A small molecule compound specifically targeting lipid droplet imaging and degradation and its preparation method and application

Abstract

The present invention discloses a small molecule compound that specifically targets lipid droplet imaging and degradation, and a preparation method and application thereof, and relates to the field of biomedical technology; the present invention provides a general structural formula of a small molecule compound, and also provides a preparation method and application of a small molecule compound that specifically targets lipid droplet imaging and degradation. The small molecule compound of the present invention can specifically target lipid droplets, and carry lipid droplets to specifically bind to microtubule-associated protein light chain 3 (LC3), and finally degrade lipid droplets through the autophagosome-lysosome pathway.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule compound specifically targeting lipid droplet imaging and degradation, and a preparation method and application thereof. Background Art

[0002] Lipid droplets are ubiquitous and unique organelles in organisms. In addition to storing neutral lipids to maintain intracellular lipid metabolism homeostasis, they are also involved in physiological processes such as protein degradation, signal transduction, membrane synthesis and transport. In addition, due to the diversity of lipid droplet functions and abnormal levels, they also play an important role in the pathological processes of various diseases, such as obesity, non-alcoholic fatty liver disease, pancreatic steatosis, infection, cancer, and cardiovascular disease. Therefore, it is of great significance to visually observe the content of lipid droplets in cells, their morphology and changes, and specifically reduce the level of lipid droplets for the study and treatment of lipid droplet-related diseases.

[0003] Fluorescence imaging provides an intuitive and real-time observation method for studying complex biological structures and processes. As an important carrier of fluorescence imaging, organic fluorescent probes play an important role in studying organelles and cell physiological processes.

[0004] As a selective autophagy process, lipophagy can regulate lipid droplet metabolism in a variety of cells by degrading lipid droplets, including macrophages, adipocytes, hypothalamic and striatal neurons, renal tubular cells, fibroblasts, etc. It has become one of the key links in regulating cellular lipid metabolism balance and energy metabolism.

[0005] The current strategies for reducing lipid droplet levels are still imperfect and have poor specificity. Therefore, based on the above background, the present invention has developed a class of small molecule compounds that can specifically bind to lipid droplets and perform imaging and can target and degrade lipid droplets. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a small molecule compound that specifically targets lipid droplets for imaging and degradation, and a preparation method and application thereof. The small molecule compound can specifically target lipid droplets, carry lipid droplets and specifically bind to microtubule-associated protein light chain 3 (LC3), and ultimately degrade lipid droplets through the autophagosome-lysosome pathway.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: a small molecule compound specifically targeting lipid droplet imaging and degradation is provided, and its general structural formula is:

[0008] or ;

[0009] Wherein, R1 is methylene, amino or carbonyl, and the number of methylene groups is 9-20;

[0010] R2 is 1,3-indandione or 1,3-bis(dicyanomethylidene)indan;

[0011] R3 is a hydrogen atom, a methyl group, a methoxy group, an amino group, a hydroxyl group or an azadimethyl group.

[0012] The method for preparing the above-mentioned small molecule compound that specifically targets lipid droplet imaging and degradation comprises the following steps:

[0013] The aromatic aldehyde compound precursor and 5,7-dihydroxy-4-phenylcoumarin are reacted in the presence of a catalyst and a condensing agent, and then sequentially extracted, dried, filtered and purified by column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0014] Further, the aromatic aldehyde compound precursor has the general structural formula:

[0015] ;

[0016] Wherein, R1 is methylene, amino or carbonyl, and the number of methylene groups is 9-20;

[0017] R2 is 1,3-indandione or 1,3-bis(dicyanomethylidene)indan;

[0018] R3 is a hydrogen atom, a methyl group, a methoxy group, an amino group, a hydroxyl group or an azadimethyl group.

[0019] Further, the structural formula of 5,7-dihydroxy-4-phenylcoumarin is:

[0020] .

[0021] Furthermore, the molar ratio of 5,7-dihydroxy-4-phenylcoumarin to the aromatic aldehyde compound precursor is 1:1-50:1.

[0022] Furthermore, the catalyst is N,N-diisopropylethylamine or triethylamine.

[0023] Further, the condensing agent is 1-hydroxybenzotriazole (HOBT), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate 4 (HCTU), 2-(endo-5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), dicyclohexylcarbonyl At least one of diimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT).

[0024] Furthermore, the molar ratio of the catalyst to 5,7-dihydroxy-4-phenylcoumarin is 2-30:1, and the molar ratio of the condensing agent to 5,7-dihydroxy-4-phenylcoumarin is 0.5-10:1.

[0025] Further, the reaction is carried out at a temperature of 4-60°C for 24-72 hours.

[0026] Further, the product was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride respectively, dried over anhydrous sodium sulfate, filtered and the liquid was purified by rapid column chromatography.

[0027] The present invention also provides the use of the above-mentioned small molecule compound specifically targeting lipid droplet imaging and degradation in lipid droplet imaging and lipid droplet degradation in lipid droplet-rich cells.

[0028] The present invention has the following beneficial effects:

[0029] 1. The small molecule compounds of the present invention have good lipid droplet-specific imaging capabilities, which can realize real-time observation of lipid droplet distribution and morphology in living cells, laying a solid foundation for biological imaging.

[0030] 2. The small molecule compounds of the present invention can effectively bind to lipid droplets and selectively degrade them, and can effectively reduce the level of intracellular lipid droplets.

[0031] 3. The preparation method of the present invention is simple, easy to operate, and the raw materials are readily available, and industrial production can be easily realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the H NMR spectrum of the product obtained in Example 1;

[0033] Figure 2 Confocal microscopy imaging results after treatment with 10 µM and 20 µM small molecule compounds;

[0034] Figure 3 Confocal microscopy imaging results after treatment with 1µM small molecule compounds. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0036] Example 1

[0037] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0038] ;

[0039] The specific process is:

[0040] 2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione (0.23 g, 0.42 mmol), (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid (0.15 g, 0.41 mmol), catalyst DIPEA (0.21 g, 1.63 mmol), condensation agent HOBT (0.062 g, 0.46 mmol) and HBTU (0.174 g, 0.46 mmol) were dissolved in 100 mL The reaction was carried out in DMF solution at room temperature for 24 hours. The reaction progress was monitored by thin layer chromatography. After the reaction, a large amount of ethyl acetate was added. The mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried with anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation (H NMR spectrum as shown in FIG. Figure 1 as shown).

[0041] Example 2

[0042] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0043] ;

[0044] The specific process is:

[0045] 2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione (0.4 g, 0.727 mmol), 6-((7-hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl)oxy)hexanoic acid (0.27 g, 0.735 mmol), catalyst DIPEA (0.3 g, 2.32 mmol), condensation agent HOBT (0.128 g, 0.95 mmol) and HBTU (0.36 g, 0.95 mmol) were dissolved in 120 mL The reaction was carried out in DMF solution at room temperature for 36 hours, and the reaction progress was monitored by thin layer chromatography. After the reaction was completed, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0046] Example 3

[0047] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0048] ;

[0049] The specific process is:

[0050] 2,2'-(2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-diylidene)diacrylonitrile (0.5 g, 0.77 mmol), (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid (0.29 g, 0.78 mmol), catalyst DIPEA (0.5 g, 3.85 mmol), condensation agent HOBT (0.16 g, 1.16 mmol) and HBTU (0.44 g, 1.16 mmol) were dissolved in 100 mL The mixture was reacted in DMF solution at room temperature for 24 hours, and the reaction progress was monitored by thin layer chromatography. After the reaction, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0051] Example 4

[0052] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0053] ;

[0054] The specific process is:

[0055] 2,2'-(2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-diylidene)diacrylonitrile (0.5 g, 0.77 mmol), 6-((7-hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl)oxy)hexanoic acid (0.29 g, 0.78 mmol), catalyst DIPEA (0.5 g, 3.85 mmol), condensation agent HOBT (0.16 g, 1.16 mmol) and HBTU (0.44 g, 1.16 mmol) were dissolved in 100 mL The mixture was reacted in DMF solution at room temperature for 24 hours, and the reaction progress was monitored by thin layer chromatography. After the reaction, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0056] Example 5

[0057] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0058] ;

[0059] The specific process is:

[0060] 2-((3-(3-aminopropoxy)-4'-(di-p-tolylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione (0.4 g, 0.69 mmol), (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid (0.25 g, 0.69 mmol), catalyst DIPEA (0.54 g, 4.14 mmol), condensation agent HOBT (0.16 g, 1.17 mmol) and HBTU (0.44 g, 1.17 mmol) were dissolved in 100 mL The reaction was carried out in DMF solution at room temperature for 48 hours, and the reaction progress was monitored by thin layer chromatography. After the reaction was completed, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0061] Example 6

[0062] A small molecule compound that specifically targets lipid droplet imaging and degradation, the preparation route of which is as follows:

[0063] ;

[0064] The specific process is:

[0065] 2-((3-(3-aminopropoxy)-4'-(di-p-tolylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione (0.4 g, 0.69 mmol), 6-((7-hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl)oxy)hexanoic acid (0.25 g, 0.69 mmol), catalyst DIPEA (0.54 g, 4.14 mmol), condensation agent HOBT (0.16 g, 1.17 mmol) and HBTU (0.44 g, 1.17 mmol) were dissolved in 100 mL The reaction was carried out in DMF solution at room temperature for 48 hours, and the reaction progress was monitored by thin layer chromatography. After the reaction was completed, a large amount of ethyl acetate was added, and the mixture was extracted twice with sodium bicarbonate, hydrochloric acid and sodium chloride, respectively, and dried over anhydrous sodium sulfate. The liquid was filtered and purified by rapid column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

[0066] Experimental example

[0067] After using oleic acid to induce lipid droplet formation in Hela cells, the experimental groups were treated with 10 and 20 μM of the small molecule compound obtained in Example 1, respectively, and the control group was a blank control. Confocal microscopy was used for imaging 24 hours later, and the results are as follows: Figure 2 shown.

[0068] Depend on Figure 2 As can be seen, a dose-dependent reduction in the number and size of lipid droplets was observed, and LDs were almost completely removed at concentrations of approximately 10-20 µM.

[0069] After using oleic acid to induce lipid droplet formation in Hela cells, 1 μM of the small molecule compound obtained in Example 1 was used for treatment for 2 h, and then lipid droplet imaging was observed using a confocal microscope. The results are as follows: Figure 3 shown.

[0070] Depend on Figure 3 It can be seen that small molecule compounds that specifically target lipid droplets for imaging and degradation can specifically target lipid droplets and achieve visualization, and have relatively good optical properties, which can better monitor the dynamic changes of lipid droplets.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A small molecule compound specifically targeting lipid droplet imaging and degradation, characterized in that: Its structural formula is: 。 2. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation according to claim 1, characterized in that: The following steps are involved: 2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione and (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid are reacted in the presence of a catalyst and a condensing agent, and then sequentially extracted, dried, filtered and purified by column chromatography to obtain a small molecule compound that specifically targets lipid droplet imaging and degradation.

3. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation as claimed in claim 2, characterized in that: The molar ratio of 2-((3-(3-aminopropoxy)-4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)methylene)-1H-indene-1,3(2H)-dione and (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid is 1:

1.

4. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation as claimed in claim 2, characterized in that: The catalyst is N,N-diisopropylethylamine or triethylamine.

5. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation as claimed in claim 2, characterized in that: The condensing agent is at least one of 1-hydroxybenzotriazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and 2-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

6. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation as claimed in claim 2, characterized in that: The molar ratio of the catalyst to (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid is 2-30:1, and the molar ratio of the condensing agent to (6-((5-hydroxy-2-oxo-4-phenyl-2H-chromen-7-yl)oxy)hexanoic acid is 0.5-10:

1.

7. The method for preparing a small molecule compound for specific targeting of lipid droplet imaging and degradation as claimed in claim 2, characterized in that: React at 4-60℃ for 24-72 h.

8. Use of the small molecule compound specifically targeting lipid droplet imaging and degradation according to claim 1 in the preparation of an agent for lipid droplet imaging and degradation in lipid droplet-rich cells.

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