Preparation method and application of pH / nitroreductase double-response type fluorescent probe

Abstract

The invention discloses a preparation method and application of a pH / nitroreductase double-response type fluorescent probe, and belongs to the technical field of fluorescence detection. The probe is specifically activated only when the probe and nitroreductase coexist in an acidic microenvironment (pH = 6-7), fluorescence Turn-On response is achieved, the emission wavelength of the probe is located at 760-850 nm (near-infrared window), and the fluorescence intensity is enhanced by 13-132 times after the probe is activated. The probe has the advantages of high specificity, near-infrared quick response (the fluorescence is the strongest within 0-30 minutes after double conditions are met at the same time) and high sensitivity. In addition, the unique double-response mechanism effectively avoids the interference of a single factor, and the detection specificity and the signal-to-noise ratio in a complex biological system are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of fine chemical technology, specifically relating to a pH / nitroreductase dual-response fluorescent probe constructed based on heptamethrin dye. It can accurately identify and monitor changes in the level of acidic microenvironment and abnormally expressed nitroreductase in cellular tissues, and achieve precise and sensitive response through changes in fluorescence. Background Technology

[0002] Precision diagnosis and treatment of tumors is a major challenge in contemporary medical research. Studies have shown that the development and progression of tumors are not only accompanied by abnormal expression and activity of various key enzymes, but also by their unique microenvironment characteristics, especially the ubiquitous acidic microenvironment (pH 6.5-7.0), which provides an important window for tumor cell recognition. Among numerous tumor-associated enzymes, nitroreductase (NTR) has attracted widespread attention due to its high expression in tumors and its crucial role in activating prodrugs (such as 5-aminosalicylic acid prodrugs used in the treatment of colon cancer). Therefore, developing smart probes that can specifically respond to tumor microenvironment characteristics (such as acidic pH) and the activity of key enzymes (such as NTR) is of great significance for real-time, in-situ research on tumor biology at the molecular and cellular levels, and ultimately for guiding precision diagnosis and treatment.

[0003] Fluorescence imaging technology, with its high sensitivity, non-invasiveness, real-time visualization, and high spatiotemporal resolution, has become a powerful tool for studying biomolecular events and guiding surgery. However, existing "activatable" fluorescent probes designed for single biomarkers (such as those responding only to a specific enzyme or only to pH changes) still have many limitations when applied to the complex and variable in vivo tumor environment. First, the specificity of single response mechanisms is insufficient: pH-activated probes may produce false positive signals in inflammatory or other physiologically acidic regions; enzyme-activated probes may produce background interference due to low enzyme expression levels in non-tumor tissues or non-specific probe responses. Second, the sensitivity and signal-to-noise ratio are low: the expression level of target enzymes within tumors may be low, and the activation efficiency or fluorescence on-ratio of existing probes may be insufficient to clearly distinguish weak signals against complex backgrounds. To address the problems of existing probes, developing highly sensitive, high signal-to-noise ratio fluorescent probes with AND gate logic response characteristics is of great research significance for achieving more precise tumor targeting and real-time imaging-guided surgery. Summary of the Invention

[0004] This invention addresses existing problems by providing a pH / nitroreductase dual-responsive fluorescent probe. The probe uses heptamethrin as its matrix, and the matrix structure is modified to enable specific recognition under conditions where both an acidic environment and nitroreductase are present. The probe itself exhibits extremely weak fluorescence in water or organic solvents. This probe is specifically and sequentially activated when tumor cells simultaneously provide an acidic intracellular / extracellular microenvironment and express sufficient amounts of nitroreductase: acidic conditions induce the probe to reconjugate, allowing it to be activated by long-wavelength light; subsequently, the nitroreductase response blocks electron transfer, preventing PET and restoring the fluorescence signal. Due to the dual-response "switch" nature of the probe's fluorescence, it can adapt to complex microenvironments and achieve highly selective, high signal-to-noise ratio, and high-sensitivity imaging of cancer cells.

[0005] The technical solution adopted in this invention is a small molecule fluorescent probe that is dual-responsive to pH and nitroreductase, and the structural formula of the fluorescent probe is as follows:

[0006]

[0007] , , , ,

[0008] ;

[0009] Where n is 1 or 2, Y is O, S or N, W is OH, SH or NH2, and Z is... - For I - ,Br - Cl - TosO - .

[0010] Furthermore, the structure of the probe is as follows:

[0011] .

[0012] The probe is prepared by the following steps:

[0013]

[0014] (1) Slowly add a solution of dichloromethane containing phosphorus oxychloride to N,N-dimethylformamide cooled in an ice bath; then add cyclohexanone; after refluxing the mixture, pour it into an ice-water mixture and let it stand to precipitate compound 1;

[0015] (2) Dissolve 2,3,3-trimethylindoline and 2-iodoethanol in acetonitrile and stir under reflux; cool the reaction solution to room temperature and filter; wash the solid with ethyl acetate to obtain compound 2;

[0016] (3) Dissolve compound 1, compound 2 and sodium acetate in acetic anhydride, heat and stir; cool and then filter and wash to obtain compound 3;

[0017] (4) Compound 3 and sodium acetate were dissolved in anhydrous N,N-dimethylformamide, heated and stirred under nitrogen protection, and after the reaction, the solvent was removed and purified to obtain compound 4;

[0018] (5) Compound 4 was dissolved in 1,4-dioxane, and sodium hydroxide aqueous solution was added dropwise under ice-water bath conditions with stirring; after the reaction was completed, the solvent was washed and removed; and compound 5 was obtained by purification.

[0019] (6) Compound 5 was dissolved in anhydrous dichloromethane and the solution was cooled to 0°C. Then, under nitrogen protection, a dichloromethane solution of nitrobenzyl chloride was added dropwise; after the reaction was complete, the solvent was removed and the solution was purified to obtain compound 6.

[0020] (7) Dissolve compound 6 in methanol and add sodium hydroxide aqueous solution dropwise. Stir until it is completely precipitated into an orange solid. Filter, wash and dry to obtain the final product CCy7-NO2.

[0021] The fluorescent probe is used for non-disease diagnostic and therapeutic purposes, and is applied to the detection of pH / nitroreductase.

[0022] Furthermore, the fluorescent probe is used to detect pH / nitro reductase in in vitro cells.

[0023] Furthermore, the specific detection process is as follows: For in vitro cells, MCF-7 cells are cultured in a hypoxic environment and then incubated with a culture medium containing fluorescent probes. The fluorescence intensity of the amylation products produced by the fluorescent probes in response to pH / nitroreductase is measured and used as an evaluation index of nitroreductase activity.

[0024] The fluorescent probe is used for non-disease diagnostic and therapeutic purposes, and is applied to fluorescence imaging.

[0025] This invention takes a pH / nitroreductase dual-responsive fluorescent probe as an example. The pH / nitroreductase dual-responsive fluorescent probe has the following molecular structural formula:

[0026]

[0027] The present invention also provides a method for preparing the pH / nitroreductase dual-responsive fluorescent probe, the steps of which are as follows:

[0028] (1) A solution of phosphorus oxychloride in dichloromethane was slowly added dropwise to an N,N-dimethylformamide solution cooled in an ice bath. After the addition was complete, cyclohexanone was added using a syringe. The mixture was refluxed for 3 h and then poured into an ice-water mixture. After standing for 12 h, yellow crystals precipitated. After filtration, yellow solid 1 was obtained directly for the next reaction. Yield: 1.65 g (yield 18.8%).

[0029] (2) Dissolve 2,3,3-trimethylindoline and 2-iodoethanol in acetonitrile and reflux with stirring overnight. After cooling the reaction solution to room temperature, filter and wash the solid with ethyl acetate. The resulting pink solid 2 was used directly in the next step of the reaction. Yield: 18.7 g (yield 90.1%).

[0030] (3) Compound 1, Compound 2, and sodium acetate were dissolved in acetic anhydride and stirred at 90°C for 2 h. After cooling to room temperature, the mixture was filtered, and the solid was successively treated with saturated... The solution was washed with deionized water and dried under vacuum to obtain a green solid 3 with a metallic luster. Yield: 1.82 g (63.3%).

[0031] (4) Compound 3 and sodium acetate were dissolved in anhydrous N,N-dimethylformamide, and the solution was heated at 80°C for 2 h under nitrogen protection. The solvent was then removed under reduced pressure. The crude product was purified by silica gel chromatography to give a red solid 4 with a metallic luster. Yield: 524.0 mg (65%).

[0032] (5) Compound 4 was dissolved in 1,4-dioxane, and sodium hydroxide aqueous solution was added dropwise under ice-water bath conditions, with stirring for 4 h. Dichloromethane and saturated sodium hydroxide were used. The solution was extracted twice with deionized water, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography to give a red solid of 5 mg. Yield: 103.4 mg (41%).

[0033] (6) Compound 5 was dissolved in anhydrous dichloroethane, and the solution was cooled to 0 °C. Then, under nitrogen protection, a dichloroethane solution of nitrobenzyl chloride was added dropwise over 10 min. The mixture was allowed to react at 0 °C for another 20 min, then the ice bath was removed, and the mixture was allowed to react at room temperature for 3.5 h. After the reaction was complete, the solvent was removed, and the crude product was purified by silica gel chromatography to give a dark red solid 6. Yield: 50.4 mg (39%).

[0034] (7) Compound 6 was dissolved in methanol, and about 2 drops of sodium hydroxide aqueous solution were slowly added dropwise while stirring continuously until the blue solution completely precipitated into an orange solid. After cooling to room temperature, the mixture was filtered, the filter residue was washed with diethyl ether, and dried under vacuum. The final product CCy7-NO2 was obtained. The yield was 42.7 mg (84%).

[0035] This invention also proposes the application of a pH / nitroreductase dual-responsive fluorescent probe, which, through its highly selective fluorescence response to pH and nitroreductase, can be used to detect live cells, tissues, and cancer cells in living organisms.

[0036] The detection process of the fluorescent probe in solution with dual response to pH / nitroreductase is as follows: three sets of experiments are designed, namely: pH group, to explore the effect of different pH on probe absorption and fluorescence; To investigate the fluorescence changes of the probe over time when only the enzyme is present; This study investigated the change in probe fluorescence over time under conditions where both pH and enzymes were present. Because the fluorescence difference of the probe before and after the response was very significant, the probe provided by this invention can detect tumor cells in complex biological systems.

[0037] The detection process of the fluorescent probe on pH / nitroreductase in in vitro cells is as follows: the fluorescent probe is dissolved in dimethyl sulfoxide solution to prepare a 5 mM stock solution. For in vitro cells, 8... The stock solution was dissolved in 2 mL of culture medium to prepare a 20 mL solution. The incubation solution was prepared for use. MCF-7 cells were incubated in a hypoxic bag for 4 h at a pH of 6.5–6.8 and a temperature of 37 °C. The cells were then incubated with a culture medium containing a fluorescent probe. The changes in fluorescence intensity of the reduction products produced by the reaction of the fluorescent probe with intracellular nitroreductase were measured over time, and this was used as an evaluation index of nitroreductase activity and level fluctuations.

[0038] The process of using the fluorescent probe to guide surgical navigation involves spraying CCy7-NO2 (20) in situ at the tumor site in mice. 100 Time-dependent in vivo fluorescence imaging was performed on the tumor. After 30 minutes, only the tumor site showed a significant fluorescence signal, clearly distinguishing the tumor margins and exhibiting a high signal-to-noise ratio compared to the surrounding normal tissue. Next, the tumor was surgically removed using a scalpel guided by fluorescence. No excess fluorescence was observed at the tumor removal site. The removed site was then sprayed with the probe CCy7-NO2 again, and its fluorescence signal was observed.

[0039] The pH-responsive ether bond cleavage mechanism of the fluorescent probe molecule CCy7-NO2 in this invention enables it to maintain a closed ring structure (yellow, 430 nm absorption) under neutral conditions. In the acidic tumor microenvironment (pH≈6.8), the ether bond hydrolyzes and opens the ring, restoring the heptamethrin conjugated chain and activating near-infrared absorption at 780 nm. The nitro group at the mid-position is a strong electron-withdrawing group, quenching the probe fluorescence through intramolecular electron transfer (PET). When the NTR reduces the nitro group to an amino group, electron transfer is inhibited, and fluorescence is restored (fluorescence is 35 times that of the quenched state).

[0040] The reaction formula is as follows:

[0041]

[0042] This probe uses heptamethrin dye as a parent compound and incorporates pH-sensitive and nitroreductase-specific recognition groups to construct a fluorescent switching system based on the photoinduced electron transfer (PET) mechanism. This fluorescent probe has the following key advantages:

[0043] (1) High specificity: This probe achieves a dual response of pH and nitroreductase by introducing oxazolidine and nitrobenzene. It activates fluorescence only when nitroreductase coexists in an acidic microenvironment, completely avoiding interference from a single factor. It does not show obvious fluorescence response to a variety of interfering substances (such as biological enzymes, biological macromolecules, and inorganic salts).

[0044] (2) Rapid imaging in the near-infrared region: The probe emission wavelength is located in the near-infrared region. The fluorescence reaches its peak within 30 minutes after dual signal triggering, and the response speed meets the requirements of real-time dynamic monitoring.

[0045] (3) High sensitivity: The dual-response fluorescence "on-off" strategy is adopted. Before being recognized by nitroreductase, the probe CCy7-NO2 has only weak fluorescence. After activation, the fluorescence is enhanced by 35 times, which greatly improves the detection sensitivity.

[0046] This probe is specifically activated only in the presence of nitroreductase in an acidic microenvironment (pH=6-7), achieving a fluorescent "turn-on" response. Its emission wavelength is located in the 760-850 nm (near-infrared window), and the fluorescence intensity increases 13-132 times after activation. This probe boasts advantages such as high specificity, rapid near-infrared response (maximum fluorescence within 0-30 min after both conditions are met), and high sensitivity. Furthermore, its unique dual-response mechanism effectively avoids interference from single factors, significantly improving detection specificity and signal-to-noise ratio in complex biological systems. Attached Figure Description

[0047] Figure 1 This is the 1H NMR spectrum of the probe CCy7-NO2.

[0048] Figure 2 It is a high-resolution mass spectrometer of the probe CCy7-NO2.

[0049] Figure 3 It is a selective test of the probe CCy7-NO2 against nitroreductase and other interfering molecules.

[0050] Figure 4 The color difference in the solution is due to the change in absorption of the probe CCy7-NO2 at different pH values.

[0051] Figure 5 The images show the (a) absorption and (b) fluorescence spectra of the probe CCy7-NO2 at different pH values.

[0052] Figure 6 The fluorescence response spectra of probe CCy7-NO2 to nitroreductase at different times are shown in (a) pH=7.4 and (b) pH=6.8.

[0053] Figure 7 This is a time-dependent fluorescence imaging of the probe CCy7-NO2 in MCF-7 cells.

[0054] Figure 8 It is a time-dependent fluorescence imaging and surgical navigation of the probe CCy7-NO2 in live mice. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0056] Example 1

[0057] The fluorescent probe of this invention is prepared in the following manner:

[0058] (1) A solution of 18.5 mL (198 mmol) in dichloromethane (17.5 mL) was slowly added dropwise to a 20 mL solution of DMF (258 mmol) in dichloromethane cooled in an ice bath. After the addition was complete, cyclohexanone (5.0 g, 50 mmol) was added via syringe. The mixture was refluxed for 3 h and then poured into a 150 mL mixture of ice and water. After standing for 12 h, yellow crystals precipitated. The crystals were filtered to obtain yellow solid 1, which was used directly for the next reaction. Yield: 1.65 g (18.8%).

[0059] (2) 2,3,3-Trimethylindoline (10.0 g, 62.8 mmol, 1 eq) and 2-iodoethanol (32.4 g, 188.4 mmol, 3 eq) were dissolved in 30 mL of acetonitrile and stirred under reflux overnight. The reaction solution was cooled to room temperature and filtered. The solid was washed with ethyl acetate, and the resulting pink solid 2 was used directly in the next step of the reaction. Yield: 18.7 g (yield 90.1%).

[0060] (3) Compound 1 (0.8 g, 4.52 mmol, 1 eq), compound 2 (3.1 g, 9.03 mmol, 2 eq), and sodium acetate (1.1 g, 13.55 mmol, 3 eq) were dissolved in 25 mL of acetic anhydride and stirred at 90 °C for 2 h. After cooling to room temperature, the mixture was filtered, and the solid was successively saturated with acetic anhydride. The solution was washed (until no more bubbles were produced) and deionized water (twice), and then vacuum dried to obtain a green solid 3 with a metallic luster. Yield: 1.82 g (63.3%).

[0061] (4) Compound 3 (1.0 g, 1.32 mmol, 1 eq) and sodium acetate (325.9 mg, 3.97 mmol, 3 eq) were dissolved in anhydrous N,N-dimethylformamide (20 mL), and the solution was heated at 80 °C for 2 h under nitrogen protection. The solvent was then removed under reduced pressure. The crude product was purified by silica gel chromatography (DCM:MeOH = 100:1) to give a red solid 4 with a metallic luster. Yield: 524.0 mg, 65%.

[0062] (5) Compound 4 (300.0 mg, 492.79 mg) Dissolve 1 eq) in 10 mL of 1,4-dioxane, and under ice-water bath conditions, add 2 mL of 0.5 M sodium hydroxide aqueous solution dropwise, stirring for 4 h. Use dichloromethane separately with saturated... The solution was washed twice with deionized water, and the organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (DCM:MeOH = 20:1) to give a red solid of 5 mg. Yield: 103.4 mg, yield: 41%.

[0063] (6) Compound 5 (103.4 mg, 190.6 mg) Dissolve 1 eq) of nitrobenzoyl chloride in anhydrous DCM (15 mL) and cool the solution to 0 °C. Then, under nitrogen protection, add 35.4 mg, 190.6 mg of nitrobenzoyl chloride dropwise over 10 min. A 1 eq) solution of DCM (5 mL) was added. The mixture was allowed to react at 0 °C for another 20 min, then the ice bath was removed, and the mixture was allowed to react at room temperature for 3.5 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solvent was removed, and the crude product was purified by silica gel chromatography (DCM:MeOH = 20:1) to give a deep red solid 6. Yield: 50.4 mg, 39%.

[0064] (7) Compound 6 (50.4 mg, 74.9 mg) Dissolved in 3 mL of methanol, approximately 2 drops of 0.5 M sodium hydroxide aqueous solution were slowly added dropwise while stirring continuously until the blue solution completely precipitated into an orange solid. After cooling to room temperature, the mixture was filtered, the filter residue was washed with diethyl ether, and dried under vacuum to obtain the final product CCy7-NO2. The yield was 42.7 mg, with a yield of 84%. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (q, J = 8.8 Hz, 4H), 7.62 (d, J = 14.2 Hz, 2H), 7.47 (d, J = 7.5Hz, 2H), 7.37 (q, J = 7.2 Hz, 4H), 7.19 (t, J = 7.3 Hz, 2H), 6.36 (d, J =14.2 Hz, 2H), 4.25 (s, 4H), 3.76 (d, J = 5.4 Hz, 4H), 2.72 (s, 4H), 1.91 (s, 2H), 1.33 (d, J = 61.1 Hz, 12H).

[0065] The probe CCy7-NO2 (5 mg) was dissolved in deuterated DMSO and measured using a Bruker Avance NEO 600 MHz NMR spectrometer, with tetramethylsilane as an internal standard for chemical shift; the results are as follows. Figure 1 As shown.

[0066] High-resolution mass spectrometry (HMS) was used to detect the probe CCy7-NO2 (0.1 mg / mL dichloromethane solution) in ESI positive ion mode, with a mass range of m / z 150-1500; the results are as follows. Figure 2 As shown, the theoretical m / z is 673.3152, and the actual m / z is 674.3227.

[0067] Example 2: Selectivity test of probe CCy7-NO2 against nitroreductase and other interfering molecules:

[0068] The NADPH reduction system of the incubation system consisted of: β-nicotinamide adenine dinucleotide phosphate (1.0 mM); glucose-6-phosphate (10.0 mM); glucose-6-phosphate dehydrogenase (1.0 unit / mL); and magnesium chloride (4.0 mM). All of the above coenzyme systems were dissolved in 0.1 M PBS buffer (pH = 6.8).

[0069] In the response test involving nitroreductase, at a final volume of 200... The above glucose-6-phosphate solution was added to the incubation system 2 β-Nicotinamide adenine dinucleotide phosphate solution 20 0.2 g of glucose-6-phosphate dehydrogenase solution , and 3 Nitroreductase (final concentration 0.15 mg / mL) and 0.8 The 5 mM probe stock solution (CCy7-NO2 final concentration in the incubation system 20) Finally, add 174.2. The mixture was then thoroughly mixed with PBS buffer. The mixture was placed in a 37°C shaker and incubated for 2 hours. After incubation, an equal volume of dimethyl sulfoxide (DMSO) was added to the system to stop the incubation and the mixture was thoroughly mixed.

[0070] In response tests involving other small biomolecules, each small biomolecule (final concentration 0.15 mg / mL) was incubated with the probe in PBS buffer at 37°C in a shaker for 2 h. After incubation, an equal volume of DMSO was added. The reaction solution was then subjected to fluorescence analysis with an excitation wavelength of 750 nm. Figure 3 It is known that the fluorescent probe CCy7-NO2 of the present invention has high selectivity for nitro reductase.

[0071] Example 3: The probe CCy7-NO2 caused color differences in the solution due to changes in absorption at different pH values.

[0072] Prepare 0.1 M phosphate-buffered saline (PBS), adjust the pH gradient to (4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0, 11.0, 12.0), and calibrate with a pH meter. (0.05% error). Take the probe stock solution (5 mM, DMSO solution) and add it to each pH buffer to a final concentration of 20. Mix well. Place the mixture in a 37 ℃ constant temperature water bath and incubate in the dark for 30 min (simulating physiological temperature). Arrange according to the pH gradient and then take pictures.

[0073] Example 4: Absorption and fluorescence spectra of probe CCy7-NO2 at different pH values.

[0074] Three mL of solutions at different pH values ​​from Example 3 were used for spectral analysis. The UV-Vis absorption and fluorescence spectra of the probe were measured using a Lambda 35 UV-Vis spectrophotometer (PerkinElmer) and a fluorescence spectrophotometer (VAEIANCARY Eclipse), respectively. =750 nm; slit width = 10 nm). The fluorescence intensity of each pH group was recorded, and the results are as follows: Figure 5As shown, the changes in the absorption spectrum confirm the pH-induced ring-opening process of the ether bond: the inverse relationship between the absorption at 430 nm and 776 nm corresponds to the decrease in the closed ring form and the increase in the open ring form, respectively. However, the significant absorption change did not trigger fluorescence enhancement at 820 nm, indicating that the ring-opening caused by pH response alone is insufficient to activate fluorescence, and the probe remains in a fluorescence-off state.

[0075] Example 5: Fluorescence response spectra of probe CCy7-NO2 to nitroreductase at different time points at pH=7.4 or 6.8

[0076] The NADPH reduction system of the incubation system consists of: β-nicotinamide adenine dinucleotide phosphate (1.0 mM); glucose-6-phosphate (10.0 mM); glucose-6-phosphate dehydrogenase (1.0 unit / mL); magnesium chloride (4.0 mM). All of the above coenzyme systems were dissolved in 0.1 M PBS buffer (pH = 7.4 or 6.8).

[0077] In the nitroreductase-mediated response test, 30 μL of the above glucose-6-phosphate solution was added to a final incubation system of 3 mL. β-Nicotinamide adenine dinucleotide phosphate solution 300 3 glucose-6-phosphate dehydrogenase solutions , and 12 The 5 mM probe stock solution (CCy7-NO2 final concentration in the incubation system 20) Finally, add 2.61 mL of PBS buffer. Quickly add 45 mL of PBS buffer. After adding nitroreductase (final concentration 0.15 mg / mL), the fluorescence intensity at 760-900 nm was immediately monitored in real time under 750 nm excitation light. Measurements were taken every 3 minutes for 30 minutes. Results are as follows: Figure 6 As shown, the probe achieved complete fluorescence activation (35-fold enhancement) within a very short time of 30 minutes only when nitroreductase was present in conjunction with an acidic pH (pH 6.8), with an intensity far exceeding that under single stimulation conditions (12-fold enhancement with only nitroreductase). This confirms the AND gate logic response characteristics, ensuring that the probe can respond rapidly and specifically in complex environments.

[0078] Example 6: Time-dependent fluorescence imaging of probe CCy7-NO2 in MCF-7 cells

[0079] MCF-7 cells were seeded into confocal cell culture dishes, and 2 mL of the appropriate culture medium (containing 10% fetal bovine serum) was added. The dishes were incubated in a constant temperature incubator (37 ℃, 5% CO2) for 24 h. When the MCF-7 cells reached 70%-80% confluence, the confocal dishes were placed in a hypoxic bag and incubated for 4 h, at which point the culture medium turned yellow. The old culture medium was discarded, and the cells were washed three times with PBS (1 mL). Then, a culture medium containing CCy7-NO2 (2 mL) was added. The cells were co-incubated with the culture medium. Fluorescence changes over time were observed using a single-photon confocal microscope. The excitation wavelength was 685 nm, and the fluorescence acquisition range was 690-850 nm. The results are as follows: Figure 7 As shown, cell uptake experiments confirmed that the probe possesses excellent cell permeability and rapid activation properties. Specific fluorescence was detected within 5 minutes of excitation at 685 nm, reaching maximum intensity at 30 minutes. This indicates that the probe can be rapidly taken up by cells and efficiently activated by endogenous nitroreductase, making it suitable for real-time tracking and imaging at the live cell level.

[0080] Example 7: Time-dependent fluorescence imaging and surgical navigation of probe CCy7-NO2 in live mice

[0081] In situ spraying of CCY7-NO2 (20) at the tumor site in mice 100 Time-dependent in vivo fluorescence imaging was performed, and after 30 minutes, only the tumor site showed a significant fluorescence signal. The tumor margin was clearly distinguishable and exhibited a high signal-to-noise ratio compared to the surrounding normal tissue. Next, the tumor was surgically removed using a scalpel guided by fluorescence. No excess fluorescence was observed at the tumor removal site. The removed site was then sprayed with the probe CCy7-NO2 again, and its fluorescence signal was observed. Figure 8 As shown, no target fluorescence was observed after the probe was sprayed again.

[0082] The above embodiments are only used to illustrate the present invention. Any equivalent transformations and improvements made on the basis of the technical solutions of the present invention should not be excluded from the protection scope of the present invention.

Claims

1. A small molecule fluorescent probe that is dual-responsive to pH and nitro reductase, characterized in that: The structural formula of the fluorescent probe is: ； in, , , , , ; n is 1 or 2, Y is O, S or N, W is OH, SH or NH2, Z is... - For I - ,Br - Cl - TosO - .

2. The fluorescent probe according to claim 1, characterized in that, The probe has the following structure: 。 3. The method for preparing the probe according to claim 2, characterized in that, The synthesis steps are as follows: ； (1) Slowly add a solution of dichloromethane containing phosphorus oxychloride to N,N-dimethylformamide cooled in an ice bath; then add cyclohexanone; after refluxing the mixture, pour it into an ice-water mixture and let it stand to precipitate compound 1; (2) Dissolve 2,3,3-trimethylindoline and 2-iodoethanol in acetonitrile and stir under reflux; cool the reaction solution to room temperature and filter; wash the solid with ethyl acetate to obtain compound 2; (3) Dissolve compound 1, compound 2 and sodium acetate in acetic anhydride, heat and stir; cool and then filter and wash to obtain compound 3; (4) Compound 3 and sodium acetate were dissolved in anhydrous N,N-dimethylformamide, heated and stirred under nitrogen protection, and after the reaction, the solvent was removed and purified to obtain compound 4; (5) Compound 4 was dissolved in 1,4-dioxane, and sodium hydroxide aqueous solution was added dropwise under ice-water bath conditions with stirring; after the reaction was completed, the solvent was washed and removed; and compound 5 was obtained by purification. (6) Compound 5 was dissolved in anhydrous dichloromethane and the solution was cooled to 0°C. Then, under nitrogen protection, a dichloromethane solution of nitrobenzyl chloride was added dropwise; after the reaction was complete, the solvent was removed and the solution was purified to obtain compound 6. (7) Dissolve compound 6 in methanol and add sodium hydroxide aqueous solution dropwise. Stir until it is completely precipitated into an orange solid. Filter, wash and dry to obtain the final product CCy7-NO2.

4. The application of the fluorescent probe according to claim 1 for non-disease diagnosis and treatment purposes, characterized in that: The fluorescent probe is used in the detection of pH / nitroreductase.

5. The application according to claim 4, characterized in that: The fluorescent probe is used for the detection of pH / nitro reductase in in vitro cells.

6. The application according to claim 5, characterized in that: The specific detection process is as follows: For in vitro cells, MCF-7 cells are cultured in a hypoxic environment and then incubated with a medium containing fluorescent probes. The fluorescence intensity of the amylation products produced by the fluorescent probes in response to pH / nitroreductase is measured and used as an evaluation index of nitroreductase activity.

7. The application of the fluorescent probe according to claim 1 for non-disease diagnosis and treatment purposes, characterized in that: The fluorescent probe is used for fluorescence imaging.

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