A class of fluorescent probes for detecting nitrite under strong acid conditions, and preparation method and application thereof

Abstract

The application belongs to the technical field of analytical chemistry, and particularly relates to a kind of fluorescent probe for detecting nitrite under strong acid conditions and a preparation method and application thereof.The fluorescent probe provided by the application has the structure shown in formula I.The fluorescent probe can maintain the stability of its own structure under strong acid conditions, and can respond to nitrite efficiently, and after reaction, obvious fluorescence and color change can be observed, which can be identified by naked eye, without the need of complex instruments, so that the detection imaging of nitrite in strong acid solution and in vivo model can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to a kind of fluorescent probe for detecting nitrite under strong acid conditions and its preparation method and application. BACKGROUND

[0002] Nitrite (sodium nitrite, NaNO2, potassium nitrite KNO2, etc.) exists widely in our daily life and environment. As one of the important inorganic anions required for plant growth, it is also the most commonly used nitrogen-containing compound in nature and plays a key role in the natural nitrogen cycle. In our daily diet, nitrite is widely used as a food preservative. However, excessive intake of nitrite can cause adverse reactions in the human body, such as central nervous system defects, congenital infant deformities, and methemoglobinemia. In addition, under the acidic conditions in the stomach, nitrite can react with secondary amines and amides in the stomach to form nitrosoamines with carcinogenicity, which can cause gastric cancer. In order to prevent the potential harm of nitrite, the World Health Organization has set the maximum allowable concentration of nitrite in drinking water to 3 ppm (65.1 μM). Therefore, it is of great significance to develop a probe that can detect nitrite in the stomach to avoid its potential harm.

[0003] To date, researchers have developed various types of detection methods to achieve effective detection of nitrite, including spectrophotometry (such as the Griess reaction), chemiluminescence, electrochemical methods, high-performance liquid chromatography, fluorescence spectroscopy, and electrochemiluminescence. Among these methods, fluorescence spectroscopy has attracted much attention due to its high sensitivity, ease of operation, and real-time nature. In addition, the development of fluorescent probes enables qualitative and quantitative detection of nitrite in vitro, providing a new option for monitoring nitrite in the environment. Since nitrite mainly exists in the stomach of the human body, in situ recognition and tracking of nitrite in the stomach is crucial for preventing diseases caused by nitrite. However, according to the prior art, there is currently no specialized probe for detecting nitrite in the stomach. This is mainly because the core recognition unit of most currently developed fluorescent probes for nitrite contains an aromatic amine structure, which is prone to diazotization reaction with nitrite under the acidic conditions in the stomach, resulting in the formation of highly toxic and unstable diazonium salts in the stomach. Therefore, there is an urgent need to develop a nitrite probe that does not rely on aromatic amine structures in order to accurately detect nitrite under strong acid conditions in the stomach. SUMMARY

[0004] In order to solve the problems existing in the prior art, the purpose of the present application is to provide a kind of fluorescent probe for detecting nitrite under strong acid conditions and its preparation method and application in solution or in vivo imaging.

[0005] The technical scheme of the present application is:

[0006] A class of fluorescent probes for detecting nitrite under strong acid conditions, wherein the fluorescent probe molecule has the structure shown in formula I:

[0007]

[0008] (1) Z is O or S;

[0009] (2) R1, R4, R5, R6, R7 and R 10 each independently selected from a hydrogen atom, a halogen or an alkyl group;

[0010] (3) R2, R3, R8 and R9 are each independently selected from a hydrogen atom, an unsubstituted alkyl group, a phenyl-substituted alkyl group, an alkoxy group or a hydroxyalkoxy group, wherein the phenyl group is unsubstituted or substituted with one or more halogen, alkoxy, saturated amino or alkyl groups;

[0011] (4) -NR2R3 and -NR8R9 groups can independently represent a saturated or unsaturated heterocyclic ring, wherein the heterocyclic ring can contain one or more additional heteroatoms selected from N, O, S, and the heterocyclic ring is unsubstituted or substituted with halogen and alkyl groups;

[0012] (5) R 11 is selected from a hydrogen atom, an unsubstituted alkyl group, a phenyl-substituted alkyl group, an alkoxy group or a hydroxyalkoxy group, wherein the phenyl group is unsubstituted or substituted with one or more halogen, alkoxy, saturated amino or alkyl groups;

[0013] (6) The halogen in the present application is selected from fluorine, chlorine, bromine and iodine atoms; and the alkyl group refers to a saturated alkane having a specific number of carbon atoms, including straight chains or branched chains, such as methyl, ethyl, propyl, butyl, pentyl, isopropyl and tert-butyl.

[0014] Further, the present application also provides a preparation method of the fluorescent probe for detecting nitrite under strong acid conditions, comprising the following synthesis route:

[0015]

[0016] Further, the specific preparation steps of the fluorescent probe compound I for detecting nitrite under strong acid conditions are as follows:

[0017] S1, Preparation of intermediate B:

[0018]

[0019] Compound A is dissolved in an organic solvent, hydrazine hydrate is added dropwise into the system for reaction, concentrated, washed by methanol, and then freeze-dried for 6-48 hours to obtain intermediate B.

[0020] The organic solvent is selected from aprotic solvents, including tetrahydrofuran, dichloromethane, etc.; the reaction temperature is 20-80℃, preferably 40℃; and the molar ratio of hydrazine hydrate to compound A is 3-10:1.

[0021] S2, preparation of fluorescent probe compound I:

[0022]

[0023] The reaction of compound B with sulfonyl chloride can be carried out in two ways:

[0024] Different types of sulfonyl chloride are dissolved in an organic solvent and added dropwise into a solution of compound B or compound B is dissolved in an organic solvent and added dropwise into a solution of different types of sulfonyl chloride dissolved in an organic solvent to obtain a crude product of compound I; the crude product is purified by silica gel column chromatography to obtain pure compound I.

[0025] The organic solvent is selected from aprotic solvents, including tetrahydrofuran, dichloromethane, etc., preferably dichloromethane; the molar ratio of sulfonyl chloride to compound B is 1.2-5:1; and the reaction temperature is -10-20℃, preferably 0℃.

[0026] Further, when the crude product is purified by silica gel column chromatography, the eluent is selected from a system of ethyl acetate and petroleum ether or a system of dichloromethane and methanol, preferably a system of ethyl acetate and petroleum ether.

[0027] The application provides an application of the fluorescent probe in detecting nitrite under strong acid conditions.

[0028] Further, the pH of the strong acid condition is less than 3; and the reaction of the fluorescent probe with nitrite has a response mechanism as shown in the following formula:

[0029]

[0030] The compound with the structure of formula I itself has no fluorescence, and after responding to nitrite, intermediate C is first generated, and then further oxidized by strong acid active oxygen to generate compound D with strong fluorescence emission.

[0031] The application also provides an application of the fluorescent probe in preparing a detection reagent for detecting nitrite in a detection solution or a living body.

[0032] In addition, the application also provides a detection method for detecting nitrite in a detection solution or a living body for non-disease diagnosis and treatment purposes, and the specific method is as follows:

[0033] The compound of formula I is dissolved in an organic solvent, then diluted into a buffer solvent containing the test substance for detection of nitrite under strong acidic conditions;

[0034] The compound of formula I is dissolved in an organic solvent, then administered in a live model of an animal and used for detection of nitrite under strong acidic conditions. The administration methods include injection, gavage, oral administration, etc.

[0035] Compared with the prior art, the fluorescent probe for detecting nitrite under strong acid conditions, the preparation method and the application thereof provided by the present application have the following advantages:

[0036] (1) The compound having the structure of formula I provided by the present application can maintain its own structure stable under strong acidic conditions (pH≤3);

[0037] (2) The compound having the structure of formula I can react with nitrite under strong acidic conditions;

[0038] (3) After the compound having the structure of formula I reacts with nitrite under strong acidic conditions, a significant color change can be observed, which can be recognized by naked eyes without the need of complex instruments;

[0039] (4) The compound having the structure of formula I can be used for in-situ detection of nitrite in the stomach. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The H NMR spectrum of the fluorescent probe DHUROS-1 described in Example 1 related to the present application is as follows: 1 The H NMR spectrum of the fluorescent probe DHUROS-1 described in Example 1 related to the present application is as follows:

[0041] Figure 2 The HRMS spectrum of the fluorescent probe DHUROS-1 described in Example 1 related to the present application is as follows:

[0042] Figure 3 The H NMR spectrum of the fluorescent probe DHUROS-2 described in Example 2 related to the present application is as follows: 1 The H NMR spectrum of the fluorescent probe DHUROS-2 described in Example 2 related to the present application is as follows:

[0043] Figure 4 The HRMS spectrum of the fluorescent probe DHUROS-2 described in Example 2 related to the present application is as follows:

[0044] Figure 5 The H NMR spectrum of the fluorescent probe DHUROS-5 described in Example 3 related to the present application is as follows: 1 The H NMR spectrum of the fluorescent probe DHUROS-5 described in Example 3 related to the present application is as follows:

[0045] Figure 6 The HRMS spectrum of the fluorescent probe DHUROS-5 described in Example 3 related to the present application is as follows:

[0046] Figure 7 The H NMR spectrum of the fluorescent probe DHUROS-8 as described in Embodiment 4 related to the present application; 1 H NMR spectrum;

[0047] Figure 8 The HRMS spectrum of the fluorescent probe DHUROS-8 as described in Embodiment 4 related to the present application;

[0048] Figure 9 The H NMR spectrum of the fluorescent probe DHUROS-11 as described in Embodiment 5 related to the present application; 1 H NMR spectrum;

[0049] Figure 10 The HRMS spectrum of the fluorescent probe DHUROS-11 as described in Embodiment 5 related to the present application;

[0050] Figure 11 The H NMR spectrum of the fluorescent probe DHUROS-12 as described in Embodiment 6 related to the present application; 1 H NMR spectrum;

[0051] Figure 12 The fluorescence spectra of the fluorescent probe DHUROS-1 as described in Embodiment 1 related to the present application before and after response to nitrite, wherein the concentration of DHUROS-1 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0052] Figure 13 The time-dependent diagram of the response of the fluorescent probe DHUROS-1 as described in Embodiment 1 related to the present application to nitrite, wherein the concentration of DHUROS-1 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0053] Figure 14 The fluorescence spectra of the fluorescent probe DHUROS-2 as described in Embodiment 2 related to the present application before and after response to nitrite, wherein the concentration of DHUROS-2 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0054] Figure 15 The time-dependent diagram of the response of the fluorescent probe DHUROS-2 as described in Embodiment 2 related to the present application to nitrite, wherein the concentration of DHUROS-2 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0055] Figure 16The fluorescence spectrum of the fluorescent probe DHUROS-5 described in Embodiment 3 of the present application before and after response to nitrite; wherein the concentration of DHUROS-5 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0056] Figure 17 The time-dependent diagram of the response of the fluorescent probe DHUROS-5 described in Embodiment 3 of the present application to nitrite; wherein the concentration of DHUROS-5 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0057] Figure 18 The fluorescence spectrum of the fluorescent probe DHUROS-8 described in Embodiment 4 of the present application before and after response to nitrite; wherein the concentration of DHUROS-8 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm.

[0058] Figure 19 The time-dependent diagram of the response of the fluorescent probe DHUROS-8 described in Embodiment 4 of the present application to nitrite; wherein the concentration of DHUROS-8 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0059] Figure 20 The fluorescence spectrum of the fluorescent probe DHUROS-11 described in Embodiment 5 of the present application before and after response to nitrite; wherein the concentration of DHUROS-11 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm.

[0060] Figure 21 The time-dependent diagram of the response of the fluorescent probe DHUROS-11 described in Embodiment 5 of the present application to nitrite; wherein the concentration of DHUROS-11 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm;

[0061] Figure 22 The photos of the response of the fluorescent probe DHUROS-11 described in Embodiment 5 of the present application to nitrite with different concentrations; wherein the concentration of DHUROS-11 is 5 μM, and the concentration of nitrite is 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM;

[0062] Figure 23 The imaging diagram of the fluorescent probe DHUROS-11 described in Embodiment 5 of the present application in a mouse in vivo;

[0063] Figure 24The fluorescence spectra of the fluorescent probe DHUROS-12 described in Embodiment 6 of the present application before and after responding to nitrite; wherein the concentration of DHUROS-12 is 5 μM, the concentration of nitrite is 50 μM, and the excitation wavelength is 620 nm. DETAILED DESCRIPTION

[0064] The present application is further described through the description of specific embodiments, but this is not a limitation of the present application. Those skilled in the art can make various modifications or improvements according to the basic idea of the present application, as long as they do not deviate from the basic idea of the present application, and they are within the protection scope of the present application.

[0065] In the following examples, the reagents not specifically mentioned are conventional reagents, which can be purchased from conventional reagent production and sales companies. The methods used are prior art, unless otherwise specified.

[0066] Embodiment 1

[0067] The preparation of the compound DHUROS-1 (R 11 and the response performance to nitrite.

[0068] The synthesis route of the compound DHUROS-1 (R 11 is as follows:

[0069]

[0070] (1) Preparation of the fluorescent probe DHUROS-1:

[0071] In a dry round-bottom flask, compound A (0.50 g, 1.46 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, and then hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq) dissolved in 10 mL of dichloromethane was added dropwise. The reaction progress was monitored by TLC, and the reaction was completed at 0.5 h. After the reaction was completed, the whole reaction system was concentrated, washed with methanol, and then freeze-dried for 12 hours to obtain compound B.

[0072] Compound B (0.50 g, 1.46 mmol, 1 eq) after freeze-drying was re-dissolved in DCM. The reaction system was placed in an ice-water bath and stirred, and then the dissolved product B DCM solution was added dropwise to the benzene sulfonyl chloride (0.39 g, 2.19 mmol, 1.5 eq) solution dissolved in 10 mL of dichloromethane under the protection of nitrogen. The whole reaction process was monitored by TLC until the reaction was completed.

[0073] The solvent was removed by evaporation on a rotary evaporator and the residue was purified by column chromatography on silica gel (ethyl acetate: petroleum ether = 1 : 3) to give the pure white compound DHUROS-1 in a yield of 286 mg, 41% of the product.

[0074] 1 H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.44 (s, 1H), 7.89 - 7.87 (m, 2H), 7.71 - 7.67 (m, 1H), 7.61 (t, J = 7.4 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 2.4 Hz, 2H), 6.63 (dd, J = 8.8, 2.8 Hz, 2H), 2.88 (s, 12H).

[0075] HRMS (ESI) calc. for C 23 H 26 N5O3S2 + [M+H + ]: 484.1472; found: 484.1487.

[0076] The fluorescent probe DHUROS-1 prepared in Example 1 of the present application has the following properties: 1 H NMR, HRMS spectra as shown in Figures 1-2 .

[0077] (2) Performance experiment of strong acidic active oxygen fluorescent probe compound

[0078] As shown in the following figure, the 5 μM DHUROS-1 buffer solution (0.1M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system has no fluorescence. But after adding 50 μM nitrite, DHUROS-1 has a strong fluorescence response, and the fluorescence intensity at 686 nm is increased by more than 2800 times. Figure 12 As shown in the following figure, when 50 μM nitrite is added to the 5 μM DHUROS-1 buffer solution (0.1M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system, the fluorescence intensity at 686 nm in the system can be observed to be significantly enhanced over time.

[0079] Figure 13 Example 2

[0080] Preparation of the compound DHUROS-2 (R 11 is p-nitrophenyl) with the structure of formula I and the response performance research with nitrite.

[0081] Preparation of the compound DHUROS-2 (R 11 is p-nitrophenyl) with the structure of formula I and the response performance research with nitrite.

[0082] The synthetic route of the DHUROS-2 (R 11 is p-nitrophenyl) is as follows:

[0083]

[0084] (1) Preparation of fluorescent probe DHUROS-2:

[0085] In a dry round bottom flask, A (0.50 g, 1.46 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, then hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq) dissolved in 10 mL of dichloromethane was added dropwise. The reaction progress was monitored by TLC, and the reaction was completed at 0.5 h. After the reaction was completed, the whole reaction system was concentrated, washed with methanol, and then freeze-dried for 12 hours to obtain compound B.

[0086] Compound B (0.50 g, 1.46 mmol, 1 eq) after freeze-drying was re-dissolved in DCM. The reaction system was placed in an ice water bath and stirred, then the dissolved product B DCM solution was added dropwise to 10 ml of dichloromethane p-nitrobenzenesulfonyl chloride (0.48 g, 2.19 mmol, 1.5 eq) solution under nitrogen protection, and the whole reaction process was monitored by TLC until the reaction was completed.

[0087] The solvent was evaporated on a rotary evaporator, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to finally obtain pure white compound DHUROS-2 with a yield of 193 mg and a product yield of 25%.

[0088] 1 H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 8.63 (s, 1H), 8.44 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 2.8 Hz, 2H), 6.50 - 6.62 (m, 2H), 2.87 (s, 12H).

[0089] HRMS (ESI) calc. for C 23 H 25 N6O5S2 + [M+H + ]: 529.1322; found: 529.1339

[0090] The fluorescent probe DHUROS-2 prepared in Example 2 of the present application has the following properties: 1 H NMR, HRMS spectra, as shown in Figures 3-4 .

[0091] (2) Response behavior of the compound to strong acidic active oxygen

[0092] As shown in Figure 2, the 5 μM DHUROS-2 buffered solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system is non-fluorescent. But after adding 50 μM nitrite, DHUROS-2 has a strong fluorescence response, and the fluorescence intensity at 686 nm is increased by more than 2500 times. Figure 14 As shown in Figure 3, when 50 μM nitrite is added to the 5 μM DHUROS-2 buffered solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system, the fluorescence intensity at 686 nm in the system is observed to be significantly enhanced over time.

[0093] Figure 15 As shown in Figure 3, when 50 μM nitrite is added to the 5 μM DHUROS-2 buffered solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system, the fluorescence intensity at 686 nm in the system is observed to be significantly enhanced over time.

[0094] Example 3

[0095] Preparation of the compound DHUROS-5 (R 11 is a thiophene heterocycle) and study of its response performance to nitrite.

[0096] The synthesis route of the compound DHUROS-5 (R 11 is a thiophene heterocycle) is as follows:

[0097]

[0098] (1) Preparation of the fluorescent probe DHUROS-5:

[0099] In a dry round-bottom flask, A (0.50 g, 1.46 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, and then hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq) dissolved in 10 mL of dichloromethane was added dropwise. The reaction progress was monitored by TLC, and the reaction was completed at 0.5 h. After the reaction was completed, the whole reaction system was concentrated, washed with methanol, and then freeze-dried for 12 h.

[0100] Compound B (0.50 g, 1.46 mmol, 1 eq) after freeze-drying was re-dissolved in DCM. The reaction system was placed in an ice water bath and stirred, and then the dissolved product B DCM solution was added dropwise to 10 mL of dichloromethane 2-thiophenesulfonyl chloride (0.40 g, 2.19 mmol, 1.5 eq) solution under nitrogen protection. The whole reaction process was monitored by TLC until the reaction was completed.

[0101] ​The solvent was removed by evaporation on a rotary evaporator and the residue was purified by column chromatography on silica gel (ethyl acetate: petroleum ether = 1 : 3) to give the pure white compound DHUROS-5 in a yield of 200 mg, 28% of the product.

[0102] 1 H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.61 (s, 1H), 8.04-8.03 (m, 1H), 7.68 (dd, J = 4.0, 1.2 Hz, 1H), 7.25-7.23 (m, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.68-6.64 (m, 4H), 2.88 (s, 12H).

[0103] HRMS (ESI) calc. for C 21 H 24 N5O3S3 + [M+H + ]: 490.1036; found: 490.1046

[0104] The fluorescent probe DHUROS-5 prepared in Example 3 of the present application has the following properties: 1 H NMR, HRMS spectra, as shown in Figures 5-6 .

[0105] (2) Performance experiment of strong acidic active oxygen fluorescent probe compound

[0106] As shown in Figure 16 , the 5 μM DHUROS-5 buffer solution (0.1M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system has no fluorescence. But after adding 50 μM nitrite, DHUROS-5 has a strong fluorescence response, and the fluorescence intensity at 686 nm is increased by more than 2900 times.

[0107] As shown in Figure 17 , when 50 μM nitrite is added to the 5 μM DHUROS-5 buffer solution (0.1M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF), the fluorescence intensity at 686 nm in the system can be observed to be significantly enhanced over time.

[0108] Example 4

[0109] Preparation of the compound DHUROS-8 (R 11 is benzyl) with the structure of formula I and research on the response performance with nitrite.

[0110] The compound DHUROS-8 (R11 The synthetic route of compound DHUROS-8 is as follows:

[0111]

[0112] (1) Preparation of fluorescent probe DHUROS-8:

[0113] In a dry flask, compound A (0.50 g, 1.46 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, and then hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq) dissolved in 10 mL of dichloromethane was added dropwise. The reaction progress was monitored by TLC, and the reaction was completed at 0.5 h. After the reaction was completed, the whole reaction system was concentrated, washed with methanol, and then freeze-dried for 12 hours to obtain compound B.

[0114] Compound B (0.50 g, 1.46 mmol, 1 eq) after freeze-drying was re-dissolved in DCM. The reaction system was placed in an ice-water bath and stirred, and then the dissolved product B DCM solution was added dropwise to 10 mL of dichloromethane solution of benzylsulfonyl chloride (0.42 g, 2.19 mmol, 1.5 eq) under nitrogen protection. The whole reaction process was monitored by TLC until the reaction was completed.

[0115] The solvent was evaporated on a rotary evaporator, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to obtain 225 mg of pure white compound DHUROS-8, and the yield of the product was 31%.

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.58 (s, 1H), 7.49-7.47 (m, 2H), 7.40-7.34 (m, 5H), 6.73 (d, J = 2.8 Hz, 2H), 6.69 (dd, J = 8.8, 2.8 Hz, 2H), 4.38 (s, 2H), 2.90 (s, 12H).

[0117] HRMS (ESI) calc. for C 24 H 28 N5O3S2 + [M+H + ]:498.1628; found:498.1647

[0118] The fluorescent probe DHUROS-8 prepared in Example 4 of the present application has the following properties: 1 H NMR, HRMS spectra, as shown in Figures 7-8 .

[0119] (2) Response behavior test of the compound to strong acidic active oxygen

[0120] As shown in Figure 1, the 5 μM DHUROS-8 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system is non-fluorescent. But after adding 50 μM nitrite, DHUROS-8 has a strong fluorescence response, and the fluorescence intensity at 686 nm is increased by more than 3000 times. Figure 18 As shown in Figure 2, when 50 μM nitrite is added to the 5 μM DHUROS-8 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system, the fluorescence intensity at 686 nm in the system can be observed to be significantly enhanced over time.

[0121] Figure 19 As shown in Figure 3, when 50 μM nitrite is added to the 5 μM DHUROS-8 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system, the fluorescence intensity at 686 nm in the system can be observed to be significantly enhanced over time.

[0122] Example 5

[0123] Preparation of the compound DHUROS-11 (R 11 is dimethylamine) and response performance research with nitrite.

[0124] The synthesis route of the compound DHUROS-11 (R 11 is dimethylamine) is as follows:

[0125]

[0126] (1) Preparation of the fluorescent probe DHUROS-11

[0127] In a dry round-bottom flask, A (0.50 g, 1.46 mmol, 1 eq) was dissolved in 10 mL of dichloromethane, and then hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq) dissolved in 10 mL of dichloromethane was added dropwise. The reaction progress was monitored by TLC, and the reaction was completed at 0.5 h. After the reaction was completed, the whole reaction system was concentrated, washed with methanol, and then freeze-dried for 12 hours to obtain compound B.

[0128] Compound B (0.50 g, 1.46 mmol, 1 eq) after freeze-drying was re-dissolved in DCM. The reaction system was placed in an ice water bath and stirred, and then the dissolved product B DCM solution was added dropwise to 10 mL of dimethylamine sulfonyl chloride (0.31 g, 2.19 mmol, 1.5 eq) in dichloromethane under nitrogen protection, and the whole reaction process was monitored by TLC until the reaction was completed.

[0129] ​The solvent was removed by evaporation on a rotary evaporator and the residue was purified by column chromatography on silica gel (ethyl acetate: petroleum ether = 1 : 3) to give the pure white compound DHUROS-11 in a yield of 197 mg, 30% of the product.

[0130] 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.33 (s, 1H), 7.29 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 2.8 Hz, 2H), 6.68 (dd, J = 8.8, 2.8 Hz, 2H), 2.90 (s, 12H), 2.80 (s, 6H).

[0131] HRMS (ESI) calc. for C 19 H 27 N6O3S2 + [M+H + ]: 451.1581; found: 451.1581.

[0132] The fluorescent probe DHUROS-11 prepared in Example 5 of the present application has the following properties: 1 H NMR, HRMS spectra as shown in Figures 9-10 .

[0133] (2) Test of response behavior of the compound to strong acidic reactive oxygen

[0134] As shown in Figure 20 , the 5 mM DHUROS-11 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system has no fluorescence. But after adding 50 mM nitrite, DHUROS-11 has a strong fluorescence response, and the fluorescence intensity at 686 nm increases by more than 3200 times.

[0135] As shown in Figure 21 , when 50 mM nitrite is added to the 5 mM DHUROS-11 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF), a significant increase in fluorescence intensity at 686 nm in the system can be observed over time.

[0136] As shown in Figure 22 , when 0 mM, 10 mM, 20 mM, 30 mM, 40 mM and 50 mM nitrite are added to the 5 mM DHUROS-11 buffer solution (0.1 M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) respectively, the color of the solution gradually changes from colorless to blue and becomes more and more blue.

[0137] (3) Application of DHUROS-11 in live mice

[0138] like Figure 23 As shown, fluorescence imaging was performed on three groups of fasted mice (groups a, b, and c). Initially, no fluorescence was observed in the stomachs of any of the three groups of mice. Then, the mice were administered sodium nitrite solution (1.34 mg / mL, 100 μL, 2 mmol / L) via gavage. Ten minutes later, no fluorescence was observed in the stomachs of any of the three groups. Next, the mice were administered DHUROS-11 dissolved in DMF (2 mmol / L, 100 μL) via gavage. Five minutes later, fluorescence was observed in the stomachs of all three groups. Further fluorescence imaging at 15, 30, and 60 minutes revealed that the fluorescence in the stomachs of all three groups gradually increased over time. Simultaneously, fluorescence also gradually appeared in the bladder areas of all three groups. This indicates that DHUROS-11 can be used for in-situ detection of nitrite in the mouse stomach, and that the probe is well metabolized by the mice and does not remain in the body.

[0139] Example 6

[0140] Compound DHUROS-12(R) with the structure of formula I 11 Preparation of p-methylphenyl and its response performance with nitrite:

[0141] The compound DHUROS-12(R) 11 The synthetic route for p-methylphenyl is as follows:

[0142]

[0143] (1) Preparation of fluorescent probe DHUROS-12

[0144] In a dry, round-bottomed flask, Al (0.50 g, 1.29 mmol, 1 eq) was added and dissolved in 10 mL of dichloromethane. Then, hydrazine hydrate (0.37 g, 7.3 mmol, 5 eq), dissolved in 10 mL of dichloromethane, was added dropwise. The reaction was monitored by TLC and completed in 0.5 h. After the reaction was complete, the entire reaction system was concentrated, washed with methanol, and compound B1 was obtained, which was then freeze-dried for 12 h.

[0145] The compound B1 (0.50 g, 1.30 mmol, 1 eq) after freeze-drying was re-dissolved with DCM. The reaction system was placed in an ice-water bath condition and stirred, then under the protection of nitrogen, p-methylbenzenesulfonyl chloride (0.37 g, 1.96 mmol, 1.5 eq) dissolved in 10 ml dichloromethane was added dropwise into the system, the whole reaction process was monitored by TLC until the reaction was completed.

[0146] The solvent was evaporated on a rotary evaporator, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3), and finally the pure white compound DHUROS-12 was obtained with a yield of 305 mg, and the product yield was 44%.

[0147] 1 H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 1H), 8.67 (s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 6.36-6.33 (m, 2H), 6.29 (d, J = 2.0 Hz, 2H), 3.29 (q, J = 6.8 Hz, 8H), 2.39 (s, 3H), 1.06 (t, J = 6.8 Hz, 12H).

[0148] The fluorescence probe DHUROS-12 prepared in Example 6 of the present application has the following properties: 1 The H NMR spectrum is shown in Figure 11 .

[0149] (2) Test of response behavior of the compound to strong acidic active oxygen

[0150] As shown in Figure 24 , the 5 μM DHUROS-12 buffer solution (0.1M H3PO4-NaH2PO4, pH = 2.0, 0.5% DMF) system has no fluorescence. But after adding 50 μM nitrite, DHUROS-12 has a strong fluorescence response, and the fluorescence intensity at 686 nm is increased by more than 560 times.

[0151] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should still be covered by the claims of the present application.

Claims

1. A class of fluorescent probes for detecting nitrite under strong acid conditions, characterized in that, The fluorescent probe molecule has a structure shown in Formula I: ， (1) Z is S; (2) R2, R3, R8, R9 are methyl or ethyl; (3) R1, R4, R5, R6, R7, R 10 is H; (4) R 11 is phenyl, p-tolyl, dimethylamino, benzyl, thiophene ring, p-nitrophenyl.

2. A method for preparing a class of fluorescent probes for detecting nitrite under strong acid conditions according to claim 1, characterized in that, The synthesis route comprises the following steps: 。 3. The method of claim 2, wherein the method is characterized by, The specific preparation steps of the fluorescent probe compound I are as follows: S1, preparation of intermediate B: compound A is dissolved in an organic solvent, and hydrazine hydrate is added dropwise to obtain intermediate B; S2, preparation of fluorescent probe compound I: the reaction of compound B and sulfonyl chloride is carried out in the following two ways: ① Different types of sulfonyl chloride are dissolved in an organic solvent, and are added dropwise to the solution of compound B for reaction; ② Compound B is dissolved in an organic solvent, and is added dropwise to a solution of different types of sulfonyl chloride dissolved in an organic solvent, and compound I is obtained after reaction; the crude product is purified by silica gel column chromatography to obtain compound I.

4. The method of claim 3, wherein the method is characterized by, In step S1, the molar ratio of hydrazine hydrate to compound A is 3-10:1; the reaction temperature is 20-80℃; in step S2, the molar ratio of sulfonyl chloride to compound B is 1.2-5:1; the reaction temperature is -10-20℃; the organic solvent in steps S1 and S2 is an aprotic solvent.

5. Use of the fluorescent probe according to claim 1 for detecting nitrite under strong acid conditions for non-disease diagnosis and treatment purposes.

6. Use according to claim 5, characterized in that, The pH of the strong acid condition is less than 3; the reaction of the fluorescent probe with nitrite has the following response mechanism: ， The compound with the structure of Formula I itself has no fluorescence, and after responding to nitrite, intermediate C is first generated, and then further oxidized by strong acid active oxygen to generate compound D with strong fluorescence emission.

7. Use of the fluorescent probe according to claim 1 for preparing a detection reagent for detecting nitrite in a solution or in vivo for non-disease diagnosis and treatment purposes.

8. A method for detecting nitrite in a solution or a living body for a purpose other than disease diagnosis and treatment, characterized by, The detection method is as follows: Dissolve the compound of Formula I according to claim 1 in an organic solvent, and then dilute it into a buffer solvent containing the to-be-detected substance, for detection of nitrite under strong acid conditions; Dissolve the compound of Formula I according to claim 1 in an organic solvent, and then administer it to an animal in vivo model for detection of nitrite under strong acid conditions.

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