WO2025112010A1 - 一种自噬调节剂及其制备方法和应用 - Google Patents

一种自噬调节剂及其制备方法和应用 Download PDF

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WO2025112010A1
WO2025112010A1 PCT/CN2023/135725 CN2023135725W WO2025112010A1 WO 2025112010 A1 WO2025112010 A1 WO 2025112010A1 CN 2023135725 W CN2023135725 W CN 2023135725W WO 2025112010 A1 WO2025112010 A1 WO 2025112010A1
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autophagy
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autophagy regulator
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宋国芬
王怀雨
李鹏辉
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to the field of probes, and in particular to an autophagy regulator and a preparation method and application thereof.
  • Autophagy is the self-digestion of lysosomes or vacuoles and the degradation and recycling of cell contents, which is essential for maintaining homeostasis and energy balance in cells. At the same time, it has a wide range of biological effects, 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 the autophagy process is related to a variety of diseases, including cancer, neurodegenerative diseases, diabetes, autoimmune diseases, and cardiovascular diseases. Therefore, autophagy regulation is of great significance for the treatment of a variety of diseases. At present, targeted drugs for autophagy in various diseases are also under further development.
  • the existing autophagy regulators include rapamycin, chloroquine, etc., which can only activate or inhibit autophagy, and do not produce fluorescence after binding to the target, so fluorescence observation is not possible. Especially when used for tumor treatment, the efficacy and tumor selectivity need to be improved.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the present invention proposes a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor and its application, aiming to solve the problem that the prior art has a single autophagy regulation target and no cell selectivity, and poor cancer cell killing efficacy.
  • the autophagy regulator is a compound having a structure as shown in formula (I), or a pharmaceutically acceptable salt thereof:
  • the R1 is selected from any one of hydrogen and C1-C4 alkyl; the R2 is selected from any one of hydrogen and C1-C4 alkyl; the R3 is selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; and the X is selected from any one of halogen atom, BF4 , and ClO4 .
  • the autophagy regulator is an autophagy activator and an autophagy inhibitor.
  • 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 the autophagy flow.
  • the mitochondrial membrane potential is reduced.
  • the pH of the lysosomes increases.
  • the autophagy regulator provided by the present invention is a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor.
  • the dual-targeted fluorescent organic small molecule of the present invention is unique in that it can simultaneously activate mitochondrial autophagy and destroy lysosomal function, inhibit autophagic flow, and thus kill cancer cells; it is also a new type of mitochondrial/lysosomal fluorescent probe, which can simultaneously target mitochondria and lysosomes compared with the existing mitochondrial and lysosomal fluorescent probes. It emits red fluorescence, images the morphology, quantity and distribution of mitochondria and lysosomes, and has good membrane permeability and good redyeing compatibility.
  • the dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor provided by the present invention can, on the one hand, be used as an autophagy regulator to activate mitochondrial autophagy and inhibit autophagy 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 mark the morphology, number 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, and has a wide range of applications and good effects.
  • the halogen atom is selected from any one of iodine, bromine and chlorine.
  • 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.
  • the C1-C4 alkyl group includes any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
  • said 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 a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • the obtained autophagy regulator independent of mitochondrial membrane potential is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide.
  • a method for preparing an autophagy regulator comprises the following steps:
  • the catalyst includes piperidine.
  • the solvent comprises ethanol.
  • the molar ratio of indole-3-carboxaldehyde to 4-methylquinoline is 1:(1.0-2.0).
  • the reflux reaction time is 3 to 4 days.
  • the reflux reaction time is 1 to 2 days.
  • 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:
  • 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.
  • an autophagy regulator is used in the preparation of a related life form for regulating cell autophagy Application in active products.
  • 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 cannot fuse with the autophagosome to form autophagic lysosomes, thereby inhibiting the completion of the autophagic flow and achieving dual regulation of autophagy.
  • an autophagy regulator is used in the preparation of a product for targeting mitochondria or lysosomes.
  • the method of using an autophagy regulator to perform mitochondrial/lysosomal fluorescence imaging 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.
  • the present invention provides an application of a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor for mitochondrial/lysosomal fluorescence imaging.
  • 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, thereby realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.
  • 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, thereby realizing the application of simultaneous fluorescence imaging of mitochondria and lysosomes.
  • a drug for treating tumors includes a cell autophagy regulating drug, and the cell autophagy regulating drug includes the autophagy regulator.
  • the tumor comprises a tumor overexpressing albumin receptor.
  • the tumors overexpressing albumin receptors include cervical cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, liver cancer, etc.
  • the medicament comprises an injectable composition or a composition for oral administration.
  • the composition includes a dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor and other pharmaceutically acceptable carriers, wherein the carrier includes but is not limited to various pharmaceutical excipients.
  • the method of using the autophagy regulator to kill cancer cells in the present invention comprises: mixing and incubating sample cells with the autophagy regulator, activating mitochondrial autophagy, inhibiting lysosomal function and autophagy, and inducing death of the sample cells.
  • the autophagy regulator of the present invention has dual-targeted fluorescent organic small molecules for autophagy activation and Autophagy inhibitors have dual targeting effects and can simultaneously target cell mitochondria and lysosomes, activating mitochondrial autophagy and inhibiting lysosomal function, ultimately leading 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 dual-targeted fluorescent organic small molecule autophagy activator and autophagy inhibitor as autophagy regulator and cancer cell killer.
  • Figure 1 is a fluorescence micrograph of HeLa cells co-stained with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide and mitochondrial green fluorescent probe (MitoTracker Green) and lysosomal deep red fluorescent probe (LysoBrite NIR) provided in Example 3 of the present application.
  • FIG2 is a fluorescence micrograph of HeLa cells stained with rhodamine 123 after being treated with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinoline iodide provided in Example 4 of the present application.
  • Figure 3 is a fluorescence micrograph of lysosomal green fluorescent probe (Lysosensor Green DND-189) staining of HeLa cells treated with (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinoline iodide provided in Example 5 of the present application.
  • Figure 5 is a fluorescence micrograph of the staining of mitochondria and lysosomes of HeLa cells after being treated with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide and CCCP, respectively, as provided in Test Example 6 of the present application.
  • FIG6 is a fluorescence micrograph of HeLa cells incubated at 37° C. and 4° C. with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-dodecylquinoline iodide provided in Test Example 7 of the present application.
  • FIG. 7 is a microscopic photograph 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.
  • the term "and/or” describes the association relationship of associated objects, indicating that there may be three relationships.
  • a and/or B can mean: A exists alone, A and B exist at the same time, 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.
  • At least one means one or more
  • plural 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.
  • at least one of a, b, or c or “at least one of a, b, and c” can all mean: 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.
  • 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, and 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.
  • the weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application.
  • the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as ⁇ g, mg, g, kg, etc.
  • the mixture is slowly cooled, filtered, and washed with a small amount of isopropanol to obtain dark green crystals or dark red powder, or excess solvent is evaporated, cooled, and the product is purified by column chromatography using dichloromethane/methanol as an eluent to obtain dark green crystals and dark red powder, with a yield of about 27%.
  • R 3 can also be selected from any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and R 2 is selected from any one of hydrogen or C1-C4 alkyl.
  • the function of the autophagy regulator in the present invention is determined by the conjugated organic cationic group and has nothing to do with the anion X- ;
  • R 2 is selected from any one of hydrogen or C1-C4 alkyl;
  • R 3 is selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.
  • indoloctyl quinoline salt has a weak binding effect on albumin and still enters the cell in a free diffusion manner.
  • the binding effect with albumin is enhanced and enters the cell in an active transport manner.
  • Cancer cells HeLa and normal cells HEK293 were cultured in complete culture medium (DMEM culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin) at 37°C in a saturated humidity incubator with 5% CO 2 , and passaged every 2 to 3 days.
  • complete culture medium DMEM culture medium containing 10% fetal bovine serum and 1% penicillin/streptomycin
  • the HeLa cell-covered slide prepared in Test Example 1 was washed twice with PBS, and then the following staining steps were performed: (1) incubated with 0.5 ⁇ M commercial mitochondrial green fluorescent probe (MitoTracker Green) solution for 30 min, and washed with PBS; (2) incubated with 0.5 ⁇ M commercial lysosomal near-infrared fluorescent probe (LysoBrite NIR) solution for 30 min, and washed with PBS; (3) incubated with 2 ⁇ M (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 for multi-channel fluorescence co-localization using a confocal fluorescence microscope.
  • 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 an overlay of Figure 1 (A), Figure 1 (B), and Figure 1 (C).
  • Figure 1 (A) covers the two regions of Figure 1 (B) and Figure 1 (C), and the sum of Figure 1 (A), Figure 1 (B), and Figure 1 (C) overlap well, indicating that the fluorescence of the molecule synthesized in Example 1 is distributed in two organelles, mitochondria and lysosomes.
  • Figure 2 is a fluorescence micrograph of the mitochondrial membrane potential probe rhodamine 123 before and after the synthetic molecule of Example 1 is treated on HeLa cells.
  • 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 synthetic molecule of Example 1;
  • Figure 2(D) is a bright field micrograph corresponding to Figure 2(C).
  • Figure 3 is a fluorescence micrograph of the lysosomal green fluorescent probe (LysoSensor Green DND-189) before and after the synthetic molecule in Example 1 treated HeLa cells.
  • 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 in Example 1;
  • Figure 3(D) is a bright field micrograph corresponding to Figure 3(C).
  • the fluorescence of LysoSensor Green DND-189 will increase with the decrease of pH.
  • Figure 4 is a fluorescence micrograph of the green live cell tracer 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 the cells in the blank control sample group
  • Figure 4 (B) is a fluorescence micrograph of the green live cell tracer probe of the cells treated with the synthetic molecule in Example 1. Comparing Figures 4 (A) and 4 (B), a large number of vacuoles (autophagic vacuoles and autophagosomes) were generated in the cytoplasm of the cells treated with the synthetic molecule in Example 1, indicating that the synthetic molecule in Example 1 can activate cell autophagy.
  • vacuoles autophagic vacuoles and autophagosomes
  • Figure 5 is a fluorescence micrograph of mitochondria and lysosomes of HeLa cells treated with the synthetic molecules of 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 synthetic molecules of Example 1
  • Figure 5 (E) is a fluorescence micrograph of lysosomes of cells treated with the synthetic molecules of Example 1
  • Figure 5 (F) is an overlay of Figure 1 (D) and Figure 1 (E).
  • 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-n-dodecylquinoline iodide at 37°C and 4°C, respectively.
  • the fluorescence intensity of Figure 6(A) is significantly greater than that of Figure 6(B), indicating that the main way in which (E)-4-(2-(5-methoxy-1H-indol-3-)vinyl)-1-n-dodecylquinoline iodide enters the cell is active transport.
  • Test Example 8 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 HEK293 cell spheres and HeLa cell sphere blank control groups after 24 hours, respectively
  • Figures 7(F) and 7(H) are bright field micrographs of HEK293 cell spheres and HeLa cell spheres after incubation and administration for 24 hours, respectively.
  • Example 2 Different concentrations of the molecule synthesized in Example 1 (0-15 ⁇ M) were added to a PBS solution of bovine serum albumin, and then the fluorescence spectrum of the albumin (280 nm EX) was measured using a fluorescence spectrometer.
  • FIG8 (A) is the fluorescence spectrum of albumin with different concentrations of the molecules synthesized in Example 1 (0-15 ⁇ M)
  • FIG8 (B) is a double logarithmic fitting curve of the fluorescence intensity reduction degree and the concentration of the molecules synthesized in Example 1 made according to FIG8 (A). It can be seen from FIG8 (A) that with the increase in the concentration of the molecules synthesized in Example 1, the fluorescence peak of albumin gradually decreases. According to the fitting curve FIG8 (B), it is concluded that the binding constant between the molecules synthesized in Example 1 and albumin is as high as 1.35 ⁇ 10 8 .
  • the present application provides a dual-targeted fluorescent organic small molecule autophagy regulator and its application.
  • 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 pH of lysosomes, preventing the fusion of mitochondria and lysosomes, and inhibiting the autophagy flow.
  • the fluorescence is greatly enhanced after targeting mitochondria and lysosomes, and the synchronous fluorescence visualization of mitochondria and lysosomes is achieved.

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Abstract

本发明公开了一种自噬调节剂及其制备方法和应用，本发明的双靶向的荧光有机小分子自噬激活兼自噬抑制剂及其应用，旨在解决现有技术中自噬调节靶向单一且无细胞选择性、杀癌细胞疗效不佳的问题。

Description

一种自噬调节剂及其制备方法和应用 技术领域
本发明涉及探针领域，尤其涉及一种自噬调节剂及其制备方法和应用。
背景技术
自噬是溶酶体或液泡的自我消化以及细胞内容物的降解和回收，对维持细胞体内平衡和能量平衡至关重要。同时它具有广泛的生物学作用，包括细胞器重塑、蛋白质和细胞器质量控制、抑制肿瘤、消灭病原体、免疫和炎症调节和细胞生存等等。研究表明自噬过程中的功能失常关系着多种疾病，包括癌症、神经退行性疾病、糖尿病、自身免疫病和心血管疾病等。因此，自噬调节对于治疗多种疾病意义重大。目前各种疾病中关于自噬的靶向药物也在进一步的研发之中。
目前已有的自噬调节剂包括雷帕霉素、氯喹等，只能单一地激活自噬或者抑制自噬，并且与靶标结合后不产生荧光，不能进行荧光观测。特别是用于肿瘤治疗时，疗效和肿瘤选择性都有待提高。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种双靶向的荧光有机小分子自噬激活兼自噬抑制剂及其应用，旨在解决现有技术中自噬调节靶向单一且无细胞选择性、杀癌细胞疗效不佳的问题。
根据本发明的第一方面实施例的一种自噬调节剂，所述自噬调节剂为具有如式(I)所示结构的化合物，或其药学上可接受的盐：
其中，所述R1选自氢或C1-C4的烷基的任意一种；所述R2选自氢或C1-C4的烷基的任意一种；所述R3选自氢、C1-C4的烷基、C1-C4的烷氧基的任意一种；所述X选自卤素原子、BF4、ClO4中的任意一种。
根据本发明的一些实施例，所述自噬调节剂为自噬激活剂和自噬抑制剂。
本发明的自噬调节剂的自噬调节的方法为：将样品细胞与所述自噬调节剂混合孵育，破坏所述样品细胞的线粒体，引发线粒体自噬；破坏所述样品细胞的溶酶体，抑制自噬流。
本发明中的自噬调节剂破坏上述样品细胞的线粒体的步骤中，所述线粒体膜电位降低。
本发明中的自噬调节剂破坏上述样品细胞的溶酶体的步骤中，所述溶酶体pH升高。
本发明提供的自噬调节剂为一种双靶向的荧光有机小分子自噬激活兼自噬抑制剂，与目前已有的自噬调节剂比，本发明所述双靶向的荧光有机小分子的独特之处在于可以同时激活线粒体自噬并破坏溶酶体功能，抑制自噬流，从而杀死癌细胞；亦是一类新型的线粒体/溶酶体荧光探针，与目前已有的线粒体和溶酶体荧光探针相比，可以同时靶向线粒体和溶酶体。并发射红色荧光，成像线粒体和溶酶体形态、数量和分布，同时膜通透性好、复染兼容性好。
本发明所提供的双靶向的荧光有机小分子自噬激活兼自噬抑制剂，一方面可作为自噬调节剂激活线粒体自噬并抑制自噬流，从而可作为抗癌药物杀癌细胞；另一方面可作为荧光探针进行标记细胞中线粒体和溶酶体的形态、数量和分布，能够为线粒体和溶酶体相关的生理和病理学研究以及临床诊断提供简捷、直观的生物检测试剂，应用广泛，作用效果佳。
根据本发明的一些实施例，所述卤素原子选自碘、溴和氯中的任意一种。
根据本发明的一些实施例，所述R1中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种。
根据本发明的一些实施例，所述R2中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种。
根据本发明的一些实施例，所述R3中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种
根据本发明的一些实施例，所述C1-C4的烷氧基包括甲氧基、乙氧基、丙氧基和丁氧基中的任意一种。
根据本发明的一些实施例，所述自噬调节剂包括(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐。
本发明中，R1选自乙基，R2选自氢，所述R3选自甲氧基，所述X选自碘时，得到的所述不依赖线粒体膜电位的自噬调节剂为(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐。
根据本发明的第二方面实施例的一种所述的自噬调节剂的制备方法，所述制备方法包括如下步骤：
S1.将4-甲基喹啉(式II)与卤代长链烷(式III)置于溶剂中，回流反应生成1-长链烷基-4-甲基喹啉盐(式IV)；
S2.将1-长链烷基-4-甲基喹啉盐、吲哚-3-甲醛(式V)和催化剂混合、回流反应后除杂；
所述催化剂包括哌啶。
根据本发明的一些实施例，所述溶剂包括乙醇。
根据本发明的一些实施例，所述吲哚-3-甲醛与4-甲基喹啉的摩尔比为1：(1.0～2.0)。
根据本发明的一些实施例，步骤S1中，所述回流反应的时间为3～4天。
根据本发明的一些实施例，步骤S2中，所述回流反应的时间为1～2天。
根据本发明的一些优选地实施例，所述吲哚-3-甲醛选自5-甲氧基-3-甲酰基吲哚；所述卤代烷选自碘十二烷，以5-甲氧基-3-甲酰基吲哚、碘十二烷作为反应物，自噬调节剂的制备方法如下：
S01.配制4-甲基喹啉和碘十二烷的乙醇混合溶液；
S02.加热搅拌回流反应三天；
S03.加入5-甲氧基-3-甲酰基吲哚的乙醇溶液；
S04.在乙醇混合溶液中加入催化剂哌啶，将加了哌啶的乙醇混合溶液于85℃加热回流搅拌反应一天，缓慢冷却至室温，得到墨绿色晶体或者待提纯有机固体产物；
S05.将待提纯有机固体产物进行柱层析色谱提纯，淋洗剂使用二氯甲烷/甲醇，干燥得到墨绿色晶体和墨红色粉末，墨绿色晶体和墨红色粉末为双靶向的荧光有机小分子自噬激活兼自噬抑制剂，双靶向的荧光有机小分子自噬激活兼自噬抑制剂为(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐。
根据本发明的第三方面实施例的一种自噬调节剂在制备应用于细胞自噬调节的相关生命 活动产品中的应用。
本发明中，所述双靶向的荧光有机小分子自噬激活兼自噬抑制剂用于非诊断和治疗方法目的的进行细胞自噬调节，本申请双靶向的荧光有机小分子，能够靶向细胞的线粒体和溶酶体，线粒体膜电位下降甚至丧失，引起线粒体自噬，产生了大量自噬小体泡；同时溶酶体pH上升，不能与自噬小体融合形成自噬溶酶体，抑制自噬流的完成，实现自噬的双重调节。
根据本发明的第四方面实施例的一种自噬调节剂在制备应用于靶向线粒体或靶向溶酶体的产品中的应用。
根据本发明的第五方面实施例的自噬调节剂在制备应用于线粒体成像或溶酶体成像的产品中的应用。
本发明中自噬调节剂进行线粒体/溶酶体荧光成像的方法包括：将样品细胞与自噬调节剂混合孵育，自噬调节剂与所述样品细胞的线粒体和溶酶体结合，荧光强度增强，实现线粒体和溶酶体的荧光成像。
本发明提供的一种双靶向的荧光有机小分子自噬激活兼自噬抑制剂进行线粒体/溶酶体荧光成像的应用，与已有的线粒体和溶酶体荧光探针相比，双靶向的荧光有机小分子能够同时靶向细胞线粒体和溶酶体，与线粒体和溶酶体结合以后，荧光大大增强，实现了同时荧光成像线粒体和溶酶体的应用。
本发明中的所述双靶向的荧光有机小分子自噬激活兼自噬抑制剂用于非诊断和治疗目的的进行线粒体/溶酶体荧光成像，本申请双靶向的荧光有机小分子本身不发荧光，与线粒体和溶酶体结合以后，荧光大大增强，实现了同时荧光成像线粒体和溶酶体的应用。
根据本发明的第六方面实施例的一种治疗肿瘤的药物，所述药物包括细胞自噬调节类药物，所述细胞自噬调节类药物包括所述的自噬调节剂。
根据本发明的一些实施例，所述肿瘤包括白蛋白受体过表达的肿瘤。
根据本发明的一些实施例，所述白蛋白受体过表达的肿瘤包括宫颈癌、乳腺癌、卵巢癌、黑色素瘤、胰腺癌和肝癌等。
根据本发明的一些实施例，所述药物包括可注射组合物或用于口服的组合物。
根据本发明的一些实施例，所述组合物包括双靶向的荧光有机小分子自噬激活兼自噬抑制剂和药学上可接受的其他载体，其中，载体包括但不限于各种药物辅料。
本发明中自噬调节剂进行杀癌细胞的方法包括：将样品细胞与自噬调节剂混合孵育，激活线粒体自噬，抑制溶酶体功能和自噬，诱导所述样品细胞死亡。
本发明中的自噬调节剂与已有的自噬调节剂相比，双靶向的荧光有机小分子自噬激活兼 自噬抑制剂具有双靶向作用，能够同时靶向细胞线粒体和溶酶体，激活线粒体自噬和抑制溶酶体功能，最终导致细胞死亡，可利用此双靶向的荧光有机小分子自噬激活兼自噬抑制剂杀癌细胞，实现了双靶向的荧光有机小分子自噬激活兼自噬抑制剂作为自噬调节和杀癌细胞的应用。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是本申请实施例3提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐与线粒体绿色荧光探针(MitoTracker Green)、溶酶体深红色荧光探针(LysoBrite NIR)对HeLa细胞的共染色荧光显微照片。
图2是本申请实施例4提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理后的罗丹明123染色的荧光显微照片。
图3是本申请实施例5提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理后的溶酶体绿色荧光探针(Lysosensor Green DND-189)染色的荧光显微照片。
图4是本申请实施例6提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理后的5-氯甲基荧光素二乙酸酯(绿色活细胞示踪探针CellTrackerTM Green CMFDA)染色的荧光显微照片。
图5是本申请测试例6提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐和CCCP分别对HeLa细胞处理后的线粒体和溶酶体的染色荧光显微照片。
图6是本申请测试例7提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞分别在37℃和4℃下孵化后的荧光显微照片。
图7是本申请测试例8提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对正常细胞球HEK293和癌细胞球HeLa孵化前后的显微照片。
图8是本申请测试例9提供的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐与白蛋白作用的荧光光谱与双对数拟合曲线。
具体实施方式
为了使本申请要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“或”的关系。
本申请中，“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“以下至少一项(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。例如，“a，b，或c中的至少一项(个)”，或，“a，b，和c中的至少一项(个)”，均可以表示：a，b，c，a-b(即a和b)，a-c，b-c，或a-b-c，其中a，b，c分别可以是单个，也可以是多个。
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。
在本申请实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。
本申请实施例说明书中所提到的相关成分的重量不仅仅可以指代各组分的具体含量，也可以表示各组分间重量的比例关系，因此，只要是按照本申请实施例说明书相关组分的含量按比例放大或缩小均在本申请实施例说明书公开的范围之内。具体地，本申请实施例说明书中所述的质量可以是μg、mg、g、kg等化工领域公知的质量单位。
实施例1
本实施例提供了一种(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐的合成：
首先将200μL4-甲基喹啉和393μL碘十二烷溶于10mL乙醇，85℃加热搅拌回流反应三天；接着加入含有0.263g5-甲氧基-3-甲酰基吲哚的乙醇溶液，搅拌均匀后加5滴哌啶，溶液逐渐变红色；85℃回流反应一天后，缓慢冷却，过滤，少量异丙醇洗涤，得墨绿色晶粒或墨红色粉末，或者蒸出多余溶剂，冷却，产物用二氯甲烷/甲醇作淋洗剂柱层析色谱提纯，得墨绿色晶体和墨红色粉末，产率约27％。
1H 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)。13C NMR(400MHz，DMSO-d6)，δ(ppm):155.71，154.17，146.15，139.13，138.31，135.12，132.60，132.44，128.80，127.28，126.96，126.15，119.29，115.08，113.87，113.81，112.95，112.80，102.69，56.18，56.07，31.76，29.66，29.47，29.36，29.33，29.18，28.98，26.27，22.57，14.43。HRMS:calculated469.32，found 469.32。
为了拓展更多具有类似功能的化合物，本发明中保持分子母核骨架不变，R3还可选自氢、C1-C4的烷基、C1-C4的烷氧基的任意一种，R2选自氢或C1-C4的烷基的任意一种。据发明人研究，本发明中的自噬调节剂的功能由共轭有机阳离子基团所决定，与阴离子X-无关；R2选自氢或C1-C4的烷基的任意一种；R3选自氢、C1-C4的烷基、C1-C4的烷氧基的任意一种，在这个范围内的改变不影响分子的功能；但是R1相连的碳链长度对分子的靶向性和蛋白结合作用影响较大，例如吲哚辛烷基喹啉盐与白蛋白结合作用弱，仍以自由扩散方式进入细胞，随着喹啉碳链的增加(由癸烷基到十四烷基)，与白蛋白的结合作用增强，以主动运输的方式进入细胞。
测试例1
HeLa和HEK293细胞和细胞球的培养：
将癌细胞HeLa和正常细胞HEK293使用完全培养基(含10％胎牛血清和1％青霉素/链霉素的DMEM培养基)，在37℃、5％CO2的饱和湿度培养箱中培养，每2～3天传代1次。
待细胞生长到对数期，转移到共聚焦皿培养：将T25细胞培养瓶中长满的细胞先用PBS洗，再用1mL胰酶消化1～2分钟(HeLa使用0.25％胰酶，HEK293使用0.025％胰酶)，去除胰酶，加入新鲜培养基吹打均匀并细胞计数，以培养基的添加量控制细胞密度，(1)接种至共聚焦玻底培养皿中，放入5％CO2培养箱中培养，待细胞生长至覆盖率约为70％用于细胞成像实验；(2)接种到低吸附U底96孔板中培养，待细胞球直径至～800μm用于药物抗肿瘤评估。
测试例2
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞的染色观察
将测试例1制备的长满HeLa细胞的爬片用PBS洗两遍后，然后进行以下染色步骤：(1)用0.5μM商业线粒体绿色荧光探针(MitoTracker Green)溶液孵化30min，PBS清洗；(2)用0.5μM商业溶酶体近红外荧光探针(LysoBrite NIR)溶液孵化30min，PBS清洗；(3)用2μM(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐荧光探针溶液孵化30min，DMEM清洗。将染色后的细胞样品使用共聚焦荧光显微镜进行多通道荧光共定位观察。
结果如图1所示，图1(A)为实施例1所合成分子的红色荧光图，图1(B)为商业线粒体绿色荧光探针的荧光图，图1(C)为商业溶酶体近红外荧光探针的荧光图，图1(D)为图1(A)和图1(B)、图1(C)的叠加图。图1(A)覆盖了图1(B)与图1(C)两个区域，图1(A)和图1(B)与图1(C)的加和重叠得很好，说明实施例1所合成分子的荧光分布于线粒体和溶酶体两个细胞器。此结果证明(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐可用于靶向线粒体和溶酶体，也可用于线粒体和溶酶体的同时荧光成像。
测试例3
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理前后的线粒体膜电位变化
将测试例1制备的分别长满HeLa细胞的两组玻底培养皿PBS洗后用5μM罗丹明123在CO2培养箱中避光孵化30min，再用PBS洗，一组用浓度为10μM的实施例1所合成分子的培养基溶液避光孵化30min，另一组置于加同等量DMSO的培养基中作空白对照样品。PBS洗后将孵化后的细胞在激光扫描共聚焦显微镜下观察，记录两组细胞中罗丹明123的荧光亮度变化。
结果分析：
测试例3的实验结果见图2，图2是实施例1所合成分子对HeLa细胞处理前后的线粒体膜电位探针罗丹明123的荧光显微照片，图2(A)为空白对照样品组细胞的荧光显微照片；图2(B)为图2(A)对应的明场显微照片；图2(C)为实施例1所合成分子处理过的细胞的罗丹明123的荧光显微照片；图2(D)为图2(C)对应的明场显微照片。从图2(A)和图2(C)中可以得知，实施例1所合成分子处理后的细胞，罗丹明123的的荧光大大减弱(图2(C))，表明实施例1所合成分子处理后的细胞线粒体膜电位显著降低，即线粒体受损。
测试例4
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理前后的溶酶体pH变化
将测试例1制备的分别长满HeLa细胞的两组玻底培养皿PBS洗后用2μM溶酶体绿色荧光探针(LysoSensor Green DND-189)在CO2培养箱中避光孵化30min，再用PBS洗，一组用浓度为10μM的实施例1所合成分子的培养基溶液避光孵化30min，另一组置于加同等量DMSO的培养基中作空白对照样品。将孵化后的细胞PBS洗后置于DMEM中，在激光扫描共聚焦显微镜下观察记录两组细胞中溶酶体绿色荧光探针的荧光亮度变化。
结果分析：
测试例4的实验结果见图3，图3是实施例1所合成分子对HeLa细胞处理前后的溶酶体绿色荧光探针(LysoSensor Green DND-189)的荧光显微照片，图3(A)为空白对照样品组细胞的荧光显微照片；图3(B)为图3(A)对应的明场显微照片；图3(C)为实施例1所合成分子处理过的细胞的溶酶体绿色荧光探针的荧光显微照片；图3(D)为图3(C)对应的明场显微照片。LysoSensor Green DND-189荧光会随pH的降低而增强，从图3(A)和图3(C)中可以得知，实施例1所合成分子处理后的细胞，溶酶体绿色荧光探针的荧光大大减弱(图3(C))，表明实施例1所合成分子处理后的细胞溶酶体pH显著升高，影响自噬流中溶酶体的消化功能。
测试例5
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理前后的自噬泡和自噬体的成像
将测试例1制备的分别长满HeLa细胞的两组玻底培养皿PBS洗后，一组用浓度为10μM的实施例1所合成分子的培养基溶液避光孵化90min，另一组置于加同等量DMSO的培养基中作空白对照样品。随后用5μM绿色活细胞示踪探针(Cell-Tracker Green CMFDA)避光孵化30min，将孵化后的细胞PBS洗后置于DMEM中，在激光扫描共聚焦显微镜下观察记录两组细胞中绿色活细胞示踪探针的荧光分布。
结果分析：
测试例5的实验结果见图4。图4是实施例1所合成分子对HeLa细胞处理前后的绿色活细胞示踪探针(Cell-Tracker Green CMFDA)的荧光显微照片，图4(A)为空白对照样品组细胞的荧光显微照片；图4(B)为实施例1所合成分子处理过的细胞的绿色活细胞示踪探针的荧光显微照片。对比图4(A)和图4(B)，实施例1所合成分子处理后的细胞，细胞质中产生大量的空泡(自噬泡和自噬体)，表明实施例1所合成分子能够激活细胞自噬。
测试例6
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HeLa细胞处理后的线粒体与溶酶体的融合
将测试例1制备的分别长满HeLa细胞的两组玻底培养皿PBS洗后，先后用1μM线粒体绿色荧光探针(MitoTracker Green)和1μM溶酶体深红探针(LysoBrite NIR)分别避光孵化30min；随后，一组用20μM CCCP的PBS溶液孵化～5h作线粒体自噬阳性对照样品，另一组用浓度为10μM的实施例1所合成分子的培养基溶液避光孵化相同时间。将孵化后的细胞PBS洗后置于DMEM中，在激光扫描共聚焦显微镜下观察记录两组细胞中线粒体绿色荧 光探针和溶酶体深红探针的荧光分布。
结果分析：
测试例6的实验结果见图5。图5是实施例1所合成分子对HeLa细胞处理后的线粒体和溶酶体的荧光显微照片，图5(A)为阳性对照样品组细胞的线粒体荧光显微照片；图5(B)为阳性对照样品组细胞的溶酶体荧光显微照片；图5(C)为图1(A)和图1(B)的叠加图；图5(D)为实施例1所合成分子处理后的细胞的线粒体荧光显微照片；图5(E)为实施例1所合成分子处理后的细胞的溶酶体荧光显微照片；图5(F)为图1(D)和图1(E)的叠加图。图5(A)和图5(B)的荧光重叠性很好，在线粒体自噬后期线粒体和溶酶体融合；图5(D)和图5(E)的荧光几乎完全不重叠，说明实施例1所合成分子处理后的细胞自噬激活以后，在自噬后期线粒体和溶酶体不能融合；对比图5(C)和图5(F)，表明实施例1所合成分子能够阻止细胞线粒体和溶酶体的融合，抑制自噬流。
测试例7
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐进入细胞的方式验证
将测试例1制备的分别长满HeLa细胞的两组玻底培养皿PBS洗后，一组用2μM实施例1所合成分子的完全培养基溶液在37℃下避光孵化30min，另一组用2μM的实施例1所合成分子的完全培养基溶液在4℃下避光孵化相同时间。将孵化后的细胞PBS洗后置于DMEM中，使用激光扫描共聚焦显微镜观察记录两组细胞中的红色荧光分布(EX561nm，EM600-700nm)。
结果分析：
测试例7的试验结果见图6。图6(A)和6(B)为分别在37℃和4℃下(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐孵化处理过的HeLa细胞的红色荧光显微照片。对比可以看出，图6(A)的荧光强度显著大于图6(B)的荧光，说明(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐进入细胞的方式主要为主动运输。
测试例8
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐对HEK293和HeLa细胞球给药后的生长变化
将测试例1制备得到的两组HEK293细胞球和两组HeLa细胞球在显微镜下拍照后，一组HEK293和HeLa细胞球置于10μM的实施例1所合成分子的完全培养基溶液培养作为实验组，另一组HEK293和HeLa细胞球置于同等体积DMSO的完全培养基溶液培养作为空白对照组，24h后再使用显微镜拍照，观察细胞球的形貌尺寸变化。
结果分析：
测试例8的试验结果见图7。图7(A)和7(B)为HEK293细胞球孵化给药前的明场显微照片；图6(C)和6(D)为HeLa细胞球孵化给药前的明场显微照片；图7(E)和7(G)分别为HEK293细胞球和HeLa细胞球空白对照组24h后的明场显微照片；图7(F)和7(H)分别为HEK293细胞球和HeLa细胞球孵化给药24h后的明场显微照片。对比图7(A)、7(B)、7(E)、7(F)可以看出，与空白对照组相比，给药实验组HEK293细胞球的尺寸和形状变化不明显，说明10μM实施例1所合成分子对正常细胞HEK293细胞球的生长抑制不明显；而对比图7(C)、7(D)、7(G)、7(H)可以看出，空白对照组HeLa细胞球24小时后尺寸明显变大，实验给药组HeLa细胞球24h后尺寸与空白对照组比要小的多，与用药之前的细胞球相比变小，而且细胞球附近有很多细胞碎片，说明10μM实施例1所合成分子对癌细胞HeLa细胞球的生长有显著的抑制和破坏效果。结果表明，实施例1所合成分子具有显著的选择性杀癌细胞的效果。
测试例9
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐与白蛋白的结合常数
向牛血清白蛋白的PBS溶液中加入不同浓度的实施例1所合成的分子(0-15μM)，然后使用荧光光谱仪测得白蛋白的荧光光谱(280nmEX)。
结果分析：
测试例9的试验结果见图8。图8(A)为加入不同浓度的实施例1所合成的分子(0-15μM)的白蛋白的荧光光谱，图8(B)为根据图8(A)做的荧光强度降低程度与实施例1所合成分子浓度的双对数拟合曲线。从图8(A)可以看出，随着加入实施例1所合成分子的浓度的增加，白蛋白的荧光峰逐渐降低，根据拟合曲线图8(B)得出实施例1所合成的分子与白蛋白的结合常数高达1.35×108。
综上，本申请提供的一种提供了一种双靶向的荧光有机小分子自噬调节剂及其应用，与已有的自噬激活剂和自噬抑制剂相比，本发明双靶向的荧光有机小分子自噬调节剂可同时靶向线粒体和溶酶体，一方面破坏线粒体激活线粒体自噬，另一方面使溶酶体pH碱化，阻止线粒体和溶酶体的融合，抑制自噬流。通过激活自噬并抑制自噬流，实现杀癌细胞的作用。另外，靶向线粒体和溶酶体后荧光大大增强，实现了线粒体和溶酶体的同步荧光可视化。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种自噬调节剂，其特征在于，所述自噬调节剂为具有如式(I)所示结构的化合物，或其药学上可接受的盐：
    其中，所述R1选自氢或C1-C4的烷基的任意一种；所述R2选自氢或C1-C4的烷基的任意一种；所述R3选自氢、C1-C4的烷基、C1-C4的烷氧基的任意一种；所述X选自卤素原子、BF4、ClO4中的任意一种。
  2. 根据权利要求1所述的自噬调节剂，其特征在于，所述R1中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种。
  3. 根据权利要求1所述的自噬调节剂，其特征在于，所述R2中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种。
  4. 根据权利要求1所述的自噬调节剂，其特征在于，所述R3中，所述C1-C4的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基中的任意一种；所述C1-C4的烷氧基包括甲氧基、乙氧基、丙氧基和丁氧基中的任意一种。
  5. 根据权利要求1～4中任一所述的自噬调节剂，其特征在于，所述自噬调节剂包括(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-正十二烷基喹啉碘盐。
  6. 一种如权利要求1～5中任一项所述的自噬调节剂的制备方法，其特征在于，所述制备方法包括如下步骤：
    S1.将4-甲基喹啉与卤代长链烷置于溶剂中，回流反应生成1-长链烷基-4-甲基喹啉盐；
    S2.将1-长链烷基-4-甲基喹啉盐、吲哚-3-甲醛和催化剂混合、回流反应后除杂；
    所述催化剂包括哌啶。
  7. 一种如权利要求1～5中任一项所述自噬调节剂在制备应用于细胞自噬调节的相关生命活动产品中的应用。
  8. 一种如权利要求1～5中任一项所述自噬调节剂在制备应用于靶向线粒体或靶向溶酶体的产品中的应用。
  9. 一种如权利要求1～5中任一项所述自噬调节剂在制备应用于线粒体成像或溶酶体成像的产品中的应用。
  10. 一种治疗肿瘤的药物，其特征在于，药物包括细胞自噬调节类药物，所述细胞自噬调节类药物包括如权利要求1～4中任一项所述的自噬调节剂。
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