Aza-hemicyanine dye and application thereof

Abstract

The invention discloses an aza-hemicyanine dye and application thereof, and compared with a dye with similar functions, the novel aza-hemicyanine dye molecule provided by the invention can effectively prevent non-specific binding of serum albumin and the hemicyanine dye; the stability, the fluorescence quantum yield and the Stokes shift of the hemicyanine dye are enhanced; the dye provided by the invention is simple in synthesis step, convenient to purify and relatively high in yield; a six-membered heterocyclic ring is introduced into a side chain of the aza-hemicyanine dye, so that the binding with protein is effectively avoided, the false positive number interference of serum albumin on a dye fluorescence signal is eliminated, meanwhile, the kidney targeting is realized, and the high fidelity of imaging is improved; the probe NA-H2S2 synthesized on the basis of the dye NA-6, provided by the invention, is used for rapidly detecting the change of the concentration of H2S2; the dye molecule is simple and convenient in synthesis step, easy to purify and excellent in imaging performance, a fluorescence signal is not interfered by serum albumin, and the dye molecule is suitable for high-fidelity imaging of kidneys.

Description

Technical Field

[0001] The invention relates to the technical field of near-infrared organic small molecule dyes, and in particular to a class of aza-hemicyanine dyes and applications thereof. Background Art

[0002] Near-infrared fluorescent probes have important application value in medical imaging, especially in the field of kidney imaging. They make in vivo imaging possible by emitting fluorescence in the near-infrared (NIR) range. The advantage of near-infrared fluorescent probes is that they have strong tissue penetration ability and can effectively reduce tissue absorption and scattering compared to visible light or ultraviolet light, thereby improving signal transmission efficiency. However, there are still some technical challenges in kidney imaging.

[0003] On the one hand, in the in vivo environment, fluorescent probes often bind to rich biomacromolecules such as serum albumin. This binding not only leads to an enhancement of the fluorescent signal, but also may mask the detection of specific signals. The kidney is a highly vascularized organ, and serum albumin occupies an important position in the blood in the body. Therefore, when imaging, its interference signal often significantly affects the clarity and accuracy of the final imaging.

[0004] On the other hand, the types of kidney-targeted molecular probes are relatively limited. Although there are some fluorescent probes targeting the kidney on the market, their targeting and selectivity still need to be further improved. This limits the accurate imaging and early diagnosis of different types of kidney diseases.

[0005] Therefore, in kidney imaging, how to prepare probes that can image without interference from serum albumin is a key research issue at present. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a class of aza-hemicyanine dyes and applications thereof to solve the problem of interference by albumin in the current high-fidelity imaging of the kidney.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] The present invention provides a class of aza-hemicyanine dyes, wherein the dyes include the following structural formula:

[0009]

[0010] The present invention also provides a method for synthesizing the above-mentioned aza-hemicyanine dye, comprising the following synthesis steps:

[0011]

[0012] S1. Synthesis of intermediate 1: Compound 1 was dissolved in a mixed organic solvent of n-butanol and toluene, followed by addition of compound 3, reflux reaction, cooling, and concentrating the organic phase to obtain a crude product which was further purified by a silica gel chromatography column to obtain intermediate 1;

[0013] S2. Synthesis of intermediate 2: Compound 2 was dissolved in a mixed organic solvent of n-butanol and toluene, followed by addition of compound 3, reflux reaction, cooling, and concentrating the organic phase to obtain a crude product which was further purified by a silica gel chromatography column to obtain intermediate 2;

[0014] S3. Synthesis of compounds 1a-1 to 3: The intermediate 1a was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The mixture was refluxed and cooled. The organic phase was concentrated to obtain a crude product which was further purified by silica gel chromatography to obtain compounds 1a-1 to 3:

[0015] S4. Synthesis of compounds 1b-1 to 3: The intermediate 1b was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The reaction was refluxed, cooled, and the organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compounds 1b-1 to 3:

[0016] S5. Synthesis of compounds 1c-1 to 3: The intermediate 1c was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The mixture was refluxed and cooled. The organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compounds 1c-1 to 3.

[0017] S6. Synthesis of compounds 2a-1 to 3: Dissolve intermediate 2 in N,N-dimethylformamide, then add compound 4, and finally add potassium carbonate, reflux reaction, cool, concentrate the organic phase, and obtain a crude product, which is further purified by silica gel chromatography to obtain compounds 2a-1 to 3;

[0018] S7, synthesis of dyes NA-1~5, NA-7~11, NA-13~17, NA-19~23: dissolve compound 1a-2~3\1b-2~3\1c-2~3\2a-2~3 in ultra-dry acetonitrile, then add compound 5, and finally add potassium carbonate and potassium iodide, reflux reaction, cool, concentrate the organic phase, and further purify the crude product with a silica gel chromatography column to obtain dyes NA-1~5, NA-7~11, NA-13~17, NA-19~23;

[0019] S8. Synthesis of compound 3NA-6\12\18\24: Compound 1a-1\1b-1\1c-1\2a-1 was dissolved in ultra-dry acetonitrile, followed by addition of compound 5, and finally potassium carbonate and potassium iodide. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compound 3NA-6\12\18\24;

[0020] S9. Synthesis of dye NA-6\12\18\24: Dissolve compound 3NA-6\12\18\24 in ultra-dry dichloromethane, add boron tribromide under ice bath conditions, react for a period of time, cool, concentrate the organic phase, and further purify the crude product using a silica gel chromatography column to obtain dye NA-6\12\18\24.

[0021] The intermediate 1 includes intermediate 1a, intermediate 1b and intermediate 1c.

[0022] Preferably, in steps S1 and S2, the mass ratio of n-butanol to toluene in the mixed organic solvent is (6-8): (2-4);

[0023] The temperature of the reflux reaction is 105-115°C, and the time of the reflux reaction is 9-11h;

[0024] The molar ratio of compound 1 to compound 3 is (1-3):1;

[0025] The molar ratio of compound 2 to compound 3 is (1-3):1.

[0026] Preferably, in steps S3 to S6, the temperature of the reflux reaction is 75 to 85° C., and the time of the reflux reaction is 5 to 7 hours;

[0027] The molar ratio of the intermediate 1a to the compound 4 is (1-3):1;

[0028] The molar ratio of the intermediate 1b to the compound 4 is (1-3):1;

[0029] The molar ratio of the intermediate 1c to the compound 4 is (1-3):1;

[0030] The molar ratio of the intermediate 2 to the compound 4 is (1-3):1.

[0031] Preferably, in steps S7 to S8, the temperature of the reflux reaction is 105 to 115° C., and the time of the reflux reaction is 11 to 13 hours;

[0032] The molar ratio of the compounds 1a-2~3\1b-2~3\1c-2~3\2a-2~3 and compound 5 is 1:(1~3);

[0033] The molar ratio of the compound 1a-1\1b-1\1c-1\2a-1 and the compound 5 is 1:(1~3).

[0034] Preferably, in step S9, the reaction temperature is 35-45°C, and the reaction time is 3-5h;

[0035] The molar ratio of the compound 3NA-6\12\18\24 to boron tribromide is 1:(1-2).

[0036] The present invention also proposes an application of the above-mentioned azahemicyanine dye in an azahemicyanine NA-H2S2 probe, wherein the probe can detect H2S2.

[0037] Preferably, the synthesis method of the aza-hemicyanine NA-H2S2 fluorescent probe comprises the following steps:

[0038]

[0039] The dye NA-6 was dissolved in N,N-dimethylformamide, and then compound 6 and potassium carbonate were added and reacted for 0.5 to 1.5 hours. The organic phase was concentrated and the obtained product was further purified by silica gel chromatography to obtain the semicyanine NA-H2S2 fluorescent probe.

[0040] Preferably, the method for detecting H2S2 comprises the following steps: dissolving the probe NA-H2S2 in DMSO to prepare a 5 mM stock solution, and then adding the NA-H2S2 stock solution to the test solution; after adding H2S2, observing the fluorescence spectrum changes of the test solution containing NA-H2S2 using a fluorescence spectrometer.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The present invention provides a novel type of aza-hemicyanine dye molecule. Compared with dyes with similar functions, the present invention can effectively prevent the nonspecific binding of serum albumin to the hemicyanine dye and enhance the stability, fluorescence quantum yield and Stoke's shift of the hemicyanine dye.

[0043] (2) The dye provided by the present invention has simple synthesis steps, convenient purification, and high yield; by introducing a six-membered heterocycle into the side chain of the aza-hemicyanine dye, the binding with protein is effectively avoided, and the false positive interference of serum albumin on the dye fluorescence signal is eliminated, while kidney targeting is achieved, thereby improving the high fidelity of imaging.

[0044] (3) The probe molecule provided by the present invention can quantitatively detect and evaluate the concentration changes of H2S2 during renal injury. The probe has a highly sensitive, rapid and selective response to hydrogen disulfide (H2S2) in the kidney, with a detection limit as low as 24.21 nM. Through in vivo fluorescence detection and in vitro urine detection, its precise imaging can be achieved, providing important diagnostic value for renal ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The dye NA-6 of Example 1 1 H NMR spectrum;

[0046] Figure 2 The dye NA-6 of Example 1 13 C NMR spectrum;

[0047] Figure 3 The ultraviolet absorption spectrum and fluorescence emission spectrum of the dye NA-6 in Example 2 in a solvent;

[0048] Figure 4 This is a graph showing the change in fluorescence emission spectrum of the dye NA-6 in Example 3 before and after the response in different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol;

[0049] Figure 5 This is a diagram showing the renal targeting and metabolism of the dye NA 1-6 in Example 4 in mice;

[0050] Figure 6 This is a graph showing the results of the stability fluorescence test of the dye NA-6 of Example 5;

[0051] Figure 7 This is a diagram showing the change in the fluorescence emission spectrum of the probe NA-H2S2 of Example 7 in response to the titration of H2S2;

[0052] Figure 8 This is a fluorescence test result diagram of the probe NA-H2S2 of Example 8 in response to different analytes;

[0053] Fig. 9 The probe NA-H2S2 of Example 9 performs fluorescence imaging of endogenous H2S2 and exogenous H2S2 in HK-2 cells, and imaging of renal ferroptosis cells induced by estin and cisplatin;

[0054] Fig.10 These are near-infrared fluorescence images of the body, kidneys and urine of mice with renal ferroptosis after the probe NA-H2S2 of Example 10 was injected into the tail vein. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0056] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0057] Example 1

[0058] The structural formula of dye NA-6 is as follows:

[0059]

[0060] The synthesis method of dye NA-6 comprises the following synthesis steps:

[0061] (1) 36 mg of compound 1 was dissolved in a mixed organic solvent of n-butanol and toluene in a mass ratio of 7:3, and then 18 mg of compound 3 was added. The mixture was refluxed at 110° C. for 10 hours. After cooling to room temperature, the organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain intermediate 1.

[0062] (2) 33 mg of intermediate 1 was dissolved in 20 mL of N,N-dimethylformamide, followed by the addition of 66 mg of compound 4 and finally 10 mg of potassium carbonate. The mixture was refluxed at 80° C. for 6 hours. After cooling to room temperature, the organic phase was concentrated to obtain a crude product which was further purified by silica gel chromatography to obtain compound 1a-1.

[0063] (3) 28 mg of compound 1a-1 was dissolved in ultra-dry acetonitrile, followed by the addition of 70 mg of compound 5, and finally potassium carbonate and potassium iodide. The mixture was refluxed at 110° C. for 12 hours. After cooling to room temperature, the organic phase was concentrated to obtain a crude product which was further purified by silica gel chromatography to obtain compound 3NA-6.

[0064] (4) 65.1 mg of compound 3NA-6 was dissolved in 5 mL of ultra-dry dichloromethane, and 2 ml of boron tribromide was added under ice bath conditions, followed by stirring at 40°C for 4 hours under nitrogen protection. After the reaction was completed, the organic phase was concentrated to obtain a crude product. The product was purified by chromatography on a silica gel (200-300 mesh) column using dichloromethane and methanol in a volume ratio of 50:1 as the eluent to obtain 64.1 mg of a green solid (yield: 93.8%). Its molecular structure was identified as follows:

[0065] Dye NA-6 1 H NMR spectrum Figure 1 shown. 1H NMR(600MHz, Methanol-d4)δ7.99(s,1H),7.77(d,J=8.8Hz,1H),7.39(d,J=2.3Hz,1H),7.29(s,1H), 7.14(d,J=2.0Hz,1H),7.12(d,J=2.0Hz,1H),7.04(d,J=2.5Hz,1H),7.02(d,J=2.4Hz,1H),6.98(d,J= 7.5Hz,1H),6.95(s,1H),3.90(q,J=7.1Hz,2H),3.53(d,J=8.2Hz,2H),2.75(t,J=5.9Hz,2H),2.17(t, J=7.5Hz,2H),1.95–1.93(m,2H),1.53–1.49(m,7H),1.26(s,6H),1.03(d,J=4.2Hz,2H),0.76(s,3H).

[0066] Dye NA-6 13 C NMR Figure 2 shown. 13 C NMR(151MHz,Methanol-d4)δ143.10,142.20,140.10,132.35,129.37,125.43,124.64,123.75,1 14.63,104.27,67.89,57.77,35.89,35.30,34.85,33.03,31.88,30.43,22.91.HRMS(ESI)Calcd for C 33 H 40 N3O2 + ([M] + ):510.3115, found,510.3138.

[0067] This indicates that the structure of the obtained target compound is consistent with the structure shown in the above structural formula.

[0068] The synthesis steps of other dyes NA-1 to 5 are different from those of NA-6 in that the selection of raw material compound 1 and intermediate 1 is different, and the other synthesis steps are the same.

[0069] Example 2

[0070] The ultraviolet absorption spectrum and fluorescence emission spectrum of the dye NA-6 prepared in Example 1 were tested.

[0071] The test method is as follows: prepare the DMSO stock solution of the dye NA-6 dye at a concentration of 5mM. Then add 1996μL of different solutions (1,4-dioxane, ACN, DCM, DMF, DMSO, MeOH, PBS) and 4μL of the stock solution to the UV and fluorescent dishes respectively, and test their UV absorption and fluorescence emission. The results are as follows Figure 3 shown.

[0072] Example 3

[0073] The fluorescence emission spectrum of the dye NA-6 prepared in Example 1 was tested to change before and after the response to different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol, verifying that the fluorescence emission spectrum of the dye NA-6 dye was not interfered by serum albumin.

[0074] The test method is as follows: prepare the DMSO stock solution of the dye at a concentration of 5mM. Then add 1996μL PBS and 4μL stock solution to the fluorescent dish, then add different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol, and test its fluorescence emission. The results are as follows: Figure 4 As shown (wherein, Figure (a) and Figure (b) are fluorescence emission diagrams of dye NA-6 for serum proteins of different concentrations; Figure (c) is the fluorescence emission diagram of dye NA-6 for hemoglobin of different concentrations; Figure (d) is the fluorescence emission diagram of dye NA-6 for polyvinyl alcohol of different concentrations; Figure (e) is the fluorescence emission diagram of dye NA-6 for glycerol of different concentrations).

[0075] Example 4

[0076] The dye NA 1-6 prepared in Example 1 was used to study kidney targeting and its metabolism in mice.

[0077] Kidney targeting and metabolism of dye NA 1-6 in mice Figure 5As shown in the figure (wherein, Figure (A) is a mouse intravenous injection diagram; Figure (B) is a structural diagram of dyes NA-1 to 6; Figure (C) is a metabolic diagram of mice after intravenous injection; Figure (D) is a fluorescence diagram of mouse urine injected with NA-6 at 0 minutes and 90 minutes; Figure (E) is a diagram of mouse organs injected with NA-6 at 90 minutes after dissection; Figure (F) is a quantitative diagram of the fluorescence intensity in the body of NA-1 to 5 60 minutes after tail vein injection; Figure (G) is a quantitative diagram of the fluorescence intensity in the body of mice injected with NA-6 at different times; Figure (H) is a quantitative diagram of the fluorescence intensity in different organs), after tail vein injection of NA 1-6, we were pleasantly surprised to find that this type of N-heterocyclic nitrogen semi-cyanine dye can target the kidney. Taking NA-6 as an example, within 1.5 hours after injection, NA-6 was rapidly metabolized by the kidney, and the fluorescence intensity in the body reached the highest at 60 minutes. After 90 minutes, part of the dye was excreted from the body with urine. This suggests that compared to dyes that may take days or even months to be metabolized, NA-6 exhibits significant biocompatibility, reducing the likelihood of accumulation in the body, thereby reducing the potential toxic effects and side effects of NA-6 and providing a basis for high-fidelity imaging of the kidneys as well as urine analysis.

[0078] Example 5

[0079] Fluorescence stability test of dye NA-6.

[0080] The test method is as follows: prepare the DMSO stock solution of the dye at a concentration of 5mM. Then add 1996μL of liquid (PBS: methanol = 7:3) and 4μL of stock solution to the UV and fluorescent dishes respectively, and then add redox agents of different concentrations to test their UV absorption spectrum and fluorescence emission spectrum. The results are as follows: Figure 6 shown.

[0081] Example 6

[0082] The probe NA-H2S2 was prepared using the dye NA-6 prepared in Example 1 as a raw material.

[0083] The NA-H2S2 fluorescent probe responding to H2S2 has the following structural formula:

[0084]

[0085] The specific synthesis steps are as follows: 63.7 mg of dye NA-6 (0.1 mmol) and 18.6 mg of 2,4-dinitrofluorobenzene (0.1 mmol) are placed in a round-bottom flask, and 5 mL of N,N-dimethylformamide is added to completely dissolve them, followed by adding 8.3 mg of potassium carbonate (0.06 mmol), stirring for 1 hour under nitrogen protection and room temperature, and after the reaction is completed, the organic phase is concentrated to obtain a crude product. Using dichloromethane and methanol in a volume ratio of 50:1 as the eluent, purify with a silica gel (200-300 mesh) chromatography column to obtain 67.1 mg of a green solid (yield: 83.5%).

[0086] Probe 1 H NMR spectrum Figure 1 . 1 H NMR (600MHz, DMSO-d6) δ9.00(s,1H),8.56(s,1H),8.24(d,J=6.1Hz,2H),8.14(d,J=8.8Hz,1H),7.57( t,J=12.1Hz,2H),7.50(d,J=8.6Hz,1H),7.44(d,J=9.2Hz,1H),7.33(t,J=7.1Hz,1H),7.23(d,J=8.0Hz ,1H),7.11(t,J=7.4Hz,1H),5.18(s,2H),4.07(q,J=14.1,10.6Hz,5H),2.79(s,2H),2.05–1.96(m,4H) ,1.46(d,J=11.5Hz,2H),1.36–1.25(m,8H),1.23(s,6H),1.04(d,J=7.8Hz,2H),0.85(t,J=6.9Hz,3H).

[0087] Probe 13 C NMR Figure 2 . 13 C NMR(151MHz,DMSO-d6)δ153.35,141.77,141.55,140.12,135.30,134.45,129.05,127.66,123.50,122.48, 121.54,120.16,118.61,108.90,47.07,34.51,29.54,28.48,26.38,25.95,24.51,20.34.HRMS(ESI)Calcd for C 36 H 47 N4 + ([M] + ):676.3139,found,676.3162.

[0088] Example 7

[0089] Fluorescence emission spectra of the test probe NA-H2S2 before and after responding to H2S2.

[0090] The test method is as follows: Prepare the DMSO mother solution of probe NA-H2S2 at a concentration of 5mM. Then add 1996μL liquid (PBS:DMSO=9:1) and 4μL mother solution to the fluorescent dish and test its fluorescence emission spectrum. Then, add 1-90μg / ml H2S2 to the dish, and the fluorescence intensity in the fluorescence spectrum is enhanced by 15 times. The results are as follows: Figure 7 shown.

[0091] Example 8

[0092] The fluorescence signal changes of the test probe NA-H2S2 under different analytes.

[0093] The test method is as follows: prepare the DMSO mother solution of probe NA-H2S2 at a concentration of 5mM. Then add 1996μL liquid (PBS:DMSO=9:1) and 4μL mother solution to the fluorescent dish, and then add different amino acids and aqueous solutions of common ions in organisms, including H2S, BSA, HAS, HB, Cys, GSH, Hcy, H2S4, SO3 2- 、S2O8 2- 、S2O3 2- , HSO 4- , Pro, Gly, Glu and other biological reagents and metal ions and other compounds. Record the changes in fluorescence signals and draw a bar graph of adding different analytes, see Figure 8 The experiment proved that other amino acids and common ions in organisms do not interfere with the detection of H2S2 by the system. That is, the probe can specifically respond to H2S2.

[0094] Example 9

[0095] The probe NA-H2S2 was used for fluorescence imaging of endogenous and exogenous H2S2 in HK-2 cells, as well as for imaging of renal ferroptosis induced by estin and cisplatin.

[0096] To demonstrate that the probe can recognize endogenous H2S2 in cells, we incubated cells with LPS for 12 h and then added the probe to the cells for direct time monitoring imaging. Fig. 9As shown in A (wherein, Figure (A) is a confocal fluorescence imaging of endogenous H2S2 in HK-2 cells at different time points), as the probe is incubated in the cell for a longer time, the red fluorescence gradually increases, and the fluorescence remains almost unchanged after 30 minutes, indicating that the probe responds to endogenous H2S2 and reacts almost completely with endogenous H2S2 in the cell in about 30 minutes. Therefore, the probe can be used for accurate monitoring of intracellular H2S2. Next, in order to investigate the ability of the probe NA-H2S2 to detect changes in H2S2 concentration in cells, we performed an exogenous H2S2 experiment on the cells. After the cells in the first group were treated with NEM, the probe was added half an hour after the addition of exogenous 100 μM H2S2, and the probe was added half an hour after the cells in the second group were treated with NEM, and the probe was added half an hour after the addition of exogenous 50 μM H2S2, and the probe was added to the cells in the third group only for incubation, and in the fourth group, N-ethylmaleimide (NEM, a thiol digestion reagent) was added to the cells in advance for 30 minutes to eliminate the H2S2 produced in the cells, and then the probe was added. All four groups of cells were observed through the red channel. Fig. 9 As shown in Figure B (Figure (B) is a confocal fluorescence image of HK-2 cells after adding different concentrations of exogenous H2S2), red fluorescence is almost invisible in cells treated with NEM. After adding 100μM or 50μM exogenous H2S2 to the cells, we found that the red fluorescence of the probe was significantly enhanced by 3.37 times, indicating that the probe can specifically recognize H2S2 in cells.

[0097] At the same time, in order to investigate the ability of the probe NA-H2S2 to detect H2S2 during renal cell ferroptosis, we conducted the following experiment. Normal HK-2 cells were divided into four groups. The first group of cells were induced to ferroptosis with 10 μM Erastin for 8 hours, and then the probe was added for 30 minutes. The second group of cells were induced to ferroptosis with 10 μM Erastin for 6 hours, and then the probe was added for 30 minutes. The third group of cells were incubated with the probe only for 30 minutes. The fourth group of cells were inhibited from ferroptosis with 5 μM ferroptosis inhibitor Fer-1 for 8 hours, and then the probe was added for 30 minutes. Each group of cells was washed three times with PBS before imaging, and all were observed using the red channel: the excitation wavelength was 638 nm, and the emission wavelength collection range was 650-780 nm. Fig. 9 As shown in C, after HK-2 was induced with Erastin for 8 h, its fluorescence intensity increased by 9.66 times compared with the inhibition group, indicating that this probe can be used for accurate monitoring of H2S2 during intracellular ferroptosis. Fig. 9 As shown in D to E (wherein, Figure (D) is the confocal fluorescence imaging of cells induced by cisplatin at different times; Figure (E) is the confocal fluorescence imaging of ferroptosis of cells 24 hours after cisplatin induction), the cells were treated with cisplatin and the same results were obtained.

[0098] Example 10

[0099] The probe NA-H2S2 was used to image H2S2 during ferroptosis in mouse kidney.

[0100] To further clarify the changes of H2S2 during ferroptosis in vivo, we established a mouse renal ferroptosis model. We injected Erastin solution into the mouse peritoneum at a dose of 25 mg / kg to induce ferroptosis in the kidneys. The injection was repeated every 12 hours for 5 days. 100 μL of 1 mM probe was injected through the tail vein, followed by fluorescence imaging. Fig.10 As shown (wherein, Figure (A) is a schematic diagram of the imaging experimental design and urine analysis of H2S2 levels in mouse kidneys after Erastin modeling; Figure (B) is a fluorescence imaging of mice after tail vein injection of probes from 0 to 90 minutes in Erastin, normal, NEM and Fer-1 group mice; Figure (C) is a fluorescence imaging of kidneys after dissection 90 minutes after probe injection in mice; Figure (D) is a quantification of fluorescence intensity in mice and kidneys; Figure (E) is a fluorescence imaging of mouse urine co-incubated with probes at different times; Figure (F) is a quantification of GSH (glutathione) and MDA (malondialdehyde) levels in kidneys of ferroptotic mice), compared with normal mice or control group mice, the fluorescence in the kidneys of ferroptotic mice was significantly increased compared with normal mice, indicating that the H2S2 concentration in the kidneys of ferroptotic mice has an upward trend. Subsequently, by stripping the kidneys of mice for imaging, we found that the fluorescence in the kidneys of the Erastin-induced group was significantly enhanced, about 30 times that of the NEM group. Moreover, as the injection time of Einstein increased, the fluorescence intensity of the urine excreted by mice increased significantly after being incubated with the probe for 30 minutes, further proving that the probe has good application potential.

[0101] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. A type of aza-hemicyanine dye, characterized in that: The dye comprises the following structural formula:

2. The method for synthesizing an aza-hemicyanine dye according to claim 1, characterized in that: The synthesis steps include: S1. Synthesis of intermediate 1: Compound 1 was dissolved in a mixed organic solvent of n-butanol and toluene, followed by addition of compound 3, reflux reaction, cooling, and concentrating the organic phase to obtain a crude product which was further purified by a silica gel chromatography column to obtain intermediate 1; S2. Synthesis of intermediate 2: Compound 2 was dissolved in a mixed organic solvent of n-butanol and toluene, followed by addition of compound 3, reflux reaction, cooling, and concentrating the organic phase to obtain a crude product which was further purified by a silica gel chromatography column to obtain intermediate 2; S3. Synthesis of compounds 1a-1 to 3: The intermediate 1a was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The mixture was refluxed and cooled. The organic phase was concentrated to obtain a crude product which was further purified by silica gel chromatography to obtain compounds 1a-1 to 3: S4. Synthesis of compounds 1b-1 to 3: The intermediate 1b was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The reaction was refluxed, cooled, and the organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compounds 1b-1 to 3: S5. Synthesis of compounds 1c-1 to 3: The intermediate 1c was dissolved in N,N-dimethylformamide, followed by the addition of compound 4 and finally potassium carbonate. The mixture was refluxed and cooled. The organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compounds 1c-1 to 3. S6. Synthesis of compounds 2a-1 to 3: Dissolve intermediate 2 in N,N-dimethylformamide, then add compound 4, and finally add potassium carbonate, reflux reaction, cool, concentrate the organic phase, and obtain a crude product, which is further purified by silica gel chromatography to obtain compounds 2a-1 to 3; S7, synthesis of dyes NA-1~5, NA-7~11, NA-13~17, NA-19~23: dissolve compound 1a-2~3\1b-2~3\1c-2~3\2a-2~3 in ultra-dry acetonitrile, then add compound 5, and finally add potassium carbonate and potassium iodide, reflux reaction, cool, concentrate the organic phase, and further purify the crude product with a silica gel chromatography column to obtain dyes NA-1~5, NA-7~11, NA-13~17, NA-19~23; S8. Synthesis of compound 3NA-6\12\18\24: Compound 1a-1\1b-1\1c-1\2a-1 was dissolved in ultra-dry acetonitrile, followed by addition of compound 5, and finally potassium carbonate and potassium iodide. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain a crude product which was further purified by a silica gel chromatography column to obtain compound 3NA-6\12\18\24; S9. Synthesis of dye NA-6\12\18\24: Dissolve compound 3NA-6\12\18\24 in ultra-dry dichloromethane, add boron tribromide under ice bath conditions, react for a period of time, cool, concentrate the organic phase, and further purify the crude product using a silica gel chromatography column to obtain dye NA-6\12\18\24.

3. The method for synthesizing an azahemicyanine dye according to claim 1, characterized in that: In steps S1 and S2, the mass ratio of n-butanol to toluene in the mixed organic solvent is (6-8):(2-4); The temperature of the reflux reaction is 105-115°C, and the time of the reflux reaction is 9-11h; The molar ratio of compound 1 to compound 3 is (1-3):1; The molar ratio of compound 2 to compound 3 is (1-3):

1.

4. The method for synthesizing an aza-hemicyanine dye according to claim 1, characterized in that: In steps S3 to S6, the temperature of the reflux reaction is 75 to 85° C., and the time of the reflux reaction is 5 to 7 hours; The molar ratio of the intermediate 1a to the compound 4 is (1-3):1; The molar ratio of the intermediate 1b to the compound 4 is (1-3):1; The molar ratio of the intermediate 1c to the compound 4 is (1-3):1; The molar ratio of the intermediate 2 to the compound 4 is (1-3):

1.

5. The method for synthesizing an aza-hemicyanine dye according to claim 1, characterized in that: In steps S7 to S8, the temperature of the reflux reaction is 105 to 115° C., and the time of the reflux reaction is 11 to 13 hours; The molar ratio of the compounds 1a-2~3\1b-2~3\1c-2~3\2a-2~3 and compound 5 is 1:(1~3); The molar ratio of the compound 1a-1\1b-1\1c-1\2a-1 and the compound 5 is 1:(1~3).

6. The method for synthesizing an aza-hemicyanine dye according to claim 1, characterized in that: In step S9, the reaction temperature is 35-45°C, and the reaction time is 3-5h; The molar ratio of the compound 3NA-6\12\18\24 to boron tribromide is 1:(1-2).

7. Use of the azahemicyanine dye according to claim 1, characterized in that: The application is the use of aza-hemicyanine dyes in detecting fluorescence signals for purposes other than disease diagnosis and treatment.

8. Use of the azahemicyanine dye according to claim 1, characterized in that: The invention is applied to an aza-hemicyanine NA-H2S2 probe, and the probe can detect H2S2.

9. The use of the azahemicyanine dye according to claim 8, characterized in that: The synthesis method of the aza-hemicyanine NA-H2S2 fluorescent probe comprises the following steps: The dye NA-6 was dissolved in N,N-dimethylformamide, and then compound 6 and potassium carbonate were added and reacted for 0.5 to 1.5 hours. The organic phase was concentrated and the obtained product was further purified by silica gel chromatography to obtain the semicyanine NA-H2S2 fluorescent probe.

10. The use of the azahemicyanine dye according to claim 8, characterized in that: The method for detecting H2S2 comprises the following steps: dissolving the probe NA-H2S2 in DMSO to prepare a 5 mM mother solution, and then adding the NA-H2S2 mother solution to a test solution; after adding H2S2, observing the fluorescence spectrum change of the test solution containing NA-H2S2 using a fluorescence spectrometer.

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