Aza-semicorin dyes and their use

Abstract

The application discloses a kind of azahemicyanine dyes and application thereof, compared with the dye similar to the function of novel azahemicyanine dye molecule provided by the application, the application can effectively prevent the non-specific combination of serum albumin and hemicyanine dye, and the stability, fluorescence quantum yield and stoke shift of the hemicyanine dye are enhanced;The dye synthesis step provided by the application is simple, easy to purify, and has high yield;By introducing six-membered heterocyclic ring into the side chain of azahemicyanine dye, the combination with protein is effectively avoided, the false positive interference of serum albumin on dye fluorescence signal is excluded, and kidney targeting is realized, and the high fidelity of imaging is improved;The probe NA-H2S2 synthesized based on the dye NA-6 provided by the application is used for rapid detection of the change of H2S2 concentration;The synthesis step of the dye molecule is simple, easy to purify, has excellent imaging performance, fluorescence signal is not interfered by serum albumin, and is suitable for high-fidelity imaging of kidney.

Description

Technical Field

[0001] This invention relates to the field of near-infrared organic small molecule dye technology, specifically to a class of azahexacyanine dyes and their applications. Background Technology

[0002] Near-infrared fluorescent probes have significant applications in medical imaging, particularly in kidney imaging. They enable in vivo imaging by emitting fluorescence in the near-infrared (NIR) range. The advantage of NIR fluorescent probes lies in their strong tissue penetration; compared to visible or ultraviolet light, they effectively reduce tissue absorption and scattering, thereby improving signal transmission efficiency. However, some technical challenges remain in kidney imaging.

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

[0004] On the other hand, the variety of kidney-targeting molecular probes is relatively limited. Currently, although some fluorescent probes targeting the kidneys are available on the market, their targeting and selectivity still need further improvement. This limits accurate imaging and early diagnosis of different types of kidney diseases.

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

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a class of azahexacyanine dyes and their applications, thereby solving the problem of albumin interference in high-fidelity imaging of the kidney.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention proposes a class of azahexacyanine dyes, wherein the dyes comprise the following structural formula:

[0009]

[0010] The present invention also proposes a method for synthesizing the azahexacyanine dyes as described above, comprising the following synthesis steps:

[0011]

[0012] Synthesis of S1 and intermediate 1: Compound 1 was dissolved in a mixed organic solvent of n-butanol and toluene, then compound 3 was added, the mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain intermediate 1.

[0013] Synthesis of S2 and intermediate 2: Compound 2 was dissolved in a mixed organic solvent of n-butanol and toluene, then compound 3 was added, the mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain intermediate 2.

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

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

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

[0017] S6. Synthesis of compounds 2a-1 to 3: Intermediate 2 was dissolved in N,N-dimethylformamide, then compound 4 was added, and finally potassium carbonate was added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compounds 2a-1 to 3.

[0018] Synthesis of dyes NA-1~5, NA-7~11, NA-13~17, and NA-19~23: Compounds 1a-2~3, 1b-2~3, 1c-2~3, and 2a-2~3 were dissolved in ultra-dry acetonitrile, followed by the 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. The crude product was further purified by silica gel chromatography to obtain dyes NA-1~5, NA-7~11, NA-13~17, and NA-19~23.

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

[0020] S9. Synthesis of dye NA-6\12\18\24: Compound 3NA-6\12\18\24 was dissolved in ultra-dry dichloromethane, boron tribromide was added under ice bath conditions, the reaction was carried out for a period of time, cooled, and the organic phase was concentrated to obtain crude product. The crude product was further purified by silica gel chromatography 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 reflux reaction temperature is 105–115°C, and the reflux reaction time is 9–11 hours.

[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 intermediate 1a to compound 4 is (1-3):1;

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

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

[0030] The molar ratio of intermediate 2 to 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 compound 1a-2~3\1b-2~3\1c-2~3\2a-2~3 to compound 5 is 1:(1~3);

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

[0034] Preferably, in step S9, the reaction temperature is 35–45°C, and the reaction time is 3–5 hours.

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

[0036] The present invention also proposes an application of the azahesine dye as described above, applied to azahesine NA-H2S2 probe, wherein the probe is capable of detecting H2S2.

[0037] Preferably, the method for synthesizing the azahexacyanin NA-H2S2 fluorescent probe includes the following steps:

[0038]

[0039] The dye NA-6 was dissolved in N,N-dimethylformamide, followed by the addition of compound 6 and potassium carbonate. The reaction was carried out for 0.5–1.5 hours, the organic phase was concentrated, and the product was further purified by silica gel chromatography to obtain the hemicyanine NA-H2S2 fluorescent probe.

[0040] Preferably, the method for detecting H2S2 includes the following steps: dissolving the probe NA-H2S2 in DMSO to prepare a 5mM stock solution, and then adding the NA-H2S2 stock solution to the test solution; after adding H2S2, observing the change in fluorescence spectrum 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 non-specific binding of serum albumin to hemicyanine dye and enhance the stability, fluorescence quantum yield and Stokes shift of this type of hemicyanine dye.

[0043] (2) The dye synthesis steps provided by the present invention are simple, the purification is convenient, and the yield is high. By introducing a six-membered heterocycle into the side chain of the azahexacyanine dye, the binding with protein is effectively avoided, the interference of serum albumin on the dye fluorescence signal is eliminated, and the kidney is targeted, thus improving the high fidelity of imaging.

[0044] (3) The probe molecule provided by the present invention can quantitatively detect and assess the changes in H2S2 concentration during kidney 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. It can achieve accurate imaging through in vivo fluorescence detection and in vitro urine detection, providing important diagnostic value for renal ferroptosis. Attached Figure Description

[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 UV absorption and fluorescence emission spectra of dye NA-6 in solvent are shown in Example 2.

[0048] Figure 4 The graph shows the changes in fluorescence emission spectra of dye NA-6 in Example 3 before and after reacting with different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol.

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

[0050] Figure 6 The image shows the fluorescence test results for the stability of dye NA-6 in Example 5.

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

[0052] Figure 8 The image shows the fluorescence test results of the probe NA-H2S2 in Example 8 in response to different analytes;

[0053] Figure 9 The images show fluorescence imaging of endogenous and exogenous H2S2 in HK-2 cells using the probe NA-H2S2 from Example 9, as well as imaging of kidney ferroptosis induced by Einstein and cisplatin.

[0054] Figure 10 Near-infrared fluorescence images of the body, kidneys, and urine of mice that died after the probe NA-H2S2 was injected via the tail vein into the kidney iron in Example 10. Detailed Implementation

[0055] The present invention will be 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, all chemical reagents involved in this 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 includes the following synthetic 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 the crude product. The crude product was further purified by silica gel chromatography to obtain intermediate 1.

[0062] (2) Dissolve 33 mg of intermediate 1 in 20 mL of N,N-dimethylformamide, then add 66 mg of compound 4, and finally add 10 mg of potassium carbonate. Reflux the mixture at 80 °C for 6 hours. After cooling to room temperature, concentrate the organic phase to obtain the crude product. Further purify the crude product using silica gel chromatography to obtain compound 1a-1.

[0063] (3) 28 mg of compound 1a-1 was dissolved in ultra-dry acetonitrile, followed by 70 mg of compound 5, and finally potassium carbonate and potassium iodide were added. The mixture was refluxed at 110 °C for 12 hours. After cooling to room temperature, the organic phase was concentrated to obtain the 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. 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 complete, the organic phase was concentrated to obtain the crude product. Purification was performed using a silica gel (200-300 mesh) column with dichloromethane and methanol (v / v) as eluents 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 as follows Figure 1 As 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 such as Figure 2 As 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 target compound obtained is consistent with the structure shown in the above structural formula.

[0068] The synthesis steps for other dyes NA-1 to NA-5 differ from those for NA-6 in the selection of starting compound 1 and intermediate 1; 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: A DMSO stock solution of dye NA-6 was prepared at a concentration of 5 mM. Then, 1996 μL of different solutions (1,4-dioxane, ACN, DCM, DMF, DMSO, MeOH, PBS) and 4 μL of the stock solution were added to UV and fluorescein dishes, respectively, and their UV absorption and fluorescence emission were measured. The results are as follows: Figure 3 As shown.

[0072] Example 3

[0073] The fluorescence emission spectra of dye NA-6 prepared in Example 1 were tested in response to different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol before and after testing, to verify that the fluorescence emission spectra of dye NA-6 are not affected by serum albumin.

[0074] The test method is as follows: Prepare a DMSO stock solution for the dye with a concentration of 5 mM. Then, add 1996 μL of PBS and 4 μL of the stock solution to a fluorescence dish, followed by different concentrations of serum protein, hemoglobin, polyvinyl alcohol, and glycerol, and test its fluorescence emission. The results are as follows. Figure 4 As shown (where Figures (a) and (b) are fluorescence emission patterns of dye NA-6 for different concentrations of serum proteins; Figure (c) is fluorescence emission pattern of dye NA-6 for different concentrations of hemoglobin; Figure (d) is fluorescence emission pattern of dye NA-6 for different concentrations of polyvinyl alcohol; and Figure (e) is fluorescence emission pattern of dye NA-6 for different concentrations of glycerol).

[0075] Example 4

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

[0077] The renal targeting and metabolism of dyes NA 1-6 in mice are as follows: Figure 5As shown in the figures (where Figure (A) shows the intravenous injection in mice; Figure (B) shows the structural formulas of dyes NA-1 to NA-6; Figure (C) shows the metabolism of mice after intravenous injection; Figure (D) shows the urine fluorescence of mice injected with NA-6 via the tail vein at 0 and 90 minutes; Figure (E) shows the dissected organs of mice injected with NA-6 via the tail vein at 90 minutes; Figure (F) shows the fluorescence intensity quantification in vivo 60 minutes after tail vein injection of NA-1 to NA-5; Figure (G) shows the fluorescence intensity quantification in mice at different time points after tail vein injection of NA-6; Figure (H) shows the fluorescence intensity quantification in different organs), we were surprised to find that these N-heterocyclic nitrogen hemicyanine dyes can target the kidneys after tail vein injection of NA 1-6. Taking NA-6 as an example, NA-6 is rapidly metabolized by the kidneys within 1.5 hours after injection, and the fluorescence intensity in vivo reaches its peak at 60 minutes. After 90 minutes, some of the dye is excreted in the urine. This indicates that NA-6 exhibits significant biocompatibility compared to dyes that may take days or even months to metabolize, reducing the likelihood of accumulation in the body and thus mitigating the potential toxicity and side effects of NA-6. It also provides a basis for high-fidelity imaging of the kidneys and urine analysis.

[0078] Example 5

[0079] Stability fluorescence test of dye NA-6.

[0080] The test method is as follows: Prepare a DMSO stock solution for the dye with a concentration of 5 mM. Then, add 1996 μL of liquid (PBS:methanol = 7:3) and 4 μL of the stock solution to UV and fluorescein dishes, respectively, and then add different concentrations of redox agents. Test the UV absorption and fluorescence emission spectra. The results are as follows: Figure 6 As 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 structure of the NA-H2S2 fluorescent probe responsive to H2S2 is as follows:

[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) were placed in a round-bottom flask, and 5 mL of N,N-dimethylformamide was added to completely dissolve them. Then, 8.3 mg of potassium carbonate (0.06 mmol) was added. The mixture was stirred for 1 hour under nitrogen protection and at room temperature. After the reaction was complete, the organic phase was concentrated to obtain the crude product. Purification was performed using a silica gel (200-300 mesh) column with dichloromethane and methanol (v / v) as eluents to obtain 67.1 mg of green solid (yield: 83.5%).

[0086] probe 1 H NMR spectrum as follows 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 such as 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] The fluorescence emission spectra of the NA-H2S2 probe before and after responding to H2S2 were tested.

[0090] The testing method is as follows: Prepare a DMSO stock solution for probe NA-H2S2 with a concentration of 5 mM. Then, add 1996 μL of liquid (PBS:DMSO = 9:1) and 4 μL of the stock solution to a fluorescence dish and test its fluorescence emission spectrum. Subsequently, add 1–90 μg / ml of H2S2 to the dish; the fluorescence intensity in the fluorescence spectrum increased 15-fold, as shown in the results. Figure 7 As shown.

[0091] Example 8

[0092] The fluorescence signal of the test probe NA-H2S2 under different analytes was observed to change.

[0093] The testing method is as follows: Prepare a DMSO stock solution for probe NA-H2S2 at a concentration of 5 mM. Then, add 1996 μL of liquid (PBS:DMSO = 9:1) and 4 μL of the stock solution to a fluorescent dish. Subsequently, add aqueous solutions of different amino acids and common ions found in vivo, including H2S, BSA, HAS, HB, Cys, GSH, Hcy, H2S4, and SO3. 2- S2O8 2- S2O3 2- HSO 4- Different biological reagents such as Pro, Gly, and Glu, as well as compounds such as metal ions, were used. Fluorescence signal changes were recorded, and bar charts were plotted for each analyte added. Figure 8 Experiments have shown that other amino acids and common ions in the body do not interfere with the system's detection of H2S2. 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 kidney ferroptosis induced by Einstein and cisplatin.

[0096] To demonstrate that the probe could recognize endogenous H2S2 in cells, we incubated cells with LPS for 12 hours, added the probe to the cells, and then performed time-lapse imaging. The results are as follows: Figure 9As shown in Figure A (where Figure (A) is a confocal fluorescence imaging of endogenous H2S2 in HK-2 cells at different time points), the red fluorescence gradually increased with the extension of probe incubation time in cells, and remained almost unchanged after 30 minutes, indicating that the probe responded to endogenous H2S2 and reacted almost completely with endogenous H2S2 in the cells around 30 minutes. Therefore, the probe can be used for precise monitoring of intracellular H2S2. Next, to investigate the ability of the NA-H2S2 probe to detect changes in H2S2 concentration in cells, we conducted an exogenous H2S2 experiment. In the first group, cells were treated with NEM, and then 100 μM exogenous H2S2 was added for half an hour before the probe was added. In the second group, cells were treated with NEM, and then 50 μM exogenous H2S2 was added for half an hour before the probe was added. In the third group, cells were incubated with the probe only. In the fourth group, cells were incubated with N-ethylmaleimide (NEM, a thiol digestion reagent) for 30 minutes beforehand to eliminate intracellular H2S2 before the probe was added. All four groups of cells were observed through the red channel. The results are as follows: Figure 9 As shown in Figure B (where Figure (B) is a confocal fluorescence image of HK-2 cells after adding different concentrations of exogenous H2S2), cells treated with NEM showed almost no red fluorescence. However, after adding 100 μM or 50 μM of 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 the cells.

[0097] To investigate the ability of the probe NA-H2S2 to detect H2S2 during renal cell ferroptosis, we conducted the following experiments. Normal HK-2 cells were divided into four groups. Group 1 cells were induced to undergo ferroptosis with 10 μM Erastin for 8 hours, followed by incubation with the probe for 30 minutes. Group 2 cells were induced to undergo ferroptosis with 10 μM Erastin for 6 hours, followed by incubation with the probe for 30 minutes. Group 3 cells were incubated with the probe alone for 30 minutes. Group 4 cells were induced to undergo ferroptosis with the 5 μM ferroptosis inhibitor Fer-1 for 8 hours, followed by incubation with the probe for 30 minutes. Cells in each group were washed three times with PBS before imaging. All observations were performed using the red channel: excitation wavelength 638 nm, and emission wavelength collection range 650-780 nm. Figure 9 As shown in Figure C, after 8 hours of ferroptosis induced by Erastin, the fluorescence intensity of HK-2 was 9.66 times higher than that of the inhibition group, indicating that this probe can be used for precise monitoring of H2S2 during intracellular ferroptosis. Meanwhile, as... Figure 9 As shown in Figures D to E (where Figure (D) is a confocal fluorescence imaging of cells induced by cisplatin at different times; Figure (E) is a confocal fluorescence imaging of ferroptosis of cells 24 hours after cisplatin induction), the same results were obtained when the cells were treated with cisplatin.

[0098] Example 10

[0099] The probe NA-H2S2 was used for imaging studies of H2S2 during ferroptosis in mouse kidneys.

[0100] To further clarify the changes in H2S2 during ferroptosis in vivo, we established a mouse model of renal ferroptosis. Ferroptosis was induced in the kidneys by continuous intraperitoneal injection of Erastin solution at a dose of 25 mg / kg, repeated every 12 hours for 5 days. 100 μL of a 1 mM probe was injected via the tail vein, followed by fluorescence imaging. Figure 10 As shown in the figures (where Figure (A) is a schematic diagram of the imaging experiment design and urine analysis of H2S2 levels in the kidneys of mice after Erastin modeling; Figure (B) is a fluorescence imaging image of mice after tail vein injection of the probe within 0 to 90 minutes in the Erastin, normal, NEM, and Fer-1 groups; Figure (C) is a fluorescence imaging image of the kidneys after dissection 90 minutes after probe injection in mice; Figure (D) is a quantitative map of fluorescence intensity in the mice and kidneys; Figure (E) is a fluorescence imaging image of mouse urine co-incubated with the probe at different time points; Figure (F) is a quantitative map of GSH (glutathione) and MDA (malondialdehyde) levels in the kidneys of ferroptosis mice), the fluorescence in the kidneys of ferroptosis mice was significantly increased compared with that of normal mice or control mice, indicating that the H2S2 concentration in the kidneys of ferroptosis mice tended to be upregulated. Subsequently, by imaging the dissected kidneys of mice, we found that the fluorescence in the kidneys of the Erastin-induced group was significantly enhanced, approximately 30 times that of the NEM group. Furthermore, as the injection time of Einstein increased, the fluorescence intensity of the urine excreted by the mice and the probe was significantly increased after incubation for 30 minutes, further demonstrating the good application potential of the probe.

[0101] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A class of azahexacyanine dyes, characterized in that, The dye comprises the following structural formula: 。 2. The method for synthesizing azahexacyanine dye according to claim 1, characterized in that, The synthesis steps include the following: ； Synthesis of S1 and intermediate 1: Compound 1 was dissolved in a mixed organic solvent of n-butanol and toluene, then compound 3 was added, the mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain intermediate 1. Synthesis of S2 and intermediate 2: Compound 2 was dissolved in a mixed organic solvent of n-butanol and toluene, then compound 3 was added, the mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain intermediate 2. Synthesis of S3 and compounds 1a-1~3: Intermediate 1a was dissolved in N,N-dimethylformamide, then compound 4 was added, and finally potassium carbonate was added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compounds 1a-1~3. Synthesis of S4 and compounds 1b-1~3: Intermediate 1b was dissolved in N,N-dimethylformamide, then compound 4 was added, and finally potassium carbonate was added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compounds 1b-1~3. S5. Synthesis of compounds 1c-1~3: Intermediate 1c was dissolved in N,N-dimethylformamide, then compound 4 was added, and finally potassium carbonate was added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compounds 1c-1~3. S6. Synthesis of compounds 2a-1~3: Intermediate 2 was dissolved in N,N-dimethylformamide, then compound 4 was added, and finally potassium carbonate was added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compounds 2a-1~3. Synthesis of dyes NA-1~5, NA-7~11, NA-13~17, and NA-19~23: Compounds 1a-2~3, 1b-2~3, 1c-2~3, and 2a-2~3 were dissolved in ultra-dry acetonitrile, followed by the 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. The crude product was further purified by silica gel chromatography to obtain dyes NA-1~5, NA-7~11, NA-13~17, and NA-19~23. Synthesis of S8 and compound 3NA-6\12\18\24: Compound 1a-1\1b-1\1c-1\2a-1 was dissolved in ultra-dry acetonitrile, then compound 5 was added, and finally potassium carbonate and potassium iodide were added. The mixture was refluxed, cooled, and the organic phase was concentrated to obtain the crude product. The crude product was further purified by silica gel chromatography to obtain compound 3NA-6\12\18\24. S9. Synthesis of dye NA-6\12\18\24: Compound 3NA-6\12\18\24 was dissolved in ultra-dry dichloromethane, boron tribromide was added under ice bath conditions, the reaction was carried out for a period of time, cooled, and the organic phase was concentrated to obtain crude product. The crude product was further purified by silica gel chromatography to obtain dye NA-6\12\18\24.

3. The method for synthesizing azahexacyanine dye according to claim 2, 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 reflux reaction temperature is 105~115 ℃, and the reflux reaction time is 9~11 h; 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 azahexacyanine dye according to claim 2, 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 h. The molar ratio of intermediate 1a to compound 4 is (1~3):1; The molar ratio of intermediate 1b to compound 4 is (1~3):1; The molar ratio of intermediate 1c to compound 4 is (1~3):1; The molar ratio of intermediate 2 to compound 4 is (1~3):

1.

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

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

7. An application of the azahexacyanine dye as described in claim 1, characterized in that, The application described is the use of azahexacyanine dyes for detecting fluorescence signals for non-disease diagnostic and therapeutic purposes.

8. An application of the azahexacyanine dye as described in claim 1, characterized in that, It is applied to the NA-H2S2 probe, which is capable of detecting H2S2.

9. The application of the azahexacyanine dye according to claim 8, characterized in that, The synthesis method of the azahexacyanin NA-H2S2 fluorescent probe includes the following steps: ； The dye NA-6 was dissolved in N,N-dimethylformamide, followed by the addition of compound 6 and potassium carbonate. The reaction was carried out for 0.5 to 1.5 hours, the organic phase was concentrated, and the product was further purified by silica gel chromatography to obtain the hemicyanine NA-H2S2 fluorescent probe.

10. The application of the azahexacyanine dye according to claim 8, characterized in that, The method for detecting H2S2 includes 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 change in fluorescence spectrum of the test solution containing NA-H2S2 using a fluorescence spectrometer.

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