Chemiluminescent small-molecule probe with ONOO <-> specific response as well as preparation method and application of chemiluminescent small-molecule probe

Abstract

The invention provides an ONOO <-> specific response chemiluminescent small-molecule probe as well as a preparation method and application thereof, and relates to the technical field of application of chemiluminescent probes. The light-emitting small molecule probe is named as CL-P, and a diphenyl phosphonate structure is used as an ONOO <-> response site; schap's dioxetane emitting 550nm is used as a chemiluminescent donor molecule, and the Schap's dioxetane and the Schap's dioxetane are linked together through an ether bond to obtain the chemiluminescent material. According to the probe, specific response and detection of ONOO <-> in vitro and in cells are realized by using a biologically responsive chemiluminescence signal for the first time, lung enrichment in APAP-induced mouse liver injury and lung injury diseases is realized, and specific response imaging of high-expression ONOO <-> is realized. The probe can also realize in-vivo responsive chemiluminescence imaging of ONOO <-> molecules of other lung diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescent probe applications, and particularly relates to a chemiluminescent small molecule probe with specific response to ONOO - and its preparation method and application. Background Art

[0002] The lung is a difficult area for optical imaging due to its complex microstructure and high optical heterogeneity. Essentially, almost all biological tissues scatter photons. The lung consists of a large number of alveoli with thin alveolar walls containing abundant blood and fluid, and the alveoli are filled with gas inside. When photons pass through the lung, scattering occurs at the interface between the alveolar wall and the gas inside the alveoli. Due to the tiny size and high density of the alveoli, this scattering significantly affects the propagation path of photons. The absorption coefficient of lung tissue is relatively low while the scattering coefficient is relatively high. This means that when photons propagate in the lung, more scattering occurs rather than absorption, resulting in a complex propagation path of photons. In recent years, significant progress has been made in advanced imaging methods. Nevertheless, there is still an urgent need to continue developing new optical imaging technologies so that researchers can utilize the powerful capabilities of molecular imaging to study lung diseases in small animal models. Chemiluminescent imaging is a luminescent imaging method that converts chemical energy into light energy. Chemiluminescent imaging technology has the advantages of not requiring real-time excitation light, being able to avoid biological autofluorescence, having a low imaging background, a high signal-to-noise ratio, good tissue penetration, and high sensitivity, providing an effective technical analysis means for obtaining high-sensitivity, specific, and real-time molecular information at the cellular and in vivo levels, and has been widely applied in research such as cell tracking, biosensing, cancer diagnosis and treatment, etc. Therefore, high-resolution and high-signal-to-noise imaging effects can be obtained by performing chemiluminescent imaging at the in vivo level.

[0003] Currently, there is only one reported chemiluminescent probe (Angew. Chem. Int. Ed. Engl. 2023, 64, e202303982) for lung disease imaging, and its administration method is intratracheal administration. The drug distribution of intratracheal administration may vary due to uneven airway branching and airflow distribution, resulting in too high drug concentration in some areas while too low in other areas, affecting the accuracy of imaging. In contrast, the method of intravenous injection via the tail vein is simple to operate, and after the drug injected via the tail vein enters the blood circulation, its metabolism and clearance in the body are relatively stable, capable of providing an imaging signal for a long time, which is beneficial for observing the dynamic changes of lung diseases. In addition, intravenous injection via the tail vein does not cause direct physical damage or irritation to the lungs or airways of mice, avoiding complications such as airway inflammation and pulmonary edema that may be caused by intratracheal administration; a chemiluminescent probe for intravenous injection via the tail vein for lung imaging has not been reported yet. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a chemiluminescent small molecule probe that specifically responds to ONOO - and its preparation method and application. Using a diphenyl phosphate structure as the ONOO - response site, and using Schaap's dioxetane that emits at 550 nm as the chemiluminescent donor molecule, the two are linked together through an ether bond. This probe can achieve in vivo responsive chemiluminescent imaging of ONOO - molecules in lung diseases.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0006] A chemiluminescent small molecule probe that specifically responds to ONOO - The structural formula of the probe is as follows:

[0007]

[0008] The preparation method of the above probe includes the following steps:

[0009] S1. Take 2-chloro-3-hydroxybenzaldehyde as compound A. Compound A, trimethyl orthoformate, and tetrabutylammonium tribromide are dissolved in methanol and stirred overnight. The reaction solution is washed three times with saturated sodium chloride, then extracted with ethyl acetate, and finally dried and concentrated by evaporation to obtain compound B;

[0010] S2. Take compound B and imidazole and dissolve them in dichloromethane. Stir at room temperature, and slowly add tert-butyldimethylsilyl chloride during this period and continue stirring for 30 min. The obtained product is filtered by suction. Take the filtrate and wash it with saturated sodium chloride solution, dry it with sodium sulfate, and concentrate by evaporation to obtain a brown oil, which is compound C;

[0011] S3. Take compound C and trimethoxy and put them into a two-necked flask. Add dichloromethane, and dropwise add titanium tetrachloride solution in an ice bath. Then quench with saturated sodium bicarbonate, and filter by suction to obtain a liquid, dry it, and concentrate by evaporation to obtain compound D;

[0012] S4. Take compound D and place it in a three-necked flask. Add tetrahydrofuran, stir at low temperature under nitrogen protection, dropwise add LDA during this period, then add adamantanone, and then concentrate by evaporation; Use column chromatography to purify the product to obtain the target product, compound 1;

[0013] S5. Dissolve compound 1 in ethyl acetate, then add tetrabutylammonium fluoride and stir at room temperature. Dilute the reaction mixture with EA and then extract. Dry and evaporate the organic layer and purify by chromatography to obtain compound 2;

[0014] S6. Dissolve Compound 2 in an acetonitrile solution, add magnesium chloride and triethylamine and mix well to obtain a mixed solution. Then add paraformaldehyde to the mixed solution for reaction. After the reaction is completed, carry out concentration under negative pressure, then dilute with EA and extract. After separating the organic layer, wash, dry, evaporate and purify by column chromatography to obtain Compound 3;

[0015] S7. Dissolve Compound 3 and methoxycarbonylmethylene triphenylphosphine in DCM and stir for reaction. Then dilute with EA, extract with brine and purify by silica gel column chromatography to obtain Compound 4;

[0016] S8. Under nitrogen protection, add Compound 4 to tetrahydrofuran and triethylamine. After stirring well in an ice bath, add 0.52 mmol of diphenylphosphoryl chloride dropwise. After the reaction is completed, add dilute hydrochloric acid dropwise to terminate the reaction. Then extract with ethyl acetate to collect the organic layer, concentrate and purify by column chromatography to obtain Compound 5;

[0017] S9. Dissolve Compound 5 and the catalyst methylene blue in DCM, then introduce oxygen and irradiate with yellow light (590 nm) for reaction. After the reaction is completed, concentrate the solvent under reduced pressure and purify by column chromatography to obtain the probe CL-P.

[0018] Preferably, in step S5, the mass ratio of Compound 1 to tetrabutylammonium fluoride is 2.5 - 3:4 - 4.5.

[0019] Preferably, in step S6, the mass ratio of Compound 2, magnesium chloride, triethylamine, and paraformaldehyde during the reaction is 2.0 - 2.5:1.5 - 1.8:1.3 - 1.6:1.6 - 2.0; and the reaction conditions are heating at 90 °C for 8 h and then cooling to room temperature; and subsequently, extract with 1M HCl, wash the separated organic layer with brine, dry with anhydrous Na2SO4, evaporate under reduced pressure, and then purify by silica gel column chromatography.

[0020] Preferably, in step S7, the mass ratio of Compound 3 to methoxycarbonylmethylene triphenylphosphine is 1.8 - 2.2:2.2 - 2.5.

[0021] Preferably, in step S8, the dosage ratio of Compound 4, tetrahydrofuran, triethylamine, and 0.52 mmol of diphenylphosphoryl chloride is 90 - 110 mg:28 - 32 mL:0.1 - 0.3 ml:115 - 125 mg.

[0022] Preferably, the addition amount of the catalyst methylene blue in step S9

[0023] The above-mentioned probe is used for the specific response of ONOO in cells - The application includes any one of the following:

[0024] (1) Use a probe to achieve lung enrichment and specific detection of highly expressed ONOO- in mouse models of APAP-induced acute liver injury and lung injury;

[0025] (2) Use a probe to achieve specific detection of highly expressed ONOO in a mouse model of LPS-induced acute lung injury; - ;

[0026] (3) Use a probe to achieve specific detection of highly expressed ONOO- in a mouse model of lung metastases;

[0027] (4) Use a probe to achieve in vivo responsive chemiluminescence imaging of ONOO- molecules in lung diseases.

[0028] The present invention provides a chemiluminescent small molecule probe that specifically responds to ONOO - , its preparation method and application. Compared with the prior art, the advantages are as follows:

[0029] The probe obtained in the present invention first uses a bioreactive chemiluminescent signal to achieve specific response and detection of ONOO in vitro and intracellularly. At the same time, it achieves lung enrichment in mouse liver injury and lung injury diseases induced by APAP, and achieves specific response imaging of highly expressed ONOO. This probe can also achieve in vivo responsive chemiluminescence imaging of ONOO molecules in other lung diseases. - ; - ; - ; BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 1H NMR data of compound 2 in Example 1 of the present invention; 1 ;

[0031] Figure 2 1H NMR data of compound 3 in Example 1 of the present invention; 1 ;

[0032] Figure 3 1H NMR data of compound 4 in Example 1 of the present invention; 1 ;

[0033] Figure 4 1H NMR data of compound 5 in Example 1 of the present invention; 1 ;

[0034] Figure 5 1H NMR data of probe CL-P in Example 1 of the present invention; 1 ;

[0035] Figure 6 1H NMR data of probe CL-P in Example 1 of the present invention; 1313C NMR data;

[0036] Figure 7 This is the 31 31P NMR data of probe CL-P in Example 1 of the present invention;

[0037] Figure 8 This is the ESI-MS data of probe CL-P in Example 1 of the present invention;

[0038] Figure 9 This is the chemiluminescence spectrum of probe CL-P in Example 2 of the present invention after the addition of ONOO - ;

[0039] Figure 10 This is the chemiluminescence (a) and imaging signal intensity (b) of different cells after incubation with CL-P in Example 3 of the present invention;

[0040] Figure 11 This is the chemiluminescence imaging signal (a) and the change in luminescence intensity (b) of CL-P in acute liver injury and lung injury in Example 4 of the present invention;

[0041] Figure 12 This is the chemiluminescence imaging signal (a) and the change in luminescence intensity (b) of CL-P in acute lung injury in Example 5 of the present invention;

[0042] Figure 13 This is the chemiluminescence imaging signal (a) and the change in luminescence intensity (b) of CL-P in mice with lung metastases in Example 6 of the present invention. Detailed implementation manners

[0043] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, rather than all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0044] Example 1:

[0045] Preparation of probe CL-P:

[0046] (1) Under room temperature and nitrogen protection, dissolve compound A (5 g, 41 mmol, 1 eq), trimethyl orthoformate (8.69 g, 82 mmol, 2 eq), and tetrabutylammonium tribromide (0.99 g, 2.05 mmol, 1 / 20 eq) in 80 mL of methanol and stir. This process requires overnight. Wash the reaction solution three times with saturated NaCl, extract with ethyl acetate (EA), dry with anhydrous sodium sulfate (Na2SO4), and then rotary evaporate to obtain compound B (6.54 g, 92.7%).

[0047] (2) Take compound B (6.54 g, 38 mmol, 1 eq) and add imidazole (3.98 g, 58 mmol, 1.5 eq) to a single-neck flask. Add 50 mL of dichloromethane and stir at room temperature. During this period, slowly add tert-butyldimethylsilyl chloride (7.03 g, 45.7 mmol, 1.2 eq) and continue stirring for 30 minutes. Filter the resulting product by suction. Wash the filtrate twice with saturated aqueous NaCl, dry with Na2SO4, and rotary evaporate to obtain a brown oily compound C (10.7 g, 99.8%).

[0048] (3) Take compound C (10.7 g, 37.9 mmol, 1 eq) and trimethoxyphosphine (7.24 g, 56.85 mmol, 1.5 eq) and place them in a two-neck flask. Add 40 mL of dichloromethane and react and stir in an ice bath for 30 minutes. During this period, dropwise add 7 mL of titanium tetrachloride (11.06 g, 56.85 mmol, 1.5 eq). Monitor with a thin-layer chromatography plate. After completion, pour the solution into saturated sodium bicarbonate at 0 °C and stir to quench. After no bubbles are generated, filter by suction. Add diatomaceous earth to the funnel during suction filtration. Extract the obtained liquid with EA (50 mL × 3 times), dry, and rotary evaporate to obtain the product (11.43 g, 83.6%).

[0049] (4) Take compound D (1 g, 2.77 mmol, 1 eq) and place it in a three-neck flask. Add tetrahydrofuran (THF) and stir in a low-temperature (-70 °C) reaction bath under nitrogen protection for 10 minutes. During this period, control the rate and dropwise add LDA (4.15 mL, 8.31 mmol, 3 eq), add adamantanone (388.30 mg, 5.55 mmol, 2 eq), and continue stirring for 40 minutes. After detecting with a thin-layer chromatography plate, rotary evaporate. Purify the product by column chromatography, collect the target product, and rotary evaporate to obtain compound 1 (242 mg, 35%).

[0050] (5) Dissolve 2.92 g of Compound 1 in 30 mL of EA, then add 8 mL of 2.0 M tetrabutylammonium fluoride and stir at room temperature. Monitor the reaction by thin-layer chromatography (TLC). After the reaction is complete, dilute the reaction mixture with 150 mL of EA and extract with water and brine. Dry the combined organic layers with Na2SO4 and evaporate under reduced pressure. Purify by silica gel column chromatography (PE:EA = 100:1) to obtain 2.36 g of white solid Compound 2 with a yield of 93%.

[0051] (6) Add 1.60 g of magnesium chloride and 2 mL of triethylamine to an 8 mL acetonitrile solution dissolving 2.36 g of Compound 2. Then add 1.84 g of paraformaldehyde to the mixture solution. Heat the reaction at 90 °C for 8 hours and then cool to room temperature. Monitor the reaction by TLC (PE:EA = 9:1). After the reaction is complete, filter the reaction solution and concentrate it under negative pressure. Dilute the reaction solution with 100 mL of EA and extract with 100 mL of 1 M HCl. After separating the organic layer, wash it with brine, then dry it with anhydrous Na2SO4 and evaporate under reduced pressure. Purify the obtained product by silica gel column chromatography (PE:EA = 100:1) to obtain 2.34 g of pale yellow solid Compound 3 (yield 91%).

[0052] (7) Dissolve 2 g of Compound 3 and 2.39 g of methoxycarbonylmethylenetriphenylphosphine in 40 mL of DCM. Stir at room temperature and monitor by TLC (PE:EA = 5:1). After complete conversion of the reaction, dilute the reaction solution with 100 mL of EA and extract three times with 150 mL of brine. Purify the product by silica gel column chromatography (PE:EA = 90:10) to obtain 2.10 g of pale yellow solid as Compound 4 with a yield of 90%.

[0053] (8) Add 100 mg of Compound 4 to a 100 mL two-necked round-bottom flask, remove oxygen and protect with nitrogen. Under nitrogen protection, add 30 mL of dry tetrahydrofuran (THF) and 0.2 mL of triethylamine (Et3N) with a syringe, ensuring that the mixture is stirred well in an ice bath. After that, drop diphenylphosphoryl chloride (121.7 mg, 0.52 mmol) into the reaction solution in an ice bath at 0 °C with a syringe. Monitor the reaction by TLC (PE:EA = 2:1) until the reaction is complete. Add 50 mL of 1 M dilute hydrochloric acid to terminate the reaction in the ice bath and extract three times with ethyl acetate. Collect the organic phase and concentrate it under reduced pressure. Purify the product by column chromatography (PE:EA = 5:1) to obtain 95.6 mg of white solid as Compound 5 with a yield of 62.5%.

[0054] (9) Dissolve 95.6 mg of Compound 5 and the catalyst methylene blue in 20 mL of DCM. Pass oxygen through the solution while irradiating with yellow light. Monitor the reaction by TLC. After the reaction is completed, concentrate the solvent under reduced pressure. Purify by column chromatography (PE:EA = 5:1) to obtain 95.7 mg of a white solid as the probe CL-P, with a yield of 95%.

[0055] The specific synthesis route is as follows:

[0056]

[0057] And the characterization data of Compound 2, Compound 3, Compound 4, Compound 5 and the probe CL-P are as Figures 1-8 shown.

[0058] Example 2:

[0059] Specific response and detection of ONOO - in vitro:

[0060] Take the pre-prepared DMSO (10%) saline solution containing the probe (50 μM); add the ONOO - (200 μM) to be measured dropwise into the above system, and then perform real-time imaging.

[0061] The specific results are shown in Figure 9 shown. The chemiluminescence spectrum data shows that after adding ONOO - , the chemiluminescence spectrum of the probe CL-P is collected by the detector, with its maximum emission peak at 550 nm, and its intensity is approximately 354 times that without adding ONOO - .

[0062] Example 3:

[0063] Specific response and detection of ONOO- in cells:

[0064] Digest the A549, HBe, 4T1-Luc cells purchased from Wuhan Ponsai Life Science Co., Ltd. with trypsin and add them to fresh medium (dulbecco's modified eagle medium, DMEM). Inoculate them in a 96-well plate and culture for 24 h until the cells adhere.

[0065] Blank group: Remove the medium, then wash three times with PBS, and add DMEM medium containing CL-P (10 μM).

[0066] LPS experimental group: Add 1 mg·mL -1 LPS for stimulation treatment for 1 h. Then wash three times with PBS, and add cell culture medium containing CL-P (10 μM).

[0067] ONOO - Experimental group: First, add 100 μM ONOO to the DMEM cell culture medium - Stimulate for 1 h, then wash three times with PBS, and add cell culture medium containing CL-P (10 μM). After treatment, perform imaging analysis in a small animal imager.

[0068] The imaging results show that for these three types of cells, the concentration of ONOO in 4T1-luc cells is the highest, followed by A549 cells, and the lowest in HBe cells. The ONOO - group has the strongest chemiluminescence intensity, the highest intracellular ONOO - concentration, and the LPS group also shows an increase compared to the Blank group. This further indicates that CL-P can specifically respond to highly expressed ONOO in tumor cells - ( - ( Figure 10 ). The small animal imaging system uses the bioluminescence mode to collect images through an open filter, and the exposure time is 10 s.

[0069] Example 4:

[0070] Imaging of APAP-induced acute liver and lung injury models in mice:

[0071] First, intraperitoneally inject APAP at 320 mg / kg into mice. After 5 h, the acute liver and lung injury models are successfully established.

[0072] Inject the probe (50 μM, 100 μL) through the tail vein, and then perform real-time imaging on the mice. As Figure 11 shown, there are obvious chemiluminescence signals in the lungs of the mice, while in contrast, the chemiluminescence signals in the liver are very weak, indicating that the probe CL-P can be enriched and retained in the lungs after intravenous injection through the tail vein, achieving specific imaging of highly expressed ONOO at the lung injury site - .

[0073] Example 5:

[0074] Imaging of LPS-induced acute lung injury models in mice:

[0075] First, instill 30 μL of 1 mg / ml LPS solution into the trachea of mice. After 24 h, the acute lung injury model is successfully established.

[0076] Inject the probe (50 μM, 100 μL) through the tail vein, and then perform real-time imaging on the lungs of LPS-treated mice and normal mice.

[0077] Specifically, as Figure 12Shown as follows: The imaging results show that there are obvious chemiluminescence signals in the LPS-treated mice, and the signal intensity is the strongest at 3 min. However, no obvious chemiluminescence signal is seen in the lungs of normal mice, indicating that the probe CL-P can achieve specific imaging of highly expressed ONOO at the acute lung injury site. - Specific imaging.

[0078] Example 6:

[0079] Imaging of a mouse model of lung metastases

[0080] First, inject 100 μL of a 4T1-Luc cell suspension with a cell density of 1×10 6 / mL into the tail vein of the mice. After 7 days, inject potassium fluorescein intraperitoneally, and verify the success of the model by means of the bioluminescence signal generated by the reaction of luciferase and potassium fluorescein.

[0081] After the model is successfully established, inject the probe (50 μM, 100 μL) through the tail vein, and then perform real-time imaging on the lungs of the 4T1-Luc-treated mice and normal mice. The imaging results show that there are obvious chemiluminescence signals in the mice injected with 4T1-Luc through the tail vein, and the positions are highly consistent with the bioluminescence positions. However, no obvious chemiluminescence signal is seen in the lungs of normal mice, indicating that the probe CL-P can achieve specific imaging of highly expressed ONOO- in lung tumors ( Figure 13 ).

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chemiluminescent small molecule probe that specifically responds to ONOO - , characterized in that The structural formula of the probe is as follows:

2. A method for preparing a probe as described in claim 1, characterized in that, The preparation method includes the following steps: S1. Take 2-chloro-3-hydroxybenzaldehyde as compound A. Compound A, trimethyl orthoformate, and tetrabutylammonium tribromide are dissolved in methanol and stirred overnight. The reaction solution is washed three times with saturated sodium chloride, then extracted with ethyl acetate, and finally dried and concentrated by rotary evaporation to obtain compound B. S2. Take compound B and imidazole and dissolve them in dichloromethane. Stir at room temperature, and slowly add tert-butyldimethylsilyl chloride during the process and continue stirring for 30 min. The obtained product is filtered by suction. Take the filtrate and wash it with saturated sodium chloride solution, dry it with sodium sulfate, and concentrate by rotary evaporation to obtain a brown oil, which is compound C. S3. Take compound C and trimethoxy and put them into a two-necked flask. Add dichloromethane, and dropwise add titanium tetrachloride solution in an ice bath. Then quench with saturated sodium bicarbonate, and filter by suction to obtain a liquid, dry it, and concentrate by rotary evaporation to obtain compound D. S4. Take compound D and place it in a three-necked flask. Add tetrahydrofuran, stir at low temperature under nitrogen protection, dropwise add LDA during the process, then add adamantanone, and then concentrate by rotary evaporation; purify the product by column chromatography to obtain the target product, compound 1. S5. Dissolve compound 1 in ethyl acetate, then add tetrabutylammonium fluoride and stir at room temperature for reaction. Dilute the reaction mixture with EA and then extract. Dry and evaporate the organic layer and then purify by chromatography to obtain compound 2. S6. Dissolve compound 2 in an acetonitrile solution, add magnesium chloride and triethylamine and mix evenly to obtain a mixed solution. Then add paraformaldehyde to the mixed solution for reaction. After the reaction is completed, concentrate under negative pressure, then dilute with EA and extract. Separate the organic layer, wash, dry, evaporate, and then purify by chromatography to obtain compound 3. S7. Dissolve compound 3 and methoxycarbonylmethylene triphenylphosphine in DCM and stir for reaction. Then dilute with EA, extract with brine, and purify by silica gel column chromatography to obtain compound 4. S8. Under nitrogen protection, add compound 4 to tetrahydrofuran and triethylamine. Stir well in an ice bath and then dropwise add 0.52 mmol of diphenylphosphoryl chloride. After the reaction is completed, dropwise add dilute hydrochloric acid to terminate the reaction. Then extract with ethyl acetate to collect the organic layer, concentrate, and then purify by column chromatography to obtain compound 5. S9. Dissolve compound 5 and the catalyst methylene blue in DCM, then introduce oxygen, and at the same time irradiate with yellow light (590 nm) for reaction. After the reaction is completed, concentrate the solvent under reduced pressure and purify by column chromatography to obtain the probe CL-P.

3. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of compound 1 to tetrabutylammonium fluoride is 2.5 - 3∶4 - 4.

5.

4. The preparation method according to claim 2, characterized in that: In step S6, during the reaction, the mass ratio of compound 2, magnesium chloride, triethylamine, and paraformaldehyde is 2.0 - 2.5∶1.5 - 1.8∶1.3 - 1.6∶1.6 - 2.0; and the reaction conditions are heating at 90 °C for 8 h and then cooling to room temperature; and subsequently, extract with 1M HCl, wash the separated organic layer with saturated brine, dry with anhydrous Na2SO4, and evaporate under reduced pressure, and then purify by silica gel column chromatography.

5. The preparation method according to claim 2, characterized in that: In step S7, the mass ratio of compound 3 to methoxycarbonylmethylene triphenylphosphine is 1.8 - 2.2∶2.2 - 2.

5.

6. The preparation method according to claim 2, wherein: In the step S8, the dosage ratio of compound 4, tetrahydrofuran, triethylamine, and 0.52 mmol of diphenylphosphoryl chloride is 90 - 110 mg∶28 - 32 mL∶0.1 - 0.3 ml∶115 - 125 mg.

7. The preparation method according to claim 2, wherein: In the step S9, the addition amount of the catalyst methylene blue is 0 - 1 mg.

8. Use of the probe as described in claim 1 for achieving specific response to ONOO within cells - .

9. The application according to claim 8, characterized in that: The application includes any one of the following: (1) Using the probe to achieve pulmonary enrichment and specific detection of highly expressed ONOO- in APAP-induced acute liver injury and lung injury mouse models; (2) Using the probe to achieve specific detection of highly expressed ONOO- in LPS-induced acute lung injury mouse models; (3) Using the probe to achieve specific detection of highly expressed ONOO- in lung metastasis tumor mouse models; (4) Using a probe to achieve in vivo responsive chemiluminescence imaging of ONOO - molecules for lung diseases.

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