Autophagy regulator, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are an autophagy regulator, a preparation method therefor, and a use thereof. A dual-targeting fluorescent organic small-molecule autophagy activator and autophagy inhibitor and the use thereof aim to solve the problems of single targeting, no cell selectivity, and poor cancer cell killing effects in autophagy regulation within the prior art.

Description

An autophagy regulator and its preparation method and application Technical Field

[0001] The present invention relates to the field of probes, and in particular to an autophagy regulator and a preparation method and application thereof. Background Art

[0002] Autophagy is the self-digestion of lysosomes or vacuoles, and the degradation and recycling of cellular contents. It is crucial for maintaining cellular homeostasis and energy balance. It also has a wide range of biological roles, including organelle remodeling, protein and organelle quality control, tumor suppression, pathogen elimination, immune and inflammatory regulation, and cell survival. Research has shown that dysfunction in the autophagy process is associated with a variety of diseases, including cancer, neurodegenerative diseases, diabetes, autoimmune diseases, and cardiovascular disease. Therefore, regulating autophagy is of great significance for the treatment of various diseases. Targeted drugs targeting autophagy are currently under development for various diseases.

[0003] Currently available autophagy regulators, including rapamycin and chloroquine, can only activate or inhibit autophagy, and do not produce fluorescence upon binding to their targets, making fluorescence observation impossible. In particular, their efficacy and tumor selectivity need to be improved for tumor treatment.

[0004] Summary of the Invention

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

[0006] According to an embodiment of the first aspect of the present invention, an autophagy regulator is a compound having a structure as shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0007] Among them, the R 1 Any one selected from hydrogen or C1-C4 alkyl; said R 2 Any one selected from hydrogen or C1-C4 alkyl; said R 3 Any one selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy; said X is any one selected from halogen atom, BF4, ClO4.

[0008] According to some embodiments of the present invention, the autophagy regulator is an autophagy activator and an autophagy inhibitor.

[0009] The autophagy regulating method of the autophagy regulator of the present invention is: mixing sample cells with the autophagy regulator and incubating them, destroying the mitochondria of the sample cells to induce mitochondrial autophagy; and destroying the lysosomes of the sample cells to inhibit autophagic flux.

[0010] In the step of destroying the mitochondria of the sample cells using the autophagy regulator of the present invention, the mitochondrial membrane potential is reduced.

[0011] In the step of destroying the lysosomes of the sample cells using the autophagy regulator of the present invention, the pH of the lysosomes increases.

[0012] The autophagy regulator provided by the present invention is a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor. Compared with existing autophagy regulators, the dual-targeted fluorescent organic small molecule of the present invention is unique in that it can simultaneously activate mitochondrial autophagy and disrupt lysosomal function, inhibiting autophagic flux and thus killing cancer cells. It is also a new type of mitochondrial / lysosomal fluorescent probe. Compared with existing mitochondrial and lysosomal fluorescent probes, it can simultaneously target both mitochondria and lysosomes. It emits red fluorescence to image the morphology, number, and distribution of mitochondria and lysosomes, and has good membrane permeability and redyeing compatibility.

[0013] The dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor provided by the present invention can, on the one hand, act as an autophagy regulator to activate mitochondrial autophagy and inhibit autophagic flux, thereby being used 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 research related to mitochondria and lysosomes and clinical diagnosis, with wide application and good effect.

[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 the embodiment, the C1-C4 alkyl group includes any one of a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group.

[0016] According to some embodiments of the present invention, the R 2 In the embodiment, the C1-C4 alkyl group includes any one of a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group.

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

[0018] According to some embodiments of the present invention, the C1-C4 alkoxy group includes any one of a methoxy group, an ethoxy group, a propoxy group and a butoxy group.

[0019] According to some embodiments of the invention, the autophagy regulator comprises (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide.

[0020] In the present invention, R 1 Selected from ethyl, R 2 is selected from hydrogen, said R 3 When the methyl group is selected from methoxy and the X is selected from iodine, the obtained autophagy regulator independent of mitochondrial membrane potential is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide.

[0021] According to a second aspect of the present invention, a method for preparing an autophagy regulator comprises the following steps:

[0022] S1. placing 4-methylquinoline (Formula II) and a long-chain alkyl halide (Formula III) in a solvent and refluxing to generate a 1-long-chain alkyl-4-methylquinoline salt (Formula IV);

[0023] S2. Mixing 1-long-chain alkyl-4-methylquinoline salt, indole-3-carboxaldehyde (Formula V) and the catalyst, reflux the reaction and remove impurities;

[0024] The catalyst includes piperidine.

[0025] According to some embodiments of the invention, the solvent comprises ethanol.

[0026] According to some embodiments of the present invention, the molar ratio of indole-3-carboxaldehyde to 4-methylquinoline is 1:(1.0-2.0).

[0027] According to some embodiments of the present invention, in step S1, the reflux reaction time is 3 to 4 days.

[0028] According to some embodiments of the present invention, in step S2, the reflux reaction time is 1 to 2 days.

[0029] According to some preferred embodiments of the present invention, the indole-3-carboxaldehyde is selected from 5-methoxy-3-formyl indole; the alkyl halide is selected from iododecane, and the autophagy regulator is prepared by using 5-methoxy-3-formyl indole and iododecane as reactants as follows:

[0030] S01. Prepare a mixed solution of 4-methylquinoline and iodododecane in ethanol;

[0031] S02. The reaction was heated with stirring at reflux for three days;

[0032] S03. Add 5-methoxy-3-formyl indole in ethanol;

[0033] S04. A piperidine catalyst was added to the ethanol mixed solution, and the piperidine-added ethanol mixed solution was heated to 85°C and refluxed with stirring for one day, and then slowly cooled to room temperature to obtain dark green crystals or an organic solid product to be purified;

[0034] S05. The organic solid product to be purified is purified by column chromatography, using dichloromethane / methanol as eluent, and dried to obtain dark green crystals and dark red powder. The dark green crystals and dark red powder are dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor. The dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide.

[0035] According to the third aspect of the present invention, an autophagy regulator is used in the preparation of related life activity products for regulating cell autophagy.

[0036] In the present invention, the dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor is used to regulate cell autophagy for non-diagnostic and therapeutic purposes. The dual-targeted fluorescent organic small molecule of the present application can target the mitochondria and lysosomes of the cells, causing the mitochondrial membrane potential to decrease or even be lost, thereby inducing mitochondrial autophagy and generating a large number of autophagosome vesicles; at the same time, the lysosomal pH rises, and it cannot fuse with the autophagosome to form autophagolysosomes, thereby inhibiting the completion of the autophagic flow and achieving dual regulation of autophagy.

[0037] According to the fourth aspect of the present invention, an autophagy regulator is used in the preparation of a product for targeting mitochondria or lysosomes.

[0038] Use of the autophagy regulator according to the fifth aspect of the present invention in the preparation of a product for mitochondrial imaging or lysosomal imaging.

[0039] The method for performing mitochondrial / lysosomal fluorescence imaging using an autophagy regulator in the present invention comprises: mixing and incubating sample cells with an autophagy regulator, wherein the autophagy regulator binds to the mitochondria and lysosomes of the sample cells, and the fluorescence intensity is enhanced to achieve fluorescence imaging of the mitochondria and lysosomes.

[0040] The present invention provides a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor for the application of mitochondrial / lysosomal fluorescence imaging. Compared with existing mitochondrial and lysosomal fluorescent probes, the dual-targeted fluorescent organic small molecule can simultaneously target cell mitochondria and lysosomes. After binding to mitochondria and lysosomes, the fluorescence is greatly enhanced, realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.

[0041] The dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor in the present invention is used for mitochondrial / lysosomal fluorescence imaging for non-diagnostic and therapeutic purposes. The dual-targeted fluorescent organic small molecule in the present application does not fluoresce itself. After binding to mitochondria and lysosomes, the fluorescence is greatly enhanced, realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.

[0042] According to a sixth aspect of the present invention, a drug for treating tumors includes a cell autophagy regulating drug, and the cell autophagy regulating drug includes the autophagy regulator.

[0043] According to some embodiments of the present invention, the tumor comprises an albumin receptor-overexpressing tumor.

[0044] According to some embodiments of the present invention, the tumors overexpressing albumin receptors include cervical cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, liver cancer, etc.

[0045] According to some embodiments of the present invention, the medicament comprises an injectable composition or a composition for oral administration.

[0046] According to some embodiments of the present invention, the composition includes a dual-targeted 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.

[0047] The method of using the autophagy regulator to kill cancer cells in the present invention comprises: mixing sample cells with the autophagy regulator and incubating them, activating mitochondrial autophagy, inhibiting lysosomal function and autophagy, and inducing death of the sample cells.

[0048] Compared with existing autophagy regulators, the autophagy regulator in the present invention is a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor with dual targeting effects. It can simultaneously target cell mitochondria and lysosomes, activate mitochondrial autophagy and inhibit lysosomal function, and ultimately lead to cell death. This dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor can be used to kill cancer cells, realizing the application of the dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor as an autophagy regulator and cancer cell killer. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0050] Figure 1 is a fluorescence micrograph of HeLa cells co-stained with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide, a mitochondrial green fluorescent probe (MitoTracker Green), and a lysosomal deep red fluorescent probe (LysoBrite NIR) provided in Example 3 of the present application.

[0051] Figure 2 is a fluorescence micrograph of HeLa cells stained with rhodamine 123 after being treated with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide provided in Example 4 of the present application.

[0052] Figure 3 is a fluorescence micrograph of HeLa cells stained with a lysosomal green fluorescent probe (Lysosensor Green DND-189) after treatment with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide provided in Example 5 of the present application.

[0053] FIG4 is a diagram of 5-chloromethylfluorescein diacetate (green live cell tracking probe CellTracker) after HeLa cells were treated with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide provided in Example 6 of the present application. TM Fluorescence micrographs of CMFDA Green staining.

[0054] Figure 5 is a fluorescence micrograph of the staining of mitochondria and lysosomes of HeLa cells after treatment with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide and CCCP, respectively, provided in Test Example 6 of the present application.

[0055] FIG6 is a fluorescence micrograph of HeLa cells incubated with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide provided in Test Example 7 of the present application at 37° C. and 4° C., respectively.

[0056] FIG7 is a micrograph of normal cell spheres HEK293 and cancer cell spheres HeLa before and after incubation with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide provided in Test Example 8 of the present application.

[0057] FIG8 is the fluorescence spectrum and double logarithmic fitting curve of the interaction between (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide and albumin provided in Test Example 9 of the present application. DETAILED DESCRIPTION

[0058] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0059] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0060] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0061] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can 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 the present application.

[0062] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0063] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0064] Example 1

[0065] This example provides a synthesis of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide:

[0066] First, 200 μL of 4-methylquinoline and 393 μL of iodododecane were dissolved in 10 mL of ethanol and heated at 85°C with stirring and reflux for three days. Then, an ethanol solution containing 0.263 g of 5-methoxy-3-formyl indole was added, stirred evenly, and 5 drops of piperidine were added, causing the solution to gradually turn red. After one day of reflux at 85°C, 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, cooled, and the product was purified by column chromatography using dichloromethane / methanol as the eluent to obtain dark green crystals and dark red powder in a yield of approximately 27%.

[0067] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 12.11 (s, 1H), 9.12 (d, J = 4.0Hz, 1H), 8.97 (d, J = 8.0Hz, 1H), 8.63 (d, J = 16.0Hz, 1H), 8.44 (m, 3H), 8.18 (t, J = 8.0Hz , 1H), 7.99 (m, 2H), 7.70 (s, 1H), 7.42 (d, J=8.0Hz, 1H), 6.90 (dd, J=4.0, 8 .0Hz, 1H), 4.85 (t, J=8.0Hz, 2H), 3.90 (s, 3H), 1.91 (m, 2H), 1.37 (m, 2H), 1.21 (m, 16H), 0.83 (t, J=8.0Hz, 3H). 13 C NMR (400MHz, 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, 119.2 9, 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.

[0068] In order to develop more compounds with similar functions, the present invention keeps the molecular core skeleton unchanged, R 3It can also be selected from any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, R 2 Any one selected from hydrogen or C1-C4 alkyl. According to the inventors' research, the function of the autophagy regulator in the present invention is determined by the conjugated organic cationic group, and the anion X - Unrelated; R 2 Any one selected from hydrogen or C1-C4 alkyl; R 3 Any one selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, changes within this range do not affect the function of the molecule; but R 1 The length of the connected carbon chain has a great influence on the targeting and protein binding of the molecule. For example, indole octyl quinoline salt has weak binding to albumin and still enters the cell by free diffusion. As the quinoline carbon chain increases (from decyl to tetradecyl), the binding to albumin is enhanced and it enters the cell by active transport.

[0069] Test Example 1

[0070] HeLa and HEK293 cell and spheroid culture:

[0071] Cancer cells HeLa and normal cells HEK293 were cultured in complete culture 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 to 3 days.

[0072] After the cells grow to the logarithmic phase, they are transferred to confocal dish for culture: the cells in the T25 cell culture flask are washed with PBS first, and then digested with 1 mL of trypsin for 1 to 2 minutes (HeLa uses 0.25% trypsin, HEK293 uses 0.025% trypsin), the trypsin is removed, fresh culture medium is added, blown evenly and the cells are counted, and the cell density is controlled by the amount of culture medium added. (1) The cells are inoculated into a confocal glass-bottom culture dish and placed in a 5% CO2 incubator for culture. When the cells grow to a coverage rate of about 70%, they are used for cell imaging experiments; (2) The cells are inoculated into a low-adsorption U-bottom 96-well plate for culture. When the diameter of the cell sphere reaches 800 μm, it is used for drug anti-tumor evaluation.

[0073] Test Example 2

[0074] Observation on the staining of HeLa cells by (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinolinium iodide

[0075] The HeLa cell-covered slide prepared in Test Example 1 was washed twice with PBS and then subjected to the following staining steps: (1) incubated with 0.5 μM commercial mitochondrial green fluorescent probe (MitoTracker Green) solution for 30 min and washed with PBS; (2) incubated with 0.5 μM commercial lysosomal near-infrared fluorescent probe (LysoBrite NIR) solution for 30 min and washed with PBS; (3) incubated with 2 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide fluorescent probe solution for 30 min and washed with DMEM. The stained cell samples were observed by multi-channel fluorescence co-localization using a confocal fluorescence microscope.

[0076] The results are shown in Figure 1. Figure 1(A) shows the red fluorescence image of the molecule synthesized in Example 1, Figure 1(B) shows the fluorescence image of a commercial mitochondrial green fluorescent probe, and Figure 1(C) shows the fluorescence image of a commercial lysosomal near-infrared fluorescent probe. Figure 1(D) is an overlay of Figure 1(A), Figure 1(B), and Figure 1(C). Figure 1(A) covers both Figures 1(B) and 1(C), and the sum of Figures 1(A), 1(B), and 1(C) overlaps well, indicating that the fluorescence of the molecule synthesized in Example 1 is distributed in both mitochondria and lysosomes. This result demonstrates that (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide can be used to target mitochondria and lysosomes, and can also be used for simultaneous fluorescence imaging of mitochondria and lysosomes.

[0077] Test Example 3

[0078] Changes of mitochondrial membrane potential in HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinolinium iodide

[0079] Two sets of glass-bottomed culture dishes, each filled with HeLa cells prepared in Test Example 1, were washed with PBS and then incubated in a CO2 incubator in the dark for 30 minutes with 5 μM rhodamine 123. The dishes were then washed again with PBS. One set was incubated in the dark for 30 minutes with a 10 μM culture medium solution containing the molecule synthesized in Example 1. The other set was placed in culture medium supplemented with an equal amount of DMSO as a blank control. After washing with PBS, the incubated cells were observed under a laser scanning confocal microscope, and changes in the fluorescence brightness of rhodamine 123 in both sets of cells were recorded.

[0080] Result analysis:

[0081] The experimental results of Test Example 3 are shown in Figure 2. Figure 2 is a fluorescence micrograph of the mitochondrial membrane potential probe Rhodamine 123 before and after treatment of HeLa cells with the molecule synthesized in Example 1. Figure 2(A) is a fluorescence micrograph of cells in the blank control sample group; Figure 2(B) is a bright field micrograph corresponding to Figure 2(A); Figure 2(C) is a fluorescence micrograph of Rhodamine 123 of cells treated with the molecule synthesized in Example 1; and Figure 2(D) is a bright field micrograph corresponding to Figure 2(C). As can be seen from Figures 2(A) and 2(C), the fluorescence of Rhodamine 123 in cells treated with the molecule synthesized in Example 1 is greatly weakened (Figure 2(C)), indicating that the mitochondrial membrane potential of cells treated with the molecule synthesized in Example 1 is significantly reduced, that is, the mitochondria are damaged.

[0082] Test Example 4

[0083] Changes in lysosomal pH of HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide

[0084] Two sets of glass-bottomed culture dishes, each filled with HeLa cells prepared in Test Example 1, were washed with PBS and then incubated in a CO2 incubator in the dark for 30 minutes with 2 μM lysosomal green fluorescent probe (LysoSensor Green DND-189). The cells were then washed again with PBS. One set was incubated in the dark for 30 minutes with a 10 μM culture medium solution containing the molecule synthesized in Example 1, and the other set was placed in culture medium supplemented with an equal amount of DMSO as a blank control sample. After incubation, the cells were washed with PBS and placed in DMEM. The changes in the fluorescence brightness of the lysosomal green fluorescent probe in the two sets of cells were observed and recorded under a laser scanning confocal microscope.

[0085] Result analysis:

[0086] The experimental results of Test Example 4 are shown in Figure 3, which is a fluorescence micrograph of the lysosomal green fluorescent probe (LysoSensor Green DND-189) before and after treatment of HeLa cells with the synthetic molecule of Example 1. Figure 3(A) is a fluorescence micrograph of cells in the blank control sample group; Figure 3(B) is a bright field micrograph corresponding to Figure 3(A); Figure 3(C) is a fluorescence micrograph of the lysosomal green fluorescent probe of cells treated with the synthetic molecule of Example 1; and Figure 3(D) is a bright field micrograph corresponding to Figure 3(C). LysoSensor Green DND-189 fluorescence increases with decreasing pH. As can be seen from Figures 3(A) and 3(C), the fluorescence of the lysosomal green fluorescent probe is greatly weakened in cells treated with the synthetic molecule of Example 1 (Figure 3(C)), indicating that the pH of the lysosomes of cells treated with the synthetic molecule of Example 1 is significantly increased, affecting the digestive function of the lysosomes in the autophagic flow.

[0087] Test Example 5

[0088] Imaging of autophagic vacuoles and autophagosomes in HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide

[0089] Two sets of glass-bottomed culture dishes, each filled with HeLa cells, prepared in Test Example 1, were washed with PBS. One set was then incubated in the dark for 90 minutes with a 10 μM culture medium solution containing the molecule synthesized in Example 1. The other set was placed in the culture medium supplemented with an equal amount of DMSO as a blank control. Subsequently, the cells were incubated in the dark for 30 minutes with a 5 μM green live cell tracking probe (Cell-Tracker Green CMFDA). The cells were washed with PBS and placed in DMEM. The fluorescence distribution of the green live cell tracking probe in both sets of cells was observed and recorded under a laser scanning confocal microscope.

[0090] Result analysis:

[0091] The experimental results of Test Example 5 are shown in Figure 4. Figure 4 is a fluorescence micrograph of the green live cell tracer probe (Cell-Tracker Green CMFDA) of HeLa cells before and after treatment with the molecule synthesized in Example 1, Figure 4 (A) is a fluorescence micrograph of cells in the blank control sample group; Figure 4 (B) is a fluorescence micrograph of the green live cell tracer probe of cells treated with the molecule synthesized in Example 1. Comparing Figure 4 (A) and Figure 4 (B), a large number of vacuoles (autophagic vacuoles and autophagosomes) are produced in the cytoplasm of cells treated with the molecule synthesized in Example 1, indicating that the molecule synthesized in Example 1 can activate cellular autophagy.

[0092] Test Example 6

[0093] Effect of (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide on the fusion of mitochondria and lysosomes in HeLa cells treated with

[0094] Two sets of glass-bottomed culture dishes, each filled with HeLa cells, prepared in Test Example 1, were washed with PBS and then incubated in the dark for 30 minutes with 1 μM mitochondrial green fluorescent probe (MitoTracker Green) and 1 μM lysosomal deep red probe (LysoBrite NIR). Subsequently, one set was incubated with a 20 μM CCCP solution in PBS for ~5 hours as a positive control for mitophagy, and the other set was incubated in the dark for the same period of time with a 10 μM culture medium solution containing the molecule synthesized in Example 1. After incubation, the cells were washed with PBS and placed in DMEM. The fluorescence distribution of the mitochondrial green fluorescent probe and lysosomal deep red probe in the two sets of cells was observed and recorded under a laser scanning confocal microscope.

[0095] Result analysis:

[0096] The experimental results of Test Example 6 are shown in Figure 5. Figure 5 is a fluorescence micrograph of mitochondria and lysosomes of HeLa cells after treatment with the molecule synthesized in Example 1. Figure 5(A) is a fluorescence micrograph of mitochondria of cells in the positive control sample group; Figure 5(B) is a fluorescence micrograph of lysosomes of cells in the positive control sample group; Figure 5(C) is an overlay of Figure 1(A) and Figure 1(B); Figure 5(D) is a fluorescence micrograph of mitochondria of cells treated with the molecule synthesized in Example 1; Figure 5(E) is a fluorescence micrograph of lysosomes of cells treated with the molecule synthesized in Example 1; and Figure 5(F) is an overlay of Figure 1(D) and Figure 1(E). The fluorescence overlap between Figure 5(A) and Figure 5(B) is very good, indicating that mitochondria and lysosomes fuse in the late stage of mitochondrial autophagy; the fluorescence between Figure 5(D) and Figure 5(E) is almost completely non-overlapping, indicating that after the activation of cell autophagy after treatment with the synthesized molecule in Example 1, mitochondria and lysosomes cannot fuse in the late stage of autophagy; comparing Figure 5(C) and Figure 5(F), it is shown that the synthesized molecule in Example 1 can prevent the fusion of cell mitochondria and lysosomes and inhibit the autophagic flow.

[0097] Test Example 7

[0098] Verification of the entry mode of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinolinium iodide into cells

[0099] Two sets of glass-bottomed culture dishes, each filled with HeLa cells, prepared in Test Example 1, were washed with PBS. One set was incubated with a 2 μM solution of the molecule synthesized in Example 1 in complete culture medium at 37°C for 30 min in the dark. The other set was incubated with a 2 μM solution of the molecule synthesized in Example 1 in complete culture medium at 4°C for the same time in the dark. The incubated cells were washed with PBS and placed in DMEM. The red fluorescence distribution (EX 561 nm, EM 600-700 nm) in both sets of cells was observed using a laser scanning confocal microscope.

[0100] Result analysis:

[0101] The experimental results of Test Example 7 are shown in Figure 6. Figures 6(A) and 6(B) are red fluorescence micrographs of HeLa cells incubated with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide at 37°C and 4°C, respectively. As can be seen by comparison, the fluorescence intensity in Figure 6(A) is significantly greater than that in Figure 6(B), indicating that (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide enters the cells primarily through active transport.

[0102] Test Example 8

[0103] Effects of (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinolinium iodide on the growth of HEK293 and HeLa cell spheres

[0104] After photographing the two groups of HEK293 cell spheroids and two groups of HeLa cell spheroids prepared in Test Example 1 under a microscope, one group of HEK293 and HeLa cell spheroids were 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 spheroids were cultured in a complete culture medium solution with an equal volume of DMSO as the blank control group. After 24 hours, the cells were photographed again using a microscope to observe the changes in the morphology and size of the cell spheroids.

[0105] Result analysis:

[0106] The test results of Test Example 8 are shown in Figure 7. Figures 7(A) and 7(B) are bright field micrographs of HEK293 cell spheres before incubation and administration; Figures 6(C) and 6(D) are bright field micrographs of HeLa cell spheres before incubation and administration; Figures 7(E) and 7(G) are bright field micrographs of the HEK293 cell spheres and HeLa cell sphere blank control groups after 24 hours; Figures 7(F) and 7(H) are bright field micrographs of the HEK293 cell spheres and HeLa cell spheres after 24 hours of incubation and administration, respectively. Comparison of Figures 7(A), 7(B), 7(E), and 7(F) shows that compared with the blank control group, the size and shape of the HEK293 cell spheres in the treatment experimental group did not change significantly, indicating that 10 μM of the molecule synthesized in Example 1 did not significantly inhibit the growth of normal HEK293 cell spheres. Comparison of Figures 7(C), 7(D), 7(G), and 7(H) shows that the size of the HeLa cell spheres in the blank control group increased significantly after 24 hours, while the size of the HeLa cell spheres in the treatment experimental group was much smaller than that of the blank control group after 24 hours, and smaller than that of the cell spheres before treatment. In addition, there were many cell fragments near the cell spheres, indicating that 10 μM of the molecule synthesized in Example 1 had a significant inhibitory and destructive effect on the growth of cancer cell HeLa cell spheres. The results show that the molecule synthesized in Example 1 has a significant selective cancer cell killing effect.

[0107] Test Example 9

[0108] Binding constants of (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinolinium iodide to albumin

[0109] Different concentrations of the molecule synthesized in Example 1 (0-15 μM) were added to a PBS solution of bovine serum albumin, and the fluorescence spectrum of the albumin (280 nm EX) was measured using a fluorescence spectrometer.

[0110] Result analysis:

[0111] The experimental results of Test Example 9 are shown in Figure 8. Figure 8(A) shows the fluorescence spectra of albumin with different concentrations of the molecule synthesized in Example 1 (0-15 μM) added, and Figure 8(B) shows a double logarithmic fitting curve based on Figure 8(A) showing the degree of fluorescence intensity reduction and the concentration of the molecule synthesized in Example 1. As can be seen from Figure 8(A), as the concentration of the molecule synthesized in Example 1 increases, the fluorescence peak of albumin gradually decreases. According to the fitting curve Figure 8(B), the binding constant between the molecule synthesized in Example 1 and albumin is as high as 1.35×10 8 .

[0112] In summary, the present application provides a dual-targeted fluorescent organic small molecule autophagy regulator and its application. Compared with existing autophagy activators and autophagy inhibitors, the dual-targeted fluorescent organic small molecule autophagy regulator of the present invention can simultaneously target mitochondria and lysosomes, on the one hand destroying mitochondria to activate mitophagy, and on the other hand alkalizing the lysosomal pH, preventing the fusion of mitochondria and lysosomes, and inhibiting the autophagic flow. By activating autophagy and inhibiting the autophagic flow, the effect of killing cancer cells is achieved. In addition, the fluorescence is greatly enhanced after targeting mitochondria and lysosomes, realizing synchronous fluorescence visualization of mitochondria and lysosomes.

[0113] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. An autophagy regulator, characterized in that, The autophagy regulator is a compound having a structure represented by formula (I), or a pharmaceutically acceptable salt thereof: Among them, the R 1 is selected from any one of hydrogen or C1-C4 alkyl; the R 2 is selected from any one of hydrogen or C1-C4 alkyl; the R 3 is selected from any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy; the X is selected from any one of halogen atoms, BF 4 , ClO 4 and the like.

2. The autophagy regulator according to claim 1, characterized in that, The R 1 Among them, the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

3. The autophagy regulator according to claim 1, characterized in that, The R 2 Among them, the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

4. The autophagy regulator according to claim 1, characterized in that, The R 3 Among them, the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl; the C1-C4 alkoxy group includes any one of methoxy, ethoxy, propoxy and butoxy.

5. The autophagy regulator according to any one of claims 1 to 4, characterized in that, the autophagy regulator comprises (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide.

6. A method for preparing an autophagy regulator according to any one of claims 1 to 5, characterized in that, the preparation method comprises the following steps: S1. 4-Methylquinoline and a long-chain alkyl halide are placed in a solvent and refluxed to react to form 1-long-chain alkyl-4-methylquinolinium salt; S2. 1-Long-chain alkyl-4-methylquinolinium salt, indole-3-carbaldehyde and a catalyst are mixed, refluxed and then the impurities are removed; the catalyst comprises piperidine.

7. An application of an autophagy regulator according to any one of claims 1 to 5 in the preparation of products for regulating related life activities applied to autophagy.

8. An application of an autophagy regulator according to any one of claims 1 to 5 in the preparation of products for targeting mitochondria or targeting lysosomes.

9. An application of an autophagy regulator according to any one of claims 1 to 5 in the preparation of products for mitochondrial imaging or lysosomal imaging.

10. A drug for treating tumors, characterized in that, the drug comprises drugs for regulating autophagy, and the drugs for regulating autophagy comprise an autophagy regulator according to any one of claims 1 to 4.

Images