High brightness and high stability fluorescent dyes based on dimethylacridine donor

Abstract

The present application relates to the technical field of fluorescent dyes, and particularly relates to a kind of high-brightness and high-stability fluorescent dyes based on dimethylacridine donor, including rhodamine fluorescent dyes, coumarin fluorescent dyes and coumarin fluoroboride fluorescent dyes.This kind of fluorescent dyes has long absorption and emission wavelength, high fluorescence quantum efficiency and high stability.The fluorescent dyes with dimethylacridine as electron donor designed in the present application generally have longer absorption and emission wavelength, higher fluorescence quantum efficiency and high stability compared with traditional diethylamine group and guanidinium group donor fluorescent dyes.These advantages make it have potential application in high-resolution biological imaging and biosensing fields.Based on the donor, symmetric and asymmetric rhodamine fluorescent dyes, coumarin fluorescent dyes and coumarin fluoroboride dyes are developed, and their photochemical physical properties are tested in detail, and they are successfully applied to long-time sustained high-resolution fluorescence imaging in cells.

Description

Technical Field

[0001] This invention relates to the field of fluorescent dye technology, and more specifically to a class of high-brightness and high-stability fluorescent dyes based on dimethylacridine donors. Background Technology

[0002] Fluorescent dyes possess advantages such as high detection sensitivity, fast response time, good biocompatibility, and high modifiability, making them widely used and highly advantageous in the biological field. Based on traditional fluorescent precursors, such as coumarin, quinoline, fluorescein, rhodamine, cyanine, squaric acid, and bodily dyes, many widely used small-molecule fluorescent dyes have been developed. However, these traditional dyes have some drawbacks, such as short wavelengths (less than 600 nm), small Stokes shifts (less than 30 nm), low fluorescence efficiency, or poor photostability. It is well known that red or NIR (near-infrared) wavelengths cause less photodamage to biological samples and have strong penetrating power. However, most red-emitting dyes have low fluorescence quantum efficiencies. Furthermore, since biological samples typically exhibit strong autofluorescence, a small Stokes shift can lead to a high signal-to-noise ratio, ultimately resulting in detection errors. Therefore, developing high-brightness and high-stability fluorescent dyes with red / NIR emission and large Stokes shifts is an urgent problem to be solved.

[0003] Fluorescent dyes typically consist of a donor and an acceptor. The development of novel and superior donors is fundamental to the development of high-performance fluorescent dyes. The amino group is a strong electron-donating group and is one of the commonly used donors for fluorescent dyes. Different amino donors also present different problems. For example, the diethylamino donor has a strong electron-donating ability, but its structure is prone to rotation, causing some of the excited-state energy of the fluorescent dye to dissipate via non-radiative transitions, leading to a decrease in the dye's fluorescence quantum efficiency. Simultaneously, the diethylamino group is prone to oxidation, causing photobleaching of the fluorescent dye. Summary of the Invention

[0004] Based on the above considerations, in this invention, we developed a novel electron-donating group, 9,10-dihydro-9,9-dimethylazanthracene (abbreviated as MA), the structure of which is shown in the figure below. This electron-donating group has a strong electron-donating ability, which can effectively increase the absorption and emission wavelengths of the dye while increasing the Stokes shift. In addition, since the MA group has a large conjugated plane that can disperse the electron density, it can maintain both strong electron-donating ability and high antioxidant stability, thus giving the fluorescent dye containing the MA donor good stability. The rigid structure of the MA group also suppresses the twisted intramolecular charge transfer (TICT) effect that is usually present in flexible donors, thereby enabling the dye to maintain high fluorescence brightness while increasing the wavelength. Based on the MA group, symmetric and asymmetric rhodamine fluorescent dyes, coumarin fluorescent dyes, and coumarin fluoroboron dyes were developed, and their photochemical and physical properties were tested in detail.

[0005] Technical solution: A novel dimethylacridine amino donor, the structural formula of which is as follows: Formula I.

[0006]

[0007] Formula I Where R1 is -H or -CHO, and R2 is -OCH3 or -OH.

[0008] The synthetic route for the donor MA in this invention is shown below: .

[0009] The synthetic route for asymmetric carboxylated rhodamine in this invention is shown below: .

[0010] The synthetic route for asymmetric ester-based rhodamine in this invention is shown below: .

[0011] The synthetic route for symmetrical carboxylated rhodamine in this invention is shown below: .

[0012] The structure and synthetic route of the coumarin dye in this invention are shown below: .

[0013] The structure and synthetic route of the coumarin fluoroboron dye in this invention are shown below: .

[0014] Table 1. Optical properties of Rhodamine dyes MAR and MARE in ethanol.

[0015]

[0016] Table 2 Optical properties of coumarin dye MACou in dichloromethane and ethanol.

[0017]

[0018] Table 3 Optical properties of coumarin fluoroboron dye MABF in dichloromethane and ethanol.

[0019]

[0020] This invention provides a class of high-brightness and high-stability fluorescent dyes based on dimethylacridine donors. Compared with existing technologies, its advantages are as follows: The donor group possesses a strong electron-donating ability, effectively increasing the absorption and emission wavelengths of the dye while simultaneously increasing the Stokes shift. Furthermore, the large conjugated plane of the MA group disperses the electron density, thus maintaining both strong electron-donating ability and high antioxidant stability, resulting in excellent stability for fluorescent dyes containing MA donors. The rigid structure of the MA group also suppresses the twisted intramolecular charge transfer (TICT) effect typically present in flexible donors, allowing the dye to maintain high fluorescence brightness while increasing wavelength. Based on the MA group, symmetric and asymmetric rhodamine fluorescent dyes, coumarin fluorescent dyes, and coumarin fluoroboron dyes were developed, and their photochemical and physical properties were tested in detail. Attached Figure Description

[0021] Figure 1 The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the carboxyrhodamine fluorescent dye MAR in ethanol as described in this invention. Figure 2 The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the ester-based rhodamine fluorescent dye MARE in ethanol according to the present invention. Figure 3 The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the coumarin fluorescent dye MACou in dichloromethane according to the present invention. Figure 4 The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the coumarin fluorescent dye MACou in ethanol according to the present invention. Figure 5 The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the coumarin fluorinated boron fluorescent dye MABF described in this invention in dichloromethane. Figure 6The normalized UV-Vis absorption spectrum (left) and emission spectrum (right) of the coumarin fluorinated boron fluorescent dye MABF described in this invention in ethanol. Figure 7 Compound 4 of the present invention 1 H NMR spectrum; Figure 8 Compound 8 of the present invention 1 H NMR spectrum; Figure 9 The compound MAR-SM described in this invention 1 H NMR spectrum; Figure 10 The compound MAR-SM described in this invention 13 C NMR spectrum; Figure 11 The compound MAR-DM described in this invention 1 H NMR spectrum; Figure 12 The compound MAR-DM described in this invention 13 C NMR spectrum; Figure 13 The compound MAR-DE described in this invention 1 H NMR spectrum; Figure 14 The compound MAR-DE described in this invention 13 C NMR spectrum; Figure 15 The compound MAR-Julo described in this invention 1 H NMR spectrum; Figure 16 The compound MAR-Julo described in this invention 13 C NMR spectrum; Figure 17 The compound MAR-SY described in this invention 1 H NMR spectrum; Figure 18 The compound MAR-SY described in this invention 13 C NMR spectrum; Figure 19 The compound MARE-SM described in this invention 1 H NMR spectrum; Figure 20 The compound MARE-SM described in this invention 13 C NMR spectrum; Figure 21 The compound MARE-DM described in this invention 1 H NMR spectrum; Figure 22 The compound MARE-DM described in this invention 13 C NMR spectrum; Figure 23 The compound MARE-DE described in this invention 1 H NMR spectrum; Figure 24 The compound MARE-DE described in this invention 13 C NMR spectrum; Figure 25 The compound MARE-Julo described in this invention 1 H NMR spectrum; Figure 26 MARE-Julo, the compound described in this invention 13 C NMR spectrum; Figure 27 The compound MARE-SY described in this invention 1 H NMR spectrum; Figure 28 The compound MARE-SY described in this invention 13 C NMR spectrum; Figure 29 The compound MACou-BI described in this invention 1 H NMR spectrum; Figure 30 The compound MACou-BI described in this invention 13 C NMR spectrum; Figure 31 The compound MACou-EE described in this invention 1 H NMR spectrum; Figure 32 The compound MACou-EE described in this invention 13 C NMR spectrum; Figure 33 The compound MACou-AC described in this invention 1 H NMR spectrum; Figure 34 The compound MACou-AC described in this invention 13 C NMR spectrum; Figure 35 The compound MACou-CN described in this invention 1 H NMR spectrum; Figure 36 The compound MACou-CN described in this invention 13 C NMR spectrum; Figure 37The compound MABF-OMe described in this invention 1 H NMR spectrum; Figure 38 The compound MABF-OMe described in this invention 13 C NMR spectrum; Figure 39 The compound MABF-Me described in this invention 1 H NMR spectrum; Figure 40 The compound MABF-Me described in this invention 13 C NMR spectrum; Figure 41 The compound MABF-Br described in this invention 1 H NMR spectrum; Figure 42 The compound MABF-Br described in this invention 13 C NMR spectrum; Figure 43 The compound MABF-CN described in this invention 1 H NMR spectrum; Figure 44 The compound MABF-CN described in this invention 13 C NMR spectrum; Figure 45 This is a stability test diagram of the compound MAR-SY described in this invention; Figure 46 The images show (a) and (b) of cells subjected to STED high-resolution imaging of the compound MAR-SY described in this invention. Figure 47 This is the structural formula of the high-brightness and high-stability fluorescent dye based on the dimethylacridine donor of the present invention. Detailed Implementation

[0022] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0024] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0025] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the communication between the inner sides of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0026] Example 1: Synthesis of donor MA Compound 1: m-Methoxyaniline (7.2 g, 58.4 mmol) and sodium amino acid (9.2 g, 234 mmol) were added to a three-necked flask, followed by 150 mL of anhydrous tetrahydrofuran. The mixture was then purged with argon gas. The mixture was heated to 50 °C and stirred for 30 min, followed by the addition of o-fluorobenzoic acid (9.8 g, 70.2 mmol). The reaction mixture was reacted at 50 °C for 16 h. After cooling to room temperature, the reaction solution was slowly poured into 200 mL of water. The pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted with DCM (5 x 150 mL). The combined organic phases were dried over anhydrous sodium sulfate and then evaporated to dryness to obtain a brownish-black solid, which was crude product 1. This product was not purified and proceeded directly to the next reaction step.

[0027] Compound 2: The crude product 1 from the previous step was dissolved in anhydrous methanol (400 mL), and thionyl chloride (30 mL, 413 mmol) was slowly added dropwise under ice bath conditions. The mixture was then refluxed at 70 °C for 13 h. The reaction was stopped, cooled to room temperature, and the reaction solution was poured into 200 mL of ice water. The pH was adjusted to 7-8 with saturated Na₂CO₃ solution, and the mixture was extracted with DCM (3 x 150 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and then purified by column chromatography (PE:DCM = 10:1) to obtain 9.6 g of orange-yellow liquid, which was product 2. The overall yield of the two steps was 64%.

[0028] Compound 3: Compound 2 (9.6 g, 37.4 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran under argon protection and cooled to -78 °C using a dry ice bath. After stirring for 30 min under dry ice bath, methyl magnesium bromide (3 M in 2-methyl THF, 60 mL, 180 mmol) was slowly added dropwise over approximately 40 minutes. The mixture was then stirred for another 20 min under dry ice bath, followed by a reaction at room temperature for 12 h. After the reaction was stopped, the reaction mixture was slowly poured into 100 mL of ice water, the pH was adjusted to neutral with dilute hydrochloric acid, and the mixture was extracted with EA (3 x 150 mL). The combined organic phases were dried over anhydrous sodium sulfate and evaporated to dryness to give 9.2 g of a yellow solid, which was crude product 3, with a yield of 96%.

[0029] Compound 4: Compound 3 (9.2 g, 35.8 mmol) was completely dissolved in a small amount of DCM, and then 100 mL of polyphosphoric acid was added. The mixture was heated to 40 °C and reacted overnight. The reaction solution was poured into 100 mL of water, and saturated Na₂CO₃ solution was added to completely dissolve the compound. The pH was then adjusted to neutral with solid Na₂CO₃. The mixture was extracted with DCM (3 x 150 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solution was evaporated to dryness and passed through a column (PE:DCM = 12:1) to give 3.15 g of white solid, yield 37%. 1 H NMR (400 MHz, CDCl3) δ 7.36 (dd, J =7.8, 1.0 Hz, 1H), 7.27 (t, J = 7.1 Hz, 1H), 7.11 – 7.05 (m, 1H), 6.90 (dd, J =10.9, 4.2 Hz, 1H), 6.67 (t, J = 6.6 Hz, 1H), 6.49 (dt, J = 8.6, 4.3 Hz, 1H), 6.24(t, J = 3.9 Hz, 1H), 6.12 (s, 1H), 3.78 (d, J = 4.6 Hz, 1H), 1.56 (d, J = 5.3 Hz, 1H). Compound 5: Compound 4 (1.00 g, 4.18 mmol), iodoethane (1.64 g, 10.5 mmol), and NaOH (0.126 g, 3.14 mmol) were dissolved in 4 mL of DMSO under argon protection and in the dark at 130 °C for 12 h. After the reaction solution cooled, it was poured into 100 mL of water, and the pH was adjusted to neutral with dilute hydrochloric acid. The solution was then extracted with EA (3 x 60 mL), and the organic phases were combined and washed with saturated NaCl solution. The organic phases were dried over anhydrous sodium sulfate. The solution was evaporated to dryness and passed through a column (PE:DCM = 20:1) to give 725 mg of a yellowish-brown liquid, with a yield of 65%.

[0030] Compound 6: Compound 5 (250 mg, 0.935 mmol) was dissolved in 40 mL of anhydrous DCM under Ar protection. 6 mL of boron tribromide solution (17% BBr3 in DCM) was slowly added to the reaction solution under dry ice bath. The reaction solution turned reddish-brown. After the dry ice sublimated, the reaction system continued to react at room temperature for approximately 13 h. The reaction solution was poured into 100 mL of ice water, the pH was adjusted to neutral, and the organic phase was separated. The aqueous phase was extracted three times with DCM. The organic phases were combined and dried over anhydrous sodium sulfate, then evaporated to dryness to obtain 200 mg of a dark green solid.

[0031] Compound 7: Anhydrous DMF (5 mL, 64.7 mmol) was placed in a 25 mL flask and stirred in an ice bath for 10 min. Then, POCl3 (2 mL, 21.4 mmol) was slowly added to the flask, turning the solution orange-red. After stirring in an ice bath for 1 h, Compound 5 (650 mg, 2.43 mmol) dissolved in 2 mL of anhydrous DMF was added to the reaction solution, and the mixture was heated to 65 °C and reacted for 2 h. The reaction solution was slowly poured into 100 mL of ice water, and the pH was adjusted to neutral with saturated Na2CO3 solution. Extraction was performed using EA (4 x 50 mL), followed by washing with saturated NaCl solution. The organic phase was dried over anhydrous sodium sulfate. The solution was evaporated to dryness to obtain 712 mg of a brownish-red oily liquid, which was the crude product 7.

[0032] Compound 8: Compound 7 (0.91 g, 3.08 mmol) was dissolved in 40 mL of anhydrous DCM under argon protection and stirred for 30 min in a dry ice bath. Boron tribromide (1 M in DCM, 18 mL, 18 mmol) was then added dropwise, turning the reaction solution a deep red color. Stirring continued for another 30 min in a dry ice bath, followed by heating to room temperature and reacting overnight. The reaction solution was slowly added to 100 mL of ice water, and the pH was adjusted to neutral with saturated Na₂CO₃ solution. Extraction was then performed with DCM (3 x 50 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solution was evaporated to dryness and column chromatography (PE:DCM = 10:1) to give 600 mg of a yellow solid, which was product 8. The overall yield of the two-step reaction was 69%.

[0033] Example 2: Synthesis of the asymmetric carboxylated rhodamine dye MAR Compound 5 (100 mg, 0.374 mmol) and compounds R1 / R2 / R3 / R4 (0.42 mmol) were dissolved in 3 mL of methanesulfonic acid, and the mixture was heated to 90 °C. The reaction was monitored by TLC, and the reaction was completed in about 2-4 hours. The reaction solution was poured into 60 mL of water, and DCM (6 x 30 mL) was added for extraction. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and filtered through a column using DCM / MeOH (100:1 to 20:1) to obtain the dye MAR.

[0034] MAR-SM: Yield 25%. 1 H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 6.5 Hz, 1H), 7.66 (d, J = 1.8 Hz, 2H), 7.31 (s, 1H), 7.26 – 7.13 (m, 2H), 7.02 (d, J = 7.9 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.86 (s, 1H), 6.72 (s, 1H), 6.64 (d, J = 8.9 Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 6.45 (dd, J = 8.9, 2.6 Hz, 1H), 4.14 – 4.04 (m,2H), 3.49 (s, 3H), 1.49 (dd, J = 8.5, 5.4 Hz, 3H), 1.32 (s, 3H), 1.29 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.38, 153.09, 152.34, 151.31, 142.59, 139.27,134.65, 131.96, 128.73, 127.60, 124.37, 123.52, 121.06, 112.64, 110.43,109.03, 106.75, 99.61, 98.37, 40.16, 35.79, 29.76, 28.25, 11.37. MAR-DM: Yield 51%. 1 H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 6.8 Hz, 1H),7.64 (dd, J = 13.7, 6.4 Hz, 2H), 7.31 (s, 1H), 7.25 – 7.12 (m, 2H), 7.02 (d, J =8.0 Hz, 1H), 6.95 (t, J = 8.9 Hz, 1H), 6.85 (s, 1H), 6.71 (d, J = 6.9 Hz, 1H),6.63 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 6.43 (d, J = 8.6 Hz, 1H), 4.08 (d, J = 7.0Hz, 2H), 2.99 (s, 6H), 1.48 (t, J = 6.9 Hz, 3H), 1.35 – 1.26 (m, 6H). 13 C NMR(100 MHz, CDCl3) δ 170.46, 153.05, 152.27, 151.28, 144.49, 142.52, 141.34,139.30, 134.68, 132.50, 131.95, 129.54, 128.72, 126.75, 124.98, 124.37,123.51, 121.04, 112.63, 110.38, 108.97, 106.66, 99.83, 99.63, 98.38, 40.64,40.16, 35.79, 29.78, 28.23, 11.37. MAR-DE: Yield 20%. 1 H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 5.3 Hz, 1H),7.64 (d, J = 5.8 Hz, 2H), 7.30 (s, 1H), 7.20 (s, 2H), 7.01 (d, J = 7.1 Hz, 1H),6.95 (d, J = 6.2 Hz, 1H), 6.84 (s, 1H), 6.70 (s, 1H), 6.58 (d, J = 6.3 Hz, 1H),6.48 (s, 1H), 6.37 (d, J = 6.3 Hz, 1H), 4.08 (d, J = 5.9 Hz, 2H), 3.37 (d, J = 5.9Hz, 4H), 1.48 (s, 3H), 1.29 (d, J = 14.5 Hz, 6H), 1.19 (d, J = 4.9 Hz, 6H). 13 C NMR(100 MHz, CDCl3) δ 170.34, 153.38, 152.49, 151.33, 149.73, 142.43, 139.38,134.60, 132.02, 129.47, 128.95, 126.75, 124.40, 123.58, 121.01, 112.62,110.54, 108.39, 105.70, 99.66, 97.48, 44.52, 40.66, 35.82, 29.90, 28.20,12.48, 11.44. MAR-Julo: Yield 23%. 1 H NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 18.3, 5.9Hz, 1H), 7.60 – 7.48 (m, 2H), 7.48 – 7.39 (m, 1H), 7.18 (d, J = 3.6 Hz, 2H),7.05 (d, J = 6.2 Hz, 1H), 7.00 (d, J= 7.5 Hz, 1H), 6.98 – 6.92 (m, 2H), 6.58 –6.53 (m, 1H), 4.09 (d, J = 12.3 Hz, 2H), 3.30 (dd, J = 14.9, 6.5 Hz, 4H), 2.70 –2.41 (m, 4H), 1.84 (d, J = 3.7 Hz, 4H), 1.56 – 1.41 (m, 6H), 0.80 (d, J = 6.5 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 170.53, 152.51, 149.90, 147.10, 146.67, 143.92,138.91, 134.09, 132.90, 132.32, 131.73, 129.83, 129.54, 126.94, 126.70,125.85, 125.65, 124.62, 124.28, 121.71, 119.89, 113.04, 112.58, 112.20,110.14, 106.52, 99.33, 45.81, 40.99, 36.03, 30.07, 28.39, 27.38, 21.45, 20.80, 11.62. Example 3: Synthesis of the symmetrical carboxylated rhodamine dye MAR-SY The crude product 6 (68 mg, 0.268 mmol) and phthalic anhydride (20 mg, 0.135 mmol) were dissolved in 5 mL of toluene, and 100 μL of methanesulfonic acid was added. The mixture was then reacted under argon protection at 110 °C for 6 h. After rotary evaporation to dryness, the product was passed through a column (DCM: MeOH = 100:0 → DCM: MeOH = 100:1) to give 19 mg of a deep blue solid.

[0035] MAR-SY: Yield 23%. 1 H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 4.4 Hz, 1H), 7.68 (dd, J = 9.8, 6.2 Hz, 3H), 7.32 (s, 1H), 7.21 (dd, J = 11.1, 4.3 Hz, 2H),7.18 – 7.13 (m, 1H), 7.04 (d,J = 8.2 Hz, 2H), 7.00 – 6.95 (m, 2H), 6.88 (d, J =6.6 Hz, 1H), 6.84 (d, J = 5.3 Hz, 1H), 6.71 (dd, J = 7.3, 5.0 Hz, 2H), 4.11 (d, J =6.9 Hz, 2H), 3.61 (s, 2H), 1.53 – 1.48 (m, 3H), 1.33 (dd, J = 12.8, 6.4 Hz,12H), 1.04 – 0.71 (m, 3H). 13 C NMR (100 MHz, CDCl3) δ 170.26, 151.11, 144.57,144.50, 142.67, 142.54, 141.28, 139.28, 139.22, 134.89, 132.48, 131.94,129.68, 126.76, 124.37, 123.55, 122.61, 121.35, 121.13, 121.07, 112.67,112.65, 112.50, 110.38, 110.31, 110.26, 110.12, 99.79, 99.59, 40.69, 36.16, 35.81, 31.84, 31.29, 29.61, 11.37. Example 4: Synthesis of the ester-based rhodamine dye MARE MAR (0.1 mmol) was dissolved in 10 mL of anhydrous ethanol, and 7 drops of concentrated sulfuric acid were added as a catalyst. The reaction was refluxed for 12 h under argon protection. After the reaction was completed, the solvent was first evaporated to dryness, and then the solid was dissolved in 50 mL of DCM. After washing with 80 mL of distilled water, the mixture was extracted three times with DCM, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and then passed through a column (using DCM / MeOH as the developing solvent) to obtain the pure esterified products MARE series.

[0036] MARE-SM: Yield 68%. 1 H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 7.7 Hz, 1H),8.00 – 7.81 (m, 2H), 7.44 (d, J = 7.7 Hz, 1H), 7.38 (d,J = 7.7 Hz, 2H), 7.35 (s,1H), 7.27 (s, 1H), 7.25 (d, J = 3.5 Hz, 1H), 7.23 (d, J = 4.5 Hz, 1H), 7.20 (s,1H), 7.04 (dd, J = 9.6, 2.4 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 4.40 – 4.24 (m,2H), 4.12 (d, J = 7.1 Hz, 2H), 3.63 (s, 3H), 1.42 (d, J = 6.0 Hz, 6H), 1.28 (t, J =7.1 Hz, 3H), 1.05 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 165.11, 159.62,158.09, 157.87, 155.89, 148.89, 136.57, 134.33, 133.00, 132.10, 131.36,130.75, 130.49, 130.07, 127.76, 125.15, 124.94, 124.33, 115.90, 115.55,114.76, 114.50, 98.51, 96.60, 61.67, 41.04, 36.35, 30.38, 30.08, 14.93,11.54. MARE-DM: Yield 76%. 1 H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 6.8 Hz, 1H),7.86 (dd, J = 17.3, 7.0 Hz, 2H), 7.44 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.6 Hz,1H), 7.26 (d, J = 3.9 Hz, 2H), 7.23 (d, J = 2.6 Hz, 1H), 7.22 – 7.15 (m, 2H),7.05 (d, J= 9.2 Hz, 1H), 6.97 (s, 1H), 4.29 (d, J = 6.6 Hz, 2H), 4.14 – 4.09 (m,2H), 3.33 – 3.00 (m, 6H), 1.40 (d, J = 4.5 Hz, 6H), 1.34 – 1.27 (m, 3H), 1.13 –1.01 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ 165.09, 159.68, 158.07, 157.82,155.89, 148.91, 136.52, 134.37, 132.99, 132.08, 131.36, 130.77, 130.47,130.03, 127.76, 125.15, 124.96, 124.36, 115.89, 115.50, 114.73, 114.50,98.45, 96.56, 61.66, 46.34, 42.37, 41.04, 36.33, 30.36, 30.08, 14.89, 13.67. MARE-DE: Yield 63%. 1 H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 7.80 (d, J =7.6 Hz, 2H), 7.36 (d, J = 8.1 Hz, 1H), 7.28 (s, 2H), 7.23 – 7.04 (m, 5H), 6.92(d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.9 Hz, 1H), 4.21 (s, 2H), 4.12 – 3.89 (m, 4H),3.50 – 3.23 (m, 2H), 1.53 (d, J = 4.4 Hz, 3H), 1.33 (d, J = 6.2 Hz, 9H), 1.21 (d, J = 4.3 Hz, 3H), 1.00 (d, J = 5.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 165.13,159.25, 158.38, 156.23, 155.74, 148.77, 136.59, 134.20, 133.04, 132.05,131.71, 131.34, 130.76, 130.43, 127.75, 125.13, 124.90, 124.28, 115.79,115.45, 114.71, 114.46, 98.46, 96.25, 61.67, 46.35, 42.34, 36.32, 30.37,30.05, 14.91, 13.70, 11.51. MARE-Julo: Yield 73%. 1 H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 7.4 Hz, 1H),7.84 (dt, J = 20.4, 7.3 Hz, 2H), 7.63 (dt, J = 25.1, 7.4 Hz, 1H), 7.42 (d, J = 7.5Hz, 1H), 7.35 (s, 1H), 7.20 (d, J = 7.2 Hz, 2H), 7.15 (d, J = 6.9 Hz, 1H), 7.13 –7.07 (m, 1H), 6.76 (s, 1H), 4.25 (d, J = 6.8 Hz, 2H), 4.09 (dt, J = 21.0, 7.0 Hz,4H), 3.65 (dd, J = 14.9, 5.9 Hz, 4H), 3.22 – 3.10 (m, 2H), 2.26 – 2.11 (m, 2H),2.10 – 1.96 (m, 2H), 1.39 (d, J = 8.5 Hz, 6H), 1.28 (t, J = 7.0 Hz, 3H), 1.12 –1.05 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 165.21, 156.85, 154.90, 153.09,153.05, 147.70, 141.39, 136.94, 133.50, 132.96, 131.92, 131.26, 130.49,130.24, 127.61, 126.67, 125.98, 125.03, 124.36, 123.79, 115.30, 114.98,114.15, 105.53, 98.41, 61.58, 51.41, 41.98, 36.24, 29.90, 27.52, 20.27, 19.74, 19.33, 14.86, 11.53. MARE-SY: Yield 26%. 1 H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 7.1 Hz, 1H), 7.93 (dt, J = 14.9, 7.5 Hz, 2H), 7.51 – 7.43 (m, 4H), 7.42 (s, 1H), 7.38 (d, J =3.0 Hz, 2H), 7.34 (s, 1H), 7.31 (s, 2H), 7.23 (dd, J = 16.5, 8.6 Hz, 3H), 4.35(d, J = 6.5 Hz, 2H), 4.13 (dd, J = 14.1, 7.0 Hz, 2H), 4.05 (dt, J = 14.8, 7.5 Hz, 2H), 1.65 (t, J = 6.6 Hz, 6H), 1.34 – 1.26 (m, 12H), 1.11 – 1.01 (m, 3H). 13C NMR(101 MHz, CDCl3) δ 165.15, 156.68, 151.49, 149.65, 138.17, 136.18, 135.61,133.09, 132.78, 132.28, 131.43, 131.08, 130.82, 130.07, 127.94, 127.83,125.27, 124.91, 124.42, 116.74, 114.94, 99.13, 98.50, 61.80, 42.77, 29.60,22.59, 14.81, 13.72, 11.62. Example 5: Synthesis of coumarin dye MACou MACou-BI: Compound 8 (30 mg, 0.107 mmol) and 2-cyanomethylbenzimazole (18 mg, 0.117 mmol) were dissolved in 5 mL of EtOH, and 30 μL of piperidine was added. The mixture was heated to 80 °C and refluxed for 2 h until the reaction was essentially complete. 5% HCl solution was added to the reaction mixture, and the mixture was heated to 100 °C and reacted for 4 h. The reaction mixture was poured into 50 mL of water and extracted with DCM (3 x 20 mL). The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and column-coated (PE:DCM = 2:1 → pure DCM) to give 14 mg of an orange-red solid, yield 31%. 1 H NMR (400 MHz, CDCl3) δ 11.29 (s, 1H), 9.10 (s, 1H), 7.64 (d, J = 4.0Hz, 1H), 7.47 (dd, J = 7.1, 3.1 Hz, 1H), 7.29 (d, J = 8.7 Hz, 4H), 7.11 (t, J = 8.6Hz, 2H), 7.08 – 7.03 (m, 1H), 6.95 (s, 1H), 4.12 (q, J = 7.1 Hz, 2H), 1.58 (s, 6H), 1.50 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.82, 154.43, 147.25,145.17, 142.91, 140.30, 138.33, 131.95, 131.06, 127.25, 124.83, 124.05,122.51, 113.40, 112.36, 99.41, 99.16, 53.48, 50.86, 41.45, 36.12, 34.27,31.46, 30.20, 29.67, 27.97, 11.49. MACou-EE: Compound 8 (20 mg, 0.0712 mmol) was dissolved in 3 mL of EtOH, followed by the addition of diethyl malonate (62 μL, 0.216 mmol) and 90 μL of piperidine. The mixture was heated to 80 °C and refluxed for 3 h until the reaction was essentially complete. The solution was evaporated to dryness to give a yellow solid, which was then passed through a column (PE:DCM = 1:1 → PE:DCM = 1:5) to give 9 mg of a yellow solid, yielding 34%. 1 H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.54 (s, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.11 (d, J = 4.3 Hz, 1H), 7.09 (s, 1H), 6.87 (s, 1H), 4.40(q, J = 7.1 Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 1.56 (s, 6H), 1.48 (t, J = 7.1 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 149.47, 138.12, 127.22,125.65, 124.68, 124.51, 123.92, 122.59, 118.91, 113.49, 111.07, 98.92, 61.46,41.42, 36.62, 34.73, 31.29, 30.07, 29.60, 14.15, 11.33. MACou-AC: Compound 8 (20 mg, 0.0712 mmol) was dissolved in 3 mL of EtOH, followed by the addition of ethyl acetoacetate (28 μL, 0.216 mmol) and 30 μL of piperidine. The mixture was heated to 80 °C and refluxed. After 2 h, the reaction was essentially complete. The solution was evaporated to dryness to give a yellow solid. Column chromatography (PE:DCM = 1:1 → PE:DCM = 1:4) yielded 15 mg of a yellow solid, with a yield of 61%. 1 H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 5.4 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 6.3 Hz, 1H), 7.15 – 7.06 (m, 2H), 6.88 (s, 1H), 4.11 (q, J =7.0 Hz, 2H), 2.71 (s, 3H), 1.56 (s, 6H), 1.49 (t, J = 7.0 Hz, 3H). 13 C NMR (100MHz, CDCl3) δ 195.68, 160.55, 156.38, 147.95, 146.21, 138.15, 132.01, 130.64,127.29, 126.37, 124.82, 122.70, 118.63, 113.53, 111.57, 98.92, 41.56, 36.06,30.75, 29.76, 28.07, 11.50. MACou-CN: Compound 8 (20 mg, 0.0712 mmol) was dissolved in 3 mL of EtOH, followed by the addition of ethyl cyanoacetate (23 μL, 0.216 mmol) and 30 μL of piperidine. The solution immediately turned orange-yellow. The mixture was heated to 80 °C and refluxed for 20 min until the reaction was complete. The solution was evaporated to dryness to obtain an orange-yellow solid. Column chromatography (PE:DCM = 1:1 → pure DCM) yielded 14 mg of an orange solid, with a yield of 60%. 1 H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.48 (s, 1H), 7.32 – 7.27 (m, 2H), 7.15 – 7.10 (m, 2H), 6.87 (s, 1H), 4.11 (q, J= 7.1 Hz, 2H), 1.56 (s, 6H), 1.49(t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 155.54, 151.36, 146.92, 137.71,131.24, 127.39, 125.35, 124.82, 123.11, 113.73, 110.57, 99.42, 95.25, 41.60,36.03, 31.33, 30.10, 29.69, 11.41. Example 6: Synthesis of coumarin fluoroboron dye MABF The dyes MABF-OMe, MABF-Me, MABF-Br, and MABF-CN were synthesized according to the methods described in the literature. Chem. Commun., [2020, 56, 2159--2162]. First, MA salicylaldehyde (compound 8) reacts with 2-cyanomethylbenzimidazole under reflux in ethanol to generate imine coumarin (compound 9). Subsequently, imine coumarin reacts with aniline monomers to generate compounds 10a-d. Compounds 10a-d, without further purification, directly undergo a complexation reaction with BF3•OEt2 to obtain the imine coumarin fluoroboron dye MABF-R.

[0037] MABF-OMe: Yield 36%. 1 H NMR (400 MHz, CDCl3) δ 9.01 (d, J = 3.7 Hz, 1H), 7.92 (q, J = 6.0, 4.4 Hz, 1H), 7.72 (dq, J = 7.9, 4.0, 3.3 Hz, 2H), 7.53 – 7.48(m, 2H), 7.33 – 7.26 (m, 3H), 7.13 (s, 4H), 7.11 (s, 1H), 6.72 – 6.67 (m,1H), 4.31 (s, 3H), 4.09 (t, J = 7.3 Hz, 2H), 1.60 (s, 6H), 1.43 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 159.74, 152.49, 146.71, 140.30, 137.81, 135.63,133.55, 132.32, 129.09, 127.97, 127.57, 126.47, 125.12, 123.51, 117.60,114.96, 114.28, 114.06, 112.93, 98.97, 55.74, 53.65, 41.63, 36.55, 34.68,29.68, 28.14, 11.77. MABF-Me: Yield 89%. 1 H NMR (400 MHz, CDCl3) δ 9.03 (s, 1H), 7.90 (q, J =3.0 Hz, 1H), 7.75 (td, J = 7.5, 4.5 Hz, 2H), 7.50 (dq, J = 7.9, 1.7 Hz, 1H), 7.41(d, J = 2.7 Hz, 4H), 7.30 (tt, J = 7.6, 1.6 Hz, 2H), 7.26 – 7.20 (m, 1H), 7.17 –7.09 (m, 2H), 6.69 (t, J = 2.2 Hz, 1H), 4.07 (q, J = 7.4 Hz, 2H), 2.52 (s, 3H),1.61 (s, 6H), 1.42 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 152.31,146.44, 139.99, 138.60, 137.80, 136.12, 133.82, 133.38, 132.25, 130.26,127.45, 126.53, 126.28, 125.03, 123.33, 123.23, 117.78, 114.16, 113.91,112.87, 98.91, 53.57, 41.51, 36.48, 29.62, 28.09, 21.35, 11.72. MABF-Br: Yield 43%. 1H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 7.99 (s,1H), 7.83 – 7.70 (m, 5H), 7.52 (d, J = 7.9 Hz, 2H), 7.42 (s, 2H), 7.21 – 7.10(m, 3H), 6.71 (d, J = 10.8 Hz, 1H), 4.16 – 4.08 (m, 2H), 1.63 (s, 6H), 1.46 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 158.65, 148.33, 146.65, 141.60,137.79, 132.30, 131.68, 129.08, 127.97, 127.58, 126.45, 125.65, 125.11,123.50, 117.62, 115.99, 114.95, 114.27, 112.94, 98.93, 55.76, 41.61, 36.56,31.09, 29.69, 28.14, 11.78. MABF-CN: Yield 39%. 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.96 (d, J =8.1 Hz, 2H), 7.79 (s, 3H), 7.70 (d, J = 7.8 Hz, 2H), 7.63 (s, 1H), 7.53 (d, J =7.5 Hz, 2H), 7.22 – 7.14 (m, 2H), 7.11 (s, 1H), 6.68 (s, 1H), 4.13 (s, 2H),1.64 (s, 6H), 1.47 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 158.65,148.33, 146.65, 141.60, 137.79, 132.30, 131.68, 129.08, 127.97, 127.58,126.45, 125.65, 125.11, 123.50, 117.62, 115.99, 114.95, 114.27, 112.94,98.93, 55.76, 41.61, 36.56, 31.09, 29.69, 28.14, 11.78. Example 7: Stability test of Rhodamine dye MAR-SY MAR-SY, along with the commercial dyes Rhodamine B and Atto 565, were exposed to a 500W xenon lamp for 85 minutes, and the changes in their absorption spectra were observed. The test system was trifluoroethanol / PBS buffer = 1 / 1. The results are as follows. Figure 45 As shown, the maximum absorption of Atto 565 decreased by 27%, the maximum absorption of Rhodamine B decreased by 18%, while the maximum absorption of MAR-SY decreased by only 3%.

[0038] Example 8: STED high-resolution imaging test of Rhodamine dye MAR-SY MAR-SY was used in conjunction with the commercial dyes Rhodamine B and Atto 565 for STED high-resolution imaging assays. Cell images showing changes over time were collected, and relative fluorescence intensities were calculated. Results are as follows: Figure 46 As shown, within 20 minutes, Atto 565 and Rhodamine B exhibited varying degrees of fluorescence decrease or quenching, while MAR-SY maintained its fluorescence imaging within cells relatively well.

[0039] The above description is merely one embodiment of the present invention, but it cannot be used to limit the scope of the present invention. Any structural changes made based on the present invention, as long as they do not lose the essence of the present invention, should be considered to fall within the protection scope of the present invention and be subject to its restrictions.

[0040] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the system described above can be found in the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0041] It should be noted that the system provided in the above embodiments is only illustrated by the division of the above functional modules. In practical applications, the above functions can be assigned to different functional modules as needed, that is, the modules or steps in the embodiments of the present invention can be further decomposed or combined. For example, the modules in the above embodiments can be merged into one module, or further divided into multiple sub-modules to complete all or part of the functions described above. The names of the modules and steps involved in the embodiments of the present invention are only for distinguishing the various modules or steps and are not considered as an improper limitation of the present invention.

[0042] Those skilled in the art will recognize that the modules and method steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, computer software, or a combination of both. The programs corresponding to the software modules and method steps can be placed in random access memory (RAM), main memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disks, removable disks, CD-ROMs, or any other form of storage medium known in the art. To clearly illustrate the interchangeability of electronic hardware and software, the components and steps of the various examples have been generally described in terms of functionality in the foregoing description. Whether these functions are implemented in electronic hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of the invention.

[0043] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent in such process, method, article, or apparatus / device.

[0044] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A class of high-brightness and high-stability fluorescent dyes based on dimethylacridine donors, with the following structural formula:

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