating the upregulation of calreticulin CRT, the release of high-mobility group protein B1 HMGB1, and the efflux of ATP, which together indicated that the light treatment of the complex Ir-1 induced the immunogenic death of A549 cells.

[0105] Example 11

[0106] Anti-tumor and immune activation applications of the cyclometallated iridium (III) complex Ir-1 prepared in Example 1:

[0107] Methods: Mouse Lewis lung cancer (LLC) cells (approximately 1×10 7 / mL) was inoculated subcutaneously in the left axilla of C57BL / 6J mice to establish a proximal tumor model. Five days later, a distal tumor model was established by subcutaneous injection in the right axilla of mice. When the tumor volume reached 80-100 mm 3 The LLC tumor-bearing mice were randomly divided into 4 groups (n=5): PBS group, PBS+light group, Ir-1 group, Ir-1+light group (light source: 630 nm LED light, 80 mW / cm 2 The proximal tumors of each group were injected with PBS and Ir-1 at a dose of 10 mg / kg. The light-exposed group was exposed to light for 5 minutes, once every 2 days for a total of 3 times. The tumor volume and mouse weight were measured every 2 days. The tumor volume was calculated as follows: Tumor volume (V) = a × b 2 / 2, where a is the primary axis of the tumor and b is the secondary axis. Ten days later, all mice were euthanized and the tumors were harvested. The maturity of dendritic cells (DCs) and different types of T cells in distant tumors and their biomarkers were also measured. Distal tumors were prepared into single-cell suspensions, and the collected lymphocytes were incubated with anti-CD-86-PE, anti-CD-80-FITC, anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE and analyzed by flow cytometry. Serum was isolated from mice for analysis. The secretion of TNF-α, IFN-γ, and IL-6 was detected using ELISA kits.

[0108] The experimental results are as follows Figure 10 The results showed that when Ir-1 was used for simultaneous illumination, both proximal and distal tumors had significant inhibitory effects. After simultaneous illumination of the proximal tumor, the expression of CD86 and CD80 in the distal tumor increased from 18.5% to 58.4%, indicating that a large number of mature dendritic cells were distributed in the distal tumor to play an immune function. In addition, flow cytometry data showed that after Ir-1 treatment of the proximal tumor, the expression of CD4 + and CD8 + The expression levels of T cells were significantly upregulated, indicating that both helper and effector T cells were formed in the distal tumor. Furthermore, the serum secretion concentrations of anti-inflammatory cytokines were the highest in the group treated with Ir-1 PDT, indicating that Ir-1 PDT can stimulate the strongest systemic anti-tumor immune response.

Claims

1. A fluoroboron dipyrrole ring metal iridium (III) complex, characterized in that: The structural formula of the complex is shown below:

2. A method for synthesizing the complex according to claim 1, characterized in that: The following steps are involved: (1) Under an inert atmosphere, a bipyridine ligand modified with fluoroborane dipyrrole 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) replacing the crude product with NH4PF6, and separating and purifying the crude product by column chromatography to obtain a fluoroboron dipyrrole-modified cyclometalated iridium (III) complex; The structural formula of the fluoroborane dipyrrole-modified bipyridine ligand is as follows: The structural formula of the cyclometallated iridium dimer is shown below:

3. The synthesis method according to claim 2, characterized in that The preparation method of the fluoroborane dipyrrole-modified bipyridine ligand is as follows: under an inert atmosphere, iodinated fluoroborane dipyrrole 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 fluoroboron dipyrrole to 5-ethynyl-2,2'-bipyridine is 1:1-1.

5.

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

6. The synthesis method according to claim 2, characterized in that In step (1), the reaction molar ratio of the fluoroboron dipyrrole-modified bipyridine ligand to the cyclometallated iridium dimer is 2:1 to 1.

2.

7. The synthesis method according to claim 2, characterized in that In step (1), the volume ratio of dichloromethane to methanol in the mixed solution is 2:1 to 1.

5.

8. The synthesis method according to claim 2, characterized in that In step (1), the reflux reaction is carried out for 12 to 13 hours at a temperature of 45 to 48°C.

9. The synthesis method according to claim 2, characterized in that In step (2), the crude product is obtained by substitution with NH4PF6, specifically, by adding a saturated NH4PF6 methanol solution and stirring for 2 to 3 hours.

10. Use of the fluoroboron dipyrrole-modified cyclometallated iridium (III) complex according to claim 1 in the preparation of antitumor drugs or antitumor drug components.

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