A fluorescent probe based on triphenylamine derivative mitochondrial-targeted nitroreductase response and a synthesis method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and relates to a fluorescent probe based on a triphenylamine derivative mitochondria-targeting nitroreductase response, and a synthesis method and application thereof. The fluorescent probe based on the triphenylamine derivative mitochondria-targeting nitroreductase response provided by the application is formed by connecting 4-butyric acid carbonyl triphenyl phosphine as a mitochondria-targeting group, 4-nitrobenzyl bromide as a nitroreductase response group, and a triphenylamine derivative as a fluorescent group through a synthesis reaction.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and relates to a mitochondrial-targeting nitroreductase-responsive fluorescent probe based on triphenylamine derivatives, its synthesis method, and its application. Background Technology

[0002] Mitochondria are membrane-bound organelles found in most cells, serving as the primary site for cellular aerobic respiration and energy supply. Their basic structure can be divided into four functional regions: the outer mitochondrial membrane, the mitochondrial membrane space, the inner mitochondrial membrane, and the mitochondrial matrix. The inner mitochondrial membrane encloses its own genetic material, mitochondrial DNA, making it a semi-autonomous organelle. Besides providing energy, mitochondria participate in processes such as cell differentiation, cell signaling, and apoptosis, and possess the ability to regulate cell growth and the cell cycle.

[0003] Programmed apoptosis is a crucial process in maintaining the balance between growth and death in normal cells. Once this balance is disrupted, normal cells can transform into tumor cells, gaining the ability to proliferate indefinitely – a classic hallmark of cancer. The Bcl-2 protein family, including anti-apoptotic, pro-apoptotic, and BH3-only members, are key regulators of this process. They initiate apoptosis by regulating mitochondrial integrity in response to cellular stress (such as DNA damage or endoplasmic reticulum stress) and mitochondrial outer membrane permeation. Furthermore, activation of caspase leads to a loss of mitochondrial inner membrane potential and ATP synthesis, as well as an increase in reactive oxygen species. During tumorigenesis, the apoptotic program can be dysregulated at multiple levels, contributing to and driving cancer development. Mitochondria, vital organelles, provide energy for cellular processes, are essential for aerobic respiration, and participate in cell differentiation, signal transduction, and apoptosis, playing a crucial role in cell growth and cell cycle regulation.

[0004] More and more scholars are focusing their research on key substances in mitochondrial metabolism as a target for cancer treatment research. Therefore, developing new technologies to study mitochondrial metabolic processes is becoming increasingly important for mitochondrial and cell biology.

[0005] Chinese patent application document (publication number: CN110204693A) discloses a high molecular covalent organic framework polymer based on triphenylamine derivatives, its preparation method and application. This probe belongs to the field of polymer materials technology. The molecular framework is a large conjugated system and ion recognition groups are added. However, it is mainly used for the detection of divalent mercury ions in sewage. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned problems in the existing technology by proposing a mitochondrial-targeting nitroreductase-responsive fluorescent probe based on triphenylamine derivatives, which enables the detection of nitroreductases in tumor cells and real-time imaging of mitochondrial metabolic activities.

[0007] The objective of this invention can be achieved through the following technical solution: a mitochondrial-targeting nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the fluorescent probe comprising the structure shown below:

[0008] .

[0009] Nitroreductase (NTR), under the action of coenzymes nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), can reduce aromatic nitro compounds and is an endogenous flavin protease with reducing activity. It is widely found in *Escherichia coli* and the brain, kidney, lung, and heart tissues of mammals, and is closely related to solid tumors. Nitroreductase is overexpressed in hypoxic tumor cells, such as HeLa cells, A549 cells, MCF-7 cells, and V79 cells. The expression level of nitroreductase in tumor cells is directly related to the hypoxic state of the cells, and the expression level increases with the severity of hypoxia. Severe oxygen deficiency can also trigger other complications, such as myocardial ischemia, stroke, and other inflammatory diseases. Therefore, by using the triphenylamine derivative-based mitochondrial-targeting nitroreductase-responsive fluorescent probe to assess the expression level of nitroreductase in biological tissues or tumor cells, this invention can be used to study the generation of hypoxia and its development in solid tumors, which has important guiding significance for the clinical prevention and treatment of diseases caused by tissue hypoxia.

[0010] In the above-mentioned fluorescent probe based on triphenylamine derivative mitochondrial-targeting nitroreductase response, the fluorescent probe uses the fluorescent group triphenylamine TPA as the parent compound and connects the nitroreductase recognition group and the mitochondrial targeting group through a chemical synthesis reaction.

[0011] The fluorescent probe of this invention is a fluorescent probe for detecting nitroreductase activity in solution and cells, and also has the function of targeting mitochondria in cells, so it can be applied to mitochondrial activity and nitroreductase responsive bioimaging in cells.

[0012] In the above-mentioned fluorescent probe based on the mitochondrial-targeted nitroreductase response of triphenylamine derivatives, the nitroreductase recognition group is p-nitrobenzyl bromide, and the mitochondrial targeting group is 4-butyric triphenylphosphine.

[0013] This invention also provides a method for synthesizing a mitochondrial-targeting nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the method comprising the following steps:

[0014] S1. Intermediate 2 was prepared by reacting 4-bromobenzoyl chloride and thiophene in an aluminum chloride catalyzed Friedels-Crafts reaction.

[0015] S2. Using the Suzuki coupling method, with a palladium complex as a catalyst, intermediate 3 was prepared in an alkaline environment using intermediate 2 and intermediate 1 as reactants, and tetrahydrofuran and water as solvents.

[0016] S3. Intermediate 3 is reacted with malononitrile in the presence of titanium tetrachloride via Knoevenagel condensation to generate intermediate 4.

[0017] S4. React intermediate 4 with boron tribromide to hydrolyze the methoxy group to obtain intermediate 5 containing two hydroxyl groups;

[0018] S5. Intermediate 5 and p-nitrobenzyl bromide are reacted to give intermediate 6, which is monosubstituted with p-nitrobenzyl.

[0019] S6. A fluorescent probe was prepared by reacting intermediate 6 with compound f in an alkaline solvent via a nucleophilic substitution reaction.

[0020] The structural formulas of intermediate 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5, and intermediate 6 are as follows:

[0021] ;

[0022] The structural formula of compound f is .

[0023] In the above-mentioned method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the palladium complex in step S2 is tetrakis(triphenylphosphine)palladium.

[0024] Preferably, the molar ratio of intermediate 1 to intermediate 2 is 1:(1-5), and the volume ratio of tetrahydrofuran to water is (2-5):1.

[0025] In the above-mentioned method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the molar ratio of intermediate 3 to malononitrile in step S3 is 1:(8-15).

[0026] In the above-mentioned method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the molar ratio of intermediate 4 and boron tribromide in step S4 is 1:(2-5).

[0027] In the above-mentioned method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, an acid-binding agent is added during step S5.

[0028] 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.

[0029] Preferably, the molar ratio of intermediate 5 and p-nitrobenzyl bromide in step S5 is 1:(1.2-1.5).

[0030] In the above-mentioned method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, the molar ratio of intermediate 6 to intermediate f in step S6 is 1:(1.1-1.5).

[0031] Preferably, the base used in step S6 is triethylamine, the solvent is anhydrous dichloromethane, the reaction is carried out at room temperature, and the product is obtained by column chromatography.

[0032] The present invention also provides an application of the above-mentioned triphenylamine derivative-based mitochondrial-targeting nitroreductase-responsive fluorescent probe in monitoring and tracking the dynamic movement of mitochondria in living cells.

[0033] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on triphenylamine derivatives. This probe uses 4-butyricotriphenylphosphine as the mitochondrial targeting group, 4-nitrobenzyl bromide as the nitroreductase responsive group, and a triphenylamine derivative as the fluorophore, which are linked together through a synthetic reaction. This probe exhibits almost no fluorescence in normal cells, but can produce a strong fluorescence signal in tumor cells. This is because, under the action of nitroreductase in tumor cells, the probe TPA-TPP-PBN undergoes a single electron transfer, generating a nitro anion free radical, which is then further reduced to an amino group. Finally, the p-nitrobenzyl amino group leaves, exposing the fluorophore, thereby producing fluorescence. This enables the detection of nitroreductase in tumor cells and real-time imaging of mitochondrial metabolic activity. Attached Figure Description

[0034] Figure 1 The synthetic route diagram for the fluorescent probe TPA-TPP-PBN prepared in Example 1 is shown.

[0035] Figure 2 The phosphorus NMR spectrum (500 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1;

[0036] Figure 3The 1H NMR spectrum (126 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1;

[0037] Figure 4 The carbon NMR spectrum (126 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1;

[0038] Figure 5 High-resolution mass spectrometry (ESI-HR-MS) of the fluorescent probe TPA-TPP-PBN prepared in Example 1.

[0039] Figure 6 The fluorescence emission spectra of the fluorescent probe TPA-TPP-PBN prepared in Example 1 in different concentrations of dimethyl sulfoxide / aqueous solution (A) and the fluorescence emission spectra of TPA-TPP-PBN in different concentrations (B) are shown.

[0040] Figure 7 The cytotoxicity of the fluorescent probe TPA-TPP-PBN prepared in Example 1: (A) Survival rate of colon cancer cells (CT26) after co-incubation with different concentrations of TPA-TPP-PBN; (B) Survival rate of renal tubular epithelial cells (HK-2) after co-incubation with different concentrations of TPA-TPP-PBN.

[0041] Figure 8 The fluorescence signal of the fluorescent probe TPA-TPP-PBN prepared in Example 1 is shown in the following images: (A) Fluorescence signal of TPA-TPP-PBN in normal renal tubular epithelial cells (HK-2), and (B) Fluorescence signal of TPA-TPP-PBN in vascular endothelial cells (HUVEC).

[0042] Figure 9 The fluorescence signal of the fluorescent probe TPA-TPP-PBN prepared in Example 1 in liver cancer cells (HepG2) is shown in the image. (A) Fluorescence signal of different concentrations of TPA-TPP-PBN (5, 20, 50, 100 μg / mL) at different times (1 h, 6 h, 24 h). (B) Semi-quantitative analysis results of fluorescence signal.

[0043] Figure 10 The fluorescence signal of the fluorescent probe TPA-TPP-PBN prepared in Example 1 in colon cancer cells (CT26) is shown in the images. (A) Fluorescence signal of different concentrations of TPA-TPP-PBN (5, 20, 50, 100 μg / mL) at different times (1 h, 6 h, 24 h). (B) Semi-quantitative analysis results of fluorescence signal.

[0044] Figure 11The fluorescence signal of the fluorescent probe TPA-TPP-PBN prepared in Example 1 in breast cancer cells (MCF-7) is shown in the images. (A) Fluorescence signal of different concentrations of TPA-TPP-PBN (5, 20, 50, 100 μg / mL) at different times (1 h, 6 h, 24 h). (B) Semi-quantitative analysis results of fluorescence signal.

[0045] Figure 12 The penetration and imaging performance of the fluorescent probe TPA-TPP-PBN prepared in Example 1 in hepatocellular carcinoma tumor spheres.

[0046] Figure 13 The fluorescent probe TPA-TPP-PBN prepared in Example 1 is shown in the fluorescence signal diagram and analysis results of co-localization with mitochondria. Detailed Implementation

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

[0048] This invention designs a mitochondrial-targeting nitroreductase-responsive fluorescent probe TPA-TPP-PBN based on a triphenylamine derivative, the structure of which is shown in formula (1).

[0049]

[0050] Formula 1

[0051] The aforementioned triphenylamine derivative-based mitochondrial-targeting nitroreductase-responsive fluorescent probe comprises a fluorescent molecule, a triphenylamine derivative (TPA), a mitochondrial-targeting group, 4-butyric acid-triphenylphosphine (TPP), and a nitroreductase recognition group, p-nitrobenzyl bromide, synthesized through a chemical reaction. This probe exhibits almost no fluorescence in normal cells but produces a strong fluorescence signal in tumor cells, allowing for the monitoring of dynamic mitochondrial activity and the investigation of nitroreductase expression levels using fluorescence imaging.

[0052] The following examples illustrate the preparation process and technical effects of this mitochondrial-targeting nitroreductase-responsive fluorescent probe.

[0053] Example 1:

[0054] The synthesis method of the fluorescent probe TPA-TPP-PBN is as follows: Figure 1 As shown, the specific steps include the following:

[0055] S1. Add 1.6 g (7.29 mmol) of 4-bromobenzoyl chloride and 2.0 g (15 mmol) of aluminum chloride to a 100 mL dry round-bottom flask. Dissolve the bromobenzoyl chloride in 10 mL of anhydrous dichloromethane under argon protection and cool to 0 °C. Add 575 µL of thiophene dropwise and stir at room temperature for 3 h. After the reaction is complete, quench with ice water and add 5.87 mL of 3 M HCl and stir for 20 min. Extract the reaction solution with dichloromethane, wash the organic phase with water and saturated brine, dry with sodium sulfate, concentrate under reduced pressure, and separate by column chromatography to obtain 1.4 g of milky white solid, i.e., intermediate 2, with a yield of 89.7%. The structure was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 HNMR (500 MHz, Chloroform-d): δ (ppm) 7.77 (dd,J= 6.6 Hz, J= 1.85 Hz, 3H), 7.67 (d, J= 8.2 Hz, 2H), 7.65 (d, J= 8.75 Hz, 1H), 7.20 (t, J = 9.55 Hz, 1H); 13 CNMR (126 MHz, Chloroform-d): δ (ppm) 187.1,143.3, 136.9, 134.8, 134.6,131.8, 130.7, 128.1, 127.3; HR-MS(ESI): C 11 H8BrOS [M+H] + m / z 266.9473, found266.9479.

[0056] S2. 4,4'-Dimethoxy-4''-boronic acid triphenylamine (1.0 g, 2.86 mmol), i.e., raw material 1 and intermediate 2 (1.52 g, 5.72 mmol), potassium carbonate (3.95 g, 28.6 mmol), and Pd(PPh3)4 (0.165 g, 0.143 mmol) were placed in a 50 mL round-bottom flask and dissolved in 16 mL of a THF:water (3:1) mixed solvent. Under argon protection, the mixture was refluxed and stirred at 60 °C for 24 hours. The solution was washed with ethyl acetate, the organic phase was washed with water and saturated brine, dried over sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 1.2 g of yellow foamy solid, i.e., intermediate 3, with a yield of 88.7%. The structure was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 H NMR (500 MHz, Chloroform-d): δ (ppm) 7.95 (dt, J =8.45 Hz, J= 2.10 Hz, 2H),7.74 (m, 2H), 7.69 (dt, J = 8.45 Hz, J = 1.7 Hz, 2H), 7.50 (dt, J = 8.80 Hz, J = 2.75Hz, 2H), 7.20 (q, J = 13.70 Hz, 1H), 7.13 (td, J = 8.95 Hz, J = 3.45 Hz, 4H), 7.02(td, J = 8.75 Hz, J = 2.55 Hz, 2H), 6.89 (td, J = 8.95 Hz, J = 3.45 Hz (4H), 3.84 (s, 6H); 13 C NMR (126 MHz, 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 ; HR-MS(ESI): C 31 H 26 NO3S [M+H] + m / z 492.1633, found492.1620.

[0057] S3. Add intermediate 3 (1.2 g, 2.86 mmol) to a dry 100 mL double-necked flask. Dissolve the intermediate in 20 mL anhydrous dichloromethane under argon protection. Add malononitrile (16.99 g, 25.74 mmol) under ice bath conditions. After 20 minutes, add 1.34 mL titanium tetrachloride. After 30 minutes, add 2.12 mL pyridine. Stir the reaction at 35 °C for 24 h. After the reaction is complete, quench the reaction with 20 mL ice water, separate the phases, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by column chromatography to obtain 1.17 g of reddish-brown solid, intermediate 4, with a yield of 88.6%. Characterize the structure using 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 H NMR (500 MHz, Chloroform-d): δ (ppm) 7.84 (t, J= 9.80 Hz, 2H), 7.70 (d, J = 8.35 Hz, 2H), 7.51 (dd, J = 21.35 Hz, J = 8.30 Hz, 4H), 7.27 (t, J = 4.90 Hz, 1H), 7.14 (d, J = 8.90 Hz, 4H), 7.02 (d, J = 8.75 Hz, 2H), 6.89 (d, J = 8.85 Hz, 4H), 3.84 (s, 6H); 13 C NMR (126 MHz, Chloroform-d): δ (ppm) 165.0 , 156.3 ,144.8 , 140.4 , 138.8 , 136.4 , 135.8 , 133.7 , 130.6 , 128.9 , 127.7 , 127.0, 126.3, 120.0, 114.8, 114.2,77.2, 55.5; HR-MS(ESI): C 34 H 25 N3O2S [M]m / z539.1667, found 539.1655.

[0058] S4. Add intermediate 4 (425 mg, 0.788 mmol) to a dry 100 mL double-necked flask. Dissolve in 10 mL of anhydrous dichloromethane under argon protection. Slowly add 2 mL of 1 M boron tribromide dichloromethane solution in an ice bath. After the addition is complete, stir the reaction at room temperature for 16 hours. After the reaction is complete, add 20 mL of water to quench the reaction, separate the liquid and liquid phase, wash the organic phase with water and saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by column chromatography to obtain 400 mg of reddish-brown solid, i.e., intermediate 5, with a yield of 99.3%. The structure was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 H NMR (500 MHz, Chloroform-d): δ (ppm)7.83 (m,2H), 7.67 (d, J = 8.35 Hz, 2H), 7.52 (d, J = 8.35 Hz, 2H), 7.45 (d, J =8.30 Hz,2H), 7.26 (m, 1H), 7.04 (d, J = 7.60 Hz, 4H), 6.97 (d,J = 7.80 Hz (2H), 6.81 (m, 4H); 13 C NMR (126 MHz, 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; HR-MS(ESI):C 32 H 20 N3O2S [MH] - m / z 510.1276, found 510.1277.

[0059] S5. Add the above 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) to a dry two-necked flask. Under argon protection, add 15 mL of anhydrous dimethylformamide to dissolve the mixture, and reflux at 60 °C with stirring for 2 h. After the reaction is complete, add 40 mL of water to quench the reaction, extract and separate the contents, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by column chromatography to obtain 145 mg of wine-red solid, i.e., intermediate 6, with a yield of 42.5%. The structure was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 H NMR (500 MHz, Chloroform-d): δ(ppm) 8.28 (d, J = 8.60 Hz, 2H), 7.84 (m, 2H), 7.67 (dd, J = 20.15 Hz, J = 8.35 Hz, 4H), 7.54 (d, J = 8.35 Hz, 2H), 7.49 (d, J = 8.40 Hz, 2H,)7.27 (t, J = 4.45 Hz, 1H), 7.13 (d, J = 8.50 Hz, 2H), 7.07 (d, J = 8.65 Hz, 2H), 7.02 (d, J = 8.25 Hz, 2H), 6.93 (d, J= 8.70 Hz, 2H), 6.82 (d, J = 7.95 Hz, 2H), 5.18(s, 2H), 13 C NMR (126 MHz, 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, 130.6, 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; HR-MS(ESI): C 39 H 25 N4O4S - [MH] - m / z 645.1602, found 645.1604.

[0060] S6. Add (4-carboxybutyl)triphenylphosphine bromide (221 mg, 0.5 mmol) to a 100 mL dry single-necked flask, dissolve it in 15 mL of anhydrous dichloromethane under argon protection, add 1 mL of thionyl chloride, reflux at 50 °C for 2 h, and after the reaction is complete, evaporate the reaction solution to dryness, and then use the oil pump to obtain intermediate f, which can be used directly in the next step of the reaction.

[0061] S7. Compound 6 (48 mg, 0.074 mmol) was added to a 50 mL round-bottom flask and dissolved in 10 mL of anhydrous dichloromethane. 200 µL of anhydrous triethylamine was then added. Compound f (67.2 mg, 0.146 mmol) was added dropwise under ice bath and argon protection. The mixture was stirred at room temperature for 24 hours. After compound 6 had reacted completely, water (20 mL × 3) was added, followed by washing with saturated brine (20 mL × 3), drying with anhydrous sodium sulfate, concentration under reduced pressure, and extraction to obtain 63 mg of a blood-red solid (yield 82.9%). The structure was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 13C NMR (126 MHz, Chloroform-d: δ (ppm) 171.9,164.9, 155.0, 148.4, 147.5,145.9,144.9, 144.6, 144.4, 140.8, 138.6,136.6, 136.2, 135.1, 135.0, 134.0, 133.7, 133.6, 132.1, 130.6, 130.5, 130.48,129.0, 128.6, 128.5, 127.9, 127.7, 127.4, 126.5, 124.7, 123.8, 122.4, 122.0,119.5, 118.6, 117.9, 117.1, 115.9, 115.6, 114.6, 114.2, 77.4, 77.4, 77.2,76.9,69.0, 33.4, 28.9 ,22.5, 22.1; HR-MS(ESI):C 62 H 48 N4O5PS + [M-Br - ] + m / z 991.3078, found 991.3073.

[0062] Figure 2 The NMR phosphorus spectrum (500 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1 shows that the phosphorus purity of the probe TPA-TPP-PBN exceeds 95%.

[0063] Figure 3 The 1H NMR spectrum (126 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1 shows that the probe TPA-TPP-PBN has five methylene peaks between 1.5 and 5.5.

[0064] Figure 4 The figure shows the carbon NMR spectrum (126 MHz, Chloroform-d) of the fluorescent probe TPA-TPP-PBN prepared in Example 1. As can be seen from the figure, the probe TPA-TPP-PBN has a characteristic peak of carbonyl group at low field.

[0065] Figure 5 This is a high-resolution mass spectrometry (ESI-HR-MS) image of the fluorescent probe TPA-TPP-PBN prepared in Example 1; as shown in the figure, the probe TPA-TPP-PBN produces [M-Br] at 991.3078. - ]+ Positive ion peak.

[0066] The fluorescent probe TPA-TPP-PBN prepared above was dissolved in dimethyl sulfoxide / water mixed solutions with different proportions. As the proportion of water in the mixed solution increased, the fluorescence intensity of the probe TPA-TPP-PBN also increased due to aggregation-induced emission. Figure 6 A), and the fluorescence is concentration-dependent ( Figure 6 B).

[0067] Using colon cancer (CT26) cells and renal tubular epithelial cells (HK-2) cells as model cells, the cytotoxicity of TPA-TPP-PBN was investigated. CT26 cells and HK-2 cells were co-incubated with different concentrations of TPA-TPP-PBN (5, 20, 50, 100 μg / mL), and cell viability was detected using the MTT assay. Results are as follows: Figure 7 As shown, cell viability was above 90% at all concentrations. These results indicate that TPA-TPP-PBN has good cell safety.

[0068] Using HK-2 cells and HUVEC cells as model cells, the nitroreductase response performance of TPA-TPP-PBN in normal cells was investigated. After co-incubation for 24 h with TPA-TPP-PBN and TPA-TPP-OH (which did not exhibit nitroreductase response), DAPI staining was performed, and observation was conducted under a fluorescence microscope. Figure 8 As shown, TPA-TPP-PBN exhibits a weak fluorescence signal in normal cells, while TPA-TPP-OH displays a significant fluorescence signal. This indicates that normal cells lack nitrosoreductase and have a low level of nitrosoreductase, which prevents the removal of the p-nitroarylbenzyl group in TPA-TPP-PBN, resulting in its weak fluorescence signal.

[0069] Using CT26 cells, breast cancer cells (MCF-7), and liver cancer cells (HepG2) as models, the fluorescence expression of nitrosoreductase in TPA-TPP-PBN was detected. Figure 9 , 10 As shown in Figure 11, the fluorescence signal of TPA-TPP-PBN in all three types of tumor cells increased in a time- and concentration-dependent manner; in addition, a significant fluorescence signal was observed in tumor spheroids cultured from HepG2 cells. Figure 12 This further demonstrates that it has good nitro reductase response performance.

[0070] Using HepG2 and MCF-7 cells as model cells, the mitochondrial targeting distribution characteristics of TPA-TPP-PBN were investigated. Results are as follows: Figure 13As shown, the fluorescence signal of TPA-TPP-PBN highly overlaps with the fluorescence signal of commercially available mitochondrial probes. Colocalization analysis using ImageJ software yielded colocalization correlation coefficients of 0.94 and 0.59, respectively, demonstrating that TPA-TPP-PBN has good mitochondrial targeting distribution characteristics.

[0071] In summary, the above-mentioned mitochondrial-targeted nitroreductase-responsive fluorescent probe based on triphenylamine derivatives provided by this invention uses 4-butyricotriphenylphosphine as the mitochondrial targeting group, 4-nitrobenzyl bromide as the nitroreductase responsive group, and a triphenylamine derivative as the fluorophore, and is synthesized through a reaction. This probe exhibits almost no fluorescence in normal cells, but can produce a strong fluorescent signal in tumor cells. This is because, under the action of nitroreductase in tumor cells, the probe TPA-TPP-PBN undergoes a single-electron transfer, generating a nitro anion free radical, which is then further reduced to an amino group. Finally, the p-nitrobenzyl amino group leaves, exposing the fluorophore and thus producing fluorescence, enabling the detection of nitroreductase in tumor cells and real-time imaging of mitochondrial metabolic activity.

[0072] The embodiments herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution) of its technical solution, and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.

[0073] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

[0074] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A mitochondrial-targeting nitroreductase-responsive fluorescent probe based on a triphenylamine derivative, characterized in that, The fluorescent probe has the following structure: 。 2. A method for synthesizing a triphenylamine derivative-based mitochondrial-targeting nitroreductase-responsive fluorescent probe as described in claim 1, characterized in that, The method includes the following steps: S1. Intermediate 2 was prepared by reacting 4-bromobenzoyl chloride and thiophene in an aluminum chloride catalyzed Friedels-Crafts reaction. S2. Using the Suzuki coupling method, with a palladium complex as a catalyst, intermediate 3 was prepared in an alkaline environment using intermediate 2 and intermediate 1 as reactants, and tetrahydrofuran and water as solvents. S3. Intermediate 3 is reacted with malononitrile in the presence of titanium tetrachloride via Knoevenagel condensation to generate intermediate 4. S4. React intermediate 4 with boron tribromide to hydrolyze the methoxy group to obtain intermediate 5 containing two hydroxyl groups; S5. Intermediate 5 and p-nitrobenzyl bromide are reacted to give intermediate 6, which is monosubstituted with p-nitrobenzyl. S6. A fluorescent probe was prepared by reacting intermediate 6 with compound f in an alkaline solvent via a nucleophilic substitution reaction. The structural formulas of intermediate 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5, and intermediate 6 are as follows: ； The structural formula of compound f is .

3. The method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative according to claim 2, characterized in that, In step S2, the palladium complex is tetra(triphenylphosphine)palladium.

4. The method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative according to claim 2, characterized in that, In step S3, the molar ratio of intermediate 3 to malononitrile is 1:(8-15).

5. The method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative according to claim 2, characterized in that, In step S4, the molar ratio of intermediate 4 to boron tribromide is 1:(2-5).

6. The method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative according to claim 2, characterized in that, An acid-binding agent is added during the reaction process in step S5.

7. The method for preparing a mitochondrial-targeted nitroreductase-responsive fluorescent probe based on a triphenylamine derivative according to claim 2, characterized in that, In step S6, the molar ratio of intermediate 6 to intermediate f is 1:(1.1-1.5).

8. The application of the triphenylamine derivative-based mitochondrial-targeting nitroreductase-responsive fluorescent probe as described in claim 1 in monitoring and tracking the dynamic movement of mitochondria in living cells.

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