Naphthalimide type fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a naphthalimide type fluorescent probe and a preparation method and application thereof.The naphthalimide type fluorescent probe is of a structure shown in the formula I. The naphthalimide type fluorescent probe takes 4-nitrobenzyl bromide as a recognition group and a fluorescence quencher, and 1, 2, 3, 4-tetramethyl-1, 3, 4-tetramethyl-1, 3, 4-tetramethyl-1, 3, 4-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1, 3-tetramethyl-1 and naphthalimide modified by 2, 3, 3-tetramethyl-3H-indole iodide is used as a fluorophore. When nitroreductase responds to the probe, a fluorescence signal is enhanced, and the probe has the advantages of low detection limit, good stability, rapid response and the like, and can be used as a tool for quantitative research of nitroreductase.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes and relates to a naphthalimide-type fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hypoxia is a key characteristic of tumor tissue. Due to the disorder of blood vessels in tumor tissue, some tissues are far away from blood vessels and have slow blood flow, so there are often areas of low oxygen concentration in tumor tissue. Studies have shown that tumor hypoxia is closely related to tumor proliferation, invasion, and drug resistance. Therefore, detecting tumor hypoxia is of great medical significance. Tumor tissue hypoxia can lead to the excessive expression of reductases such as nitrate reductase (NTR) and azoreductase. The activity of NTR is positively correlated with the degree of tumor hypoxia. Therefore, the degree of cellular hypoxia can be detected by detecting the level of NTR.

[0003] Fluorescent probes have attracted considerable attention due to their efficient and rapid detection of specific molecules in cells. Fluorescence analysis offers advantages such as high sensitivity, good selectivity, and simple operation. However, current fluorescent probes for nitroreductase still suffer from high detection limits, poor stability, and long response times. Therefore, the development of a novel fluorescent probe with a low detection limit, excellent stability, and rapid response time is crucial for the detection of nitroreductase. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a naphthalimide-type fluorescent probe and its preparation method and application. The naphthalimide-type fluorescent probe of the present invention has the advantages of low detection limit, good stability, fast response, good selectivity, and simple operation.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides a naphthalimide-type fluorescent probe having a structure shown in Formula I below:

[0007]

[0008] The naphthalimide-based fluorescent probe described in this invention uses 4-nitrobenzyl bromide as a recognition group and fluorescence quencher, and 1,2,3,3-tetramethyl-3H-indole iodide-modified naphthalimide as a fluorophore. When nitroreductase interacts with the probe, the fluorescence signal is enhanced. This probe has advantages such as a low detection limit, good stability, and a fast response, making it suitable for quantitative studies of nitroreductase.

[0009] On the other hand, the present invention provides a method for preparing the naphthalimide-type fluorescent probe as described above, the preparation method comprising the following steps:

[0010] S1, 4-bromo-1,8-naphthalene dicarboxylic anhydride react with n-butylamine to obtain product 1. The reaction formula is as follows:

[0011]

[0012] S2. Product 1 reacts in methanol to obtain product 2. The reaction formula is as follows:

[0013]

[0014] S3, product 2 reacts with hydroiodic acid to obtain product 3, and the reaction formula is as follows:

[0015]

[0016] S4, product 3 and urotropine react in trifluoroacetic acid to obtain product 4. The reaction formula is as follows:

[0017]

[0018] S5, product 4 reacts with 1,2,3,3-tetramethyl-3H-indole iodide to obtain product 5. The reaction formula is as follows:

[0019]

[0020] S6, product 5 reacts with 4-nitrobenzyl bromide to obtain the naphthaleneimide type fluorescent probe, and the reaction formula is as follows:

[0021]

[0022] In the present invention, the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to n-butylamine in step S1 is 1:1.2-1.5, for example, 1:1.2, 1:1.25, 1:3, 1:3.5, 1:4, 1:4.5, 1:4.8 or 1:1.5.

[0023] Preferably, the reaction in step S1 is carried out in a solvent, and the solvent is selected from any one of anhydrous ethanol, methanol, acetonitrile or acetone, or a combination of at least two thereof.

[0024] Preferably, the reaction in step S1 is carried out under reflux, and the reaction time is 8-12 h, such as 8 h, 9 h, 10 h, 11 h or 12 h.

[0025] In the present invention, after the reaction in step S1 is completed, the reaction solution is cooled to room temperature, and the precipitated brown product 1 is collected by filtration.

[0026] Preferably, the reaction in step S2 is carried out in the presence of an alkaline substance.

[0027] Preferably, the molar ratio of the product 1 to the alkaline substance is 1:2 to 3, for example, 1:2, 1:2.2, 1:2.5, 1:2.8 or 1:3.

[0028] Preferably, the alkaline substance is selected from any one of potassium carbonate, sodium carbonate or cesium carbonate, or a combination of at least two thereof.

[0029] Preferably, the reaction in step S2 is carried out under reflux, and the reaction time is 6-8 h, for example, 6 h, 6.5 h, 7 h, 7.5 h or 8 h.

[0030] In the present invention, after the reaction in step S2 is completed, the mixture is cooled to 0° C. in an ice bath, and the precipitate is collected by filtration to obtain a yellow product 2.

[0031] Preferably, in step S3, the reaction of the product 2 with hydroiodic acid is carried out by dissolving the product 2 in a hydroiodic acid solution for the reaction.

[0032] Preferably, the mass percentage concentration of the hydroiodic acid solution is 55-57%, such as 55%, 56% or 57%.

[0033] Preferably, the reaction in step S3 is carried out under reflux, and the reaction time is 8-14 h, for example, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h or 14 h.

[0034] In the present invention, after the reaction in step S3 is completed, deionized water is added to the reaction system until no more precipitate is precipitated, and the precipitate is collected by filtration to obtain a yellow product 3.

[0035] Preferably, the molar ratio of the product 3 to hexamethylenetetramine in step S4 is 1:2 to 3, for example, 1:2, 1:2.2, 1:2.5, 1:2.8 or 1:3.

[0036] Preferably, the reaction in step S4 is carried out under reflux, and the reaction time is 8-10 h, such as 8 h, 8.5 h, 9 h, 9.5 h or 10 h.

[0037] In the present invention, after the reaction in step S4 is completed, deionized water is added to the reaction system until no more precipitate is precipitated, and the precipitate is collected by filtration to obtain a yellow product 4.

[0038] Preferably, the molar ratio of the product 4 to 1,2,3,3-tetramethyl-3H-indole iodide in step S5 is 1:1.2 to 1.8, for example, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7 or 1:1.8.

[0039] Preferably, the reaction in step S5 is carried out in the presence of a base.

[0040] Preferably, the base is selected from piperidine and / or sodium acetate.

[0041] Preferably, the molar ratio of product 4 to base is 1:0.1 to 0.5, for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4 or 1:0.5.

[0042] In the present invention, after the reaction in step S5 is completed, column chromatography is performed using dichloromethane / methanol as the eluent and silica gel as the stationary phase. The eluate is collected and the solvent is dried to obtain a dark blue product 5.

[0043] Preferably, the volume ratio of methanol to dichloromethane in the eluent is 1:100-50, for example, 1:100, 1:99, 1:98, 1:95, 1:90, 1:88, 1:85, 1:80, 1:75, 1:73, 1:70, 1:65, 1:60, 1:55, 1:50, etc.

[0044] Preferably, the molar ratio of the product 5 to 4-nitrobenzyl bromide in step S6 is 1:1.2 to 2, for example, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.

[0045] Preferably, the reaction in step S6 is carried out in the presence of an alkaline substance.

[0046] Preferably, the alkaline substance is selected from any one of potassium carbonate, sodium carbonate or cesium carbonate, or a combination of at least two thereof.

[0047] Preferably, the molar ratio of the product 5 to the alkaline substance is 1:1 to 3, for example, 1:1, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8 or 1:3.

[0048] Preferably, the reaction in step S6 is carried out in a solvent, and the solvent is selected from any one or a combination of at least two of acetone, acetonitrile or N,N-dimethylformamide.

[0049] Preferably, in step S6, the product 5 is dissolved in a solvent, potassium carbonate is added, and the mixture is stirred at room temperature for 10-40 min (e.g., 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min), and then 4-nitrobenzyl bromide is added to react.

[0050] Preferably, the reaction in step S6 is carried out under reflux, and the reaction time is 12-24 h, for example, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h or 24 h.

[0051] In the present invention, after the reaction in step S6 is completed, column chromatography is performed using dichloromethane / methanol as an eluent and silica gel as a stationary phase, the eluate is collected, and the solvent is dried to obtain the naphthaleneimide-type fluorescent probe.

[0052] Preferably, the volume ratio of methanol to dichloromethane in the eluent is 1:250-100, for example 1:250, 1:220, 1:200, 1:180, 1:150, 1:130, 1:110 or 1:100.

[0053] In the present invention, the preparation process of the naphthalimide-type fluorescent probe is preferably as follows:

[0054]

[0055] In another aspect, the present invention provides use of the naphthalimide-type fluorescent probe described above in the detection of nitroreductase for non-therapeutic purposes.

[0056] In the present invention, the specific steps of using the naphthalimide-type fluorescent probe of the present invention for nitroreductase are as follows:

[0057] (1) Dilute the prepared naphthalimide fluorescent probe to 10-40 μM using PBS buffer solution;

[0058] (2) Add 100-500 μM NADH and 6 μg / mL nitroreductase to the nitroreductase detection system prepared in step 1, and react at 37°C. Use a fluorescence spectrometer with an excitation wavelength of 450 nm and a 2-min interval to record the changes in the fluorescence signal intensity emitted by the reaction system at 525 nm to investigate the response time of the probe to nitroreductase.

[0059] (3) Add 100-500 μM NADH to the nitroreductase detection system prepared in step 1, add different amounts (1-8 μg / mL) of nitroreductase, and react at 37°C for 30 minutes. Use a fluorescence spectrometer with an excitation wavelength of 450 nm to record the changes in the fluorescence signal intensity emitted by the reaction system at 525 nm. Plot the nitroreductase concentration as the horizontal axis and the fluorescence intensity at 525 nm as the vertical axis and perform a linear fit to obtain a standard curve of the nitroreductase probe fluorescence signal intensity and the nitroreductase concentration, so that the nitroreductase can be quantitatively analyzed.

[0060] (4) Add 100-500 μM NADH to the nitroreductase detection system prepared in step 1, and add common substances in organisms such as calcium chloride (CaCl2), magnesium sulfate (MgSO4), sodium sulfide (Na2S), potassium chloride (KCl), sodium hypochlorite (NaClO), cysteine, glutathione, β-glucan, etc. as interfering substances. Use a fluorescence spectrometer with an excitation wavelength of 450 nm to record the change in the fluorescence signal intensity emitted by the reaction system at 525 nm. Detect the anti-interference ability of the probe against common substances in organisms.

[0061] (5) Add 100-500 μM NADH and 6 μg / mL nitroreductase to the nitroreductase detection system prepared in step 1, and set up a blank detection system without adding NADH and nitroreductase. Use sodium hydroxide or hydrochloric acid solution to adjust the detection system to different pH values ​​and react at 37°C for 30 minutes. Use a fluorescence spectrometer with an excitation wavelength of 450 nm to record the change in the intensity of the fluorescence signal emitted by the reaction system at 525 nm. Detect the response of nitroreductase to the probe under different pH conditions and the stability of the probe at different pH values.

[0062] The PBS buffer solution has a concentration of 10 mM and a pH of 7.4.

[0063] The interferor concentration was 10 mM.

[0064] The concentration of the sodium hydroxide and hydrochloric acid solution is 1M / L.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] The naphthaleneimide fluorescent probe of the present invention has good selectivity and strong anti-interference ability. It is used for nitroreductase detection, has a fast response time and a low detection limit, and can achieve rapid and accurate detection of nitroreductase. In addition, the probe of the present invention has good stability under different pH conditions, is simple to prepare, and is convenient to post-process. The operation for nitroreductase detection is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1A High-resolution mass spectrometry of a fluorescent probe for nitroreductase.

[0068] Figure 1B H NMR spectrum of the fluorescent probe for nitroreductase.

[0069] Figure 2 is the response time of the nitroreductase fluorescent probe to nitroreductase.

[0070] Figure 3 The fluorescence spectrum of the nitroreductase fluorescent probe responds to different concentrations of nitroreductase.

[0071] Figure 4 The standard curve is the response of the nitroreductase fluorescent probe to different concentrations of nitroreductase.

[0072] Figure 5 The fluorescence probe of nitroreductase responds to the fluorescence spectrum of nitroreductase under different pH conditions.

[0073] Figure 6 It is a nitroreductase fluorescent probe that responds to fluorescence spectra of common substances in organisms.

[0074] Figure 7 UV-visible absorption spectrum of the nitroreductase fluorescent probe and the fluorophore.

[0075] Figure 8 It represents the color change before and after the reaction of the nitroreductase fluorescent probe with nitroreductase.

[0076] Figure 9A It is a high-resolution mass spectrometer of the product after the reaction of nitroreductase fluorescent probe and nitroreductase.

[0077] Figure 9B This is the H NMR spectrum of the product after the nitroreductase fluorescent probe reacts with nitroreductase. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0079] Example 1: A method for preparing a fluorescent probe for detecting nitroreductase, comprising the following steps:

[0080] (1) 4-Bromo-1,8-naphthalene dicarboxylic anhydride (27.70 g, 100.0 mmol) and n-butylamine (10.2 g, 140.0 mmol) were dissolved in 200 mL of anhydrous ethanol and refluxed at 78°C for 8 h. After the reaction was completed, the mixture was cooled to room temperature and the precipitated solid was filtered. The precipitate was washed three times with a small amount of anhydrous ethanol and dried to obtain a brownish-white product 1 (17.7 g, yield 53.5%), which was used directly in the next reaction.

[0081] (2) The product 1 (17.7 g, 53.5 mmol) and potassium carbonate (30 g) were added to 500 ml of methanol and refluxed at 65°C for 6 h. After the reaction was completed, the mixture was cooled to 0°C in an ice bath, and yellow needle-like crystals gradually precipitated. The mixture was kept in an ice bath for 2 h, and the precipitated solid was filtered out. The precipitate was washed with water three times and dried to obtain light yellow needle-like product 2 (5.5 g, yield 27.5%), which was used directly in the next reaction.

[0082] (3) The product 2 (5.5 g, 19.4 mmol) from the previous step was added to 80 ml of 57% hydroiodic acid solution and refluxed at 127°C for 8 h. After the reaction was completed and cooled to room temperature, 150 ml of deionized water was added to the reaction system. The precipitated solid was filtered, washed with water three times, and dried to obtain the yellow product 3 (4.5 g, yield 87%), which was used directly in the next reaction.

[0083] (4) The product 3 (4.5 g, 16.9 mmol) from the previous step and urotropine (7.10 g, 50.7 mmol) were dissolved in 100 mL of trifluoroacetic acid and refluxed at 72°C for 8 h. After the reaction was completed and cooled to room temperature, 150 mL of deionized water was added to the reaction system. The precipitated solid was filtered, washed with water three times, and dried to obtain the yellow powder product 4 (3.04 g, yield 60.6%), which was used directly in the next reaction.

[0084] (5) The product 4 (0.60 g, 2.0 mmol) and 1,2,3,3-tetramethyl-3H-indole iodide (0.72 g, 2.3 mmol) from the previous step were dissolved in 20 mL of anhydrous ethanol, 500 μL of piperidine was added, and the mixture was refluxed at 78°C for 4 h. After the reaction was completed, the solvent was dried. Column chromatography was performed using silica gel as the stationary phase and methanol / dichloromethane = 1:100-50. The dark blue eluate was collected and dried to obtain the dark blue product 5 (0.86 g, yield 73.40%). 1 H NMR (500MHz, DMSO) δ8.88–8.13(m,5H),7.70(d,J=7.4Hz,1H),7.62–7.50(m,2H),7.48(t,J=7.7Hz,1H),7.37(t,J=7.5Hz,1H) ,4.03(t,J=7.4Hz,2H),3.82(s,3H),1.76(s,6H),1.60(p,J=7.3Hz,2H),1.34(tt,J=10.9,5.6Hz,2H),0.93(t,J=7.4Hz,3H).

[0085] (6) The product 5 (0.86 g, 1.5 mmol) from the previous step was dissolved in 15 mL of acetone, and 0.62 g of potassium carbonate (4.5 mmol) was added and stirred at room temperature for 30 min. Subsequently, 0.644 g of 4-nitrobenzyl bromide (3.0 mmol) was added and the mixture was refluxed at 65°C for 24 h. After the reaction was completed, the solvent was dried. Column chromatography was performed using silica gel as the stationary phase and methanol / dichloromethane = 1:250-100. The purple-red eluent was collected and dried to obtain the desired nitroreductase detection fluorescent probe (0.19 mg, yield 18%). 1H NMR (500MHz, DMSO) δ8.60 (dd, J=8.4, 1.2Hz, 1H), 8.58–8.53 (m, 1H), 8.28 (s, 1H), 7.95–7.87 (m, 1H), 7. 74(d,J=12.4Hz,1H),7.53–7.48(m,2H),7.44–7.39(m,2H),7.33(dd,J=7.5,1.2Hz,1H),7.17(td,J=7. 7,1.3Hz,1H),6.92–6.87(m,1H),6.83(d,J=7.9Hz,1H),5.24(d,J=27.8Hz,2H),5.11(d,J=12.5Hz,1H) ,4.05(t,J=7.4Hz,2H),2.88(s,3H),1.71–1.57(m,8H),1.36(h,J=7.4Hz,2H),0.93(t,J=7.3Hz,3H)(such as Figure 1B shown).

[0086] The product was characterized by high-resolution mass spectrometry. Figure 1A As shown, it can be seen that a product peak with a molecular weight of 588.2505 appears in the high-resolution mass spectrum.

[0087] Example 2: Using a fluorescence spectrometer to detect the reaction time between the probe and nitroreductase in vitro.

[0088] 1. Dissolve the probe in DMSO to prepare a 1 mM stock solution.

[0089] 2. Take 20 μL of probe stock solution and dilute it to 2 mL with PBS buffer solution (10 Mm, pH = 7.4)

[0090] (The probe concentration in the detection system is 20 μM.) NADH and nitroreductase were added to 500 μM and 6 μg / mL, respectively, and the reaction system was incubated in a 37°C water bath. Fluorescence was measured every 2 minutes using a fluorescence spectrometer with an excitation wavelength of 450 nm and an emission wavelength of 525 nm. The reaction was considered complete when the fluorescence signal remained almost constant.

[0091] The spectrum of the probe detecting the response time of nitroreductase is as follows Figure 2 As shown, the results show that after 20 minutes, the fluorescence signal intensity almost no longer changes and the reaction is almost complete, indicating that the probe reacts quickly with nitroreductase and the detection speed is fast.

[0092] Example 3: In vitro quantitative detection of nitroreductase using a fluorescence spectrometer.

[0093] 20 μL of the probe stock solution was diluted to 2 mL with 10 μM PBS buffer (pH 7.4) (probe concentration in the detection system was 10 μM). NADH was added to 500 μM. A series of nitroreductase concentrations (0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 6 μg / mL, and 8 μg / mL) were added and reacted in a 37°C water bath for 30 min. Fluorescence signals were measured using a fluorescence spectrometer with an excitation wavelength of 450 nm and an emission wavelength of 525 nm. A plot was plotted with nitroreductase concentration as the abscissa and the fluorescence emission signal at a wavelength of 525 nm as the ordinate, and a linear fit was performed. The results showed good linearity when the nitroreductase concentration ranged from 0.1 to 8 μg / mL, indicating that the probe has the ability to quantitatively detect nitroreductase. The standard curve equation is y=3.81x+11.80(R 2 =0.991), and the detection limit of the probe was calculated to be 50 ng / mL using the detection limit calculation equation LD=3.29Σ / k.

[0094] The spectra of different concentrations of nitroreductase detected by the probe and the standard curve are as follows Figure 3 and Figure 4 shown.

[0095] Example 4: pH stability of the probe and its reaction with nitroreductase.

[0096] Adjust the pH of the PBS buffer solution to 5, 6, 7.4, 8, 9, 10, 11 and 12, take 20 μL of the probe stock solution, dilute it to 2 mL with PBS buffer solution (10 Mm) of different pH values ​​(the probe concentration in the detection system is 10 μM), add NADH and nitroreductase to 500 μM and 6 μg / mL respectively, and place the reaction system in a 37°C water bath for 30 minutes. Use 450 nm as the excitation wavelength and 525 nm as the emission wavelength to measure the fluorescence signal using a fluorescence spectrometer. The fluorescence intensity of the reaction between the probe and nitroreductase at different pH values ​​is shown in Figure 2. Figure 5 The results show that the probe itself is minimally affected by pH. The probe exhibits the highest reactivity with nitroreductase at physiological pH (pH = 7.4), and maintains good activity at pH = 7.4-11, indicating that the probe has good stability over a wide pH range.

[0097] Example 5: Probe and its reaction selectivity with nitroreductase.

[0098] Prepare twelve 1M interfering substance solutions (Na + , K + , Ca 2+ , Mg 2+ , NO2- , Cl - , SO4 2- , S 2- , SO3 2- , cysteine, glutathione, β-glucan) take 20 μL probe stock solution and dilute it to 2 mL with PBS buffer solution (10Mm, pH=7.4) (the probe concentration in the detection system is 20 μM). Add 20 μL of the above-mentioned interfering substance stock solution respectively (the interfering substance concentration in the detection system is 10 μM), and then set up a set of interference experiments in which the probe reacts with 500 μM NADH alone. Place the reaction system in a 37°C water bath for 30 minutes. Use 450 nm as the excitation wavelength and 525 nm as the emission wavelength to measure the fluorescence signal using a fluorescence spectrometer. The fluorescence intensity of the probe reacting with different interfering substances is shown as follows: Figure 6 As shown in the figure, the fluorescence of the probe hardly changes after reacting with the above-mentioned interfering substances, and none of the above-mentioned interfering substances can cause the probe to respond. This shows that the probe has strong anti-interference ability against common substances in the body.

[0099] Figure 7 is the UV-visible absorption spectrum of the nitroreductase fluorescent probe and the fluorophore (i.e., product 5 in Example 1). It can be seen that the product 5 generated after the nitroreductase fluorescent probe reacts with the nitroreductase has absorption at 450 nm, and 450 nm can be selected as the emission wavelength.

[0100] In the present invention, the nitroreductase fluorescent probe and the nitroreductase response mechanism can be expressed as follows:

[0101]

[0102] Figure 8 The color depth of the solution changes before and after the reaction of the nitroreductase fluorescent probe prepared in Example 1 with nitroreductase. It can be seen that the color becomes lighter after the reaction.

[0103] Figure 9A This is the high-resolution mass spectrum of the reaction between the nitroreductase fluorescent probe and nitroreductase. It can be seen that the reaction between the nitroreductase fluorescent probe and nitroreductase generates product 5. The nuclear magnetic hydrogen spectrum of product 5 is as follows Figure 9B shown.

[0104] The applicant declares that while the present invention uses the above-described embodiments to illustrate the naphthalimide-based fluorescent probe, its preparation method, and its application, the present invention is not limited to the above-described embodiments, nor does it necessarily rely on the above-described embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, addition of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A naphthalimide-type fluorescent probe, characterized in that: The naphthaleneimide fluorescent probe has a structure shown in the following formula I:

2. The method for preparing a naphthalimide-type fluorescent probe according to claim 1, wherein: The preparation method comprises the following steps: S1, 4-bromo-1,8-naphthalene dicarboxylic anhydride react with n-butylamine to obtain product 1. The reaction formula is as follows: S2. Product 1 reacts in methanol to obtain product 2. The reaction formula is as follows: S3, product 2 reacts with hydroiodic acid to obtain product 3, and the reaction formula is as follows: S4, product 3 and urotropine react in trifluoroacetic acid to obtain product 4. The reaction formula is as follows: S5, product 4 reacts with 1,2,3,3-tetramethyl-3H-indole iodide to obtain product 5. The reaction formula is as follows: S6, product 5 reacts with 4-nitrobenzyl bromide to obtain the naphthaleneimide type fluorescent probe, and the reaction formula is as follows:

3. The preparation method according to claim 2, characterized in that The molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to n-butylamine in step S1 is 1:1.2-1.5; Preferably, the reaction in step S1 is carried out in a solvent, and the solvent is selected from any one or a combination of at least two of anhydrous ethanol, methanol, acetonitrile or acetone; Preferably, the reaction in step S1 is carried out under reflux, and the reaction time is 8-12 hours.

4. The preparation method according to claim 2 or 3, characterized in that The reaction in step S2 is carried out in the presence of an alkaline substance; Preferably, the molar ratio of product 1 to alkaline substance is 1:2-3; Preferably, the alkaline substance is selected from any one or a combination of at least two of potassium carbonate, sodium carbonate or cesium carbonate; Preferably, the reaction in step S2 is carried out under reflux, and the reaction time is 6-8 hours.

5. The preparation method according to any one of claims 2 to 4, characterized in that In step S3, the reaction of the product 2 with hydroiodic acid is carried out by dissolving the product 2 in a hydroiodic acid solution for reaction; Preferably, the mass percentage concentration of the hydroiodic acid solution is 55-57%; Preferably, the reaction in step S3 is carried out under reflux, and the reaction time is 8-14 hours.

6. The preparation method according to any one of claims 2 to 5, characterized in that In step S4, the molar ratio of the product 3 to hexamethylenetetramine is 1:2-3; Preferably, the reaction in step S4 is carried out under reflux, and the reaction time is 8-10 h.

7. The preparation method according to any one of claims 2 to 6, characterized in that In step S5, the molar ratio of the product 4 to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1.2-1.8; Preferably, the reaction in step S5 is carried out in the presence of a base; Preferably, the base is selected from piperidine and / or sodium acetate; Preferably, the molar ratio of product 4 to base is 1:0.1-0.5; Preferably, after the reaction in step S5 is completed, column chromatography is performed using dichloromethane / methanol as the eluent and silica gel as the stationary phase, the eluate is collected, and the solvent is dried to obtain product 5; Preferably, the volume ratio of methanol to dichloromethane in the eluent is 1:100-50.

8. The preparation method according to any one of claims 2 to 7, characterized in that In step S6, the molar ratio of the product 5 to 4-nitrobenzyl bromide is 1:1.2-2; Preferably, the reaction in step S6 is carried out in the presence of an alkaline substance; Preferably, the alkaline substance is selected from any one or a combination of at least two of potassium carbonate, sodium carbonate or cesium carbonate; Preferably, the molar ratio of the product 5 to the alkaline substance is 1:1 to 3.

9. The preparation method according to any one of claims 2 to 8, characterized in that The reaction in step S6 is carried out in a solvent, wherein the solvent is selected from any one or a combination of at least two of acetone, acetonitrile or N,N-dimethylformamide; Preferably, in step S6, the product 5 is dissolved in a solvent, potassium carbonate is added, and the mixture is stirred at room temperature for 10-40 minutes, followed by addition of 4-nitrobenzyl bromide to carry out the reaction; Preferably, the reaction in step S6 is carried out under reflux, and the reaction time is 12-24 hours; Preferably, after the reaction in step S6 is completed, column chromatography is performed using dichloromethane / methanol as an eluent and silica gel as a stationary phase, the eluate is collected, and the solvent is dried to obtain the naphthaleneimide-type fluorescent probe; Preferably, the volume ratio of methanol to dichloromethane in the eluent is 1:250-100.

10. Use of the naphthalimide fluorescent probe according to claim 1 in the detection of nitroreductase for non-therapeutic purposes.

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