Triphenylamine-benzopyrylium salt derivative nir-bt-p and synthesis method and application thereof

Abstract

The application provides a triphenylamine-benzopyrylium salt derivative NIR-BT-P and a synthesis method and application thereof.The Chinese name of the derivative NIR-BT-P is (E)-6-(diethylamino)-4-(4(diphenylamino)benzylidene)-1,2,3,4 tetrahydroxanthylium, and the English name is (E)-6-(diethylamino)-4-(4(diphenylamino)benzylidene)-1,2,3,4 tetrahydroxanthylium.The derivative NIR-BT-P is used as a fluorescent probe, different fluorescent intensity signals are achieved in different organic solvents through a fluorescence spectrophotometer, and the polarity is detected.The method utilizes the difference between cancer cells and normal cells in a living body, realizes near-infrared fluorescence imaging, and is used for distinguishing and detecting tumor tissues and normal tissues in the living body.

Description

TECHNICAL FIELD

[0001] The present application relates to a triphenylamine-benzopyrylium salt derivative, and particularly belongs to the application of a triphenylamine-benzopyrylium salt derivative NIR-BT-P in near-infrared detection of polarity. BACKGROUND

[0002] Polarity, as an important index, plays a crucial role in chemistry and biology. In biological systems, especially at the cellular level, polarity determines the interaction activity of a large number of proteins and enzymes and reflects the permeability of membrane compartments. In addition, polarity is closely related to a variety of physiological processes, such as membrane fusion, protein denaturation, enzyme conformational changes, and peptide aggregation. In particular, abnormal changes in polarity are closely related to a variety of diseases such as Alzheimer's disease, diabetes, cirrhosis, and cancer. In addition, the roles played by the polarity of different subcellular compartments are also different. Mitochondria are the main energy-producing compartments in most cells and play a role in many important cellular processes, such as ATP production, calcium regulation, and oxidative and reductive signal transduction, as well as the apoptosis process of cell death. Mitochondrial polarity strongly influences protein transport within cells and interactions between biological macromolecules. Recent studies have shown that fluctuations in the mitochondrial microenvironment are closely related to cell carcinogenesis. The mitochondrial polarity level of cancer cells is lower than that of normal cells. Therefore, it is of great significance to accurately monitor the polarity in mitochondria, especially in biological systems. However, polarity is a complex factor that contains a series of non-covalent interactions, including dipolarity / polarizability and hydrogen bonding, so it is difficult to measure it in living cells, and researchers urgently need to develop effective and convenient tools to accurately detect the polarity in mitochondria.

[0003] Compared with many traditional tools, fluorescent probes have the advantages of simple operation, high selectivity and sensitivity, real-time monitoring, non-invasiveness, and high spatiotemporal resolution, and are the most commonly used means for detecting polarity in vitro and biological systems. Therefore, designing fluorescent probes with good selectivity, high sensitivity, mitochondrial targeting, and low cytotoxicity for detecting polarity in living cells and tissues has become one of the challenging frontiers in the current development of biomedical science.

[0004] In the present application, a triphenylamine-benzopyrylium salt-based compound NIR-BT-P is synthesized, and the accurate detection of the polarity in mitochondria in cells is achieved by the change in the fluorescence signal intensity of NIR-BT-P before and after the reaction under different polarities. SUMMARY

[0005] The purpose of the present application is to provide a triphenylamine-benzopyrylium salt derivative NIR-BT-P, a synthesis method thereof, and the application thereof in near-infrared detection of polarity, and the detection method is simple, convenient to operate, has good selectivity, and high sensitivity.

[0006] The application provides a triphenylamine-benzopyrylium salt derivative NIR-BT-P, which is (E)-6-(diethylamino)-4-(4(diphenylamino)benzylidene)-1,2,3,4 tetrahydroxanthylium, named as NIR-BT-P, and the structural formula is:

[0007]

[0008] The application provides a synthesis method of a triphenylamine-benzopyrylium salt derivative NIR-BT-P, and the steps are as follows:

[0009] (1) at 0℃, cyclohexanone is added dropwise into concentrated H2SO4, and the solution is stirred, then 4-(diethylamino)-2-hydroxybenzaldehyde is added; the mixture is further heated at 90℃ for 2-3 hours, after being cooled to room temperature, the mixture is slowly poured into ice water, then HClO4 is added, the mixture is filtered, washed with water, and dried in vacuum to obtain brown solid, which is 6-(diethylamino)-1,2,3,4-tetrahydrooxanthylium, which can be used in the next step without further purification, wherein the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to cyclohexanone is 1:1.5-2.5;

[0010] (2) 6-(diethylamino)-1,2,3,4-tetrahydrooxanthylium and 4-(diphenylamino) benzaldehyde are dissolved in CH3COOH; the mixture is heated at 110℃ for 2-3 hours. After the reaction is completed, the reaction product is cooled to room temperature, the solvent is removed under reduced pressure, and dried in vacuum to obtain a crude product. Then, (E)-6-(diethylamino)-4-(4(diphenylamino)benzylidene)-1,2,3,4 tetrahydroxanthylium (NIR-BT-P) is obtained by silica gel column chromatography purification with dichloromethane and methanol as eluents in a volume ratio of 20:1, wherein the molar ratio of 6-(diethylamino)-1,2,3,4-tetrahydrooxanthylium to 4-(diphenylamino) benzaldehyde is 1:1.5-2.5.

[0011] As preferred:

[0012] In step (1), the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to cyclohexanone is 1:2.

[0013] In step (2), the molar ratio of 6-(diethylamino)-1,2,3,4-tetrahydrooxanthylium to 4-(diphenylamino) benzaldehyde is 1:2.

[0014] The synthesized NIR-BT-P can be used for near-infrared detection of polarity and can be applied to preparation of a reagent for detecting polarity in mitochondria of cells.

[0015] The application provides a method for detecting polarity by using NIR-BT-P, and the steps are as follows:

[0016] (1) dissolving NIR-BT-P in DMSO to prepare a 2mM solution;

[0017] (2) taking 2mL of 1, 4-dioxane, 10ul of NIR-BT-P DMSO solution into a fluorescence cuvette, and detecting on a fluorescence spectrophotometer; with the increase of the volume ratio of distilled water in the system, the fluorescence intensity at 830nm gradually decreases;

[0018] (3) adding 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9ml of 1, 4-dioxane into the fluorescence cuvette respectively, and adding 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1ml of distilled water into the fluorescence cuvette respectively, and meanwhile, detecting the maximum fluorescence intensity F max on a fluorescence spectrometer; wherein the polarities (Delta f) are taken as the abscissa, and the fluorescence intensities F max are taken as the ordinate to draw a graph, and the working curve of the polarity is obtained; and the linear regression equation is: F max = 3297.0-10830.5Delta f.

[0019] Compared with the prior art, the application has the following advantages and effects:

[0020] 1. The triphenylamine-benzopyrylium salt derivative synthesized in the application is simple in synthesis and low in cost;

[0021] 2. The triphenylamine-benzopyrylium salt derivative synthesized in the application can realize efficient targeting of mitochondria and near-infrared detection of polarity, and has high sensitivity and good selectivity in detection results;

[0022] 3. The detection means in the application is simple, and only a fluorescence spectrometer is needed to realize the detection;

[0023] 4. The application adopts near-infrared detection, and the detection signal is obvious and has good penetration ability. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 NMR hydrogen spectrum of the triphenylamine-benzopyrylium salt derivative NIR-BT-P prepared in Example 1

[0025] Figure 2NMR carbon spectrum of triphenylamine-benzopyrylium salt derivative NIR-BT-P prepared in Example 1

[0026] Figure 3 Mass spectrum of triphenylamine-benzopyrylium salt derivative NIR-BT-P prepared in Example 1

[0027] Figure 4 Fluorescence spectrum of triphenylamine-benzopyrylium salt derivative NIR-BT-P after interaction with different volume ratios of 1,4 dioxane-water

[0028] Figure 5 Working curve for determination of polarity of triphenylamine-benzopyrylium salt derivative NIR-BT-P

[0029] Figure 6 Fluorescence column chart of triphenylamine-benzopyrylium salt derivative NIR-BT-P with various analytes

[0030] Figure 7 Colocalization cell imaging chart

[0031] Figure 8 Cell imaging chart of triphenylamine-benzopyrylium salt derivative NIR-BT-P with different types of cells

[0032] Figure 9 Imaging chart of triphenylamine-benzopyrylium salt derivative NIR-BT-P for detection and differentiation of tumor tissues in tumor-bearing mice DETAILED DESCRIPTION

[0033] The present application is further described in connection with the following examples and drawings, but the present application is not limited by the following examples.

[0034] Example 1

[0035] Preparation and characterization of NIR-BT-P

[0036] Synthetic route of NIR-BT-P:

[0037]

[0038] Synthetic method of NIR-BT-P, the steps are:

[0039] (1) Cyclohexanone (3.1 mL, 30 mmol) was added dropwise to concentrated H2SO4(40 ml) at 0 °C and the solution was stirred, then 4-(diethylamino)-2-hydroxybenzaldehyde (2.9 g, 15 mmol) was added. The mixture was further heated at 90 °C for 2 h, after cooling to room temperature, the mixture was slowly poured into ice water (300 ml), then 4 ml HCIO4 was added, the mixture was filtered, washed with water, and dried in vacuum to give 6-(diethylamino)-1,2,3,4-tetrahydrooxepin (5.2 g, 97% yield) as a brown solid, which was used in the next step without further purification.

[0040] (2) 6-(Diethylamino)-1,2,3,4-tetrahydrooxepin (1.8 g, 5 mmol) and 4-(diphenylamino)benzaldehyde (2.7 g, 10 mmol) were dissolved in CH3COOH. The mixture was heated at 110 °C for 2.5 h. After the reaction was completed, the reaction product was cooled to room temperature, the solvent was removed under reduced pressure, and dried in vacuum to give the crude product. Then, (E)-6-(diethylamino)-4-(4-(diphenylamino)benzylidene)-1,2,3,4-tetrahydrooxepin (NIR-BT-P, 1.6 g, 62% yield) was obtained by silica gel column chromatography with dichloromethane and methanol as eluents in a volume ratio of 20:1. 1 H NMR (600 MHz, DMSO) δ 8.49 (s, 1H), 8.03 (s, 1H), 7.89 (d, J = 9.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 2.2 Hz, 1H), 7.43 - 7.40 (m, 4H), 7.27 (d, J = 1.9 Hz, 1H), 7.21 (d, J = 7.4 Hz, 2H), 7.18 (t, J = 7.9 Hz, 4H), 6.96 (d, J = 8.8 Hz, 2H), 3.70 (dd, J = 13.9, 6.8 Hz, 4H), 2.95 - 2.92 (m, 2H), 2.88 - 2.85 (m, 2H), 1.86 (dd, J = 11.2, 5.4 Hz, 2H), 1.25 (t, J = 7.0 Hz, 6H). Figure 1 ) 13C NMR(151MHz,DMSO)δ163.41(s),158.89(s),156.06(s),149.57(s),148.4 7(s),146.37(s),137.01(s),133.40(s),132.28(s),130.41(s),128.26(s) ),126.29(s),126.09(s),125.43(s),123.72(s),120.09(s),118.85(s),1 18.44(s),95.94(s),45.95(s),27.34(d,J=9.8Hz),21.52(s),13.03(s).( Figure 2 )ESI-MS m / z:[NIR-BT-P+H] + Calcd.For 511.2744,Found 511.2747.( Figure 3 )

[0041] Example 2

[0042] Prepare 2mM NIR-BT-P DMSO solution, take 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9ml of 1,4-dioxane and add them to 12 fluorescence cuvettes, add 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1mL of distilled water to the cuvettes, add 10μL NIR-BT-P DMSO solution to them, and detect on the fluorescence spectrophotometer at the same time. As the volume ratio of distilled water in the system increases, the fluorescence intensity at 830nm gradually decreases. Fluorescence emission diagram is shown in Figure 4 .

[0043] Example 3

[0044] Prepare a 2mM DMSO solution of NIR-BT-P and a 2mM aqueous solution of sulfur dioxide; add 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, and 0.9ml of 1,4-dioxane to 9 cuvettes, respectively, and then add 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, and 1.1ml of distilled water, respectively, and measure the maximum fluorescence intensity F on a fluorescence spectrometer. max are 556.1, 464.4, 394.4, 314.2, 260.2, 191.9, 142.1, 94.4, and 41.1, with polarity (Δf) as the horizontal axis and fluorescence intensity F max Draw a graph for the vertical axis to obtain the polarity working curve; the linear regression equation is: Fmax =3297.0-10830.5Δf, see Figure 5 .

[0045] Example 4

[0046] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10 mM, and prepare a 2 mM NIR-BT-P DMSO solution. In a fluorescence cuvette, add 2 mL of pure PBS buffer solution and 10 μL of NIR-BT-P DMSO solution. Then add different molar amounts of analytes: water, KCl (100 μM), CaCl2 (2 mM), ZnCl2 (150 μM), CuCl2 (100 μM), MgCl2 (1 mM), Na2CO3 (2 mM), NaNO3 (2 mM), Na2S (150 μM), Na2SO4 (150 μM), NaHSO4 (100 μM), GSH (10 mM), Hcy (10 mM), and Cys (10 mM) aqueous solutions. In addition, add 2 mL of 1,4-dioxane and 10 μL of NIR-BT-P DMSO solution to another fluorescence cuvette as a control group. The fluorescence intensity of the analytes at 830 nm was measured on a fluorescence spectrophotometer in a fluorescence cuvette, and a histogram was drawn (see Figure 6 1,4-dioxane significantly increased the fluorescence intensity of the detection system at 830 nm, while other analytes did not cause any change in the fluorescence intensity of the detection system.

[0047] Example 5

[0048] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10mM, prepare a 2mM NIR-BT-P DMSO solution, add 10μL of NIR-BT-P DMSO solution to 2mL of PBS buffer solution; add the probe solution to the HeLa cell culture medium to make its concentration 10μM, and react with the HeLa cells at 37°C for 15 minutes; observe the system under a fluorescence imager; then add 0.2μM MitoTracker Green to the system and react at 37°C for 20 minutes. Observe the system under a fluorescence imager and calculate the colocalization rate of the obtained fluorescence image. Figure 7 .

[0049] Example 6

[0050] A PBS buffer solution with pH = 7.4 and a concentration of 10 mM was prepared, a DMSO solution of 2 mM NIR-BT-P was prepared, 10 μL of the DMSO solution of NIR-BT-P was added to 2 mL of the PBS buffer solution; the probe solution was added to HeLa, HepG2 and 7702 cell culture solutions respectively so that the concentration was 10 μM, and the HeLa, HepG2 and 7702 cells were reacted at 37°C for 15 min; the system was placed under a fluorescence imager to show that the red fluorescence intensity of the cancer cells (HeLa and HepG2 cells) treated with NIR-BT-P was higher than that of the normal cells (7702 cells), as shown in Fig. 2. Figure 8 .

[0051] Example 7

[0052] A DMSO solution of 2 mM NIR-BT-P was prepared, HeLa cells were subcutaneously injected into BALB / c female nude mice, and tumor blocks were obtained about two weeks later. Then 20 μL of NIR-BT-P was injected into the tumor site and the left lower limb site (normal site) of the BALB / c female nude mice. Under 710 nm excitation, it was found that the mouse tumor site in the red channel showed a clear fluorescence signal, while the normal site showed a very weak fluorescence signal (see Fig. 3). Figure 9 )

Claims

1. A triphenylamine-benzopyrylium salt derivative NIR-BT-P, characterized in that: The structural formula is: 。 2. The method for synthesizing a triphenylamine-benzopyrylium salt derivative NIR-BT-P according to claim 1, wherein: The steps include: (1) At 0°C, cyclohexanone is added dropwise to concentrated H2SO4, and the solution is stirred, and then 4-(diethylamino)-2-hydroxybenzaldehyde is added; the mixture is further heated at 90°C for 2-3 hours, cooled to room temperature, and then slowly poured into ice water, followed by the addition of HClO4, the mixture is filtered, washed with water, and vacuum dried to obtain a brown solid, which is 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine; wherein the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to cyclohexanone is 1:1.5-2.5; (2) dissolving 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine and 4-(diphenylamino)benzaldehyde in CH3COOH at a molar ratio of 1:1.5-2.5; heating the mixture at 110°C for 2-3 hours; after the reaction is completed, cooling the reaction product to room temperature, removing the solvent under reduced pressure, and vacuum drying to obtain a crude product; then, separating the crude product by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 20:1 as eluents, and purifying to obtain the triphenylamine-benzopyrylium salt derivative NIR-BT-P.

3. The method for synthesizing NIR-BT-P according to claim 2, wherein In the step (1), the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to cyclohexanone is 1:

2.

4. The method for synthesizing NIR-BT-P according to claim 2, wherein In the step (2), the molar ratio of 6-(diethylamino)-1,2,3,4-tetrahydropyrimidine to 4-(diphenylamino)benzaldehyde is 1:

2.

5. Use of the triphenylamine-benzopyrylium salt derivative NIR-BT-P according to claim 1 in preparing a reagent for detecting polarity in cell mitochondria.

Images