Phosphate-containing compounds, iridium phosphate complexes, their preparation methods and applications

Abstract

This invention discloses a phosphate-containing compound, a phosphate-containing iridium complex, its preparation method, and its applications. The complex incorporates two phosphate groups through ligand modification, yielding a class of phosphate-containing cyclic metallized iridium complexes. These iridium complexes exhibit high water solubility and structural stability, and possess aggregation-induced emission properties. Their fluorescence emission is weak in the water-soluble state but significantly enhanced in the aggregated state. The introduction of phosphate groups gives these iridium complexes a high affinity for hydroxyapatite. This invention combines the fluorescence properties of iridium complexes with the affinity of phosphate groups for bone tissue, forming a product that can bind to the calcium matrix on the bone tissue surface, generating aggregation-induced emission, and can be used for fluorescent staining of bone tissue.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent materials technology, specifically relating to phosphate-containing compounds, phosphate-containing iridium complexes, their preparation methods, and applications. Background Technology

[0002] With the improvement of living standards, increased bone fragility and decreased bone quality caused by osteoporosis have attracted increasing attention. Due to factors such as aging and accidents, cases of bone injury are also increasing year by year. Bone microstructure and micro-trauma are important factors affecting bone quality. Accurate and rapid analysis of bone structure can help to quickly assess bone quality and health levels. Furthermore, in the field of criminal investigation, the analysis of bone tissue, especially trace bone tissue samples, is often necessary.

[0003] Currently used methods for bone tissue analysis include HE staining, Masson staining, Von Kossa staining, and Alizarin Red staining. These staining methods require a series of cumbersome steps, such as paraffin embedding, decalcification, and sectioning of the bone tissue. The staining effect is also affected by factors such as temperature, pH, and staining time, requiring operation by specially trained technicians, which is time-consuming and labor-intensive. Therefore, the development and advancement of detection technologies are essential, and the development of simple and rapid bone tissue analysis methods is of great significance.

[0004] Fluorescence detection methods offer advantages such as high sensitivity, good repeatability, and ease of operation, making fluorescence microscopy a simple, rapid, and sensitive method for observing bone microstructures. Currently, basic fuchsin is commonly used as a bone fluorescent dye; however, it lacks specificity, causing severe background interference, and its staining effect is unstable. Therefore, developing bone fluorescent dyes with bone-bonding specificity and staining stability is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide phosphate-containing compounds, iridium-containing phosphate complexes, preparation methods and applications, so as to solve the problems of unstable staining effect and background signal interference caused by bone fluorescent dyes in the prior art.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] Phosphate-containing compounds, with the following structural formula:

[0008]

[0009] The preparation method of the phosphate-containing compound is as follows: 2,2'-bipyridine-4,4'-dicarboxylic acid and N-hydroxythiosuccinimide are added to N,N-dimethylformamide, then N,N'-diisopropylcarbodiimide is added, the mixture is heated to reflux until it becomes a transparent solution, and then placed at low temperature; after filtering to remove the precipitated crystals, the filtrate is collected.

[0010] (3-Aminopropane)phosphoric acid and N,N-diisopropylethylamine were added to a solvent and placed in an ice-water bath to form a mixed solution;

[0011] The filtrate was added dropwise to the mixed solution to obtain a suspension. The suspension was stirred and reacted in an ice-water bath, and then extracted with dichloromethane to obtain a crude product. The crude product was then purified to obtain a phosphate-containing ligand.

[0012] Preferably, the mixing ratio of 2,2'-bipyridine-4,4'-dicarboxylic acid, N-hydroxythiosuccinimide, N,N-dimethylformamide and N,N'-diisopropylcarbodiimide is 2.01 mmol: 5 mmol: 10 mL: 0.65 mL; and the mixing molar ratio of (3-aminopropane)phosphoric acid and N,N-diisopropylethylamine is 1:2.

[0013] The phosphate-containing iridium complex has the following chemical structural formula:

[0014]

[0015] Among them, the cyclometalated C^N is an aromatic cyclometalated ligand.

[0016] Preferably, the aromatic cyclic metal ligand is 2-phenylpyridine or 2-phenylquinoline.

[0017]

[0018] A method for preparing iridium phosphate complexes involves dissolving the [Ir(C^N)2Cl]2 precursor and the phosphate-containing ligand described in claim 1 in a mixed solvent. After the reaction, the entire reaction system is filtered, and the solvent obtained by filtration is rotary evaporated to dryness to obtain a crude product. The crude product is then dissolved in water, and sodium bicarbonate is added to adjust the pH value until the crude product is completely dissolved. After purification, the iridium phosphate complex is obtained.

[0019] Preferably, the molar ratio of the [Ir(C^N)2Cl]2 precursor to the phosphate-containing ligand is 0.5:1.1.

[0020] Preferably, the preparation process of the [Ir(C^N)2Cl]2 precursor is as follows: iridium trichloride and C^N ligand are dissolved in a solvent, heated under reflux and reacted, then cooled to room temperature, the entire reaction system is filtered, the solid obtained by filtration is washed and dried to obtain the [Ir(C^N)2Cl]2 precursor.

[0021] Preferably, the mixing equivalent ratio of iridium trichloride and C^N ligand is 1:2, and the reaction temperature is 120℃.

[0022] Applications of iridium phosphate complexes for fluorescent staining analysis of bone tissue.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention discloses a phosphate-containing compound and its preparation method. The compound contains two phosphate groups and exhibits a high affinity for hydroxyapatite (hydroxyapatite microcrystals have OH sites on their surface that are at least momentarily vacant; due to the two positively charged Ca ions, an adsorption site is formed, which can adsorb PO4). 3- (Or phosphate groups on macromolecules), which can effectively target bone tissue. At the same time, this compound has a 2,2'-bipyridine ligand structure, which can chelate with a variety of metals to form metal complexes (including iridium complexes).

[0025] This invention discloses a phosphate-containing iridium complex and its preparation method. The complex incorporates two phosphate groups through ligand modification, resulting in a class of phosphate-containing cyclic metallized iridium complexes. These iridium complexes exhibit high water solubility and structural stability, and possess aggregation-induced emission properties. Fluorescence emission is weak in the water-soluble state but significantly enhanced in the aggregated state. The introduction of phosphate groups gives these iridium complexes a high affinity for hydroxyapatite, enabling bone targeting, and also provides a fluorescence-enhanced response mechanism. This invention combines the fluorescence properties of iridium complexes with the affinity of phosphate groups for bone tissue, forming a product that can bind to the calcium matrix on the bone tissue surface, generating aggregation-induced emission, which can be used for fluorescent staining of bone tissue.

[0026] This invention also discloses an application of an iridium phosphate complex. This type of iridium complex fluorescent dye can be used for fluorescent staining of bone tissue, allowing observation of the fine structure of the sample surface and detection of bone micro-damage while obtaining morphometric parameters of bone tissue. Combined with a wash-free fluorescent dye for bone tissue, the complex of this invention can be formulated as a spray dye for rapid staining analysis of bone tissue, eliminating the need for cumbersome pretreatment and post-treatment steps. This method is simple, fast, and provides clear observation. Attached Figure Description

[0027] Figure 1 The relative fluorescence spectra of the complex Ir-BP1 of the present invention in THF / H2O mixed solvents with different THF contents.

[0028] Figure 2 The relative fluorescence spectra of the complex Ir-BP1 of the present invention after binding with different contents of hydroxyapatite.

[0029] Figure 3 Fluorescence imaging of rat femur stained with the complex Ir-BP1 of the present invention.

[0030] Figure 4 The relative fluorescence spectra of the complex Ir-BP2 of the present invention in THF / H2O mixed solvents with different THF contents.

[0031] Figure 5 The relative fluorescence spectra of the complex Ir-BP2 of the present invention after binding with different contents of hydroxyapatite.

[0032] Figure 6 Fluorescence imaging of rat femur stained with the complex Ir-BP2 of the present invention. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to the accompanying drawings:

[0034] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. The terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Furthermore, unless otherwise explicitly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection or a detachable connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal connection of two elements. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0035] One embodiment of the present invention discloses a phosphate-containing compound, abbreviated as BPA, the general structural formula of which is shown in structural formula I:

[0036]

[0037] One embodiment of the present invention discloses a method for preparing a phosphate-containing compound. This embodiment utilizes NHS and DIC to activate the carboxyl group and form an amidation reaction with the amino group to generate the phosphate-containing compound. The specific steps of this preparation method are as follows:

[0038] (1) Prepare phosphate-containing ligands (abbreviated as BPA) as shown in formula (1).

[0039]

[0040] The procedure is as follows: 2,2'-bipyridine-4,4'-dicarboxylic acid (0.49 g, 2.01 mmol) and N-hydroxythiosuccinimide (NHS) (0.575 g, 5.0 mmol) were added to 10 mL of N,N-dimethylformamide (DMF), followed by 0.65 mL of N,N'-diisopropylcarbodiimide (DIC). The suspension was stirred and refluxed at 60 °C until a clear solution was obtained. The solution was then cooled to -20 °C and allowed to stand overnight. The precipitated crystals were removed by filtration, and the filtrate was collected. (3-Aminopropane)phosphoric acid (1.11 g, 8.0 mmol) and N,N-diisopropylethylamine (DIE A, 2.65 mL, 16.0 mmol) were added to a mixture of 10 mL of DMF and 4 mL of distilled water. The mixture was placed in an ice-water bath and cooled for ten minutes. The entire filtrate from the previous step was then slowly added dropwise to this mixture. The suspension was stirred overnight in an ice-water bath. After the reaction was complete, 20 mL of distilled water was added, and DMF was removed by extraction with dichloromethane. The crude product was purified by high-performance liquid chromatography (HPLC) using acetonitrile and water containing 0.05% trifluoroacetic acid as the mobile phase. A pink powder of ligand BPA was obtained, with a yield of 62.6%. The molecular structure of ligand BPA was confirmed by proton, carbon, and phosphorus nuclear magnetic resonance (NMR) spectra.

[0041] 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=4.9Hz,2H),8.77(s,2H),7.84(dd,J=4.9,1.9Hz,2H),3.34(t,J=6.9Hz,4H),1.81–1.69(m,4H),1.61–1.52(m,4H).

[0042] 13 C NMR (151MHz, DMSO-d6) δ164.69,155.66,150.21,143.00,122.02,118.38,40.26,26.00,25.08,23.06.

[0043] 31 PNMR (162MHz, DMSO-d6) δ 24.54.

[0044] One embodiment of the present invention discloses a phosphate-containing cyclic metallized iridium complex, the general structural formula of which is shown in structural formula II:

[0045]

[0046] The cyclic metallized C^N ligand is one of the structures in Formula III. The iridium complex described in this invention is not limited to the type of cyclic metallized C^N ligand; any C^N ligand having a structure similar to any of the aromatic C^N cyclic metallized ligands in Formula III can achieve the purpose of this invention. Specifically, the aromatic C^N cyclic metallized ligand with a similar structure is 2-phenylpyridine (abbreviated as ppy) or 2-phenylquinoline (abbreviated as pq).

[0047]

[0048] One embodiment of the present invention discloses a method for preparing the above-mentioned phosphate-containing cyclic metallized iridium complex, the method comprising the following steps:

[0049] (2) Preparation of [Ir(C^N)2Cl]2 precursor: Iridium trichloride and C^N ligand were dissolved in a mixed solution of ethylene glycol ether / distilled water (volume ratio 3:1) at a 1:2 equivalent ratio. The solution was then heated under reflux at 120°C for 24 hours under argon protection. After the reaction was completed, the solution was cooled to room temperature, filtered, and the solid was washed with water, ethanol, and acetone. The solid was then dried to obtain the corresponding [Ir(C^N)2Cl]2 precursor product.

[0050] (3) Prepare the phosphate-containing cyclic metallized iridium complex [Ir(C^N)2(BPA)]Cl, as shown in formula (2).

[0051]

[0052] The procedure is as follows: The [Ir(C^N)2Cl]2 precursor (0.5 mmol) and ligand BPA (1.1 mmol) were dissolved in a 20 mL mixture of methanol and dichloromethane (1:1 v / v) and refluxed at 50 °C in the dark for 8 hours. After the reactants became a clear solution, the reaction was stopped, and the solvent was rotary evaporated to dryness. The crude product was dissolved completely in 5 mL of distilled water with 1 mol / L sodium bicarbonate solution to adjust the pH to 8. Purification was performed using high-performance liquid chromatography (HPLC) with a mobile phase of methanol and water free of trifluoroacetic acid. This process uses hydrated iridium trichloride and C^N as initial reactants to obtain a chloro-bridged dimer of iridium, yielding the phosphate-containing cyclic metallized iridium complex [Ir(C^N)2(BPA)]Cl.

[0053] The technical solutions and application examples related to Na[Ir(pq)2(BPA)] and Na[Ir(ppy)2(BPA)] in the embodiments of the present invention will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0054] Example 1: Preparation of iridium complex Na[Ir(pq)2(BPA)] (abbreviated as Ir-BP1)

[0055] Ir-Cl3·3H2O (496 mg, 1.5 mmol) and 2-phenylquinoline (abbreviated as pq) (615 mg, 3 mmol) were dissolved in a mixture of ethylene glycol diethyl ether / distilled water (3:1, v / v), and then heated under reflux at 120 °C for 24 hours under argon protection. After the reaction was completed, the solution was cooled to room temperature, filtered, and the solid was dried to give 84 mg of the precursor [Ir(pq)2Cl]2 red powder, with a yield of 58%. The structure of this precursor is shown in structural formula IV.

[0056]

[0057] The [Ir(pq)₂Cl]₂ precursor (64 mg, 0.5 mmol) and ligand BPA (53 mg, 1.1 mmol) were dissolved in a 20 mL mixture of methanol and dichloromethane (1:1 v / v) and refluxed overnight at 50 °C in the dark. After the reaction was stopped, insoluble matter was removed by filtration, and the solvent was evaporated to dryness. The crude product was dissolved completely in 5 mL of distilled water and the pH was adjusted to 8 with 1 mol / L sodium bicarbonate solution. Purification was performed by high performance liquid chromatography (HPLC) using methanol and water free of trifluoroacetic acid as the mobile phase. 57 mg of an orange-yellow cyclometalated iridium complex, Ir-BP₁, was obtained, yielding 51%. The structure of the iridium complex Ir-BP₁ is shown in structural formula V. The molecular structure of the complex Ir-BP₁ was confirmed by proton, carbon, and phosphorus nuclear magnetic resonance (NMR) spectra.

[0058] 1 H NMR (400MHz, MeOD-d4) δ8.92 (s, 2H), 8.44–8.33 (m, 6H), 8.20 (d, J = 6.6Hz, 2H), 7. 98–7.91(m,2H),7.82(dd,J=8.2,1.6Hz,2H),7.41–7.32(m,4H),7.22–7.16(m,2H ),7.06(ddd,J=8.5,6.9,1.5Hz,2H),6.83(td,J=7.3,1.2Hz,2H),6.52(dd,J=7.8 ,1.2Hz,2H),3.49–3.39(m,4H),1.98–1.81(m,4H),1.63(dt,J=17.6,7.1Hz,4H).

[0059] 13C NMR(151MHz,MeOD-d4)δ171.39,165.07,157.28,151.47,149.55,148.45,146.99,145.81,141.41,135.43,132.1 4,131.70,130.29,129.09,128.39,127.89,127.33,125.73,124.19,123.14,118.95,42.43,28.06,27.18,24.06.

[0060] 31 P NMR (162MHz, MeOD-d4) δ 25.83.

[0061]

[0062] Performance verification:

[0063] (1) Aggregation-induced emission properties of iridium complex Ir-BP1

[0064] The iridium complex Ir-BP1 exhibits excellent water solubility. 1.1 mg of Ir-BP1 was weighed and dissolved in 1 mL of distilled water to obtain a 1 mmol / L mother liquor. The mother liquor was diluted with water and tetrahydrofuran (THF) to obtain Ir-BP1 solutions containing 0%, 50%, 80%, 90%, 95%, and 98% THF, respectively, at a concentration of 20 μmol / L. The fluorescence spectra of these solutions were measured under excitation at 385 nm. The results are as follows. Figure 1 As shown, the iridium complex Ir-BP1 exhibits obvious aggregation-induced emission characteristics. The fluorescence of Ir-BP1 gradually increases with increasing THF content. When the THF content is 98%, the luminescence intensity of the iridium complex Ir-BP1 in the THF / H2O mixture is 74.7 times higher than that in the H2O solution, with a maximum emission peak at 590 nm.

[0065] (2) Fluorescence response of iridium complex Ir-BP1 to hydroxyapatite

[0066] The mother liquor of the iridium complex Ir-BP1 was diluted with distilled water to obtain a 20 μmol / L solution (3 mL). 0, 20, 40, 60, 80, 100, and 120 μg of hydroxyapatite (HAP) were added respectively, and the mixtures were thoroughly mixed. The mixtures were allowed to stand for 10 minutes, then mixed again until homogeneous. The fluorescence spectra of these solutions under 385 nm excitation were immediately measured. The results are as follows: Figure 2 As shown, the fluorescence of Ir-BP1 significantly increased with increasing HAP content. When the amount of HAP was 120 μg, the luminescence intensity of the iridium complex Ir-BP1 increased by approximately 9.5 times.

[0067] (3) Fluorescence imaging of bone tissue by iridium complex Ir-BP1

[0068] A 20 μmol / L solution of the iridium complex Ir-BP1 was prepared using physiological saline and placed in a spray bottle to make a spray dye. Fresh rat femurs were taken, washed with physiological saline, and then sprayed with the prepared iridium complex Ir-BP1. After standing for 10 minutes, no further washing was required. The samples were then observed under a confocal fluorescence microscope with an excitation wavelength of 405 nm, collecting fluorescence signals in the 600-650 nm range. The results are as follows. Figure 3 The display shows that, in addition to observing and measuring the structure of bone fossae, it can also observe minute micro-damage. Under a 10x objective (100x magnification), obvious linear micro-damage and diffuse micro-damage composed of multiple clusters of extremely small bone cell cracks can be clearly observed on the bone tissue surface, both occurring in independent areas. Under a 40x objective (400x magnification), the diameter of the linear micro-damage is approximately 2 μm, which is clearly impossible to observe using current conventional staining methods.

[0069] Example 2: Preparation of iridium complex Na[Ir(ppy)2(BPA)] (abbreviated as Ir-BP2)

[0070] In this embodiment, Ir-Cl3·3H2O (496 mg, 1.5 mmol) and 2-phenylpyridine (abbreviated as ppy) (462 mg, 3 mmol) were dissolved in a mixed solution of ethylene glycol ether / distilled water (3:1, v / v), and then heated under reflux at 120 °C for 24 hours under argon protection. After the reaction was completed, the solution was cooled to room temperature, filtered, and the solid was dried to obtain a red powder of [Ir(ppy)2Cl]2, the structure of which is shown in structural formula VI.

[0071]

[0072] The [Ir(ppy)₂Cl]₂ precursor (53 mg, 0.5 mmol) and ligand BPA (53 mg, 1.1 mmol) were dissolved in a 20 mL mixture of methanol and dichloromethane (1:1 v / v) and refluxed overnight at 50 °C in the dark. After the reaction was stopped, insoluble matter was removed by filtration, and the solvent was evaporated to dryness. The crude product was dissolved completely in 5 mL of distilled water and the pH was adjusted to 8 with 1 mol / L sodium bicarbonate solution. Purification was performed by high performance liquid chromatography (HPLC) using methanol and water free of trifluoroacetic acid as the mobile phase. An orange-yellow cyclometalated iridium complex Ir-BP₂ powder was obtained, weighing 49.2 mg, with a yield of 51%. The structure of the iridium complex Ir-BP₂ is shown in structural formula VII. The molecular structure of the complex Ir-BP₂ was confirmed by proton, carbon, and phosphorus nuclear magnetic resonance (NMR) spectra.

[0073] 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=4.9Hz,2H),8.77(s,2H),7.84(dd,J=4.9,1.9Hz,2H),3.34(t,J=6.9Hz,4H),1.81–1.69(m,4H),1.61–1.52(m,4H).

[0074] 13 C NMR (151MHz, DMSO-d6) δ164.69,155.66,150.21,143.00,122.02,118.38,40.26,26.00,25.08,23.06.

[0075] 31 PNMR (162MHz, DMSO-d6) δ 24.54.

[0076]

[0077] Performance verification:

[0078] (1) Aggregation-induced emission properties of iridium complex Ir-BP2

[0079] Weigh 1.0 mg of the iridium complex Ir-BP2 and dissolve it in 1 mL of distilled water to obtain a 1 mmol / L mother liquor. Dilute the mother liquor with water and tetrahydrofuran (THF) to obtain Ir-BP2 solutions containing 0%, 50%, 80%, 90%, 95%, and 98% tetrahydrofuran, respectively, with a concentration of 20 μmol / L. Measure the fluorescence spectra of the above solutions under excitation at 385 nm. The results are as follows. Figure 4 As shown, the iridium complex Ir-BP1 exhibits obvious aggregation-induced emission characteristics. With increasing THF content, the fluorescence of Ir-BP2 gradually increases. When the THF content is 98%, the luminescence intensity of the iridium complex Ir-BP2 in the THF / H2O mixture is 56 times higher than that in the H2O solution, with a maximum emission peak at 602 nm.

[0080] (2) Fluorescence response of iridium complex Ir-BP2 to hydroxyapatite

[0081] The mother liquor of the iridium complex Ir-BP2 was diluted with distilled water to obtain a 20 μmol / L solution (3 mL). 0, 20, 40, 60, 80, 100, and 120 μg of hydroxyapatite (HAP) were added respectively, and the mixtures were thoroughly mixed. The mixtures were allowed to stand for 10 minutes, then mixed again until homogeneous. The fluorescence spectra of these solutions under 385 nm excitation were immediately measured. The results are as follows: Figure 5As shown, the fluorescence of Ir-BP2 significantly increased with increasing HAP content. When the HAP content was 120 μg, the luminescence intensity of the iridium complex Ir-BP2 increased by approximately 2.8 times. The maximum emission wavelength redshifted from 605 nm to 625 nm.

[0082] (3) Fluorescence imaging of bone tissue by iridium complex Ir-BP2

[0083] A 20 μmol / L solution of the iridium complex Ir-BP2 was prepared using physiological saline and placed in a spray bottle to make a spray dye. Fresh rat femurs were taken, washed with physiological saline, and then sprayed with the prepared iridium complex Ir-BP2. After standing for 10 minutes, no further washing was required. The samples were then observed under a confocal fluorescence microscope with an excitation wavelength of 405 nm, collecting fluorescence signals in the 600-650 nm range. The results are as follows. Figure 6 The bone tissue surface structure can be clearly observed under a 10x objective lens (100x magnification).

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An iridium complex containing phosphate groups, characterized in that, The chemical structural formula is: (II) The ring metallization C^N is an aromatic ring metallization ligand; The aromatic ring metallization ligand is 2-phenylpyridine or 2-phenylquinoline: (III).

2. A process for the preparation of the phosphato iridium complex of claim 1, characterized in that, The [Ir(C^N)2Cl]2 precursor and the phosphate-containing ligand in claim 1 are dissolved in a mixed solvent, after reaction, the whole reaction system is filtered, the solvent obtained by filtration is rotary evaporated to dryness, and the crude product is obtained; the crude product is dissolved in water, sodium bicarbonate is added to adjust the pH value to complete dissolution of the crude product, and the phosphate-containing iridium complex is obtained after purification. The preparation process of the [Ir(C^N)2Cl]2 precursor is as follows: iridium trichloride and C^N ligand are dissolved in a solvent, heated to reflux, cooled to room temperature, the whole reaction system is filtered, the solid obtained by filtration is washed and dried, and the [Ir(C^N)2Cl]2 precursor is obtained.

3. The method of preparing a phosphato iridium complex according to claim 2, characterized in that, The mixing molar ratio of the [Ir(C^N)2Cl]2 precursor and the phosphate-containing ligand is 0.5:1.

1.

4. The method of preparing a phosphato iridium complex according to claim 2, wherein The mixing equivalent ratio of iridium trichloride and C^N ligand is 1:2, and the reaction temperature is 120 DEG C.

5. Use of the phosphato iridium complex as claimed in claim 1, characterized in that, The fluorescent staining analysis for bone tissue does not involve the diagnosis or treatment of diseases.

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