Pterostilbene-modified cyclometallated iridium (III) complexes and their preparation and application

Abstract

The invention discloses a pterostilbene-modified cyclometal iridium (III) complex and a preparation method and application thereof. The cyclometal iridium (III) complex modified based on the natural product pterostilbene has good fat solubility, tumor cell uptake ability and anti-tumor cell proliferation ability, can effectively inhibit tumor cell proliferation, induce ROS generation, reduce mitochondrial membrane potential and induce tumor cell autophagy, thereby having good anti-tumor activity; therefore, the cyclometal iridium (III) complex of the invention has a better effect than cisplatin drugs under the premise of small toxic and side effects; based on the ability of the complex of the invention to inhibit tumor cell proliferation, damage tumor cell mitochondrial function and induce tumor cell autophagy, it has broad application prospects in the preparation of anti-tumor drugs and anti-tumor drug components.

Description

Technical Field

[0001] The invention relates to a pterostilbene-modified cyclometallated iridium (III) complex, and also relates to a preparation method of the complex and application of the complex in preparing anti-tumor drugs. Technical Background

[0002] Since the discovery of cisplatin (cis-Pt), inorganic drugs have been at the forefront of chemotherapy research. However, commonly used platinum-based therapies, including cisplatin, oxaliplatin, or carboplatin, can induce a series of serious adverse reactions. Nephrotoxicity is the most common specific adverse reaction encountered by cisplatin patients, while bone marrow suppression and neurotoxicity are associated with the use of carboplatin and oxaliplatin, respectively, which has promoted the research of other metal-based anticancer drugs that retain the efficacy of platinum drugs but have fewer side effects.

[0003] Natural products or their derivatives have a variety of biological activities. Their potential medicinal value has made the anti-cancer research of natural products gradually become a hot spot in clinical anti-tumor drug research. Pterostilbene is mainly found in blueberries. It is a natural phenolic compound and a dimethyl analog of resveratrol. It has been widely studied. Studies have shown that it has anti-diabetic, anti-hyperlipidemic, anti-atherosclerotic, anti-hypertensive, anti-cardiovascular disease, anti-obesity, and anti-fatty degeneration activities. In addition, pterostilbene has been shown to have powerful biological functions in anti-inflammatory and anti-cancer processes. However, pterostilbene is only effective at high doses. Therefore, in recent years, many researchers have modified pterostilbene to improve its bioavailability and achieve better anti-tumor effects. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a pterostilbene-modified cyclometallated iridium (III) complex, which can solve the problem of low bioavailability of pterostilbene when used as an anti-tumor drug or an anti-tumor drug component, and can also solve the problem of large toxic side effects of a simple cyclometallated iridium (III) dimer (precursor) when used as an anti-tumor drug or an anti-tumor drug component. Another purpose of the present invention is to provide a method for preparing the above-mentioned complex and its use in preparing anti-tumor drugs or anti-tumor drug components.

[0005] Technical solution: The pterostilbene-modified cyclometallated iridium (III) complex of the present invention has a general structural formula as shown below:

[0006]

[0007] in, Select from any of the following structures:

[0008]

[0009] The above single key indicates the link position.

[0010] The preparation method of the above-mentioned pterostilbene-modified cyclometallated iridium (III) complex comprises the following steps:

[0011] (1) Preparation of pterostilbene-derived ligands:

[0012] Under an inert atmosphere, a pterostilbene derivative, EDCI (carbodiimide hydrochloride, which acts as an activator for carboxyl groups in the system) or DCC (dicyclohexylcarbodiimide, which acts as an activator for carboxyl groups in the system), HOBt (1-hydroxybenzotriazole, which acts as a catalyst for condensation reaction in the system), TEA (triethylamine, which acts as a basic catalyst in the system) and a bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine are dissolved in N,N-dimethylformamide, the mixture is reacted at room temperature to obtain a crude product, and the crude product is separated and purified by silica gel column chromatography to obtain a pterostilbene-derived ligand;

[0013] The chemical structural formula of the ligand is shown below:

[0014]

[0015] (2) placing the ligand and the precursor in a mixed solvent of dichloromethane / methanol, refluxing in the dark under argon protection, standing and cooling to room temperature after the reaction, adding an excess of saturated NH4PF6 solution and stirring, concentrating under reduced pressure after stirring, and then adding the concentrated solution dropwise to ether, separating and purifying the precipitate by silica gel column chromatography, and drying to obtain a complex; wherein the precursor is any one of [(ppy)2IrCl]2, [(thpy)2IrCl]2, [(dzq)2IrCl]2 or [(pq)2IrCl]2;

[0016] The structural formulas of the pterostilbene-modified cyclometallated iridium (III) complexes obtained based on different precursors are:

[0017]

[0018]

[0019] Wherein, in step (1), the molar ratio of the pterostilbene derivative, EDCI or DCC, HOBt and the bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine is 0.5:0.6:0.4:0.6.

[0020] Wherein, in step (2), the molar ratio of the ligand to the precursor is 2:1.

[0021] Wherein, in step (2), the volume ratio of dichloromethane to methanol is 1 to 3:1; preferably, the volume ratio of dichloromethane to methanol is 2:1.

[0022] Wherein, in step (2), the light-proof reflux reaction time is 6 to 10 hours, and the preferred reaction time is 6 hours.

[0023] Wherein, the structural formula of the bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine is: It was prepared using the synthesis method reported in the literature Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

[0024] Wherein, in step (3), the precursor [(ppy)2IrCl]2 has the structural formula:

[0025] The structural formula of the precursor [(thpy)2IrCl]2 is:

[0026] The precursor [(dzq)2IrCl]2 has the structural formula

[0027] The precursor [(pq)2IrCl]2 has the structural formula

[0028] The above precursors are all prepared according to the synthesis method reported in the literature Cyclometalated iridium (III) complexes as mitochondria-targeted anticancer and antibacterial agents to induce both autophagy and apoptosis.

[0029] Application of the pterostilbene-modified cyclometallated iridium (III) complex in the preparation of antitumor drugs or antitumor drug components.

[0030] The reaction scheme of the present invention is as follows:

[0031] Step (1):

[0032]

[0033] Step (2):

[0034]

[0035] Beneficial effects: The present invention is based on a cyclometal iridium (III) complex modified with the natural product pterostilbene. The four synthesized complexes have good lipid solubility, tumor cell uptake ability, and anti-tumor cell proliferation ability, and can effectively inhibit tumor cell proliferation, induce ROS production, reduce mitochondrial membrane potential, and induce tumor cell autophagy, thereby having good anti-tumor activity; therefore, the cyclometal iridium (III) complex of the present invention has a better effect than cisplatin drugs under the premise of small toxic and side effects; based on the ability of the complex of the present invention to inhibit tumor cell proliferation, damage tumor cell mitochondrial function, and induce tumor cell autophagy, it has broad application prospects in the preparation of anti-tumor drugs and anti-tumor drug components. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The confocal fluorescence imaging diagrams of drug uptake after tumor cells were incubated with the complex obtained in Example 5 for different time periods and the cell flow cytometry diagrams at different concentrations; wherein, Figure 1 a is the confocal fluorescence imaging at different time points. Figure 1 b is the flow cytometry graph of cells at different concentrations;

[0037] Figure 2 The cytometry graph and confocal fluorescence imaging graph of the complex obtained in Example 5 inducing tumor cells to produce reactive oxygen species; wherein, Figure 2 a is a confocal fluorescence imaging image; Figure 2 b is the flow cytometry graph of cells at different concentrations;

[0038] Figure 3 The cytometry graph and confocal fluorescence imaging graph of the changes in mitochondrial membrane potential of tumor cells induced by the complex obtained in Example 5; wherein, Figure 3 a is a confocal fluorescence imaging image; Figure 3 b is the flow cytometry graph of cells at different concentrations;

[0039] Figure 4 This is the immunoblot protein image of the complex obtained in Example 5 inducing autophagy in tumor cells. DETAILED DESCRIPTION

[0040] Example 1

[0041] The preparation method of the ligand of the present invention is specifically as follows:

[0042]

[0043] A pterostilbene derivative (1 mmol, 1 equiv) and EDCI (1.2 mmol, 1.2 equiv) were placed in a two-necked flask, evacuated, and argon was introduced. Then, anhydrous DMF (N,N-dimethylformamide) (4 mL) and TEA (400 μL) were added under ice bath conditions and stirred for 20 min. Then, a DMF solution containing HOBt (0.8 mmol, 0.8 equiv) was added. After stirring for 15 min, the ice bath was removed and the temperature was restored to room temperature. Then, a DMF solution containing 4-methyl-4'-aminomethyl-2,2'-bipyridine (1.2 mmol, 1.2 equiv) was added, and the reaction was continued for 48 h. After the reaction was completed, 20 mL of distilled water was added, and the mixture was extracted with dichloromethane (3×30 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The crude product was separated and purified by silica gel column chromatography. The solid obtained by drying was the ligand, and the yield was 72%. 1 H NMR (400MHz, Chloroform-d) δ (ppm): δ8.64 (dd, J=5.0, 0.9Hz, 1H), 8.55 (dd, J=5.0, 0.8Hz, 1H), 8.32 (dd, J=1.7, 0.8Hz, 1H), 8.24 (dt, J=1.7, 0.9Hz, 1H), 7. 51-7.45(m,2H),7.25(dd,J=5.0,1.8Hz,1H),7.18-6.89(m,6H),6.67(d,J=2. 3Hz, 2H), 6.41 (t, J = 2.3Hz, 1H), 4.72-4.60 (m, 4H), 3.85 (s, 6H), 2.46 (s, 3H).

[0044] Example 2

[0045] The preparation method of the complex of the present invention based on the precursor [(ppy)2IrCl]2 is specifically as follows:

[0046]

[0047] The ligand (0.25mmol, 2equiv) and the precursor [(ppy)2IrCl]2 (0.125mmol, 1equiv) were placed in a three-necked flask, and 45mL of a mixed solvent of dichloromethane / methanol (2:1, v / v) was added to the flask. The mixture was refluxed in the dark for 6 hours under argon protection. After the reaction was completed, the mixture was allowed to stand and cool to room temperature. Six times the equivalent of the ligand was added to a saturated NH4PF6 solution, and stirred for 2 hours. The mixture was then concentrated under reduced pressure to 2mL. The concentrated solution was added dropwise to 20mL of ether, and a large amount of yellow precipitate was generated. The precipitate was separated and purified by silica gel column chromatography and dried to obtain complex I with a yield of 75%. 1H NMR (400MHz, DMSO-d6) δ (ppm): δ8.84-8.62 (m, 3H), 8.26 (t, J=8.2Hz, 2H), 7.91 (dd, J=10.4, 6.5Hz, 4H ),7.74(dd,J=6.0,2.8Hz,1H),7.69(t,J=5.2Hz,1H),7.61(d,J=5.9Hz,2H),7.50(dt,J=12.0,4.8Hz,3 H), 7.23-7.10 (m, 4H), 7.06-6.96 (m, 3H), 6.95-6.85 (m, 3H), 6.70 (d, J = 2.1 Hz, 1H), 6.54-6.44 (m, 1H), 6.41-6.30 (m, 2H), 6.24-6.15 (m, 2H), 4.73-4.48 (m, 4H), 3.77 (d, J = 1.3 Hz, 3H), 2.53 (d, J = 4.6 Hz, 3H). ESI-MS (CH3OH) calculated value: m / z = 996.18, found value m / z = 996.40.

[0048] Example 3

[0049] The preparation method of the complex of the present invention based on the precursor [(thpy)2IrCl]2 is specifically as follows:

[0050]

[0051] The ligand (0.25mmol, 2equiv) and the precursor [(thpy)2IrCl]2 (0.125mmol, 1equiv) were placed in a three-necked flask, and 45mL of a mixed solvent of dichloromethane / methanol (2:1, v / v) was added to the flask. The mixture was refluxed in the dark for 6 hours under argon protection. After the reaction was completed, the mixture was allowed to stand and cool to room temperature. Six times the equivalent of saturated NH4PF6 solution of the ligand was added thereto, and stirred for 2 hours. The mixture was then concentrated under reduced pressure to 2mL. The concentrated solution was added dropwise to 20mL of ether, and a large amount of yellow precipitate was generated. The precipitate was separated and purified by silica gel column chromatography and dried to obtain complex II with a yield of 68%. 1H NMR (400MHz, DMSO-d6) δ (ppm): δ8.80 (dt, J=15.1, 6.0Hz, 1H), 8.69-8.62 (m, 2H), 7.84- 7.68(m,6H),7.66-7.62(m,3H),7.59-7.49(m,5H),7.22-7.12(m,2H),7.02-6.88(m,5H) ,6.71(d,J=2.3Hz,1H),6.54-6.45(m,1H),6.37(d,J=2.3Hz,1H),6.17(dd,J=4.8,2.5Hz,2H),4.66(s,1H),4.63-4.52(m,3H),3.78(s,3H),3.60(s,3H),2.54(d,J=5.0Hz,3H).ESI-MS(CH3OH)calculated value: m / z=1008.22, experimental value m / z=1008.20.

[0052] Example 4

[0053] The preparation method of the complex of the present invention based on the precursor [(dzq)2IrCl]2 is specifically as follows:

[0054]

[0055] The ligand (0.25mmol, 2equiv) and the precursor [(dzq)2IrCl]2 (0.125mmol, 1equiv) were placed in a three-necked flask, and 45mL of a mixed solvent of dichloromethane / methanol (2:1, v / v) was added to the flask. The mixture was refluxed in the dark for 6 hours under argon protection. After the reaction was completed, the mixture was allowed to stand and cool to room temperature. Six times the equivalent of the ligand was added to a saturated NH4PF6 solution and stirred for 2 hours. The mixture was then concentrated under reduced pressure to 2mL. The concentrated solution was added dropwise to 20mL of ether. A large amount of yellow precipitate was generated. The precipitate was separated and purified by silica gel column chromatography and dried to obtain complex III with a yield of 81%. 1H NMR (400MHz, DMSO-d6) δ (ppm): δ8.81-8.67 (m, 4H), 8.57 (ddt, J = 8.2, 6.2, 1.4Hz, 2H), 8.08-8.05 (m, 2H), 7.97 (d dd,J=8.9,3.8,2.5Hz,2H),7.91-7.86(m,2H),7.72(dd,J=5.7,3.0Hz,1H),7.67-7.57(m,4H),7.56-7.48(m,3H), 7.44-7.36 (m, 2H), 7.18-7.14 (m, 3H), 6.99-6.95 (m, 1H), 6.89-6.86 (m, 1H), 6.68 (d, J = 2.3 Hz, 1H), 6.47 (d, J = 2.7 Hz, 1H), 6.35 (d, J = 2.3 Hz, 1H), 6.23-6.19 (m, 2H), 4.61 (s, 1H), 4.56 (d, J = 7.7 Hz, 3H), 3.76 (s, 3H), 3.58 (s, 3H). ESI-MS (CH3OH) calculated value: m / z = 1044.31, found value m / z = 1044.30.

[0056] Example 5

[0057] The preparation method of the complex of the present invention based on the precursor [(pq)2IrCl]2 is specifically as follows:

[0058]

[0059] The ligand (0.25mmol, 2equiv) and the precursor [(pq)2IrCl]2 (0.125mmol, 1equiv) were placed in a three-necked flask, and 45mL of a mixed solvent of dichloromethane / methanol (2:1, v / v) was added to the flask. The mixture was refluxed in the dark for 6 hours under argon protection. After the reaction was completed, the mixture was allowed to stand and cool to room temperature. Six times the equivalent of saturated NH4PF6 solution of the ligand was added thereto, and stirred for 2 hours. The mixture was then concentrated under reduced pressure to 2mL. The concentrated solution was added dropwise to 20mL of ether, and a large amount of yellow precipitate was generated. The precipitate was separated and purified by silica gel column chromatography and dried to obtain complex IV with a yield of 70%. 1H NMR (400MHz, DMSO-d6) δ (ppm): δ8.72 (dt, J=16.7, 6.0Hz, 1H), 8.59-8.52 (m, 4H), 8.27 (t, J=7.7Hz, 4H), 7.98 (dd, J=5. 8,2.8Hz,1H),7.93(dq,J=8.5,2.3Hz,3H),7.54-7.47(m,2H),7.46-7.39(m,3H),7.27(d,J=8.8Hz,1H),7.18-7.07(m,6 H), 7.06-6.99 (m, 1H), 6.96-6.91 (m, 1H), 6.90-6.85 (m, 1H), 6.81 (tdt, J = 6.3, 3.2, 1.3 Hz, 2H), 6.70 (d, J = 2.3 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H), 6.42-6.31 (m, 4H), 4.67-4.55 (m, 2H), 4.49-4.40 (m, 2H), 3.78 (s, 3H), 3.60 (s, 3H), 2.38 (s, 3H). ESI-MS (CH3OH) calculated value: m / z = 1096.30, found value m / z = 1096.40.

[0060] The cytotoxicity of the ligand L prepared in Example 1 and the pterostilbene-modified cyclometallated iridium complexes prepared in Examples 2 to 5 on human breast cancer cells MCF-7, 4T1 and human normal cells MCF-10A:

[0061] MTT colorimetry was used to analyze the antiproliferative effects of four cyclometallated iridium (III) complexes modified with pterostilbene, pterostilbene (PTS), ligand L, and cis-Pt, a cis-Pt, on human breast cancer cells MCF-7, 4T1, and human normal cells MCF-710A. 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 570nm, so it can be ... MTT (thiazolyl blue) on human breast cancer cells MCF-7, 4T1, and MCF-710A. 570 nm To analyze cell proliferation.

[0062] The specific experimental steps are as follows:

[0063] (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 two passages;

[0064] (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;

[0065] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing four complexes with different concentration gradients, pterostilbene with different concentration gradients, ligands with different concentration gradients, and cis-Pt with different concentration gradients was added to each well, and then the cells were placed in a constant temperature box for further incubation;

[0066] (4) After incubation for 48 hours, 20 μL of MTT (5 mg / mL) was added to each well and the cells were 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. A570 nm was detected using an enzyme-linked immunosorbent assay (ELISA) to calculate the cell proliferation inhibition rate and obtain the IC 50 The MTT test results of pterostilbene, ligand L, four complexes and cis-Pt are shown in Table 1.

[0067] Table 1 shows the IC values ​​of pterostilbene, ligand L, four complexes and cis-Pt 50 Value (μM)

[0068]

[0069] The experimental results show that compared with pterostilbene, the toxicity of the four complexes to human breast cancer cell lines is greatly improved, indicating that the anti-tumor activity of complexes I to IV is improved after modification. Complex IV is more cytotoxic to MCF-7 cells than the other three complexes, IC 50 The value is 1.9±0.1μM. Meanwhile, for normal MCF-10A cells, the cytotoxicity of complex IV is slightly lower than that of the other three cells.

[0070] Uptake of complex IV prepared in Example 5 in tumor cells:

[0071] Method 1: Confocal microscopy was used to detect the uptake of complex IV by MCF-7 cells at different time periods. MCF-7 cells were seeded in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, a cell culture medium containing 4 μM complex IV was added. The incubation time was 0.5, 1, 2, 4 and 6 h, respectively. The culture medium was discarded, and the cells were washed three times with PBS and observed by confocal microscopy with an excitation wavelength of 405 nm.

[0072] Method 2: Flow cytometry was used to detect the uptake of complex IV by tumor cells. Cell culture medium containing 0, 1, 2, 4 and 6 μM concentrations of complex IV was added to 6-well plates inoculated with MCF-7 cells with good morphology and normal growth. After 24 hours of drug treatment, the cells were harvested, washed twice with PBS, and resuspended in 500 μl of PBS. The fluorescence intensity was detected by flow cytometry.

[0073] The uptake results of complex IV in MCF-7 cells are shown in Figure 1 As shown. The confocal microscopy results showed that as the incubation time increased, the intracellular drug uptake increased in a time-dependent manner. The flow cytometer results showed that compared with the control group, the fluorescence intensity of the drug-added group was significantly increased and increased in a dose-dependent manner, indicating that tumor cells showed a good uptake effect on complex IV, indicating that complex IV of the present invention has good cell uptake ability.

[0074] Application of complex IV prepared in Example 5 to inducing the generation of intracellular reactive oxygen species:

[0075] Method 1: Confocal microscopy to detect ROS in cancer cells. MCF-7 cells were seeded in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, complex IV was added at concentrations of 0, 1, 2, and 4 μM for 24 h. Then, the cells were stained with serum-free medium containing 10 μM H2DCFH-DA at 37 ° C for 30 min in the dark, and then immediately observed with a confocal microscope, with an excitation wavelength of 488 nm and an emission wavelength of 530 ± 20 nm.

[0076] Method 2: Detection of ROS in tumor cells by flow cytometry. MCF-7 cells were treated with complex IV at concentrations of 0, 1, 2, 4, and 6 μM for 24 h, and then stained with serum-free medium containing 10 μM H2DCFH-DA at 37°C for 30 min in the dark. The cells were centrifuged and the supernatant was discarded. The cells were 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 the green light was analyzed using FlowJo 7.6 (Tree Star, OR, USA) software.

[0077] The results of complex IV on inducing the generation of reactive oxygen species in MCF-7 cells are shown in Figure 2 The results showed that compared with the control group, both flow cytometry and confocal microscopy showed that green fluorescence was significantly enhanced after treatment with complex IV, indicating that complex IV can effectively induce an increase in the content of intracellular reactive oxygen species.

[0078] Application of the complex IV prepared in Example 5 to inducing changes in intracellular mitochondrial membrane potential:

[0079] Method 1: Confocal microscopy was used to detect changes in mitochondrial membrane potential in tumor cells. MCF-7 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density grew to 70%, complex IV was added at concentrations of 0, 1, 2, and 4 μM for 24 h. Then, the cells were stained with the pre-prepared JC-1 working solution at 37 °C in the dark for 30 min and then immediately observed with a confocal microscope.

[0080] Method 2: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. Cell culture medium containing complex IV at concentrations of 0, 1, 2, 4, and 6 μM was added to a 6-well plate inoculated with MCF-7 cells with good morphology and normal growth. After 24 hours of drug treatment, the cells were collected, washed with PBS, and then stained with the prepared JC-1 working solution for 30 minutes; the cells were washed with buffer and resuspended, and the samples were immediately detected using a BD FACS verse flow cytometer, and the results were processed and analyzed using Flow Jo7.6 software. The detection fluorescence channel was λ ex =488nm,λ em =530±30nm;λ ex =488nm,λ em =590±30nm.

[0081] The results of complex IV on inducing changes in intracellular mitochondrial membrane potential are as follows Figure 3 The results showed that compared with the control group, after the complex treatment, the green fluorescence was significantly enhanced and the red fluorescence was significantly weakened, indicating that the complex IV can effectively induce the decrease of mitochondrial membrane potential. The results of flow cytometry also showed the same result.

[0082] Application of complex IV prepared in Example 5 to inducing cell autophagy:

[0083] Western blotting (WB) was used to detect changes in autophagy protein content. Pre-prepared cell culture medium containing complex IV (concentrations of 0, 1, 2 and 4 μM) was added to the 100 mm culture dish of MCF-7 cells that had grown on the wall. After 24 hours of drug treatment, the cells were collected by centrifugation, washed with PBS to remove the serum in the residual culture medium, and added with Biyuntian RIPA strong lysis buffer containing PMSF for 20 minutes of whole cell lysis. During this whole process, a low temperature environment of 4°C was maintained to ensure protein invariance. After the end, centrifuged at 13400 rpm for 20 minutes under low temperature conditions, 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 content of different proteins in the sample was 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, and after the end, the membrane was placed in a 5% skim milk powder for blocking for 2 hours. Dilute the primary antibody with skim milk powder according to the instructions for use of the antibody in the corresponding proportion, and place the blocked membrane in the primary antibody incubation solution at room temperature for a period of time to allow it to specifically bind to the target protein. After completion, wash with PBST (5×6min / time). Place the washed membrane in the pre-prepared secondary antibody incubation solution for a period of time to allow it to bind to the primary antibody, and wash it with PBST. Prepare an equal volume of ECL developer, cover it on the PVDF membrane, and use the chemiluminescence imaging system to shoot after 2 minutes of treatment.

[0084] The experimental results of complex IV on the expression of autophagy-related proteins in MCF-7 cells are shown in Figure 4 The results showed that compared with the control group without drug treatment, the content of autophagy marker protein LC3-II in MCF-7 cells treated with complex IV increased in a concentration-dependent manner, indicating that the death mode of MCF-7 cells induced by complex IV was autophagy.

Claims

1. A pterostilbene-modified cyclometallated iridium (III) complex, characterized in that: The general structural formula of the complex is shown below: in, Select from any of the following structures: The above single key indicates the link position.

2. The method for preparing the pterostilbene-modified cyclometallated iridium (III) complex according to claim 1, characterized in that: The steps include: (1) Preparation of pterostilbene-derived ligands: Under an inert atmosphere, a pterostilbene derivative, EDCI or DCC, HOBt, TEA and a bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine are dissolved in N,N-dimethylformamide, the mixture is reacted at room temperature to obtain a crude product, and the crude product is separated and purified by silica gel column chromatography to obtain a pterostilbene-derived ligand; The chemical structural formula of the ligand is shown below: (2) placing the ligand and the precursor in a mixed solvent of dichloromethane / methanol, refluxing in the dark under argon protection, standing and cooling to room temperature after the reaction, adding an excess of saturated NH4PF6 solution and stirring, concentrating under reduced pressure after stirring, and then adding the concentrated solution dropwise to ether, separating and purifying the precipitate by silica gel column chromatography, and drying to obtain a complex; wherein the precursor is any one of [(ppy)2IrCl]2, [(thpy)2IrCl]2, [(dzq)2IrCl]2 or [(pq)2IrCl]2; The structural formulas of the pterostilbene-modified cyclometallated iridium (III) complexes obtained based on different precursors are:

3. The method for preparing the pterostilbene-modified cyclometallated iridium (III) complex according to claim 2, characterized in that: In step (1), the molar ratio of the pterostilbene derivative, EDCI or DCC, HOBt and the bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine is 0.5:0.6:0.4:0.

6.

4. The method for preparing the pterostilbene-modified cyclometallated iridium (III) complex according to claim 2, characterized in that: In step (2), the molar ratio of the ligand to the precursor is 2:

1.

5. The method for preparing the pterostilbene-modified cyclometallated iridium (III) complex according to claim 2, characterized in that: In step (2), the volume ratio of dichloromethane to methanol is 1 to 3:

1.

6. The method for preparing the pterostilbene-modified cyclometallated iridium (III) complex according to claim 2, characterized in that: In step (2), the light-shielded reflux reaction time is 6 to 10 hours.

7. Use of the pterostilbene-modified cyclometallated iridium (III) complex according to claim 1 for preparing anti-tumor drugs; wherein: The tumor is human breast cancer.

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