Phenoxazone derivative, preparation method and application thereof, and method for specifically recognizing NO

Abstract

The application provides a phenoxazone derivative and a preparation method and application thereof and a method for specifically recognizing NO, and belongs to the technical field of phenoxazone derivatives.The phenoxazone derivative provided by the application is a NO-activated near-infrared emission fluorescent probe, can be used for researching the complex relationship between nitric oxide and Parkinson's disease, and realizes in-vivo diagnosis and evaluation of Parkinson's disease progression.The phenoxazone derivative provided by the application has a small molecular structure and good lipophilicity, shows excellent blood-brain barrier crossing ability, so that it can monitor the fluctuation of NO in the brain of Parkinson's disease in the body, and has the advantages of high sensitivity, good selectivity, strong specificity, real-time response and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of phenoxazone derivatives, and particularly relates to a phenoxazone derivative, a preparation method and application thereof, and a method for specifically recognizing NO. BACKGROUND

[0002] Fluorescence imaging technology with light as medium has many advantages such as non-invasiveness, high sensitivity and real-time response, and has attracted wide attention in the field of chemical biology in recent years. NO is an important neurotransmitter in the brain, and its imbalance indicates the occurrence of various neurodegenerative diseases. Among them, the occurrence of Parkinson's disease is closely related to the change of the content of nitric oxide in the brain. Studies have shown that neuroinflammation caused by Parkinson's disease can cause a surge in the content of nitric oxide in the brain. Therefore, designing a fluorescence probe capable of specifically recognizing NO has great application value in studying the fluctuation of NO in the pathogenesis of Parkinson's disease. SUMMARY

[0003] Therefore, the present application aims to provide a phenoxazone derivative, a preparation method and application thereof, and a method for specifically recognizing NO. The phenoxazone derivative provided by the present application can specifically recognize NO.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a phenoxazone derivative having the structure shown in formula I:

[0006]

[0007] The present application also provides a preparation method of the phenoxazone derivative according to the above technical solutions, comprising the following steps:

[0008] mixing 2-amino-7-(diethylamino)-3H-phenoxazin-3-one, tert-butyl nitrite and CuBr to perform a Sandmeyer reaction to obtain a Sandmeyer product;

[0009] mixing the Sandmeyer product, N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(PPh3)4, K2CO3 and a solvent to perform a coupling reaction to obtain a coupling product;

[0010] mixing the coupling product, Pd / C and a solvent, and then introducing hydrogen to perform a reduction reaction to obtain the phenoxazone derivative.

[0011] Preferably, the molar ratio of the 2-amino-7-(diethylamino)-3H-phenoxazin-3-one and CuBr is 1:1-1.5.

[0012] Preferably, the temperature of the Sandmeyer reaction is 55-65℃, and the time is 5-12h.

[0013] Preferably, the molar ratio of the Sandmeyer product to N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline is 1:1-1.3.

[0014] Preferably, the temperature of the coupling reaction is 80-100℃, and the time is 4-6h.

[0015] The application also provides the application of the phenoxazone derivative in the specific identification of NO.

[0016] The application also provides the application of the phenoxazone derivative in the preparation of a drug for detecting the fluctuation of NO in the brain of Parkinson.

[0017] The application also provides a method for specifically identifying NO, comprising the following steps:

[0018] After the to-be-tested solution, the PBS buffer solution and the phenoxazone derivative solution are mixed, fluorescence detection is performed at 660nm, the phenoxazone derivative in the phenoxazone derivative solution is the phenoxazone derivative in the above technical solution or the phenoxazone derivative prepared by the preparation method in the above technical solution, and the to-be-tested solution contains NO.

[0019] The concentration of NO in the to-be-tested solution is calculated by a standard curve, the standard curve takes the concentration of NO as the abscissa and F 660 as the ordinate.

[0020] Preferably, the to-be-tested solution also contains one or more of Ca 2+ , Zn 2+ , Mg 2+ , CO3 2- , SO3 2- , NO3 2- , S2O3 2- , H2O2, ClO4 - , Cys and GSH.

[0021] The application provides a phenoxazone derivative, and compared with the prior art, the application has the following advantages and effects:

[0022] The phenoxazone derivative provided by the application is a NO activated near-infrared emission fluorescent probe, which can be used for researching the complex relationship between nitric oxide and Parkinson's disease, realizing in-vivo diagnosis and evaluation of Parkinson's disease progression; the phenoxazone derivative provided by the application has a small molecular structure and good lipophilicity, and shows excellent blood-brain barrier crossing ability, so that it can monitor the fluctuation of NO in the brain of Parkinson's disease in the body, and has the advantages of high sensitivity, good selectivity, strong specificity, real-time response and the like.

[0023] The application further provides a preparation method of the phenoxazone derivative, and the synthesis steps of the application are simple, the cost is low, and the toxicity is small.

[0024] The application further provides a method for specifically recognizing NO, and the detection means is simple and fast, and only needs to use a fluorescence spectrometer. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A nuclear magnetic hydrogen spectrum of the phenoxazone derivative PO-NH prepared for example 1;

[0026] Figure 2 A nuclear magnetic carbon spectrum of the phenoxazone derivative PO-NH prepared for example 1;

[0027] Figure 3 A nuclear magnetic hydrogen spectrum of the phenoxazone derivative PO-NO prepared for example 1;

[0028] Figure 4 Ultraviolet absorption spectrum diagrams of the phenoxazone derivative PO-NH after reacting with NO for 30 min, and PO-NO;

[0029] Figure 5 A fluorescence emission spectrum diagram of the phenoxazone derivative PO-NH after reacting with NO (100 muM) for 30 min;

[0030] Figure 6 A kinetic diagram of the phenoxazone derivative PO-NH after adding NO (0, 50, 100 muM);

[0031] Figure 7 A fluorescence emission spectrum diagram of the phenoxazone derivative PO-NH after reacting with NO (0-100 muM) for 30 min;

[0032] Figure 8 A linear relationship diagram of the fluorescence intensity F of the phenoxazone derivative PO-NH at a wavelength of 660 nm 660 and the concentration of NO (0-100 muM);

[0033] Figure 9The fluorescence emission spectrum of the phenoxazone derivative PO-NH at a wavelength of 660 nm after reacting with various interfering substances for 30 min;

[0034] Figure 10 The NO cell imaging map of the phenoxazone derivative PO-NH;

[0035] Figure 11 The cell imaging maps of the phenoxazone derivative PO-NH and the control group (untreated cells) and the PD model (cells treated with rotenone) after reaction, respectively. DETAILED DESCRIPTION

[0036] The present application provides a phenoxazone derivative, which has the structure shown in formula I:

[0037]

[0038] The Chinese name of the phenoxazone derivative PO-NH provided by the present application is 2-(3-amino-4-(methylamino)phenyl)-7-(diethylamino)-3H-phenoxazin-3-one, and the English name is 2-(3-amino-4-(methylamino)phenyl)-7-(diethylamino)-3H-phenoxazin-3-one.

[0039] The present application also provides a preparation method of the phenoxazone derivative described in the above technical solution, comprising the following steps:

[0040] Mixing 2-amino-7-(diethylamino)-3H-phenoxazin-3-one, tert-butyl nitrite and CuBr to perform Sandmeyer reaction to obtain a Sandmeyer product;

[0041] Mixing the Sandmeyer product, N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(PPh3)4, K2CO3 and a solvent to perform coupling reaction to obtain a coupling product;

[0042] Mixing the coupling product, Pd / C and a solvent, and then introducing hydrogen to perform reduction reaction to obtain the phenoxazone derivative.

[0043] In the present application, if no special description, the raw materials used are commercially available in the art.

[0044] In the present application, 2-amino-7-(diethylamino)-3H-phenoxazin-3-one, tert-butyl nitrite and CuBr are mixed to perform Sandmeyer reaction to obtain a Sandmeyer product, i.e. 2b in the general formula of the preparation method of Example 1.

[0045] In the present application, the molar ratio of the 2-amino-7-(diethylamino)-3H-phenoxazin-3-one and CuBr is preferably 1:1 to 1.5.

[0046] In the present application, the molar ratio of the 2-amino-7-(diethylamino)-3H-phenoxazin-3-one and CuBr is preferably 1:1 to 1.5.

[0047] In the present application, the temperature of the Sandmeyer reaction is preferably 55 to 65°C, more preferably 60°C, and the time is 5 to 12h, more preferably 7 to 10h.

[0048] In the present application, the Sandmeyer reaction is preferably performed in an organic solvent, and the organic solvent is preferably acetonitrile.

[0049] In the present application, the 2-amino-7-(diethylamino)-3H-phenoxazin-3-one, tert-butyl nitrite and CuBr are mixed in a flat-bottomed flask containing acetonitrile at room temperature, the mixture is stirred at room temperature for 30min to make the mixture fully dissolved, and then the Sandmeyer reaction is performed.

[0050] After the Sandmeyer reaction is completed, the present application preferably removes the solvent by distillation under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography to obtain the Sandmeyer product.

[0051] In the present application, the eluent for column chromatography is preferably a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixture is preferably 50:1.

[0052] After obtaining the Sandmeyer product, the present application mixes the Sandmeyer product, N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(PPh3)4, K2CO3 and a solvent to perform a coupling reaction, thereby obtaining a coupling product, i.e. 2c in the formula of the principle of the preparation method of Example 1.

[0053] In the present application, the molar ratio of the Sandmeyer product to N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline is preferably 1:1 to 1.3.

[0054] In the present application, the molar ratio of the Sandmeyer product to Pd(PPh3)4 is preferably 1:0.06.

[0055] In the present application, the molar ratio of the Sandmeyer product to K2CO3 is preferably 1:3.

[0056] In the present application, the temperature of the coupling reaction is preferably 80-100°C, more preferably 90°C, and the time is 4-6h, more preferably 5h.

[0057] In the present application, the solvent is preferably a mixed solution of toluene and water, and the volume ratio of toluene to water in the mixed solution is preferably 6:1.

[0058] After the completion of the coupling reaction, the present application preferably extracts the water layer of the obtained system with dichloromethane, collects the organic phase, then dries the organic phase with anhydrous sodium sulfate, removes the solvent by distillation under reduced pressure, and then separates by column chromatography to obtain the coupling product.

[0059] In the present application, the eluent for column chromatography separation is preferably a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixture is preferably 50:1.

[0060] After obtaining the coupling product, the present application mixes the coupling product, Pd / C and a solvent, and then passes hydrogen gas to perform a reduction reaction to obtain a phenoxazinone derivative having a structure shown in Formula I.

[0061] In the present application, the solvent is preferably ethanol.

[0062] In the present application, the molar ratio of the coupling product to Pd / C is preferably 1:0.4.

[0063] In the present application, the reduction reaction is preferably performed under reflux conditions, and the time is preferably 4h.

[0064] The present application preferably dissolves the coupling product in ethanol, then adds Pd / C to the system, and passes hydrogen gas to perform the reduction reaction.

[0065] After the completion of the reduction reaction, the present application preferably clarifies the obtained reaction solution, naturally cools it to room temperature, removes the solvent by distillation under reduced pressure to obtain a crude product, then performs column chromatography separation on the crude product using dichloromethane / methanol (100:1, v / v) as the eluent to obtain a dark green powder solid, which is the phenoxazinone derivative.

[0066] The present application also provides the use of the phenoxazinone derivative of the above technical solution or the phenoxazinone derivative prepared by the preparation method of the above technical solution in the specific recognition of NO.

[0067] The present application also provides the use of the phenoxazinone derivative of the above technical solution or the phenoxazinone derivative prepared by the preparation method of the above technical solution in the qualitative detection of NO.

[0068] In the present application, the use preferably includes the following steps:

[0069] After mixing the solution to be tested, the PBS buffer solution and the phenoxazone derivative solution, ultraviolet spectrophotometry is performed, and if the absorption peak at 600 nm starts to decline, it indicates that the solution to be tested contains NO.

[0070] In the present application, the application preferably comprises the following steps:

[0071] After mixing the solution to be tested, the PBS buffer solution and the phenoxazone derivative solution, fluorescence detection is performed, and if the fluorescence intensity at 660 nm is enhanced, it indicates that the solution to be tested contains NO.

[0072] The present application also provides the use of the phenoxazone derivative in the preparation of a drug for detecting NO fluctuation in the brain of Parkinson.

[0073] The present application also provides a method for specifically recognizing NO, comprising the following steps:

[0074] After mixing the solution to be tested, the PBS buffer solution and the phenoxazone derivative solution, fluorescence detection is performed at 660 nm, the phenoxazone derivative in the phenoxazone derivative solution is the phenoxazone derivative in the above technical solution or the phenoxazone derivative prepared by the preparation method in the above technical solution, and the solution to be tested contains NO.

[0075] The concentration of NO in the solution to be tested is calculated by a standard curve, the standard curve takes the concentration of NO as the abscissa and F 660 as the ordinate.

[0076] In the present application, the solution to be tested preferably further contains one or more of Ca 2+ , Zn 2+ , Mg 2+ , CO3 2- , SO3 2- , NO3 2- , S2O3 2- , H2O2, ClO4 - , Cys and GSH.

[0077] In the present application, the pH value of the PBS buffer solution is preferably 4.0-8.0, and more preferably 5.0-7.0.

[0078] In the present application, the concentration of NO in the solution to be tested is preferably 0-100 μM.

[0079] The present application does not have special limitations on the method for obtaining the standard curve, and a method well known to those skilled in the art can be used.

[0080] In the present application, the compound obtained after the phenoxazone derivative is recognized has a structure shown in Formula II.

[0081]

[0082] After obtaining the phenoxazone derivative having a structure shown in Formula I, the present application mixes the phenoxazone derivative having a structure shown in Formula I, NaNO2, hydrochloric acid and water to perform a ring-forming reaction, thereby obtaining a compound having a structure shown in Formula II.

[0083] In the present application, the molar ratio of the phenoxazone derivative having a structure shown in Formula I to NaNO2 is preferably 1:1.2.

[0084] In the present application, the ring-forming reaction is preferably performed under ice bath conditions, and the time is preferably 1 h.

[0085] In the present application, the concentration of the hydrochloric acid is preferably 3M.

[0086] In the present application, the phenoxazone derivative having a structure shown in Formula I and NaNO2 are dissolved in water, hydrochloric acid is added to the reaction solution under ice bath conditions, and after stirring for 1 h, the organic phase is extracted with DCM, dried over Na2SO4, and concentrated under reduced pressure, and the obtained crude product is purified by silica gel column chromatography to obtain the compound having a structure shown in Formula II.

[0087] In the present application, the eluent for the silica gel column chromatography purification is preferably dichloromethane.

[0088] In order to further illustrate the present application, the phenoxazone derivative provided by the present application, the preparation method and application thereof, and the method for detecting SO3 2- cannot be understood as limiting the scope of protection of the present application.

[0089] Example 1

[0090] Preparation and characterization of PO-NH and PO-NO

[0091] The principle of the preparation method is shown in the following formula:

[0092]

[0093] At room temperature, 2-amino-7-(diethylamino)-3H-phenoxazin-3-one (5.66 g, 20 mmol), tert-butyl nitrite (3.09 g, 30 mmol) and CuBr (4.25 g, 30 mmol) were mixed in a flat bottom flask containing 120 mL of acetonitrile, the mixture was stirred at room temperature for 30 min to allow complete dissolution, then the reaction was refluxed at 65 °C for 7 h. The solvent was removed by distillation under reduced pressure to obtain the crude product, which was then separated by column chromatography using dichloromethane / methanol (50:1, v / v) as eluent to obtain compound 2b by Sandmeyer reaction with a yield of 11%.

[0094] Compound 2b (0.6 g, 4 mmol), N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.45 g, 5.2 mmol), Pd(PPh3)4(0.28 g, 0.24 mmol), K2CO3(1.6 g, 12 mmol) were mixed in a mixture of toluene and water (6:1, v / v) and heated to 90 °C for 5 h. The aqueous layer was extracted with dichloromethane and the organic phase was collected. The organic phase was then dried with anhydrous sodium sulfate and the solvent was removed by distillation under reduced pressure, then the product was separated by column chromatography using dichloromethane / methanol (50:1, v / v) as eluent to obtain compound 2c by coupling reaction with a yield of 58%.

[0095] Compound 2c (0.84 g, 2 mmol) was dissolved in ethanol. Then Pd / C (0.085 g, 0.8 mmol) was added to the system and hydrogen was introduced for reduction reaction under reflux conditions for 4 h. After the reaction solution was clarified, it was cooled to room temperature and the solvent was removed by distillation under reduced pressure to obtain the crude product, which was then separated by column chromatography using dichloromethane / methanol (100:1, v / v) as eluent to obtain the target product PO-NH as a dark green powder with a yield of 43%.

[0096] Hydrogen spectrum: 1 H NMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 9.1 Hz, 1H), 7.30 (s, 1H), 7.03 - 6.93 (m, 2H), 6.84 (dd, J = 9.2, 2.7 Hz, 1H), 6.64 (d, J = 2.6 Hz, 1H), 6.42 (d, J = 8.2 Hz, 1H), 6.20 (s, 1H), 4.82 (d, J = 169.2 Hz, 3H), 3.50 (q, J = 7.1 Hz, 4H), 2.77 (s, 3H), 1.16 (t, J = 7.0 Hz, 6H). Figure 1 Carbon spectrum: 13C NMR (101 MHz, DMSO-d6) δ 184.29, 151.46, 149.63, 146.55, 141.21, 141.15, 138.74, 134.84, 131.56, 128.53, 126.52, 124.32, 120.27, 114.94, 111.32, 108.85, 105.98, 96.43, 45.07, 30.53, 13.02. Figure 2 Mass: [M+H]+calcd 400.1768, found 400.1770. + Calcd 389.1899, Found 389.1971.

[0097] PO-NH (0.39 g, 1 mmol) and NaNO2(0.08 g, 1.2 mmol) were dissolved in water. Hydrochloric acid (3M) was added to the reaction solution under ice bath condition. After stirring for 1 h, it was extracted with DCM. Then the organic phase was dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane as eluent to obtain the product PO-NO with a yield of 36%.

[0098] Hydrogen spectrum: 1 H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.80 (dd, J = 8.7, 1.5 Hz, 1H), 7.69 (s, 1H), 7.62 (d, J = 9.2 Hz, 1H), 6.94 (dd, J = 9.2, 2.6 Hz, 1H), 6.75 (d, J = 2.7 Hz, 1H), 6.33 (s, 1H), 4.34 (s, 3H), 3.56 (q, J = 6.9 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H). Figure 3 Mass: [M+H]+calcd 400.1768, found 400.1770.

[0099] Example 2

[0100] 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution was added to the cuvette, and the probe was detected on the ultraviolet spectrophotometer with 600 nm as the center, which showed a wide and strong absorption peak. After adding 5 μM NO, the absorption peak at 600 nm began to decrease, Figure 4 The ultraviolet absorption spectrum of the phenoxazone derivative PO-NH after reacting with NO for 30 min and PO-NO is shown in the following figure: Figure 4 It can be seen that the compound obtained after PO-NH specifically recognizes NO is PO-NO.

[0101] Example 3

[0102] The 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution and 100 μM NO were added into the fluorescence cuvette, and the fluorescence intensity at 660 nm increased after 30 min of reaction. The fluorescence emission spectrum is shown in Figure 5 .

[0103] Example 4

[0104] The 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution and different concentrations of NO (0, 50, 100 μM) were added into the fluorescence cuvette for kinetic study. The fluorescence intensity at 660 nm gradually increased and tended to be stable after 8 min. The fluorescence emission spectrum is shown in Figure 6 .

[0105] Example 5

[0106] The 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution and NO (0-100 μM) were added into the fluorescence cuvette, and the fluorescence intensity change within 30 min was observed. The fluorescence intensity at 660 nm gradually increased. The fluorescence emission spectrum is shown in Figure 7 .

[0107] Example 6

[0108] The 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution and different concentrations of NO were added into the fluorescence cuvette for fluorescence titration experiment. The graph was plotted with NO concentration as abscissa and F 660 as ordinate, and the linear relationship between NO concentration and fluorescence intensity (F 660 ) was obtained. The linear relationship between fluorescence intensity (F 660 ) and NO concentration is shown in Figure 8 .

[0109] Example 7

[0110] The 2 mL PBS buffer solution, 5 μM PO-NH DMSO solution and other analytes (200 μM) Ca 2+ , Zn 2+ , Mg 2+ , CO3 2- , SO3 2- , NO3 2- , S2O3 2- , H2O2, ClO4 - , Cys and GSH were added into the fluorescence cuvette, and the aqueous solution of NO (100 μM) was added. The fluorescence intensity change of PO-NH after reaction with different analytes at 660 nm was detected by fluorescence spectrophotometer, and the graph is shown in Figure 9As shown, NO makes the detection system fluorescence intensity at 660 nm enhanced, and other analytes do not cause the detection system fluorescence intensity to change.

[0111] Example 8

[0112] A 2 mL PBS buffer solution was prepared, and a 5 μM PO-NH DMSO solution was added to the 2 mL PBS buffer solution. The probe solution was added to the HeLa cell culture solution (control group) to make the concentration 5 μM, and incubated for 5 min. The Hela cells showed very weak fluorescence signals. The cells were then incubated with SNP (300 uM) for 1 h, with LPS (20 μg / mL) for 12 h, and with LPS (20 μg / mL) and L-Arg (5 mg / mL) for 12 h, respectively, and then subjected to probe staining. The fluorescence signals of channel 1: (λ em = 650-720 nm. λ ex = 633 nm) (red light) were collected, and laser confocal fluorescence imaging showed that the fluorescence signals in the three groups of cells were significantly stronger than those in the control group. When the cells were incubated with LPS (20 μg / mL) and L-NAME (0.5 mM) for 12 h and then subjected to probe staining, the fluorescence signals in the cells were found to be significantly reduced. This indicates that PO-NH can effectively detect endogenous and exogenous NO, as shown in Figure 10 .

[0113] Example 9

[0114] A 5 μM PO-NH DMSO solution was added to untreated cells (control group) and incubated for 5 min. A 5 μM PO-NH DMSO solution was added to cells treated with rotenone (1 μM) for 1 h (PD model) and incubated for 5 min. The fluorescence signals of channel 1: (λ em = 650-720 nm. λ ex = 633 nm) (red light) were collected, and laser confocal fluorescence imaging showed that the fluorescence intensity of the PD model was significantly stronger than that of the control group. This indicates that the probe can achieve in vivo diagnosis and evaluation of Parkinson's disease, as shown in Figure 11 .

[0115] The above only describes preferred embodiments of the present application and does not limit the present application in any form. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A phenoxazone derivative having a structure shown in Formula I: ###0001### Formula I.

2. The process for preparing the phenoxazone derivative according to claim 1, characterized by, comprising the steps of: mixing 2-amino-7-(diethylamino)-3H-phenoxazin-3-one, tert-butyl nitrite and CuBr to perform a Sandmeyer reaction to obtain a Sandmeyer product; mixing the Sandmeyer product, N-methyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)aniline, Pd(PPh3)4, K2CO3 and a solvent to perform a coupling reaction to obtain a coupling product; mixing the coupling product, Pd / C and a solvent and passing hydrogen gas to perform a reduction reaction to obtain the phenoxazone derivative.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the 2-amino-7-(diethylamino)-3H-phenoxazin-3-one to CuBr is 1:1-1.

5.

4. The preparation method according to claim 2, characterized in that, The Sandmeyer reaction is performed at a temperature of 55-65°C for 5-12 hours.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the Sandmeyer product to N-methyl-2-nitro-5-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)aniline is 1:1-1.

3.

6. The preparation method according to claim 2, characterized in that, The coupling reaction is performed at a temperature of 80-100°C for 4-6 hours.

7. Use of the phenoxazone derivative of claim 1 or the phenoxazone derivative prepared by the method of any one of claims 2-6 in specific recognition of NO, wherein the use is not for disease diagnosis or treatment.

8. Use of the phenoxazone derivative of claim 1 or the phenoxazone derivative prepared by the method of any one of claims 2-6 in the preparation of a medicament for detecting NO fluctuation in the brain of Parkinson's disease.

9. A method for specific recognition of NO, characterized in that, comprising the steps of: mixing a test solution, a PBS buffer solution and a phenoxazone derivative solution, wherein the phenoxazone derivative in the phenoxazone derivative solution is the phenoxazone derivative of claim 1 or the phenoxazone derivative prepared by the method of any one of claims 2-6, and the test solution contains NO, and performing fluorescence detection at 660 nm. The concentration of NO in the solution to be measured is calculated from a standard curve, the standard curve having the concentration of NO as the abscissa and F 660 as the ordinate.

10. The method of claim 9, wherein, The solution to be tested also contains one or more of Ca 2+ , Zn 2+ , Mg 2+ , CO3 2- , SO3 2- , NO3 2- , S2O3 2- , H2O2, ClO4 - , Cys and GSH.

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