Coumarin-benzopyrylium salt derivative MI-BP-CC and synthesis method and application thereof

Abstract

The application provides a coumarin-benzopyrylium salt derivative MI-BP-CC and a synthesis method and application thereof. The derivative MI-BP-CC has a Chinese name of 4-((4-chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium chromenylium perchlorate and an English name of 4-((4-chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-6-(diethylamino)-1,2,3,4-(tetrahydroxanthylium)chromenylium perchlorate. The derivative MI-BP-CC is used as a fluorescent probe, and specific detection of trace viscosity in glycerol / ethanol (v / v) solutions of different proportions is realized through a fluorescence spectrophotometer. The detection method is simple, sensitive, rapid, and has high accuracy of detection results.

Description

Technical Field

[0001] The invention relates to a coumarin-benzopyrylium salt derivative and viscosity detection, in particular to a coumarin-benzopyrylium salt derivative MI-BP-CC and a synthesis method thereof and application in viscosity detection. Background Art

[0002] Viscosity, as an important parameter in the cellular microenvironment, plays a vital role in controlling cellular processes, such as mass and signal transmission, protein aggregation, membrane fusion, and interactions between different biomolecules. Viscosity is associated with many cellular dysfunctions and the occurrence of certain diseases (such as Alzheimer's disease, hypertension, diabetes, and even cancer). Mitochondria, as one of the most complex subcellular organelles, are the main site of cellular metabolism and participate in various physiological processes such as cell growth, apoptosis, and differentiation, thereby regulating cellular functions. Generally speaking, the number of mitochondria depends on the metabolic level of the cell. However, mitochondrial metabolic capacity is closely related to changes in viscosity. Once mitochondrial function is lost and mutated, viscosity changes will occur, leading to the occurrence of a series of diseases such as Parkinson's disease, diabetes, aging, heart disease, and fatty liver. Therefore, it is urgent to design and develop a fluorescent sensor that can simultaneously detect viscosity changes in mitochondria.

[0003] In recent years, some conventional viscometers suitable for macroscopic fluids (e.g., falling ball viscometers, capillary viscometers, and rotational viscometers) cannot be used to monitor viscosity changes in cellular microenvironments. However, small molecule fluorescent probes, due to their unique advantages such as ease of operation, real-time detection, good specificity, and high sensitivity, are considered powerful tools for monitoring disease-related biomolecules.

[0004] In response to the above problems, designing fluorescent probes with good selectivity, high sensitivity and low cytotoxicity to distinguish and detect changes in mitochondrial viscosity levels has become one of the challenging frontier topics in the current development of biomedicine. Summary of the Invention

[0005] The invention provides a coumarin-benzopyrylium salt derivative MI-BP-CC, a synthesis method thereof and an application of the coumarin-benzopyrylium salt derivative in viscosity detection.

[0006] The present invention provides a coumarin-benzopyrylium salt derivative MI-BP-CC. The Chinese name of the derivative MI-BP-CC is: 4-((4-chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate; the English name is 4-((4-chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-6-(diethylamino)-1,2,3,4-(tetrahydroxanthylium)chromenyliumperchlorate, and the structural formula is:

[0007]

[0008] The present invention provides a method for synthesizing coumarin-benzopyrylium salt MI-BP-CC, comprising the following steps:

[0009] (1) Add 3-N,N-diethylaminophenol to a toluene solution of diphenyl malonate. Heat the reaction mixture under reflux for 6-8 hours. After the reaction is complete, filter and wash the filter cake. Dry the product under vacuum to obtain a light yellow solid, i.e., 4-hydroxy-7-diethylaminocoumarin. The molar ratio of diphenyl malonate to 3-N,N-diethylaminophenol is 1:1.

[0010] (2) Under a nitrogen atmosphere, DMF is added dropwise to POCl3 at 20-50°C and stirred for about 20-40 minutes to obtain a red solution; 4-hydroxy-7-diethylaminocoumarin is then dissolved in DMF and then added dropwise to the above solution to obtain a red suspension; the mixture is stirred at 55-65°C for 10-14 hours and then poured into ice water, and the pH is adjusted to obtain a large amount of precipitate; filtration, washing, drying and recrystallization are performed to obtain 4-chloro-7-diethylaminocoumarin-3-aldehyde; wherein the molar ratio of N,N-dimethylformamide, phosphorus oxychloride and 4-hydroxy-7-diethylaminocoumarin is 3:3:1;

[0011] (3) Cyclohexanone was slowly added to concentrated sulfuric acid and stirred at 0°C for 20 minutes. Then 4-diethylamino-2-hydroxybenzaldehyde was added to the reaction system. The mixture was reacted at 90°C for 2 hours and cooled to room temperature. The obtained mixture was slowly poured into ice water, and then perchloric acid was slowly added; the obtained precipitate was filtered and washed with water, and dried in a vacuum to obtain a dark purple solid, namely 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine chromium perchlorate; wherein the molar ratio of cyclohexanone to 4-diethylamino-2-hydroxybenzaldehyde was 2:1;

[0012] (4) 4-Chloro-7-diethylaminocoumarin-3-aldehyde and 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine chromium perchlorate were mixed in glacial acetic acid at a molar ratio of 1:2, and the mixture was heated at 113°C for 3 hours. After the reaction was completed, the mixture was washed, dried, and purified by column chromatography to obtain a dark purple solid (coumarin-benzopyrylium salt derivative MI-BP-CC).

[0013] A method for detecting viscosity comprises the following steps:

[0014] (1) Prepare a 2 mM MI-BP-CC solution in DMSO and glycerol / ethanol solutions of different viscosities (Table 1):

[0015]

[0016]

[0017] (2) Add 10 μL of MI-BP-CC DMSO solution to the prepared glycerol / ethanol solutions of different viscosities in a fluorescence cuvette. After thorough mixing, the change in the fluorescence intensity of the probe MI-BP-CC at 720 nm with increasing viscosity is detected on a fluorescence spectrophotometer.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The coumarin-benzopyrylium salt derivatives of the present invention are simple to synthesize and have low cost;

[0020] 2. The probe MI-BP-CC has a large Stokes shift for viscosity detection and near-infrared emission (720nm), which has the advantages of high penetration, low background fluorescence, and is convenient for cell imaging.

[0021] 3. The coumarin-benzopyrylium salt derivative MI-BP-CC of the present invention can be used as a fluorescent probe to detect trace amounts of viscosity, showing high sensitivity;

[0022] 4. The positive ions in benzopyran salts can effectively target mitochondria, thereby achieving mitochondrial-targeted viscosity;

[0023] 5. The present invention can realize the detection of viscosity at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 H NMR spectrum of MI-BP-CC prepared in Example 1

[0025] Figure 2 NMR carbon spectrum of MI-BP-CC prepared in Example 1

[0026] Figure 3 Mass spectrum of MI-BP-CC prepared in Example 1

[0027] Figure 4 Example 2 Fluorescence spectra of MI-BP-CC in glycerol-ethanol solutions with different ratios

[0028] Figure 5 Example 3 Fluorescence histogram of MI-BP-CC and various analytes

[0029] Figure 6 Example 4 Cell imaging of MI-BP-CC detecting cell viscosity

[0030] Figure 7 Example 5 Cell imaging of MI-BP-CC detecting viscosity of normal liver cells and liver cancer cells

[0031] Figure 8 Example 6 Cellular imaging of MI-BP-CC detecting viscosity in PD model

[0032] Figure 9 Example 7 MI-BP-CC Mitochondrial Localization Imaging DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0034] Example 1

[0035] Preparation and characterization of MI-BP-CC

[0036] Synthesis route of MI-BP-CC:

[0037]

[0038] The synthesis method of MI-BP-CC comprises the following steps:

[0039] (1) 3-N,N-diethylaminophenol (8.25 g, 50 mmol) was added to a solution of diphenyl malonate (12.8 g, 50 mmol) in toluene (50 mL). The reaction mixture was refluxed for 7 hours. After the reaction was complete, the filter cake was filtered and washed with hexane. The product was dried under vacuum to obtain a light yellow solid (Compound 1, 8.7 g, 74% yield);

[0040] (2) Under nitrogen atmosphere, fresh DMF (2.5 mL, 30 mmol) was added dropwise to POCl3 (2.8 mL, 30 mmol) at 20-50°C and stirred for 30 minutes to obtain a red solution. Compound 1 (2.33 g, 10 mmol) was then dissolved in 13.2 mL of DMF and added dropwise to the above solution to obtain a scarlet suspension. The mixture was stirred at 60°C for 12 hours and then poured into 100 mL of ice water. NaOH solution (20%) was added to adjust the pH of the mixture to obtain a large amount of precipitate. The crude product was filtered, washed with water, dried and recrystallized from anhydrous ethanol to obtain a brown needle-shaped solid (compound 2, 1.20 g, 88.2% yield);

[0041] (3) Cyclohexanone (6.1 mL, 60 mmol) was slowly added to concentrated sulfuric acid (80 mL) and stirred at 0°C for 20 minutes. Then 4-diethylamino-2-hydroxybenzaldehyde (5.89 g, 30 mmol) was added to the reaction system. The mixture was reacted at 90°C for 2 hours and cooled to room temperature. The obtained mixture was slowly poured into 500 mL of ice water, and then 7.8 mL of perchloric acid was slowly added. The obtained precipitate was filtered, washed with water, and dried in vacuo. Thus, a dark purple solid (Compound 3, 8.7 g, 82% yield) was obtained;

[0042] (4) A mixture of compound 2 (0.28 g, 1 mmol) and compound 3 (0.36 g, 2 mmol) was dissolved in 15 mL of glacial acetic acid. The reaction system was heated and stirred at 113°C for 2.5 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a dark purple crude product. After the reaction was completed, the mixture was washed, dried, and purified by column chromatography to obtain a dark purple solid (coumarin-benzopyrylium salt MI-BP-CC, 0.19 g, yield 32%). 1 H NMR (600MHz, DMSO) δ8.58(s,1H),7.94(d,J=9.5Hz,1H),7.70(d,J=9.1Hz,1H),7.65(s,1H) ,7.53(dd,J=9.5,2.2Hz,1H),7.36(d,J=1.5Hz,1H),6.88(dd,J=9.2,2.4Hz,1H),6.65(d,J =2.3Hz,1H),3.74(d,J=6.2Hz,4H),3.50(dd,J=14.0,6.9Hz,4H),2.90(t,J=6.0Hz,2H),2. 57–2.54(m,2H),1.83(dt,J=12.2,6.0Hz,2H),1.25(t,J=7.0Hz,6H),1.15(t,J=7.0Hz,6H). 13C NMR(151MHz,DMSO)δ160.24(s),159.24(s),158.28(s),156.95(s),155.15(s),152.37 (s),149.34(s),148.17(s),133.12(s),132.83(s),127.62(d,J=24.5Hz),127.53–127. 44(m),123.97(s),120.65(s),119.66(s),113.83(s),110.72(s),106.91(s),96.80(s) ),96.07(s),46.26(s),44.83(s),31.13(s),28.35(s),27.32(s),21.58(s),12.80(s).

[0043] Example 2

[0044] Prepare 2.0mL of glycerol and ethanol (glycerol volume ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) solutions of different viscosities, mix them thoroughly, and sonicate for half an hour to remove bubbles in the solution. Then, mix them with 10μL of 2.0mmol / L probe MI-BP-CC solution in a cuvette, and after thorough mixing, test their fluorescence intensity. With the increase of viscosity (increase of glycerol volume ratio), the fluorescence of probe MI-BP-CC at 720nm gradually increases (Ex = 595nm) ( Figure 4 ).

[0045] Example 3

[0046] Prepare 2.0 mL of glycerol and ethanol (50% glycerol by volume) viscosity solution, mix thoroughly, and ultrasonicate for half an hour to remove bubbles in the solution. Detect the changes in fluorescence intensity of other analytes at 720 nm when the glycerol volume content is 0% and 50%. In a fluorescence cuvette, add 2 mL of anhydrous ethanol solution / 2 mL of 50% glycerol and ethanol viscosity solution and 10 μM MI-BP-CC DMSO solution, respectively. Then add 20 equivalents of other analytes: (1) probe MI-BP-CC (2) Ca 2+ ,(3)Na + ,(4)K + ,(5)Mg 2+ ,(6)Ni 2+ ,(7)Cr 3+ ,(8)NO3 - ,(9)CO3 2- ,(10)Br -,(11)H2O2,(12)Cys,(13)Hcy,(14)SO3 2- ,(15)GSH,(16)S2 - The aqueous solution was tested on a fluorescence spectrophotometer and a histogram of the fluorescence intensity at 720 nm corresponding to different analytes was drawn (see Figure 5 The addition of other analytes did not cause significant fluorescence changes in the MI-BP-CC probe at 50% glycerol viscosity. Compared to 0% glycerol, the fluorescence intensity at 50% glycerol was significantly enhanced.

[0047] Example 4

[0048] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10mM, and prepare a DMSO solution of 2mM MI-BP-CC; first add 2mL of PBS to the HeLa cells, and then add the probe solution to the HeLa cells to make a concentration of 10μM. After the HeLa cells are placed at 37°C for 15 minutes, the cells are rinsed three times with PBS, and then the images are taken under a fluorescence confocal microscope. The probe shows a weak red fluorescence. Next, 10μM nystatin is added to the cells incubated with the probe, and the red channel fluorescence is significantly enhanced under a fluorescence confocal microscope. In addition, in the cell starvation experiment, HeLa cells are incubated in D-Hanks buffer solution for 12 hours, and then the probe solution is added to the HeLa cells to make a concentration of 10μM. After the HeLa cells are placed at 37°C for 15 minutes, the cells are rinsed three times with PBS, and then the images are taken under a fluorescence confocal microscope. The probe shows an enhanced red fluorescence phenomenon (see Figure 6 ).

[0049] Example 5

[0050] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10mM, and prepare a DMSO solution of 2mM MI-BP-CC; first add 2mL of PBS to 7702 cells, then add the probe solution to 7702 cells to make its concentration 10μM, place the 7702 cells at 37°C for 20 minutes, rinse the cells three times with PBS, and then image and photograph under a fluorescence confocal microscope. The probe exhibits red fluorescence. Next, add 2mL of PBS to HepG 2 cells, then add the probe solution to HepG 2 cells to make its concentration 10μM, place the HepG 2 cells at 37°C for 20 minutes, rinse the cells three times with PBS, and then image and photograph under a fluorescence confocal microscope. The probe exhibits enhanced red fluorescence (see Figure 7 ).

[0051] Example 6

[0052] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10mM, prepare a 2mM MI-BP-CC DMSO solution, prepare a 20mM L-glutamate solution and a H2O2 solution; add 10μL of the MI-BP-CC DMSO solution to 2mL of the PBS solution; add the probe solution to the PC 12 cell culture medium to make its concentration 10μM, and after placing it with the PC 12 cells at 37°C for 15 minutes, the system shows weak red fluorescence under a fluorescence imager; then, add 10μL of L-glutamate solution and H2O2 solution to the culture dish containing PC 12 cells, respectively, and treat them at 37°C in a culture environment containing 5% CO2 for 6 hours to establish a Parkinson's disease (PD) model. After culturing for 6 hours, 2 mL of PBS was added to the PC 12 cells. Then, 10 μL of the probe solution was added to the L-glutamic acid solution and H2O2 solution to make the concentration 10 μM. After the PC 12 cells were placed at 37°C for 15 minutes, the cells were washed three times with PBS. The probe showed enhanced fluorescence in the red channel (see Figure 8 ).

[0053] Example 7

[0054] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10 mM, and prepare a DMSO solution of 2 mM MI-BP-CC; first add 2 mL of PBS to the HeLa cells, then add the probe solution to the HeLa cells to make its concentration 10 μM, place the HeLa cells at 37°C for 15 minutes, rinse the cells three times with PBS, and then add the commercial mitochondrial green dye to the HeLa cells to make its concentration 50 nM. After placing the cells in a 37°C incubator for 20 minutes, rinse them three times with PBS, and then image them under a fluorescence confocal microscope (see Figure 9 The results showed that the probe had good targeting to mitochondria (Pearson coefficient 0.92).

[0055] The above experimental results indicate that MI-BP-CC is a good candidate for detecting mitochondrial viscosity.

Claims

1. A coumarin-benzopyrylium salt derivative MI-BP-CC, characterized in that The structural formula is: 。 2. The method for synthesizing a coumarin-benzopyrylium salt derivative MI-BP-CC according to claim 1, wherein: The steps include: (1) Add 3-N,N-diethylaminophenol to a toluene solution of diphenyl malonate, and heat the reaction mixture under reflux for 6-8 hours; after the reaction is completed, filter and wash the filter cake; and dry the product under vacuum to obtain a light yellow solid product, namely 4-hydroxy-7-diethylaminocoumarin; wherein the molar ratio of diphenyl malonate to 3-N,N-diethylaminophenol is 1:1; (2) Under nitrogen atmosphere, DMF is added dropwise to POCl3 at 20-50°C and stirred for 20-40 minutes to obtain a red solution; 4-hydroxy-7-diethylaminocoumarin is then dissolved in DMF and added dropwise to the above solution to obtain a red suspension; the mixture is stirred at 55-65°C for 10-14 hours and then poured into ice water, and the pH is adjusted to obtain a large amount of precipitate; filtration, washing, drying and recrystallization are performed to obtain 4-chloro-7-diethylaminocoumarin-3-aldehyde; wherein the molar ratio of N,N-dimethylformamide, phosphorus oxychloride and 4-hydroxy-7-diethylaminocoumarin is 3:3:1; (3) Cyclohexanone was slowly added to concentrated sulfuric acid and stirred at 0°C for 20 minutes; 4-diethylamino-2-hydroxybenzaldehyde was then added to the reaction system; the mixture was reacted at 90°C for 2 hours and cooled to room temperature; the obtained mixture was slowly poured into ice water, and then perchloric acid was slowly added; the obtained precipitate was filtered and washed with water, and dried in a vacuum to obtain a dark purple solid, namely 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine chromium perchlorate; wherein the molar ratio of cyclohexanone to 4-diethylamino-2-hydroxybenzaldehyde was 2:1; (4) 4-Chloro-7-diethylaminocoumarin-3-aldehyde and 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine chromium perchlorate were mixed in glacial acetic acid at a molar ratio of 1:2, and the mixture was heated at 113°C for 3 hours. After the reaction was completed, the mixture was washed, dried, and purified by column chromatography to obtain a dark purple solid, i.e., the target product, coumarin-benzopyrylium salt derivative MI-BP-CC.

3. Use of a coumarin-benzopyrylium salt derivative MI-BP-CC as claimed in claim 1 in preparing a fluorescent probe for detecting viscosity.

4. A method for detecting viscosity, comprising the following steps: (1) Prepare a 2 mM DMSO solution of the MI-BP-CC described in claim 1 and prepare glycerol / ethanol solutions of different viscosities: (2) Add 10 μL of MI-BP-CC DMSO solution to the prepared glycerol / ethanol solutions of different viscosities in a fluorescence cuvette. After thorough mixing, the change in the fluorescence intensity of the probe MI-BP-CC at 720 nm with increasing viscosity is detected on a fluorescence spectrophotometer.

5. Use of the coumarin-benzopyrylium salt derivative MI-BP-CC according to claim 1 in the preparation of a cell imaging reagent.

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