Near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline and preparation method and application thereof

Abstract

The present application provides an application of a dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe reagent for preparing tumor diagnosis. It includes a fluorescent probe having the structural formula: the application of the fluorescent probe in the preparation of ·OH detection reagent. Its preparation method includes: 3-formyl-1-methylquinoline-1-ammonium iodide, 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl) azadiacyl) dimethyl propionate and sodium hydroxide react under a protective atmosphere to obtain a near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline. The application of the near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline is used to detect hydroxyl radicals in cells. The near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline provided in the present application can detect ·OH from a variety of reactive oxygen species alone, and has excellent sensitivity, selectivity and a faster start-up response to ·OH in the sample.

Description

Technical Field

[0001] The present application relates to the field of analytical chemistry, and in particular to a near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline, and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) play an important regulatory role in many physiological and pathological processes. Studies have shown that cellular oxidative stress is caused by an imbalance between the production and elimination of reactive oxygen species in cells. ·OH is the most active and toxic ROS. It can easily react with various organic matter in the body's tissues to cause tissue lipid peroxidation, nucleic acid breakage, protein and polysaccharide decomposition, thereby inducing tissue lesions and leading to various diseases such as gastric cancer and liver cancer. Therefore, monitoring ·OH in various cancer cells is crucial to understanding its biological effects and its role in tumor occurrence and development. However, due to its short lifespan and low concentration in cancer cells, the selective detection of endogenous ·OH is highly challenging.

[0003] Compared with traditional methods, the fluorescence method is not only simple and easy to operate, but also has the advantages of high sensitivity, fast spatial analysis speed, low biological damage, and effective capture and detection. The currently developed ·OH fluorescent probes show poor selectivity for other ROS, especially ONOO-, which is also highly oxidizing. Near-infrared emitting (NIR) fluorescent probes are more suitable for in vivo imaging applications. They have little photodamage, deep tissue penetration, and strong resistance to background autofluorescence interference. Therefore, it is urgent to develop fluorescent probes with near-infrared emission that can effectively distinguish ·OH from other ROS. Summary of the invention

[0004] The purpose of the present application is to provide a near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline and a preparation method and application thereof to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solutions:

[0006] A near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline, the structural formula of which is:

[0007]

[0008] The present application also provides a method for preparing the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe, comprising:

[0009] The first reaction is carried out between quinoline-3-carboxaldehyde and methyl iodide to obtain 3-formyl-1-methylquinoline-1-ammonium iodide;

[0010] The dimethyl 3,3'-((4-formyl-3-hydroxyphenyl)azepine diyl)dipropionate, ethyl acetoacetate and piperidine are subjected to a second reaction to obtain dimethyl 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl)azepine diyl)dipropionate;

[0011] The 3-formyl-1-methylquinoline-1-ammonium iodide, the 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl)azepine diacyl)dipropionic acid dimethyl ester and sodium hydroxide are subjected to a third reaction under a protective atmosphere to obtain the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe.

[0012] Preferably, the temperature of the first reaction is 50°C and the time is 24h;

[0013] Preferably, the solvent for the first reaction comprises anhydrous acetonitrile.

[0014] Preferably, the first reaction comprises: removing the solvent and then subjecting the solid to a first recrystallization;

[0015] Preferably, the first recrystallization solvent comprises n-hexane.

[0016] Preferably, the temperature of the second reaction is 45°C and the time is 10h;

[0017] Preferably, the solvent of the second reaction comprises anhydrous ethanol.

[0018] Preferably, the second reaction comprises: filtering to obtain a yellow solid, and then performing a second recrystallization of the yellow solid;

[0019] Preferably, the solvent of the second recrystallization comprises ethanol.

[0020] Preferably, the temperature of the third reaction is room temperature and the time is 24h;

[0021] Preferably, the solvent of the third reaction includes anhydrous ethanol and deionized water;

[0022] Preferably, the volume ratio of the anhydrous ethanol to the deionized water is 1:1.

[0023] Preferably, the third reaction comprises: removing the solvent and then purifying.

[0024] The present application also provides an application of the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe for detecting hydroxyl radicals in cells.

[0025] Preferably, the detection wavelength is 525 nm.

[0026] Compared with the prior art, the beneficial effects of this application include:

[0027] The near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline provided in this application is based on a coumarin-dihydroquinoline hybrid platform, has a highly specific response to ·OH, has no response to other active substances in the body, can detect ·OH from a variety of active oxygen substances, and has excellent sensitivity, selectivity and fast start-up response to ·OH in the sample; the probe has good response speed (7min), photostability, low cytotoxicity, low detection limit, low cost and other characteristics, and can be used for specific detection of ·OH in complex biological environments such as HeLa cells. The probe is suitable for related tumor research and biological applications, and has great application prospects in technical fields such as analytical chemistry and life sciences.

[0028] The preparation method of the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe provided in the present application is simple to operate and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0030] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe provided in the present application.

[0031] Figure 2 This is a fluorescence spectrum of ·OH detected by the dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe provided in the present application.

[0032] Figure 3 This is a fluorescence imaging diagram of endogenous ·OH in HeLa cells using a dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe provided in the present application. DETAILED DESCRIPTION

[0033] As used herein:

[0034] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0035] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0036] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0037] In these examples, parts and percentages are by mass unless otherwise indicated.

[0038] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0039] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0040] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] Example

[0042] This embodiment provides a near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline, and its structural formula is as follows:

[0043]

[0044] The preparation method is as follows:

[0045] 1. Synthesis of 3-formyl-1-methylquinoline-1-ammonium iodide

[0046] a. Add 1.00 g of quinoline-3-carboxaldehyde and 0.90 g of iodomethane to 10 mL of anhydrous acetonitrile solution, react at 50 ° C for 24 hours, and spin dry the solution;

[0047] b. Add n-hexane to the dried solid for recrystallization to precipitate a yellow solid, which was filtered and dried to give 1.71 g of 3-formyl-1-methylquinoline-1-ammonium iodide with a yield of 89.85%.

[0048] The reaction equation for this step is as follows:

[0049]

[0050] 2. Synthesis of dimethyl 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl)azepine diacyl)dipropionate

[0051] c. Add 500 mg of dimethyl 3,3'-((4-formyl-3-hydroxyphenyl)azepine diyl)dipropionate and 231 mg of ethyl acetoacetate to 10 mL of anhydrous ethanol, then add 2 drops of piperidine, and stir at 45 ° C for 10 hours;

[0052] d. The reaction solution was filtered to obtain a yellow solid. The crude product was purified by recrystallization from ethanol to obtain 412 mg of dimethyl 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl)azepine diacyl)dipropionate with a yield of 67.90%.

[0053] The reaction equation for this step is as follows:

[0054]

[0055] 3. Synthesis of dimethyl 3,3'-((3-(2-(3-formyl-1-methyl-1,4-dihydroquinolin-4-yl)acetyl)-2-oxo-2H-pyran-7-yl)azepine diacetyl)dipropionate

[0056] e. Under argon protection, 200 mg of dimethyl 3,3'-((3-acetyl-2-oxo-2H-pyran-7-yl)azepine diacyl) dipropionate and 159 mg of 3-formyl-1-methylquinoline-1-ammonium iodide were added to a mixed solvent of 5 mL of anhydrous ethanol and 5 mL of deionized water, and then 21 mg of sodium hydroxide was added and reacted at room temperature for 24 hours;

[0057] f. The reaction solution was spin dried and purified by column chromatography to obtain a red solid, namely 3,3'-((3-(2-(3-formyl-1-methyl-1,4-dihydroquinolin-4-yl)acetyl)-2-oxo-2H-pyran-7-yl)azepine)dimethyl dipropionate 32 mg, with a yield of 10.99%.

[0058] The reaction equation for this step is as follows:

[0059]

[0060] The obtained near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline has a hydrogen nuclear magnetic resonance spectrum as shown in Figure 1 shown.

[0061] In order to demonstrate the effect of the obtained dihydroquinoline-based near-infrared hydroxyl radical fluorescent probe, the following experiment was conducted:

[0062] 1. Application of fluorescent molecular probes in in vitro environmental detection of OH

[0063] Experiment on the spectral properties of open-type fluorescent molecular probes: The probe was dissolved in N,N-dimethylformamide (DMF) to prepare a probe solution with a concentration of 1mM, and a 10mM ·OH aqueous solution was prepared. The specific test method is: take 20μL of 1mM probe solution, 380μL of analytical grade DMF, the required amount of 10mM ·OH aqueous solution and the required amount of deionized water solution in a 2mL sample tube. All tests maintained the volume ratio of organic phase to aqueous phase at 1:9 (the total volume of each test sample is 2mL). For example, when the fluorescence intensity of the probe after the reaction with OH is required to be tested at a concentration of 200 μM, the sample preparation is as follows: 20 μL of 1 mM probe solution, 380 μL of analytical grade DMF, 1560 μL of deionized water and 40 μL of 10 mM OH aqueous solution are placed in a 2 mL sample tube, and after shaking at 37°C for 10 minutes, the fluorescence emission intensity can be measured at an excitation wavelength of 525 nm. Other test operations are similar to the above steps. The probe molecule has excellent sensitivity, selectivity and fast start-up response to OH, and is very suitable for imaging and analysis of endogenous OH in living cells.

[0064] The fluorescence spectrum of OH detected by the near-infrared hydroxyl radical fluorescent probe based on dihydroquinoline is shown in the figure below. Figure 2 shown.

[0065] 2. Fluorescence imaging analysis of endogenous OH in HeLa cancer cells

[0066] HeLa cells were subcultured into confocal dish cell culture medium and cultured under standard growth conditions for 24 hours. Then, an appropriate amount of probe (5 μM) was added and cultured under standard growth conditions for another 30 minutes. Then, the cells were photographed under a confocal fluorescence microscope and the red fluorescence channel was used for fluorescence imaging. Endogenous ·OH in HeLa cells was detected by fluorescence imaging. Figure 3 It can be seen that the near-infrared fluorescent probe of the present invention successfully realizes high-sensitivity fluorescence imaging analysis of endogenous ·OH in cancer cells, and has good application value in the fields of tumor research, analysis and detection.

[0067] Fluorescence probe imaging of HeLa cells. Fluorescence imaging of endogenous OH in HeLa cells. Figure 3 shown.

[0068] The application of the highly selective near-infrared fluorescent probe provided in the present application in the detection of hydroxyl radicals provides an extremely highly selective and near-infrared fluorescent probe for high-sensitivity detection of ·OH, which has great application value in the fields of tumor research, analytical detection, etc.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0070] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. An application of a dihydroquinoline near-infrared hydroxyl radical fluorescent probe in the preparation of a reagent for detecting hydroxyl radicals, characterized in that: The structural formula of the fluorescent probe is: 。 2. The use of a dihydroquinoline near-infrared hydroxyl radical fluorescent probe in the preparation of a reagent for detecting hydroxyl radicals according to claim 1, characterized in that: The detection wavelength is 525 nm.

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