A pH-responsive dye platform, and preparation method and application thereof

Abstract

This invention discloses a pH-responsive dye platform, its preparation method, and its applications. The invention achieves precise pH regulation by introducing a heteroaryl amine at position 9 of a traditional rhodamine dye and replacing different heteroatoms at position 10. Based on this strategy, a pH-responsive dye platform spanning the entire physiological pH range (the platform consists of a series of pH-responsive dyes suitable for different pH ranges) is constructed. Furthermore, the pH-responsive dyes in this platform possess excellent photophysical properties, sensitive pH response capabilities, and cover a response range from pH 0.5 to 7.0, meeting the monitoring needs of different pH microenvironments in vivo.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a pH-responsive dye platform, its preparation method, and its applications. Background Technology

[0002] pH homeostasis is crucial for regulating a wide variety of physiological activities. Due to the differential distribution of protons in different cellular regions, multiple pH microenvironments exist within the cell, such as the neutral cytoplasm (pH 7.0-7.4), the alkaline mitochondria (pH ~8.0), and the acidic lysosomes (pH 4.5-5.5) and endosomes (pH 4.5-6.8). Furthermore, each organ in the human body has its optimal pH microenvironment for operation; for example, the stomach typically operates under highly acidic conditions (pH 1.0), while the intestines have a slightly alkaline pH (pH 8.0). Disruption of pH homeostasis is closely related to the progression of various diseases, such as Alzheimer's disease, Parkinson's disease, and cancer. For tumors, surgical resection remains the primary treatment, and residual tumor is a major cause of postoperative cancer recurrence. However, traditional tumor resection relies on the surgeon's experience to clean up small lesions and determine tumor boundaries, making complete removal difficult. Therefore, accurate identification of small lesions and tumor boundaries is crucial during surgery. The Warburg effect endows tumors with a slightly acidic microenvironment, so we can use sensitive pH-responsive probes to visualize tumor boundaries and assist clinicians in removing tumors during surgery.

[0003] Fluorescent probes, due to their superior biocompatibility, high sensitivity, and spatiotemporal resolution, have become real-time and non-invasive detection tools, widely used in the monitoring of physiological and pathological activities. Among numerous fluorophores, rhodamine pH-responsive dyes have become very promising pH-responsive dye cores due to their good stability, high brightness, and outstanding modifiability. In recent years, a large number of pH-responsive probes based on rhodamine cores have been developed for imaging tumors or organelles. However, unfortunately, these developed probes do not have a molecular platform to meet the requirements of pH monitoring in living organisms; they all have only a single, limited pH response range.

[0004] Therefore, it is of great significance to construct a pH-responsive dye platform with a full pH range in a simple and rapid manner. Summary of the Invention

[0005] To address the problem that existing probes lack molecular platforms capable of monitoring various pH levels in living organisms, one objective of this invention is to provide a pH-responsive dye platform. This invention utilizes heteroarylamines and heteroatoms to regulate the pH response range of pH-responsive dyes, thereby establishing a pH-responsive dye platform with a pH range of pH ≤ 7.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] A pH-responsive dye platform, comprising a pH-responsive dye, the structure of which is shown below:

[0008] Among them, R1 is a heteroarylamine, and X is... Any one of them.

[0009] Based on the above technical solution, the present invention can be further improved as follows:

[0010] Furthermore, R1 is Any one of them.

[0011] Furthermore, the pH-responsive dye platform includes pH-responsive dyes with structural formulas as shown in formulas I to VIII; wherein, the structural formula of formula I is... The structural formula of formula II is: The structural formula of Formula III is: The structural formula of Formula IV is: The structural formula for equation V is: The structural formula of Equation VI is: The structural formula for equation VII is: The structural formula of equation VIII is:

[0012] A second objective of this invention is to provide a method for preparing a pH-responsive dye platform, wherein the pH-responsive dye platform includes a pH-responsive dye, and the preparation of the pH-responsive dye includes the following steps:

[0013] Step 1: Compound 1, with the structure shown below, is thoroughly mixed with formaldehyde aqueous solution and acetic acid and reacted at room temperature for 10-14 hours to obtain intermediate 1. The synthetic route is shown below:

[0014] Wherein, R is H or Br; preferably, the molar ratio of compound 1 to formaldehyde aqueous solution and acetic acid is 1.0:0.5:0.5, and the reaction time is 12 h; more preferably, when R in compound 1 is H, compound 1 is N,N-dimethylaniline, and the synthetic route of intermediate 1 is as follows:

[0015] Specifically, N,N-dimethylaniline, formaldehyde aqueous solution and acetic acid are mixed and reacted at room temperature for 10-14 h. After adjusting to neutral with sodium hydroxide, dichloromethane is added for extraction and the organic phase is collected. The organic phase is dried, solvent is removed, purified and eluted to obtain intermediate 1.

[0016] When R in compound 1 is Br, compound 1 is 3-bromo-N,N-dimethylaniline, and the synthetic route of intermediate 1 is as follows:

[0017] Specifically, 3-bromo-N,N-dimethylaniline was mixed with formaldehyde aqueous solution and acetic acid and reacted at room temperature for 10-14 hours. The mixture was then poured into ice water to precipitate a white solid. The solid was filtered and dried to obtain intermediate 1.

[0018] Step 2: React intermediate 1 with compound 2 to obtain intermediate 2. The synthetic route of intermediate 2 is shown below:

[0019] Compound 2 is any one of dichlorodimethylsilane, acetone, dichlorophenylphosphine, and fuming sulfuric acid;

[0020] Step 3: Oxidize intermediate 2 to obtain intermediate 3. The synthetic route of intermediate 3 is shown below:

[0021]

[0022] Step 4: Dissolve intermediate 3 and oxalyl chloride in the first solvent and react at 40–50 °C for 45–80 min. Then remove the solvent and dissolve the intermediate in the second solvent. Add the solution dropwise to a mixture containing compound 3 and a weak base, and react at room temperature for 6–10 h to obtain the pH-responsive dye. The synthetic route is shown below:

[0023] Preferably, both the first solvent and the second solvent are dichloromethane and / or trichloromethane, and the reaction time at room temperature is 6 hours; more preferably, both the first solvent and the second solvent are dichloromethane.

[0024] Wherein, compound 3 is any one of 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 3-methyl-4-aminopyridine and 2-aminoquinoline;

[0025] X in intermediates 2 and 3 and the pH-responsive dye are all... Any one of them;

[0026] R1 is Any one of them.

[0027] Further, when compound 1 in step 1 is 3-bromo-N,N-dimethylaniline and compound 2 is dichlorodimethylsilane, the preparation of intermediate 2 in step 2 is as follows: intermediate 1 is dissolved in a solvent and cooled to -75 to -80°C, a strong base is added and the reaction continues for 100 to 120 min, then compound 2 is added and the temperature is raised to room temperature for 12 to 16 h, the reaction is quenched and extracted to obtain intermediate 2; wherein, the strong base is n-butyllithium or sec-butyllithium, and the synthetic route of intermediate 2 is shown below:

[0028] Preferably, the molar ratio of intermediate 1, compound 2 and strong base is 1.0:0.5:1.05, the strong base is n-butyllithium, and the solvent is tetrahydrofuran; more preferably, the mixture is cooled to -78°C, the strong base is added, and the reaction continues for 60 min, and then the reaction is carried out at room temperature for 12 h.

[0029] In step 3, intermediate 2 is oxidized with potassium permanganate to obtain intermediate 3. Specifically, intermediate 2 is first dissolved in an organic solvent, and then the system is cooled to -10 to -15°C before potassium permanganate is added in batches to react with intermediate 2 at room temperature for 5 to 8 hours. The molar ratio of intermediate 2 to potassium permanganate is 1.0:5.0, and the organic solvent is acetone. The synthetic route of intermediate 3 is shown below:

[0030] Preferably, the reaction time at room temperature is 6 hours.

[0031] Further, when compound 1 in step 1 is 3-bromo-N,N-dimethylaniline and compound 2 is acetone, the preparation of intermediate 2 in step 2 is as follows: intermediate 1 is dissolved in a solvent and cooled to -75 to -80°C, a strong base is added and the reaction continues for 100 to 120 min, then compound 2 is added and the temperature is raised to room temperature for 12 to 16 h, the reaction is quenched and extracted to obtain an extract, and finally aluminum trichloride is added to the extract and the reaction continues at room temperature for 12 to 16 h to obtain intermediate 2; wherein, the strong base is n-butyllithium or sec-butyllithium, and the synthetic route of intermediate 2 is shown below:

[0032] Preferably, the molar ratio of intermediate 1, compound 2, strong base, and aluminum trichloride is 1.0:0.55:1.1:5.0, the strong base is n-butyllithium, the solvent is tetrahydrofuran, the mixture is cooled to -78°C, the strong base is added, and the reaction continues for 60 min, and then the mixture is reacted at room temperature for 12 h.

[0033] In step 3, intermediate 2 is oxidized with potassium permanganate to obtain intermediate 3. Specifically, intermediate 2 is first dissolved in an organic solvent, and then the system is cooled to -10 to -15°C before potassium permanganate is added in batches to react with intermediate 2 at room temperature for 5 to 8 hours. The molar ratio of intermediate 2 to potassium permanganate is 1.0:5.0, and the organic solvent is acetone. The route for intermediate 3 is shown below:

[0034] Preferably, the reaction time at room temperature is 6 hours.

[0035] Further, when compound 1 in step 1 is 3-bromo-N,N-dimethylaniline and compound 2 is dichlorophenylphosphine, the preparation of intermediate 2 in step 2 is as follows: intermediate 1 is dissolved in a solvent and cooled to -75 to -80°C, a strong base is added and the reaction continues for 100 to 120 min, then compound 2 is added and the temperature is raised to room temperature for 12 to 16 h. After quenching the reaction, H2O2 is added and the reaction continues. After quenching the reaction, intermediate 2 is obtained by extraction. The strong base is n-butyllithium or sec-butyllithium. The synthetic route of intermediate 2 is shown below:

[0036] Preferably, the molar ratio of intermediate 1, compound 2 and strong base is 1.0:0.55:1.1.

[0037] In step 3, intermediate 2 is dissolved in a solvent, and then tetrabutylammonium bromide and sodium hydroxide are added. Oxygen is then introduced, and the mixture is reacted at room temperature for 12–16 hours to obtain intermediate 3. The synthetic route for intermediate 3 is shown below:

[0038] Preferably, the organic solvent is tetrahydrofuran, the molar ratio of intermediate 2, tetrabutylammonium bromide and NaOH is 1.0:0.05:3.0, and the reaction time is 12h.

[0039] Further, when compound 1 in step 1 is N,N-dimethylaniline and compound 2 is fuming sulfuric acid, the preparation of intermediate 2 in step 2 is as follows: intermediate 1 is mixed with fuming sulfuric acid and heated to 75-85°C for 30-90 minutes. After the reaction is complete, the mixture is cooled to room temperature, and the obtained substance is poured into ice water. The system is adjusted to neutral with a strong base, and a solid precipitates. The solid is filtered and dried to obtain intermediate 2. The synthetic route of intermediate 2 is shown below:

[0040] Preferably, the strong base is NaOH, the fuming sulfuric acid is fuming sulfuric acid with a sulfur trioxide concentration of 20%, and the reaction time is 60 min;

[0041] In step 3, intermediate 2 is dissolved in a solvent, and then tetrabutylammonium bromide and sodium hydroxide are added. Oxygen is then introduced, and the mixture is reacted at room temperature for 12–16 hours to obtain intermediate 3. The synthetic route for intermediate 3 is shown below:

[0042] Preferably, the organic solvent is tetrahydrofuran, the molar ratio of intermediate 2 to TBAB and sodium hydroxide is 1.0:0.05:3.0, and the reaction time is 12 h.

[0043] Furthermore, the weak base in step 4 is at least one of pyridine, triethylamine, and 4-dimethylaminopyridine; preferably, the molar ratio of intermediate 3, oxalyl chloride, weak base, and heteroarylamine is 1.0:10.0:0.1:5.0.

[0044] The third objective of this invention is the application of pH-responsive dyes with structural formulas I to VIII in tumor recognition formulations or gastric imaging formulations.

[0045] The present invention has the following beneficial effects:

[0046] 1. This invention provides a novel and simple pH regulation strategy, namely, achieving precise pH control by introducing heteroaryl amines at position 9 of traditional rhodamine dyes and replacing different heteroatoms at position 10. Based on this strategy, a pH-responsive dye platform spanning the entire physiological pH range (the pH-responsive dye platform consists of a series of pH-responsive dyes suitable for different pH ranges) is constructed. Furthermore, the pH-responsive dyes in this platform possess excellent photophysical properties, sensitive pH response capabilities, and cover a response range from pH 0.5 to 7.0, meeting the monitoring needs of different pH microenvironments in vivo.

[0047] 2. The near-infrared pH-responsive dye with the structure shown in Formula III obtained by this invention is suitable for use in the slightly acidic environment of tumors and has near-infrared emission (690nm) and a relatively sensitive pH response capability. Traditional pH probes have a mutation range of more than 2 pH units, but the smallest mutation range obtained by this invention is 1 pH unit. The mutation range of pH-responsive dye III for tumor detection is 1.5 pH units, which is beneficial to improving the signal-to-noise ratio of imaging and improving the accuracy of tumor boundary identification. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the synthesis route of the pH-responsive dye in this invention;

[0049] Figure 2 and Figure 3 The figures show the proton and carbon spectra of a pH-responsive dye with structural formula I.

[0050] Figure 4 and Figure 5The images show the proton and carbon spectra of a pH-responsive dye with structural formula II.

[0051] Figure 6 and Figure 7 The images show the proton and carbon spectra of a pH-responsive dye with structural formula III, respectively.

[0052] Figure 8 and Figure 9 The images show the proton and carbon spectra of a pH-responsive dye with structural formula IV.

[0053] Figure 10 and Figure 11 The images show the 1H NMR and 1C NMR spectra of a pH-responsive dye with structural formula V.

[0054] Figure 12 and Figure 13 The images show the proton and carbon spectra of a pH-responsive dye with structural formula VI.

[0055] Figure 14 and Figure 15 The images show the 1H NMR and 1C NMR spectra of a pH-responsive dye with structural formula VII.

[0056] Figure 16 and Figure 17 The figures show the proton and carbon spectra of a pH-responsive dye with structural formula VIII.

[0057] Figure 18 and Figure 19 The figures show the proton and carbon spectra of the pH-responsive dye in Comparative Example 1, respectively.

[0058] Figure 20 The UV absorption spectrum, fluorescence emission spectrum and pH-related normalized fluorescence spectrum of the pH-responsive dye with structural formula I-VIII and the pH-responsive dye in Comparative Example 1 in triacid buffer solution.

[0059] Figure 21 This invention provides a CCK-8 cytotoxicity assay using pH-responsive dyes with structural formulas III, VI, and VII.

[0060] Figure 22 This invention provides a laser confocal microscopy experiment for lysosomal staining of HepG2 cells with pH-responsive dyes of structural formulas III and VI.

[0061] Figure 23 This is a comparative laser confocal experiment of the pH-responsive dyes with structural formulas III and VI in this invention on cancer cells (HepG2) and normal cells (HL-7702, L929). Detailed Implementation

[0062] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0063] In the examples described below, the solvents, catalysts, ligands, and bases used were purchased from Aladdin Technology Co., Ltd., the cell lines were purchased from ATCC (American Type Culture Collection), 10% fetal bovine serum (FBS) was purchased from Hyclone, and DMEM medium was purchased from Gibco, USA.

[0064] Example 1

[0065] The synthetic route for intermediate R-1 (i.e., intermediate 1) is shown below:

[0066]

[0067] The specific method for synthesizing intermediate R-1 is as follows: 3-bromo-N,N-dimethylaniline (25.0 mmol), formaldehyde aqueous solution (37%, 12.5 mol), and acetic acid (12.5 mmol) are mixed and stirred at room temperature for 12 hours, and then poured into ice water to precipitate a white solid; the obtained solid is filtered and dried in a vacuum drying oven to obtain intermediate R-1 as a white solid with a yield of 98%.

[0068] Example 2

[0069] The synthesis of the intermediate includes the following steps:

[0070] Step 1: Synthesis of intermediate Si-1 (i.e., intermediate 2)

[0071] The synthetic route for intermediate Si-1 is shown below:

[0072]

[0073] The synthesis of intermediate Si-1 was as follows: Under nitrogen protection, intermediate R-1 (10 mmol) prepared in Example 1 was added to the reaction flask and dissolved in anhydrous tetrahydrofuran (40 mL). After the system was cooled to -78°C, n-butyllithium solution (11 mmol) was added dropwise and stirred to form a mixture. The mixture was then stirred at -78°C for about 1 hour. Dichlorodimethylsilane (5.5 mmol) was added and the temperature was raised to room temperature for 12 hours. After the reaction was monitored by thin-layer chromatography, 10 mL of water was added to the reaction system to quench the reaction. The solvent was removed by dichloromethane extraction and rotary evaporation to obtain light blue liquid Si-1.

[0074] Step 2: Synthesis of intermediate Si-2 (intermediate 3)

[0075] The synthetic route for intermediate Si-2 is shown below:

[0076]

[0077] The synthesis of intermediate Si-2 was carried out as follows: intermediate Si-1 (1 mmol) was dissolved in acetone (50 mL), and potassium permanganate was added in portions when the system was cooled to -15 °C. The total amount of potassium permanganate added was 3 mmol. The reaction was carried out at room temperature for 6 hours. After the reaction was completed by monitoring the reaction with thin-layer chromatography, manganese dioxide was removed by filtration and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography with a mesh size of 200-300 and eluted with petroleum ether / dichloromethane (1:1) to obtain a light yellow solid Si-2 with a yield of 32%.

[0078] Example 3

[0079] The synthetic route for pH-responsive dyes with structural formula I is as follows: Figure 1 The specific preparation method is as follows:

[0080] Intermediate Si-2 (1 mmol) and oxalyl chloride (10 mmol) were added to dichloromethane (20 mL) and reacted at 45 °C for 2 hours. The solvent was removed by rotary evaporation. The solution was then dissolved in dichloromethane and added dropwise to a mixture of 2-aminopyridine (5 mmol) and triethylamine (1 mL). The mixture was reacted at room temperature for 6 hours. After removing the solvent by rotary evaporation, the crude product was purified by silica gel column chromatography (200-300 mesh). The product was eluted with dichloromethane / methanol (60:1) to obtain a pH-responsive dye with the structural formula shown in Figure I, with a yield of 42%.

[0081] Example 4

[0082] The synthesis method of the pH-responsive dye with the structural formula as shown in Example II in this embodiment is the same as that in Example 3. The difference is that 2-aminopyridine is replaced with 3-aminopyridine, and the pH-responsive dye with the structural formula as shown in Example II is finally obtained with a yield of 36%.

[0083] Example 5

[0084] The synthesis method of the pH-responsive dye with the structural formula shown in Example III in this embodiment is the same as that in Example 3, except that 2-aminopyridine is replaced with 4-aminopyridine, and the pH-responsive dye with the structural formula shown in Example III is finally obtained with a yield of 80%.

[0085] Example 6

[0086] The synthesis method of the pH-responsive dye with the structural formula as shown in Example IV in this embodiment is the same as that in Example 3. The difference is that 2-aminopyridine is replaced with 3-methyl-4-aminopyridine, and the pH-responsive dye with the structural formula as shown in Example IV is finally obtained with a yield of 81%.

[0087] Example 7

[0088] The synthesis method of the pH-responsive dye with the structural formula shown in Example V in this embodiment is the same as that in Example 3. The difference is that 2-aminopyridine is replaced with 2-aminoquinoline, and the pH-responsive dye with the structural formula shown in Example V is finally obtained with a yield of 65%.

[0089] Example 8

[0090] The synthetic route for pH-responsive dyes with structural formulas as shown in VI is as follows. Figure 1 The above includes the following steps:

[0091] Step 1: Synthesis of intermediate C-1 (i.e., intermediate 2)

[0092] The synthetic route for intermediate C-1 is shown below:

[0093]

[0094] The synthesis of intermediate C-1 was carried out as follows: Under nitrogen protection, intermediate R-1 (10 mmol) prepared in Example 1 was added to the reaction flask and dissolved in anhydrous tetrahydrofuran (40 mL). After the reaction system was cooled to -78°C, n-butyllithium solution (11 mmol) was added dropwise and stirred to form a mixture. The mixture was then stirred at -78°C for about 1 hour. Acetone (5.5 mmol) was added and the temperature was raised to room temperature for 12 hours. After the reaction was monitored by thin-layer chromatography, 10 mL of water was added to the system to quench the reaction. Aluminum trichloride (50 mmol) was added to the crude product after dichloromethane extraction and the reaction was continued at room temperature for 14 hours to obtain intermediate C-1.

[0095] Step 2: Synthesis of intermediate C-2 (intermediate 3)

[0096] The synthetic route for intermediate C-2 is shown below:

[0097]

[0098] The synthesis of intermediate C-2 is as follows: intermediate C-1 (1 mmol) is dissolved in acetone (50 mL), the system is cooled to -15 °C, potassium permanganate is added in portions of 3 mmol, and the reaction is carried out at room temperature for 6 hours. After the reaction is completed by thin-layer chromatography, manganese dioxide is removed by filtration and the solvent is removed by rotary evaporation. The crude product is purified by silica gel column chromatography with 200-300 mesh and eluted with petroleum ether / ethyl acetate (2:1) to obtain intermediate C-2.

[0099] Step 3: Synthesis of pH-responsive dyes with structural formulas as shown in VI

[0100] The synthetic route for pH-responsive dyes with structural formulas as shown in VI is as follows:

[0101]

[0102] The preparation of the pH-responsive dye with the structural formula shown in VI is as follows: intermediate C-2 (1 mmol) and oxaloyl chloride (10 mmol) are added to dichloromethane (20 mL), and the reaction is carried out at 45 °C for 2 hours. The solvent is removed by rotary evaporation. Then, the solution is dissolved in dichloromethane and added dropwise to a mixture of 4-aminopyridine (5 mmol) and triethylamine (1 mL). The mixture is stirred at room temperature for 6 hours. After removing the solvent by rotary evaporation, the crude product is purified by silica gel column chromatography with a mesh size of 200-300. The pH-responsive dye with the structural formula shown in VI is obtained by elution with dichloromethane / methanol (60:1) with a yield of 47%.

[0103] Example 9

[0104] The synthetic route for pH-responsive dyes with structural formulas as shown in VII is as follows. Figure 1 The above includes the following steps:

[0105] Step 1: Synthesis of intermediate P-1 (intermediate 2)

[0106] The synthetic route for intermediate P-1 is shown below:

[0107]

[0108] The preparation of intermediate P-1 was as follows: Under nitrogen protection, intermediate R-1 (10 mmol) prepared in Example 1 was added to the reaction flask and dissolved in anhydrous tetrahydrofuran (40 mL). After the system was cooled to -78°C, n-butyllithium solution (11 mmol) was added dropwise and stirred to form a mixture. The mixture was then stirred at -78°C for about 1 hour. Dichlorophenylphosphine (5.5 mmol) was added and the temperature was raised to room temperature for 12 hours. The reaction was quenched with water and then 10 mL of hydrogen peroxide (H2O2) was added. After the reaction was monitored by thin-layer chromatography, 10 mL of water was added to the system to quench the reaction. The mixture was extracted with dichloromethane and the solvent was removed by rotary evaporation to obtain a light yellow liquid, P-1.

[0109] Step 2: Synthesis of intermediate P-2 (i.e., intermediate 3)

[0110] The synthetic route for intermediate P-2 is shown below:

[0111]

[0112] The synthesis of intermediate P-2 is as follows: intermediate P-1 (1 mmol), tetrabutylammonium bromide (0.05 mmol) and sodium hydroxide (3.0 mmol) are added to tetrahydrofuran (10 mL) to form a mixture. The mixture is then stirred with oxygen at room temperature for 12 hours. After the reaction is completed by monitoring the reaction with thin-layer chromatography, the solvent is removed by rotary evaporation. The crude product is then purified by silica gel column chromatography with a 200-300 mesh filter and eluted with petroleum ether / ethyl acetate (5:1) to obtain intermediate P-2.

[0113] Step 3: Synthesis of pH-responsive dyes with structural formulas as shown in VII

[0114] The synthetic route for pH-responsive dyes with structural formulas as shown in VII is as follows:

[0115]

[0116] The preparation of the pH-responsive dye with the structural formula shown in VII is as follows: intermediate P-2 (1 mmol) and oxaloyl chloride (10 mmol) were dissolved in dichloromethane (20 mL), and reacted at 45 °C for 2 hours. The solvent was removed by rotary evaporation. Then, the solution was dissolved in dichloromethane and added dropwise to a mixture of 4-aminopyridine (5 mmol) and triethylamine (1 mL). The mixture was stirred at room temperature for 6 hours. After removing the solvent by rotary evaporation, the crude product was purified by silica gel column chromatography with a 200-300 mesh screen and eluted with dichloromethane / methanol (60:1) to obtain compound VII in 30% yield.

[0117] Example 10

[0118] The synthetic route for pH-responsive dyes with structural formulas as shown in Figure VIII is as follows. Figure 1 The above includes the following steps:

[0119] Step 1: Synthesis of intermediate R-2 (i.e., intermediate 1)

[0120] The synthetic route for intermediate R-2 is shown below:

[0121]

[0122] The synthesis of intermediate R-2 was as follows: N,N-dimethylaniline (25.0 mmol), formaldehyde aqueous solution (37%, 12.5 mol), and acetic acid (12.5 mmol) were mixed and stirred at room temperature for 12 hours. The reaction was monitored by thin-layer chromatography. After the reaction was completed, the mixture was adjusted to neutral with sodium hydroxide, extracted with 100 mL of dichloromethane, and the organic phase was collected. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography with a mesh size of 200-300 and eluted with petroleum ether / ethyl acetate to obtain intermediate R-2 in a yield of 49%.

[0123] Step 2: Synthesis of intermediate SO-1 (intermediate 2)

[0124] The synthetic route for intermediate SO-1 is shown below:

[0125]

[0126] The synthesis of intermediate SO-1 is as follows: 5 mmol of intermediate R-2 was weighed into a 50 mL round-bottom flask. Under vigorous stirring, 8 mL of fuming sulfuric acid with a sulfur trioxide concentration of 20% was poured into the system. The mixture was heated to 80 °C and reacted for 1 hour. Then, it was cooled to room temperature and poured into ice water. The system was adjusted to neutrality with sodium hydroxide, and a large amount of white solid precipitated. After filtration, the solid was dried in a vacuum drying oven to obtain intermediate SO-1.

[0127] Step 3: Synthesis of intermediate SO-2

[0128] The synthetic route for intermediate SO-2 is shown below:

[0129]

[0130] The synthesis of intermediate SO-2 was carried out as follows: intermediate SO-1 (1 mmol), tetrabutylammonium bromide (0.05 mmol), and sodium hydroxide (3.0 mmol) were added to tetrahydrofuran (10 mL) to form a mixture. The mixture was then stirred with oxygen at room temperature for 12 hours. After the reaction was monitored by thin-layer chromatography, the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography with a mesh size of 200-300 and eluted with dichloromethane / methanol to obtain intermediate SO-2 in a yield of 47%.

[0131] Step 4: Synthesis of pH-responsive dyes with structural formulas as shown in VIII

[0132] The synthetic route for pH-responsive dyes with structural formulas as shown in VIII is as follows:

[0133]

[0134] The synthesis of the pH-responsive dye with the structural formula shown in VIII is as follows: intermediate SO-2 (1 mmol) and oxaloyl chloride (10 mmol) were added to dichloromethane (20 mL), and the reaction was carried out at 45 °C for 2 hours. The solvent was removed by rotary evaporation. The solution was then dissolved in dichloromethane and added dropwise to a mixture of 4-aminopyridine (5 mmol) and triethylamine (1 mL). The mixture was stirred at room temperature for 6 hours. After removing the solvent by rotary evaporation, the crude product was purified by silica gel column chromatography with a 200-300 mesh screen and eluted with dichloromethane / methanol (60:1) to obtain compound VIII in 60% yield.

[0135] Comparative Example 1

[0136] This embodiment describes a method for preparing a pH-responsive dye of formula O-Py, comprising the following steps:

[0137] Step 1: Synthesis of intermediate O-1

[0138] The synthetic route for intermediate O-1 is shown below:

[0139]

[0140] The synthesis of intermediate O-1 was as follows: Pyronin Y (0.2 mmol) was dissolved in MeOH (15 mL) and heated to 60 °C; after the solid dissolved, sodium borohydride (1.0 mmol) was slowly added; the solution was then stirred for 30 minutes, cooled to room temperature, and concentrated to obtain a dark purple solid; the solid was extracted into dichloromethane (3 × 10 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain pure intermediate O-1.

[0141] Step 2: Synthesis of intermediate O-2

[0142] The synthetic route for intermediate O-2 is shown below:

[0143]

[0144] The synthesis of intermediate O-2 was as follows: Compound O-1 (1 mmol) was added to acetone (50 mL). After the system was cooled to zero degrees Celsius, potassium permanganate was added in portions, with a total amount of 3 mmol of potassium permanganate added. The mixture was then stirred at room temperature for about 6 hours. After the reaction was monitored by thin-layer chromatography, manganese dioxide was removed by filtration and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (200-300 mesh) and eluted with petroleum ether / ethyl acetate (2:1) to obtain intermediate O-2.

[0145] Step 3: Synthesis of pH-responsive dye O-Py

[0146] The synthetic route of the pH-responsive dye O-Py is shown below:

[0147]

[0148] The synthesis of the pH-responsive dye O-Py was as follows: intermediate O-2 (1 mmol) and oxalyl chloride (10 mmol) were added to dichloromethane (20 mL), and the reaction was carried out at 45 °C for 2 hours. The solvent was removed by rotary evaporation. Then, the solution was dissolved in dichloromethane and added dropwise to a mixture of 4-aminopyridine (5 mmol) and triethylamine (1 mL). The mixture was stirred at room temperature for 6 hours. After removing the solvent by rotary evaporation, the crude product was purified by silica gel column chromatography with a 200-300 mesh screen and eluted with dichloromethane / methanol (60:1) to obtain compound O-Py in 78% yield.

[0149] Test Analysis:

[0150] 1. UV absorption spectra of pH-responsive dyes at different pH levels

[0151] The pH-responsive dyes prepared in the above examples and comparative examples were each formulated into 10 mM DMSO stock solutions. Triacid buffer solutions with different pH values ​​(0.3-10.0) were then prepared at 10 μM, and the UV absorbance values ​​were scanned and plotted. Figure 20 As shown, the long-wavelength absorptions of pH-responsive dyes with structural formulas I to VIII and the pH-responsive dye in Comparative Example 1 under acidic conditions are 650, 650, 654, 650, 650, 630, 700, 720, and 560 nm, respectively.

[0152] 2. Fluorescence spectra of pH-responsive dyes at different pH levels

[0153] The pH-responsive dyes prepared in the examples and comparative examples were prepared as 10 mM DMSO stock solutions. Fluorescence spectra were measured by adding triacid buffer solutions of different pH values, and fluorescence emission curves were obtained. Furthermore, the equation for the change in fluorescence intensity with pH was fitted, as shown below. Figure 20As shown, the fluorescence emission wavelengths of the pH-responsive dyes with structural formulas I-VIII and the pH-responsive dye in Comparative Example 1 are 690, 680, 695, 690, 690, 660, 750, 760, and 590 nm, respectively, and their pKa values ​​are 2.20, 2.30, 5.60, 4.00, 3.70, 6.50, 1.60, 0.80, and 6.93, respectively.

[0154] 3. CCK-8 cytotoxicity assay

[0155] HepG2 cells in logarithmic growth phase were seeded in 96-well plates at approximately 10,000 cells per well and cultured for 24 hours at 37°C and 5% CO2 in DMEM(H) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1000 KU / L). After complete cell adhesion, different concentration gradients (0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) of pH-responsive dyes (structures III, VI, and VII) were added, with three replicates for each concentration. A blank control group was also included. Cells were cultured for another 24 hours after adding the pH-responsive dyes, and cell viability was assessed using a CCK-8 toxicity assay kit. The results are shown below. Figure 21 As shown; from Figure 21 It can be seen that the cell survival rate is greater than 80% when the concentration is less than 5 μM.

[0156] 4. Laser confocal imaging of lysosomal staining in HepG2 cells

[0157] HepG2 cells were cultured overnight in 1 cm confocal glass-bottomed culture dishes. After staining with pH-responsive dyes of different concentrations (2 μL of 1 mM pH-responsive dye stock solution added to 1 mL PBS) for 25–30 min, the pH-responsive dyes were imaged under a laser confocal microscope using appropriate excitation and emission filters. The excitation wavelength and absorption range of both pH-responsive dyes of structure III and VI were λex = 543 nm and λem = 600–740 nm, respectively. (See details...) Figure 22 The reason is that, since the probe is water-soluble, it is expected to be uniformly dispersed in the aqueous solution, and the positive charge of the probe allows it to quickly enter the lysosome; based on this, lysosome imaging can be achieved.

[0158] 5. Contrastive laser confocal imaging of cancer cells (HepG2) and normal cells (HL-7702, L929)

[0159] HepG2, HL-7702, and L929 cells were cultured overnight in 1 cm confocal glass dishes. After staining with pH-responsive dyes of specific concentrations (2 μL of 1 mM pH-responsive dye stock solution added to 1 mL PBS) for 25–30 min, the pH-responsive dyes were imaged under a laser confocal microscope using appropriate excitation and emission filters. The excitation wavelength and absorption range of both pH-responsive dyes of structure III and VI were λex = 543 nm and λem = 600–740 nm, respectively. (See details...) Figure 23 As shown, the pH-responsive dye with structural formula III has better cancer cell recognition ability.

[0160] In summary, this invention provides a novel and simple pH regulation strategy, namely, achieving precise pH control by introducing a heteroaryl amine at position 9 and replacing different heteroatoms at position 10 of a rhodamine pH-responsive dye. Based on this strategy, a full-pH-range pH-responsive dye platform was constructed. The pH-responsive dyes in this platform possess excellent photophysical properties, sensitive pH response capabilities, and cover a response range from pH 0.5 to 7.0, meeting the monitoring needs of different pH microenvironments in vivo. Furthermore, the near-infrared pH-responsive dye III prepared in this invention is suitable for the slightly acidic environment of tumors and exhibits near-infrared emission (690 nm), relatively sensitive pH response capabilities, and a mutation range of 1.5 pH units, which is beneficial for improving the signal-to-noise ratio of imaging and enhancing the accuracy of tumor boundary identification.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pH-responsive dye platform characterized in that, The pH-responsive dye platform comprises a pH-responsive dye, the structure of which is shown as follows: wherein R1 is , , , and X is .

2. The pH-responsive dye platform of claim 1, wherein, The pH-responsive dye platform comprises a pH-responsive dye with a structural formula as shown in formula I-V; wherein the structural formula of formula I is , the structural formula of formula II is , the structural formula of formula III is , the structural formula of formula IV is , and the structural formula of formula V is .

3. A method of preparing the pH-responsive dye platform of any one of claims 1-2, wherein, The pH-responsive dye platform comprises a pH-responsive dye, the preparation of which comprises the following steps: Step 1, an intermediate 1 is prepared by mixing a compound 1 with formaldehyde aqueous solution and acetic acid and then reacting at room temperature, the synthesis route of which is shown as follows: wherein R is Br; Step 2, the intermediate 1 is reacted with a compound 2 to obtain an intermediate 2, the synthesis route of which is shown as follows: ; wherein the compound 2 is dichlorodimethylsilane; Step 3, the intermediate 2 is oxidized to obtain an intermediate 3, the synthesis route of which is shown as follows: ； Step 4, the intermediate 3 and oxalyl chloride are dissolved in a first solvent and reacted at 40-50°C for 45-80 min, then the solvent is removed and dissolved in a second solvent, and then a mixture containing a compound 3 and a weak base is added dropwise and reacted at room temperature for 6-10 h to obtain a pH-responsive dye, the synthesis route of which is shown as follows: , the compound 3 is any one of 2-aminopyridine, 3-aminopyridine, 4- aminopyridine, 3-methyl-4-aminopyridine, and 2-aminoquinoline; X in the intermediate 2, the intermediate 3 and the pH-responsive dye is all ; The R1 is , , , and .

4. The production method according to claim 3, characterized by, The preparation of the intermediate 2 in the step 2 is as follows: the intermediate 1 is dissolved in a solvent and cooled to-75--80°C, a strong base is added and the reaction is continued for 100-120 min, then the compound 2 is added and the temperature is increased to room temperature and reacted for 12-16 h, the reaction is quenched and extracted to obtain the intermediate 2; wherein the strong base is n-butyllithium or sec-butyllithium; the intermediate 2 in the step 3 is oxidized with potassium permanganate to obtain the intermediate 3.

5. The preparation method according to claim 3, characterized in that, The weak base in the step 4 is at least one of pyridine, triethylamine and 4-dimethylaminopyridine.

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