Benzoxazine derivative as well as preparation method, application and detection method thereof

Abstract

The invention belongs to the technical field of chemical detection, and particularly relates to a benzoxazine derivative as well as a preparation method, application and a detection method thereof. The benzoxazine derivative provided by the invention is used as a fluorescent probe, the benzsulfamide group of the benzoxazine derivative can efficiently target an endoplasmic reticulum, dynamic rapid detection of oxidation reduction in the endoplasmic reticulum in a living body is realized for the first time, and the benzoxazine derivative is high in sensitivity, capable of performing specific Mechal addition with GSH, excellent in selectivity, good in biocompatibility, small in toxicity and low in cost. The invention further provides a method for detecting the redox dynamic state in the endoplasmic reticulum of the living body. The benzoxazine derivative provided by the invention selectively responds to environmental changes and is quick in response, so that the detection method provided by the invention has the advantage of real-time in-situ monitoring, is convenient and quick to operate, can be realized only by virtue of an ultraviolet and fluorescence spectrophotometer, and is low in equipment dependence degree.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and specifically relates to a benzoxazine derivative and a preparation method, application and detection method thereof. Background Art

[0002] Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and also acts as an antioxidant. Fluctuations in its levels are closely related to a variety of diseases and cell functions. GSH maintains intracellular redox homeostasis mainly by removing cytotoxic reactive oxygen species (ROS) generated by mitochondrial respiration. Therefore, GSH plays a vital role in maintaining redox homeostasis in biological systems and other physiological and pathological processes.

[0003] Intracellular redox reactions mainly occur in some major organelles, such as mitochondria, endoplasmic reticulum and Golgi apparatus. Among them, the endoplasmic reticulum has the strongest basic redox capacity and participates in a variety of physiological processes. Various external factors can induce endoplasmic reticulum stress, causing cells to produce various bioactive molecules, thereby leading to changes in cell physiological activity and causing cancer and other diseases. Monitoring the changes in active species in the endoplasmic reticulum has a great driving force in conquering certain metabolic diseases and cancers.

[0004] Fluorescence probe method is an important optical analysis method. Its mechanism of action is mainly that the molecular recognition part binds or reacts with the target to be detected, thereby causing the fluorescence performance of the system to change, thereby achieving the purpose of detection. As an important tool of fluorescence analysis, fluorescent probe has the characteristics of high sensitivity and low toxicity, and is widely used in many fields such as reactive oxygen detection.

[0005] However, the existing fluorescent probe method is mainly aimed at static detection and cannot achieve dynamic detection of redox in the endoplasmic reticulum of living organisms. Summary of the invention

[0006] The object of the present invention is to provide a benzoxazine derivative and a preparation method, application and detection method thereof. The benzoxazine derivative provided by the present invention can be used as a fluorescent probe to realize dynamic detection of redox in the endoplasmic reticulum of living organisms.

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

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

[0009]

[0010] The present invention also provides a method for preparing the benzoxazine derivatives described in the above scheme, comprising the following steps:

[0011] (1) mixing 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one, selenium dioxide and an organic solvent for oxidation reaction to obtain compound 1;

[0012]

[0013] (2) mixing the compound 1, cyanoacetic acid, piperidine and an organic solvent to carry out a first condensation reaction to obtain a compound 2;

[0014]

[0015] (3) The compound 2, N-(2-aminoethyl)-4-methylbenzenesulfonamide, N,N-diisopropylethylamine, N,N-dimethylformamide and O-benzotriazole-tetramethyluronium hexafluorophosphate are mixed for a second condensation reaction to obtain the benzoxazine derivative.

[0016] Preferably, the molar ratio of 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one to selenium dioxide is 1:1 to 1.5.

[0017] Preferably, the molar ratio of the compound 1 to cyanoacetic acid is 1:1-1.5; the molar ratio of the compound 1 to piperidine is 1:0.1-0.2.

[0018] Preferably, the molar ratio of the compound 2 to N-(2-aminoethyl)-4-methylbenzenesulfonamide is 1:1-1.5; the molar ratio of the compound 2 to N,N-diisopropylethylamine is 1:1-1.2; the molar ratio of the compound 2 to O-benzotriazole-tetramethyluronium hexafluorophosphate is 1:1-1.2; and the volume ratio of N,N-diisopropylethylamine to N,N-dimethylformamide is 1:25-30.

[0019] The present invention also provides the use of the benzoxazine derivatives described in the above scheme or the benzoxazine derivatives obtained by the preparation method described in the above scheme in the field of redox detection for non-diagnostic and non-therapeutic purposes.

[0020] The present invention also provides a method for detecting the redox dynamics in the endoplasmic reticulum of a living being, comprising the following steps:

[0021] A buffer solution containing a benzoxazine derivative was added to a culture medium containing HeLa cells, and then glutathione ethyl ester and N-ethylmaleimide were added to the culture medium in sequence, and changes in the fluorescence intensity of the red channel and the yellow channel were observed using a fluorescence confocal microscope.

[0022] Preferably, the preparation method of the buffer solution containing benzoxazine derivatives is: mixing a dimethyl sulfoxide solution containing benzoxazine derivatives and a buffer solution; the volume ratio of the dimethyl sulfoxide solution containing benzoxazine derivatives to the buffer solution is 1:100-400; the concentration of the dimethyl sulfoxide solution containing benzoxazine derivatives is 1-2 mmol / L; the buffer solution includes a phosphate buffer solution and acetonitrile; the volume ratio of the phosphate buffer solution to acetonitrile in the buffer solution is 1-4:1.

[0023] Preferably, the concentration of glutathione ethyl ester in the observation mixed solution is 0 to 20 mmol / L; the concentration of N-ethylmaleimide in the observation mixed solution is 0 to 20 mmol / L.

[0024] Preferably, the N-ethylmaleimide is an N-ethylmaleimide acetonitrile solution; the concentration of the N-ethylmaleimide acetonitrile solution is 1-2 mol / L.

[0025] The present invention provides a benzoxazine derivative. The benzoxazine derivative provided by the present invention is used as a fluorescent probe, and its benzenesulfonamide group can efficiently target the endoplasmic reticulum, realizing for the first time the dynamic rapid detection of redox in the endoplasmic reticulum in living organisms, with high sensitivity, specific Mecheal addition with GSH, excellent selectivity, good biocompatibility, low toxicity and low cost. When glutathione ethyl ester (GSH-OEt) is added, the ultraviolet absorption spectrum shows that the absorption at 445nm is enhanced, the absorption at 565nm is weakened, and the solution changes from red to light yellow; the fluorescence emission spectrum shows that the fluorescence at 560nm is enhanced, the fluorescence at 610nm is reduced, and the fluorescence of the solution changes from orange-red to yellow (such as Fig.15 When N-ethylmaleimide (NEM) was added, the UV absorption spectrum showed that the absorption at 445 nm decreased, and the absorption at 565 nm increased; the fluorescence emission spectrum showed that the fluorescence at 560 nm decreased, and the fluorescence at 610 nm increased.

[0026] The present invention also provides a method for preparing the benzoxazine derivatives described in the above scheme. The preparation method provided by the present invention has simple steps, convenient operation and good feasibility.

[0027] The present invention also provides the use of the benzoxazine derivatives described in the above scheme or the benzoxazine derivatives obtained by the preparation method described in the above scheme in the field of redox detection. The benzoxazine derivatives provided by the present invention selectively respond to environmental changes and respond quickly, and are suitable for use in the field of redox detection, especially for dynamic detection of redox in the endoplasmic reticulum of living organisms.

[0028] The present invention also provides a method for detecting the redox dynamics in the endoplasmic reticulum of a living body. The benzoxazine derivatives provided by the present invention selectively respond to environmental changes and respond quickly, so that the detection method provided by the present invention has the advantages of real-time in-situ monitoring, convenient and fast operation, and can be realized with the help of ultraviolet and fluorescence spectrometers, with low equipment dependence. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is the H NMR spectrum (600 MHz, DMSO-d6) of the benzoxazine derivative prepared in Example 1;

[0031] Figure 2 This is the NMR carbon spectrum (151 MHz, DMSO-d6) of the benzoxazine derivative prepared in Example 1;

[0032] Figure 3 It is the UV-visible absorption spectrum of the benzoxazine derivatives and GSH (0-20mmol / L);

[0033] Figure 4 It is the UV-visible absorption spectrum of the reaction product of benzoxazine derivative and GSH with N-ethylmaleimide (0-20mmol / L);

[0034] Figure 5 This is the fluorescence emission diagram of benzoxazine derivatives reacting with GSH (0-20 mmol / L) at 560 nm;

[0035] Figure 6 This is the fluorescence emission diagram of benzoxazine derivatives reacting with GSH (0-20 mmol / L) at 610 nm;

[0036] Figure 7 It is the fluorescence emission diagram of the reaction product of benzoxazine derivative and GSH with N-ethylmaleimide (0-20mmol / L) at 560nm;

[0037] Figure 8 It is the fluorescence emission diagram of the reaction product of benzoxazine derivative and GSH with N-ethylmaleimide (0-20mmol / L) at 610nm;

[0038] Fig. 9It is a benzoxazine derivative with Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - , GSH fluorescence intensity ratio (F 560nm / F 610nm ) Selective interference histogram;

[0039] Fig.10 It is a benzoxazine derivative in Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - The fluorescence intensity ratio of the response effect to GSH under the influence of 560nm / F 610nm ) Selective interference histogram;

[0040] Fig.11 This is the reversible cycle diagram of benzoxazine derivatives under the repeated action of GSH and N-ethylmaleimide;

[0041] Fig.12 is the co-localization map of benzoxazine derivatives and ER-Green;

[0042] Fig.13 This is a cell imaging diagram of a benzoxazine derivative after adding glutathione ethyl ester (GSH-OEt) to the cell and then adding N-ethylmaleimide;

[0043] Fig.14 The redox diagram of benzoxazine derivatives in zebrafish after addition of glutathione ethyl ester (GSH-OEt) and N-ethylmaleimide;

[0044] Fig.15 Schematic diagram of the detection principle of benzoxazine derivatives. DETAILED DESCRIPTION

[0045] The present invention provides a benzoxazine derivative, the structure of which is shown in Formula I:

[0046]

[0047] The benzoxazine derivative provided by the present invention has a Chinese name of (E)-2-cyano-3-(7-(diethylamino)-2-oxy-2H-benzo[b][1,4]oxazin-3-yl)-N-(2-((4-methylphenyl)sulfonamido)ethyl)acrylamide and an English name of (E)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)-N-(2-((4-methylphenyl)sulfonamido)ethyl)acrylamide, referred to as BSOA.

[0048] The present invention also provides a method for preparing the benzoxazine derivatives described in the above scheme, comprising the following steps:

[0049] (1) mixing 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one, selenium dioxide and an organic solvent for oxidation reaction to obtain compound 1;

[0050]

[0051] (2) mixing the compound 1, cyanoacetic acid, piperidine and an organic solvent to carry out a first condensation reaction to obtain a compound 2;

[0052]

[0053] (3) The compound 2, N-(2-aminoethyl)-4-methylbenzenesulfonamide, N,N-diisopropylethylamine, N,N-dimethylformamide and O-benzotriazole-tetramethyluronium hexafluorophosphate are mixed for a second condensation reaction to obtain the benzoxazine derivative.

[0054] In the present invention, 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one, selenium dioxide and an organic solvent (referred to as the first organic solvent) are mixed (referred to as the first mixture) for oxidation reaction to obtain compound 1. In the present invention, the molar ratio of the 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one to selenium dioxide is preferably 1:1 to 1.5, specifically 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0055] In the present invention, the first organic solvent preferably includes 1,4-dioxane; the mass ratio of the 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one to the first organic solvent is preferably 1:1 to 30, specifically 1:1, 1:5, 1:10, 1:15, 1:20, 1:25 or 1:30.

[0056] In the present invention, the temperature of the first mixing is preferably room temperature.

[0057] In the present invention, the temperature of the oxidation reaction is preferably 120-180°C, specifically 120°C, 140°C, 150°C, 160°C or 180°C, and the insulation time is preferably 20-28h, specifically 20h, 22h, 24h, 26h or 28h.

[0058] In the present invention, after the oxidation reaction, the obtained oxidation reaction system is preferably cooled, desolventized, purified and recrystallized in sequence.

[0059] In the present invention, the final temperature of the cooling is preferably room temperature.

[0060] In the present invention, the solvent removal is preferably performed by rotary evaporation; and the equipment for rotary evaporation is preferably a rotary evaporator.

[0061] In the present invention, the purification is preferably separation by chromatographic column; the eluent used for separation by chromatographic column is preferably dichloromethane.

[0062] In the present invention, the reagent used for the recrystallization is preferably ethyl acetate.

[0063] After obtaining compound 1, the present invention mixes compound 1, cyanoacetic acid, piperidine and an organic solvent (referred to as the second organic solvent) (referred to as the second mixture) to carry out a first condensation reaction to obtain compound 2. In the present invention, the molar ratio of compound 1 to cyanoacetic acid is preferably 1:1 to 1.5, specifically 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0064] In the present invention, the molar ratio of the compound 1 to piperidine is preferably 1:0.1 to 1:0.2, specifically 1:0.1, 1:0.13, 1:0.16 or 1:0.2.

[0065] In the present invention, the second organic solvent preferably includes one or more of dichloromethane, methanol, ethanol, tetrahydrofuran and N,N-dimethylformamide, more preferably dichloromethane; the molar ratio of the compound 1 to the second organic solvent is preferably 1:130 to 1:150, specifically 1:130, 1:140 or 1:150.

[0066] In the present invention, the temperature of the second mixing is preferably room temperature.

[0067] In the present invention, the temperature of the first condensation reaction is preferably room temperature, and the reaction time is preferably 3 to 8 hours, specifically 3 hours, 5 hours, 7 hours or 8 hours; the first condensation reaction is preferably carried out under stirring conditions.

[0068] In the present invention, after the first condensation reaction, the method preferably further comprises distilling the obtained condensation reaction system under reduced pressure, removing the solvent and purifying the obtained condensation reaction system.

[0069] In the present invention, the temperature of the reduced pressure distillation is preferably 35 to 45°C, specifically 37°C, 40°C or 43°C, the vacuum degree is preferably -0.07 to -0.10MPa, specifically -0.08MPa or -0.09MPa, and the fraction collection temperature is preferably 0 to -10°C, specifically -1°C, -3°C, -5°C, -7°C or -9°C.

[0070] In the present invention, the solvent removal is preferably performed by rotary evaporation.

[0071] In the present invention, the purification is preferably chromatographic column separation; the eluent used for the chromatographic column separation is preferably dichloromethane and methanol; the volume ratio of dichloromethane to methanol is preferably 10:1.

[0072] After obtaining compound 2, the present invention mixes the compound 2, N-(2-aminoethyl)-4-methylbenzenesulfonamide, N,N-diisopropylethylamine, N,N-dimethylformamide and O-benzotriazole-tetramethyluronium hexafluorophosphate (referred to as the third mixing) for a second condensation reaction to obtain the benzoxazine derivative. In the present invention, the molar ratio of the compound 2 to N-(2-aminoethyl)-4-methylbenzenesulfonamide is preferably 1:1 to 1.5, specifically 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0073] In the present invention, the molar ratio of the compound 2 to N,N-diisopropylethylamine is preferably 1:1-1.2, specifically 1:1.05, 1:1.1, 1:1.15 or 1:1.2.

[0074] In the present invention, the molar ratio of the compound 2 to O-benzotriazole-tetramethyluronium hexafluorophosphate is preferably 1:1-1.2, specifically 1:1.05, 1:1.1, 1:1.15 or 1:1.2.

[0075] In the present invention, the N,N-dimethylformamide is preferably super dry N,N-dimethylformamide; the volume ratio of the N,N-diisopropylethylamine to N,N-dimethylformamide is preferably 1:25 to 30, specifically 1:25, 1:26, 1:27, 1:28, 1:29 or 1:30.

[0076] In the present invention, the third mixing is preferably: compound 2, N,N-diisopropylethylamine, O-benzotriazole-tetramethyluronium hexafluorophosphate and part of N,N-dimethylformamide are premixed to obtain a first premix, N-(2-aminoethyl)-4-methylbenzenesulfonamide and the remaining part of N,N-dimethylformamide are premixed to obtain a second premix, and then the first premix and the second premix are mixed.

[0077] In the present invention, the mass ratio of the part of N,N-dimethylformamide to the remaining part of N,N-dimethylformamide is preferably 25 to 30:1, specifically 25:1, 26:1, 27:1, 28:1, 29:1 or 30:1.

[0078] In the present invention, the third mixing is preferably carried out in a protective atmosphere; the protective atmosphere is preferably argon; the temperature of the third mixing is preferably -5 to 5°C, specifically -5°C, -3°C, -1°C, 0°C, 1°C, 3°C or 5°C, and the mixing time is preferably 0.5 to 2h, specifically 0.5h, 1h, 1.5h or 2h; the third mixing is preferably stirring mixing.

[0079] In the present invention, the temperature of the second condensation reaction is preferably room temperature, and the reaction time is preferably 8 to 16 hours, specifically 8 hours, 10 hours, 12 hours, 14 hours or 16 hours; the second condensation reaction is preferably carried out under stirring conditions.

[0080] In the present invention, after the second condensation reaction, the method preferably further comprises extracting, removing solvent and purifying the obtained condensation reaction liquid in sequence.

[0081] In the present invention, the extraction is preferably performed by pouring the condensation reaction liquid into ice water and then extracting with dichloromethane; the number of extractions is preferably 3 or more.

[0082] In the present invention, the solvent removal is preferably performed by rotary evaporation; and the equipment for rotary evaporation is preferably a rotary evaporator.

[0083] In the present invention, the purification is preferably silica gel column chromatography; the eluent used in the silica gel column chromatography preferably includes dichloromethane and methanol; the volume ratio of dichloromethane to methanol is preferably 15:1.

[0084] The present invention also provides the use of the benzoxazine derivatives described in the above scheme or the benzoxazine derivatives obtained by the preparation method described in the above scheme in the field of redox detection for non-diagnostic and non-therapeutic purposes.

[0085] The benzoxazine derivatives provided by the present invention selectively respond to environmental changes and respond quickly, and are suitable for use in the field of redox detection, and are particularly suitable for dynamic detection of redox in the endoplasmic reticulum of living organisms.

[0086] The present invention also provides a method for detecting the redox dynamics in the endoplasmic reticulum of a living being, comprising the following steps:

[0087] A buffer solution containing a benzoxazine derivative was added to a culture medium containing HeLa cells, and then glutathione ethyl ester and N-ethylmaleimide were added to the culture medium in sequence to obtain an observation mixed solution, and changes in the fluorescence intensity of the red channel and the yellow channel were observed using a fluorescence confocal microscope.

[0088] In the present invention, the method for preparing the buffer solution (probe solution) containing benzoxazine derivatives is preferably: mixing a dimethyl sulfoxide solution containing benzoxazine derivatives and a buffer solution.

[0089] In the present invention, the concentration of the dimethyl sulfoxide solution containing the benzoxazine derivative is preferably 1-2 mmol, specifically 1 mmol / L, 1.4 mmol / L, 1.7 mmol / L or 2 mmol / L.

[0090] In the present invention, the buffer solution preferably includes a phosphate buffer solution and acetonitrile; the phosphate in the phosphate buffer solution preferably includes disodium hydrogen phosphate and sodium dihydrogen phosphate; the pH value of the phosphate buffer solution is preferably 7.4; the volume ratio of the phosphate buffer solution to acetonitrile in the buffer solution is preferably 1 to 4:1, specifically 1:1, 2:1, 7:3, 3:1 or 4:1.

[0091] In the present invention, the volume ratio of the dimethyl sulfoxide solution containing the benzoxazine derivative to the buffer solution is preferably 1:100-400, specifically 1:400, 1:300, 1:200, 1:150 or 1:100.

[0092] In the present invention, the glutathione ethyl ester is preferably an aqueous solution of glutathione ethyl ester; the concentration of the aqueous solution of glutathione ethyl ester is preferably 1-2 mol / L, specifically 1 mol / L, 1.4 mol / L, 1.7 mol / L or 2 mol / L.

[0093] In the present invention, the concentration of glutathione ethyl ester in the observation mixed solution is preferably 0-20mmol / L, specifically 1mmol / L, 3mmol / L, 5mmol / L, 7mmol / L, 9mmol / L, 12mmol / L, 15mmol / L, 17mmol / L or 20mmol / L.

[0094] In the present invention, the N-ethylmaleimide is preferably an N-ethylmaleimide acetonitrile solution; the concentration of the N-ethylmaleimide acetonitrile solution is preferably 1-2 mol / L, specifically 1 mol / L, 1.4 mol / L, 1.7 mol / L or 2 mol / L.

[0095] In the present invention, the concentration of N-ethylmaleimide in the observation mixed solution is preferably 0-20 mmol / L, specifically 1 mmol / L, 3 mmol / L, 5 mmol / L, 7 mmol / L, 9 mmol / L, 12 mmol / L, 15 mmol / L, 17 mmol / L or 20 mmol / L.

[0096] The benzoxazine derivatives provided by the present invention selectively respond to environmental changes and respond quickly, so that the detection method provided by the present invention has the advantage of real-time in-situ monitoring, is easy and quick to operate, can be achieved with the help of ultraviolet and fluorescence spectrometers, and has low equipment dependence.

[0097] In order to further illustrate the present invention, the scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0098] Example 1

[0099] Preparation and characterization of BSOA

[0100] At room temperature, 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazine-2-one (1.16 g, 5 mmol) and selenium dioxide (0.67 g, 6 mmol) were dissolved in 1,4-dioxane (25 mL). The reaction mixture was heated at 150 ° C for 24 h. After the reaction solution was cooled to room temperature, the solvent was evaporated using a rotary evaporator to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane as eluent), and then recrystallized from ethyl acetate to obtain compound 1, a black-red solid 0.8 g, yield: 65%.

[0101] At room temperature, compound 1 (0.74 g, 3 mmol), cyanoacetic acid (0.26 g, 3 mmol) and piperidine (25 mg, 0.3 mmol) were dissolved in 25 mL of dichloromethane and stirred at room temperature for 5 h. After the reaction was completed, the mixture was distilled under reduced pressure in vacuum, the solvent was removed by rotation, and the crude product was purified by column chromatography (eluent: dichloromethane / methanol, V / V=10:1) to obtain compound 2 (0.66 g) with a yield of 70%.

[0102] Compound BSOA: Add 50 mL of ultra-dry N,N-dimethylformamide to a 100 mL two-necked flask, then add compound 2 (0.63 g, 2 mmol), N,N-diisopropylethylamine (1.37 g, 2.4 mmol) and O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (0.91 g, 2.4 mmol). Stir at 0 ° C for 1 h under argon protection. Dissolve N-(2-aminoethyl)-4-methylbenzenesulfonamide (0.43 g, 2 mmol) in 2 mL of ultra-dry N,N-dimethylformamide and then add it to the flask, and continue to stir the mixture at room temperature for 12 h. Subsequently, pour the reaction solution into 100 mL of ice water and extract it with dichloromethane (3×50 mL). The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol, V / V=15:1) to obtain 0.41 g of BSOA solid. The yield was 40%.

[0103] The H NMR spectrum of the product is shown in Figure 1 The hydrogen spectrum data are as follows: 1 HNMR (600MHz, DMSO-d6) δ8.36(t,J=5.8Hz,1H),8.13(s,1H),7.68(d,J=7.5Hz,3H),7.49(d,J=9.2Hz,1H),7.39(d,J=7.9Hz,2H),6.93(dd,J=9 .3,2.7Hz,1H),6.67(d,J=2.7Hz,1H),3.55(q,J=7.1Hz,4H),3.26(q,J=6.4Hz,2H),2.88(q,J=6.5Hz,2H),2.36(s,3H),1.17(t,J=7.0Hz,6H).

[0104] The NMR carbon spectrum of the product is as follows Figure 2 As shown, the carbon spectrum data are as follows: 13 C NMR (151MHz, DMSO-d6) δ161.37,154.17,152.96,151.14,143.20,141.37,137.99,134.95,132.01,130.22,127. 07,124.12,116.38,112.10,106.88,97.21,60.30,45.38,42.04,21.49,21.31,14.62,13.04.HR-MS:calculated for C 25 H 28 N5O5S + 510.1733(M+H + ),found510.1798(M+H +).

[0105] Example 2

[0106] Prepare a buffer solution with a pH value of 7.4 (disodium hydrogen phosphate and sodium dihydrogen phosphate): acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L GSH aqueous solution, take 10μL of the BSOA DMSO solution and add it to a cuvette containing 2mL of phosphate buffer solution / acetonitrile buffer solution, detect it on a UV spectrophotometer, and then add 20mmol / L GSH. The obtained UV-visible absorption spectrum is as shown in the figure Figure 3 As shown. Figure 3 It can be seen that the probe presents a broad and strong absorption peak centered at 565nm. With the addition of GSH (0-20mmol / L), the UV absorption intensity at 445nm increases, while that at 565nm decreases. The solution can be observed to change from red to yellow by the naked eye.

[0107] Example 3

[0108] Prepare a phosphate buffer solution (disodium hydrogen phosphate and sodium dihydrogen phosphate) with a pH value of 7.4: a buffer solution with a volume ratio of 7:3 of acetonitrile, prepare a 2mmol / L BSOA DMSO solution, and prepare a 1mol / L N-ethylmaleimide acetonitrile solution. Take 10μL of BSOA DMSO solution and add it to a cuvette containing 2mL of phosphate buffer solution / acetonitrile buffer solution, then add 20mmol / L GSH, and then gradually add 20mmol / L N-ethylmaleimide to the reaction system. Detect on a UV spectrophotometer, and the obtained UV-visible absorption spectrum is as follows Figure 4 As shown. Figure 4 It can be seen that with the addition of N-ethylmaleimide (0-20 mmol / L), the ultraviolet absorption intensity at 445 nm weakened, and the ultraviolet absorption intensity at 565 nm gradually increased.

[0109] Example 4

[0110] Prepare a buffer solution with a pH value of 7.4 (disodium hydrogen phosphate and sodium dihydrogen phosphate): acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L GSH aqueous solution, take 10μL BSOA DMSO solution and add it to a fluorescence cuvette containing 2mL of phosphate buffer solution / acetonitrile buffer solution, then add 20mmol / L GSH, and detect the changes in fluorescence intensity at 560nm and 610nm on a fluorescence spectrophotometer. The obtained fluorescence emission graph is as shown in the figure. Figures 5-6 As shown. Figures 5-6It can be seen that with the addition of GSH (0-20 mmol / L), the fluorescence intensity at 610 nm decreased, and the fluorescence intensity at 560 nm gradually increased.

[0111] Example 5

[0112] Prepare a buffer solution with a pH value of 7.4 (disodium hydrogen phosphate and sodium dihydrogen phosphate): acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L N-ethylmaleimide acetonitrile solution, take 10μL of the BSOA DMSO solution and add it to a fluorescence cuvette containing 2mL of phosphate buffer solution / acetonitrile buffer solution, then add 20mmol / L GSH, and then add 20mmol / L N-ethylmaleimide to the reaction system, and detect the changes in fluorescence intensity at 560nm and 610nm on a fluorescence spectrophotometer, respectively. The obtained fluorescence emission graph is as follows: Figures 7-8 As shown. Figures 7-8 It can be seen that as N-ethylmaleimide (0-20 mmol / L) is added to the reaction system, the fluorescence intensity at 560 nm decreases, and the fluorescence intensity at 610 nm increases.

[0113] Example 6

[0114] Prepare a phosphate buffer solution with a pH value of 7.4 (disodium hydrogen phosphate and sodium dihydrogen phosphate): acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L GSH aqueous solution, prepare 2mmol / L Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - Solution, take 10 μL of BSOA DMSO solution and add it to a cuvette containing 2 mL of phosphate buffer solution / acetonitrile buffer solution, and then add Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - , GSH, detected on a fluorescence spectrophotometer, BSOA reacted with different analytes 565 / F 610 Fluorescence intensity ratio changes as Fig. 9 As shown. Fig. 9 It can be seen that the addition of GSH makes F 560nm / F 610nm The fluorescence intensity ratio was enhanced, and other analytes basically did not cause changes in the fluorescence intensity of the detection system.

[0115] Example 7

[0116] Prepare a phosphate buffer solution with a pH value of 7.4 (disodium hydrogen phosphate and sodium dihydrogen phosphate): acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L GSH aqueous solution, prepare 2mmol / L Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - Solution, take 10 μL of BSOA DMSO solution and add it to a cuvette containing 2 mL of phosphate buffer solution / acetonitrile buffer solution, and then add Cys, Hcy, Arg, Lys, Trp, Ser, His, NO3 - 、NO2 - , ClO - 、SO3 2- 、ClO4 - 、S2 - ,AcO - Then GSH was added to each group, and the fluorescence intensity ratio (F 560nm / F 610nm ) Histogram Fig.10 As shown. Fig.10 It can be seen that other analytes did not cause any change in the fluorescence intensity of the detection system, but after the addition of GSH, the fluorescence intensity of 560nm / F 610nm The fluorescence intensity ratios were significantly enhanced, indicating that the benzoxazine derivatives had high selectivity for GSH.

[0117] Example 8

[0118] Prepare a phosphate buffer solution (disodium hydrogen phosphate and sodium dihydrogen phosphate) with a pH value of 7.4: a buffer solution with an acetonitrile volume ratio of 7:3, prepare a 2mmol / L BSOA DMSO solution, prepare a 1mol / L N-ethylmaleimide acetonitrile solution, take 10μL of the BSOA DMSO solution and add it to a fluorescence cuvette containing 2mL of phosphate buffer solution / acetonitrile buffer solution, add GSH, and F 560nm / F610nm The fluorescence intensity ratio will gradually increase with time. Then, when N-ethylmaleimide is added, the fluorescence intensity ratio will gradually decrease with time. GSH and N-ethylmaleimide are added repeatedly to test the reversible cycle of BSOA under the repeated action of GSH and N-ethylmaleimide. The results are as follows: Fig.11 As shown. Fig.11 It can be seen that after five cycles, BSOA still has a good response to GSH and N-ethylmaleimide.

[0119] Example 9

[0120] The probe solution was added to the HeLa cell culture medium to make the concentration of the probe solution 10 μmol / L, and the co-localization map of BSOA and ER-Green was obtained. The results are as follows: Fig.12 As shown. Fig.12 It can be seen that the colocalization coefficient of BSOA and ER-Green is R=0.86.

[0121] Example 10

[0122] In the cell experiment, the probe solution was added to the HeLa cell culture medium to make the concentration of the probe solution 10μmol / L and incubated at 37℃. The results are as follows Fig.13 As shown. Fig.13 It can be seen that after incubation, BSOA generates signals in both the red and yellow channels. After the addition of glutathione ethyl ester, the yellow channel fluorescence signal is enhanced, and the red channel fluorescence signal disappears. Subsequently, after the addition of N-ethylmaleimide, the yellow channel fluorescence signal disappears, and the red channel fluorescence signal appears.

[0123] Embodiment 11

[0124] In the zebrafish experiment, the probe solution was incubated with zebrafish so that the concentration of the probe solution was 10 μmol / L. Fig.14 As shown. Fig.14 It can be seen that BSOA exhibits red fluorescence and yellow fluorescence. After the addition of glutathione ethyl ester, the yellow channel fluorescence signal of zebrafish is enhanced, and the red channel fluorescence signal is significantly weakened; then after the addition of N-ethylmaleimide, the yellow channel fluorescence signal is significantly reduced, and the red channel fluorescence signal is enhanced.

[0125] It can be seen from the above examples that the benzoxazine derivatives provided by the present invention selectively respond to environmental changes and respond quickly, so that the detection method provided by the present invention has the advantage of real-time in-situ monitoring and is convenient and fast to operate.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

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

2. The method for preparing the benzoxazine derivative according to claim 1, characterized in that: The following steps are involved: (1) mixing 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one, selenium dioxide and an organic solvent for oxidation reaction to obtain compound 1; (2) mixing the compound 1, cyanoacetic acid, piperidine and an organic solvent to carry out a first condensation reaction to obtain a compound 2; (3) The compound 2, N-(2-aminoethyl)-4-methylbenzenesulfonamide, N,N-diisopropylethylamine, N,N-dimethylformamide and O-benzotriazole-tetramethyluronium hexafluorophosphate are mixed for a second condensation reaction to obtain the benzoxazine derivative.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the 7-(diethylamino)-3-methyl-2H-benzo[b][1,4]oxazine-2-one to selenium dioxide is 1:1 to 1.

5.

4. The preparation method according to claim 2 or 3, characterized in that: The molar ratio of the compound 1 to cyanoacetic acid is 1:1 to 1.5; The molar ratio of the compound 1 to piperidine is 1:0.1 to 1:0.

2.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the compound 2 to N-(2-aminoethyl)-4-methylbenzenesulfonamide is 1:1 to 1.5; The molar ratio of the compound 2 to N,N-diisopropylethylamine is 1:1 to 1.2; The molar ratio of the compound 2 to O-benzotriazole-tetramethyluronium hexafluorophosphate is 1:1 to 1.2; The volume ratio of the N,N-diisopropylethylamine to N,N-dimethylformamide is 1:25-30.

6. Use of the benzoxazine derivative according to claim 1 or the benzoxazine derivative obtained by the preparation method according to any one of claims 2 to 5 in the field of redox detection for non-diagnostic and non-therapeutic purposes.

7. A method for detecting redox dynamics in endoplasmic reticulum in living organisms, characterized in that: The following steps are involved: A buffer solution containing a benzoxazine derivative was added to a culture medium containing HeLa cells, and then glutathione ethyl ester and N-ethylmaleimide were added to the culture medium in sequence to obtain an observation mixed solution, and changes in the fluorescence intensity of the red channel and the yellow channel were observed using a fluorescence confocal microscope.

8. The detection method according to claim 7, characterized in that: The preparation method of the buffer solution containing benzoxazine derivatives is as follows: mixing a dimethyl sulfoxide solution containing benzoxazine derivatives and a buffer solution; The volume ratio of the dimethyl sulfoxide solution containing the benzoxazine derivative to the buffer solution is 1:100-400; The concentration of the dimethyl sulfoxide solution containing the benzoxazine derivative is 1 to 2 mmol / L; The buffer solution comprises a phosphate buffer solution and acetonitrile; The volume ratio of the phosphate buffer solution to acetonitrile in the buffer solution is 1 to 4:

1.

9. The detection method according to claim 7 or 8, characterized in that: The concentration of glutathione ethyl ester in the observation mixed solution is 0 to 20 mmol / L; The concentration of N-ethylmaleimide in the observation mixed solution is 0-20 mmol / L.

10. The detection method according to claim 7 or 8, characterized in that: The N-ethylmaleimide is an N-ethylmaleimide acetonitrile solution; The concentration of the N-ethylmaleimide acetonitrile solution is 1-2 mol / L.

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