A class of phenoxazine coumarin derivatives and preparation method and application thereof

Abstract

The present invention relates to a class of phenoxazine coumarin derivatives, their preparation methods and applications. The phenoxazine coumarin derivatives have the following structure of Formula I: #imgabs0# (Formula I); wherein, R is one of -COOEt, -COCH3, #imgabs1#; by introducing a phenoxazine electron donor into the coumarin derivative, the present invention can effectively improve its spectral properties.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and more specifically to a class of phenoxazine coumarin derivatives and preparation methods and applications thereof. Background Art

[0002] Phenoxazine is a nitrogen-anthracene heterocyclic compound connected by an oxygen bridge bond. The ring structure expands its conjugated system. It also contains electron-donating nitrogen and oxygen atoms. Its electron-donating capacity is much greater than that of diethyl and julolidine electron donors. Therefore, phenoxazine is often used as an electron donor in the development of fluorescent dyes. The conjugated system of phenoxazine-based fluorescent dyes is significantly expanded, and the electron delocalization area of ​​the excited state is also larger. Therefore, the wavelength of fluorescent dyes based on phenoxazine donors will produce a significant red shift.

[0003] Traditional coumarin fluorescent dyes are widely used as fluorescent imaging tools in biomedical research due to their excellent photophysical properties and good biocompatibility. However, the relatively short absorption and emission wavelengths of traditional coumarin fluorescent dyes limit their application to a certain extent (short-wavelength photons have weak penetration into biological tissues). Summary of the invention

[0004] Based on the above technical problems existing in the prior art, the present invention provides a phenoxazine coumarin derivative, which can effectively improve the spectral performance of phenoxazine by modifying the phenoxazine with a specific molecular structure.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] A phenoxazine coumarin derivative has the following structure: (Formula I);

[0007] Where R is -COOEt, -COCH 3 , One of them.

[0008] The present invention also provides a method for preparing the phenoxazine coumarin derivative of the above-mentioned formula I, which comprises the following steps:

[0009] S1, adding compound 6 and anhydrous potassium carbonate into a first organic solvent, heating and reacting to obtain compound 7;

[0010] S2, adding compound 7, compound A and piperidine into a second organic solvent, heating to react, and obtaining a phenoxazine coumarin derivative having a structure of formula I;

[0011] Wherein, the structure of the compound 6 is: The compound A is one of diethyl malonate, ethyl acetoacetate, and 2-cyanomethylbenzimidazole; the structure of the phenoxazine coumarin derivative having the structure of formula I is: .

[0012] In some embodiments, in step S1, the reaction temperature is 100 - 140 °C.

[0013] In some embodiments, in step S1, the reaction time is 10 - 24 h.

[0014] In some embodiments, in step S2, the reaction temperature is 90 - 100 °C.

[0015] In some embodiments, in step S2, the reaction time is 10 - 24 h.

[0016] In some embodiments, the first organic solvent is N, N - dimethylformamide (DMF), and the second organic solvent is anhydrous ethanol (EtOH).

[0017] In some embodiments, the preparation of compound 6 is carried out by using compound 1 as a raw material, sequentially through electrophilic substitution reaction, amine nucleophilic substitution reaction, N -alkylation reaction, Vilsmeier-Haack formylation reaction and demethylation reaction to obtain the compound 1; the compound 1 is 1,4-dimethoxybenzene.

[0018] In some embodiments, the preparation of compound 6 comprises the following steps:

[0019] (1) Add the compound 1 to a reaction vessel, then add acetonitrile, cool the reaction vessel at a low temperature below -40°C, and then add N- After iodosuccinimide reacts at low temperature for 10-40 min, trifluoroacetic acid is added to react to obtain compound 2;

[0020] (2) adding the compound 2, 2-fluoroaniline, sodium tert-butyl phosphine, and tris(dibenzylideneacetone)dipalladium-chloroform adduct into a reaction vessel, then adding toluene, and heating to react to obtain compound 3;

[0021] (3) Add the compound 3, iodoethane and sodium hydride into a reaction vessel, and then add N, N - dimethylformamide, in the dark, heated to react to obtain compound 4;

[0022] (4) dissolving the compound 4 in a third organic solvent to prepare a solution a; adding phosphorus oxychloride to a fourth organic solvent, placing the mixture in an ice-water bath for reaction to prepare a solution b; reacting the solution b with the solution a to obtain a compound 5;

[0023] (5) Add the compound 5 to a reaction container, then add dichloromethane (DCM), cool the reaction container at a low temperature below -78°C, and then dropwise add boron tribromide to react to obtain compound 6;

[0024] Wherein, the structure of the compound 2 is: ; The structure of compound 3 is: ; The structure of compound 4 is: ; The structure of compound 5 is: ; The structure of compound 6 is: .

[0025] In some embodiments, in step (2), the reaction is carried out at 110-140°C.

[0026] In some embodiments, in step (2), the reaction time is 10-24 hours.

[0027] In some embodiments, in step (3), the temperature is raised to 90-130°C to react in the dark.

[0028] In some embodiments, in step (3), the reaction time is 10-24 hours.

[0029] In some embodiments, in step (4), the third organic solvent is N, N - dimethylformamide; the fourth organic solvent is N, N -Dimethylformamide.

[0030] The present invention also provides the use of the phenoxazine coumarin derivative as a fluorescent dye.

[0031] The present invention also provides the use of the above-mentioned phenoxazine coumarin derivative as a fluorescent dye in the fields of biological imaging and biomedicine.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention can effectively improve the spectral performance of phenoxazine coumarin derivatives by introducing phenoxazine electron donors.

[0034] The phenoxazine electron donor-based coumarin compounds of the present invention can be successfully used as fluorescent dyes in fluorescence imaging experiments of living cells. In particular, due to the strong "donating-withdrawing" electron effect within the phenoxazine coumarin derivative molecules, they are more sensitive to environmental polarity and can be applied to dual-channel imaging of different polarity positions in cells, showing excellent imaging properties, and have broad application prospects in the fields of cell imaging, biomarkers, and disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The normalized absorption and emission spectra of compound POZ-Cou-1 in different solvents; (a) is the absorption spectrum, and (b) is the emission spectrum;

[0036] Figure 2 The normalized absorption and emission spectra of compound POZ-Cou-2 in different solvents; (a) is the absorption spectrum, and (b) is the emission spectrum;

[0037] Figure 3 The normalized absorption and emission spectra of compound POZ-Cou-3 in different solvents; (a) is the absorption spectrum, and (b) is the emission spectrum;

[0038] Figure 4 The absorption spectra and normalized absorption spectra of compound POZ-Cou-1 in different polar solvents; (a) is the absorption spectrum; (b) is the normalized absorption spectrum;

[0039] Figure 5 The emission spectra and normalized emission spectra of compound POZ-Cou-1 in different polar solvents; (a) is the emission spectrum; (b) is the normalized emission spectrum;

[0040] Figure 6 is the fluorescence spectra of probe POZ-Cou-1 (10 μM) in different ethanol / glycerol solvent systems;

[0041] Figure 7 The fluorescence spectra of probe POZ-Cou-1 (10 μM) in different 1, 4-dioxane / water solvent systems;

[0042] Figure 8 is the fluorescence intensity ratio of 1,4-dioxane / water mixture (I 563 nm / I 613 nm ) and polarity (Δƒ); Excitation wavelength: 464 nm;

[0043] Fig. 9 To study the toxicity of compound POZ-Cou-1 to HeLa cells and 4T1 cells;

[0044] Fig.10 HeLa cells were incubated with the dye POZ-Cou-1 (5 µM) for 30 min. Scale bar = 50 μm. Green channel range 500-550 nm, red channel range 600-700 nm, excitation wavelength 488 nm. DETAILED DESCRIPTION

[0045] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. Example 1

[0047] The synthetic route of compound 6 is as follows:

[0048]

[0049] Its preparation comprises the following steps:

[0050] Preparation of compound 2: In a 500 mL single-necked round-bottom flask, compound 1 (30.0 g, 217.1 mmol) was dissolved in 150 mL acetonitrile and stirred at -40 °C for 30 min. N -Iodosuccinimide (39.1 g, 173.7 mmol) was added to the reaction system in batches within 20 min. After the addition, the mixture was stirred at low temperature for 20 min. Trifluoroacetic acid (5.0 mL, 65.2 mmol) was added and the mixture was reacted at room temperature for 40 h. The reaction was stopped. The reaction solution was dried to obtain a brown-red oily crude product. The crude product was purified by column chromatography (silica gel 200-300 mesh, eluent: V 石油醚 / V 乙酸乙酯 = 40 / 1 to 15 / 1) to separate compound 2, 26.0 g of brown-red oily liquid, with a yield of 45.4%. Characterization data are as follows: 1 H NMR (400 MHz, CDCl 3 )δ 7.34 (d, J =3.0 Hz, 1H), 6.84 (s, 1H), 6.77 – 6.74 (m, 1H), 3.82 (s, 3H), 3.75 (s, 3H).

[0051] Preparation of compound 3: Compound 2 (40.0 g, 151.5 mmol), 2-fluoroaniline (15.3 g, 137.7 mmol), sodium tert-butoxide (17.5 g, 181.7 mmol), tri-tert-butylphosphine (4.2 g, 20.7 mmol) and tris(dibenzylideneacetone)dipalladium-chloroform adduct (2.8 g, 2.8 mmol) were added to a 500 mL three-necked round-bottom flask, and 100.0 mL of dry toluene was added. After heating under reflux at 110°C for 12 h, the reaction was stopped and cooled to room temperature. The reaction solution was directly purified by column chromatography (silica gel 200-300 mesh, eluent: V 石油醚 / V 乙酸乙酯 = 30 / 1 to 10 / 1) to separate compound 3, 30.0 g of light yellow oily liquid, with a yield of 88.1%. Characterization data are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 7.44 (td, J = 8.3,1.4 Hz, 1H), 7.13 – 7.02 (m, 2H), 6.87 (d, J = 2.9 Hz, 1H), 6.84 (s, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.39 (dd, J = 8.8, 2.9 Hz, 1H), 3.86 (s, 3H), 3.75 (s, 3H).

[0052] Preparation of compound 4: Compound 3 (3.0 g, 12.1 mmol), ethyl iodide (2.9 g, 18.1 mmol), and sodium hydride (0.6 g, 24.2 mmol) were added to a 250 mL round-bottom flask, and 60.0 mL of dry N, N -dimethylformamide, the outer wall of the round-bottom flask was covered with tin foil to avoid light, and the reaction was carried out at 100 °C for 12 h, and the reaction was stopped; the reaction solution was poured into 100.0 mL of saturated NaCl solution, extracted with dichloromethane (40.0 mL × 4), dried over anhydrous sodium sulfate, and dried under reduced pressure to obtain a crude product, which was then purified by column chromatography (eluent: V 石油醚 / V 二氯甲烷 = 5 / 1 to 2 / 1) to obtain compound 4, 2.1 g of yellow oily liquid, with a yield of 63.2%. The characterization data are as follows: 1 H NMR (400 MHz, CDCl 3) δ 7.02 – 6.98 (m, 1H), 6.98 – 6.96(m, 1H), 6.95 (t, J = 1.9 Hz, 1H), 6.84 (d, J = 3.7 Hz, 1H), 6.83 – 6.80 (m,1H), 6.62 (d, J = 3.0 Hz, 1H), 6.58 (dd, J = 8.7, 3.0 Hz, 1H), 3.74 – 3.66(m, 8H), 1.20 (t, J = 7.1 Hz, 3H).

[0053] Preparation of compound 5: Compound 4 (1.0 g, 3.6 mmol) was added to a 50 mL round-bottom flask and 20 mL of dry N, N - dimethylformamide was dissolved, and the reaction system was placed in an ice-water bath and stirred to cool; N, N -Dimethylformamide (3.4 mL, 43.6 mmol) was added to a 10 mL round-bottom flask, and the mixture was placed in an ice-water bath and stirred for 10 min. Phosphorus oxychloride (2.1 mL, 21.8 mmol) was added to the 10 mL round-bottom flask, and the reaction solution gradually turned into an orange-red viscous liquid. After continuing to stir in the ice-water bath for 30 min, the reaction intermediate was transferred to the reaction system by aspiration, and the reaction system instantly turned purple, and then quickly turned into yellow-green. After the reaction system was stirred in the ice water bath for 10 min, it was transferred to an oil bath and reacted at 60°C for 10 h. The reaction was stopped and the reaction solution was poured into 200.0 mL of saturated NaCl solution and extracted with dichloromethane (40.0 mL × 4). The organic phase was washed with saturated NaCl solution 6 times, and the organic phase was dried over anhydrous sodium sulfate and dried under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (eluent: V petroleum ether / V dichloromethane = 2 / 1 to 1 / 1) to obtain compound 5, 0.65 g of yellow solid product, and the yield was 61.5%. The characterization data are as follows: 1 H NMR (400 MHz, CDCl3 δ 10.30 (s, 1H), 7.31 (s, 1H), 7.12 – 7.05 (m, 4H), 6.49 (s, 1H), 3.86 – 3.80 (m, 5H), 3.62 (s, 3H), 1.26(s, 3H).

[0054] Preparation of compound 6: Compound 5 (0.1 g, 0.3 mmol) was added to a 50 mL round-bottom flask, 10 mL of dry dichloromethane was added, the reaction system was placed in a -78°C low-temperature stirrer for cooling, and boron tribromide (2.0 mL, 4.0 mmol) was added dropwise. The reaction system gradually turned yellow-green. After the addition was completed, stirring was continued at low temperature for 15 min, and then the reaction system was transferred to room temperature and stirred for 10 h. The reaction was stopped, and the reaction solution was poured into 150.0 mL of ice water, fully shaken, and the liquid was quickly separated. The organic phase was blown dry with Ar gas to obtain 0.09 g of a green solid product with a yield of 88.2%. Characterization data are as follows: 1 H NMR (400MHz, CDCl 3 ) δ 10.89 (s, 1H), 9.70 (s, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 4H),7.03 (s, 1H), 6.65 (s, 1H), 3.65 (q, J = 7.0 Hz, 2H), 1.22 (t, J = 7.1 Hz,3H). Example 2

[0055] The synthetic route of phenoxazine coumarins is as follows:

[0056]

[0057] The preparation method is as follows:

[0058] Preparation of compound POZ-Cou-1: In a 100 mL two-necked round-bottom flask, compound 6 (300 mg, 0.9 mmol) and anhydrous potassium carbonate (200 mg, 1.5 mmol) were added, and 15.0 mL of dry N, N -dimethylformamide, a double-necked round-bottom flask connected to a condenser, reacted at 120 °C for 12 h, stopped the reaction, cooled to room temperature, poured the reaction solution into 150.0 mL of saturated NaCl solution, quickly extracted with dichloromethane (50.0 mL × 2), dried the organic phase with anhydrous sodium sulfate, and dried the organic phase with Ar to obtain a brown-yellow viscous liquid compound 7; the obtained compound 7 and diethyl malonate (300 mg, 1.9 mmol) were added to a 100 mL single-mouth round-bottom flask, dissolved in 40.0 mL of anhydrous ethanol, added 120.0 μL of piperidine, heated under reflux for 12 h, stopped the reaction, cooled to room temperature, and the reaction solution was dried under reduced pressure to obtain a crude product, which was purified by column chromatography (eluent: V 石油醚 :V 二氯甲烷= 2 / 1 to 0 / 1) to obtain the product, POZ-Cou-1, an orange solid, 45 mg, with a two-step total yield of 14.9%. The characterization data are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 8.29 (s, 1H), 6.87 (td, J = 7.8, 1.5 Hz, 1H), 6.80 (td, J = 7.7, 1.3 Hz, 1H), 6.70 (dd, J = 6.9, 2.4 Hz, 2H), 6.64 (dd, J =7.9, 1.0 Hz, 1H), 6.39 (s, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.67 (q, J = 7.1Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz,CDCl 3 ) δ 162.71, 156.51, 153.88, 147.00, 143.32, 140.93, 138.75, 128.60,123.10, 122.22, 114.82, 111.37, 111.34, 111.13, 109.64, 96.91, 60.50, 38.36,13.31, 9.19. HRMS (ESI) m / z calcd for C 20 H 17 NNaO 5 + [M+Na] + , 374.0999; found,374.0923.

[0059] Preparation of compound POZ-Cou-2: In a 100 mL two-necked round-bottom flask, compound 6 (75 mg, 0.23 mmol) and anhydrous potassium carbonate (50 mg, 0.38 mmol) were added, and 15.0 mL of dry N, N-dimethylformamide, a double-necked round-bottom flask connected to a condenser, reacted at 120 °C for 12 h, stopped the reaction, cooled to room temperature, poured the reaction solution into 100.0 mL of saturated NaCl solution, quickly extracted with dichloromethane (30.0 mL × 2), dried the organic phase with anhydrous sodium sulfate, and dried the organic phase with Ar to obtain a brown-yellow viscous liquid compound 7; the obtained compound 7 and ethyl acetoacetate (50 mg, 0.38 mmol) were added to a 100 mL single-mouth round-bottom flask, dissolved in 30.0 mL of anhydrous ethanol, added 120.0 μL of piperidine, heated under reflux for 12 h, stopped the reaction, cooled to room temperature, and the reaction solution was dried under reduced pressure to obtain a crude product, which was purified by column chromatography (eluent: V 石油醚 :V 二氯甲烷 = 2 / 1 to 0 / 1) to obtain the product, POZ-Cou-2, orange solid, 10 mg, with a two-step total yield of 13.8%. The characterization data are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 8.29 (s, 1H), 6.88 (t, J = 7.4 Hz, 1H), 6.81(t, J = 7.4 Hz, 1H), 6.74 – 6.68 (m, 2H), 6.65 (d, J = 7.8 Hz, 1H), 6.39 (s,1H), 3.68 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.31 (t, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 195.51, 160.12, 155.31, 146.74, 144.40, 142.09, 140.02,129.53, 124.14, 123.42, 118.93, 115.93, 112.93, 112.47, 111.20, 97.80, 39.45,30.62, 10.30. HRMS (ESI) m / z calcd for C 19 H 15 NNaO 4 + [M+Na] + , 344.0894; found,344.0823.

[0060] Preparation of compound POZ-Cou-3: In a 100 mL two-necked round-bottom flask, compound 6 (100 mg, 0.36 mmol) and anhydrous potassium carbonate (60 mg, 0.44 mmol) were added, and 20.0 mL of dry N, N -dimethylformamide, a double-necked round-bottom flask connected to a condenser, reacted at 120 °C for 12 h, stopped the reaction, cooled to room temperature, poured the reaction solution into 150.0 mL of saturated NaCl solution, quickly extracted with dichloromethane (40.0 mL × 2), dried the organic phase with anhydrous sodium sulfate, and dried the organic phase with Ar to obtain a brown-yellow viscous liquid compound 7; the obtained compound 7 and 2-cyanobenzimidazole (60 mg, 0.03 mmol) were added to a 100 mL single-necked round-bottom flask, dissolved in 40.0 mL of anhydrous ethanol, added 120.0 μL of piperidine, heated under reflux for 12 h, stopped the reaction, cooled to room temperature, and the reaction solution was dried under reduced pressure to obtain a crude product, which was purified by column chromatography (eluent: V 石油醚 :V 二氯甲烷 = 2 / 1 to 0 / 1) to obtain the product, POZ-Cou-3, yellow solid, 10 mg, with a total yield of 10.1% in two steps. The characterization data are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 8.01 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.35 (s, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.93 (s, 1H), 6.87 (t, J = 7.4 Hz,1H), 6.74 (dd, J = 14.8, 7.2 Hz, 1H), 6.67 (s, 1H), 6.60 (d, J = 7.8 Hz, 1H), 3.31 (dd, J = 11.2, 5.5 Hz, 1H), 0.87 (d, J = 5.9 Hz, 1H). 13 C NMR (101 MHz, DMSO) δ 151.96, 151.48, 144.14, 143.98, 141.07, 134.46, 131.37, 130.12,124.93, 122.20, 118.83, 115.73, 113.03, 112.98, 105.03, 95.46, 91.15, 31.77,31.62, 30.28, 29.51, 29.18, 22.58, 9.97. HRMS (ESI) m / z calcd for C 24 H18 N 3 O 3 + [M+H] + , 396.1343; found, 396.1276.

[0061] MTT cytotoxicity test:

[0062] The cytotoxicity of compound POZ-Cou-1 was analyzed by MTT method. The experimental operation process was as follows: 1. HeLa cells were inoculated into 96-well plates; 2. Incubate at 37 °C, 5% CO 2 After incubation for 24 h under the conditions, the dye POZ-Cou-1 was added at concentrations of 0.0, 5.0, 10.0, 15.0 and 20.0 µM, respectively. Twelve replicate wells were set for each concentration gradient, and the incubation continued for 24 h; 3. The culture medium was aspirated, and 100 µL of MTT solution (0.5 mg / mL, the solvent was the culture medium) was added to each well, and the culture was continued for 4 h; 4. The culture medium was aspirated, and 100 µL of DMSO was added to each well, and the cells were placed on a shaker for 10 min to fully dissolve the crystals; 5. 490 nm was selected as the detection wavelength, and the absorbance value of each well was measured on an enzyme immunoassay monitor.

[0063] Cell culture and fluorescence imaging:

[0064] HeLa cells were cultured in a humidified cell culture incubator (37 °C and 5% CO 2 ), the culture medium is DMEM containing 10% fetal bovine serum and 2% penicillin and streptomycin. The cells were inoculated into the confocal culture dish and cultured for 24 h before the cells adhered to the wall for fluorescence imaging experiments. Before use, the cells were rinsed with PBS buffer.

[0065] Laser confocal microscopy imaging: The cells were incubated with the dye POZ-Cou-1 (5.0 µM) at 37 °C for 30 min, and the cells were rinsed with PBS buffer before laser confocal imaging experiments. Dye POZ-Cou-1, red fluorescence channel: λ em =600-700 nm, green fluorescence channel: λ em = 500-550 nm, λ ex = 488 nm.

[0066] Study on the Spectral Properties of Compounds POZ-Cou-1~3

[0067] Firstly, the compounds POZ-Cou-1~3 were studied in different solvents (DCM, EtOH, CH 3 CN and DMSO), the results are shown in Table 1 and Figure 1-Figure 3 shown.

[0068] Table 1 Photophysical properties of compounds POZ-Cou-1~3 in different solvents

[0069]

[0070] As shown in Table 1 and Figure 1-Figure 3 As shown in the figure, the absorption peaks of compounds POZ-Cou-1 and POZ-Cou-2 are around 470 nm, while the absorption peak of POZ-Cou-3 is at 430 nm, indicating that compound POZ-Cou-3 has a 40 nm blue shift relative to POZ-Cou-1 and POZ-Cou-2. The emission light of compounds POZ-Cou-1, POZ-Cou-2 and POZ-Cou-3 are all in the orange-yellow to red region, and have high quantum yields (in dichloromethane, their fluorescence quantum yields are 67.0%, 65.0%, and 60.0%, respectively) and high molar extinction coefficients (about 10 4 M -1 cm -1 ).

[0071] The results of the study on the spectral properties of compounds POZ-Cou-1, POZ-Cou-2 and POZ-Cou-3 in different solvents show that the emission spectrum exhibits a slight red shift with the increase of solvent polarity. This series of compounds has a large Stokes shift, especially in solvents with greater polarity, such as the Stokes shift of POZ-Cou-3 in acetonitrile can reach 159nm.

[0072] Research has shown that a large Stokes shift means that fluorescent molecules release less energy after being excited, thereby reducing potential photodamage to biological samples, which is particularly important for bioimaging and biomedical research.

[0073] Subsequently, the changes in the absorption and emission spectra of the compound POZ-Cou-1 in different polar solvents were investigated. Figure 4 and Figure 5 As shown. Figure 4 , the compound POZ-Cou-1 has a maximum absorption at 455 nm in solvents with lower polarity, such as 1, 4-dioxane, and the maximum absorption is red-shifted to 469 nm in solvents with higher polarity, such as MeOH and DMSO. The maximum absorption wavelength of the compound POZ-Cou-1 in solvents with different polarity does not change significantly; however, the maximum emission wavelength of the compound POZ-Cou-1 in solvents with different polarity has a larger red shift, such as Figure 5 As shown, in highly polar organic solvents such as DMSO, CH 3CN and others exhibit red fluorescence, while in solvents with lower polarity, such as 1,4-dioxane and THF, they exhibit yellow-green fluorescence.

[0074] Based on the above-mentioned spectral property research, it was found that the emission spectrum of compound POZ-Cou-1 would change significantly in different polar solvents. Therefore, we conducted an in-depth study on the relationship between compound POZ-Cou-1 and polar response. First, viscosity, as one of the key elements of the cell microenvironment, often has a significant effect on fluorescence intensity. Therefore, in order to eliminate the effect of cell viscosity on polarity, we tested compound POZ-Cou-1 in systems with different ethanol and glycerol ratios. The results are as follows: Figure 6 As shown in the figure, both ethanol and glycerol have great polarity, but the viscosity of glycerol is much greater than that of ethanol. As the glycerol content in the system increases, the fluorescence intensity of the compound POZ-Cou-1 is always at a low level. The above results show that the response of the compound POZ-Cou-1 to polarity is largely independent of the environmental viscosity.

[0075] Subsequently, the fluorescence behavior of compound POZ-Cou-1 in solutions of different polarities was investigated to explore its polarity effect. Figure 7 As shown in the figure, the fluorescence intensity of the compound POZ-Cou-1 increases with the increase of the proportion of 1, 4-dioxane in water (from 10% to 100%), and its fluorescence color also changes from red to yellow. Since dyes with D-π-A structure usually produce a large dipole moment, the dipole moment will also show significant differences in different polar environments as the solvation energy changes. The study found that the fluorescence intensity ratio (I 563nm / I 613nm ) has a good linear relationship with the polarity Δƒ in the range of 0.0205 (100% 1, 4-dioxane) to 0.2774 (70% 1, 4-dioxane) (R 2 = 0.994) (e.g. Figure 8 ). The above research results show that compound POZ-Cou-1 can perform quantitative ratiometric fluorescence detection on solvents of different polarities.

[0076] Cell imaging

[0077] Based on the above excellent optical properties, phenoxazine coumarin POZ-Cou-1 was selected for fluorescence imaging of HeLa cells and fluorescence imaging experiments of different polar regions of cells. First, the cytotoxicity of the two dyes was investigated, such as Fig. 9 As shown, after incubating cells with 20 µM POZ-Cou-1 for 24 h, the cells still maintained viability and the survival rate could reach over 85%, proving that the cytotoxicity of both was relatively low.

[0078] After the compound POZ-Cou-1 was co-stained and incubated with the cells for 30 min, dual-channel imaging images were collected under a confocal imaging microscope in different fields of view. Fig.10 The green channel shows obvious granular fluorescence, which may be due to the fact that there are more lipid-rich areas and the dye shows green light when the polarity is low. In sharp contrast, the red channel fluorescence is more dispersed, which may be due to the fact that there are more aqueous phase areas in the cells. The compound POZ-Cou-1 shows red fluorescence in areas with high polarity. The above experiments show that dual-channel polarity region difference imaging can give us a deeper understanding of the polarity distribution in cells, which helps to visualize the changes in polarity distribution in cells under disease conditions.

[0079] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A phenoxazine coumarin derivative, characterized in that: It has the following structure: (Formula I); Wherein, R is one of -COOEt and -COCH3.

2. The method for preparing the phenoxazine coumarin derivative according to claim 1, characterized in that: The following steps are involved: (1) Compound 1 was added to a reaction vessel, followed by acetonitrile. The reaction vessel was cooled at a low temperature below -40°C, and then the following were added in batches: N- After iodosuccinimide reacts at low temperature for 10-40 min, trifluoroacetic acid is added to react to obtain compound 2; (2) adding the compound 2, 2-fluoroaniline, sodium tert-butyl phosphine, and tris(dibenzylideneacetone)dipalladium-chloroform adduct into a reaction vessel, then adding toluene, and heating to react to obtain compound 3; (3) Add the compound 3, ethyl iodide and sodium hydride into a reaction vessel, and then add N, N - dimethylformamide, in the dark, heated to react to obtain compound 4; (4) dissolving the compound 4 in a third organic solvent to prepare a solution a; adding phosphorus oxychloride to a fourth organic solvent, placing the mixture in an ice-water bath for reaction to prepare a solution b; reacting the solution b with the solution a to obtain a compound 5; (5) Add the compound 5 to a reaction container, then add dichloromethane, cool the reaction container at a low temperature below -78°C, and then dropwise add boron tribromide to react to obtain compound 6; (6) adding compound 6 and anhydrous potassium carbonate into a first organic solvent and heating the mixture to react to obtain compound 7; (7) adding compound 7 obtained in step (6), compound A and piperidine into a second organic solvent and heating the mixture to react to obtain a phenoxazine coumarin derivative having a structure of formula I; Wherein, the compound 1 is 1,4-dimethoxybenzene; the structure of the compound 2 is: ; The structure of compound 3 is: ; The structure of compound 4 is: ; The structure of compound 5 is: ; The structure of the compound 6 is: ; The structure of compound 7 is: ; The compound A is one of diethyl malonate and ethyl acetoacetate; the structure of the phenoxazine coumarin derivative having the structure of formula I is: .

3. The method for preparing a phenoxazine coumarin derivative according to claim 2, characterized in that: The first organic solvent is N, N -dimethylformamide; the second organic solvent is anhydrous ethanol.

4. The method for preparing a phenoxazine coumarin derivative according to claim 2, characterized in that: In step (6), the reaction temperature is 100-140°C; and / or the reaction time is 10-24 h.

5. The method for preparing the phenoxazine coumarin derivative according to claim 2, characterized in that: In step (7), the reaction temperature is 90-100°C; and / or the reaction time is 10-24 h.

6. Use of the phenoxazine coumarin derivative according to claim 1 as a fluorescent dye in the field of bioimaging and biomedicine for purposes other than disease diagnosis or treatment.

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