A near-infrared carbon monoxide fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a near-infrared carbon monoxide fluorescent probe and a preparation method and application thereof, wherein the probe has a structure shown in formula I; a fluorescent unit of the probe is constructed by connecting 2-pyridine acetonitrile and hydroxyl xanthene derivative through an olefin bond; the probe itself has weak fluorescence and can release strong near-infrared fluorescence signals after responding to carbon monoxide. The probe has the advantages of rapid response, high sensitivity and good selectivity. The probe can sensitively detect expression changes of endogenous carbon monoxide levels, realize direct diagnosis of pathological degrees of drug-induced liver injury, and realize visual evaluation of liver-protecting effects of active components of traditional Chinese medicines. The application integrates disease diagnosis and drug efficacy evaluation functions in a single probe system, and provides an efficient and intuitive molecular imaging tool for early warning of drug-induced liver injury and screening of liver-protecting components of traditional Chinese medicines.

Description

Technical Field

[0001] This invention relates to the field of biomedical imaging technology, and in particular to a near-infrared carbon monoxide fluorescent probe, its preparation method, and its application. Background Technology

[0002] Drug-induced liver injury (DILI), caused by various chemical drugs, biological products, traditional Chinese medicines, and other drugs managed as prescription or over-the-counter medications, or their metabolites, is a common type of liver injury in clinical practice. Currently, the diagnosis of DILI mainly relies on exclusionary strategies based on causal relationship assessment, such as history taking, symptom and sign analysis, serum biochemical tests, imaging, and histological examinations. These strategies suffer from low diagnostic reliability, poor timeliness, and difficulties in subsequent management. Therefore, developing a real-time and sensitive detection strategy is urgently needed for timely diagnosis of DILI. Carbon monoxide (CO) is an endogenous signaling molecule produced by the degradation of heme oxidase (HO) in mammals. It plays an important antioxidant regulatory role in various pathological / physiological processes in vivo. Notably, under oxidative stress from DILI, hepatocytes can maintain cellular / tissue homeostasis by inducing CO expression and inhibiting oxidative stress responses. Therefore, monitoring changes in CO activity during drug-induced liver injury can be applied to the development of early diagnostic strategies for drug-induced liver injury (DILI).

[0003] Traditional Chinese medicine (TCM), as an important component of my country's traditional medicine, possesses unique advantages in disease prevention, treatment, and regulation of bodily functions. Research on the activity of TCM components is fundamental to the modernization of TCM and the development of innovative drugs. Currently, the evaluation of the activity of hepatoprotective components in TCM largely relies on traditional molecular biology methods, which are cumbersome, time-consuming, and costly. Therefore, there is an urgent need to develop a rapid, sensitive, and real-time detection tool that can reflect the efficacy of hepatoprotective components in TCM. In recent years, fluorescence imaging technology has shown broad application prospects in disease diagnosis and progression monitoring due to its high sensitivity and visualization advantages. Developing a new method that can assess the degree of drug-induced liver injury (DILI) and the efficacy of hepatoprotective components in TCM by detecting CO levels is of great significance for preventing drug-induced liver injury, optimizing drug treatment strategies, and improving the safety of TCM use.

[0004] CO has low activity concentration and short half-life in organisms. Therefore, an ideal fluorescent probe for accurately assessing the efficacy of drug-induced liver injury (DILI) and related drugs should have the following characteristics: (1) rapid response; (2) high sensitivity, capable of detecting low concentrations of endogenous CO; and (3) strong specificity, effectively eliminating interference from other reactive oxygen species / nitrogen species. Currently, many fluorescent probes have been used to detect CO. However, most of the reported cases still have some defects, such as slow reaction speed (greater than 20 min), fluorescence emission band located in the visible light region, and susceptibility to background fluorescence interference from biological macromolecules, which can lead to false positives in the detection signal and make it impossible to achieve accurate diagnosis of the disease and assessment of drug efficacy. In contrast, fluorescent probes with near-infrared wavelength emission (650-900 nm) can reduce biological background fluorescence interference, enhance tissue penetration depth, and improve the accuracy of fluorescent probes in diagnosing and imaging deep biological pathological tissues when used in biological applications. Based on this, developing a near-infrared fluorescent probe that can efficiently and specifically respond to CO is of great value for improving the early diagnosis of drug-induced liver injury and the accuracy and reliability of the efficacy assessment of hepatoprotective components of traditional Chinese medicine. Summary of the Invention

[0005] In response to the above situation, and to better address the problems encountered in the detection of drug-induced liver injury and the evaluation of the efficacy of hepatoprotective components of traditional Chinese medicine, this invention provides a near-infrared carbon monoxide fluorescent probe, its preparation method, and its application.

[0006] The primary objective of this invention is to provide a near-infrared carbon monoxide fluorescent probe (BMC-CO), wherein the molecular structure of this probe uses allyl carbonate as the recognition group and a 2-pyridineacetonitrile-oxanthracene derivative as the fluorescent signal group. Its detection mechanism is as follows: when Pd is present in the probe system... 2+ At that time, CO first put Pd 2+ Reduced to zero-valent palladium (Pd) 0 ), then Pd 0 The Tsuji-Trost reaction is mediated, triggering the breakage of the allyl carbamate bond in the probe structure, releasing a fluorescent group, thereby generating a near-infrared fluorescence signal. The structural formula of the fluorescent probe (BMC-CO) is shown in Formula I:

[0007]

[0008] Formula I.

[0009] The second objective of this invention is to provide a method for preparing a near-infrared carbon monoxide fluorescent probe, comprising the following steps:

[0010] 1) 0 oPhosphorus tribromide was added dropwise to a mixed solvent of N,N-dimethylformamide and dichloromethane under C conditions. After stirring for 0.5 h, cyclohexanone was added dropwise, and stirring was continued at room temperature (25 °C) for 8 h to 12 h. The reaction solution was then placed in an ice-water mixture, and sodium carbonate powder was added under stirring until no more bubbles were generated. The mixture was then extracted with dichloromethane, and the organic phases were combined and concentrated under reduced pressure to obtain an oily compound 1.

[0011] 2) 2-hydroxy-4-methoxy-benzaldehyde and cesium carbonate were added to a mixture of N,N-dimethylformamide and acetonitrile in which compound 1 was dissolved. The mixture was stirred at 25 °C for 18 h to 24 h. Cesium carbonate was removed by filtration, and the organic solvent was removed by vacuum concentration. The mixture was extracted with dichloromethane, and the organic phase was washed with distilled water. After concentration to remove the solvent, the crude product was purified by silica gel column chromatography to obtain solid compound 2.

[0012] 3) Compound 2 was dissolved in anhydrous dichloromethane and stirred at 0 °C. Boron tribromide was added dropwise and stirred at 25 °C for 8 h to 12 h. The reaction solution was then slowly placed into a saturated sodium carbonate solution until no more bubbles were generated. The solution was then extracted with a dichloromethane / methanol mixed solvent. After the organic solvent was removed by vacuum concentration, the crude product was purified by silica gel column chromatography to obtain solid compound 3.

[0013] 4) Add 2-pyridineacetonitrile and compound 3 to a mixed solvent of n-butanol and toluene, and heat the reaction mixture to 70°C. o After stirring for 6-8 hours, the organic solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain solid compound 4.

[0014] 5) Under ice bath conditions, allyl chloroformate was added to a dichloromethane solution containing compound 4 and triethylamine. The mixture was stirred at 25 °C for 1 h to 3 h. The solvent was removed by vacuum concentration. The crude product was purified by silica gel column chromatography to obtain the fluorescent probe compound BMC-CO.

[0015] The method for preparing a near-infrared carbon monoxide fluorescent probe according to claim 2 is characterized in that:

[0016] In step 1), the molar ratio of phosphorus tribromide to cyclohexanone is 3-6:1, and the volume ratio of N,N-dimethylformamide to dichloromethane is 2:5.

[0017] In step 2), the molar ratio of 2-hydroxy-4-methoxy-benzaldehyde, cesium carbonate and compound 1 is 1:1~2:1~2, the volume ratio of N,N-dimethylformamide and acetonitrile is 1:1, and the silica gel column chromatography eluent is petroleum ether and ethyl acetate in a volume ratio of 4:1.

[0018] In step 3), the molar ratio of compound 2 to boron tribromide is 1:10~30, the volume ratio of dichloromethane to methanol is 9:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0019] In step 4), the molar ratio of compound 3 to 2-pyridineacetonitrile is 1:1~2, the volume ratio of n-butanol to toluene is 1:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 6:1.

[0020] In step 5), the molar ratio of allyl chloroformate to compound 4 is 1~2:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 8:1.

[0021] A third objective of this invention is the application of the near-infrared carbon monoxide fluorescent probe in a reagent or kit for detecting drug-induced liver injury, wherein the reagent or kit comprises the fluorescent probe and a pharmaceutically acceptable carrier.

[0022] The fourth objective of this invention is the application of the near-infrared carbon monoxide fluorescent probe in reagents or kits for evaluating the hepatoprotective efficacy of active ingredients in traditional Chinese medicine, wherein the reagents or kits comprise the fluorescent probe and a pharmaceutically acceptable carrier.

[0023] The beneficial technical effects of this invention are as follows:

[0024] 1. The fluorescent core of the fluorescent probe described in this invention is constructed by linking 2-pyridine acetonitrile and a hydroxyoxanthracene derivative through olefinic bonds. Through ingenious molecular design, this fluorescent core constructs a "D-π-A" type push-pull electron system with pyridine and nitrile groups as electron-withdrawing units and hydroxyl groups as electron-donating units, exhibiting a significant intramolecular charge transfer effect. This structural characteristic enables the probe to emit a strong fluorescence signal in the near-infrared region (fluorescence emission wavelength of 660 nm) upon response to CO, offering advantages such as large tissue penetration depth, minimal photodamage, and low background interference, thus making it more suitable for pathological diagnostic research in biological systems.

[0025] 2. The fluorescent probe described in this invention exhibits excellent response performance, with a short response time to CO (less than 3 minutes), meeting the timeliness requirements for real-time and dynamic monitoring. It can respond to endogenous CO, and simultaneously possesses high sensitivity (lowest detection limit of 44 nM) and high selectivity, effectively eliminating interference from other reactive molecules in the environment and ensuring the accuracy and reliability of the detection results.

[0026] 3. The fluorescent probe described in this invention can be applied to the imaging diagnosis of drug-induced liver injury by responding to endogenous CO, providing an intuitive and visual detection method for the early detection and assessment of the severity of drug-induced liver injury. Furthermore, by directly observing the fluorescence changes caused by endogenous CO levels, this fluorescent probe can perform rapid and intuitive quantitative and qualitative analysis of the hepatoprotective efficacy of active ingredients in traditional Chinese medicine (represented in this experiment by baicalin, baicalein, oridonin, chlorogenic acid, and gentiopicrin), providing an efficient and intuitive detection tool for evaluating the activity of traditional Chinese medicine and assessing drug safety. Attached Figure Description

[0027] Figure 1 This is a synthetic route for the fluorescent probe BMC-CO.

[0028] Figure 2 This is the 1H NMR spectrum of the fluorescent probe BMC-CO.

[0029] Figure 3 This is a high-resolution mass spectrum of the fluorescent probe BMC-CO.

[0030] Figure 4 The fluorescent probe BMC-CO (5 μM) itself, or separately with Pd 2+ (5 μM), Pd 2+ Fluorescence emission spectrum after the reaction of (5 μM) + CO (20 μM). CORM-3 (tricarbonyl chloride (glycinyl)ruthenium) was used as the CO source donor.

[0031] Figure 5 The fluorescent probe system (5 μM BMC-CO, 5 μM Pd) 2+ Fluorescence emission spectra and fluorescence emission intensity (660 nm) of the response to 20 μM CO at different times (0-7 min).

[0032] Figure 6 The fluorescent probe system (5 μM BMC-CO, 5 μM Pd) 2+ ) and different concentrations of CO Fluorescence emission spectra after treatment with (0-20 μM) and linear relationship between fluorescence intensity (660 nm) and corresponding CO (0-20 μM) concentration.

[0033] Figure 7 To investigate the response specificity of the fluorescent probe BMC-CO. Probe system (5 μM BMC-CO, 5 μM Pd) 2+ The graphs show the changes in fluorescence emission intensity at 660 nm after co-incubation with CO or other analytes for 3 min. The experiment was repeated three times. 1, blank; 2-25. Na+ K + Ca 2+ Cu 2+ Mg 2+ Zn 2+ Fe 3+ Fe 2+ Cl - ,Br - I - SO3 2- HSO3 - HCO3 - H2PO4 - NO2 - NO3 - H2O2, Cys, Hcy, GSH, Arg, Tyr, Lys: 30 μM; 26. CO (20 μM). GSH is reduced glutathione, Cys is cysteine, Hcy is homocysteine, Arg is arginine, Tyr is tyrosine, and Lys is lysine.

[0034] Figure 8 Cellular imaging images of AML12 cells induced by acetaminophen (APAP) using the fluorescent probe BMC-CO. AML12 cells were first co-incubated with different concentrations of APAP (0, 125, 250, 500 μM) for 12 h, and then incubated with probe BMC-CO (5 μM) + Pd. 2+ (5 μM) Co-incubated for 0.5 h. Cell locations were marked using nuclear imaging (DAPI), scale bar: 40 μm.

[0035] Figure 9 Cellular imaging images showing the efficacy of different traditional Chinese medicine components (baicalin, baicalein, oridonin A, chlorogenic acid, and gentiopicrin) in alleviating drug-induced liver injury using the fluorescent probe BMC-CO. Cells were first co-incubated with 20 μM of each of the different traditional Chinese medicine components for 6 h, then co-incubated with APAP (500 μM) for 12 h, and finally incubated with probe BMC-CO (5 μM) + Pd. 2+ (5 μM) Co-incubated for 0.5 h. Cell locations were marked using nuclear imaging (DAPI), scale bar: 40 μm. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] A method for synthesizing a near-infrared carbon monoxide fluorescent probe, the synthetic route is as follows: Figure 1 As shown, the structural identification is as follows Figure 2 , Figure 3 As shown, the specific steps include:

[0039] (1) Synthesis of compound 1

[0040] 0 o Phosphorus tribromide (8.46 mL, 90 mmol) was slowly added dropwise to a mixed solvent of N,N-dimethylformamide (DMF, 8 mL) and dichloromethane (20 mL). After stirring for 0.5 h, cyclohexanone (3.11 mL, 30 mmol) was added dropwise, and stirring was continued at room temperature (25 °C) for 12 h. The reaction mixture was then slowly placed in an ice-water mixture, and sodium carbonate powder was added under stirring until no more bubbles were generated. The mixture was then extracted with dichloromethane, and the organic phase was removed by concentration under reduced pressure to obtain an oily compound 1 (4.51 g, yield 79.5%), which was directly used in the next reaction.

[0041] (2) Synthesis of compound 2

[0042] 2-Hydroxy-4-methoxy-benzaldehyde (3.04 g, 20 mmol) and cesium carbonate (6.52 g, 20 mmol) were added to a mixture of N,N-dimethylformamide and acetonitrile (volume ratio 1:1) containing compound 1 (3.78 g, 20 mmol). The reaction system was stirred at 25 °C for 24 h. Cesium carbonate was removed by filtration, and the organic solvent was removed by concentration under reduced pressure. The mixture was extracted with dichloromethane, washed with distilled water, and then the organic solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain solid compound 2 (3.06 g, yield 63.1%).

[0043] (3) Synthesis of compound 3

[0044] Compound 2 (2.35 g, 9.7 mmol) was dissolved in 30 mL of anhydrous dichloromethane. Boron tribromide (9.35 mL, 97 mmol) was added dropwise to the reaction solution under stirring at 0 °C. The mixture was stirred at room temperature (25 °C) for 12 h. The reaction solution was then slowly placed in a saturated sodium carbonate solution. When no more bubbles were generated, the mixture was extracted with a mixed solvent of dichloromethane and methanol (9:1 v / v). The organic solvent was removed by vacuum concentration. Finally, the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain solid compound 3 (1.89 g, yield 85.3%).

[0045] (4) Synthesis of compound 4

[0046] Compound 3 (1.76 g, 7.71 mmol) and 2-pyridineacetonitrile (0.95 g, 8 mmol) were added to 20 mL of a mixed solvent of n-butanol and toluene (volume ratio 1:1). The reaction solution was heated to 70 °C. o C. Stir for 6 h. After the reaction is complete, remove the organic solvent by vacuum distillation, and purify the crude product by silica gel column chromatography (petroleum ether: ethyl acetate = 6:1) to give solid compound 4 (2.06 g, yield 81.3%).

[0047] (5) Synthesis of fluorescent probe compound BMC-CO

[0048] Under ice bath conditions, allyl chloroformate (850 μL, 8 mmol) was added to 25 mL of dichloromethane containing compound 4 (1.86 g, 5.66 mmol) and triethylamine (100 μL). The mixture was stirred at room temperature (25 °C) for 1 h. After removing the solvent by concentration under reduced pressure, the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain the fluorescent probe compound BMC-CO (1.95 g, yield 83.5%).

[0049] Fluorescent probe BMC-CO structural characterization spectral data: 1 H NMR (600 MHz, CDCl3): δ H8.78 (s,1H), 8.64-8.61 (m, 1H), 7.73-7.65 (m, 2H), 7.2-7.14 (m, 1H), 7.10-7.04 (m,2H), 6.86 (dd, J = 8.3, 2.3 Hz, 1H), 6.50 (s, 1H), 6.05-5.96 (m, 1H), 5.45(dq, J = 17.1, 1.5 Hz, 1H), 5.36 (dd, J = 10.4, 1.6 Hz, 1H), 4.75 (dd, J =5.8, 1.5 Hz, 2H), 3.03 (t, J = 6.1 Hz, 2H), 2.56 (dd, J = 9.1, 3.8 Hz, 2H),1.86-1.82 (m, 2H). HRMS: m / z [M + H] + Calculated value C 25 H 21 N2O4 413.1423, found value 413.1501.

[0050] The fluorescent probe (BMC-CO) of this invention consists of a fluorescent core composed of 2-pyridine acetonitrile and an oxanthracene derivative linked by an olefinic bond. Its hydroxyl groups are protected by an allyl carbamate. This structure exhibits weak fluorescence in its initial state. (The last sentence appears to be incomplete and possibly refers to a specific technology or process.) 2+ In its presence, CO can reduce it to Pd. 0 This triggers the Tsuji-Trost reaction, specifically cleaving the allyl carbamate group and releasing hydroxyl groups, thereby activating and generating a significant near-infrared fluorescence emission signal (maximum emission wavelength of 660 nm). This fluorescent probe responds rapidly to CO, reaching fluorescence response equilibrium within 3 minutes. It exhibits high detection sensitivity, with a detection limit for CO as low as 44 nM, and excellent selectivity, effectively eliminating interference from common bioactive species. The fluorescent probe achieved the expected results in a drug-induced hepatocyte injury model, sensitively reflecting the dynamic changes of endogenous CO at different pathological degrees, enabling intuitive and visual monitoring of the severity of drug-induced liver injury. Furthermore, this probe can intuitively and visually detect changes in CO expression levels, efficiently evaluating the therapeutic and alleviating effects of active ingredients in traditional Chinese medicine (represented by baicalin, baicalein, oridonin A, chlorogenic acid, and gentiopicrin) on drug-induced liver injury, providing an intuitive and reliable molecular imaging tool for evaluating the efficacy of traditional Chinese medicine components. Relevant experimental data are as follows:

[0051] I. Detection efficacy of the fluorescent probe BMC-CO for CO

[0052] Weigh an appropriate amount of the fluorescent probe BMC-CO and dissolve it in DMSO to prepare a stock solution (5 mM). Dissolve PdCl2 in deionized water to prepare a 5 mM stock solution, using CORM-3 (tricarbonyl chloride (glycinyl)ruthenium) dissolved in deionized water as the CO donor. The buffer solution prepared for the spectral test was a PBS buffer (10 mM) containing 1% DMSO at pH = 7.4, and the fluorescence excitation wavelength was 530 nm.

[0053] First, the spectral characteristics of the fluorescent probe BMC-CO in response to CO were investigated. Fluorescence spectra were tested under physiological conditions for three different scenarios: BMC-CO (5 μM), BMC-CO (5 μM) + Pd 2+ (5 μM), BMC-CO (5 μM) + Pd 2+ (5 μM) + CO donor CORM-3 (20 μM). Results are as follows: Figure 4 As shown, under 530 nm excitation, the probe BMC-CO itself exhibits weak fluorescence signal, while BMC-CO reacts with Pd... 2+ After co-incubation, the fluorescence signal did not change significantly, indicating that the probe cannot interact with Pd alone. 2+ A reaction occurs. When the CO donor CORM-3 is added to the probe system and co-incubated, the fluorescence intensity (660 nm) of the test system increases significantly, indicating that the probe BMC-CO can only be effectively activated and release near-infrared fluorescence signals in the presence of CO.

[0054] When performing response time spectroscopy testing, measure 3 μL of the stock solution (fluorescent probes BMC-CO and Pd). 2+ Add an appropriate volume of CORM-3 stock solution to PBS buffer (pH = 7.4, 1% DMSO, 10 mM) to make the probe concentration 5 μM. 2+ The concentration of CO was 5 μM, the concentration of CO was 20 μM, and the test solution volume was 3 mL. After incubation for different times (0-7 min), the fluorescence emission spectra were measured. The changes in the fluorescence spectra are shown below. Figure 5 As shown, the fluorescence intensity at 660 nm is time-dependent, gradually increasing with the extension of the reaction time, and after about 3 min, the fluorescence intensity no longer shows significant changes, indicating that the probe has a rapid time response capability to CO, and the response time is better than most reported CO fluorescent probes. BMC-CO has the ability to rapidly detect CO in biological samples.

[0055] When performing fluorescence titration experiments, a 5 μM probe system (BMC-CO + Pd) is used. 2+The sample was reacted with different concentrations of CO (0-20 μM) in PBS buffer for 3 min, followed by fluorescence emission spectroscopy detection. Figure 6 As shown, the fluorescence intensity (660 nm) of the test buffer gradually increases with increasing CO concentration, and the fluorescence intensity has a good linear relationship with CO concentration (R0). 2 = 0.9978), according to the detection limit equation (LOD = 3σ / k), the lowest detection limit of the probe response to CO can be calculated to be 44 nM, indicating that the fluorescent probe BMC-CO has excellent detection sensitivity and strong recognition ability.

[0056] II. Environmental Specificity Study of Fluorescent Probe BMC-CO

[0057] The 5 μM probe system (BMC-CO + Pd) 2+ ) and prepared CO (20 μM) or various analytes (Na) at a concentration of 30 μM. + K + Ca 2+ Cu 2+ Mg 2+ Zn 2+ Fe 3+ Fe 2+ Cl - ,Br - I - SO3 2- HSO3 - HCO3 - H2PO4 - NO2 - NO3 - (H₂O₂, Cys, Hcy, GSH, Arg, Tyr, Lys) were reacted in PBS buffer for 3 min, followed by fluorescence spectroscopy detection. Results are as follows: Figure 7 As shown, only CO can cause a significant change in the fluorescence intensity of the test system, while the fluorescence changes caused by other analytes are negligible. This indicates that the fluorescent probe system has excellent selective recognition performance for CO and good specificity.

[0058] III. Application of the fluorescent probe BMC-CO in detecting the degree of drug-induced hepatocyte damage and evaluating the hepatoprotective efficacy of active ingredients in traditional Chinese medicine.

[0059] This invention uses the fluorescent probe BMC-CO to detect changes in CO activity and diagnoses drug-induced hepatocellular injury using cell imaging. Liver AML12 cells were co-incubated with different concentrations of acetaminophen (APAP, 0, 125, 250, 500 μM) for 12 h, and then incubated with the probe BMC-CO (5 μM) + Pd.2+ Cells were co-incubated with 5 μM for 0.5 h, and then imaged using an ImageXpress Micro Confocal high-content imaging system. Results are as follows: Figure 8 As shown, the cellular fluorescence intensity increases with increasing APAP concentration, indicating that the upregulated CO expression induced by APAP-induced hepatocyte injury can respond to the probe BMC-CO, thereby releasing a visible fluorescent signal. This fluorescent probe can conveniently and intuitively detect the pathological degree of drug-induced liver injury through fluorescence signals.

[0060] Next, using the changes in fluorescence signal induced by CO as a detection index, the therapeutic efficacy of representative active ingredients of traditional Chinese medicine, baicalin, baicalein, oridonin, chlorogenic acid, and gentiopicroside, in alleviating drug-induced liver injury was evaluated using the probe BMC-CO. Liver AML12 cells were co-cultured with each active ingredient (20 μM) for 6 h, then incubated with 500 μM APAP for 12 h, and finally with the probe system (5 μM BMC-CO + 5 μM Pd). 2+ After co-incubation for 0.5 h, cell imaging was performed. Figure 9 As shown, the fluorescence intensity of cells decreased significantly after treatment with traditional Chinese medicine (TCM) components, indicating that when TCM components with hepatoprotective effects alleviate liver damage, CO expression activity in the microenvironment decreases, thereby reducing the fluorescence signal generated by the probe. The experiment demonstrates that the fluorescent probe BMC-CO can evaluate the efficacy of hepatoprotective TCM components in treating drug-induced liver injury by responding to the visualized fluorescence signal generated by CO in the microenvironment, showing significant practical application value.

[0061] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A near-infrared carbon monoxide fluorescent probe, characterized in that, The chemical structural formula is shown in Formula I: Formula I.

2. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 1, characterized in that, Includes the following steps: 1) 0 o Phosphorus tribromide was added dropwise to a mixed solvent of N,N-dimethylformamide and dichloromethane under C conditions. After stirring for 0.5 h, cyclohexanone was added dropwise, and stirring was continued at room temperature (25 °C) for 8 h to 12 h. The reaction solution was then placed in an ice-water mixture, and sodium carbonate powder was added under stirring until no more bubbles were generated. The mixture was then extracted with dichloromethane, and the organic phases were combined and concentrated under reduced pressure to obtain an oily compound 1. 2) 2-hydroxy-4-methoxy-benzaldehyde and cesium carbonate were added to a mixture of N,N-dimethylformamide and acetonitrile in which compound 1 was dissolved. The mixture was stirred at 25 °C for 18 h to 24 h. Cesium carbonate was removed by filtration, and the organic solvent was removed by vacuum concentration. The mixture was extracted with dichloromethane, and the organic phase was washed with distilled water. After concentration to remove the solvent, the crude product was purified by silica gel column chromatography to obtain solid compound 2. 3) Compound 2 was dissolved in anhydrous dichloromethane and stirred at 0 °C. Boron tribromide was added dropwise and stirred at 25 °C for 8 h to 12 h. The reaction solution was then slowly placed into a saturated sodium carbonate solution until no more bubbles were generated. The solution was then extracted with a dichloromethane / methanol mixed solvent. After the organic solvent was removed by vacuum concentration, the crude product was purified by silica gel column chromatography to obtain solid compound 3. 4) Add 2-pyridineacetonitrile and compound 3 to a mixed solvent of n-butanol and toluene, and heat the reaction mixture to 70°C. o After stirring at C for 6-8 hours, the organic solvent was removed by vacuum distillation. The crude product was then purified by silica gel column chromatography to obtain solid compound 4. 5) Under ice bath conditions, allyl chloroformate was added to a dichloromethane solution containing compound 4 and triethylamine. The mixture was stirred at 25°C for 1 h to 3 h. The solvent was removed by vacuum concentration. The crude product was purified by silica gel column chromatography to obtain the fluorescent probe compound BMC-CO.

3. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 2, characterized in that, In step 1), the molar ratio of phosphorus tribromide to cyclohexanone is 3-6:1, and the volume ratio of N,N-dimethylformamide to dichloromethane is 2:

5.

4. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 2, characterized in that, In step 2), the molar ratio of 2-hydroxy-4-methoxy-benzaldehyde, cesium carbonate and compound 1 is 1:1~2:1~2, the volume ratio of N,N-dimethylformamide and acetonitrile is 1:1, and the silica gel column chromatography eluent is petroleum ether and ethyl acetate in a volume ratio of 4:

1.

5. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 2, characterized in that, In step 3), the molar ratio of compound 2 to boron tribromide is 1:10~30, the volume ratio of dichloromethane to methanol is 9:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 3:

1.

6. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 2, characterized in that, In step 4), the molar ratio of compound 3 to 2-pyridineacetonitrile is 1:1~2, the volume ratio of n-butanol to toluene is 1:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 6:

1.

7. The method for preparing the near-infrared carbon monoxide fluorescent probe according to claim 2, characterized in that, In step 5), the molar ratio of allyl chloroformate to compound 4 is 1~2:1, and the eluent for silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 8:

1.

8. The use of the near-infrared carbon monoxide fluorescent probe according to any one of claims 1-7 in a reagent or kit for detecting drug-induced liver injury, wherein the reagent or kit comprises the fluorescent probe and a pharmaceutically acceptable carrier.

9. The use of the near-infrared carbon monoxide fluorescent probe according to any one of claims 1-7 in a reagent or kit for evaluating the hepatoprotective efficacy of active ingredients in traditional Chinese medicine, wherein the reagent or kit comprises the fluorescent probe and a pharmaceutically acceptable carrier.

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