A lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, its synthesis method, and application

Abstract

The present invention belongs to the field of medicinal chemistry technology and relates to a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine backbone, its synthesis method, and its application. The triphenylamine derivative-based lysosomal-targeted nitroreductase-responsive fluorescent probe provided by the present invention does not emit light upon initial entry into cells. However, after a period of culture in cancer cells, the electron-withdrawing nitro group is reduced by the intracellular nitroreductase. Under laser irradiation, the charge within the fluorophore shifts, resulting in fluorescence. The present invention also provides a method for synthesizing the lysosomal-targeted nitroreductase-responsive fluorescent probe and its use in bioimaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry and relates to a lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, a synthesis method thereof, and an application thereof. Background Art

[0002] Nitroreductase (NTR) is an endogenous flavoprotein protease with reducing properties. Under the action of coenzymes nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), it metabolizes nitro compounds in cells. Nitroreductase is highly expressed in Escherichia coli, but also appears in brain tissue, liver tissue, and kidney tissue of mammals, and is closely related to solid tumors in organisms. Nitroreductase is overexpressed in tumor cells in hypoxic conditions, such as breast cancer and lung cancer cells. Based on the basic theory of magnetic resonance, Ugurbil et al. used a high-field magnetic resonance scanner to evaluate the NAD in hypoxic living cells and normal cells. + The study also examined the levels of nitroreductase and NADH metabolites, demonstrating that hypoxia in tissues can lead to increased expression of nitroreductase within cells. Furthermore, numerous studies have demonstrated that nitroreductase expression levels in tumor cells increase with decreasing intracellular oxygen concentrations. Therefore, assessing nitroreductase expression levels in tissues or tumor cells can help investigate the generation of hypoxia and its progression within solid tumors, providing valuable insights into the clinical prevention and treatment of diseases associated with tissue hypoxia.

[0003] Lysosomes are essential organelles in eukaryotic cells, performing functions such as phagocytosis, digestion, defense, and autolysis. Recent studies have also revealed that lysosomes play important roles in cell signaling, cell migration, cholesterol homeostasis, activation of apoptosis, and tissue remodeling. Disruption of the lysosomal environment can lead to cellular dysfunction and various diseases, including neurodegenerative diseases, rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, and cancer. Lysosomal hydrolases activate enzymes and degrade macromolecules such as proteins. During cell carcinogenesis, lysosomes undergo changes in their location and membrane permeability, prompting the secretion of large amounts of cathepsins into the cytoplasm. These proteases, along with zymogen activation systems such as fibrin, contribute to the degradation of these proteins, thereby enhancing cancer cell motility and invasiveness, and promoting tumor growth. Therefore, monitoring lysosomal activity in cells is crucial for studying their physiological functions, organ survival, and growth, and also provides a more effective tool for the clinical diagnosis of diseases associated with lysosomal dysfunction.

[0004] Lysosomal imaging technologies have developed rapidly in recent years. Commonly used methods include magnetic resonance imaging, computed tomography, positron emission tomography, single-photon emission computed tomography, and ultrasound imaging. However, radioactive imaging techniques not only carry the inevitable risk of radiation but also have limitations in specificity, sensitivity, and resolution. Optical imaging, represented by fluorescence imaging, is a non-invasive technique with reduced risk to the human body, higher sensitivity, and shorter response time, and is attracting increasing attention from researchers. Compared with these traditional analytical methods, fluorescence detection technology has the advantages of ease of use, rapid response, and high sensitivity. In recent years, researchers have modified various small molecule dyes to investigate various substances within lysosomes, such as pH, viscosity, various metal ions, anions, small and large biomolecules, and so on. These dyes have the advantages of high spatial resolution, low biotoxicity, high fluorescence quantum yield, and good photostability. However, few fluorescent probes targeting lysosomal nitroreductase have been reported.

[0005] A Chinese patent application document (publication number: CN109456264A) discloses a fluorescent probe for detecting nitroreductase, its preparation method, and its application in enzymatic reactions. However, the probe belongs to the field of industrial analysis and detection technology and can only detect and analyze NTR activity in enzymatic reactions. Summary of the Invention

[0006] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and propose a lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton to achieve the detection of intracellular nitroreductase and real-time imaging of lysosomal activity.

[0007] The purpose of the present invention can be achieved by the following technical solution: a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the fluorescent probe comprising the following structure:

[0008]

[0009] In the above-mentioned lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the fluorescent probe uses the fluorescent molecule triphenylamine derivative TPA as a matrix and connects the nitroreductase recognition group and the lysosome targeting group through a chemical synthesis reaction.

[0010] The triphenylamine skeleton-based lysosome-targeted nitroreductase-responsive fluorescent probe of the present invention is a fluorescent probe used to detect NTR activity in solutions and cells. It also has the function of targeting lysosomes in cells, so it can be applied to lysosome activity and NTR-responsive biological imaging in cells.

[0011] In the above-mentioned lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the nitroreductase recognition group is p-nitrobenzyl bromide, and the lysosome-targeting group is N-(2-chloroethyl)morpholine hydrochloride.

[0012] The present invention also provides a method for synthesizing the above-mentioned lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the method comprising the following steps:

[0013] S1, 4-bromobenzoyl chloride and thiophene undergo Friedels-Crafts reaction in the presence of aluminum chloride to produce intermediate 2;

[0014] S2. Using the Suzuki coupling method, intermediate 2 and intermediate 1 are reactants in an alkaline environment with a metal palladium complex as a catalyst, and tetrahydrofuran and water as solvents to obtain intermediate 3;

[0015] S3, reacting intermediate 3 with malononitrile in the presence of titanium tetrachloride to generate intermediate 4 through Knoevenagel condensation reaction;

[0016] S4, placing intermediate 4 and boron tribromide in an ice bath, and hydrolyzing the methoxy group to obtain intermediate 5 containing two hydroxyl groups;

[0017] S5, reacting intermediate 5 with p-nitrobenzyl bromide in an alkaline environment to obtain p-nitrobenzyl monosubstituted intermediate 6;

[0018] S6, reacting the intermediate 6 with N-(2-chloroethyl)morpholine hydrochloride in a solvent containing a base and a catalyst through a nucleophilic substitution reaction to obtain a responsive fluorescent probe;

[0019] The structural formulas of raw material 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5 and intermediate 6 are as follows:

[0020]

[0021] Preferably, the molar ratio of intermediate 1 to intermediate 2 in step S1 is 1:(1.5-2.5).

[0022] In the above-mentioned method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the metal palladium complex in step S2 is tetrakis(triphenylphosphine)palladium.

[0023] Preferably, the volume ratio of tetrahydrofuran to water in step S2 is (2-4):1.

[0024] In the above-mentioned method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the molar ratio of the intermediate 3 to malononitrile in step S3 is 1:(8-15).

[0025] Preferably, the molar ratio of intermediate 4 to boron tribromide in step S4 is 1:(2-5).

[0026] In the above-mentioned method for synthesizing a lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, an acid-binding agent should be added in step S5.

[0027] Preferably, the acid-binding agent is at least one of an inorganic base and an organic base, wherein the inorganic base includes at least one of potassium carbonate, sodium carbonate, and sodium bicarbonate, and the organic base includes at least one of triethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0028] Preferably, the molar ratio of the intermediate 5 to p-nitrobenzyl bromide in step S5 is 1:(1-1.5).

[0029] In the above-mentioned method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the molar ratio of intermediate 6 in step S6 to N-(2-chloroethyl)morpholine hydrochloride is 1:(1.1-1.5).

[0030] In the above-mentioned method for synthesizing a lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, the catalyst in step S6 is potassium iodide or sodium iodide.

[0031] Preferably, the base in step S6 is cesium carbonate.

[0032] Preferably, in step S6, the product is heated to reflux in the presence of acetonitrile as a solvent and separated by column chromatography.

[0033] The present invention also provides an application of the above-mentioned lysosome-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton in monitoring and tracking the dynamic movement of lysosomes in living cells.

[0034] Compared with existing technologies, the present invention has the following advantages: the triphenylamine derivative-based lysosomal-targeted nitroreductase-responsive fluorescent probe provided by the present invention does not emit light upon initial entry into cells. However, after a period of culture in cancer cells, the electron-withdrawing nitro group is reduced by the intracellular nitroreductase. Under laser irradiation, the charge within the fluorophore shifts, resulting in fluorescence. The present invention also provides a method for synthesizing the lysosomal-targeted nitroreductase-responsive fluorescent probe and its use in bioimaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is the pathway diagram of the fluorescent probe TPA-MP-PBN prepared in Example 1.

[0036] Figure 2 This is the H NMR spectrum (500 MHz, Chloroform-d) of the fluorescent probe TPA-MP-PBN prepared in Example 1;

[0037] Figure 3 This is the NMR spectrum (126 MHz, Chloroform-d) of the fluorescent probe TPA-MP-PBN prepared in Example 1;

[0038] Figure 4 High-resolution mass spectrometry (ESI-HRMS) of the fluorescent probe TPA-MP-PBN prepared in Example 1;

[0039] Figure 5 Fluorescence emission spectra of the fluorescent probe TPA-MP-PBN prepared in Example 1 in dimethyl sulfoxide / water solutions at different concentrations (A) and fluorescence emission spectra of TPA-MP-PBN at different concentrations (B);

[0040] Figure 6 Cell viability of the fluorescent probe TPA-MP-PBN prepared in Example 1, (A) the viability of colon cancer cells (CT26) after co-incubation with different concentrations of TPA-MP-PBN, (B) the viability of renal tubular epithelial cells (HK-2) after co-incubation with different concentrations of TPA-MP-PBN.

[0041] Figure 7 The nitroreductase response performance of the fluorescent probe TPA-MP-PBN prepared in Example 1, (A) fluorescence signal diagram of TPA-MP-PBN in normal renal tubular epithelial cells (HK-2), (B) fluorescence signal diagram of TPA-MP-PBN in vascular endothelial cells (HUVEC).

[0042] Figure 8 Figure 1 shows the fluorescence signal of the fluorescent probe TPA-MP-PBN prepared in Example 1 in CT26 cells, (A) fluorescence graphs of different concentrations (5, 20, 50, 100 μg / mL) of TPA-MP-PBN at different times (1 h, 6 h, 24 h), and (B) fluorescence semi-quantitative analysis bar graph.

[0043] Figure 9 Figure 1 shows the fluorescence signal of the fluorescent probe TPA-MP-PBN prepared in Example 1 in liver cancer cells (HepG2), (A) fluorescence graphs of different concentrations (5, 20, 50, 100 μg / mL) of TPA-MP-PBN at different times (1 h, 6 h, 24 h), and (B) fluorescence semi-quantitative analysis bar graph.

[0044] Figure 10 Figure 1 shows the fluorescence signal of the fluorescent probe TPA-MP-PBN prepared in Example 1 in breast cancer cells (MCF-7), (A) fluorescence graphs of different concentrations (5, 20, 50, 100 μg / mL) of TPA-MP-PBN at different times (1 h, 6 h, 24 h), and (B) fluorescence semi-quantitative analysis bar graph.

[0045] Figure 11 The penetration and imaging performance of the fluorescent probe TPA-MP-PBN prepared in Example 1 in liver cancer tumor spheres.

[0046] Figure 12 This is the co-localization fluorescence signal diagram and analysis results of the fluorescent probe TPA-MP-PBN prepared in Example 1 and lysosomes. DETAILED DESCRIPTION

[0047] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0048] The present invention designs a lysosomal-targeted nitroreductase-responsive fluorescent probe TPA-MP-PBN based on a triphenylamine derivative, the structure of which is shown in formula (1):

[0049]

[0050] The lysosome-targeted nitroreductase-responsive probe is synthesized through a chemical reaction: a fluorescent molecule, TPA; a lysosome-targeting group, N-(2-chloroethyl)morpholine hydrochloride (MP); and a nitroreductase-recognizing group, p-nitrobenzyl bromide. This probe exhibits virtually no fluorescence in normal cells but produces a strong fluorescence signal in tumor cells. Fluorescence imaging can be used to monitor the dynamic activity of lysosomes in cells and investigate nitroreductase expression levels.

[0051] The following examples illustrate the preparation process and technical effects of the lysosome targeting probe.

[0052] Example 1:

[0053] The synthesis method of fluorescent probe TPA-MP-PBN is as follows Figure 1 As shown, the specific steps include:

[0054] S1. Add 4-bromobenzoyl chloride (1.6 g, 7.29 mmol) and aluminum chloride (2.0 g, 15 mmol) to a 100 mL dry round-bottom flask. Under argon protection, dissolve the mixture in 10 mL of anhydrous dichloromethane and cool to 0°C. Add 575 μL of thiophene dropwise and stir at room temperature for 3 h. After the reaction is completed, quench with ice water, add 5.87 mL of 3 M hydrochloric acid and stir for 20 minutes. The reaction solution is extracted with dichloromethane, and the organic phase is washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 1.4 g of an off-white solid, namely intermediate 2, with a yield of 89.7%. The structure of the intermediate 2 was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1 H NMR (500MHz, Chloroform-d): δ (ppm) 7.77 (dd, J = 6.6Hz, J = 1.85Hz, 3H), 7.67 (d, J = 8.2Hz, 2H), 7.65 (d, J = 8.75Hz, 1H), 7.20 (t, J = 9.55Hz, 1H); 13 C NMR (125MHz, Chloroform-d): δ (ppm) 187.1, 143.3, 136.9, 134.8, 134.6, 131.8, 130.7, 128.1, 127.3; HRMS (ESI): C 11 H8BrOS[M+H] + m / z 266.9473,found266.9479.

[0055] S2. 4,4'-dimethoxy-4"-boronic acid triphenylamine (1.0 g, 2.86 mmol), i.e., raw material 1, intermediate 2 (1.52 g, 5.72 mmol), potassium carbonate (3.95 g, 28.6 mmol), and tetrakistriphenylphosphine palladium (0.165 g, 0.143 mmol) were placed in a 50 mL round-bottom flask, and 16 mL of a mixed solvent of tetrahydrofuran / water (3 / 1, v / v) was added to dissolve the mixture. Under argon protection, the mixture was refluxed at 60°C with stirring for 24 h, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 1.2 g of a yellow foamy solid, i.e., intermediate 3, with a yield of 88.7%. The structure was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1H NMR (500MHz, Chloroform-d): δ (ppm) 7.95 (dt, J = 8.45Hz, J = 2.10Hz, 2H), 7.74 (m, 2H), 7.69 (dt, J = 8.45Hz, J = 1.7Hz, 2H), 7.50 (dt, J = 8.80Hz, J = 2.75Hz ,2H),7.20(q,J=13.70Hz,1H),7.13(td,J=8.95Hz,J=3.45Hz,4H),7.02(td ,J=8.75Hz,J=2.55Hz,2H),6.89(td,J=8.95Hz,J=3.45Hz,4H),3.84(s,6H); 13 C NMR (125MHz, Chloroform-d): δ (ppm) 187.7, 156.2, 149.1, 144.9, 143.9, 140.5, 135. 9,134.5,133.9,131.1,130.0,127.9,127.7,127.0,126.2,114.2,55.5; HRMS(ESI):C 31 H 26 NO3S[M+H] + m / z492.1633, found 492.1620.

[0056] S3, to a dry 100mL two-necked flask, intermediate 3 (1.2g, 2.86mmol) was added, 20mL of anhydrous dichloromethane was added under argon protection to dissolve, malononitrile (16.99g, 25.74mmol) was added under an ice bath, 1.34mL of TiCl4 was added after 20 minutes, 30 minutes later, 2.12mL of pyridine was added, and the reaction was stirred at 35°C for 24h. After completion of the reaction, 20mL of ice water was added to quench the reaction, the liquid was separated, the organic phase was extracted with dichloromethane, washed with saturated salt water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to give 1.17g of a reddish-brown solid, i.e., intermediate 4, with a yield of 88.6%. The structure was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1 H NMR (500MHz, Chloroform-d): δ (ppm) 7.84 (t, J = 9.80Hz, 2H), 7.70 (d, J = 8.35Hz, 2H), 7.51 (dd, J = 21.35Hz, J = 8.30H z,4H),7.27(t,J=4.90Hz,1H),7.14(d,J=8.90Hz,4H),7.02(d,J=8.75Hz,2H),6.89(d,J=8.85Hz,4H),3.84(s,6H); 13C NMR (125MHz, Chloroform-d): δ (ppm) 165.0,156.3,144.8,140.4,138.8,136.4,135.8,1 33.7,130.6,128.9,127.7,127.0,126.3,120.0,114.8,114.2,77.2,55.5; HRMS(ESI):C 34 H 25 N3O2S[M]m / z539.1667, found 539.1655.

[0057] S4. To a dry two-necked 100 mL flask was added intermediate 4 (425 mg, 0.788 mmol), dissolved in 10 mL of anhydrous dichloromethane under argon protection, and slowly added dropwise 2 mL of 1 M boron tribromide in dichloromethane solution under an ice bath. After the addition was complete, the reaction was stirred at room temperature for 16 hours. After the reaction was completed, 20 mL of water was added to quench the reaction, the liquid was separated, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 400 mg of a reddish-brown solid, i.e., intermediate 5, with a yield of 99.3%. The structure was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1 H NMR (500MHz, Chloroform-d): δ (ppm) 7.83 (m, 2H), 7.67 (d, J = 8.35Hz, 2H), 7.52 (d, J = 8.35Hz, 2H), 7.45(d,J=8.30Hz,2H),7.26(m,1H),7.04(d,J=7.60Hz,4H),6.97(d,J=7.80Hz,2H),6.81(m,4H); 13 CNMR (125MHz, Chloroform-d): δ (ppm) 156.2, 152.3, 144.7, 140.1, 138.7, 136.5, 136.0, 133.7, 132.4, 128.6, 131.0,130.6,128.9,127.7,127.2,126.3,120.4,114.5,119.9,116.3,114.6,114.3,77.3,68.3; HRMS(ESI):C 32 H 20 N3O2S[MH] - m / z 510.1276, found 510.1277.

[0058] S5. To a dry two-necked flask were added the above-mentioned intermediate 5 (270 mg, 0.528 mmol), 4-nitrobenzyl bromide (150 mg, 0.687 mmol added in 3 batches), and potassium carbonate (59 mg, 0.4224 mmol). Under argon protection, 15 mL of anhydrous DMF was added to dissolve the mixture, and the mixture was refluxed and stirred at 60°C for 2 h. After the reaction was completed, 40 mL of water was added to quench the reaction. The liquid was extracted, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 145 mg of a wine-red solid, i.e., intermediate 6, with a yield of 42.5%. The structure was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1 H NMR (500MHz, Chloroform-d): δ (ppm) 8.28 (d, J = 8.60Hz, 2H), 7.84 (m, 2H), 7.67 (dd, J = 20.15Hz, J = 8.35Hz, 4H), 7.54 (d, J = 8.35Hz, 2H), 7.49 (d, J = 8.40Hz, 2H, )7.27(t,J=4.45Hz,1H),7.13(d,J=8.50Hz,2H),7.07(d,J=8.65Hz,2H),7.02( d,J=8.25Hz,2H),6.93(d,J=8.70Hz,2H),6.82(d,J=7.95Hz,2H),5.18(s,2H); 13 C NMR (125MHz, Chloroform-d): δ (ppm) 165.0, 154.6, 152.5, 147.6, 144.6, 141.3, 140.3, 138.1, 136.4, 136.0, 133.8, 1 30.6,129.0,127.8,127.6,127.4,126.7,126.3,123.9,120.4,116.4,115.8,114.6,114.2,77.1,69.0; HRMS(ESI):C 39 H 25 N4O4S - [MH] - m / z 645.1602, found 645.1604.

[0059] S6. To a dry two-necked 50 mL flask was added intermediate 6 (46 mg, 0.071 mmol), which was dissolved in 10 mL of anhydrous acetonitrile under argon protection. N-(2-chloroethyl)morpholine hydrochloride (15.9 mg, 0.085 mmol), cesium carbonate (36.5 mg, 0.112 mmol), and KI (11 mg, 0.066 mmol) were added sequentially. The mixture was refluxed at 80°C for 24 h. After the reaction was completed, the reaction solution was dried and 20 mL of dichloromethane and 20 mL of water were added. The organic phase was extracted and washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 16 mg of a wine-red solid, i.e., TPA-MP-PBN, with a yield of 29.6%. The structure was characterized by H NMR, C NMR, and high-resolution mass spectrometry. 1 H NMR (600MHz, Chloroform-d): δ (ppm) 8.30 (d, J = 10.35Hz, 2H), 7.86 (m, 2H), 7. 68(q,J=10.10Hz,4H),7.53(m,4H),7.28(m,1H),7.14(t,J=9.75Hz,4H),7.04 (d,J=10.40Hz,2H),6.93(dd,J=12.85Hz,J=10.60Hz,4H),5.20(s,2H),4.17( t,J=7.20Hz,2H),3.80(t,J=7.20Hz,4H),2.90(t,J=6.55Hz,2H),2.69(s,4H); 13 C NMR (200MHz, Chloroform-d): δ (ppm) 164.9, 155.3, 154.6, 149.0, 144.6, 144.5, 141.3, 140.6, 138.7, 136.4, 135.9, 130.9, 130.6, 128.9, 128 .8,127.8,127.6,127.0,126.7,126.3,123.9,120.5,115.8,115.5,11 4.2,77.3,77.1,76.9,69.1,68.2,66.6,65.7,57.6,53.9; HRMS(ESI):C 45 H 37 N5O5S[M+H] + m / z760.2594,found760.2575.

[0060] Figure 2: This is the H NMR spectrum (500 MHz, Chloroform-d) of TPA-MP-PBN in the example. As can be seen from the figure, the probe compound TPA-MP-PBN of the present invention is very pure, almost free of impurities, and exhibits seven methylene peaks between 2.5 and 5.5.

[0061] Figure 3 : is the NMR carbon spectrum (126 MHz, Chloroform-d) of TPA-MP-PBN in the example; it can be seen from the figure that the probe compound TPA-MP-PBN of the present invention is very pure and contains almost no impurities.

[0062] Figure 4 The high-resolution mass spectrum (ESI-HRMS) of TPA-MP-PBN in the embodiment is shown in FIG. As can be seen from the figure, the probe compound TPA-MP-PBN of the present invention shows [M+H] at 760.2575. + Positive ion peak.

[0063] The TPA-MP-PBN prepared above was dissolved in different proportions of dimethyl sulfoxide / water mixed solutions. As the proportion of water in the mixed solution continued to increase, the fluorescence intensity of the probe compound TPA-MP-PBN also increased due to the aggregation-induced emission effect ( Figure 5 A), and the fluorescence was concentration-dependent ( Figure 5 B).

[0064] Breast cancer cells (MCF-7) and vascular endothelial cells (HUVEC) were used as model cells to investigate the cellular safety of TPA-MP-PBN by co-incubation with TPA-MP-PBN. MCF-7 cells and HUVEC cells were co-incubated with different concentrations of TPA-MP-PBN (5, 20, 50, 100 μg / mL), and the cell survival rate was determined using the MTT assay. Figure 6 As shown in the figure, the cell survival rate at each concentration was above 90%. The results showed that TPA-MP-PBN has good cell safety.

[0065] HK-2 cells and HUVEC were used as model cells to investigate the nitroreductase responsiveness of TPA-MP-PBN. TPA-MP-PBN and TPA-MP-OH, which does not have nitroreductase responsiveness, were added and incubated for 24 hours, followed by DAPI staining and observation under a fluorescence microscope. Figure 7 As shown in the figure, the fluorescence signal of TPA-MP-PBN in normal cells is very weak, while TPA-MP-OH has an obvious fluorescence signal, indicating that normal cells do not have a low level of nitroso reductase and the p-nitrosoarylbenzyl group in TPA-MP-PBN cannot be removed, resulting in a weak fluorescence signal.

[0066] CT26 cells, MCF-7 cells, and hepatocellular carcinoma cells (HepG2) were used as model cells to detect the fluorescence expression of nitroso reductase in response to TPA-MP-PBN. Figure 8 、 9 As shown in Figures 10 and 11, the fluorescence signals of TPA-MP-PBN in the three tumor cells were enhanced in a time- and concentration-dependent manner; in addition, obvious fluorescence signals were observed in the tumor spheres cultured with HepG2 cells ( Figure 11 ), further proving that it has good nitroreductase response performance.

[0067] MCF-7 cells and HepG2 cells were used as model cells to investigate the lysosomal targeting distribution characteristics of TPA-MP-PBN. Figure 12 As shown in the figure, the fluorescence signal of TPA-MP-PBN highly overlaps with the fluorescence signal of the commercially available lysosomal probe. The co-localization analysis was performed using ImageJ software, and the co-localization correlation coefficients of the two were 0.99 and 0.97, respectively, proving that TPA-MP-PBN has good lysosomal targeting distribution characteristics.

[0068] In summary, the present invention provides a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative. The probe does not emit light upon initial entry into cells. However, after a period of incubation in cancer cells, the electron-withdrawing nitro group is reduced by the intracellular nitroreductase. Upon laser irradiation, the charge within the fluorophore shifts, resulting in fluorescence. The present invention also provides a method for synthesizing this lysosomal-targeted nitroreductase-responsive fluorescent probe and its use in bioimaging.

[0069] The parts of the embodiment herein that are not exhaustive of the midpoint values ​​of the technical scope claimed for protection by the present invention and the new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope claimed for protection by the present invention; at the same time, in all the embodiments listed or not listed in the solutions of the present invention, each parameter in the same embodiment merely represents an example of its technical solution (i.e., a feasible solution), and there is no strict coordination and limitation relationship between the parameters, wherein the parameters can be replaced with each other without violating the axioms and the claims of the present invention, unless otherwise stated.

[0070] The technical means disclosed in the solutions of the present invention are not limited to the technical means disclosed in the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific embodiment of the present invention. It should be noted that for those skilled in the art, various improvements and modifications can be made without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention.

[0071] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton, characterized in that: The fluorescent probe has the following structure:

2. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 1, characterized in that: The method comprises the following steps: S1, 4-bromobenzoyl chloride and thiophene undergo Friedels-Crafts reaction in the presence of aluminum chloride to produce intermediate 2; S2. Using the Suzuki coupling method, intermediate 2 and intermediate 1 are reactants in an alkaline environment with a metal palladium complex as a catalyst, and tetrahydrofuran and water as solvents to obtain intermediate 3; S3, reacting intermediate 3 with malononitrile in the presence of titanium tetrachloride to generate intermediate 4 through Knoevenagel condensation reaction; S4, placing intermediate 4 and boron tribromide in an ice bath, and hydrolyzing the methoxy group to obtain intermediate 5 containing two hydroxyl groups; S5, reacting intermediate 5 with p-nitrobenzyl bromide in an alkaline environment to obtain p-nitrobenzyl monosubstituted intermediate 6; S6, reacting the intermediate 6 with N-(2-chloroethyl)morpholine hydrochloride in a solvent containing a base and a catalyst through a nucleophilic substitution reaction to obtain a responsive fluorescent probe; The structural formulas of raw material 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5 and intermediate 6 are as follows:

3. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 2, characterized in that: In step S2, the metal palladium complex is tetrakis(triphenylphosphine)palladium.

4. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 2, wherein: The molar ratio of intermediate 3 to malononitrile in step S3 is 1:(8-15).

5. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 2, wherein: In step S5, an acid binding agent is added during the process.

6. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 2, wherein: The molar ratio of intermediate 6 in step S6 to N-(2-chloroethyl)morpholine hydrochloride is 1:(1.1-1.5).

7. The method for synthesizing a lysosomal-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine skeleton according to claim 2, wherein: The catalyst in step S6 is potassium iodide or sodium iodide.

8. A use of the triphenylamine-based lysosomal-targeted nitroreductase-responsive fluorescent probe as claimed in claim 1 for monitoring and tracking the dynamic movement of lysosomes in living cells, wherein the purpose of the use is not to diagnose or treat diseases.

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