A metal iridium complex-nitroimidazole conjugate, its preparation method and application

Abstract

The present invention provides a metal iridium complex-nitroimidazole conjugate, a preparation method thereof and an application thereof, belonging to the technical field of compounds. The metal iridium complex-nitroimidazole conjugate of the present invention has a structural formula as shown in Formula 1. The present invention also provides a preparation method of the above-mentioned metal iridium complex-nitroimidazole conjugate. The present invention also provides an application of the above-mentioned metal iridium complex-nitroimidazole conjugate Ir-TPA-NI as a photosensitizer drug molecule in the preparation of drugs for treating breast cancer. For the Ir-TPA-NI of the present invention, 2-nitroimidazole is introduced as a motif for realizing RDH, and the metal iridium complex is used as a photodynamic therapy motif, realizing a photodynamic therapy effect enhanced by RDH.

Description

Technical Field

[0001] The present invention belongs to the technical field of compounds, and particularly relates to a metal iridium complex-nitroimidazole conjugate, a preparation method thereof and an application thereof. Background Art

[0002] The tumor microenvironment (TME) usually has mild acidity, hypoxia, elevated cytoplasmic glutathione concentration and high levels of intracellular ROS, and the two are in a highly dynamic balance. In cancer cells, ROS, as an important oxidative species, its level remains balanced between production and elimination. Glutathione (GSH) is the main reductant for scavenging intracellular ROS, and its presence significantly reduces the intracellular ROS level, thereby weakening ROS-mediated tumor therapy. Redox homeostasis, as an inherent cellular defense mechanism, not only contributes to the malignant transformation and metastasis of tumors, but also severely limits ROS-mediated tumor therapy. Redox homeostasis imbalance (RDH), as a novel cancer treatment strategy, can make cancer cells more sensitive to different ROS-mediated tumor treatment modes by disrupting the balance between intracellular oxidative and reductive species, thereby effectively improving the treatment effect. By regulating the level of the reductant GSH, the redox homeostasis can be disrupted, thereby achieving the purpose of tumor treatment. 2-Nitroimidazole, as an electrophilic ligand, can disrupt the intracellular redox homeostasis by consuming GSH in the hypoxic environment of cancer cells, thereby reducing the consumption of ROS by GSH and promoting the accumulation of ROS, and finally effectively enhancing the effect of ROS-mediated tumor therapy. However, at present, most 2-nitroimidazoles enter cells by encapsulation, and there are few reports on the design of 2-nitroimidazole and other drug molecules into single molecule drugs.

[0003] Photodynamic therapy (PDT) can selectively kill primary and recurrent tumors with less damage to normal tissues by using the targeting of light source irradiation, and has been successfully regarded as a new method for clinical tumor treatment. PDT mainly consists of three important parts: light of a specific wavelength, a photosensitizer and oxygen. The three interact with each other to generate cytotoxic reactive oxygen species, which kill cancer cells through apoptosis or necrosis mechanisms. Traditional photosensitizers basically generate singlet oxygen through the type II pathway. However, the hypoxia phenomenon, which is a significant feature of solid tumors, severely limits the effective implementation of the PDT process. In addition, hypoxic tumors are highly resistant to various ROS-related treatment methods. Therefore, there is an urgent need for novel hypoxic highly active molecules to improve the sensitivity of hypoxic tumors to ROS treatment. A large amount of experimental evidence shows that disrupting redox homeostasis is an effective means to make tumor cells more vulnerable to ROS damage induced by exogenous substances. Therefore, effectively using the redox imbalance (RDH) strategy to further enhance the ROS killing ability in the PDT process is of great research significance for improving the treatment effect of tumors.

[0004] Iridium metal complexes have become a current research hotspot due to their relatively long excited state lifetimes, high photoluminescence efficiencies, large Stokes shifts, tunable emission ranges, and strong intersystem crossing (ISC) capabilities, and have become potential highly efficient photosensitizers (PSs) with clinical application value, which are applied in the PDT process. SUMMARY OF THE INVENTION

[0005] The present invention provides an iridium metal complex-nitroimidazole conjugate, a preparation method thereof, and an application thereof. The iridium metal complex-nitroimidazole conjugate Ir-TPA-NI of the present invention uses 2-nitroimidazole as the element for realizing RDH and the iridium metal complex as the photodynamic therapy element, thereby realizing the enhanced photodynamic therapy effect of RDH.

[0006] The present invention first provides an iridium metal complex-nitroimidazole conjugate, and its structural formula is shown in Formula 1:

[0007]

[0008] The present invention also provides a preparation method of an iridium metal complex-nitroimidazole conjugate, including the following steps:

[0009] Step 1: Using cesium carbonate as a catalyst, reacting 2-nitroimidazole with N-Boc-3-aminopropyl bromide to obtain NI-Boc;

[0010] Step 2: Under nitrogen protection, dissolving the NI-Boc obtained in Step 1 in a solvent, and then adding a mixture of trifluoroacetic acid and dichloromethane to react to obtain NI-NH2;

[0011] Step 3: Under nitrogen protection, using tetrakis(triphenylphosphine)palladium as a catalyst, heating 4-boronic acid triphenylamine and 1-chloroisoquinoline under reflux to prepare the cyclometalated ligand TPA;

[0012] Step 4: Under nitrogen protection, heating IrCl3·3H2O and the TPA obtained in Step 3 under reflux to obtain [Ir(TPA)2Cl]2;

[0013] Step 5: Under nitrogen protection, placing [Ir(TPA)2Cl]2 in Step 4 and the 2,2'-bipyridine-4,4'-dicarboxylic acid ligand under the action of a solvent in the dark and carrying out a reflux reaction. After the reaction is completed, adding potassium hexafluorophosphate and continuing to stir to obtain Ir-TPA-COOH;

[0014] Step 6: Under nitrogen protection, first mix the Ir-TPA-COOH obtained in Step 5 with the condensing agent 1-hydroxybenzotriazole, triethylamine, and the reaction solvent, then add NI-NH2 obtained in Step 2, and finally add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for reaction to obtain the metal iridium complex-nitroimidazole conjugate Ir-TPA-NI.

[0015] Preferably, the reaction temperature in Step 3 is 110°C - 120°C, and the reaction time is 36 - 48 h.

[0016] Preferably, the molar ratio of 4-borotriphenylamine, 1-chloroisoquinoline, and tetrakis(triphenylphosphine)palladium in Step 3 is: 1.1:1:(0.045 - 0.05).

[0017] Preferably, the reaction temperature in Step 4 is 110°C - 120°C, and the reaction time is 24 - 30 h.

[0018] Preferably, the molar ratio of IrCl3·3H2O and the TPA ligand in Step 4 is 1:(2 - 3).

[0019] Preferably, the molar ratio of [Ir(TPA)2Cl]2, 2,2'-bipyridine-4,4'-dicarboxylic acid ligand, and potassium hexafluorophosphate in Step 5 is 1:2:(10 - 15).

[0020] Preferably, the reaction temperature in Step 6 is room temperature, and the reaction time is 24 - 48 h.

[0021] Preferably, the molar ratio of Ir-TPA-COOH, NI-NH2, 1-hydroxybenzotriazole, triethylamine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Step 6 is 1:(2 - 3):5.2:(2 - 3):(2 - 3).

[0022] The present invention also provides the application of the above metal iridium complex-nitroimidazole conjugate Ir-TPA-NI as a photosensitizer drug molecule in the preparation of drugs for treating breast cancer.

[0023] Advantages of the present invention

[0024] The present invention provides a metal iridium complex-nitroimidazole conjugate, a preparation method and an application thereof. The conjugate introduces 2-nitroimidazole onto the auxiliary ligand of the metal iridium complex to synthesize a conjugate of the metal iridium complex and 2-nitroimidazole. The conjugate introduces a 2-nitroimidazole derivative that can consume GSH under hypoxic conditions and a photosensitizing metal iridium complex into the drug molecule, realizing an excellent aggregation-induced emission (AIE) effect, effectively overcoming the aggregation-caused quenching (ACQ) effect of traditional photosensitizers, achieving efficient delivery and release in cells, being able to efficiently consume GSH in 4T1 cells for Ir-TPA-NI, having good biocompatibility and high phototoxicity, and realizing the PDT treatment effect enhanced based on RDH. Description of the Drawings

[0025] Figure 1 It is the ultraviolet absorption spectrogram of Ir-TPA-NI prepared in Example 1 of the present invention in solution;

[0026] Figure 2 It is the photoluminescence spectrogram of Ir-TPA-NI prepared in Example 1 of the present invention in solution;

[0027] Figure 3 It is the AIE spectrogram of Ir-TPA-NI prepared in Example 1 of the present invention in solution;

[0028] Figure 4 It is the ultraviolet absorption spectrogram and the time-dependent generation of 1 the kinetic curve graph of O2 generation of Ir-TPA-NI prepared in Example 1 of the present invention under illumination and ICG conditions;

[0029] Figure 5 It is the cell survival rate graph of Ir-TPA-NI prepared in Example 1 of the present invention after being cultured in 4T1 cells for 24 h;

[0030] Figure 6 It is the live / dead cell staining graph of Ir-TPA-NI prepared in Example 1 of the present invention after being cultured in 4T1 cells for 24 h;

[0031] Figure 7 It is the relative GSH graph of Ir-TPA-NI prepared in Example 1 of the present invention after being cultured in 4T1 cells for 24 h;

[0032] Figure 8 It is the ROS graph of Ir-TPA-NI prepared in Example 1 of the present invention after being cultured in 4T1 cells for 24 h;

[0033] Figure 9 It is the endocytosis graph of Ir-TPA-NI prepared in Example 1 of the present invention after being cultured in 4T1 cells for 24 h;

[0034] Figure 10 1H NMR spectrum of Ir-TPA-NI prepared in Example 1 of the present invention;

[0035] Figure 11 1H mass spectrum of Ir-TPA-NI prepared in Example 1 of the present invention. Detailed implementation manners

[0036] The present invention first provides a metal iridium complex-nitroimidazole conjugate, with the molecular formula C 136 H 126 IrN6O 30 , with a relative molecular mass of 2515.81 g / mol, and its structural formula is shown in Formula 1:

[0037]

[0038] The present invention also provides a preparation method of a metal iridium complex-nitroimidazole conjugate, including the following steps:

[0039] Step 1: Using cesium carbonate as a catalyst, reacting 2-nitroimidazole with N-Boc-3-aminopropyl bromide to obtain NI-Boc;

[0040] Step 2: Under nitrogen protection, dissolving the NI-Boc obtained in Step 1 in a solvent, then adding a mixture of trifluoroacetic acid and dichloromethane to react to obtain NI-NH2;

[0041] Step 3: Under nitrogen protection, using tetrakis(triphenylphosphine)palladium as a catalyst, heating 4-boronic acid triphenylamine and 1-chloroisoquinoline to reflux to prepare the cyclometalated ligand TPA;

[0042] Step 4: Under nitrogen protection, heating IrCl3·3H2O and the TPA obtained in Step 3 to reflux to obtain [Ir(TPA)2Cl]2;

[0043] Step 5: Under nitrogen protection, placing [Ir(TPA)2Cl]2 from Step 4 and the 2,2'-bipyridine-4,4'-dicarboxylic acid ligand under the action of a solvent in the dark and refluxing. After the reaction, adding potassium hexafluorophosphate and continuing to stir to obtain Ir-TPA-COOH;

[0044] Step 6: Under nitrogen protection, first mixing the Ir-TPA-COOH obtained in Step 5 with the condensing agent 1-hydroxybenzotriazole, triethylamine, and the reaction solvent, then adding the NI-NH2 obtained in Step 2, and finally adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to react to obtain the metal iridium complex-nitroimidazole conjugate Ir-TPA-NI.

[0045] The specific reaction process is as follows:

[0046]

[0047] According to the present invention, 2-nitroimidazole and cesium carbonate are mixed and dissolved in a solvent, and N-Boc-3-aminopropyl bromide is added thereto for reaction. Preferably, stirring is carried out at room temperature conditions, and the reaction time is preferably 24-30 h. The reaction is stopped, and filtration treatment is carried out. The obtained solid is continuously washed with methanol. After purification by column chromatography, the obtained pale yellow solid is dried to obtain NI-Boc. The solvent is preferably ultra-dry N,N-dimethylformamide. The molar ratio of cesium carbonate, 2-nitroimidazole, and N-Boc-3-aminopropyl bromide is preferably: 1.5:1:1;

[0048] According to the present invention, the above-obtained NI-Boc is dissolved in a solvent. The solvent is preferably a mixture of dichloromethane, trifluoroacetic acid, and dichloromethane. In the mixture, the volume ratio of trifluoroacetic acid to dichloromethane is preferably 2:1. Under the condition of sufficiently filling inert gas N2, a reaction is carried out at room temperature. The reaction time is preferably 1-3 h. After the reaction is completed, the solvent is removed by rotary evaporation under reduced pressure to obtain a pale yellow oily liquid. After co-evaporation with methanol 3 times, a white solid is obtained, which is NI-NH2. The mass mg of NI-Boc: the total volume ml of trifluoroacetic acid and dichloromethane is preferably 284:6;

[0049] According to the present invention, 4-borotriphenylamine and 1-chloroisoquinoline are added to a reaction vessel, and then a solvent, a catalyst, and a sodium carbonate solution are added. The solvent is preferably toluene, and the catalyst is preferably tetrakis(triphenylphosphine)palladium. Under the condition of sufficiently filling inert gas N2, a reflux reaction is carried out. The reaction temperature is preferably 110-120 °C, and the time is preferably 36-48 h. After the reaction is completed, it is cooled to room temperature, and then extracted with dichloromethane and water. The organic layer is separated, and the organic phase is dried with anhydrous magnesium sulfate, filtered, and the organic phase is rotary evaporated to dryness. The crude product is purified by silica gel column chromatography to obtain a solid product, which is quinoline triphenylamine cyclometalated ligand TPA. The molar ratio of 4-borotriphenylamine, 1-chloroisoquinoline, and tetrakis(triphenylphosphine)palladium is preferably: 1.1:1:(0.045-0.05).

[0050] According to the present invention, IrCl3·3H2O and the above-mentioned quinoline triphenylamine cyclometalated ligand TPA are added to a reaction vessel containing a solvent and water. Under the atmosphere of N2 protection, the reaction is heated under reflux. The reaction temperature is preferably 110 - 120 °C, and the time is preferably 24 - 30 h. After the reaction is cooled to room temperature, a large amount of poor solvent water is added thereto to precipitate a solid and then filtered, and the solid is washed repeatedly with a large amount of solvents such as water and ethanol. The obtained solid is dried to obtain bis(μ-chloro)[iridium(III)bis(2-(diphenylamino)phenylquinoline)] ([Ir(TPA)2Cl2]). The solvent is preferably 2-ethoxyethanol. The molar ratio of IrCl3·3H2O to the quinoline triphenylamine cyclometalated ligand TPA is preferably 1:(2 - 3), more preferably 1:2.5.

[0051] According to the present invention, [Ir(TPA)2Cl]2 and 2,2'-bipyridine-4,4'-dicarboxylic acid ligand obtained above are added to a reaction vessel, and then a solvent is added. The solvent is preferably a mixture of anhydrous methanol and dichloromethane. In the mixture, the volume ratio of anhydrous methanol to dichloromethane is preferably 1:1. Under the atmosphere of inert gas N2, the reaction is refluxed in the dark. The reaction temperature is preferably 60 - 70 °C, and the time is preferably 6 - 8 h. When the reaction is completed and cooled to room temperature, potassium hexafluorophosphate solid is added to the solution in the flask, and stirring is continued at room temperature. The stirring time is preferably 40 - 60 min. After filtration, the solvent in the system is removed by a rotary evaporator, and purification is carried out by column chromatography to obtain a dark red solid, which is Ir-TPA-COOH. The molar ratio of [Ir(TPA)2Cl]2, 2,2'-bipyridine-4,4'-dicarboxylic acid ligand, and potassium hexafluorophosphate is preferably 1:2:(10 - 15).

[0052] According to the present invention, the obtained Ir-TPA-COOH is added to a reaction vessel, a reaction solvent is added thereto, and then the condensing agent 1-hydroxybenzotriazole and triethylamine are added. The reaction solvent is preferably ultra-dry N,N-dimethylformamide, then NI-NH2 is added, and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added. Under sufficient nitrogen protection, the reaction is preferably stirred at room temperature. The stirring time is preferably 24-48 h. After the reaction is completed, extraction is carried out with water and dichloromethane, the organic layer is separated, dried with anhydrous magnesium sulfate, filtered, the solvent is removed under reduced pressure, and purification is carried out by column chromatography to obtain a reddish-brown solid product Ir-TPA-NI. The molar ratio of Ir-TPA-COOH, NI-NH2, 1-hydroxybenzotriazole, triethylamine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is preferably 1:(2-3):5.2:(2-3):(2-3), and the molar ratio of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.

[0053] The present invention also provides the application of the above-mentioned metal iridium complex-nitroimidazole conjugate Ir-TPA-NI as a photosensitizer drug molecule in the preparation of drugs for treating breast cancer.

[0054] The present invention is further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0055] Example 1

[0056] 2-Nitroimidazole (1 g, 8.84 mmol) and cesium carbonate (4.31 g, 13.26 mmol) were mixed and dissolved in 5 mL of ultra-dry DMF. While stirring, N-Boc-3-aminopropyl bromide (2.1 g, 8.84 mmol) was added dropwise thereto, and the reaction was stirred at room temperature for 24 h. After the reaction was stopped, filtration was carried out, and the obtained solid was continuously washed with methanol. After the solvent was removed, the crude product was purified by silica gel column chromatography, and the obtained pale yellow solid was NI-Boc (1).

[0057] First, NI-Boc (284 mg, 1.05 mmol) was dissolved in 2 mL of dichloromethane, and purged with nitrogen twice. Under nitrogen protection, a mixed solution of 4 mL of trifluoroacetic acid and 2 mL of dichloromethane was added thereto, and the reaction was carried out at room temperature for 2 h. After the solvent was removed by rotary evaporation under reduced pressure, a pale yellow oily liquid was obtained. After co-evaporation with methanol three times, the obtained white solid was NI-NH2 (2).

[0058] 4 - (Triphenylamino)boronic acid (0.972 g, 3.36 mmol) and 1 - chloroisoquinoline (0.496 g, 3.06 mmol) were mixed and dissolved in 30 mL of toluene. Tetrakis(triphenylphosphine)palladium(0) (0.177 g, 0.15 mmol) was added as a catalyst. 20 mL of 2 mol / L sodium carbonate solution was added, and the reaction was carried out under nitrogen atmosphere and refluxed at 110 °C for 48 h. After the reaction was completed, the mixture was cooled and extracted with dichloromethane and water. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the organic phase was rotary evaporated. The crude product was purified by silica gel column chromatography, and the obtained pale yellow solid was TPA(3).

[0059] TPA (0.930 g, 2.5 mmol) and IrCl3·H2O (0.317 g, 1 mmol) were dissolved in a mixed solution of 30 mL of ethylene glycol monoethyl ether and 10 mL of water. The reaction was carried out under nitrogen protection and refluxed at 120 °C for 24 h. The reaction was stopped, and the mixture was allowed to stand and cooled to room temperature. 15 mL of water was added to the round - bottom flask, and the mixture was stirred for another 40 min. Filtration was carried out using a Buchner funnel. The obtained solid was placed in an oven for 24 h, and the obtained dark red solid was [Ir(pqy)2Cl2]2(4).

[0060] TPA dichloro - bridged (0.1490 g, 0.1 mmol)(4) and 2,2'-bipyridine - 4,4'-dicarboxylic acid ligand (0.2 mmol, 0.049 g) were added to a 100 - mL single - necked flask. 20 mL of anhydrous methanol and 20 mL of dichloromethane were mixed as the solvent for this reaction system. A mixed solution of 20 mL of methanol and 20 mL of dichloromethane was added thereto, and the reaction was refluxed for 6 h under nitrogen and light - protected conditions. After completion, the mixture was cooled to room temperature. 10 - fold amount of potassium hexafluorophosphate solid (1 mmol, 0.184 g) was added to the round - bottom flask, and the mixture was stirred at room temperature for 30 min. Filtration was carried out, and the filtrate was rotary evaporated. The crude product was purified by silica gel column chromatography, and the obtained brown - red solid was Ir - TPA - COOH(5).

[0061] Under N2 protection, first dissolve Ir-TPA-COOH (0.15 mmol, 0.177 g) in ultradry N,N-dimethylformamide as the solvent. Cool the solution to 0 °C in an ice-water bath, then add 1-hydroxybenzotriazole (0.78 mmol, 0.1044 g) and triethylamine (0.3 mmol, 46 μL) to it. Stir for 30 min at room temperature, and then continue to add NI-NH2 (0.3 mmol, 0.051 g) to the above reaction system. Stir for 30 min at room temperature, and slowly add a solution of EDCl (0.3 mmol, 0.063 g) in ultradry DMF dropwise to it. After returning to room temperature, continue the reaction for 48 h. After the reaction is completed, extract with dichloromethane and water, separate the organic layer, dry it with anhydrous magnesium sulfate, filter, and evaporate the solvent under reduced pressure. Purify the crude product by silica gel column chromatography to obtain a dark red solid, namely Ir-TPA-NI(6). The mass of the obtained powder is 0.0734 g, the yield is 33%, and the molecular formula is C 78 H 62 IrN 14 O6, with a relative molecular mass of 1483.46 g / mol. The 1H NMR spectrum of the metal iridium complex-nitroimidazole conjugate Ir-TPA-NI prepared in Example 1 is as shown in Figure 10 shown, and the mass spectrum is as shown in Figure 11 shown.

[0062] Example 2

[0063] The reaction conditions and steps are the same as those in Example 1, except that the reaction time in Step 5 is 8 h. The mass of the obtained Ir-TPA-NI(6) powder is 0.0668 g, the yield is 30%, and the molecular formula is C 78 H 62 IrN 14 O6, with a relative molecular mass of 1483.46 g / mol.

[0064] Example 3

[0065] The reaction conditions and steps are the same as those in Example 1, except that the reaction time in Step 5 is 8 h and the reaction temperature in Step 6 is 28 °C. The mass of the obtained Ir-TPA-NI(6) powder is 0.0779 g, the yield is 35%, and the molecular formula is C 78 H 62 IrN 14 O6, with a relative molecular mass of 1483.46 g / mol.

[0066] Example 4

[0067] The reaction conditions and steps are the same as those in Example 1, except that the reaction time in Step 5 is 8 h, the reaction temperature in Step 6 is 20 °C, and the reaction time is 40 h. The mass of the obtained Ir-TPA-NI(6) powder is 0.0623 g, and the yield is 28%. The molecular formula is C 78 H 62 IrN 14 O6, and the relative molecular mass is 1483.46 g / mol.

[0068] Example 5

[0069] The reaction conditions and steps are the same as those in Example 1, except that the reaction time in Step 5 is 8 h, the reaction temperature in Step 6 is 30 °C. The mass of the obtained Ir-TPA-NI(6) powder is 0.0801 g, and the yield is 36%. The molecular formula is C 78 H 62 IrN 14 O6, and the relative molecular mass is 1483.46 g / mol.

[0070] Example 6

[0071] The reaction conditions and steps are the same as those in Example 1, except that the reaction time in Step 5 is 8 h, the feed of triethylamine in Step 6 becomes triethylamine (0.4 mmol, 61 μL), and the reaction temperature is 28 °C. The mass of the obtained Ir-TPA-NI(6) powder is 0.0690 g, and the yield is 31%. The molecular formula is C 78 H 62 IrN 14 O6, and the relative molecular mass is 1483.46 g / mol.

[0072] The metal iridium complex-nitroimidazole conjugate prepared in Example 1 was characterized for its properties as follows:

[0073] 1. Photophysical properties of Ir-TPA-NI

[0074] In the present invention, the photophysical properties of Ir-TPA-NI were measured in DMSO:H2O (v:v = 1:99). Figure 1 is the ultraviolet absorption spectrum of Ir-TPA-NI of the present invention. Its ultraviolet absorption spectrum shows two typical absorption bands of metal iridium complexes. The strong ultraviolet absorption band is mainly attributed to the ligand spin-allowed π-π* transition, while the relatively weak absorption band originates from the metal-to-ligand charge transfer ( 3 MLCT). Figure 2 is the photoluminescence spectrum of Ir-TPA-NI of the present invention. As can be seen from the figure, Ir-TPA-NI shows bright red light emission in aqueous solution, and its emission peak position is 660 nm.

[0075] 2. AIE Performance of Ir-TPA-NI

[0076] Molecules with AIE performance hardly emit light under dilute solution conditions, but their luminescence significantly enhances under aggregated conditions. In this invention, the AIE property of Ir-TPA-NI was measured under DMSO:H2O (v:v = 1:99). Figure 3 This is the AIE spectrogram of Ir-TPA-NI of this invention. Ir-TPA-NI hardly emits any light in pure methanol. As the water content of the poor solvent increases, the luminescence of Ir-TPA-NI gradually enhances. When the water content in the mixed solution reaches 70%, Ir-TPA-NI emits bright red luminescence, indicating that Ir-TPA-NI has typical AIE characteristics.

[0077] 3. Singlet Oxygen Generation Ability of Ir-TPA-NI

[0078] Indocyanine green (ICG) was used as an indicator to evaluate the 1 O2 generation ability of the photosensitizer in DMSO / H2O = 1 / 99. After the photosensitizer was irradiated with light for a certain time, the absorbance of ICG at 790 nm decreased significantly, indicating that the photosensitizer sensitized the generation of 1 O2. Figure 4 This is the in vitro singlet oxygen generation experiment of Ir-TPA-NI of this invention, where Figure 4 a is the ultraviolet absorption spectrogram, Figure 4 b is the kinetic curve of the generation of 1 O2 over time; under light irradiation, when ICG and PSs coexist, the characteristic absorption peak of ICG at 790 nm continuously decreases, which also proves that PSs can effectively generate 1 O2 under light irradiation. Its ability to generate 1 O2 conforms to the first-order kinetic equation; the larger the slope, the stronger the singlet oxygen generation ability. The order of singlet oxygen generation efficiency from high to low is Ir-TPA-NI > methylene blue (MB). Taking methylene blue as a reference, Ir-TPA-NI has a singlet oxygen quantum yield of up to 90%, indicating that Ir-TPA-NI can efficiently generate singlet oxygen, and they will surely play an important role in the application of PDT for cancer as PSs. 1

[0079] 4. Cytotoxicity Experiment of Ir-TPA-NI

[0080] ​Double labeling of live and dead cells was achieved using a Calcein-AM / Propidium Iodide (PI) double staining kit. Calcein-AM, which has no fluorescence itself, is used to label live cells. After entering the cells, Calcein-AM is hydrolyzed by endogenous esterases in the cells to generate Calcein, which remains in the cells and emits a green fluorescence signal. PI can stain DNA and is used to label dead cells. PI can only penetrate the cell membrane of dead cells and cannot penetrate the cell membrane of live cells. PI reaches the cell nucleus and then embeds into double-stranded DNA to release a red fluorescence signal. Live and dead cells are distinguished according to the distribution of red and green fluorescence in cell imaging. Figure 5 This is the cell survival rate graph of the Ir-TPA-NI of the present invention after being cultured in mouse breast cancer (4T1) cells for 24 h; the MTT experiment of the Ir-TPA-NI of the present invention was used to study the potential toxicity of the photosensitizer to 4T1 cells. It can be seen from the figure that even when the concentration of the photosensitizer reaches a relatively high concentration of 100 μM, under dark conditions, after being cultured in cells for 24 hours, the survival rate of cells incubated with Ir-TPA-NI is as high as 85%. Under light conditions, as the concentration increases, the number of live cells gradually decreases, indicating that Ir-TPA-NI has concentration-dependent phototoxicity. The MTT experiment proved that Ir-TPA-NI has good phototoxicity and low dark toxicity as PSs.

[0081] 5. Live / Dead cell staining experiment of Ir-TPA-NI

[0082] Figure 6 This is the live / dead cell staining graph of the Ir-TPA-NI of the present invention after being cultured in mouse breast cancer (4T1) cells for 24 h. The live / dead cell staining imaging experiment of the Ir-TPA-NI of the present invention was used to visually observe the apoptotic state of 4T1 cells induced by Ir-TPA-NI. It can be seen from the figure that in the absence of Ir-TPA-NI, the 4T1 cells are in a healthy green fluorescence state after light irradiation. After light treatment of the cells added with Ir-TPA-NI, the cells show bright red fluorescence, indicating that the ROS generated by Ir-TPA-NI causes large-scale cell death, showing excellent phototoxicity. The experimental results of live / dead cell staining are consistent with the MTT results, indicating that Ir-TPA-NI can be used as a photosensitizer for PDT research.

[0083] 6. Intracellular GSH consumption experiment of Ir-TPA-NI

[0084] Figure 7This is a graph showing the intracellular GSH levels of Ir-TPA-NI of the present invention after culturing in mouse breast cancer (4T1) cells for 24 h. The effects of Ir-TPA-NI on the content of GSH in cells were detected using a GSH and GSSG detection kit. As can be seen from the graph, in 4T1 cells treated with Ir-TPA-NI, the relative GSH level showed a concentration-dependent decrease with the increase in the concentration of the photosensitizer. When the concentration of Ir-TPA-NI was 100 μM, the GSH level decreased to below 50%, indicating that Ir-TPA-NI has good ability to consume GSH in 4T1 cells, predicting that Ir-TPA-NI may be a promising photosensitizer for realizing RDH.

[0085] 7. Intracellular Reactive Oxygen Species Generation Experiment of Ir-TPA-NI

[0086] Figure 8 This is a graph showing the intracellular reactive oxygen species of Ir-TPA-NI of the present invention after culturing in mouse breast cancer (4T1) cells for 24 h. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to generate fluorescent DCF, and the ability of the photosensitizer to generate singlet oxygen in cells can be judged by observing the intensity of the green fluorescence in the cells. As can be seen from the graph, in the absence of the photosensitizer Ir-TPA-NI, there was no green fluorescence signal in the cells under light illumination, that is, no singlet oxygen was generated. After light illumination, obvious green fluorescence signals could be observed in 4T1 cells under a confocal microscope, proving that Ir-TPA-NI can effectively generate singlet oxygen under light illumination. It is predicted that Ir-TPA-NI can be used as a qualified PSs in photodynamic therapy.

[0087] 8. Intracellular Endocytosis Experiment of Ir-TPA-NI

[0088] Figure 9 This is a graph showing the endocytosis experiment of Ir-TPA-NI of the present invention in mouse breast cancer (4T1) cells. As can be seen from the graph, after incubating Ir-TPA-NI with 4T1 cells for 2 h, 6 h, and 12 h respectively, we characterized the uptake ability of PSs using CLSM. With the extension of the culture time, the red fluorescence of Ir-TPA-NI in the cells gradually increased in a time-dependent manner, indicating that 4T1 has a high uptake ability for the photosensitizer.

Claims

1. A metal iridium complex-nitroimidazole conjugate, characterized in that, Its structural formula is shown in Formula 1:

2. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 1, characterized in that, It includes the following steps: Step 1: Using cesium carbonate as a catalyst, react 2-nitroimidazole with N-Boc-3-aminopropyl bromide to obtain NI-Boc; Step 2: Under nitrogen protection, dissolve the NI-Boc obtained in Step 1 in a solvent, then add a mixture of trifluoroacetic acid and dichloromethane to react to obtain NI-NH2; Step 3: Under nitrogen protection, using tetrakis(triphenylphosphine)palladium as a catalyst, heat 4-borotriphenylamine and 1-chloroisoquinoline under reflux to prepare the cyclometalated ligand TPA; Step 4: Under nitrogen protection, heat IrCl3·3H2O and the TPA obtained in Step 3 under reflux to obtain [Ir(TPA)2Cl]2; Step 5: Under nitrogen protection, place [Ir(TPA)2Cl]2 from Step 4 and 2,2'-bipyridine-4,4'-dicarboxylic acid ligand under the action of a solvent in the dark and carry out a reflux reaction. After the reaction is completed, add potassium hexafluorophosphate and continue stirring to obtain Ir-TPA-COOH; Step 6: Under nitrogen protection, first mix the Ir-TPA-COOH obtained in Step 5 with the condensing agent 1-hydroxybenzotriazole, triethylamine and the reaction solvent, then add the NI-NH2 obtained in Step 2, and finally add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to react to obtain an iridium metal complex-nitroimidazole conjugate.

3. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, The reaction temperature in Step 3 is 110°C - 120°C, and the reaction time is 36 - 48 h.

4. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, In Step 3, the molar ratio of 4-borotriphenylamine, 1-chloroisoquinoline, and tetrakis(triphenylphosphine)palladium is: 1.1:1:(0.045 - 0.05).

5. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, The reaction temperature in Step 4 is 110°C - 120°C, and the reaction time is 24 - 30 h.

6. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, In Step 4, the molar ratio of IrCl3·3H2O and TPA ligand is 1:(2 - 3).

7. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, In Step 5, the molar ratio of [Ir(TPA)2Cl]2, 2,2'-bipyridine-4,4'-dicarboxylic acid ligand, and potassium hexafluorophosphate is 1:2:(10 - 15).

8. A method for preparing a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, The reaction temperature in Step 6 is room temperature, and the reaction time is 24 - 48 h.

9. The preparation method of a metal iridium complex-nitroimidazole conjugate according to claim 2, characterized in that, In Step 6, the molar ratio of Ir-TPA-COOH, NI-NH2, 1-hydroxybenzotriazole, triethylamine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(2 - 3):5.2:(2 - 3):(2 - 3).

10. Use of the iridium metal complex-nitroimidazole conjugate according to claim 1 in the preparation of a photosensitizer drug molecule for treating breast cancer.

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