A pyridone derivative and its preparation method and application

Abstract

The present invention provides a pyridone derivative and a preparation method and application thereof, which belong to the field of biological detection technology. The pyridone derivative provided by the present invention solves the problem that the singlet oxygen detection fluorescent probe in the prior art cannot simultaneously achieve tracer detection and quantitative detection, is susceptible to interference, and has insufficient detection specificity and sensitivity. The pyridone derivative provided by the present invention has high selectivity for detecting singlet oxygen and a single feature of recognition. The fluorescence change produced is ratiometric, with an excitation wavelength of 461 nm, an emission wavelength of 626 nm, and a Stokes shift of up to 165 nm, further giving the probe the characteristics of low background interference, little damage to biological samples, strong sample penetration, and high detection sensitivity. The pyridone derivative provided by the present invention has a rapid reaction time with singlet oxygen, and the ratiometric fluorescence signal change has a good linear relationship with the singlet oxygen concentration, and is suitable for dual detection of singlet oxygen by the naked eye and instrument.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a pyridone derivative and a preparation method and application thereof. Background Art

[0002] Singlet oxygen ( 1 O2) is an excited molecular oxygen (also known as triplet oxygen: 3 O2), its OO bond has the characteristics of both π bond and σ bond. 1 The energy of O2 is much higher than that of molecular oxygen, and its π system is highly active. 1 O2, as an extremely important reactive oxygen species (ROS), has attracted increasing attention from scientific researchers. 1 O2 is highly selective for the oxidation of electron-rich substances (such as drugs, unsaturated biomolecules and aromatic organic compounds). 1 O2 plays an important role in pathological and physiological processes. Several enzyme systems (e.g., lipoxygenases, peroxidases, and even eosinophil peroxidase) have been identified as 1 Biochemical sources of O2. On the one hand, 1 O2 has been proposed to be involved in cell signal transduction and induction of gene expression; on the other hand, 1 O2 can oxidize a range of biomolecules, such as DNA, proteins, and lipids, which can lead to degenerative diseases or damage malignant cells. 1 Sensitive detection and accurate quantification of O2 production, especially inside cells and tissues, is crucial for studying or monitoring the aforementioned therapies.

[0003] In addition, the important application of singlet oxygen in tumor photodynamic therapy has also attracted widespread attention. Photodynamic therapy (PDT) is a tumor treatment method based on the use of molecular oxygen in the tissue to produce reactive oxygen species that can kill diseased tissue cells after photosensitizer (PS) absorbs photon energy, thereby achieving therapeutic effects. It has been widely used in the treatment of various tumors and non-tumor diseases. 1 The production of O2 is of great significance for studying the killing effect of PDT on tumors. Real-time and quantitative analysis of the 1 The spatial distribution and kinetic characteristics of O2 production and its application in monitoring the disease process during PDT treatment will become an important research direction in the future. How to dynamically detect, accurately calculate, and adjust the O2 production in real time during treatment 1 O2 production has become a challenging problem that needs to be solved urgently in clinical applications and is currently at the forefront of international PDT research.

[0004] Chinese patent CN107099165A discloses a method for synthesizing a dye compound for detecting singlet oxygen. The probe exhibits a good response to singlet oxygen. This singlet oxygen probe can be used for singlet oxygen detection in environmental, food, and biomedical applications. It can also be used in molecular probe fabrication, research on singlet oxygen and disease phototherapy, and the development of singlet oxygen detection kits.

[0005] However, the singlet oxygen detection fluorescent probes in the existing technology cannot achieve tracer detection and quantitative detection at the same time, are easily interfered with, and have insufficient detection specificity and sensitivity. Summary of the Invention

[0006] The present invention aims to provide a pyridone derivative and a preparation method and application thereof, which can be used for tracer detection and quantitative detection of singlet oxygen, is less susceptible to interference, and has higher detection specificity and sensitivity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a pyridone derivative, the structure of which is shown in Formula 1:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned pyridone derivative, comprising the following steps:

[0011] S1, 1,2-anthracene dicarboxylic anhydride and n-butylamine are mixed and subjected to a first reflux reaction, and then the reaction product is extracted and purified to obtain an intermediate imine;

[0012] S2. The intermediate imine is prepared into an intermediate imine solution, which is then mixed with a catalyst, triphenylphosphine and alkyne to undergo a second reflux reaction. The reaction product is then extracted and purified to obtain the pyridone derivative.

[0013] Preferably, the molar ratio of the 1,2-anthracene dicarboxylic anhydride to n-butylamine is 2 to 3:10;

[0014] And / or, the first reflux reaction time is 7 to 9 hours.

[0015] Preferably, the solvent used in the intermediate imine solution contains triethylamine and / or tetrahydrofuran;

[0016] and / or, the concentration of the intermediate imine solution is 0.1 to 0.2 mol / L;

[0017] and / or, the catalyst comprises a palladium catalyst and / or a copper catalyst;

[0018] And / or, the weight ratio of the intermediate imine to the catalyst is 7.5:0.5-5.

[0019] Preferably, the palladium catalyst is bistriphenylphosphine palladium dichloride;

[0020] The copper catalyst is cuprous iodide;

[0021] and / or, the weight ratio of the intermediate imine to triphenylphosphine is 20 to 40:1;

[0022] And / or, the weight ratio of the intermediate imine to the alkyne is 1 to 2:1.

[0023] Preferably, the alkyne is hexadecyne.

[0024] Preferably, the second reflux reaction is carried out in an inert gas atmosphere;

[0025] And / or, the extraction uses dichloromethane as an extraction agent;

[0026] And / or, the purification in S2 is performed by column chromatography;

[0027] The column chromatography method uses petroleum ether and ethyl acetate as eluents;

[0028] The volume ratio of petroleum ether to ethyl acetate in the eluent is 1:4-6.

[0029] The present invention also provides the use of the pyridone derivative or the pyridone derivative prepared by the preparation method as a fluorescent probe.

[0030] The present invention also provides the use of the above-mentioned pyridone derivative or the pyridone derivative prepared by the above-mentioned preparation method in the preparation of a singlet oxygen detection product.

[0031] Preferably, the product is a quantitative detection product for singlet oxygen or a tracer detection product for singlet oxygen.

[0032] Beneficial effects of the present invention:

[0033] The pyridone derivatives provided by the present invention have a unique [2+4] cycloaddition reaction due to the pyridone recognition of singlet oxygen mechanism, thus avoiding other common active oxygen species in the body such as hydroxyl radicals (HO·), hypochlorite (ClO - ), peroxynitrite (ONOO - ), superoxide anion radical (O2 -· ), hydrogen peroxide (H2O2), biological thiols and other interferences, and has the characteristics of high selectivity in detecting singlet oxygen and single identification.

[0034] The fluorescence change produced by the pyridone derivatives provided by the present invention when detecting singlet oxygen is ratiometric, and the detection effect is superior to currently reported fluorescence turn-on or enhancement probes, the detection results of which are easily affected by photobleaching, imaging conditions, instrument stability, etc.

[0035] The pyridone derivative provided by the present invention has a large Stokes shift, with an excitation wavelength of 461 nm, an emission wavelength of 626 nm, and a Stokes shift of up to 165 nm, which further endows the probe with the characteristics of low background interference, little damage to biological samples, strong sample penetration, and high detection sensitivity.

[0036] The pyridone derivatives provided by the present invention react rapidly with singlet oxygen, and the ratiometric fluorescence signal changes in a good linear relationship with the singlet oxygen concentration. After reacting with singlet oxygen, the emission band of the probe gradually shifts from the orange region (626 nm) to the green region (542 nm). 542 / F 626 The fluorescence ratio of the irradiation time (0-25 min) showed a good linear relationship (R 2 =0.982) indicating that the probe can be used for ratiometric detection of singlet oxygen.

[0037] The pyridone derivative provided by the present invention exhibits a large ratiometric fluorescence change (84 nm) due to a change in the degree of intramolecular charge transfer (ICT) of the anthracenimide fluorescent core caused by a cycloaddition reaction of the pyridine group. The solution color changes from orange-red to yellow before and after reaction with singlet oxygen, and the fluorescence emission changes from orange (Ø = 0.35) to yellow (Ø = 0.65). The derivative is suitable for both visual and instrumental detection of singlet oxygen.

[0038] The preparation method of the pyridone derivative provided by the present invention has the advantages of mild conditions, high yield and stable method. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram showing the principle of the fluorescent probe of the present invention generating ratiometric fluorescence changes;

[0040] Figure 2 The synthetic route of the fluorescent probe of the present invention is as follows;

[0041] Figure 3 The figures show the changes in the ultraviolet spectrum of the fluorescent probe solution of the present invention after adding singlet oxygen, including: (a) the ultraviolet titration spectra of the fluorescent probe of the present invention to different concentrations of singlet oxygen; (b) the linear fitting curve of the fluorescent probe of the present invention and singlet oxygen;

[0042] Figure 4The graphs show the fluorescence spectrum changes after adding singlet oxygen to the fluorescent probe solution of the present invention, including: (a) fluorescence titration spectra of the fluorescent probe of the present invention to different concentrations of singlet oxygen; (b) linear fitting curves of the fluorescent probe of the present invention and singlet oxygen;

[0043] Figure 5 is the response of the fluorescent probe (10 μM) of the present invention to various reactive oxygen species, wherein: 1-20 are Na + , K + , Ca 2+ , Mg 2+ , Cu 2+ , Hg 2+ , Zn 2+ , Fe 3+ , Fe 2+ ,H2O2,·OH,O 2. - ,ROO·,NO·,TBHP,·O t Bu, Hcy, Cys, GSH and 1 O2. DETAILED DESCRIPTION

[0044] The present invention provides a pyridone derivative, the structure of which is shown in Formula 1:

[0045]

[0046] In the present invention, the pyridone derivative is based on pyridone as a recognition group. The probe itself emits orange fluorescence, and after responding to singlet oxygen, it emits yellow fluorescence, showing a ratiometric fluorescence change, such as Figure 1 shown.

[0047] The present invention also provides a method for preparing the above-mentioned pyridone derivative, comprising the following steps:

[0048] S1, 1,2-anthracene dicarboxylic anhydride and n-butylamine are mixed and subjected to a first reflux reaction, and then the reaction product is extracted and purified to obtain an intermediate imine;

[0049] S2. The intermediate imine is prepared into an intermediate imine solution, which is then mixed with a catalyst, triphenylphosphine and alkyne to undergo a second reflux reaction. The reaction product is then extracted and purified to obtain the pyridone derivative.

[0050] In the present invention, the 1,2-anthracene dicarboxylic anhydride is preferably used in the first reflux reaction in the form of a 1,2-anthracene dicarboxylic anhydride solution, and the 1,2-anthracene dicarboxylic anhydride solution preferably uses ethanol as a solvent. The molar ratio of the 1,2-anthracene dicarboxylic anhydride solution to n-butylamine is preferably 2 to 3:10, and more preferably 2.4 to 2.6:10.

[0051] In the present invention, the time of the first reflux reaction is preferably 7 to 9 hours; the product of the first reflux reaction is extracted and purified to obtain an intermediate imine; the extraction preferably uses dichloromethane as an extractant; after extraction, the organic phase is collected and purified; the purification in the present invention is preferably carried out using a silica gel column, and the solvent used in the purification is preferably dichloromethane; in the present invention, the intermediate imine obtained in S1 is a brown powder.

[0052] In the present invention, the solvent used in the intermediate imine solution preferably contains triethylamine and / or tetrahydrofuran, and more preferably contains both triethylamine and tetrahydrofuran. When the solvent contains both triethylamine and tetrahydrofuran, the volume ratio of triethylamine to tetrahydrofuran is preferably 1:0.5-2; the concentration of the intermediate imine solution is preferably 0.1-0.2 mol / L.

[0053] In the present invention, the intermediate imine solution is mixed with a catalyst, triphenylphosphine and an alkyne to carry out a second reflux reaction. The weight ratio of the intermediate imine to the catalyst is preferably 7.5:0.5-5. The catalyst preferably includes a palladium catalyst and / or a copper catalyst. It is further preferred that both a palladium catalyst and a copper catalyst are used in the reaction. The weight ratio of the palladium catalyst to the copper catalyst is preferably 5:2-4. The palladium catalyst of the present invention is preferably bistriphenylphosphine palladium dichloride; and the copper catalyst is preferably cuprous iodide.

[0054] In the present invention, the weight ratio of the intermediate imine and triphenylphosphine is preferably 20 to 40:1; the weight ratio of the intermediate imine and alkyne is preferably 1 to 2:1; the alkyne is preferably hexadecene; the second reflux reaction is preferably carried out in an inert gas atmosphere, and the inert gas is preferably argon.

[0055] In the present invention, the product of the second reflux reaction is extracted and purified to obtain the pyridone derivative, and the extraction preferably uses dichloromethane as the extractant; the purification preferably uses column chromatography; the column chromatography preferably uses petroleum ether and ethyl acetate as eluents; the volume ratio of petroleum ether to ethyl acetate in the eluent is preferably 1:4-6; in the present invention, the pyridone derivative obtained after the purification is a red solid.

[0056] The present invention also provides the use of the pyridone derivative or the pyridone derivative prepared by the preparation method as a fluorescent probe.

[0057] The present invention also provides the use of the above-mentioned pyridone derivative or the pyridone derivative prepared by the above-mentioned preparation method in the preparation of a singlet oxygen detection product; the product is preferably a quantitative detection product for singlet oxygen or a tracer detection product for singlet oxygen.

[0058] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] To a solution of 1,2-anthracenedicarboxylic anhydride (0.82 g, 2.5 mmol) in ethanol, add n-butylamine (0.99 mL, 10 mmol). Stir for 10 minutes and reflux for 8 hours. The mixture is extracted and concentrated with CHCl / HO multiple times. The organic phase is collected and purified on a silica gel column (DCM) to obtain the intermediate imine as a brown powder in a 64% yield.

[0061] Under argon protection, to a mixed solution of the intermediate tert-butyl-6-bromoanthracenimide (594 mg, 1.56 mmol) in Et3N (5 mL) and THF (5 mL) were added Pd(PPh3)2Cl2 (50 mg, 5 mol%), CuI (30 mg, 10 mol%), and PPh3 (20 mg, 5 mol%). Stirring was continued under argon protection for 5 min. Alkyne (453 mg, 2.03 mmol) was added at room temperature and stirred at reflux for 12 h.

[0062] After the reaction mixture reached room temperature, 20 mL of dichloromethane was added to dilute the mixture, washed with 50 mL of water, and then washed three times with saturated NH4Cl solution (50 mL) and Brine (50 mL), respectively. Finally, the obtained organic layer was dried over anhydrous Na2SO4, filtered, and then evaporated under reduced pressure. The product was separated by column chromatography (PE:EA=1:5v / v) to obtain the fluorescent probe as a red solid with a yield of 65%.

[0063] Product characterization data: 1 H NMR(400MHz,Chloroform-d)δ9.97(d,J=9.1Hz,1H),8.67(d,J=7.7Hz,2H),8.52(d,J=8.4Hz,1H),7.81-7.72(m,2H),7.71-7.58(m,2H),7.51( s,1H),7.45-7.33(m,4H),5.27(s,2H),4.25-4.20(m,2H),2.29(s,3H), 1.76(dt,J=15.1,7.5Hz,2H),1.63-1.37(m,2H),1.00(t,J=7.4Hz,3H). 13C NMR (101MHz, CDCl3) δ164.8,163.5,162.1,139.5,138.3,136.0,133.4,133.3,133.0,132.6,131.0,130.9,129.4,129.2,128.5,128.4 ,127.8,127.7,127.4,127.3,127.0,126.1,122.9,115.1,102.5,101.6,86.3,53.0,40.7,30.4,20.7,17.5,14.0.HRMS(ESI)Calcd.for C 35 H 29 N2O3[M+H] + :525.2187,found:525.2173

[0064] Synthesis route diagram Figure 2 shown.

[0065] Experimental example

[0066] Test the changes in the UV spectrum after adding singlet oxygen to the fluorescent probe solution:

[0067] UV spectra were measured in a phosphate buffer solution-dimethyl sulfoxide system (10 mM PBS, pH 7.4, DMSO = 5%, V / V). A fluorescent probe (10 μM) was prepared in a 5 mL cuvette, and tetraphenylporphyrin (10 μM) was added. The mixed system was shaken evenly and allowed to stand at 37°C for 10 minutes before UV data was measured. Singlet oxygen generated by irradiating the photosensitizer tetraphenylporphyrin was used as the source of singlet oxygen in the experiment. The results are shown in Figure 2. Figure 3 shown.

[0068] from Figure 3 It can be clearly seen that the maximum absorption wavelength of the probe itself is at 487nm. After adding singlet oxygen, the maximum absorption wavelength blue-shifts to 461nm. 487 / F 461 The maximum absorption ratio of the irradiation time (0-25 min) showed a good linear relationship (R 2 =0.997), indicating that the probe can be used for ratiometric detection of singlet oxygen.

[0069] Test the fluorescence spectrum changes after adding singlet oxygen to the fluorescent probe solution:

[0070] Fluorescence spectra were measured in a phosphate buffered saline-dimethyl sulfoxide (DMSO) system (10 mM PBS, pH 7.4, 5% DMSO, v / v). The fluorescent probe (10 μM) was prepared in a 5 mL cuvette, and tetraphenylporphyrin (10 μM) was added. The mixed system was shaken and allowed to stand at 37°C for 10 minutes before measuring fluorescence changes. Singlet oxygen, generated by irradiating the photosensitizer tetraphenylporphyrin, served as the source of singlet oxygen in the experiment. The excitation wavelength was set to 475 nm, and the fluorescence emission spectrum was collected over a wavelength range of 500 to 750 nm. The slit width for both excitation and emission was set to 5.0 nm.

[0071] The results are as follows Figure 4 As shown, from Figure 4 It can be clearly seen that the maximum emission wavelength of the probe itself is at 626 nm. After adding singlet oxygen, the maximum emission wavelength blue-shifts to 542 nm. 542 / F 626 The fluorescence ratio of the irradiation time (0-25 min) showed a good linear relationship (R 2 =0.982) indicating that the probe can be used for ratiometric detection of singlet oxygen.

[0072] Testing the selectivity of fluorescent probes:

[0073] The selectivity of the fluorescent probe was measured in a phosphate buffer solution-dimethyl sulfoxide system (10 mM PBS, pH 7.4, DMSO = 5%, V / V). Multiple parallel samples (10 μM) were prepared in 5 mL cuvettes and then different analytes (analytes were: 1-20: Na + , K + , Ca 2+ , Mg 2+ , Cu 2+ , Hg 2+ , Zn 2+ , Fe 3+ , Fe 2+ ,H2O2,·OH,O 2. - ,ROO·,NO·,TBHP,·O t Bu, Hcy, Cys, GSH and 1 O2). The excitation wavelength was set to 475 nm, the fluorescence emission spectrum was collected in the wavelength range of 500-750 nm, and the slit widths for both excitation and emission were set to 5.0 nm. Figure 5 shown.

[0074] from Figure 5 It can be clearly seen that only when singlet oxygen is added can probe F be induced 542 / F 626The fluorescence ratio of the probe changes significantly, while the influence of other analytes is almost negligible. Experiments have shown that the probe has high selectivity for singlet oxygen, which is conducive to the specific detection of singlet oxygen.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A pyridone derivative, characterized in that The structure of the pyridone derivative is shown in Formula 1: Formula 1.

2. The method for preparing the pyridone derivative according to claim 1, characterized in that: The following steps are involved: S1, 1,2-anthracene dicarboxylic anhydride and n-butylamine are mixed and subjected to a first reflux reaction, and then the reaction product is extracted and purified to obtain an intermediate imine; S2. The intermediate imine is prepared into an intermediate imine solution, which is then mixed with a catalyst, triphenylphosphine and alkyne, and then the reaction product is subjected to a second reflux reaction, extracted, and purified to obtain the pyridone derivative.

3. The preparation method according to claim 2, characterized in that The molar ratio of the 1,2-anthracene dicarboxylic anhydride to n-butylamine is 2 to 3:10; And / or, the first reflux reaction time is 7 to 9 hours.

4. The preparation method according to claim 2, characterized in that The solvent used in the intermediate imine solution contains triethylamine and tetrahydrofuran; and / or, the concentration of the intermediate imine solution is 0.1-0.2 mol / L; And / or, the catalyst is selected from palladium catalysts and copper catalysts; And / or, the weight ratio of the intermediate imine to the catalyst is 7.5:0.5~5.

5. The preparation method according to claim 4, characterized in that The palladium catalyst is bistriphenylphosphine palladium dichloride; The copper catalyst is cuprous iodide; and / or, the weight ratio of the intermediate imine to triphenylphosphine is 20-40:1; And / or, the weight ratio of the intermediate imine to the alkyne is 1-2:

1.

6. The preparation method according to claim 2 or 5, characterized in that The structural formula of the alkyne is as follows: 。 7. The preparation method according to claim 2, characterized in that The second reflux reaction is carried out in an inert gas atmosphere; And / or, the extraction uses dichloromethane as an extraction agent; And / or, the purification in S2 is performed by column chromatography; The column chromatography method uses petroleum ether and ethyl acetate as eluents; The volume ratio of petroleum ether to ethyl acetate in the eluent is 1:4-6.

8. Use of the pyridone derivative according to claim 1 or the pyridone derivative prepared by the preparation method according to any one of claims 2 to 7 in the preparation of a fluorescent probe.

9. Use of the pyridone derivative according to claim 1 or the pyridone derivative prepared by the preparation method according to any one of claims 2 to 7 in the preparation of a singlet oxygen detection product.

10. The use according to claim 9, characterized in that The product is a quantitative detection product for singlet oxygen or a tracer detection product for singlet oxygen.

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