Rhodamine derivative as well as preparation method and application thereof

Abstract

The invention discloses a rhodamine derivative and a preparation method and application thereof.The rhodamine derivative has the structure shown in the following formula, R is selected from one of R1, R2 or R3: # imgabs0. The rhodamine derivative has the advantages of being longer in ultraviolet absorption wavelength, longer in fluorescence emission wavelength and the like compared with rhodamine; the fluorescent probe can be used for research in the fields of fluorescent probes and fluorescent imaging.

Description

Technical Field

[0001] The present invention belongs to the field of chemical technology, and particularly relates to a rhodamine derivative, a preparation method thereof and an application thereof. Background Art

[0002] With the continuous innovation of life science and technology, various new imaging and analysis technologies have emerged. Among them, as one of the emerging analysis technologies, fluorescence imaging technology has the excellent characteristics of non-invasive, real-time observation, high sensitivity and high selectivity, and has been widely used in the fields of biology, medicine and environmental science. The key to this technology is to design and synthesize fluorescent dye molecular materials that meet various requirements.

[0003] Among various traditional fluorescent dyes, rhodamine series fluorescent dyes have attracted much attention due to their high molar absorption coefficient, high fluorescence quantum yield, good photostability and good cell membrane permeability, and are widely used in research fields such as fluorescent probes and biomolecule labeling. However, typical rhodamine dyes, such as rhodamine B, rhodamine 6G, and rhodamine 110, have their absorption and emission wavelengths in the visible region (500 - 600 nm), resulting in the disadvantage of poor tissue penetration in imaging applications, especially in in vivo imaging, the situation is more serious.

[0004] Therefore, how to synthesize new rhodamine fluorescent dyes through chemical synthesis means to red-shift their absorption and emission wavelengths and have good photophysical properties to meet the detection of small molecules in vivo and in vivo fluorescent imaging labeling has very important scientific significance and practical value. Summary of the Invention

[0005] Based on the above reasons, the present invention proposes a rhodamine derivative, a preparation method thereof and an application thereof. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0006] On the one hand, the present invention relates to a rhodamine derivative having the structure shown in the following formula, wherein R is selected from one of R1, R2 or R3:

[0007]

[0008] In a preferred embodiment of the present invention, the R is R1. When R is R1, the dye has a high fluorescence quantum yield in water, so it is preferred.

[0009] On the other hand, the present invention relates to a preparation method of the above rhodamine derivative, which comprises the following steps:

[0010]

[0011] In a preferred embodiment of the present invention, the reaction conditions for the first step in the above steps are as follows: Dissolve 3-bromo-N,N-diethylaniline, 3-diethylaminophenol, copper(I) iodide, 2-pyridinecarboxylic acid, and potassium phosphate in dimethyl sulfoxide. Under a nitrogen protection condition, heat the reaction solution to 85 - 95 °C and react for 20 - 24 hours; after the reaction solution is cooled to room temperature, neutralize it with saturated ammonium chloride solution, extract it with ethyl acetate for several times, combine the organic phases and dry them with anhydrous magnesium sulfate; remove the solvent by vacuum distillation, and perform silica gel column separation with ethyl acetate and petroleum ether to obtain Compound 1.

[0012] In a preferred embodiment of the present invention, the reaction conditions for the second step in the above steps are as follows: Dissolve Compound 1 in acetonitrile under an ice bath, and then add N-bromosuccinimide in batches and stir well for 20 - 40 minutes; after quenching the reaction with saturated sodium bicarbonate solution, extract it with dichloromethane, combine and dry the organic phases, and then perform column separation with ethyl acetate and petroleum ether to obtain Compound 2.

[0013] In a preferred embodiment of the present invention, the reaction conditions for the third step in the above steps are as follows: Dissolve Compound 2 in dry tetrahydrofuran, cool it to below -70 °C with an acetone - liquid nitrogen bath, then dropwise add n-butyllithium solution and stir the reaction for 0.8 - 1.5 hours; dropwise add a tetrahydrofuran solution containing N,N-dimethylformamide, slowly warm it back to room temperature and react for 1.5 - 2.5 hours, and then acidify it with dilute hydrochloric acid; extract the reaction solution with dichloromethane, combine the organic phases and dry them with anhydrous sodium sulfate; perform column chromatography separation with ethyl acetate and petroleum ether to obtain Compound 3.

[0014] In a preferred embodiment of the present invention, the reaction conditions for the fourth step in the above steps are as follows: Dissolve N-methylbenzimidazole in dry tetrahydrofuran, cool it to below -70 °C with an acetone - liquid nitrogen bath, then dropwise add n-butyllithium solution and stir the reaction for 0.8 - 1.5 hours; dropwise add a tetrahydrofuran solution containing Compound 3, slowly warm it back to room temperature, react for 1.5 - 2.5 hours, and then acidify it with dilute hydrochloric acid; extract the reaction solution with dichloromethane three times, combine the organic phases, and dry them with anhydrous sodium sulfate; perform column chromatography separation with dichloromethane and methanol to obtain the target compound.

[0015] On the other hand, the present invention also relates to the application of rhodamine derivatives in fluorescence imaging.

[0016] The beneficial effects of the present invention are as follows: The rhodamine derivatives of the present invention show advantages such as a longer ultraviolet absorption wavelength and a longer fluorescence emission wavelength compared to rhodamine, and can be used in the research fields of fluorescence probes and fluorescence imaging. Description of the Drawings

[0017] Figure 1 : The 1 1H NMR spectrum of Compound OBM1;

[0018] Figure 2 : 13 C NMR spectrum of compound OBM1;

[0019] Figure 3 : HRMS spectrum of compound OBM1;

[0020] Figure 4 : 1 H NMR spectrum of compound OBM2;

[0021] Figure 5 : 13 C NMR spectrum of compound OBM2;

[0022] Figure 6 : HRMS spectrum of compound OBM2;

[0023] Figure 7 : [[ID= thirty]] 1 H NMR spectrum of compound OBM3;

[0024] Figure 8 : 13 C NMR spectrum of compound OBM3;

[0025] Figure 9 : HRMS spectrum of compound OBM3;

[0026] Figure 10 : UV absorption spectra of compounds OBM1, OBM2 and OBM3;

[0027] Figure 11 : Fluorescence emission spectra of compounds OBM1, OBM2 and OBM3. Detailed implementation mode

[0028] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0030] Example 1:

[0031] The preparation method of the dye is carried out according to the following steps:

[0032]

[0033] 3-Bromo-N,N-diethylaniline (6.0 g, 36 mmol), 3-diethylaminophenol (6.84 g, 30 mmol), copper(I) iodide (576 mg, 3 mmol), 2-pyridinecarboxylic acid (739 mg, 6 mmol), and potassium phosphate (12.7 g, 60 mmol) were dissolved in dimethyl sulfoxide (DMSO, 50 mL), and nitrogen was purged. The reaction mixture was heated to 90 °C and stirred for 20 - 24 hours. After the reaction was completed, the cooled reaction mixture was poured into 500 mL of saturated ammonium chloride solution, and extracted 5 times with ethyl acetate (100 mL). The combined organic phases were dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography using a mixture of ethyl acetate and petroleum ether (volume ratio 1:100) as the eluent, and the solvent was removed under reduced pressure to obtain compound 1 (3.2 g, yield 38%).

[0034]

[0035] Compound 1 (3.12 g, 10 mmol) was dissolved in acetonitrile (100 mL) under an ice bath, and then N-bromosuccinimide (NBS, 3.74 g, 21 mmol) was added portionwise with vigorous stirring for 30 minutes. After the reaction was quenched with saturated sodium bicarbonate solution, the mixture was extracted with dichloromethane (100 mL × 3). The combined dried organic phases were separated by column chromatography using a mixture of ethyl acetate and petroleum ether (volume ratio 1:15) as the eluent, and the solvent was removed under reduced pressure to obtain compound 2 (4.2 g, yield 89%).

[0036]

[0037] Compound 2 (4.7 g, 10 mmol) was dissolved in 100 ml of dry tetrahydrofuran. The solution was cooled to -78 °C in an acetone - liquid nitrogen bath, and then n-butyllithium solution (1.0 mol / L THF solution, 47 ml) was added dropwise. The mixture was stirred for 1 hour. A solution of N,N-dimethylformamide (DMF, 730 mg, 10 mmol) in tetrahydrofuran (5 ml) was added dropwise, and the mixture was slowly warmed to room temperature (25 °C) and stirred for 2 hours. Then the reaction mixture was acidified with 2 mol / L dilute hydrochloric acid. The reaction mixture was extracted with dichloromethane (100 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate. The residue was separated by column chromatography using a mixture of ethyl acetate and petroleum ether (volume ratio 1:5) as the eluent, and the solvent was removed under reduced pressure to obtain compound 3 (1.2 g, yield 35%).

[0038]

[0039] Dissolve N-methylbenzimidazole (0.39 g, 3 mmol) in 10 mL of dry tetrahydrofuran. Cool the solution to -78 °C in an acetone-nitrogen liquid bath, then add n-butyllithium solution (1.0 mol / L THF solution, 3.3 mL) dropwise and stir the reaction for 1 hour. Dropwise add a tetrahydrofuran solution (5 mL) containing compound 3 (0.34 g, 0.3 mmol), slowly warm the reaction mixture to room temperature, and react for 2 hours. Then acidify the reaction mixture with 2 mol / L dilute hydrochloric acid. Extract the reaction solution with dichloromethane three times, combine the organic phases, and dry over anhydrous sodium sulfate. Separate by column chromatography using dichloromethane and methanol with a volume ratio of 1:20 as the eluent, and remove the solvent under reduced pressure to obtain compound OBM1 (0.15 g, yield 33%).

[0040] The characterization results of OBM1 are as follows:

[0041] 1 H NMR (600 Hz, CDCl3) δ 7.91 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.37 (d, J = 9.6 Hz, 2H), 7.01 (d, d, J1 = 1.8 Hz, J2 = 9.0 Hz, 2H), 6.94 (d, J = 2.4 Hz, 2H), 3.77 (s, 3H), 3.71 (q, J = 7.2 Hz, 8H), 1.36 (t, J = 7.2 Hz, 12H); 13 C NMR (150 MHz, CDCl3) δ 157.8, 155.8, 144.6, 143.3, 142.2, 136.0, 131.4, 124.7, 123.6, 120.5, 115.2, 113.8, 110.5, 97.0, 46.5, 31.9, 12.7; ESI-MS [M] + : calcd for 453.2654, Found 453.2648.

[0042] Synthesize OBM2 and OBM3 in the same way.

[0043]

[0044] The yield of OBM2 is 49%, and its characterization results are as follows: 11H NMR (600 Hz, CDCl3) δ 7.92 (d, J = 10.8 Hz, 1H), 7.64 (d, J = 11.4 Hz, 1H), 7.48 (m, 2H), 7.34 (d, J = 14.4 Hz, 2H), 7.20 (d, J = 9.6 Hz, 2H), 7.03 (d, d, J1 = 3.6 Hz, J2 = 11.4 Hz, 2H), 6.93 (d, J = 3.6 Hz, 2H), 6.85 (d, d, J1 = 2.4 Hz, J2 = 8.4 Hz, 2H), 5.53 (s, 2H), 4.35 (s, 2H), 3.63 (q, J = 7.2 Hz, 8H), 1.33 (t, J = 7.2 Hz, 12H); 13 13C NMR (150 MHz, CDCl3) δ 157.8, 155.8, 144.6, 143.2, 136.1, 135.3, 131.2, 128.1, 127.7, 125.2, 123.9, 120.59, 114.9, 113.9, 111.2, 97.0, 73.4, 70.7, 46.3, 12.7; ESI-MS [M] + : calcd for 559.3067, Found 559.3068.

[0045] The yield of OBM3 was 60%, and its characterization results were as follows: 1 1H NMR (600 Hz, CDCl3) δ 7.92 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.2 Hz, 2H), 7.44 (t, J = 7.2 Hz, 1H), 7.32 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.6 Hz, 2H), 6.90 (s, 2H), 4.16 (t, J = 7.2 Hz, 2H), 3.74 (s, 4H), 3.66 (m, 8H), 1.70 (t, J = 7.2 Hz, 2H), 1.70 (s, 2H), 1.36 (t, J = 6.0 Hz, 12H); 13 13C NMR (150 MHz, CDCl3) δ 158.0, 155.9, 144.3, 143.7, 143.5, 135.0, 131.0, 124.6, 123.5, 120.6, 115.1, 112.9, 111.0, 97.0, 65.1, 57.2, 52.7, 46.2, 44.8, 27.1, 22.1, 12.6; ESI-MS [M] + : calcd for 580.3646, Found 580.3649.

[0046] Example 2: Determination of the Optical Properties of Benzimidazole-Functionalized Rhodamine Fluorescent Dyes

[0047] 1. Take an appropriate amount of OBM1-3 product and dissolve it in dimethyl sulfoxide to prepare a stock solution with a concentration of 2 mmol / L. -1 Take 2 μl of the stock solution into a sample cell and dilute it to 2 ml with the solvent to obtain a test solution with a concentration of 2 μmol / L. Measure its absorption spectrum and emission spectrum in solvents (water, acetonitrile, ethanol, dichloromethane) and perform normalization. The absorption and emission spectra of dyes OBM1-3 in water are as shown in -1 and Figure 10 and 11 . The photophysical properties in other solvents are shown in Table 1.

[0048] 2. Determination of the Relative Fluorescence Quantum Yield of Benzimidazole-Functionalized Rhodamine Fluorescent Dyes

[0049] Take an appropriate amount of OBM1-3 product and dissolve it in dimethyl sulfoxide to prepare a stock solution with a concentration of 2 mmol / L. -1 Take an appropriate amount of the stock solution into a sample cell, dilute it with the solvent to control its absorbance intensity to be less than 0.05, then measure its emission spectrum with an excitation wavelength of 590 nm and a collection wavelength range of 600 - 700 nm, and calculate the integral area of its emission spectrum. Using cresyl violet dye as a reference (Φs = 0.57 in ethanol), measure the quantum yield of the rhodamine fluorescent dye in different solvents. The experimental results are shown in Table 1.

[0050] Table 1: Determination Results of the Optical Properties of Dyes

[0051]

[0052]

[0053] The above experimental results show that introducing an electron-withdrawing benzimidazole derivative at the 9-position of the xanthene skeleton of the rhodamine dye to synthesize benzimidazole-functionalized rhodamine fluorescent dyes not only has the advantages of rhodamine dyes, such as large molar absorption coefficients and high fluorescence quantum yields, but also has a significant red shift in absorption and emission wavelengths compared with traditional rhodamines.

[0054] The preferred embodiments of the present invention have been described above, but they are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A rhodamine derivative having a structure represented by the following formula, wherein, R is selected from one of R1, R2 or R3:

2. The rhodamine derivative according to claim 1, wherein R is R1.

3. A method for preparing the rhodamine derivative according to claim 1 or 2, comprising the following steps:

4. For the preparation method according to claim 3, the reaction conditions for the first step in the above steps are: Dissolve 3-bromo-N,N-diethylaniline, 3-diethylaminophenol, copper(I) iodide, 2-pyridinecarboxylic acid, and potassium phosphate in dimethyl sulfoxide. Under nitrogen protection, heat the reaction solution to 85 - 95 °C and react for 20 - 24 hours; After the reaction solution is cooled to room temperature, neutralize it with saturated ammonium chloride solution, extract it with ethyl acetate once, combine the organic phases and dry them with anhydrous magnesium sulfate; Distill off the solvent under reduced pressure, and perform silica gel column separation with ethyl acetate and petroleum ether to obtain compound 1.

5. For the preparation method according to claim 3, the reaction conditions for the second step in the above steps are: Dissolve compound 1 in acetonitrile under an ice bath, then add N-bromosuccinimide in batches and stir well for 20 - 40 minutes; After the reaction is quenched with saturated sodium bicarbonate solution, extract it with dichloromethane, combine and dry the organic phases, and then perform column separation with ethyl acetate and petroleum ether to obtain compound 2.

6. For the preparation method according to claim 3, the reaction conditions for the third step in the above steps are: Dissolve compound 2 in dry tetrahydrofuran, cool it to below -70 °C with an acetone - liquid nitrogen bath, then dropwise add n-butyllithium solution and stir for 0.8 - 1.5 hours; Dropwise add a tetrahydrofuran solution containing N,N-dimethylformamide, slowly warm it to room temperature and react for 1.5 - 2.5 hours, then acidify it with dilute hydrochloric acid; Extract the reaction solution with dichloromethane, combine the organic phases and dry them with anhydrous sodium sulfate; Perform column chromatography with ethyl acetate and petroleum ether to obtain compound 3.

7. For the preparation method according to claim 3, the reaction conditions for the fourth step in the above steps are: Dissolve N-methylbenzimidazole in dry tetrahydrofuran, cool it to below -70 °C with an acetone - liquid nitrogen bath, then dropwise add n-butyllithium solution and stir for 0.8 - 1.5 hours; Dropwise add a tetrahydrofuran solution containing compound 3, slowly warm it to room temperature, react for 1.5 - 2.5 hours, then acidify it with dilute hydrochloric acid; Extract the reaction solution with dichloromethane 3 times, combine the organic phases, and dry them with anhydrous sodium sulfate; Perform column chromatography with dichloromethane and methanol to obtain the target compound.

8. The application of the rhodamine derivative according to claim 1 or 2 in fluorescence imaging.

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