Non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe and application thereof

Abstract

The application belongs to the field of mitochondrial super-resolution fluorescent probes, and relates to a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe and application thereof. The probe comprises a compound containing a nitrobenzofuran or a pharmaceutically acceptable salt, hydrate, solvate, optical isomer, racemate, etc. of the compound, wherein the chemical structural formula of the compound containing the nitrobenzofuran is shown as formula (I). The fluorescent probe can stain mitochondria in normal living cells, and especially when the mitochondrial membrane potential is reduced or disappears, the probe can still be fixed on the mitochondria, indicating that the probe can track the dynamic changes of mitochondria. The probe can also realize the visualization of mitochondrial cristae changes in physiological and pathological processes. Compared with other existing probes, the probe has the characteristics of high brightness, strong light stability, low cytotoxicity, etc. The probe can be a useful tool for further studying the morphology of mitochondrial cristae under physiological and pathological conditions, and can promote the basic research in the related field.

Description

Technical Field

[0001] This invention belongs to the field of mitochondrial super-resolution fluorescent probes, and relates to a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Mitochondrial cristae maintain a dynamic structure to adapt to various physiological and pathological processes, such as mitophagy and ferroptosis. Therefore, visualizing and tracking changes in cristae is of great significance for physiological and pathological research. Creistae are densely packed ultrastructures within mitochondria, with the distance between adjacent cristae typically less than 100 nm. Consequently, for many years, methods and techniques for visualizing mitochondrial cristae have been limited to transmission electron microscopy (TEM). Although researchers have gained in-depth understanding of mitochondrial cristae using TEM, the inability to observe live samples and obtain dynamic information has limited its widespread application. In the past few decades, fluorescent probe-assisted fluorescence imaging techniques have been widely used for labeling and visualizing mitochondrial cristae due to their unique advantages of being suitable for live samples, near-non-destructive detection, and real-time and in-situ visualization.

[0004] In recent years, super-resolution imaging technology has attracted increasing attention due to its nanoscale ultra-high resolution, making it suitable for observing the ultrastructure of living cells. Currently, there are three main super-resolution imaging techniques: single-molecule localization microscopy (SMLM), structured illumination microscopy (SIM), and stimulated emission depletion microscopy (STED). SMLM improves resolution to a few nanometers, but it requires collecting a large number of images for data analysis and reconstruction, resulting in high computational costs and time consumption, limiting its application in tracking cellular dynamics. While SIM enables rapid imaging, its spatial resolution of 90-120 nm is insufficient for observing individual mitochondrial cristae. In contrast, STED microscopy can provide a spatial resolution of ~50 nm and a temporal resolution of 1 s, making it a useful tool for capturing mitochondrial cristae. However, strong dissipative lasers can easily cause photobleaching of probes, placing higher demands on fluorescent probes.

[0005] Many mitochondrial-related physiological and pathological processes are accompanied by a decrease in mitochondrial membrane potential (MMP), thus monitoring changes in mitochondrial cristae during these processes requires MMP-independent probes. Several MMP-independent probes have been developed to observe mitochondrial cristae. Tian et al. reported a unique probe, Py-BODIPY, to reveal how the mitochondrial 3D ultrastructure changes under oxidative phosphorylation conditions. Yamaguchi et al. observed cristae fusion in single mitochondria using the ultralight-resistant probe MitoPB Yellow. Xi et al. rationally designed MitoESq-635 to observe cristae morphology during mitochondrial fusion and fission. These MMP-independent probes can be immobilized in mitochondria through reactions with the thiol groups of mitochondrial proteins and exhibit high cytotoxicity. Compared to reaction-based probes, non-reactive probes that bind to phospholipids through hydrophobic interactions theoretically have better biocompatibility. According to the inventor's research, there are no reports on the application of non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probes containing NBD, their use in labeling and displaying the distribution of mitochondrial cristae in normal living cells, in mitochondria of living cells with reduced mitochondrial membrane potential, or in observing intracellular mitophagy and ferroptosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a non-reactive near-infrared super-resolution fluorescent probe for tracking mitochondrial cristae and its applications. This fluorescent probe can be used to label or display the morphology and spacing of mitochondrial cristae during physiological and pathological processes under an STED microscope, and can also be used to observe intracellular mitochondrial autophagy and ferroptosis.

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

[0008] Firstly, a compound containing nitrobenzenefuran, the chemical structure of which is shown in formula (I);

[0009]

[0010] The compound is named: 1-ethyl-2-((Z)-(3-(Z)-1-ethyl-3,3-dimethylindoline-2-ylidene)methyl)-2-((3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propyl)thio)-4-oxocyclobut-2-ene-1-ylidene)methyl)-3,3-dimethyl-3H-indoline-1-bromosalt, abbreviated as CSN.

[0011] In a second aspect, a method for preparing the above-mentioned nitrobenzenefuran-containing compound includes the step of preparing the target compound using 2,3,3-trimethyl-3H-indole and bromoethane as reactants according to the following reaction route;

[0012]

[0013] Specifically, the process is as follows: 2,3,3-trimethyl-3H-indole and bromoethane undergo a quaternization reaction to generate N-1; then N-1 reacts with squaric acid to generate N-2; then N-2 reacts with Lawson's reagent to generate N-3; N-3 and N-4 react at room temperature to obtain CSN.

[0014] More specifically, 2,3,3-trimethyl-3H-indole and bromoethane react at 80–90 °C to produce N-1.

[0015] More specifically, the reaction of N-1 with squaric acid to produce N-2 is carried out at a temperature of 91–99°C.

[0016] More specifically, the solvent used for the reaction of N-1 and squaric acid to produce N-2 is an ethanol solution.

[0017] More specifically, N₂ and Lawson's reagent react at room temperature to generate N₃. The room temperature mentioned in this invention refers to indoor ambient temperature, which can be 15–30°C, typically 25°C.

[0018] More specifically, 4-chloro-7-nitro-1,2,3-benzoxadiazole reacts with bromopropylamine hydrobromide to generate N-4.

[0019] Thirdly, a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe includes the above-mentioned compounds containing nitrobenzofuran or their pharmaceutically acceptable salts, hydrates, solvates, optical isomers, racemates, etc.

[0020] The fluorescent probe CSN of this invention is constructed through a novel and ingenious design: 1. A cationic dimethylindole salt can provide a positive charge to the molecule; 2. The large conjugated system composed of cationic indole salt, vinyl group, and squaric acid ensures that CSN has a large ionic radius and an emission wavelength suitable for STED microscopy, thereby ensuring the good membrane permeability and mitochondrial targeting of the probe CSN; 3. The introduction of the lipophilic group NBD enables it to generate hydrophobic interactions with the phospholipid bilayer of the inner mitochondrial membrane, thereby ensuring the MMP-independent nature of the probe CSN.

[0021] The pharmaceutically acceptable salts described in this invention include, for example, non-toxic salts or quaternary ammonium salts formed from non-toxic inorganic acids or organic acids; wherein, inorganic acids include sulfuric acid, phosphoric acid, nitric acid, etc., and organic acids include acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, etc.

[0022] In some implementations, pharmaceutically acceptable excipients are also included. Specifically, these excipients include, but are not limited to, pH adjusters, stabilizers, antioxidants, buffers, and preservatives.

[0023] Fourthly, a detection kit includes the aforementioned compound containing nitrobenzofuran or a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe, and a solvent.

[0024] Fifthly, the application of the above-mentioned nitrobenzofuran-containing compound or non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe or detection kit in monitoring cellular mitochondria or preparing detection products for monitoring cellular mitochondria.

[0025] In some embodiments, mitochondria are monitored using a laser confocal fluorescence microscope or a stimulated emission depletion microscope; or, the detection product is used in conjunction with a light confocal fluorescence microscope or a stimulated emission depletion microscope.

[0026] In some embodiments, the detection product is used to label or display mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with absent mitochondrial membrane potential.

[0027] Specifically, the normal cells refer to animal cells or cancer cells whose mitochondrial membrane potential is within the normal range.

[0028] Among them: animal cells: primary mouse fibroblasts, African green monkey kidney fibroblast-like cell line (COS-7), mouse embryonic fibroblasts (3t3) or normal human mammary epithelial cells (MCF-10A); cancer cells: cervical cancer cells (HeLa), human breast cancer cells (MCF-7) or human non-small cell lung cancer cells (A549).

[0029] Experiments have shown that the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe containing NBD described in this invention can highly selectively label mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with absent mitochondrial membrane potential.

[0030] In some embodiments, the detection product is used to label or display the distribution of mitochondrial cristae within mitochondria in normal living cells.

[0031] Experimental results confirm that CSN can image mitochondrial cristae in different cell lines at a resolution of 52 nm.

[0032] In some embodiments, the detection product is used to observe changes in mitochondrial cristae during cellular ferroptosis and mitophagy.

[0033] Experimental results confirmed that during autophagy, mitochondria first undergo swelling and cristae breakage, then partial vacuolation, and finally complete vacuolation. In contrast, during ferroptosis, mitochondria experience a reduction in the number of cristae, followed by partial breakage, and finally vacuolation.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention discloses a non-reactive near-infrared super-resolution fluorescent probe (CSN) containing NBD for tracking mitochondrial cristae and its biological applications. Extensive experimental results demonstrate that the CSN can stain mitochondria in normal living cells, and remains immobilized even when the mitochondrial membrane potential is reduced or absent, indicating that the CSN can track dynamic changes in mitochondria. Simultaneously, under a STED microscope, the CSN clearly shows mitochondrial cristae, and the distance between cristae varies in different cell lines. The non-reactive mitochondrial tracking fluorescent probe of this invention exhibits low toxicity and can track changes in mitochondrial cristae in real time during mitophagy and ferroptosis. Using the CSN, it was successfully observed that during mitophagy, mitochondria first undergo swelling and cristae breakage, then partial vacuolation, and finally complete vacuolation. During ferroptosis, mitochondria experience a decrease in cristae number, followed by partial breakage, and finally vacuolation. These results suggest that the CSN may be a useful tool for further research on mitochondrial cristae under physiological and pathological conditions and can promote basic research in related fields. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 Confocal images obtained by staining A549 cells, HeLa cells, and MCF-7 cells with CSN (150 nM) and Mito Tracker GreenFM (200 nM). Scale bar: 10 μm. Pearson colocalization coefficients are shown in the right column. Excitation wavelengths: CSN 640 nm, MTG 488 nm; Acquisition bands: CSN 650-700 nm, MTG 505-550 nm. These results confirm that the staining behavior of the CSN probe is not affected by cell type.

[0038] Figure 2 Confocal fluorescence images of normal viable HeLa cells stained with CSN and mitochondrial tracing red probe (MTR), CCCP-treated HeLa cells, and paraformaldehyde-fixed HeLa cells. Colocalization coefficients of CSN and MTR are labeled in the merged images. Excitation wavelengths: CSN 640 nm, MTR 561 nm; Acquisition bands: CSN 650-700 nm, MTR 575-625 nm. This demonstrates that CSN can image mitochondria in cells with normal membrane potential, cells with decreased membrane potential, and cells with absent membrane potential.

[0039] Figure 3 The survival rate of live HeLa cells incubated with different concentrations of CSN and MTDR indicates that CSN has low cytotoxicity.

[0040] Figure 4 a. STED image of mitochondrial cristae in CSN-labeled live HeLa cells. Scale bar: 2 μm. Enlarged view of the central white area (i) in (a). Enlarged view of the yellow area (right) in (a). Scale bar: 1 μm. b. Fluorescence intensity analysis at the labeling location in (a), and Gaussian fitting calculation of the half-width at half-maximum. Confocal (left) and STED (right) images of mitochondria in different cells labeled with CSN: MCF-7 (c); MCF-10A (d); HeLa (e); COS-7 (f); primary mouse fibroblasts (PMF) (g) and 3t3 (h). Scale bar: 2 μm. All STED data were deconvolutioned. The above results confirm that CSN can clearly depict mitochondrial cristae.

[0041] Figure 5 a) Changes in mitochondrial morphology and cristae after treatment with carbonyl cyanide m-chlorobenzene (CCCP) for different time periods. The first row is a confocal image; the second row is a STED image; b) a magnified image of the region of interest. All STED data were deconvolved. Scale bar: 2 μm. The results show that during mitophagy, mitochondria first undergo swelling and cristae breakage, then partial vacuolation, and finally complete vacuolation. This indicates that CSN can be used to track changes in mitochondrial cristae during physiological processes.

[0042] Figure 6 a. Changes in mitochondrial morphology and cristae after treatment with Erastin at different time points. The first row is a confocal image; the second row is an STED image. The third row is a magnified image of the region of interest. b. Mitochondrial length and cristae number in control and Erastin-treated cells. All STED data were deconvolved. Scale bar: 2 μm. The results show that during ferroptosis, mitochondria undergo a decrease in cristae number, followed by partial breakage, and finally vacuolation. This indicates that CSN can be used to track changes in mitochondrial cristae during pathological processes. Detailed Implementation

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] Example 1: Synthesis of probe CSN.

[0046] The synthetic route for probe CSN is shown below:

[0047]

[0048] The specific process is as follows:

[0049] (1) Synthesis of N-1:

[0050] 2,3,3-trimethyl-3H-indole (3.18 g, 20 mmol) and iodoethane (2.16 g, 20 mmol) were mixed in 50 mL of ethanol and refluxed under an argon atmosphere for 12 hours. Heating was then stopped and the mixture was cooled. The precipitate was then filtered through a Buchner funnel, the solid product was washed with diethyl ether, and dried under vacuum to give a pink product.

[0051] (2) Synthesis of N-1:

[0052] Squaric acid (1.14 g, 10 mmol) was placed in a 250 mL round-bottom flask, and then anhydrous ethanol (100 mL) was added. The resulting reaction mixture was heated under argon atmosphere and refluxed until the white squaric acid was completely dissolved and the solution became clear. Then, N-ethyl-2,3,3-trimethylindolium bromide (5.34 g, 20 mmol) was added to the reaction mixture until all the reactants were consumed, yielding a deep blue solution. N-2 was purified by silica gel column chromatography using a DCM (dichloromethane) / MeOH (methanol) gradient eluent, yielding a blue solid.

[0053] (3) Synthesis of N-3:

[0054] Lawson's reagent (2.2 g, 5 mmol) was added to a mixed solvent of DCM (5 mL) and THF (10 mL) containing N₂ (1.3 g, 3 mmol). The solution was stirred at 40 °C for 5 hours. After cooling to room temperature, the solution was removed under reduced pressure; no further purification was required for the next reaction.

[0055] (4) Synthesis of N-4:

[0056] First, 4-chloro-7-nitro-1,2,3-benzoxadiazole (2 g, 10 mmol) was dissolved in methanol and stirred for 10 minutes, followed by the addition of 3-bromopropionamide (2.19 g, 10 mmol). The reaction mixture was then stirred at room temperature for 8 hours. The mixture was then extracted from water with dichloromethane. Subsequently, the organic compound was washed twice with water and dried overnight with anhydrous sodium sulfate. Finally, the purified product was obtained by column chromatography using ethyl acetate / petroleum ether (1:10, v / v) as the eluent.

[0057] (5) Synthesis of CSN:

[0058] (Z)-4-((1-ethyl-3,3-dimethyl-3H-indol-1-onthium-2-yl)methylene)-2-(((Z)-1-ethyl-3,3-dimethylindololin-2-yl)methyl)-3-oxocyclobutene-1-sulfate (N-3, 500 mg, 1 mmol) and MeCN (acetonitrile, 5 mL) were added to N-(3-bromopropyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (N-4, 300 mg, 1 mmol) overnight at 85 °C. CSN was purified by silica gel column chromatography using a DCM (dichloromethane) / MeOH (methanol) gradient elution to obtain a green solid.

[0059] 1 H NMR (400MHz, CDCl3) δ7.39-7.30(m,6H),7.25(d,J=7.4Hz,2H),7.14(d,J=7.9Hz,2H),5.70(s,2H),4.27(d,J=7.1Hz,4H),3.98( t,J=7.1Hz,2H),3.78(dd,J=7.2,2.0Hz,1H),2.35(d,J=6.4Hz,2H),1.62(s,12H),1.19(d,J=8.4Hz,6H),0.81(t,J=6.9Hz,2H). 13 C NMR (101MHz, CDCl3) δ174.65,173.02,141.40,139.78,127.61,125.26,121.37,110.27 ,87.44,49.36,39.44,28.84,28.67,24.91,24.83,11.40.HRMS(m / z):[M]+calculated for C 39 H 41 N6O4S + ,689.2905;found,689.2780.

[0060] Example 2: Preparation of live cell samples for testing.

[0061] MCF-7, COS-7, 3t3, and HeLa cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C and 5% CO2. Primary mouse fibroblasts were cultured in Dulbecco modified Eagle medium containing 50% Ham's F12 nutrient medium, 10% FBS, and 1% penicillin and streptomycin at 37°C and 5% CO2. A549 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin at 37°C and 5% CO2. MCF-10A cells were cultured in MCF-10A-specific medium. Once the cells have grown to the logarithmic growth phase, proceed with slide culture: ① Soak coverslips in anhydrous ethanol for 30 minutes, dry them under an alcohol lamp, and place them in a disposable 35mm culture dish for later use; ② Wash the confluent cells in a 100mL cell culture flask three times with PBS, digest with 1mL of 0.25% trypsin for 3-5 minutes, carefully pour off the trypsin, add fresh culture medium, mix well, and count the cells. Control the cell density by adding culture medium to achieve a final cell concentration of 1×10⁻⁶ cells / mL. 5 / mL, and then seeded into the culture dish containing the coverslip, and placed in a 5% CO2 incubator at 37°C to allow the cells to grow on the coverslip. After the cells have grown to a confluence with the coverslip, live cell samples of HeLa cells, MCF-7 cells, COS-7 cells, 3t3 cells, primary mouse fibroblasts, A549 cells, and MCF-10A cells were obtained and used for experiments in subsequent examples.

[0062] Example 3: Testing and verifying the staining effect of probe CSN on MCF-7 cells, HeLa cells and A549 cells.

[0063] First, prepare a 1 mM CSN in DMSO solution as the stock solution. After the cells have grown to cover the coverslip, stain the cells with 150 nM CSN and 200 nM MTG (Mito Tracker Green FM) and incubate in a CO2 incubator for 30 min. After staining, wash away excess probe, place the cells face down on the slide, and observe the staining results under a laser confocal fluorescence microscope, including the location and intensity of cell staining.

[0064] The experimental results are shown in Figure 1 The experimental results show that the CSN probe can stain mitochondria in A549 cells, and the same results were observed in HeLa cells and MCF-7 cells, indicating that the staining behavior of CSN is not affected by cell type and can be used as a useful tool for imaging mitochondria.

[0065] Example 4 verifies the mitochondrial membrane potential independence of the probe CSN through a colocalization experiment.

[0066] In live cells: viable HeLa cells were first incubated with 0.2 μM MTR (mitochondrial dye, Mito-Tracker Red CMXRos) for 30 min, followed by incubation with 150 nM CSN for 30 min. In live cells with decreased mitochondrial membrane potential: viable HeLa cells were first incubated with 200 nM MTR for 90 min, followed by incubation with 150 nM CSN for 30 min, and then treated with 10 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone) for 90 min. In cells with absent mitochondrial membrane potential: viable HeLa cells were first incubated with 200 nM MTR for 90 min, followed by incubation with 150 nM CSN for 30 min, and then fixed with 4% paraformaldehyde for 30 min. Cells were washed three times with PBS before each staining with a different probe.

[0067] The experimental results are shown in Figure 2 Experimental results showed that the fluorescence signals of CSN and MTR overlapped well, with a colocalization coefficient of 0.95 in live cells, 0.86 in CCCP-treated cells, and 0.85 in fixed cells. This indicates that CSN can be firmly immobilized in mitochondria regardless of whether the mitochondrial membrane potential is decreased or absent. Therefore, CSN can be used to image mitochondria in cells with normal mitochondrial membrane potential, cells with decreased mitochondrial membrane potential, and cells with absent mitochondrial membrane potential.

[0068] Example 5: Cytotoxicity test experiment.

[0069] The toxicity test of CSN was performed using MTT reagent.

[0070] HeLa cells were seeded in 96-well plates at an average cell density of 10,000 cells / mL and grown for 24 h. Then, 200 μL of 2, 1, and 0.5 μM CSN and MTDR (MitoTracker Deep Red) were added to the experimental groups, respectively, while the control group received 200 μL of DMEM high-glucose medium per well. The 96-well plates were then incubated at 37°C with 5% CO2 for 24 h. Next, 10 μL of MTT (thiazolyl blue, 5 mg / mL) was added to both the experimental and control groups. After 4 h of incubation, the liquid in each well was removed, and 200 μL of DMSO was added to dissolve the resulting purple crystals. After 20 min, the absorbance of each well at 490 nm was measured using a microplate reader.

[0071] The experimental results are shown in Figure 3 The experimental results show that CSN has lower cytotoxicity than MTDR.

[0072] Example 6: Imaging mitochondrial cristae of different cell lines under an STED microscope using CSN.

[0073] HeLa cells, COS-7 cells, MCF-7 cells, MCF-10A cells, 3t3 cells, and mouse primary fibroblasts were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, excess probe was washed away, and the staining results were observed under a STED microscope.

[0074] The experimental results are shown in Figure 4 Experimental results showed that the CSN probe clearly depicted mitochondrial cristae in HeLa cells at a resolution of 52 nm. Furthermore, mitochondrial cristae were also observed using the CSN probe in different cell lines.

[0075] Example 7: Imaging changes in mitochondrial cristae during mitophagy using CSN under an STED microscope.

[0076] HeLa cells were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, excess probe was washed away, and the cells were treated with 10 μM CCCP for different times. The staining results were then observed under a STED microscope. The CCCP must not be removed during fluorescence imaging.

[0077] The results are as follows Figure 5 As shown in the diagram, mitochondrial cristae were clearly observed in the control group, while in cells treated with CCCP for 40 minutes, mitochondria swelled and cristae ruptured. With prolonged treatment, some mitochondria vacuolated, and cristae disappeared. After 150 minutes of treatment, mitochondria were completely vacuolated. These experimental results indicate that mitophagy is typically accompanied by mitochondrial vacuolation and cristae loss.

[0078] Example 8: Imaging changes in mitochondrial cristae during ferroptosis using CSN under a STED microscope.

[0079] HeLa cells were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, excess probe was washed away, and the cells were treated with 10 μM erastin for different times. The staining results were then observed under a STED microscope. Erastin must not be removed during fluorescence imaging.

[0080] Experimental results are as follows Figure 6As shown in the figure. The experimental results showed that, compared with the control group, the overall number of mitochondrial cristae gradually decreased during ferroptosis after cell treatment with erastin. Statistical analysis of the number and length of mitochondrial cristae in the control and erastin-treated cells revealed that mitochondrial length gradually shortened and the number of cristae decreased with prolonged treatment time. Subsequently, mitochondrial fragmentation was observed after 90 min of treatment, and complete vacuolization of mitochondria was observed after 100 min. These experimental results indicate that ferroptosis leads to a significant reduction in the number of mitochondrial cristae, but the cristae persist until mitochondrial vacuolization.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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 compound characterized by, The chemical structural formula is shown as formula (I); 。 2. A process for the preparation of a compound according to claim 1, characterized in that, The steps for preparing the target compound include using 2,3,3-trimethyl-3H-indole and bromoethane as the reaction raw materials according to the following reaction route; 。 3. The production method according to claim 2, wherein The process is as follows: 2,3,3-trimethyl-3H-indole and bromoethane are subjected to quaternary ammonium reaction to generate N-1; then N-1 and squaric acid are subjected to reaction to generate N-2; then N-2 and Lawesson's reagent are subjected to reaction to generate N-3; N-3 and N-4 are subjected to reaction at room temperature, and CSN is obtained.

4. The production method according to claim 3, wherein 2,3,3-trimethyl-3H-indole and bromoethane are subjected to reaction at 80-90℃ to generate N-1; Or, the reaction of N-1 and squaric acid to generate N-2 is carried out at a temperature of 91-99 ℃; Or, the reaction of N-1 and squaric acid to generate N-2 uses an ethanol solution as the solvent; Or, the reaction of N-2 and Lawesson's reagent at room temperature generates N-3; Or, 4-chloro-7-nitro-1,2,3-benzoxadiazole and bromopropylamine hydrobromide are subjected to reaction to generate N-4.

5. A non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe, characterized by, The compound or the pharmaceutically acceptable salt thereof is also included.

6. The non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe as described in claim 5, characterized in that, The pharmaceutically acceptable adjuvant is also included.

7. A test kit, characterized in that, The compound of claim 1 or the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe of claim 5 or 6, and the solvent are also included.

8. Use of the compound of claim 1 or the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe of claim 5 or 6 or the detection kit of claim 7 in the preparation of a detection product for monitoring mitochondria of cells.

9. Use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The detection product is used in cooperation with a confocal fluorescence microscope or a stimulated emission depletion microscope.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The detection product is used for labeling or displaying mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with disappeared mitochondrial membrane potential; Or, the detection product is used for labeling or displaying the intramitochondrial distribution of mitochondrial cristae in normal living cells; Or, the detection product is used for observing the changes of mitochondrial cristae in the process of ferroptosis and mitochondrial autophagy. The chemical structural formula is shown as formula (I);

Images