CN110746423B

CN110746423B - Synthesis of aryl imidazophenanthroline fluorescent dye and identification of metal ions

Info

Publication number: CN110746423B
Application number: CN201911097220.XA
Authority: CN
Inventors: 张志强; 侯慧青; 柯方; 周孙英; 许贻文; 林媚; 陈艳
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2022-03-18
Anticipated expiration: 2039-11-11
Also published as: CN110746423A

Abstract

The invention discloses synthesis of an arylimidazophenanthroline fluorescent dye and identification of metal ions. The method can synthesize the aryl imidazo [4,5-f ] by a series of steps by taking cheap and easily available 1, 10-phenanthroline as a raw material]A method for preparing phenanthroline oxa-crown ether compounds. By para-arylimidazo [4,5-f]The fluorescence performance of the phenanthroline compound is screened, the complexing performance of the compound and metal ions is verified through screening experiments on different metal ions and experiments on ion concentration and the like, and the metal ion Zn in tumor cells is realized²⁺Identification of (1). The fluorescent probe has the following technical effects: 1. the compounds show Zn on a wide range of metal ions respectively²⁺High selectivity of (2); 2. can realize Zn pair in MCF-7 cells²⁺The response fluorescence quenching molecular switch plays a large role in paving the application of the system in more fields such as biological recognition and the like.

Description

Synthesis of aryl imidazophenanthroline fluorescent dye and identification of metal ions

Technical Field

The invention discloses a method for synthesizing aryl imidazo [4,5-f ] phenanthroline oxa-crown ether compounds by using cheap and easily available 1, 10-phenanthroline as a raw material, which is used for screening the fluorescence property of aryl imidazo [4,5-f ] phenanthroline compounds and identifying metal ions by using the fluorescence property of the aryl imidazo [4,5-f ] phenanthroline compounds.

Background

Phenanthroline is used as a chelating ligand, and many derivatives of phenanthroline can be used as important chelating agents to be widely applied to various fields such as analytical chemistry and homogeneous catalysis, and the complex of phenanthroline is also an important electron transfer agent in life science. Many complexes of phenanthroline show good electrochemical, photochemical and catalytic properties, and have a certain application prospect in the aspect of antitumor activity. From the structure, the phenanthroline is an aromatic conjugated system consisting of three six-membered rings, two nitrogen atoms on the 1 and 10 positions of the phenanthroline have higher electron cloud density, are easy to coordinate with metal ions and form a stable five-membered chelate ring, and are bidentate ligands with good performance. In view of the good coordination characteristics of phenanthroline and the advantage of structural rigidity plane conjugation, novel fluorescent complexes derived from phenanthroline as a parent are reported in succession. In recent years, researchers have been mainly developing complexes using phenanthroline and its derivatives as ligands to develop fluorescent materials and using its derivatives as ligands to prepare rare earth metal chelates, etc. The aryl imidazo [4,5-f ] phenanthroline compound has a large planar structure, high electron capturing capacity, high electron transmission capacity, high thermal stability and high fluorescence performance, and derivatives of the aryl imidazo [4,5-f ] phenanthroline compound are widely used as luminescent materials and biomolecule fluorescent probes.

The aryl imidazo [4,5-f ] phenanthroline compound has a large pi bond conjugated system, good photochemical stability, thermal stability and an easily modified structure. In recent years, multiple studies prove that the aryl imidazo [4,5-f ] phenanthroline compound has strong fluorescence property, and as a fluorescence functional group, a plurality of aryl imidazo [4,5-f ] phenanthroline with different structures are used in a fluorescence probe. The aryl imidazo [4,5-f ] phenanthroline is strong in fluorescence, easy to synthesize and less in report of the fluorescence crown ether containing the aryl imidazo [4,5-f ] phenanthroline. Aryl imidazo [4,5-f ] phenanthroline is taken as a raw material and is connected to a oxa-crown ether ring so as to obtain fluorescent crown ether, and the fluorescent performance of the fluorescent crown ether is researched on the basis to search for a metal ion probe with a simple and effective structure.

Disclosure of Invention

The invention aims to provide a method for synthesizing aryl imidazo [4,5-f ] phenanthroline oxa-crown ether compound by using cheap and easily available 1, 10-phenanthroline as raw material, which has the characteristics of environmental protection, simple and convenient operation, safety and low cost.

Another objective of the invention is to provide a new medicine for treating tumor cells with metal ions Zn²⁺The synthesis method of the identified small molecule fluorescent probe realizes the Zn pair in MCF-7 cells²⁺The responsive fluorescence quenching molecular switch has the characteristics of high luminous efficiency, small background interference and the like, and is a fluorescent probe with wide prospect.

The technical scheme adopted by the invention is as follows: aryl imidazo [4, 5-f)]The phenanthroline oxa-crown ether fluorescent probe is characterized by comprising the following components in parts by weight: the fluorescent probe has the following structure, and can be used for identifying Zn with high selectivity²⁺：

The above-mentioned invention for high-selectivity recognition of Ni²⁺The method for synthesizing the fluorescent probe comprises the following steps:

(1) synthesis of intermediate Compound I (triethylene glycol di-p-toluenesulfonate):

triethylene glycol (10 mmol) and p-toluenesulfonyl chloride (10 mmol) were dissolved in 150 mL of Tetrahydrofuran (THF), and 70 mmol of a potassium hydroxide solution was added thereto, followed by stirring at room temperature for 7 hours. After the reaction was completed, it was extracted with dichloromethane, and then MgSO₄Drying, distilling off the solvent under reduced pressure, purifying the crude product by chromatography on a column (V)_{Ethyl acetate}：V_{Petroleum ether}=1: 2) elution to obtain compound I (triethylene glycol)Di-p-toluenesulfonate ester);

(2) synthesis of intermediate Compound II (benzo-12-crown-4):

in a 100mL flask, catechol (5 mmol) and 50% mmol tetrabutylammonium bromide (TBAB) were dissolved in 30 mL of a toluene solution, and 10 mL of a 50% aqueous NaOH solution was added. The mixture was stirred at 50-60 ℃ for 30 min, then a solution of triethylene glycol di-p-toluenesulfonate (5 mmol) in 30 mL of toluene was added. The resulting mixture was stirred vigorously at this temperature under reflux for 16 h. After the reaction is finished, H is used₂O (3X 200 mL), the organic layer was separated, washed with brine and MgSO₄And (5) drying. The solvent was removed in vacuo and the residue was extracted with ethyl acetate (3X 50 mL). After cooling, the product is separated by column chromatography using (V)_{Ethyl acetate}：V_{Petroleum ether}=2: 1) elution to give compound II (benzo-12-crown-4);

(3) synthesis of intermediate Compound III (4-nitrobenzo-12-crown-4):

adding benzo-12-crown-4 (2 mmol), 10 mL chloroform and 6 mL acetic acid into a 100mL round-bottom flask, slowly dropping 4 mL concentrated nitric acid with a concentration of 68% under ice bath condition, stirring at room temperature for 24 h, neutralizing the reaction solution with alkali, separating a chloroform layer, extracting an aqueous layer with chloroform, drying the combined chloroform layers with anhydrous magnesium sulfate, evaporating the chloroform under reduced pressure to obtain a yellow crude product, separating by column chromatography, and purifying with (V)_{Ethyl acetate}：V_{Petroleum ether}=1: 2) to obtain a compound III (4-nitrobenzo-12-crown-4);

(4) synthesis of intermediate Compound IV (4-aminobenzo-12-crown-4):

putting 4-nitrobenzo-12-crown-4 (2 mmol) and 10% Pd/C into a 50mL round-bottom flask, adding 20 mL absolute ethyl alcohol, pumping out air, introducing hydrogen, stirring for 24 h, filtering Pd/C, and concentrating under reduced pressure to obtain a compound IV (4-aminobenzo-12-crown-4);

(5) synthesizing an intermediate compound V (2- (4-bromophenyl) imidazo [4,5-f ] phenanthroline):

respectively adding 1, 10-phenanthroline-5, 6-diketone (0.315 g, 1.5 mmol), ammonium acetate (2.31 g, 30 mmol) and p-bromobenzaldehyde (1.5 mmol) into a 100mL round-bottom flask, dissolving with 30 mL glacial acetic acid, and carrying out reflux reaction at 120 ℃ for 2 h under magnetic stirring. After the reaction is finished, cooling to room temperature, diluting with 60 ml of ice water, slowly dropwise adding concentrated ammonia water, neutralizing acid until the pH =7 to obtain a large amount of light yellow precipitate, performing suction filtration, washing with a small amount of water, drying a filter cake, and then recrystallizing with ethanol to obtain a compound V (2- (4-bromophenyl) imidazo [4,5-f ] phenanthroline);

(6) synthesizing a fluorescent probe VI (N- (4- (1H-imidazole- [4,5-f ] phenanthroline) benzo-12-crown-4):

in a 100mL round bottom flask, 2- (4-bromophenyl) imidazo [4,5-f ] was added]Phenanthroline (1 mmol), 4-aminobenzo-12-crown-4 (1.2 mmol), CuI (0.1 mmol), Cs₂CO₃(2 mmol), N, N-dimethylethylenediamine (DMDEA, 0.2 mmol) was dissolved in 30 mL of N, N-Dimethylformamide (DMF), N₂The reaction is refluxed for 24 hours at 120 ℃ under protection. After the reaction is finished, water is added for extraction with ethyl acetate, ethyl acetate is further dried by spinning, and the ethyl acetate is separated by a chromatographic column and is treated with (V)_{Ethyl acetate}：V_{Petroleum ether}The eluent is used for eluting the mixture to obtain a fluorescent probe VI (N- (4- (1H-imidazole- [4, 5-f))]Phenanthroline) benzo-12-crown-4).

The invention has the following beneficial effects:

the method for synthesizing the aryl imidazo [4,5-f ] phenanthroline two-class compound by taking cheap and easily available 1, 10-phenanthroline as a raw material through a series of steps has the advantages of simple synthetic route, mild reaction conditions and simple and convenient post-treatment.

Two types of fluorescent probes can identify Zn with high selectivity²⁺Can realize Zn in MCF-7 cells²⁺The responsive fluorescence quenching molecule switches. The system plays a large role in paving more fields such as biological identification and the like.

Drawings

FIG. 1 is an arylimidazo [4,5-f ] of the invention]The ultraviolet spectrum of the phenanthroline oxacrown ether compound and the metal ion identification is compared with the fluorescence spectrum, the abscissa is the wavelength, the ordinate is the light intensity, the concentration of the compound is 10 mu M, and the metal ion Ag is⁺, Cu2⁺, Al³⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Zn²⁺, Li⁺, Ni⁺, Ca²⁺And Na⁺The concentration of (D) is 10. mu.M;

FIG. 2 is an arylimidazo [4,5-f ] of the invention]The fluorescence spectra of the phenanthroline oxa-crown ether fluorescent probe and metal ions with different concentrations are identified, the abscissa is the wavelength, the ordinate is the fluorescence intensity, the concentration of the fluorescent probe is 10 mu M, and Ni is⁺The concentration of (b) is 0. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M;

FIG. 3 is an arylimidazo [4,5-f ] of the invention]Application of phenanthroline fluorescent probe in identifying Zn in MCF-7²⁺In the figure, A is a fluorescence image of MCF-7 cells. In the figure, B is a fluorescence image (λ ex: 500nm) of MCF-7 cells incubated with a fluorescent probe (1 μ M) for 1 h. C in the figure is the incubation of MCF-7 cells with a fluorescent probe (1 mu M) for 1h, three times of washing, and then further incubation with 1 mu M Zn^{2 +}Fluorescence image (λ ex: 500nm) after incubation for 30 minutes. In the figure, D is a transmission image of MCF-7 cells in a bright field. In the figure, E is a transmission image (λ ex: 500nm) in a bright field of 1h of incubation of MCF-7 cells with a fluorescent probe (1 μ M). In the figure, F is a transmission image under a bright field of 1h of MCF-7 cells incubated with a fluorescent probe (1 mu M), washed three times, and then further incubated with 1 mu M Zn^{2 +}Fluorescence image (λ ex: 500nm) after incubation for 30 minutes.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.

Example 1: synthesis of phenylimidazo [4,5-f ] phenanthroline oxa-crown ether fluorescent probe

dissolving triethylene glycol (10 mmol) and p-toluenesulfonyl chloride (10 mmol) in 150 mL tetrahydrofuran(THF), potassium hydroxide (70 mmol) solution was added thereto, and stirred at room temperature for 7 hours. After the reaction was completed, the reaction mixture was extracted with dichloromethane and MgSO₄Drying, spin-drying the dichloromethane, purifying the crude product on a chromatographic column using (V)_{Ethyl acetate}：V_{Petroleum ether}=1: 2) elution to obtain a compound I (triethylene glycol di-p-toluenesulfonate) with a yield of 90%;

(2) synthesis of intermediate Compound II (benzo-12-crown-4)

In a 100mL flask, catechol (5 mmol), 2.5 mmol of tetrabutylammonium bromide (TBAB) was dissolved in 30 mL of a toluene solution, and a 50 wt% aqueous NaOH solution (10 mL) was further added. The mixture was stirred at 50-60 ℃ for 30 minutes, and then a toluene solution containing triethylene glycol di-p-toluenesulfonate (5 mmol, also called triethylene glycol di-p-toluenesulfonate) (30 mL) was added. The resulting mixture was stirred vigorously at this temperature under reflux for 16 h. After the reaction is finished, H is used₂O (3X 200 mL), the organic layer was separated, washed with brine and MgSO₄And (5) drying. The solvent was removed in vacuo and the residue was extracted with ethyl acetate (3X 50 mL). After cooling, the product is separated by column chromatography using (V)_{Ethyl acetate}：V_{Petroleum ether}=2: 1) elution to give compound II (benzo-12-crown-4) in 52% yield;

(3) synthesis of intermediate compound III (4-nitrobenzo-12-crown-4)

Adding benzo-12-crown-4 (2 mmol) prepared in the step (2), 10 mL of chloroform and 6 mL of acetic acid into a 100mL round-bottom flask, slowly dropping 4 mL of concentrated nitric acid with the concentration of 68wt% under the ice bath condition, stirring at room temperature for 24 h, neutralizing the reaction solution with alkali, separating a chloroform layer, extracting a water layer with chloroform, drying the combined chloroform layer with anhydrous magnesium sulfate,distilling off chloroform under reduced pressure to obtain yellow crude product, separating by column chromatography, and purifying with (V)_{Ethyl acetate}：V_{Petroleum ether}=1: 2) to obtain a compound III (4-nitrobenzo-12-crown-4) with a yield of 75%;

(4) synthesis of intermediate compound IV (4-aminobenzo-12-crown-4)

Putting the 4-nitrobenzo-12-crown-4 (2 mmol) prepared in the step (3) into a 50mL round-bottom flask with the mass fraction of 10% Pd/C, adding 20 mL absolute ethyl alcohol, pumping out air, introducing hydrogen, stirring for 24 h, filtering out Pd/C, and concentrating under reduced pressure to obtain a compound IV (4-aminobenzo-12-crown-4), wherein the yield is 70%;

4-aminobenzo-12-crown-4:¹H NMR (400 MHz, DMSO-d₆) δ 4-aminobenzo-12-crown-4:¹H NMR (400 MHz, DMSO-d₆) δ 6.40 (s, 1H), 5.90 – 5.85 (mm, 6H),6.40 (s, 1H) 4.11 (d, J = 3.28 Hz, 2H), 4.01 (s, 1H), 3.78 (d, J = 3.02 Hz, 2H), 3.54 (d, J = 2.83 Hz, 2H).

(5) synthesis of intermediate compound V (2- (4-bromophenyl) imidazo [4,5-f ] phenanthroline)

Respectively adding 1, 10-phenanthroline-5, 6-diketone (0.315 g, 1.5 mmol), ammonium acetate (2.31 g, 30 mmol) and p-bromobenzaldehyde (1.5 mmol) into a 100mL round-bottom flask, dissolving with 30 mL glacial acetic acid, and carrying out reflux reaction at 120 ℃ for 2 h under magnetic stirring. After the reaction is finished, cooling to room temperature, diluting with 60 ml of ice water, slowly dropwise adding concentrated ammonia water, neutralizing acid until the pH =7 to obtain a large amount of light yellow precipitate, performing suction filtration, washing with a small amount of water, drying a filter cake, and then recrystallizing with ethanol to obtain a compound V (2- (4-bromophenyl) imidazo [4,5-f ] phenanthroline) with the yield of 80%;

2- (4-bromophenyl) benzeneYl) imidazo [4,5-f]Phenanthroline:¹H NMR (500 MHz, DMSO-d₆): 9.02 (dd, J = 4.3, 1.8 Hz, 2H), 8.89 (d, J = 1.8 Hz, 2H), 8.88 (d, J = 1.8Hz, 2H), 8.23–8.21 (m, 1H), 8.21–8.18 (m, 2H), 7.85–7.79 (m, 2H). LC-MS (ESI) m/z: 375.0 [M+H]⁺.

(6) synthesis of fluorescent probe VI (N- (4- (1H-imidazole- [4,5-f ] phenanthroline) benzo-12-crown-4)

In a 100mL round-bottom flask, the 2- (4-bromophenyl) imidazo [4,5-f ] prepared in step (5) is added]Phenanthroline (1 mmol), 4-aminobenzo-12-crown-4 (1.2 mmol) prepared in step (4), CuI (0.1 mmol), and Cs₂CO₃(2 mmol), N, N-dimethylethylenediamine (DMDEA, 0.2 mmol) was dissolved in 30 mL of N, N-Dimethylformamide (DMF), N₂The reaction is refluxed for 24 hours at 120 ℃ under protection. After the reaction is finished, water is added for extraction with ethyl acetate, ethyl acetate is further dried by spinning, and the ethyl acetate is separated by a chromatographic column and is treated with (V)_{Ethyl acetate}：V_{Petroleum ether}The eluent is used for eluting the mixture to obtain a fluorescent probe VI (N- (4- (1H-imidazole- [4, 5-f))]Phenanthroline) benzo-12-crown-4), yield 44%.

N- (4- (1H-imidazole- [4, 5-f)]Phenanthroline) benzo-12-crown-4:¹H NMR (400 MHz, DMSO-d₆) δ 12.60 (s, 1H), 8.51 (s, 1H), 8.27 (d, J = 5.2 Hz, 2H), 8.00 – 7.83 (m, 5H), 7.50 – 7.21 (m, 6H), 4.37 (d, J = 16.8 Hz, 8H), 3.65 (s, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 151.66, 150.00, 145.64, 135.37, 134.43, 134.36, 133.58, 131.61, 129.93, 125.43, 124.09, 58.05, 20.58. LC-MS (ESI) m/z: 534.2 [M+H]+.

example 2: measurement of ultraviolet spectrum and fluorescence spectrum

The N- (4- (1H-imidazole- [4, 5-f) obtained in example 1 was added]Phenanthroline) benzo-12-crown-4 (abbreviation: phenyl imidazo [4, 5-f)]Phenanthroline oxa-crown ether compound) with the concentration dilution of 2 multiplied by 10^-5mol/L, is selected13 different metal ions (Ag)⁺, Cu²⁺, Al³⁺, Fe³⁺, Hg²⁺, K+, Mg²⁺, Mn²⁺, Zn²⁺, Li⁺, Ni⁺, Ca²⁺And Na⁺) Is configured to be 1 x 10^-5mol/L solution. Adding 1 mL of different metal ion solutions into a cuvette, measuring the fluorescence spectra under the action of different metal ions, and screening out a probe which can specifically identify a certain metal ion, wherein the figure 1 shows that the aryl imidazo [4,5-f ] in the invention]The ultraviolet spectrum and the fluorescence spectrum of the phenanthroline oxacrown ether compound and metal ion identification are shown, the abscissa is the wavelength, the ordinate is the light intensity, the concentration of the compound is 10 mu M, and the metal ion Ag is⁺, Cu2⁺, Al³⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Mn²⁺, Zn²⁺, Li⁺, Ni⁺, Ca²⁺And Na⁺The concentration of (2) was 10. mu.M.

Example 3: determination of ultraviolet titration spectrum and fluorescence titration spectrum of phenylimidazo [4,5-f ] phenanthroline oxa-crown ether fluorescent probe and metal ion identification

The metal ions with the best response of different compounds are screened out, and then a solution (1 mu M, 2 mu M, 3 mu M, 4 mu M, 5 mu M, 6 mu M, 7 mu M, 8 mu M, 9 mu M and 10 mu M) with 10 concentration gradients of metal ions is weighed for detection. The fluorescence emission spectra of the screened N- (4- (1H-imidazole- [4,5-f ] phenanthroline) benzo-12-crown-4 compound (abbreviated as phenylimidazo [4,5-f ] phenanthroline oxa-crown ether compound) prepared in example 1 and the metal ions with different concentrations with the best response of the compound are measured, and are shown in FIG. 2.

Example 4: experiment for identifying metal ions in phenyl imidazo [4,5-f ] phenanthroline oxa-crown ether fluorescent probe cells

Firstly, respectively dissolving a certain amount of N- (4- (1H-imidazole- [4,5-f ] phenanthroline) benzo-12-crown-4 compound (abbreviated as phenylimidazo [4,5-f ] phenanthroline oxa-crown ether compound) and metal ions prepared in example 1 in Phosphate Buffered Saline (PBS) solution for later use, washing cultured cells with PBS three times, then adding the washed cells into the prepared compound solution, incubating the cells in a constant-temperature incubator at 37 ℃ for 30 min, then washing the cells with PBS for three times, exciting the cells with blue light under an inverted fluorescence microscope, taking pictures, adding the metal ion PBS solution into the just-obtained cells after photographing, placing the cells in a constant-temperature incubator at 37 ℃ for 30 min, taking out the cells, washing the cells with PBS for three times, exciting the cells with blue light under an inverted fluorescence microscope, taking pictures, see fig. 3.

Claims

1. Aryl imidazo [4, 5-f)]The phenanthroline oxa-crown ether fluorescent probe is characterized by comprising the following components in parts by weight: the fluorescent probe has the following structure, and can identify Zn with high selectivity²⁺：

。

2. High-selectivity Zn identification method²⁺Aryl imidazo [4,5-f ] of]The synthesis method of the phenanthroline oxa-crown ether fluorescent probe comprises the following steps:

dissolving 10 mmol of triethylene glycol and 10 mmol of p-toluenesulfonyl chloride in 150 mL of Tetrahydrofuran (THF), adding 70 mmol of potassium hydroxide solution, stirring at normal temperature for 7 h, extracting with dichloromethane after the reaction is finished, and extracting with MgSO₄Drying, distilling under reduced pressure, purifying the crude product with chromatographic column, and purifying with V_{Ethyl acetate}：V_{Petroleum ether}Elution with a ratio of 1: 2 to obtain a compound I (triethylene glycol di-p-toluenesulfonate);

(2) synthesis of intermediate Compound II (benzo-12-crown-4):

in a 100mL flask, 5 mmol of catechol, 2.5 mmol of tetrabutylammonium bromide (TBAB) were dissolved in 30 mL of a toluene solution, 10 mL of a 50 wt% aqueous NaOH solution was further added to obtain a mixture, the mixture was stirred at 50 to 60 ℃ for 30 minutes, then 30 mL of a mixture containing 5 mmol of triethylene glycol di-p-toluenesulfonate in toluene was further added, the obtained mixture was vigorously stirred at 50 to 60 ℃ for 16 hours under reflux, and 200 mLH was used each time after completion of the reaction₂O the organic layer was separated 3 times in total, and the 3 separated organic layers were combined and washed with 100mL of brine and MgSO₄Drying, removing the solvent in vacuo, extracting the residue 3 times with 50mL ethyl acetate, cooling, and separating the product by column chromatography using V_{Ethyl acetate}：V_{Petroleum ether}Elution with =2: 1 gave compound II (4 benzo-12-crown-4);

(3) synthesis of intermediate Compound III (4-nitrobenzo-12-crown-4):

adding 2 mmol of benzo-12-crown-4, 10 mL of chloroform and 6 mL of acetic acid into a 100mL round-bottom flask, slowly dropping 4 mL of concentrated nitric acid under ice bath condition, stirring at room temperature for 24 h, neutralizing the reaction solution with alkali, separating a chloroform layer, extracting a water layer with chloroform, drying the combined chloroform layer with anhydrous magnesium sulfate, evaporating the chloroform under reduced pressure to obtain a yellow crude product, separating by column chromatography, and performing V-column chromatography_{Ethyl acetate}：V_{Petroleum ether}Eluting by a ratio of 1: 2 to obtain a compound III (4-nitrobenzo-12-crown-4);

(4) synthesis of intermediate Compound IV (4-aminobenzo-12-crown-4):

putting 2 mmol of 4-nitrobenzo-12-crown-4 and 10% of Pd/C substance in mass fraction into a 50mL round-bottom flask, adding 20 mL of absolute ethyl alcohol, pumping out air, introducing hydrogen, stirring for 24 h, filtering Pd/C, and concentrating under reduced pressure to obtain a compound IV (4-aminobenzo-12-crown-4);

taking 1.5 mmol of 1, 10-phenanthroline-5, 6-diketone, 30 mmol of ammonium acetate and 1.5 mmol of p-bromobenzaldehyde, respectively adding the 1, 10-phenanthroline-5, 6-diketone and the 30 mmol of ammonium acetate into a 100mL round-bottom flask, dissolving the mixture with 30 mL of glacial acetic acid, performing reflux reaction for 2 hours at 120 ℃ under magnetic stirring, cooling the mixture to room temperature after the reaction is finished, diluting the mixture with 60 mL of icy water, slowly dropwise adding concentrated ammonia water, neutralizing the mixture to PH =7 to obtain a large amount of light yellow precipitate, performing suction filtration, washing with a small amount of water, drying a filter cake, and then recrystallizing with ethanol to obtain a compound V (2- (4-bromophenyl) imidazo [4,5-f ] phenanthroline);

(6) synthesizing a fluorescent probe VI (N- (4-1H-imidazole- [4,5-f ] phenanthroline) benzo-12-crown-4):

in a 100mL round-bottom flask, 1 mmol of 2- (4-bromophenyl) imidazo [4,5-f ] was added]Phenanthroline, 1.2 mmol of 4-aminobenzo-12-crown-4, 0.1 mmol of CuI, and 2 mmol of Cs₂CO₃0.2 mmol of N, N-dimethylethylenediamine (DMDEA) was dissolved in 30 mL of N, N-Dimethylformamide (DMF), and N was added₂Carrying out reflux reaction for 24 hours at 120 ℃ under protection; after the reaction, water is added and ethyl acetate is extracted, ethyl acetate is dried by spinning, and the ethyl acetate is separated by a chromatographic column and is treated by V_{Ethyl acetate}：V_{Petroleum ether}Eluting with the solvent in a ratio of 1: 1 to obtain the fluorescent probe VI (N- (4-1H-imidazole- [4, 5-f)]Phenanthroline) benzo-12-crown-4).