Autophagy modulator and use thereof

Abstract

This invention discloses an autophagy regulator, its preparation method, and its applications, aiming to solve the problems of single-target autophagy regulation, lack of cell selectivity, and poor efficacy in killing cancer cells in existing technologies. The autophagy regulator has the following structural formula: [structural formula would be inserted here]. This autophagy regulator can simultaneously target mitochondria and lysosomes, inducing mitophagy; it can also disrupt lysosomes, inhibiting autophagic flux; it can be used for mitochondrial and lysosomal imaging, and can also be used as an anticancer drug.

Description

[0001] This application is a divisional application of Chinese application No. 202311636324.X, filed on November 30, 2023, entitled "An autophagy regulator and its preparation method and application". Technical Field

[0002] This invention relates to the field of probes, and more particularly to an autophagy regulator, its preparation method, and its application. Background Technology

[0003] Autophagy, the self-digestion of lysosomes or vacuoles and the degradation and recycling of cellular contents, is crucial for maintaining homeostasis and energy balance within the cell. It also has a wide range of biological functions, including organelle remodeling, protein and organelle quality control, tumor suppression, pathogen elimination, immune and inflammatory regulation, and cell survival. Studies have shown that dysfunction in autophagy is associated with various diseases, including cancer, neurodegenerative diseases, diabetes, autoimmune diseases, and cardiovascular diseases. Therefore, the regulation of autophagy is of great significance for the treatment of various diseases. Currently, targeted drugs for various diseases related to autophagy are under further development.

[0004] Current autophagy modulators, including rapamycin and chloroquine, can only activate or inhibit autophagy individually, and they do not produce fluorescence after binding to the target, making fluorescence observation impossible. Especially when used in tumor treatment, their efficacy and tumor selectivity need improvement. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor, and its application, aiming to solve the problems of single-target autophagy regulation, lack of cell selectivity, and poor efficacy in killing cancer cells in the prior art.

[0006] An autophagy regulator according to a first aspect of the present invention, said autophagy regulator being a compound having the structure shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0007] Formula I; Wherein, the R 1 Selected from hydrogen or C1-C4 alkyl groups; the R 2 Selected from hydrogen or C1-C4 alkyl groups; the R 3 The atom is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; X is selected from halogen atoms, BF4, and ClO4.

[0008] According to some embodiments of the present invention, the autophagy regulator is a dual-targeted autophagy activator and autophagy inhibitor.

[0009] The method for autophagy regulation by the autophagy regulator of the present invention is as follows: mixing and incubating sample cells with the autophagy regulator to destroy the mitochondria of the sample cells and induce mitophagy; destroying the lysosomes of the sample cells and inhibiting autophagy flux.

[0010] In the step of the autophagy regulator in this invention disrupting the mitochondria of the above-mentioned sample cells, the mitochondrial membrane potential decreases.

[0011] In the step of the autophagy regulator in this invention destroying the lysosomes of the above-mentioned sample cells, the pH of the lysosomes is increased.

[0012] The autophagy regulator provided by this invention is a dual-targeting fluorescent organic small molecule that acts as both an autophagy activator and inhibitor. Compared with existing autophagy regulators, the unique feature of this dual-targeting fluorescent organic small molecule is that it can simultaneously activate mitophagy and disrupt lysosomal function, inhibiting autophagic flux and thus killing cancer cells. It is also a novel mitochondrial / lysosomal fluorescent probe, capable of simultaneously targeting both mitochondria and lysosomes compared to existing mitochondrial and lysosomal fluorescent probes. It emits red fluorescence, imaging the morphology, number, and distribution of mitochondria and lysosomes, while exhibiting good membrane permeability and good counterstain compatibility.

[0013] The dual-targeted fluorescent small organic molecule autophagy activator and autophagy inhibitor provided by this invention can, on the one hand, act as an autophagy regulator to activate mitochondrial autophagy and inhibit autophagic flux, thereby serving as an anticancer drug to kill cancer cells; on the other hand, it can be used as a fluorescent probe to label the morphology, number, and distribution of mitochondria and lysosomes in cells, providing a simple and intuitive biological detection reagent for physiological and pathological studies related to mitochondria and lysosomes as well as clinical diagnosis. It has wide applications and excellent effects.

[0014] According to some embodiments of the present invention, the halogen atom is selected from any one of iodine, bromine and chlorine.

[0015] According to some embodiments of the present invention, the R 1 In this context, the C1-C4 alkyl groups include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0016] According to some embodiments of the present invention, the R 2 In this context, the C1-C4 alkyl groups include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0017] According to some embodiments of the present invention, the R 3In this context, the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. According to some embodiments of the present invention, the C1-C4 alkoxy group includes any one of methoxy, ethoxy, propoxy, and butoxy.

[0018] According to some embodiments of the present invention, the autophagy regulator includes (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0019] In this invention, R 1 Selected from ethyl, R 2 Selected from hydrogen, the R 3 When X is selected from methoxy groups and iodine, the resulting autophagy regulator independent of mitochondrial membrane potential is: (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0020] According to a second aspect of the present invention, a method for preparing an autophagy regulator includes the following steps: S1. Place 4-methylquinoline (Formula II) and a long-chain haloalkane (Formula III) in a solvent and reflux to generate a 1-long-chain alkyl-4-methylquinoline salt (Formula IV). S2. Mix 1-long-chain alkyl-4-methylquinoline salt, indole-3-carboxaldehyde (Formula V) and catalyst, reflux and then remove impurities; The catalyst includes piperidine.

[0021] According to some embodiments of the present invention, the solvent includes ethanol.

[0022] According to some embodiments of the present invention, the molar ratio of indole-3-carboxaldehyde to 4-methylquinoline is 1:(1.0~2.0).

[0023] According to some embodiments of the present invention, in step S1, the reflux reaction time is 3 to 4 days.

[0024] According to some embodiments of the present invention, in step S2, the reflux reaction time is 1 to 2 days.

[0025] .

[0026] According to some preferred embodiments of the present invention, the indole-3-carboxaldehyde is selected from 5-methoxy-3-formylindole; the haloalkane is selected from iododecane. Using 5-methoxy-3-formylindole and iododecane as reactants, the autophagy regulator is prepared as follows: S01. Prepare an ethanolic mixed solution of 4-methylquinoline and iododecane; S02. Heat and stir under reflux for three days; S03. Add an ethanolic solution of 5-methoxy-3-formylindole; S04. Add the catalyst piperidine to the ethanol mixture, heat the ethanol mixture with piperidine to reflux at 85°C and stir for one day, and slowly cool to room temperature to obtain dark green crystals or organic solid products to be purified. S05. The organic solid product to be purified is subjected to column chromatography, using dichloromethane / methanol as the eluent. Drying yields dark green crystals and a dark red powder. The dark green crystals and dark red powder are dual-targeted fluorescent organic small molecule autophagy activators and inhibitors. (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0027] The application of an autophagy regulator according to a third aspect of the present invention in the preparation of products for regulating cellular autophagy-related life activities.

[0028] In this invention, the dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor is used for cell autophagy regulation for non-diagnostic and therapeutic purposes. The dual-targeting fluorescent organic small molecule of this application can target mitochondria and lysosomes of cells, causing a decrease or even loss of mitochondrial membrane potential, inducing mitochondrial autophagy, and producing a large number of autophagosome vesicles; at the same time, the pH of lysosomes increases, preventing them from fusing with autophagosomes to form autophagolysosomes, inhibiting the completion of autophagic flux, and achieving dual regulation of autophagy.

[0029] The use of an autophagy regulator according to a fourth aspect of the present invention in the preparation of a product for use in targeting mitochondria or targeting lysosomes.

[0030] The use of an autophagy regulator according to a fifth aspect of the invention in the preparation of products for use in mitochondrial imaging or lysosomal imaging.

[0031] The method for mitochondrial / lysosomal fluorescence imaging using autophagy regulators in this invention includes: mixing and incubating sample cells with autophagy regulators, whereby the autophagy regulators bind to the mitochondria and lysosomes of the sample cells, enhancing the fluorescence intensity, and thus achieving fluorescence imaging of mitochondria and lysosomes.

[0032] This invention provides a dual-targeting fluorescent organic small molecule that acts as both an autophagy activator and an autophagy inhibitor for mitochondrial / lysosomal fluorescence imaging. Compared with existing mitochondrial and lysosomal fluorescent probes, the dual-targeting fluorescent organic small molecule can simultaneously target cellular mitochondria and lysosomes. After binding to mitochondria and lysosomes, the fluorescence is greatly enhanced, enabling simultaneous fluorescence imaging of mitochondria and lysosomes.

[0033] The dual-targeting fluorescent small organic molecule autophagy activator and autophagy inhibitor described in this invention is used for mitochondrial / lysosomal fluorescence imaging for non-diagnostic and therapeutic purposes. The dual-targeting fluorescent small organic molecule of this application does not fluoresce on its own, but after binding with mitochondria and lysosomes, the fluorescence is greatly enhanced, realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.

[0034] According to a sixth aspect of the present invention, a medicament for treating tumors includes an autophagy-regulating drug, wherein the autophagy-regulating drug includes the aforementioned autophagy regulator.

[0035] According to some embodiments of the present invention, the tumor includes tumors that overexpress albumin receptors.

[0036] According to some embodiments of the present invention, the tumors overexpressing the albumin receptor include cervical cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, and liver cancer.

[0037] According to some embodiments of the present invention, the medicament comprises an injectable composition or a composition for oral administration.

[0038] According to some embodiments of the present invention, the composition comprises a dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor and other pharmaceutically acceptable carriers, wherein the carriers include, but are not limited to, various pharmaceutical excipients.

[0039] The method for killing cancer cells using autophagy regulators in this invention includes: mixing and incubating sample cells with autophagy regulators to activate mitophagy, inhibit lysosomal function and autophagy, and induce cell death.

[0040] Compared with existing autophagy regulators, the autophagy regulator in this invention is a dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor. It has a dual-targeting effect, simultaneously targeting mitochondria and lysosomes, activating mitophagy and inhibiting lysosomal function, ultimately leading to cell death. This dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor can be used to kill cancer cells, realizing the application of dual-targeting fluorescent organic small molecule autophagy activator and autophagy inhibitor as autophagy regulator and cancer cell killer. Attached Figure Description

[0041] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is provided in Test Example 2 of this application. (E) A fluorescence micrograph of HeLa cells co-stained with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide, mitochondrial green fluorescent probe (MitoTracker Green), and lysosomal deep red fluorescent probe (LysoBrite NIR).

[0042] Figure 2 This is provided in Test Example 3 of this application. E Fluorescent micrographs of HeLa cells treated with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide and stained with rhodamine 123.

[0043] Figure 3 This is provided in Test Example 4 of this application. E Fluorescent micrographs of HeLa cells stained with lysosomal green fluorescent probe (Lysosensor Green DND-189) after treatment with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0044] Figure 4 This is provided in Test Example 5 of this application. E Fluorescent micrographs of HeLa cells stained with 5-chloromethylfluorescein diacetate (CellTracker™ Green CMFDA) after treatment with iodine salt of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0045] Figure 5 This is provided in Test Example 6 of this application. E Fluorescent micrographs of mitochondria and lysosomes in HeLa cells treated with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide and CCCP, respectively.

[0046] Figure 6 This is provided in Test Example 7 of this application. E Fluorescence micrographs of HeLa cells after incubation at 37°C and 4°C with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0047] Figure 7 This is provided in Test Example 8 of this application. EMicrographs of normal cell spheres HEK293 and cancer cell spheres HeLa before and after incubation with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

[0048] Figure 8 This is provided in Test Example 9 of this application. E Fluorescence spectra and double logarithmic fitting curves of the interaction between 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide and albumin. Detailed Implementation

[0049] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0050] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0051] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0052] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0053] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0054] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0055] Example 1 ( E Synthesis of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide First, 200 μL of 4-methylquinoline and 393 μL of iodododecane were dissolved in 10 mL of ethanol and refluxed at 85 °C with stirring for three days. Then, an ethanol solution containing 0.263 g of 5-methoxy-3-formylindole was added, stirred until homogeneous, and 5 drops of piperidine were added. The solution gradually turned red. After refluxing at 85 °C for one day, the solution was slowly cooled, filtered, and washed with a small amount of isopropanol to obtain dark green crystals or dark red powder. Alternatively, excess solvent was evaporated, the product was cooled, and purified by column chromatography using dichloromethane / methanol as eluent to obtain dark green crystals and dark red powder, with a yield of approximately 27%.

[0056] 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 12.11 (s, 1H), 9.12 (d, J = 4.0 Hz, 1H), 8.97 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 16.0 Hz, 1H), 8.44 (m, 3H), 8.18(t, J = 8.0 Hz, 1H), 7.99 (m, 2H), 7.70 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 6.90 (dd, J = 4.0, 8.0 Hz, 1H), 4.85 (t, J = 8.0 Hz, 2H), 3.90 (s, 3H), 1.91 (m, 2H), 1.37 (m, 2H), 1.21 (m, 16H), 0.83 (t, J = 8.0 Hz, 3H). 13 C NMR (400 MHz, DMSO- d6), δ (ppm): 155.71, 154.17, 146.15, 139.13, 138.31, 135.12, 132.60, 132.44, 128.80, 127.28, 126.96, 126.15, 119.29, 115.08, 113.87, 113.81, 112.95, 112.80, 102.69, 56.18, 56.07, 31.76, 29.66, 29.47, 29.36, 29.33, 29.18, 28.98, 26.27, 22.57, 14.43. HRMS: calculated 469.32, found 469.32.

[0057] To develop more compounds with similar functions, the molecular core skeleton remains unchanged in this invention, R 3 It can also be selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, R 2 Selected from hydrogen or C1-C4 alkyl groups. According to the inventors' research, the function of the autophagy regulator in this invention is determined by the conjugated organic cation group, and its interaction with the anion X. - Irrelevant; R 2 Selected from hydrogen or any C1-C4 alkyl group; R 3 The molecules can be selected from any of hydrogen, C1-C4 alkyl, or C1-C4 alkoxy groups; changes within this range do not affect the molecule's function; however, R... 1 The length of the linked carbon chains has a significant impact on the targeting and protein binding of molecules. For example, indo-octyl quinoline salts have a weak binding to albumin and enter cells via free diffusion. As the carbon chain of the quinoline increases (from decyl to tetradecyl), the binding to albumin is enhanced, and the quinoline salts enter cells via active transport.

[0058] Test Example 1 Culture of HeLa and HEK293 cells and cell spheroids Cancer cells HeLa and normal cells HEK293 were cultured in complete medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin) in a saturated humidity incubator at 37°C and 5% CO2, and passaged every 2-3 days.

[0059] Once the cells have grown to the logarithmic phase, they are transferred to confocal plates for culture: The cells that have grown to the top of the T25 cell culture flask are washed with PBS and then digested with 1 mL of trypsin for 1-2 minutes (0.25% trypsin for HeLa and 0.025% trypsin for HEK293). After removing the trypsin, fresh culture medium is added, the cells are pipetted and counted. The cell density is controlled by the amount of culture medium added. (1) The cells are seeded into confocal glass-bottom culture dishes and placed in a 5% CO2 incubator for culture. The cells are cultured until the coverage is about 70% for cell imaging experiments. (2) The cells are seeded into low-adsorption U-bottom 96-well plates for culture. The cells are cultured until the diameter of the cell spheres reaches ~800 µm for drug anti-tumor evaluation.

[0060] Test Example 2 (E) Observation of staining of HeLa cells with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide After washing the HeLa cell-covered slides prepared in Test Example 1 twice with PBS, the following staining steps were performed: (1) incubation with 0.5 µM commercial mitochondrial green fluorescent probe (MitoTracker Green) solution for 30 min, followed by washing with PBS; (2) incubation with 0.5 µM commercial lysosomal near-infrared fluorescent probe (LysoBrite NIR) solution for 30 min, followed by washing with PBS; (3) incubation with 2 µM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide fluorescent probe solution for 30 min, followed by washing with DMEM. The stained cell samples were then observed using a confocal fluorescence microscope for multi-channel fluorescence co-localization.

[0061] The results are as follows Figure 1 As shown, Figure 1 (A) is a red fluorescence image of the molecule synthesized in Example 1. Figure 1 (B) is a fluorescence image of a commercial mitochondrial green fluorescent probe. Figure 1 (C) is a fluorescence image of a commercial lysosomal near-infrared fluorescent probe. Figure 1 (D) is Figure 1 (A) and Figure 1 (B) Figure 1 (C) overlay diagram. Figure 1 (A) covered Figure 1 (B) and Figure 1 (C) Two regions, Figure 1 (A) and Figure 1 (B) and Figure 1 The addition and overlap of (C) are very good, indicating that the fluorescence of the molecule synthesized in Example 1 is distributed in both the mitochondria and lysosomes. This result proves that ( E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide can be used to target mitochondria and lysosomes, and can also be used for simultaneous fluorescence imaging of mitochondria and lysosomes.

[0062] Test Example 3 (E) Changes in mitochondrial membrane potential before and after treatment of HeLa cells with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide Two groups of glass-bottomed culture dishes, each confluent with HeLa cells prepared in Test Example 1, were washed with PBS and incubated with 5 µM Rhodamine 123 in a CO2 incubator for 30 min in the dark. After washing with PBS, one group was incubated for 30 min in a culture medium solution containing 10 µM of the molecule synthesized in Example 1, while the other group served as a blank control sample in a medium containing an equal volume of DMSO. After washing with PBS, the incubated cells were observed under a laser scanning confocal microscope, and the changes in the fluorescence intensity of Rhodamine 123 in the cells of both groups were recorded.

[0063] Results analysis: The experimental results of test example 3 are shown below. Figure 2 , Figure 2 These are fluorescence micrographs of the mitochondrial membrane potential probe Rhodamine 123, synthesized in Example 1, before and after treatment of HeLa cells. Figure 2 (A) is a fluorescence micrograph of cells in the blank control sample group; Figure 2 (B) is Figure 2 (A) Corresponding bright-field photomicrograph; Figure 2 (C) A fluorescence micrograph of Rhodamine 123 in cells treated with the molecule synthesized in Example 1; Figure 2 (D) is Figure 2 (C) Corresponding bright-field photomicrograph. From Figure 2 (A) and Figure 2 As can be seen from (C), the fluorescence of Rhodamine 123 was significantly reduced in cells treated with the molecules synthesized in Example 1. Figure 2 (C) indicates that the mitochondrial membrane potential of cells treated with the molecules synthesized in Example 1 was significantly reduced, i.e., mitochondria were damaged.

[0064] Test Example 4 ( E Changes in lysosomal pH of HeLa cells before and after treatment with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide. Two groups of glass-bottomed culture dishes containing HeLa cells, prepared in Test Example 1, were washed with PBS and incubated in a CO2 incubator for 30 min in the dark with a 2 µM lysosomal green fluorescent probe (LysoSensor Green DND-189). After washing with PBS, one group was incubated for 30 min in a culture medium solution containing 10 µM of the molecule synthesized in Example 1, while the other group served as a blank control in a medium containing an equal volume of DMSO. After incubation, the cells were washed with PBS and placed in DMEM. The changes in fluorescence intensity of the lysosomal green fluorescent probe in the two groups of cells were observed and recorded under a laser scanning confocal microscope.

[0065] Results analysis: The experimental results of test example 4 are shown below. Figure 3 , Figure 3 These are fluorescence micrographs of the lysosomal green fluorescent probe (LysoSensor Green DND-189) synthesized in Example 1 before and after treatment of HeLa cells. Figure 3 (A) is a fluorescence micrograph of cells in the blank control sample group; Figure 3 (B) is Figure 3 (A) Corresponding bright-field photomicrograph; Figure 3 (C) is a fluorescence micrograph of the lysosomal green fluorescent probe of cells treated with the molecule synthesized in Example 1; Figure 3 (D) is Figure 3 (C) Corresponding bright-field micrograph. The fluorescence of LysoSensor Green DND-189 increases with decreasing pH. Figure 3 (A) and Figure 3 As can be seen from (C), the fluorescence of the lysosomal green fluorescent probe was greatly reduced in cells treated with the molecules synthesized in Example 1. Figure 3 (C) indicates that the pH of lysosomes in cells treated with the molecules synthesized in Example 1 increased significantly, affecting the digestive function of lysosomes in autophagic flux.

[0066] Test Example 5 (E) Imaging of autophagosomes and autophagosomes in HeLa cells before and after treatment with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide. Two groups of glass-bottomed culture dishes, each confluent with HeLa cells prepared in Test Example 1, were washed with PBS. One group was incubated in a culture medium solution containing 10 µM of the molecule synthesized in Example 1 for 90 min in the dark, while the other group served as a blank control sample in a culture medium containing an equal volume of DMSO. Subsequently, the cells were incubated with 5 µM of the green live-cell tracer probe (Cell-Tracker Green CMFDA) in the dark for 30 min. After incubation, the cells were washed with PBS and placed in DMEM. The fluorescence distribution of the green live-cell tracer probe in the two groups of cells was observed and recorded under a laser scanning confocal microscope.

[0067] Results analysis: The experimental results of test example 5 are shown below. Figure 4 . Figure 4 These are fluorescence micrographs of the green live-cell tracking probe (Cell-Tracker Green CMFDA) synthesized in Example 1 before and after treatment of HeLa cells. Figure 4 (A) is a fluorescence micrograph of cells in the blank control sample group; Figure 4 (B) A fluorescence micrograph of the green live-cell tracking probe used to treat cells with the molecules synthesized in Example 1. (Comparison) Figure 4 (A) and Figure 4 (B) Cells treated with the molecule synthesized in Example 1 produced a large number of vacuoles (autophagosomes and autophagosomes) in the cytoplasm, indicating that the molecule synthesized in Example 1 can activate cell autophagy.

[0068] Test Example 6 (E) Mitochondrial-lysosomal fusion of HeLa cells treated with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide Two groups of glass-bottomed culture dishes containing HeLa cells, prepared in Test Example 1, were washed with PBS and then incubated sequentially for 30 min in the dark with 1 µM MitoTracker Green and 1 µM LysoBrite NIR probes. Subsequently, one group was incubated with 20 µM CCCP in PBS solution for ~5 h as a positive control for mitophagy, while the other group was incubated for the same amount of time in a culture medium solution containing 10 µM of the molecules synthesized in Example 1. After incubation, the cells were washed with PBS and placed in DMEM. The fluorescence distribution of the MitoTracker Green and LysoBrite NIR probes in the two groups of cells was observed and recorded under a laser scanning confocal microscope.

[0069] Results analysis: The experimental results of test example 6 are shown below. Figure 5 . Figure 5These are fluorescence micrographs of mitochondria and lysosomes in HeLa cells after treatment with the molecules synthesized in Example 1. Figure 5 (A) is a fluorescence micrograph of mitochondria in cells of the positive control sample group; Figure 5 (B) is a fluorescence micrograph of lysosomes in the positive control sample group; Figure 5 (C) is Figure 5 (A) and Figure 5 (B) overlay image; Figure 5 (D) is a fluorescence micrograph of mitochondria in cells treated with the molecules synthesized in Example 1; Figure 5 (E) is a fluorescence micrograph of lysosomes in cells treated with the molecules synthesized in Example 1; Figure 5 (F) is Figure 5 (D) and Figure 5 (E) overlay. Figure 5 (A) and Figure 5 (B) exhibits excellent fluorescence overlap, indicating mitochondrial-lysosomal fusion during the later stages of mitophagy; Figure 5 (D) and Figure 5 The fluorescence of (E) is almost completely non-overlapping, indicating that after autophagy activation following treatment with the molecules synthesized in Example 1, mitochondria and lysosomes cannot fuse in the later stages of autophagy; in contrast... Figure 5 (C) and Figure 5 (F) indicates that the molecule synthesized in Example 1 can prevent the fusion of mitochondria and lysosomes in cells and inhibit autophagic flux.

[0070] Test Example 7 (E) Verification of the cell entry mechanism of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide Two groups of glass-bottomed culture dishes, each confluent with HeLa cells prepared in Test Example 1, were washed with PBS. One group was incubated with 2 µM of the complete culture medium solution containing the molecule synthesized in Example 1 at 37°C in the dark for 30 min, while the other group was incubated with 2 µM of the complete culture medium solution containing the molecule synthesized in Example 1 at 4°C in the dark for the same amount of time. After incubation, the cells were washed with PBS and placed in DMEM. The distribution of red fluorescence in the cells of both groups was observed and recorded using a laser scanning confocal microscope (EX 561 nm, EM 600-700 nm).

[0071] Results analysis: The test results for Test Example 7 are shown below. Figure 6 . Figure 6 (A) and 6 (B) are at 37℃ and 4℃ respectively. EA red fluorescence micrograph of HeLa cells treated with 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide. The contrast shows that... Figure 6 The fluorescence intensity of (A) is significantly greater than that of (A). Figure 6 The fluorescence of (B) indicates (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide enters cells primarily via active transport.

[0072] Test Example 8 (E) Growth changes of HEK293 and HeLa cells after administration of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide After photographing the two groups of HEK293 cell spheres and two groups of HeLa cell spheres prepared in Test Example 1 under a microscope, one group of HEK293 and HeLa cell spheres was cultured in a 10 µM complete culture medium solution of the molecule synthesized in Example 1 as the experimental group, and the other group of HEK293 and HeLa cell spheres was cultured in an equal volume of DMSO complete culture medium solution as the blank control group. After 24 h, the cell spheres were photographed again under a microscope to observe the changes in morphology and size.

[0073] Results analysis: The test results for Test Example 8 are shown below. Figure 7 . Figure 7 (A) and 7(B) are bright-field micrographs of HEK293 cells before incubation and drug administration; Figure 7 (C) and 7(D) are bright-field micrographs of HeLa cell spheres before drug administration after incubation; Figure 7 (E) and 7 (G) are bright-field micrographs of HEK293 cell spheres and HeLa cell spheres, respectively, after 24 h of blank control group. Figure 7 (F) and (H) are bright-field micrographs of HEK293 and HeLa cell spheres 24 h after incubation and drug administration, respectively. (Comparison) Figure 7 As can be seen from (A), 7 (B), 7 (E), and 7 (F), compared with the blank control group, the size and shape of HEK293 cell spheres in the drug-treated experimental group did not change significantly, indicating that the 10µM molecule synthesized in Example 1 did not significantly inhibit the growth of normal HEK293 cell spheres; while in contrast... Figure 7As shown in (C), 7 (D), 7 (G), and 7 (H), the size of HeLa cell spheroids in the blank control group increased significantly after 24 hours. The size of HeLa cell spheroids in the experimental drug-treated group was much smaller than that in the blank control group after 24 hours, and also smaller than before drug administration. Furthermore, many cell fragments were present near the cell spheroids, indicating that the 10 µM molecule synthesized in Example 1 has a significant inhibitory and destructive effect on the growth of cancer cell spheroids, HeLa. The results demonstrate that the molecule synthesized in Example 1 has a significant selective cancer cell killing effect.

[0074] Test Example 9 (E) The binding constant of 4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide to albumin Different concentrations of the molecule synthesized in Example 1 (0-15µM) were added to a PBS solution of bovine serum albumin, and the fluorescence spectrum of albumin (280nmEX) was then measured using a fluorescence spectrometer.

[0075] Results analysis: The test results for Test Example 9 are shown below. Figure 8 . Figure 8 (A) Fluorescence spectra of albumin synthesized in Example 1 at different concentrations (0-15 µM). Figure 8 (B) is based on Figure 8 (A) A double logarithmic fitting curve of the degree of fluorescence intensity reduction versus the concentration of the molecule synthesized in Example 1. From Figure 8 (A) It can be seen that as the concentration of the molecules synthesized in Example 1 increases, the fluorescence peak of albumin gradually decreases. Figure 8 The fitting curve in (B) shows that the binding constant between the molecule synthesized in Example 1 and albumin is as high as 1.35 × 10⁻⁶. 8 .

[0076] In summary, this application provides a dual-targeting fluorescent small organic molecule autophagy regulator and its application. Compared with existing autophagy activators and inhibitors, this invention's dual-targeting fluorescent small organic molecule autophagy regulator can simultaneously target mitochondria and lysosomes. On the one hand, it disrupts mitochondria and activates mitophagy; on the other hand, it alkalinizes the pH of lysosomes, preventing the fusion of mitochondria and lysosomes and inhibiting autophagic flux. By activating autophagy and inhibiting autophagic flux, it achieves the effect of killing cancer cells. In addition, the fluorescence is greatly enhanced after targeting mitochondria and lysosomes, realizing synchronous fluorescence visualization of mitochondria and lysosomes.

[0077] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An autophagy regulator, characterized in that, The autophagy regulator is a compound having the structure shown in formula (I): Formula I; Wherein, the R 1 Selected from hydrogen, methyl, n-propyl, or n-butyl; the R 2 Selected from methyl or ethyl; the R 3 The X is selected from ethoxy groups; the X is selected from halogen atoms, BF4, and ClO4.

2. The autophagy regulator according to claim 1, characterized in that, The autophagy regulator can simultaneously target mitochondria and lysosomes, activating mitophagy and disrupting lysosomal function, thereby inhibiting autophagic flux.

3. The autophagy regulator according to claim 1, characterized in that, When the autophagy regulator targets mitochondria, the mitochondrial membrane potential decreases; and when the autophagy regulator targets lysosomes, the pH of the lysosomes increases.

4. The autophagy regulator according to claim 1, characterized in that, The autophagy regulator targets the mitochondria and / or lysosomes of the sample cells, enhancing fluorescence intensity and enabling fluorescence imaging of the mitochondria and / or lysosomes.

5. The autophagy regulator according to any one of claims 1 to 4, characterized in that, The autophagy regulator includes (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide.

6. The autophagy regulator according to claim 5, characterized in that, The (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-dodecylquinoline iodide has an affinity for albumin, enters cells via active transport, and can selectively kill cancer cells.

7. The use of an autophagy regulator as described in any one of claims 1 to 6 in the preparation of products for regulating cell autophagy and related life activities.

8. The use of an autophagy regulator as described in any one of claims 1 to 6 in the preparation of a product for use in targeting mitochondria or targeting lysosomes.

9. The use of an autophagy regulator as described in any one of claims 1 to 6 in the preparation of a product for use in mitochondrial imaging or lysosomal imaging.

10. A drug for treating tumors, characterized in that, The drugs include autophagy regulators, which include autophagy regulators as described in any one of claims 1 to 6.

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