CN109021030B - Novel phosphorescent ruthenium complex and preparation method and application thereof - Google Patents
Novel phosphorescent ruthenium complex and preparation method and application thereof Download PDFInfo
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- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0046—Ruthenium compounds
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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Abstract
The invention discloses a novel phosphorescent ruthenium complex and a preparation method and application thereof, the complex is composed of a polypyridyl ligand, a metal center and an auxiliary ligand containing a donor functional group, and the donor group in the complex has strong electron donating capability, low oxidation potential, strong reducibility and strong electron losing capability; the C ^ N ligand for electronic cyclometallization improves the dp (Ru) orbit energy level, the electron transfer capacity is obviously improved, the ruthenium complex synthesized by the invention has low biotoxicity, can be used for I-type photodynamic therapy in an hypoxic environment, improves the photodynamic therapy effect, and has important application prospect in the aspect of photodynamic therapy; and the compound has simple synthesis method and mild synthesis conditions, and is suitable for large-scale production and use.
Description
Technical Field
The invention belongs to the technical field of organic photoelectric and biological materials. In particular to a phosphorescent ruthenium complex with I-type photodynamic therapy effect and a preparation method and application thereof.
Background
Photodynamic therapy (PDT) is a safe and effective noninvasive treatment method developed in recent years, has small damage to tissues, no obvious and long-term adverse reaction, simple and convenient operation, economy and practicability, and is easily accepted by most patients.
During PDT chemical reactions, photosensitizers that absorb light of specific wavelengths may be excited from a ground state to a singlet state with an extremely short lifetime, which may return to the ground state by liberating energy via radiative fluorescence. Or through an intersystem transition to the triplet state. Since triplet excitons have a longer lifetime, they interact more strongly with surrounding molecules. Cytotoxic substances are produced during this stage of PDT. The triplet state of the photosensitizer can directly generate electron transfer with substrate molecules, namely I-type reaction, generate radical ions or radicals of the substrate and the sensitizer, and further react with ambient oxygen to finally form oxide; or through energy transfer with ground state oxygen molecules to generate singlet state oxygen, namely type II reaction. Singlet oxygen has high reactivity and is electrophilic, because it can efficiently oxidize intracellular substances such as biomolecules, proteins, unsaturated fatty acids, nucleic acids, etc. to damage cells and finally kill the cells.
Generally, the mechanism of photodynamic therapy is based on the transfer of energy to excite a Photosensitizer (PS) to molecular oxygen to produce singlet oxygen ((ii))1O2) Which can immediately damage biomolecules to cause cell death (type ii photodynamic therapy). Molecular oxygen is essential for type II photodynamic therapy, and1O2is influenced by the ambient oxygen concentration. Thus, the therapeutic efficacy of photodynamic type ii therapy depends strongly on the oxygen level. However, since tumors grow rapidly and oxygen is poorly supplied, the primary microenvironment of solid tumors is a hypoxic environment. In addition, the rapid consumption of oxygen during PDT further exacerbates the hypoxic state, limiting therapeutic efficacy, which has become a major obstacle to PDT. In type I photodynamic therapy chemistry charge transfer occurs between the excited PS and the adjacent substrate, forming reactive free radical ion-damaged biomolecules, this type of PDT works well under hypoxic conditions and addresses the limitations of hypoxia during photodynamic type ii therapy.
Therefore, in order to maintain excellent type I photodynamic therapy effects under hypoxic conditions, it is important to develop a photosensitizer that can generate active oxygen efficiently under hypoxic conditions.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and the phosphorescent ruthenium complex capable of providing effective active oxygen under the hypoxic condition is synthesized by introducing an electron-donating functional group to an auxiliary ligand, so that the compound has a good application prospect in the aspect of I-type photodynamic therapy.
The technical scheme of the invention is as follows: the invention discloses a novel phosphorescent ruthenium complex which specifically comprises Ru1, Ru2 and Ru3, and the structural formulas are respectively as follows:
further, the synthesis route of the novel phosphorescent ruthenium complex is as follows:
further, the preparation method of the novel phosphorescent ruthenium complex comprises the following specific synthesis steps:
1) preparation of compound 2: stirring and mixing 1mmol of compound 1 and 1.3mmol of iodine oxide at normal temperature, adding acetic acid, and heating and refluxing for 2-6 h to obtain a purple black solution; cooling to room temperature, adding deionized water for sedimentation, and standing at room temperature overnight; vacuum filtration gave a yellow solid which was then combined with chloroform to give a dark red solution which was then diluted with saturated NaHCO3And saturated Na2S2O3Washing, drying the organic phase by using anhydrous sodium sulfate, and finally performing rotary evaporation to obtain a dark brown solid compound 2;
2) preparation of compound 4: reacting the compound 2, ammonium acetate and the compound 3 with glacial acetic acid at 60 ℃ overnight, cooling to room temperature, dropwise adding ammonia water for neutralization, precipitating a brown solid from a product, performing suction filtration, washing with water, and performing vacuum drying to obtain a brown powdery compound 4;
3) preparation of compound 6: reacting the compound 2, ammonium acetate, benzaldehyde and glacial acetic acid at 60 ℃ overnight, cooling to room temperature, dropwise adding ammonia water for neutralization, precipitating a brown solid from a product, performing suction filtration, washing with water, and performing vacuum drying to obtain a brown powdery compound 6;
4) preparation of compound Ru 1: stirring and mixing 1mmol of compound 5, 1mmol of compound 4, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 1;
5) preparation of compound Ru 2: stirring and mixing 1mmol of compound 5, 1mmol of compound 6, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 2;
6) preparation of compound Ru 3: stirring and mixing 1mmol of compound 7, 1mmol of compound 4, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 3.
Further, the temperature for performing the light shielding reaction in the steps 4), 5) and 6) is 115-130 ℃.
Further, the novel phosphorescent ruthenium complexes may be used for type I photodynamic therapy in hypoxic conditions.
Further, the novel phosphorescent ruthenium complexes can be used for cell imaging.
The invention has the beneficial effects that:
according to the invention, an electron-donating functional group is introduced to an auxiliary ligand to synthesize a novel phosphorescent ruthenium complex, the phosphorescent ruthenium complex can provide effective active oxygen under the condition of hypoxia, the photodynamic therapy effect is ensured, and the novel phosphorescent ruthenium complex has a good application prospect in the aspect of I-type photodynamic therapy; the preparation method disclosed by the invention is simple in process, mild in reaction conditions, rich in raw materials and capable of realizing large-scale production.
Drawings
FIG. 1 is an ultraviolet-visible absorption spectrum of phosphorescent ruthenium complexes Ru1, Ru2 and Ru3 obtained in example 3;
FIG. 2 is a plot of the change of the phosphorescent ruthenium complexes Ru1, Ru2 and Ru3 obtained in example 4 as a function of DCFH at 520nm in the presence of hypoxia;
FIG. 3 is a data statistics graph of the concentration of phosphorescent ruthenium complexes Ru1, Ru2 and Ru3 obtained by MTT dark toxicity test in example 5 as a function of cell activity;
FIG. 4 is a data statistical chart of the relationship between the concentrations of the phosphorescent ruthenium complexes Ru1, Ru2 and Ru3 and the cell activity, which is obtained by performing the MTT cell phototoxicity experiment in example 6;
FIG. 5 is a confocal imaging diagram of phosphorescent ruthenium complexes Ru1, Ru2 and Ru3 obtained in example 7 under hypoxic condition for detecting active oxygen change in cells.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit of the invention.
Example 1: preparation of ancillary ligands
Preparation of compound 2: stirring compound 1(180mg, 1mmol) and iodine oxide 41mg (1.3mmol) at room temperature, adding acetic acid 2.5mL, heating and refluxing for 3h to obtain purple black solution, cooling to room temperature, adding deionized water for settling, standing at room temperature overnight, vacuum filtering to obtain yellow solid, adding chloroform to obtain dark red solution, and adding saturated NaHCO3And saturated Na2S2O3The organic phase was washed, dried over anhydrous sodium sulfate and finally rotary evaporated to give compound 2 as a dark brown solid in yield: 93 percent.
1H NMR(400MHz,CDCl3)8.89(dd,J＝2Hz,1.6Hz,1H),8.69(dd,J＝0.8Hz,0.4Hz,1H),8.41(dd,J＝2Hz,2Hz,1H),8.20(dd,J＝1.2Hz,0.8Hz,1H),7.83-7.78(m,1H),7.59(dt,J＝1.2Hz,8.8Hz,1H),7.43(dd,J＝4.8Hz,4.8Hz,1H).
Preparation of compound 4: compound 2(833mg, 0.8mmol), ammonium acetate (3130mg, 40mmol), compound 3(221mg, 0.8mmol) were reacted with glacial acetic acid (40mL) at 60 ℃ overnight, the dark red liquid was cooled to room temperature and then neutralized by dropwise addition of aqueous ammonia, the product precipitated as a brown solid which was filtered off with suction to give a solid which was washed with water and dried in vacuo to give compound 4 as a brown powder in yield: 56 percent.
1H NMR(400MHz,(CD3)2SO)：9.24(dd,J＝8.0Hz,8.0Hz,1H),8.92(dd,J＝1.6Hz,2Hz,1H),8.85(dd,J＝1.6Hz,1.6Hz,1H),8.55-8.505(m,1H),8.17-8.14(m,2H),7.83-7.63(m,3H),7.37-7.33(m,4H),7.13-7.09(m,9H).
Preparation of compound 6: compound 2(833mg, 0.8mmol), ammonium acetate (3130mg, 40mmol), benzaldehyde (85mg, 0.8mmol) reacted with glacial acetic acid (40mL) at 60 ℃ overnight, the dark red liquid cooled to room temperature and then neutralized by dropwise addition of aqueous ammonia until a brown solid precipitated, which was filtered off with suction to give a solid which was washed with water and dried in vacuo to give compound 6 as a brown powder in 69% yield.
1H NMR(400MHz,CDCl3)9.20(dd,J＝1.6Hz,4.4Hz,1H),8.87(dd,J＝1.6Hz,1.6Hz,1H),8.66(dd,J＝1.2Hz,1.2Hz,1H),8.13-8.11(m,1H),7.97(dd,J＝2Hz,1.2Hz,2H),7.61-7.58(m,2H),7.45-7.42(m,1H),7.29-7.27(m,3H).
Example 2: preparation of Ru1, Ru2 and Ru3
Preparation of compound Ru 1: compound 5(100mg), compound 4(92mg), 1mL triethylamine, with 3mL ethylene glycol at room temperature with stirring, 120 ℃ in the dark for 24h, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, then removing ethylene glycol and triethylamine by distillation under reduced pressure, settling with ethyl acetate, purifying by column chromatography, the polarity is dichloromethane: the acetonitrile is 10: 1 and then recrystallized three times from dichloromethane and diethyl ether to give the complex Ru1 in 26% yield.
1H NMR(400MHz,(CD3)2SO)8.77(d,J＝8Hz,1H),8.70-8.61(m,4H),8.58(d,J＝8Hz,1H),8.15-8.09(m,3H),7.96-7.94(m,1H),7.92-7.90(m,1H),7.87-7.78(m,6H),7.74(dd,J＝1.2Hz,5.2Hz,1H),7.61-7.58(m,1H),7.45(dd,J＝2.6Hz,8.4Hz,1H),7.42-7.39(m,1H),7.37-7.33(m,4H),7.29-7.24(m,1H),7.22-7.18(m,3H),7.12-7.09(m,8H).13C NMR(100MHz,CD3CN)158.70,158.04,157.69,156.31,156.24,155.26,151.35,151.27,151.08,150.41,150.08,148.47,148.10,140.80,137.38,135.87,134.86,134.58,130.61,129.07,128.88,128.20,128.01,127.32,127.12,126.92,126.19,124.99,124.32,124.16,123.93,123.85,122.79,122.56,114.00.
Preparation of compound Ru 2: compound 5(100mg), compound 6(59mg), 1mL triethylamine, with 3mL ethylene glycol at room temperature with stirring, 120 ℃ in the dark for 24h, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure to remove ethylene glycol and triethylamine, settling with ethyl acetate, purifying by column chromatography, the polarity is dichloromethane: the acetonitrile is 10: 1 and then recrystallized three times from dichloromethane and diethyl ether to give the complex Ru2 in 30% yield.
1H NMR(400MHz,CD3CN)8.58(dd,J＝1.2Hz,8Hz,1H),8.39(d,8Hz,1H),8.27-8.25(m,2H),8.17-8.14(m,3H),7.96-7.92(m,3H),7.84(d,J＝5.2Hz,1H),7.81-7.76(m,3H),7.62-7.58(m,2H),7.53(dt,J＝1.2Hz,8Hz,1H),7.48(t,J＝7.2Hz,2H),7.43-7.39(m,2H),7.26-7.20(m,3H),6.91-6.87(m,2H),6.64(dd,J＝0.8Hz,7.2Hz,1H)13C NMR(100MHz,CD3CN)158.52,157.85,157.48,156.28,156.10,155.05,151.11,151.05,150.17,148.47,140.76,137.23,135.68,134.72,134.35,132.60,131.17,130.31,129.86,138.94,127.86,127.18,127.03,126.90,126.75,124.14,124.00,123.76,123.67,122.49,113.74.
Preparation of compound Ru 3: compound 7(100mg), compound 4(92mg), 1mL triethylamine, with 3mL ethylene glycol at room temperature with stirring, 120 ℃ in the dark for 24h, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure to remove ethylene glycol and triethylamine, settling with ethyl acetate, purifying by column chromatography, the polarity is dichloromethane: the acetonitrile is 10: 1 and then recrystallized three times from dichloromethane and diethyl ether to give the complex Ru3 in 20% yield.
1H NMR(400MHz,(CD3)2SO)8.71(dd,J＝1.2Hz,0.8Hz,1H),8.66(dd,J＝1.2Hz,1.2Hz,1H)8.54-8.48(m,3H),8.33-8.23(m,7H),8.15(d,J＝8.8Hz,2H),8.09-8.07(m,1H),8.03-8.02(m,1H),7.86-7.82(m,2H),7.77(dd,J＝0.8Hz,1.2Hz,1H),7.68-7.63(m,3H),7.39-7.35(m,5H),7.14-7.10(m,8H).13C NMR(100MHz,CD3CN)156.44,155.90,151.74,151.63,150.95,150.90,149.85,149.79,149.77,149.76,149.10,148.69,148.01,147.18,140.80,136.17,134.81,133.67,133.45,132.54,131.50,131.36,131.17,131.07,130.49,138.86,128.72,128.53,128.51,128.35,127.99,126.50,126.01,125.85,125.69,125.68,124.81,124.34,122.74,122.18,113.72.
Example 3: UV-VISIBLE ABSORPTION SPECTRUM TESTING OF Ru1, Ru2, and Ru3
The concentration of the spectrum test adopted by the invention is 10 mu M, and the test solvent is methanol solution.
The absorption spectra of Ru1, Ru2 and Ru3 are shown in FIG. 1, where 240-325nm is the π π π π π transition centered on an N ligand, 325-425nm is the π π π transition centered on a C ligand, 425-650nm is the Ru center and the N ligand charge transfer (MLCT) transition.
Example 4:
DCFH-DA was converted to DCFH, DCFH-DA was added to 0.5mL of ethanol solution to make a 1mM solution, then the made up DCFH-DA ethanol solution was added to 2mL of 0.01M NaOH aqueous solution and allowed to stand at room temperature for 30 minutes, the hydrolysate was neutralized with 10mL of 25mM sodium phosphate buffer, the final hydrolysate had a pH of 7.4, then stored in a refrigerator and kept dark, and the final concentration of DCFH base-activated solution was 40 mM. 10mL in each case-42mL of activated DCFH solution was added to M solutions of Ru1, Ru2, and Ru3, and the fluorescence signal of DCF was monitored by fluorescence spectrometer at 5% O2The solution was irradiated with white light (400-800nm) under atmosphere for testing, the fluorescence spectrum of DCF was recorded in the range of 490-610nm, and the excitation wavelength was 480 nm.
The functional change curves of the ruthenium complexes Ru1, Ru2 and Ru3 at 520nm under the condition of hypoxia are shown in FIG. 2, and according to the characteristic that the yield of active oxygen is increased as the slope of the functional curve is larger, the three ruthenium complexes subjected to in-vitro experiments under the condition of hypoxia can generate active oxygen and the active oxygen generated by Ru1 is the most.
Example 5: MTT cell dark toxicity test of Ru1, Ru2 and Ru3
The digested cells were seeded in 96-well plates at a density of 10 per well4One/well at 37 ℃ 5% CO2The culture was continued for 24 hours under the conditions described in (1), after sucking up the stale culture medium, the cells were continued for 24 hours in cell culture media of different concentrations of Ru1, Ru2 and Ru3(2.5, 5, 7.5, 10, 12.5, 15, 20. mu.M), after adding 10. mu.L of MTT (5mg/mL) per well and continuing for 4 hours, the culture was terminated, after sucking up the culture medium, 150. mu.L of DMSO per well was added, and after shaking for 10 minutes, OD570 was measured using a microplate reader.
MTT cell dark toxicity test results are shown in figure 3, when the concentration of the ruthenium complex is 0-20 mu M, the cell survival rate after 24 hours of culture is more than 60%, and the results prove that the Ru1, Ru2 and Ru3 complexes have low cytotoxicity and can be used for cell imaging.
Example 6: MTT cell phototoxicity assay for Ru1, Ru2 and Ru3
The digested cells were seeded in 96-well plates at a density of 10 per well4One/well at 37 ℃ 5% CO2The culture was continued for 24 hours under the conditions of (1) A, after sucking up stale culture medium, cells were continued for 1 hour with cell culture medium of different concentrations of Ru1, Ru2 and Ru3(2.5, 5, 7.5, 10, 12.5, 15, 20. mu.M), and then irradiated with xenon lamp (lambda.)ex＝400-800nm，30mW/cm2) After 10 minutes, the cells were cultured for 24 hours, 10. mu.L of MTT (5mg/mL) was added to each well, the culture was terminated after 4 hours, the culture medium was aspirated, 150. mu.L of DMSO was added to each well, and OD570 was measured by a microplate reader after shaking for 10 minutes.
MTT cell phototoxicity experiment results are shown in figure 4, when the concentration of the ruthenium complex is 1-20 mu M, the survival rate of cells after 24 hours of illumination culture is obviously reduced, the results prove that the Ru1, Ru2 and Ru3 complexes can kill the cells after illumination, and active oxygen is effectively generated to kill the cells after illumination.
Example 7: hypoxic confocal imaging experiment of ruthenium complexes Ru1, Ru2 and Ru3 and active oxygen indicator DCFH-DA
The cells adopted by the invention are all human cervical carcinoma HeLa cells. The digested cells were seeded in petri dishes at 37 ℃ in 5% CO2The culture was continued for 24 hours under the conditions of (1) to allow adhesion. After washing the cell culture medium with PBS solution, the cell culture medium was washed with 5% O at 37 ℃2The cells were incubated with cell culture solutions of Ru1, Ru2 and Ru3 (5. mu.M) for 2 hours. Further, the cells were cultured in a cell culture medium containing DCFH-DA (10. mu.M) for 20 minutes, and then the cells were irradiated with xenon lamp (. lamda.)ex＝400-800nm，30mW/cm2)10 minutes, then imaged with confocal imaging
The imaging chart of the cell hypoxia confocal images of the complexes Ru1, Ru2 and Ru3 and the active oxygen indicator DCFH-DA is shown in FIG. 5. As the more active oxygen, the stronger the fluorescence intensity of the active oxygen indicator, as can be seen from fig. 5, the confocal imaging graphs of the three ruthenium complexes disclosed in the embodiment of the present invention all have higher brightness and the imaging graph of Ru1 has the strongest brightness, which indicates that the three ruthenium complexes can effectively generate active oxygen and Ru1 generates the most active oxygen, and the confocal imaging graphs conform to the theoretical values shown in fig. 2, which indicates that the compound disclosed in the present invention has a good I-type photodynamic therapy effect, and the Ru1 effect is the most obvious.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. However, the above description is only an example of the present invention, the technical features of the present invention are not limited thereto, and any other embodiments that can be obtained by those skilled in the art without departing from the technical solution of the present invention should be covered by the claims of the present invention.
Claims (5)
1. A novel phosphorescent ruthenium complex is characterized in that the ruthenium complex is Ru1, Ru2 or Ru3, and the specific structural formulas are as follows:
2. the method for preparing the novel phosphorescent ruthenium complex as claimed in claim 1, wherein the ruthenium complex is synthesized by the following route:
3. the preparation method of the novel phosphorescent ruthenium complex as claimed in claim 2, which is characterized by comprising the following specific synthesis steps:
1) preparation of compound 2: stirring and mixing 1mmol of compound 1 and 1.3mmol of iodine oxide at normal temperature, adding acetic acid, and heating and refluxing for 2-6 h; cooling to room temperature, adding deionized water for settling, and standing at room temperatureStanding overnight; vacuum filtering, adding chloroform, and adding saturated NaHCO3And saturated Na2S2O3Washing, drying the organic phase, and performing rotary evaporation to obtain a dark brown solid compound 2;
2) preparation of compound 4: reacting the compound 2, ammonium acetate, the compound 3 and glacial acetic acid at 60 ℃ overnight, cooling to room temperature, dropwise adding ammonia water for neutralization, precipitating a brown solid from a product, performing suction filtration, washing with water, and performing vacuum drying to obtain a brown powdery compound 4;
3) preparation of compound 6: reacting the compound 2, ammonium acetate, benzaldehyde and glacial acetic acid at 60 ℃ overnight, cooling to room temperature, dropwise adding ammonia water for neutralization, precipitating a brown solid from a product, performing suction filtration, washing with water, and performing vacuum drying to obtain a brown powdery compound 6;
4) preparation of compound Ru 1: stirring and mixing 1mmol of compound 5, 1mmol of compound 4, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 1;
5) preparation of compound Ru 2: stirring and mixing 1mmol of compound 5, 1mmol of compound 6, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 2;
6) preparation of compound Ru 3: stirring and mixing 1mmol of compound 7, 1mmol of compound 4, triethylamine and ethylene glycol at normal temperature, reacting for 24h in a dark place, cooling to room temperature, adding potassium hexafluorophosphate solution, stirring for 2h, filtering, distilling under reduced pressure, settling with ethyl acetate, purifying by column chromatography, and recrystallizing for three times to obtain compound Ru 3.
4. The method for preparing the novel phosphorescent ruthenium complex as claimed in claim 3, wherein the temperature for the light shielding reaction in the steps 4), 5) and 6) is 115-130 ℃.
5. Use of a novel class of phosphorescent ruthenium complexes as claimed in any one of claims 1 to 4 in cellular imaging.
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