Cyanine fluorescent compound as well as preparation method and application thereof

Abstract

The invention discloses a cyanine fluorescent compound as well as a preparation method and application thereof, and belongs to the technical field of fine chemical engineering. The structure of the cyanine fluorescent compound is shown as a formula (I), and the cyanine fluorescent compound can be used for detecting ONOO. The luminescent mechanism of the cyanine fluorescent compound is an intramolecular charge transfer (ICT) mechanism, the emission wavelength is 800nm, the cyanine fluorescent compound is connected with a diphenyl hypophosphite group, in the presence of peroxynitrite ONOO, the diphenyl hypophosphite group leaves through a high-selectivity addition-elimination way, the ICT effect is changed, the emission wavelength is blue-shifted to 633nm, and the cyanine fluorescent compound has the advantages that the fluorescence intensity of the cyanine fluorescent compound is improved; ratio-type fluorescence detection of ONOO is realized, and the detection limit reaches 0.57 mu M. (I)

Description

Technical Field

[0001] This invention belongs to the field of fine chemical technology, specifically relating to a cyanin-based fluorescent compound, its preparation method, and its application. Background Technology

[0002] In recent years, hepatobiliary diseases have received considerable attention in fields such as biology and medicine, including cholestasis, hepatitis, and cirrhosis. These diseases are caused by damage or fibrosis of hepatocytes, severely impacting human health. Drug-induced liver injury (DILI) refers to damage to hepatocytes caused by drugs and their metabolites during chemotherapy, characterized by its unpredictability, severity, and high mortality. Clinical studies have shown that serological testing cannot provide real-time monitoring of disease progression, potentially delaying treatment. Therefore, there is an urgent need to develop real-time monitoring tools using biomarkers directly related to DILI. One such biomarker is peroxynitrite (ONOO). ONOO is a reactive nitrogen species (RNS) with strong oxidizing and nucleophilic properties. It can react with a variety of biomolecules, including proteins, nucleic acids, and lipids, thereby damaging cell structure and function. It has been used as a sensitive biomarker for the early diagnosis of DILI.

[0003] To date, numerous in vitro detection methods for DILI during ONOO have been developed both domestically and internationally. Common detection methods include enzyme cycling assays, electrochemical analysis, high-performance liquid chromatography (HPLC), and capillary electrophoresis. However, these methods cannot detect ONOO in live cells and tissues in situ and in real time. The level changes. Fluorescent probes have received widespread attention from the scientific community due to their advantages such as high sensitivity, high selectivity, fast reaction speed, low detection limit, and simple operation. During the detection of DILI, ONOO The field of visualization research has enormous application potential. Therefore, utilizing fluorescent probes to realize ONOO Activated precision imaging is an important means to achieve timely diagnosis of DILI and to study the pathogenesis of DILI. Summary of the Invention

[0004] To address the shortcomings of existing technologies for detecting peroxynitrite, such as short and singular emission wavelengths, this invention aims to provide a cyanine-based fluorescent compound, its preparation method, and its applications. This invention forms a heptamethyl cyanine dye structure by linking two indole salt groups and introducing a diphenyl phosphite group to regulate the intramolecular charge transfer effect of the cyanine dye, achieving an emission wavelength of 800 nm. (The last sentence appears to be incomplete and possibly refers to a separate invention.) In its presence, the diphenyl hypophosphite group leaves via a highly selective addition-elimination pathway, resulting in a blue shift of the emission wavelength to 633 nm. Based on the logarithm of the emission ratio lg(I... 633nm / I 800nm The concentration of peroxynitrite was determined, and the detection limit reached 0.57 μM.

[0005] In a first aspect of the invention, a cyanine fluorescent compound is provided, the structure of which is shown in formula (I), wherein two indole salt groups are connected by conjugated double bonds to form a heptamethine cyanine dye, and a diphenyl hypophosphite group is introduced, the fluorescence signal of which changes before and after the response of peroxynitrite.

[0006] (I).

[0007] The anthocyanin fluorescent compounds in peroxynitrite ONOO In its presence, an addition-elimination reaction occurs, and the diphenyl phosphite group leaves, altering the intramolecular charge transfer effect of cyanine fluorescent compounds. The emission wavelength blue-shifts from 800 nm to 633 nm, and the detection limit reaches 0.57 μM.

[0008] In a second aspect of the invention, a method for preparing the cyanine fluorescent compound is provided, wherein the cyanine fluorescent compound is prepared from compound (II) and compound (V), and the reaction formulas for preparing the cyanine fluorescent compound (I) from compounds (II) and (V) are shown below:

[0009] In one or more embodiments of the present invention, the method for preparing the cyanine fluorescent compound includes the following steps: under an N2 or inert gas atmosphere, compound (II), compound (V), organic base, and triphosgene are reacted in an organic solvent to obtain the cyanine fluorescent compound. The organic base includes one or more of N,N-diisopropylethylamine, triethylamine, pyridine, piperidine, etc., preferably N,N-diisopropylethylamine. The molar ratio of compound (II), compound (V), organic base, and triphosgene is preferably 1.0-1.2:1.0-1.5:2.5-4.0:0.8-1.1; the organic solvent preferably includes one or more of DCM, THF, chloroform, diethyl ether, etc.; the reaction temperature is preferably 15-35°C, and further, the reaction conditions are preferably room temperature stirring for 8-16 hours. Further, the step also includes separating and purifying the reaction product, preferably by using one or more of the following methods: desolventizing under reduced pressure, column chromatography, and concentration under reduced pressure.

[0010] In one or more embodiments of the present invention, the method for preparing the cyanine fluorescent compound includes the following steps: adding compound (II) to a reaction vessel, dehydrating and deoxygenating the reaction system, adding ultra-dry DCM and N,N-diisopropylethylamine under N2 atmosphere, lowering the temperature to 0°C under ice bath conditions, adding triphosgene dissolved in ultra-dry DCM, stirring at 0°C for 10-30 min, raising the temperature to room temperature and reacting for 1-3 h, then adding compound (V) dissolved in ultra-dry DCM, and stirring at room temperature for 8-16 h. After the reaction is completed, the product solvent is removed under reduced pressure, purified by silica gel column chromatography, and finally concentrated under reduced pressure to obtain the cyanine fluorescent compound.

[0011] In one or more embodiments of the present invention, compound (II) is prepared from compound (III), and the reaction formula for the preparation of compound (II) from compound (III) is shown below:

[0012] In one or more embodiments of the present invention, the preparation of compound (II) from compound (III) includes the following steps: under a N2 or inert gas atmosphere, compound (III) and a base are refluxed in an organic solvent to obtain compound (II). The base includes one or more of sodium acetate, potassium acetate, triethylamine, etc., preferably sodium acetate; the molar ratio of compound (III) to the base is preferably 1:2-5; the organic solvent is preferably DMF; the reflux reaction is preferably refluxed at 85-95°C for 3-5 hours. Further, the step also includes separating and purifying the reaction product, preferably by using one or more of the following methods: desolventizing under reduced pressure, column chromatography, or concentration under reduced pressure.

[0013] In one or more embodiments of the present invention, the preparation of compound (II) from compound (III) includes the following steps: adding compound (III) and sodium acetate to a reaction vessel; heating the reaction system under vacuum to remove water and oxygen, adding ultra-dry DMF under N2 atmosphere, and refluxing at 90°C for 4 hours. After the reaction is completed, the product solvent is removed under reduced pressure, purified by silica gel column chromatography, and finally compound (II) is obtained by concentration under reduced pressure.

[0014] In a third aspect of the invention, the application of the cyanin-based fluorescent compound in the detection of peroxynitrite is provided, the application being based on the logarithm of the luminescence ratio lg(I 633nm / I 800nm To determine the concentration of peroxynitrite.

[0015] In a fourth aspect of the invention, the use of the cyanine fluorescent compound in the preparation of a ratiometric fluorescent probe for detecting peroxynitrite is provided.

[0016] In a fifth aspect of the invention, a ratiometric fluorescent probe for detecting peroxynitrite is provided, comprising the cyanide fluorescent compound.

[0017] In a sixth aspect of the invention, the use of the cyanin fluorescent compound or the ratiometric fluorescent probe in the preparation of diagnostic reagents or kits for drug-induced liver injury (DILI) is provided.

[0018] In a seventh aspect of the invention, a diagnostic reagent or kit for drug-induced liver injury (DILI) is provided, comprising the cyanide fluorescent compound or the ratiometric fluorescent probe.

[0019] The present invention has the following advantages and beneficial effects: The anthocyanin-based fluorescent compounds prepared in this invention exhibit ONOO In its presence, diphenyl hypophosphite undergoes an addition-elimination reaction, altering the ICT effect of the fluorescent compound and causing a blue shift in emission wavelength to 633 nm. This is because diphenyl hypophosphite reacts with ONOO... The fluorescent compound exhibits specificity in response to ONOO in aqueous solution. High selectivity of the detection.

[0020] The anthocyanin-based fluorescent compounds prepared in this invention can help in the development of a kit for detecting peroxynitrite in drug-induced liver injury. Attached Figure Description

[0021] Figure 1 The probe CCy7-DP (2 μM) was reacted with ONOO in ACN / PBS buffer (ACN / PBS (10 mM, pH 7.4) = 1 / 3, V / V). The time-dependent fluorescence spectrum changes at (100 μM) excitation wavelength are shown in Figure A. Figure A shows the fluorescence spectrum changes at an excitation wavelength of 540 nm, with wavelength (nm) on the x-axis and fluorescence intensity (au) on the y-axis. Figure B shows the fluorescence spectrum changes at an excitation wavelength of 740 nm, with wavelength (nm) on the x-axis and fluorescence intensity (au) on the y-axis. Figure C shows the fluorescence intensity changes of the probe at 633 nm and 800 nm over time.

[0022] Figure 2 The probe CCy7-DP (2 μM) was reacted with different concentrations of ONOO in ACN / PBS buffer (ACN / PBS (10 mM, pH 7.4) = 1 / 3, V / V). Fluorescence spectra after 45 min of treatment (0-100 μM). In the graph, A represents the fluorescence spectrum change at an excitation wavelength of 540 nm, with the horizontal axis representing wavelength (nm) and the vertical axis representing fluorescence intensity (au); B represents the fluorescence spectrum change at an excitation wavelength of 740 nm, with the horizontal axis representing wavelength (nm) and the vertical axis representing fluorescence intensity (au); C represents the logarithm of the probe fluorescence ratio lg(I0). 633nm / I 800nm ) with ONOO Standard curve of concentration change.

[0023] Figure 3 A is probe CCy7-DP (2 μM) in ACN / PBS buffer (ACN / PBS (10 mM, pH 7.4) = 1 / 3, V / V) with ONOO (100 μM) and other competing species (1: Blank; 2: ONOO) 3: H2O2; 4: ClO ;5:O2 • 6: 1 O2; 7: •OH; 8: Cys; 9: HCy; 10: GSH; 11: Trp; 12: Tyr; 13: Gly; 14: Glu; 15: Lys; 16: ATP; 17: NAC; 18: Cellulase; 19: Lysozyme; 20: Fe 2+ ;21:K + ;22:Mg 2+ ;23:Ca 2+ Fluorescence intensity at 633 nm after 45 min of treatment. Figure 3 B represents the fluorescence intensity of probe CCy-DP (2 μM) at 633 nm in ACN / PBS buffers at different pH values ​​(ACN / PBS (10 mM) = 1 / 3, V / V) and its correlation with ONOO. (C) The fluorescence intensity change curve at 633 nm after 45 min of treatment with (100 μM); (C) shows the reaction of probe CCy7-DP with ONOO. Normalized absorption spectra of CCy7 before and after the response. Detailed Implementation

[0024] The present invention will be further illustrated below through examples, the purpose of which is solely to provide a better understanding of the invention. Therefore, the examples given do not limit the scope of protection of the present invention.

[0025] Example 1 A cyanine-based fluorescent compound, in which two indole salt groups are linked by a conjugated double bond to form a heptamethyl cyanine dye, and a diphenyl hypophosphite group is introduced, exhibits a change in fluorescence signal before and after the response to peroxynitrite. The structure of the cyanine-based fluorescent compound is shown in formula (I): (I) The anthocyanin fluorescent compounds in peroxynitrite ONOO In its presence, an addition-elimination reaction occurs, and the diphenyl phosphite group leaves, altering the intramolecular charge transfer effect of cyanine fluorescent compounds. The emission wavelength blue-shifts from 800 nm to 633 nm, and the detection limit reaches 0.57 μM.

[0026] The preparation method of the cyanin fluorescent compound includes the following steps: (1) Add 2,3,3-trimethyl-3H-indole (400 mg, 1.02 mmol) and iodoethane (432.6 mg, 1.02 mmol) to a 50 mL double-necked flask. Dehydrate and deoxygenate the reaction system by injecting 5 mL of ultra-dry acetonitrile with a syringe under N2 atmosphere and stirring at 83 °C for 8 h. Cool to room temperature, and slowly add the original reaction solution dropwise to an EA / PE (2:1, v / v) mixed solution for recrystallization. Filter using a Buchner funnel, and dry the filter cake to obtain a pink powder product (IV) (163.4 mg, yield 78.5%). The specific process parameters for dehydration and deoxygenation are: vacuum pump for 10-20 minutes, maintain room temperature for 5-10 minutes, and then replace with nitrogen gas into the system.

[0027]

[0028] 1 H NMR (400 MHz, DMSO- D 6) δ 8.01 – 7.93 (m, 1H), 7.88 – 7.81 (m, 1H), 7.67 – 7.59 (m, 2H), 4.50 (q, J = 7.3 Hz, 2H), 2.83 (s, 3H), 1.53 (s, 6H), 1.45 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, DMSO- D 6) δ 196.11, 141.96, 140.74,129.38, 128.96, 123.57, 115.31, 54.12, 43.07, 21.89, 13.86, 12.69. HRMS-ESI( m / z ): [M+H] + Calcd for C 13 H 18 N + 188.1434, found 188.1459. (2) Compound (IV) (228.26 mg, 2.50 mmol), 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (50 mg, 1.02 mmol), and sodium acetate (68.7 mg, 3.0 mmol) were added to a 50 mL Shrek tube to remove water and oxygen from the reaction system. 2 mL of ultra-dry ethanol was injected using a syringe under N2 atmosphere, and the mixture was stirred at 83 °C for 3 h. After cooling to room temperature, the original reaction solution was slowly added dropwise to an EA / PE (2:1, v / v) mixed solution for recrystallization. The solution was filtered using a Buchner funnel, and the filter cake was dried to obtain a green powdery product (III) (163.4 mg, yield 78.5%).

[0029]

[0030] 1 H NMR (400 MHz, DMSO- D 6) δ 8.23 ​​(d, J = 14.1 Hz, 2H), 7.60 (d, J = 1.1Hz, 2H), 7.47 – 7.35 (m, 4H), 7.29 – 7.22 (m, 2H), 6.29 (d, J = 14.2 Hz, 2H), 4.22 (q, J = 7.1 Hz, 4H), 2.69 (t, J = 6.1 Hz, 4H), 1.63 (s, 12H), 1.52 – 1.48(m, 2H), 1.27 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, DMSO- D 6) δ 172.32, 148.52,143.62, 142.19, 141.74, 129.19, 126.64, 125.72, 123.13, 111.87, 101.85,49.55, 27.92, 26.42, 20.90, 12.78. HRMS-ESI (m / z): [M] + Calcd for C 34 H40 ClN2 + 511.2875, found 511.2882. (3) Add compound (III) (100 mg, 0.17 mmol) and sodium acetate (668.7 mg, 0.68 mmol) to a 50 mL Shrek tube. Dehydrate and deoxygenate the reaction system by injecting 4 mL of ultra-dry DMF under a N2 atmosphere and refluxing at 90 °C for 4 h. After the reaction is complete, remove the solvent from the product under reduced pressure. Purify by column chromatography using PE / EA (3:1, v / v) as the eluent on a silica gel column. Finally, concentrate under reduced pressure to obtain the red product (II) (36.6 mg, yield 42.9%), which is designated CCy7. This step must be performed under strict light protection. Post-processing should involve wrapping the product in tin foil before removing the solvent under reduced pressure, ensuring strict light protection throughout the process. Finally, purify by column chromatography.

[0031]

[0032] 1 H NMR (400 MHz, CHLOROFORM- D ) δ 8.20 (d, J = 13.0 Hz, 2H), 7.25 –7.15 (m, 4H), 6.92 (t, J = 7.4 Hz, 2H), 6.69 (d, J = 7.9 Hz, 2H), 5.48 (d, J =13.3 Hz, 2H), 3.75 (d, J = 7.6 Hz, 4H), 2.61 (t, J = 6.2 Hz, 4H), 1.87 (p, J =6.5 Hz, 2H), 1.67 (s, 12H), 1.29 (t, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, CHLOROFORM- D) δ 161.93, 143.82, 139.92, 132.94, 127.74, 126.57, 121.92,120.53, 106.50, 92.21, 46.66, 37.13, 33.93, 32.02, 29.79, 29.46, 29.26,29.05, 28.78, 25.93, 22.79, 22.64, 14.23, 11.24. HRMS-ESI (m / z): [M+H] + Calcdfor C 34 H 40 N2O 493.3213, found 493.3219. (4) Add p-hydroxybenzyl alcohol (100 mg, 0.11 mmol) to a 50 mL three-necked flask to remove water and oxygen from the reaction system. Add 6 mL of ultra-dry DCM using a syringe under an ice bath and N2 atmosphere, stir at 0 °C for 10 min, and then inject triethylamine (122.27 mg, 1.5 mmol). Slowly add a DCM solution of diphenylphosphine chloride (232 mg, 1.2 mmol) through a constant pressure dropping funnel. Stop adding when white fumes are generated. After the reaction is finished, add 15 mL of DI (deionized water), wash with dilute hydrochloric acid, concentrate the product under reduced pressure, and purify by column chromatography with DCM / MeOH (50:1, v / v) as the eluent. Finally, concentrate under reduced pressure to obtain a white product (V) (32 mg, yield 53.2%).

[0033]

[0034] 1 H NMR (400 MHz, DMSO- d 6) δ 7.93-7.87 (m, 4H), 7.66-7.57 (m, 2H), 7.57-7.51 (m, 4H), 7.23 (s, 4H), 5.13 (t, J = 5.7 Hz, 1H), 4.39 (d, J = 5.7 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 149.42, 149.34, 138.97, 132.78, 132.75,131.64, 131.53, 130.17, 129.02, 128.89, 127.88, 120.25, 120.20, 62.24. HRMS-ESI(m / z): [M+H]+ Calculated for C 19 H 18 O3P + 325.0988, found 325.0997. (5) Compound (II) (50 mg, 1.01 mmol) was added to a 50 mL Shrek tube to remove water and oxygen from the reaction system. Under N2 atmosphere, 4 mL of ultra-dry DCM and N,N-diisopropylethylamine (40 mg, 3.0 mmol) were added using a syringe. The mixture was stirred at 0 °C for 10 min. Triphosgene (30 mg, 1.0 mmol) was dissolved in ultra-dry DCM and injected into the Shrek tube. After reacting at room temperature for 2 h, compound (V) (33 mg, 1.0 mmol) dissolved in ultra-dry DCM was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent of the product was removed under reduced pressure. Column chromatography was performed using DCM / MeOH (10:1, v / v) as the eluent. The product was purified by silica gel column chromatography. Finally, the green product (I) (14.3 mg, yield 43.3%) was obtained by concentration under reduced pressure. This is the cyanin-based fluorescent compound of the present invention, denoted as the ratiometric fluorescent probe CCy7-DP.

[0035]

[0036] 1 H NMR (400 MHz, DMSO- D 6) δ 7.93 – 7.85 (m, 4H), 7.62 – 7.54 (m, 6H), 7.51 – 7.42 (m, 10H), 7.41 – 7.36 (m, 2H), 7.30 – 7.24 (m, 2H), 6.22 (d, J =14.2 Hz, 2H), 5.28 (s, 2H), 4.22 (q, J = 7.2 Hz, 4H), 2.64 (t, J = 6.2 Hz, 4H),1.88 – 1.77 (m, 2H), 1.41 (s, 12H), 1.28 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, DMSO- D6) δ 171.76, 157.86, 152.76, 151.75, 151.67, 142.19, 141.62, 139.21,133.35, 132.03, 131.92, 131.54, 131.42, 130.51, 129.55, 129.41, 129.23,125.62, 123.08, 121.40, 121.34, 111.82, 101.20, 70.52, 55.47, 49.26, 27.75,24.38, 24.24, 20.89, 12.74, 0.64. HRMS-ESI (m / z): [M] + Calcd for C 54 H 56 N2O5P + 843.3912, found 843.3924. Example 2 Probe CCy7-DP and ONOO Time-dependent fluorescence spectra of the response: The probe CCy7-DP prepared in Example 1 was dissolved in ACN / PBS buffer (ACN / PBS (10mM, pH 7.4) = 1 / 3, V / V), and the interaction between CCy7-DP and ONOO was tested. Time-dependent fluorescence curve of the response. 100 μM ONOO was added to the solution containing 2 μM probe. Subsequently, the response time was increased from 0 min to 45 min (0, 0.5, 3, 8, 14, 22, 35, 45 min). With the extension of the response time, the fluorescence emission peak at 633 nm was significantly enhanced. Figure 1 A), the fluorescence emission peak at 800 nm is weakened ( Figure 1 B), the trend of fluorescence intensity change at both locations changes from fast to slow ( Figure 1 C).

[0037] Example 3 Probe CCy7-DP and ONOO The concentration-dependent fluorescence spectrum and detection limit of the response: The probe CCy7-DP prepared in Example 1 was dissolved in ACN / PBS buffer (ACN / PBS (10mM, pH 7.4) = 1 / 3, V / V), and the probe CCy7-DP was tested with different concentrations of ONOO. The response status can be observed after 45 minutes, as ONOO... As the concentration increased from 0 μM to 100 μM (0, 20, 40, 60, 80, 100 μM), the fluorescence emission peak at 633 nm significantly increased. Figure 2 A), the fluorescence emission peak at 800 nm is weakened ( Figure 2 B). The logarithm of the fluorescence intensity ratio was observed to be lg( I 633 nm / I 800 nm ) with ONOO There is a good linear correlation between concentrations (R0). 2 = 0.9884), and the limit of detection (LOD) is 0.57 μM, indicating that this probe has the sensitivity to detect low concentrations of ONOO. Potential ( Figure 2 C).

[0038] Example 4 Selectivity, pH stability, and sensing mechanism of probe CCy7-DP: The probe CCy7-DP (2 μM) prepared in Example 1 was mixed with common analytes in biological systems (including 1: Blank; 2: ONOO). 3: H2O2; 4: ClO ;5:O2 • 6: 1 O2; 7: •OH; 8: Cys; 9: HCy; 10: GSH; 11: Trp; 12: Tyr; 13: Gly; 14: Glu; 15: Lys; 16: ATP; 17: NAC; 18: Cellulase; 19: Lysozyme; 20: Fe 2+ ;21:K + ;22:Mg 2+ ;23:Ca 2+ (Except for Cellulase and Lysozyme, which were at 100 U / mL, all others were at 100 μM.) During incubation, the fluorescence intensity of the probe CCy7-DP at 633 nm was only at ONOO. The fluorescence enhancement effect of the probe is enhanced in the presence of ONOO, while the fluorescence enhancement effect of other analytes is negligible, indicating that the probe enhances the fluorescence of ONOO. It has good selectivity, such as Figure 3 As shown in (A), the fluorescence intensity of probe CCy-DP (2 μM) at 633 nm remained essentially unchanged in ACN / PBS buffer (ACN / PBS (10 mM) = 1 / 3, V / V) at different pH values ​​(4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9), while it decreased with ONOO. The fluorescence intensity curve at 633 nm after 45 min of treatment with (100 μM) shows that the fluorescence intensity was highest at around pH 7.4, while the fluorescence intensity was lower in acidic and alkaline pH environments. This indicates that under physiological pH conditions, ONOO... The response effect is optimal, such as Figure 3 As shown in Figure B. Probe CCy7-DP and ONOO After the interaction, the absorption spectra of CCy7-DP and CCy7 showed good overlap, indicating that the CCy7-DP structure underwent hydrolysis to produce the luminescent substance CCy7. This confirms the addition-elimination response mechanism of diphenyl hypophosphite, such as... Figure 3 As shown in C.

[0039] The present invention and its embodiments have been described above illustratively. This description is not restrictive, and the figures shown are only one embodiment of the present invention; the actual structure is not limited thereto. Therefore, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A cyanin-based fluorescent compound, characterized in that, The structure is shown in equation (I): （I）。 2. The method for preparing the cyanine fluorescent compound according to claim 1, characterized in that: The cyanin-based fluorescent compounds were prepared from compounds (II) and (V), and the structures of compounds (II) and (V) are shown below: 、 。 3. The method for preparing cyanine fluorescent compounds according to claim 2, characterized in that, The process includes the following steps: under an N2 or inert gas atmosphere, compound (II), compound (V), organic base, and triphosgene are reacted in an organic solvent to obtain the cyanine fluorescent compound.

4. The method for preparing cyanine fluorescent compounds according to claim 2, characterized in that: Compound (II) was prepared from compound (III); the structure of compound (III) is shown below: 。 5. The method for preparing cyanine fluorescent compounds according to claim 4, characterized in that: The preparation of compound (II) from compound (III) includes the following steps: under N2 or an inert gas atmosphere, compound (III) is reacted with an organic solvent under reflux to obtain compound (II).

6. The application of the cyanine fluorescent compound of claim 1 or the cyanine fluorescent compound obtained by the preparation method of any one of claims 2-5 in the detection of peroxynitrite.

7. The application of the cyanine fluorescent compound of claim 1 or the cyanine fluorescent compound obtained by the preparation method of any one of claims 2-5 in the preparation of a ratiometric fluorescent probe for detecting peroxynitrite.

8. A ratiometric fluorescent probe for detecting peroxynitrite, characterized in that: The compound comprises the cyanine fluorescent compound of claim 1 or the cyanine fluorescent compound obtained by the preparation method of any one of claims 2-5.

9. The use of the cyanine fluorescent compound of claim 1, the cyanine fluorescent compound obtained by the preparation method of any one of claims 2-5, or the ratiometric fluorescent probe of claim 8 in the preparation of diagnostic reagents or kits for drug-induced liver injury.

10. A diagnostic reagent or kit for drug-induced liver injury, characterized in that: It includes the cyanine fluorescent compound of claim 1, the cyanine fluorescent compound obtained by the preparation method of any one of claims 2-5, or the ratiometric fluorescent probe of claim 8.

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