A metal iridium complex for inducing mutp53 degradation, and a preparation method and application thereof

Abstract

The application discloses a metal iridium complex for inducing Mutp53 degradation, a preparation method and application thereof, and relates to the technical field of medicine, in particular to a metal iridium complex for inducing Mutp53 degradation, a preparation method and application thereof. The application introduces an aggregation-induced emission molecule and a triphenyl phosphorus group with mitochondrial targeting into a metal iridium complex precursor, and constructs a complex with cell fluorescence imaging and anticancer activity. Meanwhile, the complex can also generate ROS to destroy the intracellular redox homeostasis through photoactivation, up-regulate the influx copper transporter 1 (SLC31A1), destroy the copper homeostasis, further cause the occurrence of ferroptosis through significant down-regulation of glutathione peroxidase 4 (GPX4), and activate the ferroptosis to drive the degradation of Mutp53 and eliminate the gain-of-function (GOF) of Mutp53.

Description

TECHNICAL FIELD

[0001] The present application relates to a metal iridium complex for inducing Mutp53 degradation, and also relates to a preparation method of the metal iridium complex and application of the metal iridium complex in preparation of an antitumor drug or an antitumor drug component. BACKGROUND

[0002] Tumor suppressor p53 is known as the "guardian of the genome", which plays a key role in apoptosis regulation and genome stability. However, as the most frequently altered gene, p53 is often directly inactivated by mutation, which occurs in more than 50% of human cancers. In addition to losing tumor suppressor function, p53 mutation can express stable Mutp53 protein and accumulate in large quantities in cancer cells, which indicates that Mutp53 exhibits gain-of-function (GOF), including promoting tumor chemoresistance, promoting tumor growth and metastasis, etc. Therefore, attempts to restore p53 function in tumors have been developed for decades. However, so far, p53 is still considered as an "undruggable" target, and no p53-targeting drug has been approved for clinical treatment and used in I / II / III phase clinical trials.

[0003] According to the important role of Mutp53 in tumorigenesis, several strategies targeting p53 mutants have been proposed, such as restoring the expression of wild-type p53 (WTp53), converting Mutp53 into WTp53, or eliminating Mutp53 protein. The most direct p53-targeting treatment strategy is to induce Mutp53 degradation. The stability of Mutp53 is closely related to the intracellular redox state or zinc ion level of cancer cells. The successful cases of inducing Mutp53 degradation are attributed to the significant increase of Zn 2+ delivery level, which disrupts the intracellular zinc homeostasis, induces oxidative stress, and is accompanied by an increase in intracellular reactive oxygen species (ROS). However, since the mechanism of Zn 2+ and ROS degrading Mutp53 is not clear, it is not certain whether Zn 2+ is the only ion that can activate Mutp53 degradation. Therefore, more research is needed for tumor treatment targeting p53. SUMMARY

[0004] Invention purposes: The purpose of the present application is to provide a metal iridium complex for inducing Mutp53 degradation, which can generate ROS to destroy the intracellular redox homeostasis through photoactivation, up-regulate the influx copper transporter 1 (SLC31A1), destroy the copper homeostasis, further cause the occurrence of ferroptosis through significant down-regulation of glutathione peroxidase 4 (GPX4), and activated ferroptosis can drive the degradation of Mutp53 and eliminate the gain-of-function (GOF) exhibited by Mutp53; another purpose of the present application is to provide a preparation method of the above metal iridium complex and its application in preparing an antitumor drug or an antitumor drug component.

[0005] Technical scheme: The metal iridium complex for inducing Mutp53 degradation provided by the present application has the following chemical structure:

[0006]

[0007] The present application constructs a complex with cell fluorescence imaging and anticancer activity by introducing an aggregation-induced emission molecule and a triphenylphosphine group with mitochondrial targeting into the metal iridium complex precursor.

[0008] The preparation method of the above metal iridium complex for inducing Mutp53 degradation comprises the following steps:

[0009] (1) Synthesis of triphenylphosphine-modified aggregation-induced emission ligand:

[0010] (1.1) Dissolve the aggregation-induced emission molecule TPE ((E)-1,2-bis(4-bromophenyl)-1,2-diphenylethene), (4-hydroxyphenyl)boronic acid, potassium bicarbonate and Pd(PPh3)4 in a mixed solvent of THF and water; heat the mixture to reflux; after cooling to room temperature, extract the mixture with dichloromethane, wash the organic phase with water and brine and dry it over MgSO4; after removing the solvent under reduced pressure, purify the crude product on silica gel by column chromatography using dichloromethane / hexane as the eluent, and obtain the solid product TPE-1;

[0011] (1.2) Compound TPE-1, compound 1 (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[c][1,2,5]thiadiazole-4-carbaldehyde), potassium carbonate and Pd(PPh3)4 were mixed, under vacuum and in an oxygen-free environment, to which a mixture solvent of THF and H2O was added, the mixture was heated to reflux overnight, after the reaction was cooled, water was added, the mixture was extracted with dichloromethane, and then the organic phase was combined and dried with MgSO4; after removing the solvent under low pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / hexane as eluent, and the obtained solid product OH-TPE-A was obtained;

[0012] (1.3) OH-TPE-A and 4-ethynylphenylacetonitrile were dissolved in anhydrous methanol, then 3 drops of piperidine were added, the mixture was heated to reflux, after removing the solvent under low pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / petroleum ether as eluent, and the obtained solid product OH-ALK was obtained;

[0013] (1.4) The aggregation-induced emission molecule OH-ALK, (4-bromobutyl)-triphenylphosphonium bromide and K2CO3 were dissolved in acetonitrile; the mixture was stirred and refluxed under an inert gas protection atmosphere; after the reaction was completed, it was cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated under low pressure; then, the solid product PPh3-ALK was obtained by column chromatography purification;

[0014]

[0015] (2) Synthesis of metal iridium complex: PPh3-ALK, Ir-N3, CuI and sodium ascorbate were added to DMF in a Schlenk reaction tube, stirred at room temperature under nitrogen atmosphere in the dark; then the reaction solvent (DMF) was removed on a rotary evaporator; then, the obtained crude mixture was purified by column chromatography on silica gel (dichloromethane / methanol as eluent, v / v=95:5), and a red powder was obtained, named Ir-PPh3;

[0016]

[0017] In step (1), the molar ratio of TPE to (4-hydroxyphenyl)boronic acid is 1:1.2.

[0018] In step (1), the molar ratio of TPE-1 to 1 is 1:1.2.

[0019] In step (1), the molar ratio of OH-TPE-A to 4-ethynylphenylacetonitrile is 1:2.

[0020] In step (1), the molar ratio of OH-ALK to (4-bromobutyl)-triphenylphosphonium bromide is 1:2.

[0021] In step (1), the reflux reaction temperature is 80℃, and the reflux reaction time is 48h.

[0022] In step (2), the molar ratio of PPh3-ALK to Ir-N3 is 1:1.

[0023] In step (2), the reaction temperature is 25℃, and the stirring time is 24h.

[0024] The metal iridium complex is used for preparing an antitumor drug or an antitumor drug component.

[0025] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: (1) The complex Ir-PPh3 of the present application can solve the problems of low drug efficacy, large drug dosage, large side effects and difficulty in degrading Mutp53 when a single metal complex precursor or an AIE molecule is used as an antitumor drug; (2) The complex Ir-PPh3 of the present application has good active oxygen generation capacity, and the fluorescence intensity increases by several hundred times when an active oxygen probe is used for in vitro detection, which can be suitable for photodynamic therapy; (3) The complex Ir-PPh3 of the present application is non-toxic in dark conditions, and shows high cytotoxicity to a variety of tumor cells (including mutant p53 (Mutp53) cells and wild-type p53 (WTp53) cells) after light irradiation, and the IC 50 value of the complex Ir-PPh3 of the present application to A549R, which is a Mutp53 high-expression human lung cancer cell line resistant to cisplatin, is 2.7μM, which can effectively inhibit the proliferation of tumor cells; (4) The complex Ir-PPh3 of the present application can self-assemble into nanospheres, and after light irradiation, the mitochondria-targeted Ir-PPh3 can increase the generation of ROS in cancer cells, and make the redox balance unbalanced, and oxidative stress leads to the up-regulation of SLC31A1 expression, and the exogenous copper is transported into the cells, which further induces the occurrence of ferroptosis through the significant down-regulation of GPX4, and ferroptosis drives the degradation of Mutp53 through the proteasome degradation pathway, and eliminates the GOF performance of Mutp53 by degrading Mutp53, including reducing the chemotherapeutic resistance, inhibiting the migration of cancer cells, and reducing the formation of tumor cell colonies and spheres; in the mutp53-overexpressing MDA-MB-231 mouse tumor, it is further proved that the complex Ir-PPh3 has strong inhibitory capacity in vivo; (5) The complex Ir-PPh3 of the present application can achieve the effect of chemotherapeutic resistance and tumor elimination by degrading Mutp53 through photoactivation. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1Fluorescence image of the ability of the complex Ir-PPh3 of the present application to detect reactive oxygen species production in vitro by fluorescence change of reactive oxygen species indicator;

[0027] Figure 2 (a) TEM and (b) DLS images of the self-assembly of the complex Ir-PPh3 of the present application into nanoparticles;

[0028] Figure 3 Cytotoxicity images of the complex Ir-PPh3 of the present application on different cells in (a) darkness and (b) light;

[0029] Figure 4 (a) Confocal images of the complex Ir-PPh3 of the present application inducing Mutp53 degradation in A549R cells and (b) Western blot images of the complex Ir-PPh3 of the present application inducing Mutp53 degradation in A549R cells, (c) and (d) Western blot images of the complex Ir-PPh3 of the present application inducing Mutp53 degradation in MDA-MB-231 and MCF-7ADR cells;

[0030] Figure 5 Images of the complex Ir-PPh3 of the present application (a) inducing reactive oxygen species rise and (b) GSH drop;

[0031] Figure 6 Images of the complex Ir-PPh3 of the present application (a) inducing copper transporter SLC31A1 rise and (b) intracellular copper ion content rise;

[0032] Figure 7 Images of the complex Ir-PPh3 of the present application (a) inducing iron death characteristic protein GPX4 drop and (b) and (c) GPX4 protein images with the addition of reactive oxygen species scavenger NAC and copper ion chelator TTM;

[0033] Figure 8 Images of the complex Ir-PPh3 of the present application with the addition of (a) iron death inhibitor, (b) reactive oxygen species scavenger NAC and (c) copper ion chelator TTM Mutp53 protein images;

[0034] Figure 9 Images of the complex Ir-PPh3 of the present application (a) and (b) reducing chemotherapy resistance, (c) and (d) inhibiting cancer cell migration scratch test and (e) and (f) Transwell test, (g) and (h) reducing tumor cell colony formation;

[0035] Figure 10The changes of tumor tissue volume, weight, mouse weight and Mutp53 in tumor of tumor-bearing mice after treatment with the iridium complex Ir-PPh3 of the application; (a) is a schematic diagram of mouse in vivo experiment; (b) is the change of mouse tumor weight; (c) is the anatomical diagram of mouse tumor after treatment; (d) is the change of mouse tumor volume; (e) is the change of mouse weight; (f) is the HE staining and p53 expression of tumor tissue section. DETAILED DESCRIPTION

[0036] Example 1

[0037] The preparation method of the iridium complex (Ir-PPh3) of the application comprises the following steps:

[0038] (1) Synthesis of compound PPh3-ALK:

[0039] (1.1) In a 250 mL three-necked round-bottom flask equipped with a condenser, polymeric light-emitting molecule TPE ((E)-1,2-bis(4-bromophenyl)-1,2-diphenylethene) (2.45 g, 5 mmol) (synthesized according to the literature Angew. Chem. Int. Ed. 2018, 57, 13662-13665), (4-hydroxyphenyl)boronic acid (1.32 g, 6 mmol), potassium bicarbonate (5 g, 50 mmol) and Pd(PPh3)4(0.17 g, 0.15 mmol) were dissolved in a mixed solvent composed of 90 mL of distilled THF and 30 mL of water; the mixture was heated to reflux for 36 h; after cooling to room temperature, the mixture was extracted with dichloromethane for 3 times, and the organic phase was washed with water and brine and dried over MgSO4; after removing the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / hexane (v / v = 35:65) as eluent to obtain a white solid, named as TPE-1; 1 H NMR (400 MHz, CDCl3-d) δ (ppm): 7.50-7.43 (m, 2H), 7.37-7.29 (m, 2H), 7.25 (t, J = 8.3 Hz, 2H), 7.18-7.03 (m, 12H), 6.99-6.85 (m, 4H), 4.86 (s, 1H).

[0040] (1.2) Compound TPE-1 (503 mg, 1 mmol), compound 1 (7-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzo[c][1,2,5]thiadiazole-4-carbaldehyde) (350 mg 1.2 mmol) (compound 1 purchased from Beijing Huawei Ruikexingke Chemical Co., Ltd.), potassium carbonate (1 g, 10 mmol) and Pd(PPh3)4(120 mg, 0.1 mmol) were added into a 100 mL two-necked round-bottom flask; the flask was vacuumed and purged with dry nitrogen three times; then THF (30 mL) and H2O (10 mL) were added, the mixture was heated to reflux and stirred for 24 h; water was added, the mixture was extracted with dichloromethane three times, the organic phases were combined and dried over MgSO4; after the solvent was removed under low pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / hexane (v / v = 80:20) as eluent to obtain a red solid, named as OH-TPE-A; 1 HNMR (400 MHz, DMSO-d6) δ (ppm): 10.63 (s, 1H), 9.56 (s, 1H), 8.32 (d, J = 7.4 Hz, 1H), 8.04 (d, J = 7.4 Hz, 1H), 7.93-7.86 (m, 2H), 7.47-7.35 (m, 4H), 7.23-7.07 (m, 12H), 7.04-6.97 (m, 2H), 6.83-6.76 (m, 2H).

[0041] (1.3) A mixture of OH-TPE-A (148 mg, 0.25 mmol) and 4-ethynylphenylacetonitrile (71 mg, 0.5 mmol) was dissolved in anhydrous methanol (10 mL), then 3 drops of piperidine were added, and heated to reflux at 90 °C for 72 h; after the solvent was removed under low pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / petroleum ether (v / v: 70 / 30) as eluent to obtain a red solid, named as OH-ALK; 1 HNMR (400 MHz, DMSO-d6) δ (ppm): 9.56 (s, 1H), 8.60 (s, 1H), 8.53 (dd, J = 7.6, 0.9 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.95-7.83 (m, 4H), 7.71-7.63 (m, 2H), 7.49-7.36 (m, 4H), 7.25-7.07 (m, 12H), 7.01 (d, J = 8.4 Hz, 2H), 6.84-6.77 (m, 2H), 4.43 (s, 1H). 13CNMR (101 MHz, DMSO-d6) d (ppm): 157.60, 154.51, 152.69, 144.59, 143.64, 141.57, 141.41, 140.27, 138.53, 137.19, 134.90, 134.53, 134.10, 133.13, 131.70, 131.52, 131.28, 130.46, 129.12, 128.73, 128.53, 128.16, 127.94, 127.32, 127.22, 126.63, 125.65, 125.51, 123.46, 117.75, 116.15, 112.39, 83.58, 83.31. ESI-MS (negative mode, m / z) found (calcd) for [M-H] - : 708.10 (708.21).

[0042] (1.4) OH-ALK (106 mg, 0.15 mmol), (4-bromobutyl)-triphenylphosphonium bromide (143.4 mg, 0.3 mmol, 478 g / mol) and K2CO3(35 mg, 0.25 mmol) were added to CH3CN (5 mL); the resulting mixture was stirred at 80 °C for 48 h under argon atmosphere; after cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure; then, the obtained crude mixture was purified by column chromatography on silica gel (dichloromethane / methanol as eluent, v / v = 94:6) to give red powder PPh3-ALK (65 mg, 56.0% yield); characterized by NMR, mass: 1 H NMR (400 MHz, DMSO-d6) d (ppm): 8.61 (s, 1H), 8.56-8.51 (m, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.93-7.76 (m, 19H), 7.67 (d, J = 8.4 Hz, 2H), 7.59-7.53 (m, 2H), 7.46-7.40 (m, 2H), 7.24-7.15 (m, 8H), 7.11 (ddd, J = 7.6, 3.0, 1.4 Hz, 4H), 7.06-7.02 (m, 2H), 6.95-6.90 (m, 2H), 4.44 (s, 1H), 4.07 (t, J = 6.1 Hz, 2H), 3.74-3.65 (m, 2H), 1.93 (t, J = 6.7 Hz, 2H), 1.72 (m, 2H). 13C NMR (101 MHz, DMSO-d6) d (ppm): 158.50, 154.51, 152.66, 144.52, 143.60, 141.97, 141.33, 140.40, 138.10, 137.09, 135.39, 135.36, 134.86, 134.54, 134.12, 134.07, 134.02, 133.12, 132.14, 131.75, 131.50, 131.27, 130.77, 130.64, 129.11, 128.70, 128.53, 128.14, 127.92, 127.34, 127.25, 126.60, 125.75, 125.61, 123.48, 119.38, 118.53, 117.74, 115.33, 112.36, 83.62, 83.29, 66.45, 55.42, 29.51, 18.94. ESI-MS (positive mode, m / z) found (calcd) for [M-Br] + : 1026.40 (1026.36).

[0043]

[0044] (2) Synthesis of complex Ir-PPh3: In a 50 mL Schlenk tube, PPh3-ALK (28 mg, 0.025 mmol), Ir-N3 (19 mg, 0.025 mmol), Cul (1 mg) and sodium ascorbate (1 mg) were added to 3 mL of DMF and stirred at room temperature for 24 h under nitrogen atmosphere in the dark; then the reaction solvent was removed on a rotary evaporator; subsequently, the obtained crude mixture was purified by column chromatography on silica gel (dichloromethane / methanol as eluent, v / v = 95:5) to obtain red powder Ir-PPh3 (25 mg, 53.5% yield). Characterized by NMR, mass spectrometry, 1H NMR (400 MHz, DMSO-d6) d (ppm): 8.97 (s, 1H), 8.86 (s, 1H), 8.77 (s, 1H), 8.62 (d, J = 3.4 Hz, 1H), 8.55 (dd, J = 7.9, 4.0 Hz, 1H), 8.30-8.23 (m, 2H), 8.12-7.69 (m, 28H), 7.65 (t, J = 6.6 Hz, 2H), 7.57 (dd, J = 8.4, 5.0 Hz, 3H), 7.51-7.41 (m, 3H), 7.26-6.86 (m, 22H), 6.18 (dd, J = 7.4, 4.9 Hz, 2H), 5.95 (s, 2H), 4.07 (m, 2H), 3.76-3.58 (m, 2H), 2.55 (s, 3H), 1.98-1.89 (m, 2H), 1.72 (m, 2H).13C NMR (101 MHz, DMSO-d6) d (ppm): 167.25, 158.51, 156.26, 155.05, 150.85, 149.37, 148.54, 144.26, 139.22, 135.39, 134.89, 134.10, 134.00, 131.52, 131.25, 130.78, 130.65, 130.00, 127.95, 127.09, 126.55, 125.53, 124.37, 122.70, 120.50, 119.36, 118.51, 117.96, 115.35, 99.97, 65.38, 29.50, 22.56, 21.46, 15.64. ESI-MS (positive mode, m / z) found (calcd) for [M-Cl-Br] / 2 + : 876.2777 (876.2797).

[0045]

[0046] Comparative Example 1

[0047] A preparation method of a metal iridium complex (Ir-OH), specifically:

[0048] In a 50 mL Schlenk reaction tube, OH-ALK (36 mg, 0.05 mmol), Ir-N3 (30 mg, 0.04 mmol), Cul (1 mg) and sodium ascorbate (1 mg) were added to 3 mL of DMF and stirred at room temperature for 24 h under nitrogen atmosphere in the dark; then the reaction solvent was removed on a rotary evaporator; subsequently, the obtained crude mixture was purified by column chromatography on silica gel (dichloromethane / methanol as eluent, v / v = 93:7) to obtain red powder Ir-OH (55 mg, 74.8% yield). Characterized by nuclear magnetic resonance, mass spectrometry, 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.55 (d, J = 4.6 Hz, 1H), 8.96 (s, 1H), 8.85 (s, 1H), 8.76 (s, 1H), 8.61 (s, 1H), 8.54 (dd, J = 7.6, 3.1 Hz, 1H), 8.25 (t, J = 7.2 Hz, 2H), 8.12-7.83 (m, 12H), 7.70 (d, J = 5.5 Hz, 1H), 7.64 (t, J = 6.5 Hz, 2H), 7.57-7.53 (m, 1H), 7.49-7.35 (m, 5H), 7.24-6.98 (m, 18H), 6.92-6.77 (m, 4H), 6.17 (dd, J = 7.6, 4.4 Hz, 2H), 5.94 (s, 2H), 2.55 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ (ppm): 157.56, 157.11, 156.30, 155.04, 152.06, 150.85, 144.20, 143.64, 141.52, 140.29, 140.24, 139.21, 131.67, 131.50, 131.25, 130.68, 129.15, 128.51, 127.93, 127.08, 126.51, 125.52, 124.35, 122.70, 120.49, 116.15, 89.52, 21.45. ESI-MS (positive mode, m / z) found (calcd) for [M-Cl] + : 1435.5 (1435.41).

[0049]

[0050] The complex Ir-PPh3 prepared in Example 1 was used to detect the production of reactive oxygen species in vitro by the change in fluorescence of the reactive oxygen species indicator:

[0051] The ROS production of Ir-PPh3 under blue light (450 nm, 20 mW cm -2 ) irradiation was detected by using the common ROS indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in PBS buffer containing 1% DMSO. 0.5 mL of DCFH-DA ethanol solution (1 x 10 -3 M) was added to 2 mL of 1 x 10 -2 M NaOH and stirred at room temperature for 30 min. The hydrolysis product was then neutralized with 10 mL of 1 x PBS at Ph = 7.4 and stored in the dark before use. At this time, DCFH-DA was hydrolyzed to DCFH. The ROS indicator (4 x 10 -5 M) in PBS was further diluted to 1 x 10 -5 M in the Ir-PPh3 (1 μM) sample solution and measured by a fluorescence instrument. The fluorescence intensity of 2',7'-dichlorofluorescein triggered by ROS produced by Ir-PPh3 under blue light irradiation was detected at different time points. The PL spectrum was measured under 488 nm excitation and the emission was collected from 508 to 650 nm. The fluorescence intensity at 525 nm was recorded to represent the total ROS production rate. From the fluorescence emission spectrum of Ir-PPh3 containing DCFH ( Figure 1 ), it can be seen that the fluorescence emission of DCFH at 525 nm increased significantly with the increase of irradiation time, which indicated that DCFH was oxidized by the active oxygen produced by Ir-PPh3 under irradiation, and Ir-PPh3 had significant in vitro active oxygen production ability.

[0052] From the transmission electron microscopy image and dynamic light scattering image, it can be seen that Ir-PPh3 is in an aggregated state in PBS buffer containing 1% DMSO. Figure 2 (a) The TEM image shows that Ir-PPh3 can self-assemble into spherical nanoparticles, Figure 2 (b) The DLS measures the average particle size of the nanoparticles to be about 70 nm.

[0053] Cellular toxicity of complex Ir-PPh3 to different cells under (a) dark and (b) light:

[0054] A549, A549R, MCF-7, MCF-7ADR, MDA-MB-231 and HLF cells were obtained from the Experimental Animal Center of Sun Yat-Sen University. The cisplatin-sensitive cells A549, HLF and cisplatin- and doxorubicin-sensitive cells MCF-7 were routinely cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco BRL) containing 10% fetal bovine serum (FBS), 100 pg / mL streptomycin and 100 U / mL penicillin (Gibco BRL). The cisplatin-resistant A549R cells were cultured in RPMI 1640 medium containing 1 pg / mL cisplatin to maintain drug resistance, and the MDA-MB-231 cells were cultured in DMEM medium. The MCF-7ADR cells were cultured in RPMI 1640 medium containing 1 pg / mL doxorubicin to maintain drug resistance. The cells were cultured in a humidified incubator (BB 150 CO2incubator, Thermo Fisher Scientific, USA) at 37 °C with 5% CO2.

[0055] MTT assay was used to detect the toxicity of Ir-PPh3 to the above cancer cells under normoxic conditions. Cells of appropriate density (about 10,000 cells / well) were pre-incubated in the corresponding medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 96-well plate. Then fresh medium containing the required concentration of Ir-PPh3 was added. After 4 h of cell culture, phototoxicity experiments were performed by irradiating the cells with a laser at 450 nm (20 mW cm -2 , 20 min). After 24 h of treatment, 20 pL of MTT solution (5 mg / mL) was added to each well, and incubation was continued for another 4 h. The medium was carefully removed, and 150 pL of DMSO was added to each well. The culture dish was incubated for 10 min with shaking. The absorbance at 570 nm was measured using a microplate reader (LabServ K3, Thermo Fisher Scientific, USA). The average value was obtained from three replicates. The IC 50 values quoted are the mean ± standard deviation. The highest concentration of DMSO was controlled at 1% (v / v) in all experiments.

[0056] Figure 3 (a) shows that Ir-PPh3 has no cytotoxicity to all tested cell lines in the dark, with an IC 50 > 50 pM, which indicates that Ir-PPh3 is safe for normal cells without radiation. Figure 3(b) shows that Ir-PPh3 can overcome the chemoresistance under irradiation, indicating that A549R and MDA-MB-231 resistant to cisplatin, and MCF-7ADR resistant to doxorubicin have higher photo-cytotoxicity. This indicates that Ir-PPh3 can effectively inhibit cell proliferation and achieve the purpose of avoiding cytotoxicity to normal cells.

[0057] A549R cells were cultured on confocal dishes, then treated with Ir-PPh3, and then fixed with 4% paraformaldehyde for 15 min, then permeabilized with 0.3% Triton X-100 (1% BSA) for 60 min. p53 antibody (1 / 200 dilution) can specifically recognize p53 protein after staining overnight at 4°C. The secondary antibody (fluorescein-labeled affinity-pure goat anti-mouse IgG (H+L), 1:80) was detected at room temperature for 1 h. The confocal dishes were washed 3 times in PBS, stained with DAPI (1 μg / mL) for 10 min, and images were acquired under a confocal microscope.

[0058] Cells treated as indicated were collected and treated with RIPA lysis buffer on ice for 30 min. The lysate (13400 rpm) was centrifuged for 20 min and the precipitate was treated. The protein concentration was determined by protein assay reagent, and equal amounts of protein were separated on SDS-PAGE gel and transferred to PVDF membrane (Millipore). The membrane was incubated with specific antibodies at 4°C overnight, then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washing with PBST, the signal was observed with Tanon High-sig ECL Western Blotting Substrate and Tanon 5200 Multi.

[0059] Figure 4 (a) Laser confocal microscopy (CLSM) observed that the stained p53 protein in A549R cells had obvious green fluorescence. The green fluorescence intensity in the A549R cells treated with Ir-PPh3 after irradiation decreased significantly, while under the same conditions, there was no obvious change except irradiation. The total p53 protein expression level in the irradiated Ir-PPh3-treated A549R cells decreased significantly, suggesting that p53 protein was degraded. Figure 4(b) The expression levels of Mutp53 and WTp53 in A549R cells were detected after irradiation, respectively. The expression of Mutp53 in A549R cells was significantly higher than that in A549 cells, while the expression of WTp53 in A549 cells was higher. This indicates that the drug resistance of A549R cells to cisplatin is highly related to the expression of Mutp53. After treatment with Ir-PPh3 irradiation, the level of Mutp53 was significantly down-regulated compared with A549R cells irradiated only. However, PPh3-ALK and Ir-OH did not show effective regulation ability on the level of Mutp53 in A549R cells, which indicates that the degradation of Mutp53 in A549R cells depends on the synergistic effect of PPh3 fragment and polypyridyl Ir(III) fragment. It must be mentioned that the expression of endogenous Mutp53 and WTp53 in A549 cells was not affected by the treatment, which indicates that Ir-PPh3 can selectively act on the chemotherapy-resistant cells with high expression level of Mutp53. Figure 4 (c) The expression level of Mutp53 in cisplatin-resistant MDA-MB-231 cells and doxorubicin-resistant MCF-7ADR cells is also higher compared with MCF-7 cells. After treating MDA-MB-231 and MCF-7ADR cells with Ir-PPh3 under light, dose-dependent degradation of Mutp53 was found, which further indicates that Ir-PPh3 has no preference for chemotherapy-resistant cells with high Mutp53 level.

[0060] A549R cells were seeded in 6-well plates for 24 h, then treated with 6 μM of Ir-OH and Ir-PPh3 for 4 h, then irradiated with 20 mW / cm2of blue light (450 nm) for 20 min. After that, 10 μM of ROS probe DCFH-DA was added to the cell samples, incubated for 30 min, then carefully washed with PBS. Then the cell samples were analyzed using BD FACS Calibur flow cytometer, with excitation wavelength of 488 nm and emission wavelength of 530 ± 20 nm. Data analysis was performed using FlowJo 7.6.1 software. 10,000 cells were collected for each sample. 2 A549R cells were seeded in 6-well plates for 24 h, then treated with 6 μM of Ir-OH and Ir-PPh3 for 4 h, then irradiated with 20 mW / cm

[0061] GSH and GSSG assay kit was used to detect the intracellular GSH level. First, the standard curve of GSSG level and GSH level was measured according to the instruction. The A549R cells after drug treatment were collected and homogenized. The total GSH level was carefully determined with glutathione reductase and 5,5-dithiobis(2-nitrobenzoic acid). The thiol group of GSH reacts with DTNB to generate yellow 5-thio-2-nitrobenzoic acid (TNB) with an absorbance of 405-414 nm. The value of TNB (A405) can be detected by the microplate reader, and the reduced GSH level is calculated by subtracting the GSSG level from the total GSH (GSH = total GSH - 2 x GSSG).

[0062] Figure 5 (a) Flow cytometry showed that intracellular ROS increased by 19 times after Ir-PPh3 treatment, Figure 5 (b) Excessive ROS level is closely related to the intracellular antioxidant glutathione (GSH) level, which can destroy the cellular redox homeostasis, induce oxidative stress, and oxidize GSH to oxidized glutathione (GSSG). After irradiation, the ratio of GSH / GSSG in Ir-PPh3 treated A549R cells decreased significantly, further indicating that the redox balance of A549R cells after treatment was destroyed.

[0063] ICP-MS detection of total copper content in A549R cells, A549R cells were inoculated and incubated overnight under standard growth conditions. Ir-PPh3 (6 μM) treated A549R cells. After 12 h of incubation, the cells were collected with trypsin, washed with PBS and counted. Then digested with concentrated nitric acid (100 μL) for 2 h, 30% hydrogen peroxide (50 μL) for 1.5 h, and concentrated hydrochloric acid (50 μL) for 1.5 h to obtain a completely homogenized solution. Finally, the solution was diluted to a final volume of 2 mL with MiliQ water for copper measurement.

[0064] Figure 6 (a) The intracellular copper transporter SLC31A1 was significantly up-regulated and highly dependent on ROS level. Once the ROS inhibitor NAC (antioxidant N-acetyl cysteine) was added, the level of SLC31A1 was significantly inhibited, indicating that photoactivated ROS might be the cause of the disruption of intracellular copper homeostasis. Figure 6 (b) ICP-MS results further showed that the copper content in irradiated Ir-PPh3 treated A549R cells increased by more than 6 times compared with only irradiated control A549R cells. Therefore, due to photoactivated ROS, the copper homeostasis in A549R cells after irradiation with Ir-PPh3 was disrupted, which resulted in the transport of exogenous copper into A549R cells.

[0065] Figure 7(a) glutathione peroxidase 4 (GPX4) as an antioxidant enzyme and a major regulator of ferroptosis, the expression level of GPX4 was significantly inhibited after light irradiation in the presence of Ir-PPh3. Figure 7 (b) (c) To illustrate the activation of ferroptosis, the expression level of GPX4 in the presence of inhibitors NAC and TTM was studied. The level of GPX4 in TTM-treated A549R cells was significantly up-regulated than that in NAC-treated A549R cells, which indicated that the high copper content and light-induced ROS in the cells after treatment would be the main reason for the activation of ferroptosis.

[0066] In order to further study the potential relationship between high copper content and ferroptosis and Mutp53 degradation, inhibitor experiments were carried out. Figure 8 (a) Mutp53 degradation was inhibited in the presence of NAC, which indicated that light-induced ROS would be attributed to Mutp53 degradation after irradiation. Figure 8 (b) (c) Similar phenomena were observed whether TTM or Fer-1 was added, since Mutp53 degradation after irradiation was inhibited, which indicated that high copper content mediated ferroptosis in the cells was the reason for the activation of Mutp53 degradation in A549R cells after Ir-PPh3 treatment under irradiation.

[0067] A549R cells were placed in 12-well plates for culture, and the monolayer cells were scraped manually with a 100 μL pipette tip, and washed with 1640 medium for 3 times. The wound images were collected, and then the cells were treated with PBS, Ir-OH or Ir-PPh3, and treated for 24 h with or without light irradiation. At this time, images were collected for measuring the migration of cells to the wound.

[0068] The 1640 medium with 10% FBS was added in the lower chamber. A549R cells (5 x 10 4 ) were added to the upper chamber using serum-free 1640 medium. After incubation with PBS, 6 μM Ir-OH or Ir-PPh3 for 24 h with or without light irradiation, the cells migrated to the lower surface of the membrane were fixed with methanol and stained with 0.5% crystal violet. After washing with PBS for 3 times, the microscope (Nikon, Ci-L) was imaged. Among them, after complex culture for 4 h, irradiation with a laser with a wavelength of 450 nm, 20 J cm -2 for 20 min.

[0069] A549R cells (500 / well) were seeded in 6-well plates overnight, treated with PBS, 0.3 μΜ Ir-OH or Ir-PPh3 for 24 h, then replaced with fresh 1640 medium containing 10% FBS. After another 10 d culture, the colonies were fixed with 4% paraformaldehyde for 20 min, washed with PBS for 3 times, stained with 0.5% crystal violet and imaged.

[0070] After irradiation, Mutp53 removal in Ir-PPh3 chemotherapy-resistant cells can effectively eliminate the GOF phenotype caused by Mutp53. (a) (b) Cisplatin-resistant A549R cells after co-incubation with Ir-PPh3 and cisplatin under irradiation, the cell viability is much lower than that of Ir-PPh3 or cisplatin alone treatment under irradiation, indicating that the cisplatin resistance of A549R cells is effectively reversed. Due to Mutp53 degradation after Ir-PPh3 irradiation, (b) (c) cell scratch test and (d) (e) Transwell test show that A549R cells are difficult to migrate after treatment with Ir-PPh3 under irradiation, (e) (f) colony test shows that A549R cell growth is inhibited and cannot form colonies after treatment with Ir-PPh3 under irradiation. In summary, Ir-PPh3 can effectively kill p53 mutant cells by eliminating the GOF caused by mutp53, including chemotherapy resistance, cell migration and colony formation.

[0071] In vivo anticancer activity studies were carried out on MDA-MB-231 tumor-bearing nude mouse models. Balb / c nude mice (6-8 weeks old, 18-20 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. 2x10 6 The method of MDA-MB-231 cells was used to establish a nude mouse subcutaneous tumor model. When the tumor grew to 80-100 mm 3 The mice were randomly divided into 5 groups (5 mice / group). Each group was injected with normal saline (dark), normal saline (light), Ir-PPh3 (dark), Ir-PPh3 (light), and Ir-PPh3 + cisplatin (light) complex (Ir-PPh3: 2.5 mg / kg -1 body weight, cisplatin: 5 mg / kg -1 body weight) every other day for a total of 12 days. The growth of subcutaneous tumors was observed, and the body weight of mice was recorded every 2 days. The tumor volume was measured and calculated according to the following formula: volume = width 2 x length x 0.5. After the end of the experiment, the mice were sacrificed, and the tumor tissue and heart, liver, lung, spleen, and kidney tissues were stained with hematoxylin-eosin (HE).

[0072] Immunohistochemical staining was performed on mouse tumor tissues, and paraffin sections were deparaffinated and rehydrated. The sections were stained with p53 antibody by immunohistochemical method. Cell nuclei were stained with hematoxylin; images were collected using the Pannoramic MIDI system.

[0073] After 12 days of treatment, the tumors of MDA-MB-231 tumor-bearing nude mice were surgically removed and photographed. In the normal saline treatment group, the invasive growth of the tumor was observed even under light. The Ir-PPh3 group also obtained similar vigorous growth in the dark, which was consistent with the in vitro cytotoxicity evaluation. The tumor growth rate of the irradiation combined with Ir-PPh3 treatment group was significantly inhibited, and the volume decreased from 1380 mm 3 to 480 mm 3 . After the addition of cisplatin, the tumor inhibition rate was further improved, and the tumor volume decreased to 350 mm 3 . This indicates that the combination of Ir-PPh3 can reverse the cisplatin resistance of MDA-MB-231 cells under irradiation.

[0074] The present application complex Ir-PPh3 has no obvious cytotoxicity to normal cells and cancer cells without irradiation. Ir-PPh3 shows high cytotoxicity under irradiation and can effectively degrade Mutp53 to overcome chemoresistance. Under irradiation, Ir-PPh3 can break the redox balance by generating a large amount of ROS, and then the intracellular copper content is significantly increased with the up-regulation of copper transporter SLC31A1, leading to the occurrence of copper-mediated ferroptosis, and then inducing ferroptosis-mediated Mutp53 degradation, Mutp53-mediated GOF is also reduced because it reduces the chemoresistance, inhibits the migration of cancer cells, and reduces the formation of tumor cell colonies and spheres.

Claims

1. A metal iridium complex for inducing the degradation of Mutp53, characterized in that, Its chemical structural formula is shown below:

2. The method for preparing the metallic iridium complex according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of triphenylphosphine-modified aggregation-induced emission ligands: Aggregation-induced emission molecules OH-ALK, (4-bromobutyl)-triphenylphosphine bromide and K2CO3 were dissolved in acetonitrile; the mixture was stirred and refluxed under an inert gas atmosphere; after the reaction was completed, the mixture was cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated under low pressure; subsequently, the solid product PPh3-ALK was obtained by column chromatography. (2) Synthesis of the iridium complex: PPh3-ALK, Ir-N3, CuI and sodium ascorbate were added to DMF and stirred in the dark under an inert gas atmosphere at room temperature; then the reaction solvent was removed on a rotary evaporator; subsequently, the iridium complex Ir-PPh3 was obtained by column chromatography.

3. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (1), OH-ALK is prepared using the following method, the specific steps of which are as follows: (1.1) The aggregation-induced emission molecules TPE, (4-hydroxyphenyl)boronic acid, potassium bicarbonate and Pd(PPh3)4 were dissolved in a mixed solvent of THF and water; the mixture was heated to reflux; after cooling to room temperature, the mixture was extracted with dichloromethane, and the organic phase was washed with water and brine and dried with MgSO4; after removing the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel with dichloromethane / hexane as the eluent to obtain the solid product TPE-1; (1.2) Compound TPE-1, compound 1, potassium carbonate and Pd(PPh3)4 were mixed and a mixed solvent of THF and H2O was added to the mixture under vacuum and oxygen-free conditions. The mixture was heated and refluxed overnight. After the reaction cooled, water was added and the mixture was extracted with dichloromethane. The organic compounds were then combined and dried with MgSO4. After removing the solvent under low pressure, the crude product was purified by column chromatography on silica gel using dichloromethane / hexane as the eluent to obtain the solid product OH-TPE-A. (1.3) OH-TPE-A and 4-ethynylphenylacetonitrile were dissolved in anhydrous methanol, and then piperidine was added. The mixture was heated under reflux and the solvent was removed under low pressure. The crude product was purified by column chromatography on silica gel using dichloromethane / petroleum ether as the eluent to obtain the solid product OH-ALK.

4. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (1), the molar ratio of OH-ALK to (4-bromobutyl)-triphenylphosphine bromide is 1 to 1.5:

2.

5. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (1), the reflux reaction temperature is 80-90℃ and the reflux reaction time is 36-48h.

6. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (1), the inert gas is argon.

7. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (2), the molar ratio of PPh3-ALK to Ir-N3 is 1:1 to 1.

2.

8. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (2), the reaction temperature is 25-30℃ and the stirring time is 20-24h.

9. The method for preparing the metallic iridium complex according to claim 2, characterized in that: In step (2), the inert gas is nitrogen.

10. The use of the iridium complex of claim 1 in the preparation of antitumor drugs or antitumor drug components.

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