A coumarin benzothiazole salt derivative, and a preparation method and application thereof

Abstract

The application provides a coumarin benzothiazole salt derivative, a preparation method and application thereof, and belongs to the technical field of coumarin derivatives. The coumarin benzothiazole salt derivative provided by the application has good water solubility, long fluorescence emission wavelength, and small background interference; can realize mitochondrial targeting, can simultaneously respond to sulfur dioxide and viscosity, has high sensitivity, and has good selectivity.

Description

Technical Field

[0001] This invention relates to the field of coumarin derivatives technology, and in particular to a coumarin benzothiazole salt derivative, its preparation method, and its application. Background Technology

[0002] Sulfur dioxide (SO2) is a common environmental pollutant, and its main form in aqueous solution is sulfite (SO3). 2- ) and bisulfite (HSO3) - Sulfur dioxide and its derivatives are frequently used as bleaching agents, preservatives, and anti-fading agents in food processing and storage. Further research indicates that sulfur dioxide, as an endogenous gaseous signaling substance in living organisms, is closely linked to various physiological and pathological processes, playing a crucial role in regulating the cardiovascular system and maintaining the body's redox balance. In mitochondria, endogenous sulfur dioxide can be generated from thiol-containing amino acids and hydrogen sulfide (H2S) under enzymatic oxidation, and it participates as a gaseous neurotransmitter in various physiological processes such as antioxidation, vascular smooth muscle relaxation, and blood insulin regulation. Abnormal expression of sulfur dioxide and its derivatives in organisms is associated with many diseases, such as lung cancer, cardiovascular disease, and neurological disorders. Therefore, developing highly sensitive and specific fluorescent probes that target sulfur dioxide in mitochondria to achieve visualized monitoring of sulfur dioxide in mitochondria is crucial for the prevention and diagnosis of certain diseases.

[0003] Viscosity plays a crucial role in maintaining intermolecular interactions, cell signal transduction, nutrient transport, and metabolism. Abnormal viscosity often disrupts normal cellular physiological processes, inducing a series of pathological changes. Mitochondria, as one of the most important organelles in cells, play a vital role in cellular metabolism. Abnormal mitochondrial viscosity often directly disrupts cellular metabolic processes, leading to malignant tumors and neurodegenerative diseases.

[0004] Developing methods for real-time detection of sulfur disulfide and viscosity in living systems is of great significance. Fluorescent probes offer advantages such as low detection cost, simple operation, high sensitivity, and good selectivity. However, currently developed fluorescent probes often have a single response site and lack the ability to simultaneously detect sulfur disulfide and viscosity; they also exhibit low selectivity, often being susceptible to interference from sulfur disulfide analogues; and their sensitivity is low, requiring relatively high concentrations to produce a fluorescence response. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a coumarin benzothiazole salt derivative, its preparation method, and its application. The coumarin benzothiazole salt derivative provided by this invention can target mitochondria, respond simultaneously to sulfur dioxide and viscosity, and exhibits high sensitivity and good selectivity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a coumarin benzothiazole salt derivative having the structure shown in Formula I:

[0008]

[0009] Where X is a halogen.

[0010] The present invention also provides a method for preparing the coumarin benzothiazole salt derivative described in the above technical solution, comprising the following steps:

[0011] The first condensation reaction was carried out by mixing 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde, (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide, cesium carbonate and an organic solvent to obtain the condensation product;

[0012] An addition reaction was carried out by mixing 2-methylbenzothiazole, haloethane, and an organic solvent to obtain the addition product;

[0013] The condensation product, addition product, piperidine, and organic solvent are mixed and subjected to a second condensation reaction to obtain the coumarin benzothiazole salt derivative.

[0014] Preferably, the molar ratio of 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde to (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide is 1:1.1.

[0015] Preferably, the molar ratio of 2-methylbenzothiazole to haloethane is 1:1.5.

[0016] Preferably, the molar ratio of the condensation product to the addition product is 1:1.2.

[0017] Preferably, the first condensation reaction takes 24 to 48 hours.

[0018] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in the detection of sulfur disulfide.

[0019] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in viscosity detection.

[0020] This invention also provides an application for the simultaneous dual detection of sulfur disulfide and viscosity of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the above technical solutions.

[0021] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in the preparation of reagents for simultaneous detection of sulfur disulfide and viscosity.

[0022] This invention provides a coumarin benzothiazole salt derivative, which has the following advantages and effects compared with the prior art:

[0023] The coumarin benzothiazole salt derivative provided by this invention has good water solubility, long fluorescence emission wavelength, and low background interference; it can target mitochondria, respond to sulfur dioxide and viscosity simultaneously, and has high sensitivity and good selectivity.

[0024] The present invention also provides a method for preparing the above-mentioned coumarin benzothiazole salt derivative. The synthesis steps of the present invention are simple, the cost is low, and the toxicity is low.

[0025] This invention also provides an application for the simultaneous dual detection of sulfur disulfide and viscosity using the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the above technical solutions. The detection method is convenient and quick to operate, and can be achieved with the help of ultraviolet and fluorescence spectrometers. Attached Figure Description

[0026] Figure 1 The 1H NMR spectrum of the coumarin benzothiazole salt derivative prepared in Example 1;

[0027] Figure 2 The carbon NMR spectrum of the coumarin benzothiazole salt derivative prepared in Example 1;

[0028] Figure 3 The UV-Vis absorption spectrum of the reaction between coumarin benzothiazole salt derivative and SO2;

[0029] Figure 4 The fluorescence emission patterns at 500 nm and 690 nm are shown for the interaction of coumarin benzothiazole salt derivatives with SO2.

[0030] Figure 5 The fluorescence intensity ratio of coumarin benzothiazole salt derivatives (F) 500 / F 690 Linear relationship between SO2 concentration and SO2 concentration;

[0031] Figure 6 The fluorescence spectra at 500 nm show the reactions of coumarin benzothiazole salt derivatives with various interfering substances.

[0032] Figure 7The fluorescence spectra of coumarin benzothiazole salt derivatives at 690 nm in glycerol / PBS solutions of different viscosities are shown.

[0033] Figure 8 Mitochondrial localization diagram of coumarin benzothiazole salt derivative;

[0034] Figure 9 SO2 cell imaging image for coumarin benzothiazole salt derivatives;

[0035] Figure 10 This is a cell imaging image of a coumarin benzothiazole salt derivative under the influence of viscosity. Detailed Implementation

[0036] This invention provides a coumarin benzothiazole salt derivative having the structure shown in Formula I:

[0037]

[0038] Where X is a halogen.

[0039] In this invention, X is preferably F, Cl, Br or I, and more preferably I.

[0040] In this invention, the coumarin benzothiazole salt derivative preferably has the structure shown in Formula II:

[0041]

[0042] The present invention also provides a method for preparing the coumarin benzothiazole salt derivative described in the above technical solution, comprising the following steps:

[0043] The first condensation reaction was carried out by mixing 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde, (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide, cesium carbonate and an organic solvent to obtain the condensation product;

[0044] An addition reaction was carried out by mixing 2-methylbenzothiazole, haloethane, and an organic solvent to obtain the addition product;

[0045] The condensation product, addition product, piperidine, and organic solvent are mixed and subjected to a second condensation reaction to obtain the coumarin benzothiazole salt derivative.

[0046] Unless otherwise specified, all raw materials used in this invention are commercially available products in the field.

[0047] In this invention, 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde, (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide, cesium carbonate, and an organic solvent are mixed to carry out a first condensation reaction to obtain the condensation product.

[0048] In this invention, the molar ratio of 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde to (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide is preferably 1:1.1.

[0049] In this invention, the molar ratio of 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde to cesium carbonate is preferably 1:1.1.

[0050] In this invention, the organic solvent is preferably DMF.

[0051] In this invention, the temperature of the first condensation reaction is preferably room temperature, and the time is preferably 24 to 48 hours.

[0052] After the first condensation reaction is completed, the present invention preferably pours the obtained reaction solution into hydrochloric acid, stirs it at room temperature for 0.5 h, adds distilled water, filters it, then purifies the obtained liquid by column chromatography, and then dries it to obtain the condensation product.

[0053] In this invention, the eluent used for column chromatography purification is preferably a dichloromethane-petroleum ether mixture, and the volume ratio of dichloromethane to petroleum ether in the dichloromethane-petroleum ether mixture is preferably 1:5.

[0054] After obtaining the condensation product, the present invention mixes 2-methylbenzothiazole, haloethane and organic solvent to carry out an addition reaction to obtain the addition product.

[0055] In this invention, the molar ratio of 2-methylbenzothiazole to haloethane is preferably 1:1.5.

[0056] In this invention, the haloethane is preferably fluoroethane, chloroethane, bromoethane, or iodoethane.

[0057] In this invention, the addition reaction is preferably carried out under reflux conditions for a time of 2 to 5 hours.

[0058] In this invention, the organic solvent is preferably toluene.

[0059] After the addition reaction is completed, the present invention preferably cools the resulting system naturally to room temperature, and filters the precipitated solid to obtain the addition product.

[0060] After obtaining the addition product, the present invention mixes the condensation product, the addition product, piperidine and an organic solvent to carry out a second condensation reaction to obtain the coumarin benzothiazole salt derivative.

[0061] In this invention, the molar ratio of the condensation product to the addition product is preferably 1:1.2.

[0062] In this invention, the second condensation reaction is preferably carried out under reflux conditions for a time of 17 to 24 hours.

[0063] In this invention, piperidine is used as a catalyst. There is no special limitation on the amount of piperidine used in this invention, and any amount known to those skilled in the art can be used.

[0064] In this invention, the organic solvent is preferably anhydrous ethanol.

[0065] After the second condensation reaction is completed, the present invention preferably cools the obtained second condensation product to room temperature naturally, distills it under reduced pressure, removes the solvent, obtains the crude product, and then recrystallizes it with ethanol to obtain the coumarin benzothiazole salt derivative.

[0066] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in the detection of sulfur disulfide.

[0067] In this invention, the method for detecting sulfur disulfide is preferably ultraviolet light detection or fluorescence detection.

[0068] In this invention, the method for detecting sulfur disulfide is preferably qualitative or quantitative.

[0069] In this invention, the qualitative detection preferably includes the following steps:

[0070] The coumarin benzothiazole salt derivative was dissolved in DMSO to obtain a coumarin benzothiazole salt derivative solution. The coumarin benzothiazole salt derivative solution, the test solution, and PBS buffer solution were mixed and subjected to UV detection. If the UV absorption intensity at 540 nm decreased and the UV absorption intensity at 420 nm increased, it indicates that the test solution contains sulfur dioxide; or

[0071] The coumarin benzothiazole salt derivative was dissolved in DMSO to obtain a coumarin benzothiazole salt derivative solution. The coumarin benzothiazole salt derivative solution, the test solution, and PBS buffer solution were mixed for fluorescence detection. If the fluorescence intensity at 690 nm decreased and the fluorescence intensity at 500 nm increased, it indicates that the test solution contains sulfur dioxide.

[0072] In this invention, the sulfur dioxide in the test solution is in the form of SO3. 2- It exists in the form of.

[0073] In this invention, the test solution preferably also contains Cys, GSH, Hcy, and NO3. - AcO - S 2- CO3 2-Cl - I - ,ClO - H2O2, NO2 - and ClO4 - One or more of them.

[0074] In this invention, the pH value of the PBS buffer solution is preferably 7.4.

[0075] In this invention, the quantitative detection preferably includes the following steps:

[0076] The coumarin benzothiazole salt derivative was dissolved in DMSO to obtain a coumarin benzothiazole salt derivative solution. The coumarin benzothiazole salt derivative solution, the test solution, and PBS buffer solution were mixed and fluorescence detection was performed to obtain the fluorescence intensity of the test solution.

[0077] The concentration of sulfur dioxide in the test solution is obtained based on the fluorescence intensity of the test solution and a predetermined standard equation.

[0078] In this invention, the predetermined standard equation uses SO2 concentration as the abscissa and F as the ordinate. 500nm / F 690nm The vertical axis is denoted by .

[0079] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in viscosity detection.

[0080] In this invention, the method for detecting viscosity is preferably fluorescence detection.

[0081] In this invention, the method for detecting viscosity preferably includes the following steps:

[0082] The coumarin benzothiazole salt derivative was dissolved in DMSO to obtain a coumarin benzothiazole salt derivative solution. The coumarin benzothiazole salt derivative solution, the test solution, and PBS buffer solution were mixed for fluorescence detection. As the viscosity of the test solution increased, the fluorescence intensity at 690 nm increased.

[0083] This invention also provides an application for the simultaneous dual detection of sulfur disulfide and viscosity of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the above technical solutions.

[0084] The present invention also provides the application of the coumarin benzothiazole salt derivatives described in the above technical solutions or the coumarin benzothiazole salt derivatives prepared by the preparation method described in the above technical solutions in the preparation of reagents for simultaneous detection of sulfur disulfide and viscosity.

[0085] The present invention does not impose any special limitations on the preparation method, and any method known to those skilled in the art can be used.

[0086] To further illustrate the present invention, the methods provided by the present invention will be described in detail below with reference to examples, but these should not be construed as limiting the scope of protection of the present invention.

[0087] Example 1

[0088] Preparation and characterization of CMBT

[0089] The principle of the preparation method is shown in the following formula:

[0090]

[0091] At room temperature, 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde (5.58 g, 20.00 mmol), (1,3-dioxolane-2-yl)methyltriphenylbromide (9.42 g, 22.00 mmol), and cesium carbonate (7.17 g, 22.00 mmol) were dissolved in 50 mL of DMF and reacted at room temperature for 24 h. The reaction solution was poured into hydrochloric acid (2 M, 90 mL), stirred at room temperature for 0.5 h, and 200 mL of distilled water was added. The mixture was filtered and purified by column chromatography (dichloromethane / petroleum ether = 1 / 5, V / V) to give compound 1, a brownish-yellow solid, 2.72 g, yield 44.6%.

[0092] 2-Methylbenzothiazole (2.98 g, 2 mmol) and iodoethane (4.65 g, 3 mmol) were dissolved in 20 mL of toluene, heated under reflux for 2 h, cooled to room temperature, and a solid precipitated. The solid was filtered to give compound 2, 4.8 g of white solid, with a yield of 80%.

[0093] Compound 1 (0.61 g, 2.00 mmol) and compound 2 (0.67 g, 2.40 mmol) were added to 60 mL of anhydrous ethanol, followed by the addition of a catalytic amount of piperidine. The mixture was heated under reflux for 17 h, cooled to room temperature, and distilled under reduced pressure. The solvent was removed by rotary evaporation to give the crude product. Ethanol washings yielded the target compound CMBT, a dark green solid, 0.98 g, in a yield of 44.6%.

[0094] Hydrogen spectrum: 1H NMR (600MHz, DMSO-d6) δ8.37(d,J=8.2,1H),8.22(d,J=8.5Hz,1H),8.16(dd,J =14.6,11.1Hz,1H),8.02(dd,J=14.8,11.1Hz,1H),7.81(t,J=8.5Hz,1H),7.75 -7.64(m,4H),6.87(dd,J=9.3,2.5Hz,1H),6.61(d,J=2.4Hz,1H),4.79(q,J=7. 3Hz, 2H), 3.48 (q, J = 7.0Hz, 5H), 1.40 (t, J = 7.2Hz, 3H), 1.12 (t, J = 7.1Hz, 6H). ( Figure 1 ) 13 C NMR(101MHz,DMSO-d6)δ170.91,158.38,154.69,152.87,151.75,149.16,141.32,138.11,131.73,129.9 9,128.70,128.34,124.91,116.85,112.49,111.35,107.67,96.56,45.06,44.79,14.62,12.95,12.83.( Figure 2 )

[0095] Example 2

[0096] Prepare a PBS buffer solution with pH 7.4, a 2 mM CMBT DMSO solution, and a 2 mM SO2 aqueous solution. Add 10 μL of the CMBT DMSO solution to a cuvette containing 2 mL of PBS buffer solution. Detect the absorbance on a UV spectrophotometer. With the addition of sulfur dioxide (0–100 μM), the UV absorption intensity at 540 nm gradually decreases, while the UV absorption intensity at 420 nm gradually increases. The UV absorption visible spectrum is shown below. Figure 3 As shown.

[0097] Example 3

[0098] Prepare a PBS buffer solution with pH 7.4, a 2 mM CMBT DMSO solution, and a 2 mM SO2 aqueous solution. Add 10 μL of the CMBT DMSO solution to a fluorescence cuvette containing 2 mL of PBS buffer solution. Detect the fluorescence on a fluorescence spectrophotometer. With the addition of sulfur dioxide (0–100 μM), the fluorescence intensity at 690 nm gradually decreases, while the fluorescence intensity at 500 nm gradually increases. Figure 4 ); Plot SO2 concentration on the x-axis and F 500nm / F 690nmPlotting the graph on the ordinate yields the ratio of SO2 concentration to fluorescence intensity (F). 500nm / F 690nm The linear relationship between fluorescence intensity ratio (F) and fluorescence intensity ratio (F) 500nm / F 690nm The linear relationship between SO2 concentration and SO2 concentration is shown in the graph. Figure 5 As shown.

[0099] Example 4

[0100] Prepare a PBS buffer solution with pH 7.4, a 2 mM CMBT DMSO solution, and a 2 mM SO2 aqueous solution. Add 10 μL of the CMBT DMSO solution to a fluorescent cuvette containing 2 mL of PBS buffer solution, and then add 10 equivalents of the other analytes (Cys, GSH, Hcy, NO3-). - AcO - S 2- CO3 2- Cl - I - ClO - H2O2, NO2 - ClO4 - ) and SO3 2- The fluorescence intensity change at 500 nm was detected using a fluorescence spectrophotometer, as shown in the graph. Figure 6 As shown.

[0101] Example 5

[0102] Prepare a 2 mM CMBT solution in DMSO, and prepare glycerol / PBS solutions of different viscosities. Add 10 μL of the CMBT / DMSO solution to each of the glycerol / PBS solutions containing different viscosities in a fluorescence cuvette. Detect the fluorescence intensity changes at 690 nm using a fluorescence spectrophotometer. (See the graph below.) Figure 7 As shown.

[0103] Example 6

[0104] Prepare a PBS buffer solution with pH 7.4 and a 2 mM CMBT DMSO solution. Add 10 μL of the CMBT DMSO solution to a 2 mL PBS buffer solution. Add the probe solution to the HeLa cell culture medium to a concentration of 10 μM, and incubate with the HeLa cells at 37°C for 10 min. Observe the system under a fluorescence imager. Immediately afterwards, add 0.2 μM mitochondrial staining reagent to the prepared system and incubate at 37°C for 10 min. Observe the system under a fluorescence imager and calculate the colocalization rate of the obtained fluorescence images. The colocalization coefficient is 0.97. Figure 8 As shown.

[0105] Example 7

[0106] Prepare a PBS buffer solution with pH 7.4, a 2 mM CMBT DMSO solution, and a 2 mM SO2 aqueous solution. Add 10 μL of the CMBT DMSO solution to a 2 mL PBS buffer solution. Add the probe solution to the HeLa cell culture medium to achieve a concentration of 10 μM. After incubation for 10 min and 30 min, collect fluorescence signals from the red and blue channels, respectively. Laser confocal fluorescence imaging showed increased fluorescence in the blue channel and decreased fluorescence in the red channel, indicating that the probe can detect SO2 in mitochondria. Figure 9 As shown.

[0107] Example 8

[0108] Prepare a PBS buffer solution with pH 7.4 and a 2 mM CMBT DMSO solution. Add 10 μL of the CMBT DMSO solution to a 2 mL PBS buffer solution. Add the probe and nystatin solution to the HeLa cell culture medium to achieve a concentration of 10 μM. After incubation for 30 min, collect the fluorescence signal in the red channel. Laser confocal fluorescence imaging showed increased fluorescence in the red channel, indicating that the probe can detect viscosity in mitochondria. Figure 10 As shown.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A coumarin benzothiazole salt derivative having the structure shown in Formula I: Equation I, Where X is I.

2. The method for preparing the coumarin benzothiazole salt derivative according to claim 1, characterized in that, Includes the following steps: The first condensation reaction was carried out by mixing 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde, (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide, cesium carbonate and an organic solvent to obtain the condensation product; An addition reaction was carried out by mixing 2-methylbenzothiazole, a haloethane, and an organic solvent to obtain the addition product; the haloethane was iodoethane. The condensation product, addition product, piperidine, and organic solvent are mixed and subjected to a second condensation reaction to obtain the coumarin benzothiazole salt derivative.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 7-(N,N-diethylamino)-4-chlorocoumarin-3-carboxaldehyde to (1,3-dioxolane-2-yl)methyltriphenylphosphine bromide is 1:1.

1.

4. The preparation method according to claim 2, characterized in that, The molar ratio of 2-methylbenzothiazole to haloethane is 1:1.

5.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the condensation product to the addition product is 1:1.

2.

6. The preparation method according to claim 2, characterized in that, The first condensation reaction takes 24 to 48 hours.

7. The use of the coumarin benzothiazole salt derivative of claim 1 or the coumarin benzothiazole salt derivative prepared by any one of claims 2 to 6 in the detection of sulfur dioxide for non-diagnostic and / or therapeutic purposes.

8. The use of the coumarin benzothiazole salt derivative of claim 1 or the coumarin benzothiazole salt derivative prepared by any one of claims 2 to 6 in the detection of viscosity for non-diagnostic and / or therapeutic purposes.

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