Autophagy regulator as well as preparation method and application thereof

Abstract

The invention discloses an autophagy regulator and a preparation method and application thereof, relates to a dual-targeting fluorescent organic small molecule autophagy activation and autophagy inhibitor and application thereof, and aims to solve the problems of single autophagy regulation targeting, no cell selectivity and poor cancer cell killing effect in the prior art.

Description

Technical Field

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

[0002] Autophagy is the self-digestion of lysosomes or vacuoles, as well as the degradation and recycling of cellular contents, and is crucial for maintaining cellular homeostasis and energy balance. At the same time, it 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, etc. Research shows that dysfunctions in the autophagy process are related to various diseases, including cancer, neurodegenerative diseases, diabetes, autoimmune diseases, and cardiovascular diseases, etc. Therefore, autophagy regulation is of great significance for the treatment of various diseases. Currently, targeted drugs for autophagy in various diseases are also under further research and development.

[0003] Currently existing autophagy regulators include rapamycin, chloroquine, etc., which can only activate autophagy or inhibit autophagy singly, and do not produce fluorescence after binding to the target, and cannot be observed by fluorescence. Especially when used for tumor treatment, both the curative effect and tumor selectivity need to be improved. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor and its application, aiming to solve the problems of single autophagy regulation target, no cell selectivity, and poor curative effect on killing cancer cells in the prior art.

[0005] An autophagy regulator according to an embodiment of the first aspect of the present invention, the autophagy regulator is a compound having a structure shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Wherein, the R 1 is selected from any one of hydrogen or a C1-C4 alkyl; the R 2 is selected from any one of hydrogen or a C1-C4 alkyl; the R 3 is selected from any one of hydrogen, a C1-C4 alkyl, and a C1-C4 alkoxy; the X is selected from any one of a halogen atom, BF 4 , ClO 4 .

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

[0009] The method for autophagy regulation by the autophagy regulator of the present invention is as follows: Mix and incubate sample cells with the autophagy regulator, disrupt the mitochondria of the sample cells to trigger mitophagy; disrupt the lysosomes of the sample cells to inhibit autophagic flux.

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

[0011] In the step of the autophagy regulator of the present invention disrupting the lysosomes of the above sample cells, 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 the existing autophagy regulators, the unique feature of the dual-targeted fluorescent organic small molecule of the present invention is that it can simultaneously activate mitophagy and disrupt lysosomal function, inhibit autophagic flux, thereby killing cancer cells; it is also a new type of mitochondrial / lysosomal fluorescent probe. Compared with the existing mitochondrial and lysosomal fluorescent probes, it can target mitochondria and lysosomes simultaneously, emit red fluorescence, image the morphology, quantity and distribution of mitochondria and lysosomes, and has good membrane permeability and good compatibility for counterstaining.

[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 mitophagy and inhibit autophagic flux, thereby acting as an anti-cancer drug to kill cancer cells; on the other hand, it can act as a fluorescent probe to label the morphology, quantity and distribution of mitochondria and lysosomes in cells, and can provide 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, in the R 1 , the C1-C4 alkyl includes 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, in the R 2 , the C1-C4 alkyl includes 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, in the R 3 , the C1-C4 alkyl 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 methoxy, ethoxy, propoxy, and butoxy.

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

[0020] In the present invention, R 1 is selected from ethyl, R 2 is selected from hydrogen, and when the R 3 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-indol-3-yl)vinyl)-1-n-dodecylquinolinium iodide.

[0021] According to an embodiment of the second aspect of the present invention, a preparation method of the autophagy regulator includes the following steps:

[0022] S1. 4-Methylquinoline (Formula II) and a long-chain alkyl halide (Formula III) are placed in a solvent and refluxed to react to generate 1-long-chain alkyl-4-methylquinolinium salt (Formula IV);

[0023] S2. The 1-long-chain alkyl-4-methylquinolinium salt, indole-3-carboxaldehyde (Formula V), and a catalyst are mixed and refluxed, and then impurities are removed;

[0024] The catalyst includes piperidine.

[0025] According to some embodiments of the present invention, the solvent includes 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]

[0030] According to some preferred embodiments of the present invention, the indole-3-carboxaldehyde is selected from 5-methoxy-3-formylindole; the alkyl halide is selected from dodecyl iodide. Using 5-methoxy-3-formylindole and dodecyl iodide as reactants, the preparation method of the autophagy regulator is as follows:

[0031] S01. Prepare an ethanol mixed solution of 4-methylquinoline and dodecyl iodide;

[0032] S02. Heat, stir and reflux for three days;

[0033] S03. Add an ethanol solution of 5-methoxy-3-formylindole;

[0034] S04. Add the catalyst piperidine to the ethanol mixed solution, heat the ethanol mixed solution with piperidine at 85 °C under reflux with stirring for one day, and slowly cool to room temperature to obtain dark green crystals or the organic solid product to be purified;

[0035] S05. Purify the organic solid product to be purified by column chromatography, use dichloromethane / methanol as the eluent, and dry to obtain dark green crystals and dark red powders. The dark green crystals and dark red powders are dual-targeted fluorescent organic small molecule autophagy activators and autophagy inhibitors. The dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor is (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinoline iodide.

[0036] Use of an autophagy regulator according to an embodiment of the third aspect of the present invention in the preparation of a product for use in related life activities of cell autophagy regulation.

[0037] In the present invention, the dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor is used for cell autophagy regulation for non-diagnostic and therapeutic method purposes. The dual-targeted fluorescent organic small molecule of the present application can target the mitochondria and lysosomes of cells. The mitochondrial membrane potential decreases or even disappears, causing mitophagy and generating a large number of autophagosome vesicles; at the same time, the pH of lysosomes rises, and they cannot fuse with autophagosomes to form autophagolysosomes, inhibiting the completion of the autophagic flux and achieving dual regulation of autophagy.

[0038] Use of an autophagy regulator according to an embodiment of the fourth aspect of the present invention in the preparation of a product for use in targeting mitochondria or targeting lysosomes.

[0039] Use of an autophagy regulator according to an embodiment of the fifth aspect of the present invention in the preparation of a product for use in mitochondrial imaging or lysosomal imaging.

[0040] The method for mitochondrial / lysosomal fluorescence imaging with the autophagy regulator in the present invention includes: mixing and incubating the sample cells with the autophagy regulator. The autophagy regulator binds to the mitochondria and lysosomes of the sample cells, and the fluorescence intensity increases, realizing fluorescence imaging of mitochondria and lysosomes.

[0041] Application of a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor for mitochondrial / lysosomal fluorescence imaging. Compared with existing mitochondrial and lysosomal fluorescent probes, the dual-targeted fluorescent organic small molecule can target both the cell mitochondria and lysosomes simultaneously. After binding to the mitochondria and lysosomes, the fluorescence is greatly enhanced, realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.

[0042] 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 non-therapeutic purposes. The dual-targeted fluorescent organic small molecule itself in this application does not fluoresce. After binding to the mitochondria and lysosomes, the fluorescence is greatly enhanced, realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.

[0043] A drug for treating tumors according to an embodiment of the sixth aspect of the present invention, the drug includes a cell autophagy regulating drug, and the cell autophagy regulating drug includes the autophagy regulator described above.

[0044] According to some embodiments of the present invention, the tumor includes tumors with overexpression of albumin receptor.

[0045] According to some embodiments of the present invention, the tumors with overexpression of albumin receptor include cervical cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, liver cancer, etc.

[0046] According to some embodiments of the present invention, the drug includes an injectable composition or a composition for oral administration.

[0047] 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.

[0048] The method for killing cancer cells by the autophagy regulator in the present invention includes: mixing and incubating sample cells with the autophagy regulator, activating mitochondrial autophagy, inhibiting lysosomal function and autophagy, and inducing the death of the sample cells.

[0049] Compared with existing autophagy regulators, the dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor in the present invention has a dual-target effect, can target both the cell mitochondria and lysosomes simultaneously, 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 a cancer cell killer. Description of the Drawings

[0050] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

[0051] Figure 1 is a fluorescence microscopy photograph of the co-staining of HeLa cells with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide provided in Example 3 of the present application, mitochondrial green fluorescent probe (MitoTracker Green), and lysosomal deep red fluorescent probe (LysoBrite NIR).

[0052] Figure 2 is a fluorescence microscopy photograph of the rhodamine 123 staining of HeLa cells after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide provided in Example 4 of the present application.

[0053] Figure 3 is a fluorescence microscopy photograph of the lysosomal green fluorescent probe (Lysosensor Green DND-189) staining of HeLa cells after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide provided in Example 5 of the present application.

[0054] Figure 4 is a fluorescence microscopy photograph of the 5-chloromethylfluorescein diacetate (green live cell tracer probe CellTracker TM Green CMFDA) staining of HeLa cells after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide provided in Example 6 of the present application.

[0055] Figure 5 is a fluorescence microscopy photograph of the staining of mitochondria and lysosomes of HeLa cells after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide and CCCP respectively provided in Test Example 6 of the present application.

[0056] Figure 6 is a fluorescence microscopy photograph of HeLa cells after incubation with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide at 37 °C and 4 °C respectively provided in Test Example 7 of the present application.

[0057] Figure 7Micrographs of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide provided by Test Example 8 of the present application before and after incubation with normal cell spheres HEK293 and cancer cell spheres HeLa.

[0058] Figure 8 Fluorescence spectrum and double logarithmic fitting curve of the interaction between (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide and albumin provided by Test Example 9 of the present application. Detailed implementation manners

[0059] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application clearer and more understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0060] In the present application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B may be singular or plural. The character " / " generally represents an "or" relationship between the front and rear associated objects.

[0061] In the present application, "at least one" means one or more, and "a plurality" means two or more. "At least one (item)" or its similar expression below refers to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can both represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively.

[0062] It should be understood that in various embodiments of the present application, the magnitude of the sequence numbers of the above processes does not mean 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 to the implementation process of the embodiments of the present application.

[0063] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "the" and "said" used in the embodiments of the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0064] In the description of the embodiments of the present application, the weights of the relevant components not only can refer to the specific contents of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down proportionally, they are within the scope disclosed in the description of the embodiments of the present application. Specifically, the mass described in the description of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0065] Example 1

[0066] This example provides the synthesis of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide:

[0067] First, 200 μL of 4-methylquinoline and 393 μL of dodecyl iodide were dissolved in 10 mL of ethanol, and the mixture was heated under stirring and refluxed at 85 °C for three days; then an ethanol solution containing 0.263 g of 5-methoxy-3-formylindole was added. After stirring evenly, 5 drops of piperidine were added, and the solution gradually turned red; after refluxing at 85 °C for one day, it was slowly cooled, filtered, and washed with a small amount of isopropanol to obtain dark green crystals or dark red powder, or the excess solvent was distilled off, cooled, and the product was purified by column chromatography using dichloromethane / methanol as the eluent to obtain dark green crystals and dark red powder, with a yield of about 27%.

[0068] 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-d 6),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.

[0069] In order to develop more compounds with similar functions, in the present invention, the molecular parent nucleus skeleton is kept unchanged, and R 3 may also be optionally selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, and R 2 is optionally selected from any one of hydrogen or C1-C4 alkyl. According to the research of the inventors, the function of the autophagy regulator in the present invention is determined by the conjugated organic cation group and has nothing to do with the anion X-; R 2 is optionally selected from any one of hydrogen or C1-C4 alkyl; R 3 is optionally selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy. Changes within this range do not affect the function of the molecule; however, R 1 The length of the carbon chain connected has a greater impact on the targeting and protein-binding of the molecule. For example, indole octyl quinoline salt has a weak binding effect with albumin and still enters cells by free diffusion. As the carbon chain of quinoline increases (from decyl to tetradecyl), the binding effect with albumin enhances and enters cells by active transport.

[0070] Test Example 1

[0071] Culture of HeLa and HEK293 cells and cell spheres:

[0072] The cancer cells HeLa and normal cells HEK293 were cultured in a complete medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin) in a saturated humidity incubator at 37 °C and 5% CO 2 and passaged once every 2 - 3 days.

[0073] When the cells grow to the logarithmic phase, transfer them to a confocal dish for culture: Wash the confluent cells in a T25 cell culture flask with PBS first, then digest them with 1 mL of trypsin for 1 - 2 minutes (use 0.25% trypsin for HeLa and 0.025% trypsin for HEK293), remove the trypsin, add fresh medium, pipette gently to make it uniform and count the cells. Control the cell density by the amount of medium added. (1) Inoculate the cells into a confocal bottom glass dish and place it in a 5% CO 2 incubator for culture. Wait until the cell coverage reaches about 70% for cell imaging experiments; (2) Inoculate the cells into a low - attachment U - bottom 96 - well plate for culture. Wait until the cell sphere diameter reaches ~800 μm for anti - tumor drug evaluation.

[0074] Test Example 2

[0075] Observation of the staining of (E)-4-(2-(5 - methoxy - 1H - indol - 3 - yl)vinyl)-1 - dodecylquinolinium iodide on HeLa cells

[0076] Wash the coverslip with confluent HeLa cells prepared in Test Example 1 twice with PBS, and then perform the following staining steps: (1) Incubate with 0.5 μM commercial mitochondrial green fluorescent probe (MitoTracker Green) solution for 30 min, and wash with PBS; (2) Incubate with 0.5 μM commercial lysosome near - infrared fluorescent probe (LysoBrite NIR) solution for 30 min, and wash with PBS; (3) Incubate with 2 μM (E)-4-(2-(5 - methoxy - 1H - indol - 3 - yl)vinyl)-1 - dodecylquinolinium iodide fluorescent probe solution for 30 min, and wash with DMEM. Observe the multi - channel fluorescence co - localization of the stained cell samples using a confocal fluorescence microscope.

[0077] The results are as Figure 1 shown. Figure 1 (A) is the red fluorescence image of the molecule synthesized in Example 1. Figure 1 (B) is the fluorescence image of the commercial mitochondrial green fluorescent probe. Figure 1 (C) is the fluorescence image of the commercial lysosome near - infrared fluorescent probe. Figure 1 (D) is Figure 1 (A) and Figure 1 (B), Figure 1 (C) superposition image. Figure 1 (A) covers Figure 1 (B) and Figure 1 (C) two regions. Figure 1 (A) and Figure 1 (B) and Figure 1The sum of (C) overlaps well, indicating that the fluorescence of the molecule synthesized in Example 1 is distributed in two organelles, mitochondria and lysosomes. This result proves that (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinoline iodide can be used to target mitochondria and lysosomes, and can also be used for simultaneous fluorescence imaging of mitochondria and lysosomes.

[0078] Test Example 3

[0079] Changes of mitochondrial membrane potential in HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinoline iodide

[0080] Two sets of glass-bottomed culture dishes prepared in Test Example 1, each covered with HeLa cells, were washed with PBS and then incubated with 5 μM rhodamine 123 in CO 2 Incubate in an incubator in the dark for 30 minutes, then wash with PBS, one group is incubated in the dark for 30 minutes with a culture medium solution of the molecule synthesized in Example 1 at a concentration of 10 μM, and the other group is placed in a culture medium with an equal amount of DMSO as a blank control sample. After washing with PBS, the incubated cells are observed under a laser scanning confocal microscope, and the changes in the fluorescence brightness of rhodamine 123 in the two groups of cells are recorded.

[0081] Result analysis:

[0082] 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 the synthetic molecule in Example 1 was treated on HeLa cells. Figure 2 (A) is a fluorescence micrograph of cells in the blank control sample group; Figure 2 (B) Figure 2 (A) Corresponding bright-field micrograph; Figure 2 (C) is a fluorescence micrograph of rhodamine 123 in cells treated with the synthetic molecule of Example 1; Figure 2 (D) Figure 2 (C) Corresponding bright field micrograph. Figure 2 (A) and Figure 2 As can be seen from (C), the fluorescence of Rhodamine 123 is greatly weakened in cells treated with the synthetic molecules of Example 1 ( Figure 2 (C) ), indicating that the mitochondrial membrane potential of cells treated with the synthetic molecules of Example 1 was significantly reduced, that is, the mitochondria were damaged.

[0083] Test Example 4

[0084] Changes of lysosomal pH in HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinoline iodide

[0085] Two sets of glass-bottomed culture dishes filled with HeLa cells prepared in Test Example 1 were washed with PBS and then incubated with 2 μM lysosomal green fluorescent probe (LysoSensor Green DND-189) in CO 2 Incubate in an incubator in the dark for 30 minutes, then wash with PBS, one group is incubated in the dark for 30 minutes with a culture medium solution of the molecule synthesized in Example 1 at a concentration of 10 μM, and the other group is placed in a culture medium with an equal amount of DMSO as a blank control sample. After incubation, the cells are washed with PBS and placed in DMEM, and the changes in the fluorescence brightness of the lysosomal green fluorescent probe in the two groups of cells are observed and recorded under a laser scanning confocal microscope.

[0086] Result analysis:

[0087] The experimental results of Test Example 4 are shown in Figure 3 , Figure 3 This is a fluorescence microscopic photograph of the lysosomal green fluorescent probe (LysoSensor Green DND-189) before and after the HeLa cells were treated 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) Figure 3 (A) Corresponding bright-field micrograph; Figure 3 (C) is a fluorescence micrograph of the lysosomal green fluorescent probe of cells treated with the synthetic molecule of Example 1; Figure 3 (D) 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 weakened in cells treated with the synthetic molecules of Example 1 ( Figure 3 (C) ), indicating that the pH of the cell lysosomes treated with the synthetic molecules of Example 1 increased significantly, affecting the digestive function of the lysosomes in the autophagic flow.

[0088] Test Example 5

[0089] Imaging of autophagic vacuoles and autophagosomes in HeLa cells before and after treatment with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinoline iodide

[0090] After washing two groups of glass-bottomed culture dishes covered with HeLa cells prepared in Test Example 1 with PBS, one group was incubated in the dark for 90 minutes with a culture medium solution of the molecule synthesized in Example 1 at a concentration of 10 μM, and the other group was placed in a culture medium with an equal amount of DMSO as a blank control sample. Subsequently, 5 μM green live cell tracking probe (Cell-Tracker Green CMFDA) was incubated in the dark for 30 minutes, and the incubated cells were washed with PBS and placed in DMEM, and the fluorescence distribution of the green live cell tracking probe in the two groups of cells was observed and recorded under a laser scanning confocal microscope.

[0091] Result analysis:

[0092] The experimental results of Test Example 5 are shown in Figure 4 . Figure 4 This is a fluorescence microscopic photograph of the green live cell tracking probe (Cell-Tracker Green CMFDA) before and after the synthetic molecule in Example 1 treated HeLa cells. 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 tracing probe of cells treated with the synthetic molecules of Example 1. Figure 4 (A) and Figure 4 (B) After the cells were treated with the molecule synthesized in Example 1, a large number of vacuoles (autophagic vacuoles and autophagosomes) were generated in the cytoplasm, indicating that the molecule synthesized in Example 1 can activate cellular autophagy.

[0093] Test Example 6

[0094] Effect of (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-dodecylquinoline iodide on the fusion of mitochondria and lysosomes in HeLa cells treated with

[0095] After washing the two groups of glass-bottomed culture dishes covered with HeLa cells prepared in Test Example 1 with PBS, they were 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) respectively; then, one group was incubated with 20 μM CCCP in PBS solution for ~5 hours as a positive control sample for mitochondrial autophagy, and the other group was incubated in the dark for the same time with a culture medium solution of the molecule synthesized in Example 1 at a concentration of 10 μM. After the incubation, the cells were washed with PBS and placed in DMEM, and the fluorescence distribution of the mitochondrial green fluorescent probe and the lysosomal deep red probe in the two groups of cells was observed and recorded under a laser scanning confocal microscope.

[0096] Result analysis:

[0097] The experimental results of Test Example 6 are shown inFigure 5 . Figure 5 It is a fluorescence micrograph of mitochondria and lysosomes after treating the synthesized molecule in Example 1 with HeLa cells, 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 Figure 1 (A) and Figure 1 (B)'s superimposed image; Figure 5 (D) is a fluorescence micrograph of mitochondria of cells after treating with the synthesized molecule in Example 1; Figure 5 (E) is a fluorescence micrograph of lysosomes of cells after treating with the synthesized molecule in Example 1; Figure 5 (F) is Figure 5 (D) and Figure 5 (E)'s superimposed image. Figure 5 (A) and Figure 5 (B)'s fluorescence overlap is very good, and mitochondria and lysosomes fuse in the late stage of mitophagy; Figure 5 (D) and Figure 5 (E)'s fluorescence hardly overlaps at all, indicating that after autophagy is activated in cells treated with the synthesized molecule in Example 1, mitochondria and lysosomes cannot fuse in the late stage of autophagy; comparison Figure 5 (C) and Figure 5 (F) shows that the synthesized molecule in Example 1 can prevent the fusion of cell mitochondria and lysosomes and inhibit autophagic flux.

[0098] Test Example 7

[0099] Verification of the entry mode of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide into cells

[0100] After washing two groups of glass-bottom culture dishes filled with HeLa cells prepared in Test Example 1 with PBS, one group was incubated in the dark at 37 °C for 30 min with a complete medium solution containing 2 μM of the synthesized molecule in Example 1, and the other group was incubated in the dark at 4 °C for the same time with a complete medium solution containing 2 μM of the synthesized molecule in Example 1. After washing the incubated cells with PBS, they were placed in DMEM, and the distribution of red fluorescence in the two groups of cells was observed and recorded using a laser scanning confocal microscope (EX561 nm, EM600 - 700 nm).

[0101] Result analysis:

[0102] The test results of Test Example 7 are shown in Figure 6 . Figure 6(A) and 6 (B) are red fluorescence micrographs of HeLa cells incubated with (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide at 37 °C and 4 °C respectively. It can be seen from the comparison that Figure 6 the fluorescence intensity of (A) is significantly greater than Figure 6 that of (B), indicating that the main way for (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide to enter cells is active transport.

[0103] Test Example 8

[0104] Growth changes of HEK293 and HeLa cell spheres after administration of (E)-4-(2-(5-methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide

[0105] After photographing 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 complete medium solution of the synthesized molecule of Example 1 at 10 μM as the experimental group, and the other group of HEK293 and HeLa cell spheres was cultured in a complete medium solution of the same volume of DMSO as the blank control group. After 24 h, they were photographed again under a microscope to observe the morphological and dimensional changes of the cell spheres.

[0106] Result analysis:

[0107] The test results of Test Example 8 are shown in Figure 7 . Figure 7 (A) and 7 (B) are bright-field micrographs of HEK293 cell spheres before incubation and administration; Figure 6 (C) and 6 (D) are bright-field micrographs of HeLa cell spheres before incubation and administration; Figure 7 (E) and 7 (G) are bright-field micrographs of the blank control groups of HEK293 cell spheres and HeLa cell spheres after 24 h respectively; Figure 7 (F) and 7 (H) are bright-field micrographs of HEK293 cell spheres and HeLa cell spheres after incubation and administration for 24 h respectively. By comparing Figure 7 (A), 7 (B), 7 (E), 7 (F), it can be seen that compared with the blank control group, the size and shape changes of the HEK293 cell spheres in the experimental group after administration are not obvious, indicating that the synthesized molecule of Example 1 at 10 μM has no obvious growth inhibition on normal HEK293 cell spheres; while by comparing Figure 7It can be seen from (C), 7 (D), 7 (G), and 7 (H) that the size of the HeLa cell spheres in the blank control group increased significantly after 24 hours. The size of the HeLa cell spheres in the experimental drug administration group was much smaller than that in the blank control group after 24 hours and became smaller compared with the cell spheres before drug administration. Moreover, there were many cell debris near the cell spheres, indicating that the molecule synthesized in Example 1 at 10 μM had a significant inhibitory and destructive effect on the growth of cancer cells, HeLa cell spheres. The results showed that the molecule synthesized in Example 1 had a significant effect of selectively killing cancer cells.

[0108] Test Example 9

[0109] (E)-4-(2-(5-Methoxy-1H-indol-3-yl)vinyl)-1-dodecylquinolinium iodide binding constant with albumin

[0110] Different concentrations (0 - 15 μM) of the molecule synthesized in Example 1 were added to the PBS solution of bovine serum albumin, and then the fluorescence spectrum of albumin (280 nm EX) was measured using a fluorescence spectrometer.

[0111] Result analysis:

[0112] The test results of Test Example 9 are shown in Figure 8 . Figure 8 (A) shows the fluorescence spectra of albumin with different concentrations (0 - 15 μM) of the molecule synthesized in Example 1 added, Figure 8 (B) is the double-logarithmic fitting curve of the fluorescence intensity reduction degree and the concentration of the molecule synthesized in Example 1 based on Figure 8 (A). It can be seen from Figure 8 (A) that as the concentration of the molecule synthesized in Example 1 added increased, the fluorescence peak of albumin gradually decreased. According to the fitting curve Figure 8 (B), the binding constant of the molecule synthesized in Example 1 with albumin was as high as 1.35×10 8 .

[0113] 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 target mitochondria and lysosomes simultaneously. On the one hand, it destroys mitochondria to activate mitophagy, and on the other hand, it alkalizes the pH of lysosomes to prevent the fusion of mitochondria and lysosomes and inhibit autophagic flux. By activating autophagy and inhibiting autophagic flux, the effect of killing cancer cells is achieved. In addition, the fluorescence is greatly enhanced after targeting mitochondria and lysosomes, realizing the synchronous fluorescence visualization of mitochondria and lysosomes.

[0114] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. An autophagy regulator, characterized in that, the autophagy regulator is a compound having the structure shown in formula (I), or a pharmaceutically acceptable salt thereof: Wherein, the R 1 is selected from any one of hydrogen or an alkyl group having 1 to 4 carbon atoms; the R 2 is selected from any one of hydrogen or an alkyl group having 1 to 4 carbon atoms; the R 3 is selected from any one of hydrogen, an alkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms; the X is selected from any one of a halogen atom, 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 a product for regulating related life activities of autophagy.

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

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

Images