Near-infrared active oxygen fluorescent probe, preparation method thereof and application of near-infrared active oxygen fluorescent probe in high-throughput screening of anti-pan-apoptosis drugs

Abstract

The invention discloses a near-infrared active oxygen fluorescent probe, the structural formula of which is as follows: the probe has excellent low detection limit (0.42 mu M), high sensitivity and high selectivity, not only monitors the level of hydrogen peroxide in cells in real time in the LL-IRI process, but also successfully screens out anti-pan apoptosis drugs with remarkable effects based on a high-throughput screening platform. Therefore, an ideal tool is provided for deeply researching the pathogenesis of the lower limb ischemia reperfusion injury.

Description

Technical Field

[0001] This invention relates to the field of medical fluorescent probes, and more particularly to a near-infrared reactive oxygen species fluorescent probe, its preparation method, and its application in high-throughput screening of anti-apoptotic drugs. Background Technology

[0002] Lower extremity ischemia-reperfusion injury (LL-IRI) refers to a systemic immune response following vascular recanalization treatment of acute or chronic lower extremity arterial embolism. Although global epidemiological data on LL-IRI are currently lacking, this disease is clinically common and seriously endangers patients' lives, especially acute bilateral lower extremity ischemia caused by saddle emboli in the abdominal aorta-iliac artery. This often results in limb tissue necrosis, and with the reperfusion of blood flow after thrombectomy, the release of reactive oxygen species (ROS) and the resulting fulminant inflammation due to increased systemic oxidative stress can lead to death. Although relevant guidelines and expert consensus do not recommend drug treatment, given the current urgent clinical need, it is imperative to explore the pathogenesis of LL-IRI and find non-invasive, cost-effective, and easily administered treatment methods.

[0003] Studies have shown that reactive oxygen species (ROS) play a dual role in LL-IRI, not only acting as crucial agents in cell signal transduction but also inducing oxidative damage, further leading to programmed cell death in forms such as apoptosis, pyroptosis, and necroptosis. The concept of pan-apoptosis, first proposed in 2019, refers to the processes associated with multiple forms of cell death, including pyroptosis, apoptosis, and necroptosis; it is a novel mode of cell death associated with the occurrence and progression of various diseases. Currently, the mechanisms of ROS-mediated pan-apoptosis in LL-IRI are poorly understood, and high-throughput screening platforms are lacking to screen for relevant anti-pan-apoptotic drugs. Hydrogen peroxide (H₂O₂) is one of the most important reactive oxygen species in LL-IRI processes. H₂O₂ not only directly oxidizes proteins, lipids, and DNA but also acts as a signaling molecule, activating multiple inflammatory pathways and exacerbating tissue damage and death.

[0004] Plantain (PW) is abundant in Guangdong, Guangxi, and Hainan provinces. It is the dried whole herb of *Plantago asiatica* or *Plantago depressa*, belonging to the Plantaginaceae family. The entire herb has medicinal value and was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) as a superior-grade medicine. It possesses heat-clearing, detoxifying, anti-inflammatory, and antioxidant properties, making it a potential drug for treating LL-IRI. PW contains various chemical components, including flavonoids, glycosides, triterpenes, and iridoids. This study used high-performance liquid chromatography (HPLC) to identify over 90 compounds in PW. To rapidly explore the most effective monomers and optimal drug concentrations for anti-oxidative stress in PW, it is urgent to develop near-infrared reactive oxygen species (ROS) fluorescent probes for high-throughput screening of anti-pan-apoptotic drugs. Summary of the Invention

[0005] In view of this, the present invention provides a near-infrared reactive oxygen species fluorescent probe, the structural formula of which is shown in Formula 1:

[0006]

[0007] This invention provides an application of a near-infrared reactive oxygen species fluorescent probe in high-throughput screening of anti-pan-apoptotic drugs.

[0008] Preferably, the fluorescent probe can monitor intracellular H2O2 levels in real time.

[0009] Preferably, this fluorescent probe is used to monitor reactive oxygen species levels in an LL-IRI mouse model.

[0010] Another aspect of the present invention provides a method for preparing a near-infrared reactive oxygen species fluorescent probe, comprising the following steps: weighing (E)-2-((E)-3-(4-(dimethylamino)phenyl)prop-2-enyl)methylene)-6-hydroxybenzofuran-3(2H)-one, potassium carbonate, and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane and sequentially adding them to an N,N-dimethylformamide solution, heating and stirring the reaction, and after purification, obtaining (E)-2-((E)-3-(4-(dimethylamino)phenyl)allyl)-6-((4-(4,4,5,5-tetramethylpyridin-1,3,2-dioxaborane-2-yl)benzyl)oxy)benzofuran-3(2H)-one.

[0011] Preferably, (E)-2-((E)-3-(4-(dimethylamino)phenyl)prop-2-enyl)methylene)-6-hydroxybenzofuran-3(2H)-one is prepared by the following method: 6-hydroxybenzofuran-3(2H)-one is dissolved in ethanol, (E)-3-(4-(dimethylamino)phenyl)propenal and NaOH solution are added at room temperature, the mixture is heated and stirred, diluted with distilled water and the pH is adjusted with HCl, and the obtained solid precipitate is washed and dried.

[0012] This study designed a near-infrared reactive oxygen species (ROS) fluorescent probe with a low detection limit (0.42 μM), high sensitivity, and high selectivity for hydrogen peroxide. It not only enables real-time monitoring of intracellular H2O2 levels but also successfully achieved real-time monitoring of H2O2 during LL-IRI. Furthermore, based on a high-throughput screening platform using BFP-H2O2, the probe successfully screened 16 major active ingredients from Plantago asiatica extract. Among them, aucubin (AU) showed the strongest ability to scavenge ROS (H2O2). Subsequent in vivo and in vitro experiments confirmed that aucubin can increase the level of antioxidant enzymes, scavenge ROS, reduce oxidative stress, inhibit the expression of pan-apoptotic proteins, and alleviate acute LL-IRI. Attached Figure Description

[0013] Figure 1 The image shows the 1H NMR spectrum of BFP-H2O2, a near-infrared reactive oxygen species fluorescent probe.

[0014] Figure 2 High-resolution mass spectrometry of BFP-H2O2, a near-infrared reactive oxygen species fluorescent probe;

[0015] Figure 3 The graph shows the response of the fluorescent probe BFP-H2O2 (10 μM) to H2O2 in vitro. 3A represents the UV-Vis spectra of BFP-H2O2 before and after the addition of H2O2 (100 μM); 3B represents the fluorescence response spectra of BFP-H2O2 at different concentrations of H2O2 (0-300 μM); 3C represents the fluorescence spectral changes of BFP-H2O2 before and after the addition of H2O2 (100 μM); 3D represents the fluorescence intensity versus time relationship of BFP-H2O2 after the addition of H2O2; 3E represents the pH-dependent changes of BFP-H2O2 before and after the addition of H2O2 (100 μM); and 3F represents the fluorescence intensity of BFP-H2O2 in the presence of different substances (1. blank, 2. K). + 3.Zn 2+ 4.Mg 2+ 5.Fe 2+ 6.Cu 2+ 7.Ca 2+ 8.PO4 3- 9.NO2 - 10.SO4 2- , 11.CO2, 12.Glu, 13.Arg, 14.Cys, 15.Hcy, 16.GSH, 17. 1 O2, 18.ClO - 19.ONOO - The fluorescence response spectrum of ,20.·OH)(100μM).

[0016] Figure 4 The near-infrared fluorescent probe BFP-H2O2 is used to image H2O2 in rat skeletal muscle cells. 4A represents rat skeletal muscle cells incubated with different concentrations of exogenous H2O2 inducers (0, 25, 50, 100, 150, 200 μM) of H2O2, stained with BFP-H2O2 (10 μM) for 30 min, and then imaged at the cell level. B represents cells incubated with different concentrations of endogenous H2O2 inducers (0, 0.5, 1, 5, 10, 20 μg / mL) of LPS for 3 h, stained with BFP-H2O2 (10 μM) for 30 min, and then imaged at the cell level.

[0017] Figure 5The near-infrared fluorescent probe BFP-H2O2 was used to image H2O2 in a panapoptotic model of rat skeletal muscle cells. 5A represents different amounts of panapoptotic inducers TAK1i / LPS (0.0 / 0.0, 0.01 / 0.01 μM / μg / mL, 0.03 / 0.03 μM / μg / mL, 0.05 / 0.05 μM / μg / mL, 0.1 / 0.1 μM / μg / mL, 0.15 / 0.15 μM / μg / mL) followed by incubation with BFP-H2O2 (10 μM) for 30 min and then imaging at the cellular level. 5B represents the normalized fluorescence intensity of 5A.

[0018] Figure 6 This diagram illustrates the screening of anti-apoptotic natural compounds using the near-infrared fluorescent probe BFP-H2O2. 6A represents cell images obtained after 12 h of pretreatment with 16 major components (100 μM) of Plantago asiatica, followed by 1 h of stimulation with TAK1i (0.1 μM), 3 h of LPS (0.1 μg / mL), and 30 min of staining with BFP-H2O2 (10 μM). 6B represents the normalized mean fluorescence intensity of 6A.

[0019] Figure 7 The images show the cell images obtained after pretreating rat skeletal muscle cells with different concentrations of aucubin for 12 h, followed by stimulation with TAK1i (0.1 μM) for 1 h, then stimulation with LPS (0.1 μg / mL) for 3 h, and finally staining with BFP-H2O2 (10 μM) for 30 min. 7B represents the mean fluorescence intensity after normalization of 7A.

[0020] Figure 8 The images show the anti-apoptotic effect of aucubin in the LL-IRI mouse model, where 8A is a schematic diagram of the experimental procedure; 8B represents the fluorescence imaging image after in situ injection of BFP-H2O2 (250μM, 200μL) in the LL-IRI mouse model; and 8C is the average fluorescence intensity of each mouse in 8B.

[0021] Figure 9 This is a schematic diagram illustrating the application of a near-infrared reactive oxygen species fluorescent probe in high-throughput screening of natural compounds in an LL-IRI mouse model. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1: A near-infrared reactive oxygen species fluorescent probe BFP-H2O2, as shown in Formula 1:

[0024]

[0025] Example 2: A near-infrared reactive oxygen species fluorescent probe, prepared by the following methods (such as Formulas 2 and 3):

[0026] (1) Dissolve 300 mg (2 mmol) of 6-hydroxybenzofuran-3(2H)-one in 30 mL of ethanol. Add 385.5 mg (2.2 mmol) of (E)-3-(4-(dimethylamino)phenyl)propenal and 4 mL of (1 M) NaOH solution at room temperature. Heat and stir the reaction mixture at 60 °C for 3 h. After the reaction is complete, dilute with distilled water and adjust the pH to 7 with 1 N HCl. A large amount of solid is generated during this process. Wash the obtained solid with water and dry it to obtain (E)-2-((E)-3-(4-(dimethylamino)phenyl)prop-2-enyl)methylene)-6-hydroxybenzofuran-3(2H)-one (BFP), which can be used for the next reaction without purification.

[0027]

[0028] (2) As in Formula 3, weigh the BFP (307 mg, 1.0 mmol), potassium carbonate (276 mg, 2.0 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (445 mg, 1.5 mmol) prepared in step (1) and add them sequentially to the N,N-dimethylformamide (10.0 mL) solution. The reaction mixture was heated to 80°C and stirred for 6 hours. The solvent was then removed, and the crude product was redissolved in dichloromethane. Using dichloromethane as the eluent, the product was purified by silica gel column chromatography to obtain (E)-2-((E)-3-(4-(dimethylamino)phenyl)allyl)-6-((4-(4,4,5,5-tetramethylpyridin-1,3,2-dioxoboron-2-yl)benzyl)oxy)benzofuran-3(2H)-one BFP-H2O2 (183 mg, 35%). 1HNMR(400MHz, CDCl3):7.85(d,J=8.0Hz,2H),7.67(d,J=8.6Hz,1H),7.44(d,J=8.1Hz,4H),7.07(dd,J=15.4,11.5Hz,1H),6.92 13C NMR (100MHz, CDCl3):181.72,167.11,165.75,151.06,147.12,141.93,138.76,135.17,131.01,129.02 ,126.53,125.46,124.56,116.42,116.05,115.32,112.23,111.98,97.38,83.89,70.51,40.18,24.84. HRMS m / z:C 32 H 34 BNO5[M+H] + The mass-to-charge ratio is 524.2608. (From...) Figure 1 The nuclear magnetic hydrogen spectrum and Figure 2 High-resolution mass spectrometry results indicate that BFP-H2O2 was successfully prepared.

[0029]

[0030] Example 3: Study on the spectral properties of the fluorescent probe BFP-H2O2

[0031] like Figure 3 As shown in the UV absorption spectrum of A, a strong absorption peak was observed at 485 nm for BFP-H2O2. Upon addition of 10 μM H2O2, the absorption peak blue-shifted to 460 nm. (As shown in the image...) Figure 3 The fluorescence spectra of BFP-H2O2 in H2O2 solutions of different concentrations show that the fluorescence intensity of BFP-H2O2 at 650 nm gradually increases with increasing H2O2 concentration (5-80 μM). When the H2O2 concentration is 100 μM, the fluorescence intensity approaches saturation. The limit of detection (LOD) calculated based on the 3σ / slope method is determined to be 0.42 μM, indicating that the fluorescent probe BFP-H2O2 has high detection sensitivity. Figure 3 As shown in C and 3D, the fluorescence intensity of single BFP-H2O2 at 650 nm remained stable within 60 min; however, after adding H2O2, the fluorescence intensity significantly increased and stabilized within 45 min, indicating that the probe's retention time is stable and its detection efficiency is good. Figure 3As shown in Figure E, BFP-H2O2 exhibited only minor changes over a wide pH range from 2.0 to 12.0; upon the addition of H2O2, the fluorescence intensity of BFP-H2O2 gradually increased across the pH range of 3.0 to 12.0. The fluorescence response of BFP-H2O2 was particularly pronounced at pH 7.4, indicating its potential for detecting H2O2 in physiological settings. Figure 3 As shown in Figure F, BFP-H2O2 detects H2O2 in complex environments, including in the presence of other substances such as metal ions (K+). + Zn 2+ Mg 2+ Fe 2+ Cu 2+ Ca 2+ ), anion (PO4) 3- NO2 - SO4 2- Carbon dioxide), amino acids (Glu, Arg, Cys, Hcy, GSH) and reactive oxygen species (1O2, ClO) - ONOO - The results showed that BFP-H2O2 is a reliable near-infrared probe with high selectivity, which is beneficial for monitoring H2O2 changes in complex environments.

[0032] Example 4: Studying the imaging capability of the fluorescent probe BFP-H2O2 at the cellular level

[0033] (1) As Figure 4 As shown in Figure A, rat skeletal muscle cells were induced by incubation with different concentrations of exogenous H2O2 (0, 25, 50, 100, 150, 200 μM) and then stained with BFP-H2O2 (10 μM) for 30 min. As the H2O2 concentration increased, the fluorescence intensity of BFP-H2O2 in the cells increased, and the fluorescence brightness reached its strongest at a H2O2 concentration of 150 μM. This indicates that BFP-H2O2 can achieve real-time dynamic monitoring of changes in extracellular H2O2 levels.

[0034] (2) Rat skeletal muscle cells (passaged <10) were cultured at 37℃ in 5% CO2 / 5% O2. Pan-apoptosis was induced by stimulation with different concentrations of endogenous H2O2 inducer-LPS (0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL) for 3 h. Cellular imaging was then performed using BFP-H2O2 (10 μM) for 30 min. As shown in 4B, when cells were stimulated by endogenous H2O2 inducer-LPS, the fluorescence intensity increased in a concentration-dependent manner within the range of 0-20 μg / mL, reaching its peak at a concentration of 10 μg / mL. This indicates that BFP-H2O2 can achieve real-time dynamic monitoring of intracellular H2O2 levels.

[0035] To further determine the relationship between pan-apoptosis and H2O2, a pan-apoptotic cell model was established using a classic pan-apoptotic inducer: a TAK1 inhibitor combined with LPS. Rat skeletal muscle cells were pretreated with 0.01 / 0.03 / 0.05 / 0.1 / 0.5 μM TAK1 inhibitors for 1 h, and then stimulated with different concentrations (0.01 / 0.03 / 0.05 / 0.1 / 0.5 μg / ml) of LPS for 3 h to induce pan-apoptosis. BFP-H2O2 (10 μM) staining was performed for 30 min, and changes in fluorescence signal were monitored. Figure 5 Results A and 5B indicate that the fluorescence signal gradually increased with increasing TAK1i / LPS concentration. After treatment with 0.1 μM / 0.1 μg / mL TAK1i / LPS, the fluorescence intensity tended to stabilize, indicating that TAK1i / LPS-induced pan-apoptosis is positively correlated with intracellular H2O2 levels. These results suggest that intracellular H2O2 levels can be used as a basis for screening anti-pan-apoptotic drugs.

[0036] Example 5: Application of fluorescent probe BFP-H2O2 in high-throughput screening of anti-pan-apoptotic drugs

[0037] (1) Sixteen monomeric compounds extracted from Plantago asiatica were selected as screening drugs, including Skimmin, Ergosterol, Bilobetin, Isorhamnetin, Paeonol, Hecogenin acetate, Zeaxanthin, Geniposidic acid, Aucubin, Cepharanthine, Dehydroabietic acid, Fucoxanthin, Bergapten, Kaempferide, Astragalin, and Nepetin.

[0038] (2) Panapoptosis experimental design: First, a rat skeletal muscle cell line was used, divided into:

[0039] CON group cells were cultured normally;

[0040] The DMSO group was pretreated with 0.1 μM TAK1 inhibitor for 1 h, and then stimulated with 0.1 μg / mL lipopolysaccharide for 3 h to induce pan-apoptosis.

[0041] In the treatment groups, cells were pre-incubated for 12 hours with 16 different natural chemical drugs (100 μM), and pan-apoptotic induction was performed using the same procedure as the DMSO group. Cells were then stained with 10 μM BFP-H2O2 for 30 minutes before CLSM imaging. Among the tested compounds, Au exhibited the most significant anti-PANoptotic activity. Figure 6 B). Further research results, such as Figure 7 This indicates that aucubin has a dose-dependent antioxidant effect in a TAK1i / LPS-induced panapoptotic cell model.

[0042] Example 6: Application of fluorescent probe BFP-H2O2 in monitoring ROS in an LL-IRI mouse model

[0043] H2O2 dynamics in the LL-IRI mouse model were monitored using BFP-H2O2. Mice were divided into three groups: control group (CON), LL-IRI group (bilateral lower limb groin ligation with orthodontic elastic bands for 3 h of ischemia, followed by reperfusion for 0.5, 1.0, or 2.0 h), and Au pretreatment group (intraperitoneal injection of 5, 10, or 15 mg / kg Au 2 h before modeling). Figure 8 A). Figure 8B shows that the fluorescence signal in the hind limbs of the CON group mice was weak, while the fluorescence intensity in the LL-IRI group mice gradually increased with the extension of reperfusion time, indicating an increase in H2O2 levels. This demonstrates that the fluorescent probe BFP-H2O2 can monitor changes in H2O2 levels in the LL-IRI mouse model, further indicating that aucubin can effectively inhibit the accumulation of H2O2 during LL-IRI.

[0044] In summary, this invention provides a near-infrared reactive oxygen species fluorescent probe BFP-H2O2, which has a low detection limit (0.42 μM), high sensitivity, and high selectivity, such as... Figure 9 As shown, BFP-H2O2 provides an ideal tool for real-time monitoring of intracellular H2O2 levels and high-throughput screening of anti-apoptotic drugs.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A near-infrared reactive oxygen species fluorescent probe, characterized in that, Its structural formula is as shown in Formula 1:

2. The application of the near-infrared reactive oxygen species fluorescent probe as described in claim 1 in high-throughput screening of anti-apoptotic drugs.

3. The application of a near-infrared reactive oxygen species fluorescent probe according to claim 2 in high-throughput screening of anti-apoptotic drugs, characterized in that, The fluorescent probe can monitor intracellular H2O2 levels in real time.

4. The application of the near-infrared reactive oxygen species fluorescent probe according to claim 1 in monitoring reactive oxygen species levels in an LL-IRI mouse model.

5. The method for preparing a near-infrared reactive oxygen species fluorescent probe according to claim 1, characterized in that... Includes the following steps: Weigh (E)-2-((E)-3-(4-(dimethylamino)phenyl)prop-2-enyl)methylene)-6-hydroxybenzofuran-3(2H)-one, potassium carbonate, and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane and add them sequentially to N,N-dimethylformamide solution. Heat and stir the reaction mixture. After purification, (E)-2-((E)-3-(4-(dimethylamino)phenyl)allyl)-6-((4-(4,4,5,5-tetramethylpyridin-1,3,2-dioxaborane-2-yl)benzyl)oxy)benzofuran-3(2H)-one is obtained.

6. The method for preparing a near-infrared reactive oxygen species fluorescent probe according to claim 1, characterized in that, The (E)-2-((E)-3-(4-(dimethylamino)phenyl)prop-2-enyl)methylene)-6-hydroxybenzofuran-3(2H)-one was prepared by the following method: 6-hydroxybenzofuran-3(2H)-one was dissolved in ethanol, (E)-3-(4-(dimethylamino)phenyl)propenal and NaOH solution were added at room temperature, and the mixture was heated and stirred to react. The mixture was diluted with distilled water and the pH was adjusted with HCl. The obtained solid precipitate was washed and dried.

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