Cyclometalated iridium (III) complex for targeting glycometabolism as well as synthesis method and application of cyclometalated iridium (III) complex

Abstract

The invention discloses a cyclometalated iridium (III) complex for targeting glycometabolism and a synthetic method and application thereof.The cyclometalated iridium (III) complex for targeting glycometabolism has good cell uptake capacity and capacity of targeting mitochondria in tumor cells, can inhibit PDK2 expression level in the tumor cells, meanwhile, reduces the pPDH / PDH ratio, and has the advantages of being capable of inhibiting tumor cell proliferation and inhibiting tumor cell proliferation. Therefore, the glycolytic pathway of tumor cells is inhibited; besides, the complex disclosed by the invention also can cause mitochondrial membrane potential reduction and intracellular ATP level reduction by inducing generation of active oxygen, so that tumor cell apoptosis is induced, and the complex has good anti-tumor activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a cyclometalated iridium (III) complex targeting glycometabolism, and also relates to a synthesis method of the cyclometalated iridium (III) complex and application thereof in preparation of an antitumor drug. BACKGROUND

[0002] In the 1920s, Otto Warburg proposed that the metabolic characteristics of tumor cells have Warburg effect. That is, under aerobic conditions, most differentiated cells mainly oxidize pyruvate produced by glycolysis into carbon dioxide through mitochondrial tricarboxylic acid (TCA) cycle. The reaction produces NADH, promotes oxidative phosphorylation, can maximize ATP (adenosine triphosphate) synthesis, and at the same time generates minimal lactic acid. Only under anaerobic conditions, differentiated cells will produce a large amount of lactic acid. Most tumor cells do not further oxidize through mitochondrial TCA cycle after glucose metabolism to pyruvate, but convert into a large amount of lactic acid through lactate dehydrogenase, regardless of aerobic or anaerobic conditions. Therefore, the metabolism of tumor cells is usually called "aerobic glycolysis". Dichloroacetic acid (DCA) is an inhibitor of glycolysis, however, due to inefficient cellular uptake and lack of mitochondrial targeting ability, the bioavailability of DCA is very low. SUMMARY

[0003] The present application aims to provide a cyclometalated iridium (III) complex targeting glycometabolism, which can target mitochondria after entering cells, inhibit glycolysis of tumor cells, and induce generation of active oxygen, and has good antitumor activity. Another object of the present application is to provide a synthesis method of the cyclometalated iridium (III) complex and application thereof in preparation of an antitumor drug.

[0004] The cyclometalated iridium (III) complex targeting glycometabolism has the following structural formula: .

[0005] The preparation method of the cyclometalated iridium (III) complex comprises the following steps: (1) under an inert atmosphere, dichloroacetic acid (DCA) modified bipyridine ligand and cyclometalated iridium dimer are heated to reflux in an organic solvent, and the solvent is removed by reduced pressure distillation; (2) NH4PF6 is used for replacement, and after centrifugation, a crude product is obtained, and the cyclometalated iridium (III) complex targeting glycometabolism is obtained by separation and purification; The reaction equation is as follows: .

[0006] In step (1), the preparation method of the dichloroacetic acid modified bipyridine ligand is as follows: dichloroacetic acid and bridged ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine (the bridged ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine used in the application is prepared according to the method reported in the literature: Q. Wu, K. Y. Zhang, P. Dai, et al., Bioorthogonal “labeling after recognition” affording an FRET-based luminescent probe for detecting and imaging caspase-3 via photoluminescence lifetime imaging. J. Am. Chem. Soc., 2020, 142 (2), 1057-1064.) is dissolved in anhydrous methanol, and the mixture is stirred at room temperature after being catalyzed by DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride) to obtain a crude product, which is separated and purified by column chromatography to obtain the dichloroacetic acid modified bipyridine ligand; the molar ratio of dichloroacetic acid to 4-methyl-4'-aminomethyl-2,2'-bipyridine is 1:1~1.2, and the molar ratio of DMTMM to dichloroacetic acid is 1:1~1.2.

[0007] The structure of the dichloroacetic acid modified bipyridine ligand is as follows: .

[0008] In step (1), the structure of the cyclometalated iridium dimer is as follows: .

[0009] The molar ratio of the dichloroacetic acid modified bipyridine ligand to the cyclometalated iridium dimer is 2:1~1.2, the mixed solvent is a mixed solvent composed of dichloromethane and methanol in a volume ratio of 3~2:1, and the reflux reaction temperature is 50~60℃, and the reaction time is 6~8h.

[0010] In step (2), the crude product is obtained by replacing NH4PF6, specifically, a saturated NH4PF6 methanol solution is added and stirred for 2~3h.

[0011] The above-mentioned cyclometalated iridium (III) complex targeting glycometabolism is used for preparing an antitumor drug, and the tumor is a human cervical cancer cell.

[0012] The cyclometalated iridium (III) complex targeting glycometabolism of the application has good cell uptake capacity and the ability of targeting mitochondria in tumor cells, can inhibit the expression level of PDK2 in tumor cells, and at the same time, reduce the pPDH / PDH ratio, so as to inhibit the glycolysis pathway of tumor cells; in addition, the complex of the application can also induce the generation of reactive oxygen species, cause the decline of mitochondrial membrane potential and the decline of intracellular ATP level, so as to induce tumor cell apoptosis.

[0013] Beneficial effects: compared with the prior art, the complex of the application has the following remarkable advantages: after entering the cell, the complex of the application can target mitochondria, inhibit the glycolysis of tumor cells, and induce the generation of reactive oxygen species, causing mitochondrial function damage, and realizing good antitumor activity; it shows high cytotoxicity to various tumor cells, especially the IC 50 value of human cervical cancer cells (HeLa) reaches 3.8 μM, indicating that it can effectively inhibit the proliferation of tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the liposolubility diagram of the complex Ir-DCA of the application; Figure 2 is the subcellular organelle distribution of the complex Ir-DCA of the application; Figure 3 is the inhibition of PDK2 protein by the complex Ir-DCA of the application; Figure 4 is the generation of reactive oxygen species induced by the complex Ir-DCA of the application in HeLa cells; Figure 5 is the mitochondrial membrane potential damage induced by the complex Ir-DCA of the application in HeLa cells; Figure 6 is the reduction of intracellular ATP level induced by the complex Ir-DCA of the application in HeLa cells; Figure 7 is the apoptosis induced by the complex Ir-DCA of the application in HeLa cells; Figure 8 is the death of HeLa 3D cell spheres induced by the complex Ir-DCA of the application. DETAILED DESCRIPTION

[0015] Example 1 The preparation method of the cyclometalated iridium (III) complex (referred to as Ir-DCA) targeting glycometabolism of the application comprises the following steps:

[0016] (1) Under argon atmosphere, DCA (0.129 g, 1 mmol) and 4-methyl-4'- aminomethyl-2,2'-bipyridine (0.199 g, 1 mmol) were dissolved in anhydrous methanol, stirred at room temperature for 15 min, then DMTMM (0.278 g, 1 mmol) was added, and the reaction was continued to stir at room temperature for 2 h; after the reaction was completed, the mixture was concentrated under reduced pressure, and column chromatography was used for separation and purification, with a mixture of dichloromethane and methanol as the developing agent, to obtain light pink solid DCA-bpy with a yield of 47%. 1 H NMR (400 MHz, Methanol- d 4 ) δ (ppm): 8.61 (d, J = 5.1 Hz, 1H), 8.51 (d, J = 5.1Hz, 1H), 8.26-8.18 (m, 1H), 8.18-8.10 (m, 1H), 7.38 (dd, J = 5.1, 1.7 Hz,1H), 7.31 (dd, J = 5.0, 1.6 Hz, 1H), 6.38 (s, 1H), 4.58 (s, 2H), 2.48 (s,3H). (2) Under argon atmosphere, [Ir(ppy)2Cl]2(53.6 mg, 0.05 mmol) and ligand DCA-bpy (31.0 mg, 0.1 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (2:1, v:v), stirred at 60°C under reflux for 6 h; after the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure, and a saturated NH4PF6 methanol solution was added, stirred for 2 h; the obtained crude product was separated and purified by column chromatography, with a mixture of dichloromethane and methanol as the developing agent, to obtain yellow solid Ir-DCA with a yield of 52%. 1 H NMR (400 MHz, Methanol- d 4) δ (ppm): 8.57 (d, J = 1.6 Hz, 1H), 8.51 (s, 1H), 8.15-8.07 (m, 2H), 7.94 (d, J = 5.7 Hz, 1H), 7.92-7.78 (m, 5H), 7.62 (dd, J = 5.8, 1.4 Hz, 2H), 7.40 (ddd, J = 7.7, 5.7, 1.7 Hz, 2H), 7.09-6.98 (m, 4H), 6.88 (tt, J = 7.4, 1.5 Hz, 2H), 6.37 (s, 1H), 6.29 (ddd, J = 7.6, 4.1, 1.1 Hz, 2H), 4.67-4.57 (m, 2H), 2.58 (s, 3H). The cyclometalated iridium dimer [Ir(ppy)2Cl]2 was prepared as follows (cyclometalated iridium dimer [Ir(ppy)2Cl]2 used in the present application was prepared based on the method reported in Inorg. Chem. 1994, 33(1), 9-14): phenylpyridine and iridium chloride hydrate were dissolved in a mixed solution of ethylene glycol ethyl ether and water under argon atmosphere, and the mixture was reacted at reflux temperature to obtain a crude product, which was purified by filtration and water washing.

[0017] Comparative Example 1 A preparation method of a cyclopentadienyl iridium (III) complex (referred to as IrCp-DCA) includes the following steps:

[0018] Under argon atmosphere, [IrCp*Cl2]2 (39.8 mg, 0.05 mmol) and ligand DCA-bpy (31.0 mg, 0.1 mmol) were dissolved in anhydrous methanol, and stirred at room temperature under reflux for 6 h; after the reaction was completed, the mixture was concentrated under reduced pressure, saturated NH4PF6 in methanol was added, and stirred for 2 h; the obtained crude product was separated and purified by column chromatography, using a mixture of dichloromethane and methanol as the developing agent, to obtain yellow-green solid IrCp-DCA, with a yield of 58%. 1 H NMR (400 MHz, Methanol- d 4) δ (ppm): 8.91 (d, J = 5.9 Hz, 1H), 8.80 (d, J = 5.8 Hz, 1H),8.53-8.50 (m, 1H), 8.47-8.44 (m, 1H), 7.74-7.66 (m, 2H), 6.44 (s, 1H), 4.73(d, J = 3.5 Hz, 2H), 2.68 (s, 3H), 1.70 (s, 15H). Comparative Example 2 A preparation method of a cyclometalated iridium (III) complex (referred to as Ir-NH2), the specific steps are:

[0019] Under an argon atmosphere, [Ir(ppy)2Cl]2(53.6 mg, 0.05 mmol) and 4-methyl-4'- aminomethyl-2,2'-bipyridine (19.9 mg, 0.1 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (2:1, v:v), and stirred at 60°C under reflux for 6 h; after the reaction was completed, it was cooled to room temperature, the mixture was concentrated under reduced pressure, a saturated NH4PF6 methanol solution was added, and stirred for 2 h; the obtained crude product was separated and purified by column chromatography, and a mixture of dichloromethane and methanol was used as the developing agent, to obtain yellow solid Ir-NH2, with a yield of 60%. 1 H NMR (400 MHz, Methanol- d 4 ) δ (ppm):8.74-8.69 (m, 1H), 8.54 (s, 1H), 8.14-8.08 (m, 2H), 8.00 (d, J = 5.7 Hz, 1H),7.92-7.76 (m, 5H), 7.63 (d, J = 5.9 Hz, 2H), 7.53 (dd, J = 6.0, 1.5 Hz, 1H),7.44-7.37 (m, 1H), 7.04 (q, J = 6.9 Hz, 3H), 6.89 (ddd, J = 11.9, 5.9, 2.9Hz, 2H), 6.29 (t, J = 8.1 Hz, 2H), 4.25 (s, 2H), 2.59 (s, 3H). Comparative Example 3 A preparation method of a cyclopentadienyl iridium (III) complex (referred to as IrCp-bpy), the specific steps are:

[0020] [IrCp*Cl2]2(39.8 mg, 0.05 mmol) and 4,4'-dimethyl-2,2'-bipyridine (18.4 mg, 0.1 mmol) were dissolved in anhydrous methanol under argon atmosphere, and stirred at room temperature under reflux for 6 h; after the reaction was completed, the mixture was concentrated under reduced pressure, a saturated NH4PF6 solution in methanol was added, and stirred for 2 h; the obtained crude product was separated and purified by column chromatography with a mixture of dichloromethane and methanol as the developing agent to obtain a yellow-green solid IrCp-bpy with a yield of 67%. 1 H NMR (400MHz, DMSO- d 6 ) δ (ppm): 8.80 (d, J = 5.8 Hz, 2H), 8.65-8.60 (m, 2H), 7.71-7.64(m, 2H), 2.62 (s, 6H), 1.64 (s, 15H). The complex Ir-DCA prepared in Example 1 and the complexes IrCp-DCA prepared in Comparative Example 1, Ir-NH2 prepared in Comparative Example 2 and IrCp-bpy prepared in Comparative Example 3 were subjected to the following experiments: Cytotoxicity on human cervical cancer cells HeLa, human breast cancer cells MCF-7, human lung cancer cells A549 and human lung fibroblast-like cells HLF: The anti-proliferation effects of the compounds DCA, IrCp-bpy, IrCp-DCA, Ir-NH2, Ir-DCA and cisplatin CDDP were analyzed by MTT colorimetry. MTT (thiazolyl blue) is a tetrazolium salt which can be reduced by succinate dehydrogenase in the mitochondria of living cells to form a blue-violet product formazan (the product is soluble in DMSO) which has an absorption peak at 490 nm, so A490 nm can be used to analyze cell proliferation.

[0021] The specific experimental steps are as follows: (1) One tube of tumor cells was first revived, and was cultured with fresh culture medium (DMEM culture medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and was used after being subcultured for 3 times; (2) When the cells reached the logarithmic growth phase, they were inoculated into a 96-well plate at a cell density of 5000 cells / well (100 μL of culture medium per well), and were then placed in an incubator (37°C, 5% CO2) for culture; (3) After the cells adhered, 100 μL of fresh culture medium containing different concentration gradients of the compounds DCA, IrCp-bpy, IrCp-DCA, Ir-NH2, Ir-DCA and cisplatin CDDP was added to each well, respectively, and then the plate was placed in an incubator for further incubation. (4) After 48 hours of incubation, 20 μL MTT (5 mg / mL) was added to each well, and the incubation was continued at 37 °C for 4 hours. The supernatant was then removed, 150 μL dimethyl sulfoxide (DMSO) was added to each well, and the A490 nm was detected using an enzyme-linked immunoassay instrument. The cell proliferation inhibition rate was calculated, and the IC 50 value (the drug concentration corresponding to an inhibition rate of 50%) was determined.

[0022] The MTT test results (IC 50 values (μM)) of the compounds DCA, IrCp-bpy, IrCp-DCA, Ir-NH2, Ir-DCA, and cisplatin CDDP are shown in Table 1.

[0023] Table 1

[0024] The results show that the complex Ir-DCA has higher proliferation inhibition activity on HeLa cells than the compounds DCA, the complexes IrCp-bpy, IrCp-DCA, Ir-NH2, and cisplatin CDDP, indicating that the complex Ir-DCA has high cytotoxicity on tumor cells.

[0025] Example 2 The cyclometalated iridium (III) complex Ir-DCA prepared in Example 1 has good fat solubility: Method: The fat solubility of the complexes IrCp-bpy and Ir-DCA was determined by the shake flask method. The complexes were dissolved in water saturated with n-octanol, and an equal amount of water saturated organic phase was added and mixed uniformly. Under the conditions of 37 °C and 800 rpm, the mixture was shaken overnight, and then centrifuged at 8000 rpm for 8 min. The absorbance of the complexes in the organic and aqueous phases was detected by a UV-visible spectrometer, and the fat-water partition coefficient was calculated.

[0026] The fat solubility of the cyclometalated iridium (III) complex Ir-DCA synthesized in Example 1 is shown in Table 1. Figure 1 Compared with the cyclopentadienyl iridium complex IrCp-DCA, the cyclometalated iridium complex Ir-DCA has better fat solubility, indicating that Ir-DCA is easier to accumulate in cells than IrCp-DCA.

[0027] Example 3 The cyclometalated iridium (III) complex Ir-DCA prepared in Example 1 was applied to the subcellular organelle localization in cells.

[0028] Methods: HeLa cells were seeded in 35 mm Corning laser confocal culture dishes, when the cell density grew to 70%, 8 μM Ir-DCA was added and incubated for 6 h, then the medium was aspirated, washed twice with PBS, and then stained with Mito-Tracker Green for 30 min, the probe was aspirated, washed twice with PBS, and then immediately observed with confocal microscope. Complex Ir-DCA: λ ex = 405 nm, λ em = 620 ± 30 nm; Mito-Tracker Green: λ ex = 488 nm, λ em = 525 ± 30 nm. The colocalization coefficient was analyzed by ImageJ software.

[0029] The intracellular localization of the synthesized cyclometalated iridium (III) complex Ir-DCA after co-incubation with mitochondrial probe is shown in Figure 2 The results show that the cyclometalated iridium (III) complex Ir-DCA is mainly distributed in mitochondria after being taken up by HeLa cells, and the colocalization coefficient is 0.876, indicating that Ir-DCA has excellent ability to target mitochondria.

[0030] Example 4 The cyclometalated iridium (III) complex Ir-DCA prepared in Example 1 inhibits the expression of PDK2 in cells: Method: Western blot was used to detect the changes of PDK2, PDH and pPDH protein content. The pre-prepared cell culture medium containing Ir-DCA (4, 8 μΜ), Ir-NH2 (8 μΜ) and DCA (8 μΜ) was added to the 100 mm culture dish of HeLa cells which had been adherent growth, and incubated for 24 h. The cells were collected by centrifugation, washed twice with PBS, and then added with RIPA strong lysis solution containing PMSF for whole cell lysis at 4°C for 25 min. After the end, the supernatant obtained by centrifugation at 4°C at 13400 rpm for 20 min was the cell whole protein sample required for the experiment. The protein concentration in the above protein sample was determined by using 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 by wet method, and after the end, the membrane was blocked in 5% skim milk powder for 2 h. According to the instructions of the antibody, the primary antibody was diluted with skim milk powder according to the corresponding proportion, and the blocked membrane was incubated in the primary antibody incubation solution at 4°C overnight to specifically bind to the target protein. After the end, wash with PBST (5 x 6 min / time). The washed membrane was placed in the pre-prepared secondary antibody incubation solution for 2 h to bind with the primary antibody, and was also washed with PBST. Equal volume of ECL developing solution was prepared and covered on the PVDF membrane, and after 2 min of treatment, the chemical luminescence imaging system was used for shooting.

[0031] The inhibition effect of the synthesized cyclometalated iridium (III) complex Ir-DCA on PDK2 in cells is shown in Figure 3 The results show that compared with the control group (blank group), Ir-NH2 and DCA group, the complex Ir-DCA can significantly inhibit the expression level of PDK2 in cells after being taken up by HeLa cells, and reduce the pPDH / PDH ratio.

[0032] Example 5 Application of the cyclometalated iridium (III) complex Ir-DCA prepared in Example 1 to generate reactive oxygen species in cells: Method 1: Confocal microscope was used to detect ROS in cancer cells. HeLa cells were inoculated in a 35 mm Corning laser confocal culture dish, and when the cell density grew to 70%, Ir-DCA, Ir-NH2 and DCA were added for incubation for 24 h. Then, the cells were stained with 10 μΜ DCFH-DA in serum-free culture medium at 37°C in the dark for 30 min, washed twice with PBS, and then immediately observed by confocal microscope. The fluorescence channel was λ ex = 488 nm, λem = 530±30 nm.

[0033] Method 2: Flow cytometry detection of ROS in tumor cells. HeLa cells were seeded in 6-well plates and allowed to adhere overnight. After incubation with Ir-DCA, Ir-NH2, and DCA for 24 h, cells were stained with serum-free culture medium containing 10 μM DCFH-DA at 37°C in the dark for 30 min. Uninfected DCFH-DA was removed by washing with PBS. Cells were collected by trypsin digestion, washed with PBS, resuspended, and immediately analyzed by flow cytometry to determine the green fluorescence intensity. The fluorescence detection channel was λ. ex = 488 nm, λ em = 530±30 nm. The average fluorescence intensity of green light was analyzed using FlowJo 7.6 software.

[0034] The results of reactive oxygen species in the cyclic iridium(III) complex Ir-DCA are as follows: Figure 4 As shown in the figure. The results indicate that, compared with the control group and Ir-NH2 and DCA, the green fluorescence was significantly enhanced by confocal and flow cytometry after Ir-DCA treatment, indicating that the Ir-DCA complex can generate a large amount of reactive oxygen species in HeLa cells.

[0035] Example 6 The cyclic iridium(III) complex Ir-DCA prepared in Example 1 induced intracellular mitochondrial damage: Method 1: Confocal microscopy was used to detect changes in mitochondrial membrane potential in tumor cells. HeLa cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, Ir-DCA, Ir-NH2, and DCA were added and incubated for 24 h. Cells were then stained with pre-prepared JC-1 working solution at 37°C in the dark for 30 min, washed with 1× buffer, and immediately observed under a confocal microscope. The fluorescence detection channel was λ. ex = 488 nm, λ em = 530±30 nm; λ ex = 561 nm, λ em = 590±30 nm.

[0036] Method 2: Flow cytometry was used to detect changes in mitochondrial membrane potential in tumor cells. HeLa cells were seeded in 6-well plates and allowed to adhere overnight. After incubation with Ir-DCA, Ir-NH2, and DCA for 24 h, cells were harvested, washed with PBS, stained with JC-1 working solution for 30 min, washed with 1× buffer, and resuspended. The samples were immediately analyzed using a BD C6 flow cytometer, and the results were processed and analyzed using FlowJo 7.6 software. The fluorescence detection channel was λ. ex = 488nm, λ em = 530±30 nm; λ ex = 561 nm, λ em = 590±30 nm.

[0037] The results of the cyclic iridium(III) complex Ir-DCA on the induced changes in intracellular mitochondrial membrane potential are as follows: Figure 5 As shown in the figure. The results showed that compared with the control group and Ir-NH2 and DCA, Ir-DCA treatment resulted in a decrease in red fluorescence and an increase in green fluorescence in the cells, indicating that Ir-DCA effectively induced a decrease in mitochondrial membrane potential. Flow cytometry results also showed similar conclusions.

[0038] Example 7 The cyclic iridium(III) complex Ir-DCA prepared in Example 1 induces a decrease in intracellular ATP levels: Methods: HeLa cells were seeded in 6-well culture dishes and cultured until the cells reached 70% cell growth. Ir-DCA, Ir-NH2, and DCA were added, and the cells were incubated for 24 h. Cells were washed three times with PBS, and 400 μL of lysis buffer was added to each well. The cell lysis buffer was centrifuged at 12000 g at 4℃ for 5 min. Cells were cultured in 96-well white plates according to the instructions. The cell lysis supernatant was added to ATP working solution. The luminescence of each well was immediately detected using a multi-functional microplate reader.

[0039] The effects of the cyclic iridium(III) complex Ir-DCA on the induction of intracellular ATP changes are as follows: Figure 6 As shown in the figure. The results indicate that, compared with the control group and Ir-NH2 and DCA, a decrease in ATP content was observed after Ir-DCA treatment, indicating that the Ir-DCA complex effectively induced mitochondrial dysfunction.

[0040] Example 8 The cyclic iridium(III) complex Ir-DCA prepared in Example 1 induced apoptosis in HeLa cells: Method 1: Flow cytometry was used to detect the apoptosis of tumor cells. HeLa cells were seeded in 6-well culture dishes and cultured until the cells grew to 70%. Then, Ir-DCA, Ir-NH2, and DCA were added and incubated for 24 h. The cells were collected by trypsin digestion, washed with PBS, resuspended with binding buffer, and then stained with 5 μL of Annexin V-FITC working solution for 5 min in the dark. Then, 5 μL of PI was added and incubated for 5-10 min in the dark. The samples were immediately detected by a BD C6 flow cytometer, and the results were processed and analyzed by FlowJo 7.6 software. The fluorescence channels were λ ex = 488 nm, λ em = 530 ± 30 nm, λ ex = 561 nm, and λ em = 590 ± 30 nm.

[0041] Method 2: Western blotting (WB) was used to detect the changes in the content of apoptosis-related proteins. HeLa cells were seeded in 100 mm culture dishes and cultured until the cells grew to 70%. Then, Ir-DCA (4, 8 μM), Ir-NH2 (8 μM), and DCA (8 μM) were added and incubated for 24 h. The cells were collected by centrifugation, washed twice with PBS, and lysed in RIPA lysis buffer containing PMSF at 4°C for 25 min. After centrifugation at 13400 rpm for 20 min at 4°C, the supernatant was collected as the cell protein sample. The protein concentration in the sample was determined by a BCA protein content assay kit. The expression levels of different proteins in the sample were detected by SDS-PAGE gel electrophoresis. After the gel was prepared, the same volume of protein sample was added to each well for gel electrophoresis. After appropriate separation, the electrophoresis was stopped immediately. The target protein was transferred to a PVDF membrane using a wet method. After the transfer, the membrane was blocked with 5% skim milk for 2 h. According to the instructions of the antibody, the primary antibody was diluted with skim milk at a certain ratio. The blocked membrane was incubated with the primary antibody overnight at 4°C to specifically bind to the target protein. After washing with PBST (5 x 6 min each time), the washed membrane was incubated with the secondary antibody for 2 h. The same washing was performed with PBST. Equal volumes of ECL developing solution were prepared and covered on the PVDF membrane. After 2 min of treatment, the chemical luminescence imaging system was used for imaging.

[0042] The related experimental results of the cyclometalated iridium (III) complex Ir-DCA in inducing HeLa cell apoptosis are shown in FIG. 2. Figure 7The results showed that the proportion of apoptotic cells increased significantly after treatment with Ir-DCA, while the Bcl2 and Caspase 3 proteins were down-regulated, indicating that the complex Ir-DCA can induce apoptosis of HeLa cells.

[0043] Example 9 Application of the cyclometalated iridium (III) complex Ir-DCA prepared in Example 1 to induce growth inhibition of HeLa 3D cell spheres: Method: HeLa cells were seeded into ultra-low attachment 96-well round-bottom microwell plates at a density of 2500 cells per well, and 24 h later HeLa MCTs with a diameter of 500 μm were formed. In the 3D model, HeLa MCTs were incubated in medium containing Ir-DCA (20, 40 μM), Ir-NH2 (40 μM), and DCA (40 μM), respectively. The medium was replaced every two days, and the volume, morphology, and growth state of the cell spheres were monitored every other day using a microscope. After 6 days of incubation, the spheres were washed twice with PBS, stained with Calcein AM / PI, and fixed in 4% paraformaldehyde. The spheres were placed in a confocal dish and imaged at different depths using a confocal scanning microscope system. Calcein AM (λ ex = 488 nm, λ em = 525±30 nm); PI (λ ex = 561 nm, λ em = 590±30 nm).

[0044] Application of the cyclometalated iridium (III) complex Ir-DCA to inhibit the growth of HeLa 3D cell spheres is shown in Figure 8 The results showed that the cell spheres died after treatment with the complex Ir-DCA, compared with the control group without drug treatment and the Ir-NH2 and DCA groups.

Claims

1. A cyclometallated iridium (III) complex targeting glycometabolism, characterized in that, The structural formula is: .

2. Process for the preparation of the cyclometalated iridium (III) complex according to claim 1, characterized in that, The method comprises the following steps: (1) heating dichloroacetic acid modified bipyridine ligand and cyclometalated iridium dimer in an organic solvent under an inert atmosphere and refluxing, and removing the solvent under reduced pressure; (2) replacing with NH4PF6, centrifuging to obtain a crude product, and separating and purifying to obtain the cyclometalated iridium (III) complex targeting sugar metabolism; The structural formula of the dichloroacetic acid modified bipyridine ligand is: ; The structural formula of the cyclometalated iridium dimer is: .

3. The method of claim 2, wherein: In step (1), the dichloroacetic acid modified bipyridine ligand is prepared by dissolving dichloroacetic acid and bridged ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine in anhydrous methanol, catalyzing by DMTMM, and reacting the mixture at room temperature to obtain a crude product, which is separated and purified by column chromatography.

4. The method of claim 3, wherein: The molar ratio of dichloroacetic acid to 4-methyl-4'-aminomethyl-2,2'-bipyridine is 1:1-1.

2.

5. The method of claim 3, wherein: The molar ratio of DMTMM to dichloroacetic acid is 1:1-1.

2.

6. The method of claim 2, wherein: In step (1), the molar ratio of the dichloroacetic acid modified bipyridine ligand to the cyclometalated iridium dimer is 2:1-1.

2.

7. The method of claim 2, wherein: In step (1), the mixed solvent is a mixed solvent composed of dichloromethane and methanol in a volume ratio of 2-3:

1.

8. The method of claim 2, wherein: The temperature of the refluxing reaction is 50-60℃, and the time is 6-8 h.

9. The method of claim 2, wherein: In step (2), the crude product is obtained by replacing with NH4PF6, specifically, a saturated NH4PF6 methanol solution is added and stirred for 2-3 h.

10. Use of the cyclometalated iridium (III) complex targeting sugar metabolism in claim 1 in the preparation of an antitumor drug.

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