Preparation and application of acridine fluorescent ligand targeting mitochondrial G-quadruplex DNA

Abstract

The invention relates to the technical field of biological materials, in particular to preparation and application of acridine fluorescent ligands targeting mitochondrial G-quadruplex DNA. The chemical structural formula of the mitochondrial G-quadruplex DNA fluorescent ligand is # imgabs0 #. The fluorescent ligand can specifically detect and recognize the mitochondrial G-quadruplex DNA structure, and the detection process is not interfered by other components. Meanwhile, the mitochondrial G-quadruplex DNA has good chemical stability, light stability, solubility and biological compatibility, and shows extremely wide application prospects in the field of research on mitochondrial G-quadruplex DNA biological functions.

Description

Technical Field

[0001] The present invention relates to the technical field of mitochondrial G - quadruplex DNA detection, and more specifically, to the preparation and application of an acridine - based fluorescent ligand targeting mitochondrial G - quadruplex DNA, belonging to the technical field of anti - tumor drug development. Background Art

[0002] As a crucial organelle within cells, mitochondria undertake core physiological functions such as energy metabolism and apoptosis regulation, playing a key role in cell life activities. Because it is closely related to the occurrence and development of various major diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer, it has gradually become a highly potential therapeutic target, providing a new direction for conquering these intractable diseases.

[0003] Research in bioinformatics and other fields has shown that the G - quadruplex (G - quadruplex) structure widely exists in the human genome and transcriptome, including chromosome telomere ends, oncogene promoter regions, ribosomal DNA, mRNA untranslated regions, and mitochondrial DNA, etc. Mitochondrial G - quadruplex DNA is formed by a DNA sequence rich in guanine (G) through special Hoogsteen hydrogen - bond interactions, folding into a quadruplex structure with a unique topological structure. Mitochondrial G - quadruplex plays an important role in biological processes such as gene expression regulation, DNA replication, and repair, and is crucial for maintaining the normal physiological functions of mitochondria. Taking mitochondrial G - quadruplex DNA as a research target has significant advantages. On the one hand, it can deeply understand the pathogenesis of mitochondrial - related diseases from a new perspective, providing more accurate molecular markers for early disease diagnosis; on the other hand, it is expected to develop more targeted and effective innovative treatment strategies, bringing new hope to patients. Summary of the Invention

[0004] The object of the present invention is to solve the deficiency of the prior art in fluorescent ligands that specifically recognize mitochondrial G - quadruplex DNA, and provide a fluorescent ligand targeting mitochondrial G - quadruplex DNA. The compounds of the embodiments of the present invention have good biocompatibility, have good selectivity for mitochondrial G - quadruplex DNA, are suitable for the detection of mitochondrial G - quadruplex DNA in cells (especially living cells), with strong detection accuracy, high sensitivity, good stability, and simple operation. Research shows that this fluorescent ligand can induce mitochondrial dysfunction in human cervical squamous carcinoma cells, thereby achieving the purpose of treating cervical cancer.

[0005] To achieve the above - mentioned invention object, one of the technical solutions adopted by the present invention is:

[0006] The present invention provides a fluorescent ligand targeting mitochondrial G-quadruplex DNA - an acridine derivative. This acridine derivative can be used as a fluorescent ligand with specific selectivity in an in vitro environment for the detection of mitochondrial G-quadruplex DNA and for anti-human cervical squamous cell carcinoma. Among them, the mitochondrial G-quadruplex DNA fluorescent ligand effectively enhances the stability and selectivity of the derivative for mitochondrial G-quadruplex by introducing an amino side chain on the acridine nucleus, thereby endowing it with mitochondrial-related targeting ability;

[0007] The chemical structural formula of this mitochondrial G-quadruplex DNA fluorescent ligand is shown in Formula (I):

[0008]

[0009] The second technical solution adopted by the present invention is:

[0010] S1. Under nitrogen protection, dissolve Compound 1 and methyl iodide in 5 mL of acetonitrile, and reflux the reaction at 85 °C for 6 h. Cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution to obtain Compound 2.

[0011] S2. Dissolve Compound 3 and ethyl iodide in 5 mL of tetrahydrofuran, add sodium hydride to the above reaction solution, reflux the reaction for 8 h, monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution to obtain Compound 4.

[0012] S3. Stir DMF and POCl3 in an ice bath, add the DMF solution of Compound 4 to the above reaction solution, and heat the reaction. After the reaction is completed, pour the reaction solution into ice water, extract with ethyl acetate, and separate by column chromatography to obtain the product Compound 5.

[0013] S4. Dissolve Compound 2 and Compound 5 in a mixed solution of absolute ethanol and pyridine, raise the reaction temperature to 85 °C and heat the reaction for 6 h, monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution to obtain the product Compound of Formula (I);

[0014] The reaction formula is as follows:

[0015]

[0016] Furthermore, in step S1, the molar ratio of 2,3,3-trimethylindole to methyl iodide is 1:4.

[0017] Further, in step S2, the molar ratio of 9,9-dimethyl-9,10-dihydroacridine to iodoethane is 1:3. The molar ratio of 9,9-dimethyl-9,10-dihydroacridine to sodium hydride is 1:2.4.

[0018] Further, in step S3, the feeding ratio of compound 4 to DMF is 1:26 - 27 equivalents. The feeding ratio of compound 4 to POCl3 is 1:8 - 9 equivalents.

[0019] Further, in step S4, the feeding ratio of compound 5 to compound 2 is 1:1.9 equivalents. The feeding ratio of compound 5 to pyridine is 1:1 equivalent.

[0020] Further, in steps S1, S2, and S3, the heating reaction, and in step S4, the alkylation reaction are all carried out at 70 - 85 °C for 6 - 8 h.

[0021] The third technical solution adopted by the present invention is:

[0022] The provided mitochondrial G-quadruplex DNA fluorescent ligand has low biotoxicity, phototoxicity, and photobleaching properties. In practical applications, the adverse effects it causes to organisms are relatively small, which can effectively ensure the safety of the experimental process and the reliability of the results. In addition, the photostability of this ligand is excellent, and it can maintain stable performance under different lighting conditions, providing a solid foundation for long-term experimental observation and in-depth research.

[0023] Further, the mitochondrial G-quadruplex DNA fluorescent ligand has good water solubility and can be fully dissolved in water, which provides great convenience for its application in various aqueous environments. At the same time, this ligand has good cell membrane permeability and can easily cross the cell membrane and enter the cell interior for detection and research, creating favorable conditions for research in the field of cell biology.

[0024] Further, the mitochondrial G-quadruplex DNA fluorescent ligand can selectively bind to the G-quadruplex structure in mitochondria and promote its maintenance in a stable state, thereby mediating a decrease in the ATP content and the generation of reactive oxygen species in mitochondria, ultimately leading to mitochondrial dysfunction.

[0025] Further, the above-mentioned mitochondrial G-quadruplex DNA fluorescent ligand has the ability to specifically recognize mitochondrial DNA in cells.

[0026] The above solutions of the present invention can bring the following beneficial effects:

[0027] 1. The synthesis method of the mitochondrial G-quadruplex DNA fluorescent ligand in the present invention is simple.

[0028] 2. In vitro experiments showed that the mitochondrial G-quadruplex DNA fluorescent ligand compound 6 of the present invention exhibited good binding ability to various mitochondrial G-quadruplex DNAs.

[0029] 3. Treatment with the mitochondrial DNA fluorescent ligand of the present invention led to a concentration-dependent increase in the ROS level and a decrease in the ATP level in SiHa cells, indicating mitochondrial dysfunction in SiHa cells. The above phenomena are key events triggering programmed cell death.

[0030] 4. After treating SiHa cells with compound 6 in the present invention, the apoptotic characteristic marker proteins Cleaved caspase-9, Cleaved caspase-3, Cleaved caspase-7, and Cleaved caspase-PARP in SiHa cells were observed to be upregulated in a concentration-dependent manner. The above experimental data prove that compound 6 activates the mitochondrial-related apoptotic pathway. Description of the Drawings

[0031] Figure 1 Fluorescence intensity values (A) when compound 6 in Example 1 was incubated with DNA sequences of different secondary structures including single-stranded DNA, double-stranded DNA, and G4 quadruplex DNA. Quantification values (B).

[0032] Figure 2 Fluorescence titration of compound 6 on different types of DNA in Example 1. (A) Fluorescence titration diagram of adding compound 6 solution to G-quadruplex mt1015. (B) Fluorescence titration diagram between the fluorescence intensity of compound 6 at 660 nm and the concentration of the sample to be measured.

[0033] Figure 3 Detection limit experiment of compound 6 in Example 1.

[0034] Figure 4 Competition experiment of compound 6 in Example 1.

[0035] Figure 5 Cytotoxicity experiment of compound 6 in Example 1.

[0036] Figure 6 Experiment on apoptosis induction (A) and cell cycle arrest (B) of SiHa cells by compound 6 in Example 1.

[0037] Figure 7 Experiment on the change of ATP level in SiHa cells induced by different concentrations of compound 6 in Example 1 (A) and quantification (B).

[0038] Figure 8 Experiment on the change of cellular ROS level induced by different concentrations of compound 6 in Example 1 (A) and quantification (B).

[0039] Figure 9 For the long-term anti-proliferative activity of Compound 6 in Example 1 against Siha (colony formation assay (A)) and its quantification (B). The wound healing assay of Compound 6 on Siha cells incubated for 72 hours (C).

[0040] Figure 10 Western blot of different concentrations of Compound 6 in Example 1 in SiHa cells incubated for 24 hours. Detailed implementation manners

[0041] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, where the same

[0042] or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below by referring to the accompanying drawings

[0043] are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0044] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0045] Example 1:

[0046] Preparation of a fluorescent ligand for mitochondrial DNA, the chemical structure formula of the compound is as follows:

[0047]

[0048] The specific synthesis route is as follows:

[0049]

[0050] The specific synthesis steps are as follows:

[0051] S1. Under nitrogen protection, dissolve Compound 1 (0.5 g, 3.14 mmol) and methyl iodide (1.48 g, 12.56 mmol) in 5 mL of acetonitrile and reflux at 85 °C. After cooling to room temperature, separate the layers and remove the organic solvent by rotary evaporation under reduced pressure. Then, purify by silica gel column chromatography to obtain a yellow solid. Wash the yellow solid 3 times with a mixed solvent of petroleum ether and ethyl acetate (v / v = 3:1). The obtained product is Compound 2 with a yield of 86%. 1 H NMR (400 MHz, DMSO d6)δ 7.94 - 7.80 (m, 2H), 7.65 - 7.57 (m, 2H), 3.96 (d, J = 1.1 Hz, 3H), 2.76 (d, J = 1.0 Hz, 3H), 1.52 (s, 6H).

[0052] S2. Dissolve compound 3 (0.2 g, 0.96 mmol) and iodoethane (0.45 g, 2.87 mmol) in 5 mL of tetrahydrofuran. Add sodium hydride (0.09 g, 2.28 mmol) to the above reaction. Under nitrogen protection, react at 85 °C for 8 h. Monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature. Pour the reaction solution into 100 mL of ice water, extract with ethyl acetate, and separate by column chromatography (PE:EA = 80:1) to obtain compound 4 as the product with a yield of 71.43%. 1 HNMR (500 MHz, CDCl3) δ 7.41 (dd, J = 7.7, 1.6 Hz, 2H), 7.21 (td, J = 7.8, 1.6 Hz, 2H), 7.01 - 6.93 (m, 3H), 4.06 (q, J = 7.0 Hz, 2H), 1.53 (s, 6H), 1.43 (t, J = 7.0 Hz, 3H).

[0053] S3. Stir DMF (0.83 g, 11.38 mmol) and POCl3 (0.52 g, 3.69 mmol) in an ice bath for 1 h. Add the DMF solution of compound 4 (0.1 g, 0.42 mmol) to the above reaction solution and react at 85 °C for 6 h. After stopping the reaction, pour the reaction solution into 100 mL of ice water, extract with ethyl acetate, and separate by column chromatography to obtain compound 5 as the product with a yield of 39.75%. 1 H NMR (500 MHz, CDCl3) δ 9.91 (s, 2H), 7.97 (d, J = 2.0 Hz, 2H), 7.77 (dd, J = 8.6, 1.9 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H), 4.24 (dd, J = 6.0, 3.9 Hz, 1H), 4.17 (d, J = 7.1 Hz, 1H), 1.61 (s, 6H), 1.42 (s, 3H).

[0054] S4. Under nitrogen protection, dissolve compound 2 (0.19 g, 0.65 mmol) and compound 5 (0.1 g, 0.34 mmol) in a mixed solution of anhydrous ethanol and pyridine (0.027 g, 0.34 mmol). React at 85 °C for 6 h. Slowly cool to room temperature and then add a large amount of EA to obtain a large amount of precipitate. Let it stand in ice water for 30 min and then filter by suction to obtain a yellow solid. The product obtained is compound 6 with a yield of 77.12%. 1¹H NMR (400 MHz, DMSO) δ 8.47 (s, 1H), 8.34 (d, J = 2.0 Hz, 2H), 8.23 (dd, J = 9.0, 1.9 Hz, 2H), 7.91 - 7.80 (m, 5H), 7.66 - 7.53 (m, 8H), 7.43 (d, J = 9.1 Hz, 2H), 4.05 (d, J = 67.6 Hz, 9H), 1.82 (s, 12H), 1.52 (s, 3H), 1.40 (t, J = 6.9 Hz, 3H).

[0055] Example 2: Detection of DNA Samples

[0056] 1. Preparation of Samples

[0057] DNA Samples: The nucleic acid samples were purchased from Sangon Biotech Co., Ltd. An appropriate amount of nucleic acid was dissolved in a Tris-HCl buffer solution (pH 7.2, 100 mM KCl). The concentration was measured with a ultra-micro ultraviolet spectrometer. After heating at 95 °C for 5 min, it was slowly cooled and annealed to room temperature as the storage solution, and stored at 4 °C.

[0058] The DNA sample sequences for testing included:

[0059]

[0060]

[0061] Example 3:

[0062] Taking the mitochondrial G-quadruplex DNA fluorescent ligand prepared in Example 1 as an example, the photophysical properties of the mitochondrial G-quadruplex DNA fluorescent ligand were characterized. The specific steps were as follows:

[0063] The synthetic compound 6 in Example 1 was diluted to a concentration of 100 μmol / L, and then different types of DNA sequences were added. The fluorescence intensity was measured with a fluorescence spectrophotometer (slit width = 10, scanning speed = 200 nm, compound 6: Ex = 545 nm). Figure 1 For the selective screening of compound 6 in Example 2 for different types of G-quadruplex DNA. The DNA types are shown in Table 1, which are Bcl-2, Pu27, Pu22, VEGF, ds26, ss26, mt1015, mt10252, mt12086, mt16250, and mt377, a total of 11 types. From Figure 1 It can be seen that the fluorescence response of compound 6 to the hybrid mitochondrial G-quadruplex is high in in vitro experiments. Therefore, it has good nucleic acid discrimination.

[0064] Example 4: Fluorescence Titration Spectra of the Compound in Example 1 as a Fluorescent Ligand for Different DNAs

[0065] As Figure 2 shown in A, when compound 6 exists alone, a weak fluorescence emission phenomenon appears at 660 nm. After adding mt1015 to the system, the intensity of the emission peak at 660 nm increases significantly, and with the increase of the concentration of mt1015, the fluorescence intensity continues to increase and reaches saturation when the G-quadruplex reaches 10-fold equivalents. Similarly, fluorescence titration experiments were also carried out for other G-quadruplexes, such as Pu22, Bcl-2, etc. As Figure 2 shown in B, with the addition of different types of G-quadruplex DNA, the fluorescence intensity of compound 6 increases significantly, and there are certain differences in the fluorescence responses of compound 6 to different G-quadruplexes. Its fluorescence response to the hybrid G-quadruplex is high. From Figure 2 B, it is found that the fluorescence intensity of mt16250 gradually increases, but the increase amplitude is less than that of mt1015. The corresponding fluorescence intensities of ds26 and Pu27 do not increase significantly. The experimental results show that compound 6 can be used as a specific G-quadruplex fluorescent ligand.

[0066] Example 5: Determination of the detection limit of the compound synthesized in Example 1 as a fluorescent ligand

[0067] The detection performance of compound 6 for G4 was evaluated ( Figure 3 ). First, a study on the detection limit of G-quadruplex in solution was carried out. According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), the detection limit (LOD) refers to the lowest concentration of the analyte that can be detected in the background signal when the signal reaches three times the noise. Its calculation formula is: LOD = K × S / m. Among them, according to the suggestion of IUPAC, K usually takes the value of 3; Sb represents the standard deviation of the noise signal of 20 solutions of compound 4 at a specific concentration; m is the slope of the standard curve, which can be obtained by fitting the linear part of the fluorescence titration curve of compound 6 and G-quadruplex. In this study, the hybrid mt1015 was selected as the research object. The results show that the detection limit of compound 6 for mt1015 is lower than 31 nM.

[0068] Example 6 Competitive fluorescence titration experiment of the compound synthesized in Example 1 as a fluorescent ligand

[0069] To verify the selectivity of compound 6 for G-quadruplex, a competitive fluorescence titration experiment was carried out ( Figure 4) 5 μM of Compound 6 and 2 equivalents of mt1015 were placed in a mixed solution system, and then double-stranded ct-DNA was added dropwise. During the addition process, as the concentration of ct-DNA gradually increased, the fluorescence intensity of the complex formed by Compound 6 and mt1015 was not disturbed. Even when 200 μM of ct-DNA was present, when mt1015 was added dropwise to the solution of Compound 6, its fluorescence intensity still showed a steady increasing trend, similar to the increase when there was no ct-DNA in the system. In summary, Compound 6 can specifically recognize G-quadruplex in a competitive environment.

[0070] Example 7 Cytotoxicity experiment of the compound synthesized in Example 1 ( Figure 5 )

[0071] Cells were seeded in 96-well plates, 7000 cells per well, and the volume of cell culture medium was 100 μL. After culturing for 24 hours until the cells adhered and grew, the culture medium was aspirated, and different concentrations of drugs prepared with the culture medium were added, and then cultured in a cell incubator containing 5% CO2 for 24 hours. At the measurement time, CCK8: culture medium = 1:10 was added, incubated for 30 min, and after thoroughly mixing for 15 seconds, the absorbance at 450 nm was measured with a multifunctional microplate reader, and 3 - 6 replicates were set for each well. The absorbance was the average value of three samples. Cell survival rate = (OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells) * 100%. Finally, a graph of cell survival rate against drug concentration was plotted to obtain the half-maximal inhibitory concentration IC 50 value.

[0072] Example 8 Cell cycle arrest and apoptosis induction effects of the compound synthesized in Example 1

[0073] Study on the effect of different concentrations of Compound 6 on the apoptosis of SiHa cells ( Figure 6 A), the experimental results showed that the apoptosis rate of SiHa cells was positively correlated with the concentration of Compound 6. When the concentration of Compound 6 was 4 μM, the apoptosis rate of SiHa cells reached 39.42%. Study on the effect of different concentrations of Compound 6 on the cell cycle arrest of SiHa cells ( Figure 6 B), the experimental results showed that as the concentration of Compound 4 increased, the cell cycle of SiHa cells was arrested in the S phase. When the concentration of Compound 6 was 4 μM, the S-phase arrest rate of SiHa cells reached 11.97%.

[0074] Example 9: Determination of ATP content in SiHa cells of the compound synthesized in Example 1

[0075] The ATP levels in SiHa cells treated with different concentrations of Compound 6 for 24 hours were measured. As Figure 7As shown, compared with untreated cells, the ATP level in cells treated with Compound 6 was significantly decreased, indicating mitochondrial dysfunction in the cells.

[0076] Example 10: Determination of ROS Content of the Compound Synthesized in Example 1 in SiHa Cells

[0077] Collect cells in the logarithmic growth phase during subculture and seed them in a 6-well plate at 5×10 4 cells / well, and culture them under appropriate conditions for 24 hours. According to the experimental requirements, treat the cells with drugs at different concentrations, or set up a blank group, and culture. Remove the cell culture medium, add the DHE ligand diluted with serum-free culture medium, and incubate in a 37°C, 5% CO2 incubator for 40 minutes. Wash 2-3 times with PBS. Immediately observe and photograph under a fluorescence microscope. ( Figure 8 A) Statistical analysis chart of the results Figure 8 B)

[0078] Example 11: Long-term Antiproliferative Activity of the Compound Synthesized in Example 1

[0079] Seed the plates according to the grouping, with a cell density of about 5×10 5 cells / well, ensuring that the seeding density of each group of cells is the same and the confluence reaches more than 95% on the second or third day. Using a ruler / 6-well plate cover as a guide, make the 200 μL pipette tip perpendicular to the well plate and draw two perpendicular lines to the marked line to form several intersection points as fixed detection points. Discard the old culture medium, gently rinse with PBS two to three times until the detached cells are washed clean. Add the drug-containing culture medium according to the grouping. The photographing time points can be selected from 0 - 72 hours Figure 9 A)

[0080] Inoculate SiHa cells with good growth status in a 6-well plate, 200 cells per well, and culture in a 5% CO2 cell incubator for 24 h to allow the cells to adhere. Dilute the compound to the target concentration with fresh culture medium, and add an equal volume of DMSO to the blank control. Add the culture medium containing DMSO or the compound to the culture dish and continue to culture for 7 days to form clones. Discard the culture medium, carefully wash twice with PBS to remove the serum-containing culture medium. Fix the cells with absolute methanol for 15 min, add 0.1% crystal violet methanol staining solution, and stain at room temperature for 30 min. After discarding the staining solution, gently wash away the excess staining solution with a water stream and air-dry in an inverted position. Photograph the culture dish under white light and store the results. ( Figure 9 B) Statistical analysis chart of the results Figure 9 C)

[0081] Example 12: Determination of the Expression Content of Key Proteins of Compound 6 Synthesized in Example 1 in SiHa Cells

[0082] The Western blot assay further demonstrated that the cellular levels of key proteins in the apoptotic pathway, Cleaved caspase-3, Cleaved caspase-7, Cleaved caspase-9, and Cleaved caspase-PARP, were all upregulated after 24 hours of treatment with compound 6( Figure 10 ).

Claims

1. A fluorescent ligand for mitochondrial G-quadruplex DNA, characterized in that, Its chemical structural formula is shown in formula (I).

2. The fluorescent ligand of mitochondrial G-quadruplex DNA according to claim 1, wherein It exhibits low biotoxicity, phototoxicity, and photobleaching properties. In practical application scenarios, the adverse effects it produces on organisms are relatively minor, effectively ensuring the safety of the experimental process and the reliability of the results. In addition, the ligand has excellent photo-stability and can maintain the stability of its performance under different lighting conditions, providing a strong guarantee for long-term experimental observation and in-depth research.

3. The fluorescent ligand for mitochondrial G-quadruplex DNA according to claim 1, characterized in that, It has good water solubility and can be fully dissolved in water, providing convenient conditions for its application in various aqueous environments. At the same time, it has good cell membrane permeability and can easily pass through the cell membrane and enter the cell interior for detection and research, facilitating research in the field of cell biology.

4. The fluorescent ligand for mitochondrial G-quadruplex DNA according to claim 1, wherein It can selectively induce the formation of G-quadruplex structures in mitochondria and make them tend to a stable state, thereby mediating a decrease in mitochondrial ATP content and the production of reactive oxygen species, ultimately resulting in mitochondrial dysfunction.

5. The fluorescent ligand for mitochondrial G-quadruplex DNA according to claim 1, wherein It has a specific recognition ability for mitochondrial G-quadruplex DNA in cells.

6. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 1, characterized in that, Its preparation process is as follows:

7. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 6, wherein, It includes the following operation steps: S1. Under nitrogen protection, dissolve compound 1 and methyl iodide in 5 mL of acetonitrile, reflux the reaction at 85 °C, cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution of (ethyl acetate: petroleum ether = 1:3) to obtain compound 2. S2. Under nitrogen protection, dissolve compound 3 and iodoethane in 5 mL of tetrahydrofuran, add sodium hydride to the above reaction solution, and reflux the reaction for 8 h. Monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution to obtain compound 4. S3. Under nitrogen protection, stir DMF and POCl3 in an ice bath for 1 h, add the DMF solution of compound 4 to the above reaction solution, and heat the reaction. After the reaction is completed, pour the reaction solution into ice water, extract with ethyl acetate, and separate by column chromatography to obtain compound 5. S4. Under nitrogen protection, dissolve compound 2 and compound 5 in a mixed solution of absolute ethanol and pyridine, raise the reaction temperature to 85 °C and heat the reaction for 6 h. Monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature, filter the reaction solution to obtain a yellow solid, and wash the yellow solid 3 times with a mixed solution to obtain the product, the compound of formula (I).

8. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 7, wherein In step S1, the molar ratio of 2,3,3-trimethylindole to methyl iodide is 1:

4.

9. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 7, characterized in that, In step S2, the molar ratio of 9,9-dimethyl-9,10-dihydroacridine to iodoethane is 1:

3. The molar ratio of 9,9-dimethyl-9,10-dihydroacridine to sodium hydride is 1:2.

4.

10. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 7, wherein In step S3, the molar ratio of compound 4 to DMF in the feed is 1:

26. The molar ratio of compound 4 to POCl3 in the feed is 1:

8.

11. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 7, characterized in that, In step S4, the molar ratio of compound 5 to compound 2 in the feed is 1:1.

9. The molar ratio of compound 5 to pyridine in the feed is 1:1 equivalent.

12. The preparation method of the mitochondrial G-quadruplex DNA fluorescent ligand according to claim 7, characterized in that, The heating reactions in steps S1, S2, and S3 and the alkylation reaction in step S4 are all carried out at a temperature of 70 - 85 °C, and the reaction duration is 6 - 8 hours.

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