A class of small molecule fluorescent probes for detecting nitric oxide and its application

Abstract

The present invention discloses a class of small molecule fluorescent probes for detecting nitric oxide and their applications. The probe is a probe construction strategy based on the optimization of the o-phenylenediamine structure. The connection structure of the o-phenylenediamine reaction group and the fluorophore and the different substituents of o-phenylenediamine are changed to improve the sensitivity and response rate to nitric oxide; the structure of the fluorophore is changed to obtain a nitric oxide fluorescent probe with different fluorescent colors in the green visible light and near-infrared light regions, which is used for nitric oxide detection in buffer and nitric oxide fluorescence imaging in living cells. The modularity and universality of the nitric oxide fluorescent probe construction strategy designed in this application have been systematically verified. In the future, the o-phenylenediamine reaction group can also be connected to more fluorophores with different wavelengths and optical properties through a carbonamide structure, so as to conveniently and quickly develop a series of nitric oxide fluorescent probes.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis and fluorescent probe detection, and in particular to a class of small molecule fluorescent probes for detecting nitric oxide in biological samples and applications thereof. Background Art

[0002] Nitric oxide (NO) is a colorless, transparent, harmful gas that is often listed as an environmental pollutant, but it plays an indispensable role in the human body. In the body, NO is a multifunctional signaling molecule. In addition to its well-known vasodilatory effect, it is also closely related to platelet viscosity, cell proliferation, immune response, apoptosis, angiogenesis, neurotransmission, etc. Under normal physiological conditions, the concentration of NO in the human body is relatively stable, but under the stimulation of inflammation, tumor necrosis factor, interleukin, etc., excessive NO will be produced in the human body, thus endangering human health. Specifically, in the case of neuronal damage, the concentration of nitric oxide in the brain will surge by several orders of magnitude; excessive NO itself will weaken the production of cellular energy by interacting with mitochondria; secondly, NO can also produce reactive nitrogen and reactive oxygen species, further affecting cell function.

[0003] Although NO is of great significance to human health, its complex biological roles in cell signaling pathways and various diseases have not yet been fully elucidated. One of the main reasons is the lack of effective tools and methods for non-destructive real-time detection of NO in biological samples. Therefore, developing efficient NO detection methods and realizing real-time and rapid monitoring of NO concentration in biological samples will help explore the role of NO in the occurrence and development of related diseases and understand the various pathophysiological functions and roles of NO.

[0004] Currently developed NO detection methods include: electrochemical method, chemiluminescence method, spectrophotometry, electron paramagnetic resonance spectroscopy, fiber optic sensing technology and fluorescent probe method, etc. Among them, the fluorescent probe method can not only realize the specific detection of NO, but also has the advantages of low cost, high sensitivity, good selectivity, simple operation, non-invasiveness and spatiotemporal imaging ability, and has been widely used in NO detection.

[0005] Based on this, we invented a class of organic small molecule fluorescent probes based on the optimized structure of o-phenylenediamine for the specific detection and imaging of nitric oxide in biological samples. Summary of the invention

[0006] The purpose of the present invention is to provide a small molecule fluorescent probe and application for detecting nitric oxide in biological samples, so as to solve the problems that the existing o-phenylenediamine NO fluorescent probes have poor selectivity and the design strategy cannot meet the requirements of high modularity and universality.

[0007] In order to solve these problems, the present invention connects the core fluorophore to the o-phenylenediamine structure through its phenolic hydroxyl group or aromatic amine group with different linkers, and develops a universal probe template. NO undergoes a nitrosylation reaction with the amine group on the o-phenylenediamine structure, and then forms a benzotriazole structure through intramolecular cyclization and removal of a molecule of water, and then hydrolyzes and removes the benzotriazole to generate a free fluorophore, accompanied by significant fluorescence enhancement, thereby realizing the detection of NO, which can be used for the specific detection and imaging of nitric oxide in biological samples.

[0008] The following reaction process is the NO fluorescent probe design strategy of this application:

[0009]

[0010] The purpose of the present invention can be achieved by the following technical solutions:

[0011] A type of small molecule fluorescent probe for detecting nitric oxide, characterized in that: its general structural formula I is:

[0012]

[0013] In the general formula I:

[0014] R1 is selected from O, NH, NCH3, R2 is selected from H, CH3, R3 is selected from H, CH3, OCH3, F, N(CH3)2, OH, R4 is selected from H, CH3, OCH3, F, N(CH3)2, OH, N(CH2CH3)2, N(CH2CH2)2CH, NCH3CH2Ph, N(CH2)2CH2, N(CH2CH2)2, N(CH2CH2)2O, NCH3Ph, NHCHO, NCH3CHO;

[0015] FL is selected from fluorophore, which replaces fluorophore; fluorophore refers to a molecule that is excited at a specific wavelength and emits fluorescence at a specific wavelength;

[0016] Preferably, when R1 is NH and R2 is H, R3 is selected from H, CH3, OCH3, F, N(CH3)2, OH, and R4 is selected from H, CH3, OCH3, F, N(CH3)2, OH, N(CH2CH3)2, N(CH2CH2)2CH, NCH3CH2Ph, N(CH2)2CH2, N(CH2CH2)2, N(CH2CH2)2O, NCH3Ph, NHCHO, NCH3CHO;

[0017] Preferably, the parent structure of the fluorophore is selected from coumarin, naphthylimide, merocyanine and hemicyanine dye fluorophores.

[0018] Preferably, the structure comprises one of the following:

[0019]

[0020]

[0021] The present invention also provides a class of small molecule fluorescent probes for detecting nitric oxide and their application in detecting nitric oxide.

[0022] The present invention also provides a class of small molecule fluorescent probes for detecting nitric oxide and their application in the detection of nitric oxide in solutions, living cells and living animals.

[0023] The present invention also provides the use of a class of small molecule fluorescent probes for detecting nitric oxide in the preparation of a nitric oxide detection kit or test paper.

[0024] The biggest advantage of the design strategy of this application is its high modularity and universality. Since the detection performance of the probe to NO is determined by the structure of the o-phenylenediamine reactive group, the core fluorophore can be replaced with any fluorophore in theory to obtain a series of NO fluorescent probes, thereby greatly simplifying the development of the probe. The connection mode of the o-phenylenediamine reactive group to the core fluorophore is fixed to facilitate the synthesis of the probe. In addition, this modular design strategy greatly accelerates the screening and optimization of the optimal o-phenylenediamine reactive group structure. Finally, in this design strategy, one of the amine groups of o-phenylenediamine is connected to the fluorophore through a carbonyl group, which will help reduce the reactivity of the probe to potential interferents (such as MGO, DHA and AA, etc.), thereby improving the selectivity of the probe to NO.

[0025] Furthermore, the small molecule optical probe provided by the present invention only emits weak fluorescence before reacting with NO; in the presence of NO, the o-phenylenediamine unit in the probe structure can effectively react with NO to generate a fluorophore matrix through processes such as nitrosylation, intramolecular cyclization, and hydrolysis, accompanied by strong fluorescence enhancement, thereby realizing fluorescence detection of NO, which is beneficial to studying the homeostasis, signal transduction, and function of NO in biological systems;

[0026] Furthermore, the application of fluorescent probes in the detection of nitric oxide has resulted in NO fluorescent probes of various colors with wavelengths ranging from visible light to near-infrared regions. The probes have good NO selectivity and sensitivity, and the NO detection limit can be as low as about 0.16 μM.

[0027] Furthermore, regarding the application of fluorescent probes in the detection of nitric oxide, both the green light region and the near-infrared region probes can be applied to the detection of NO in living cells.

[0028] Furthermore, the application of fluorescent probes in the detection of nitric oxide, the near-infrared fluorescent probes can also be used for in vivo imaging.

[0029] The beneficial effects of the present invention are embodied in the following embodiments and will not be described again. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments will be clearly introduced below. 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 creative work.

[0031] Figure 1 This is the experimental result diagram of the fluorescence response degree of the NO fluorescent probe molecule library reacting with NO;

[0032] Figure 2 The reaction flow chart of the fluorescent probe WNO26 of the present invention

[0033] Figure 3 This is a reaction flow chart of the fluorescent probe WNO27 of the present invention;

[0034] Figure 4 This is a reaction flow chart of the fluorescent probe WNO28 of the present invention;

[0035] Figure 5 This is the fluorescence spectrum of the probe WNO26 (10 μM) after reacting with NO donor (0-2 mM) at 37°C;

[0036] Figure 6 This is the fluorescence spectrum of the probe WNO27 (10 μM) after reacting with NO donor (0-2 mM) at 37°C;

[0037] Figure 7 This is the fluorescence spectrum of the probe WNO28 (10 μM) after reacting with NO donor (0-2 mM) at 37°C;

[0038] Figure 8 The kinetics and selectivity characterization diagram of the probe WNO26 for detecting NO. (A) The fluorescence response kinetics of the probe WNO26 (10 μM) to the NO donor (1 mM), NO donor (DEA NONOate, 1 mM). (B) The fluorescence response intensity of the probe WNO26 (10 μM) to NO and other potential interferents;

[0039] Fig. 9 The characterization diagram of the probe WNO26 for detecting NO in vitro, Figure (A) shows the linear relationship between the fluorescence intensity at 554 nm and the NO donor concentration after the reaction of the probe WNO26 (10 μM) with the NO donor (0-2.0 μM); Figure (B) shows the fluorescence response of the probe WNO26 (10 μM) to the NO donor (1 mM) at different pH values;

[0040] Fig.10 The kinetics and selectivity characterization diagram of the probe WNO27 for detecting NO. (A) The fluorescence response kinetics of the probe WNO27 (10 μM) to the NO donor (1 mM), NO donor (DEA NONOate, 1 mM). (B) The fluorescence response intensity of the probe WNO27 (10 μM) to NO and other potential interferents;

[0041] Fig.11 The characterization diagram of the probe WNO27 for detecting NO in vitro, Figure (A) The linear relationship between the fluorescence intensity at 559nm and the NO donor concentration after the probe WNO27 (10μM) reacts with the NO donor (0-2.0μM); Figure (B) The fluorescence response of the probe WNO27 (10μM) to the NO donor (1mM) at different pH;

[0042] Fig.12 The kinetics and selectivity characterization diagram of the probe WNO28 in detecting NO; (A) The fluorescence response kinetics of the probe WNO28 (10 μM) to the NO donor (1 mM), NO donor (DEA NONOate, 1 mM). (B) The fluorescence response intensity of the probe WNO28 (10 μM) to NO and other potential interferents;

[0043] Fig.13 Characterization diagram of probe WNO28 detecting NO in vitro; (A) Linear relationship between the fluorescence intensity at 700nm and the NO donor concentration after the reaction of probe WNO28 (10μM) with NO donor (0-1.2μM); (B) Fluorescence response of probe WNO28 (10μM) to NO donor (1mM) at different pH;

[0044] Fig.14 Confocal fluorescence imaging of exogenous NO by probe WNO27 in HeLa cells;

[0045] Fig.15 Confocal fluorescence imaging of the probe WNO28 in response to exogenous NO in HeLa cells. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] For the chemical synthesis experiment, if not otherwise specified, all reagents were purchased from reagent companies and used directly without any purification treatment. All reactions were operated under anhydrous conditions protected by nitrogen or argon. Solvents were redistilled before use, THF was treated with a sodium-benzophenone system, and dichloromethane was treated with calcium hydride. Ultrapure water was prepared by a Thermo Fisher ultrapure water device. The reaction was detected by TLC, and the thin layer silica gel plate used was GF 254 (60-F250, 0.2mm, Qingdao Ocean Chemical Plant), which was colored with UV (wavelength 254nm and 365nm) or iodine, or soaked with acidic potassium permanganate and ninhydrin solution, and then heated with a hot air gun for color development. The silica gel used for rapid column chromatography came from Qingdao Ocean Silica Gel 60 (300-400mesh ASTM). Ethyl acetate and petroleum ether or methanol and dichloromethane are generally used as eluents, and the ratio used in the operation is the volume ratio of the two. 1HNMR and 13C NMR were measured using Bruker Avance-300 (1H: 300 MHz, 13C: 75 MHz) or Bruker Avance-400 (1H: 400 MHz, 13C: 100 MHz) or Quantum-I Plus 400 MHz (1H: 400 MHz, 13C: 100 MHz) or Bruker Avance-500 (1H: 500 MHz, 13C: 125 MHz) nuclear magnetic resonance instruments, and abbreviations such as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet) were used to explain the splitting of the NMR peaks. Deuterated reagents used were deuterated chloroform, deuterated dimethyl sulfoxide, deuterated methanol, etc. In the nuclear magnetic resonance spectrum, the solvent peaks of the hydrogen spectrum were 7.26 (deuterated chloroform), 3.31 (deuterated methanol) or 2.50 (deuterated dimethyl sulfoxide), and the solvent peaks of the carbon spectrum were 77.16 (deuterated chloroform), 49.00 (deuterated methanol) or 40.00 (deuterated dimethyl sulfoxide). High-resolution mass spectra were measured by ABIQ-starElite high-resolution mass spectrometer using ESI conditions.

[0048] 1. Preparation of NO fluorescent probe molecule library

[0049] Example 1 Preparation of NO fluorescent probe molecule library WNO1-WNO25:

[0050] Synthesis of coumarin carbonyl chloride:

[0051]

[0052] Dissolve triphosgene (177 mg, 0.6 mmol) in anhydrous tetrahydrofuran (6 mL). Then add 7-hydroxy-4-methylcoumarin (264 mg, 1.5 mmol) or 7-amino-4-methylcoumarin (262 mg, 1.5 mmol) or 7-methylamino-4-methylcoumarin.

[240] (283mg, 1.5mmol) was dissolved in anhydrous tetrahydrofuran (4mL), and then anhydrous pyridine (0.096mL, 1.2mmol) was added, and then the tetrahydrofuran solution of coumarin and pyridine was added to the tetrahydrofuran solution of triphosgene. The reaction was stirred at room temperature for 4h, and then the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was evacuated on an oil pump for 2h to remove excess triphosgene and phosgene produced as a byproduct of the reaction. The main component of the remaining solid was 7-hydroxy-4-methylcoumarin carbonyl chloride or 7-amino-4-methylcoumarin carbonyl chloride or 7-methylamino-4-methylcoumarin carbonyl chloride. The crude product prepared by this method does not need to be further purified and can be directly used in the next step of the reaction.

[0053] Synthesis of compound WNO1:

[0054]

[0055] Dissolve 5-methoxy-2-nitroaniline (3.36 g, 20 mmol) in anhydrous tetrahydrofuran (200 mL), and add Boc2O (8.72 g, 40 mmol) and NaH (1.6 g, 40 mmol, dispersed in mineral oil, mass fraction 60%) in sequence. Reflux the reaction overnight. After the reaction is complete, cool the reaction solution to room temperature, and then add water (400 mL) to quench the reaction. Ethyl acetate (3×50 mL) is added to extract, and the organic phases are combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate solid, and filtered. After filtration, the filtrate is concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-1 (1.75 g, 33%). This compound is a known compound, 1 H NMR analysis was consistent with that reported in the literature.

[0056] Compound 3-1 (1.69 g, 1.30 mmol) was dissolved in methanol (25 mL), 10% Pd / C (169 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-2 (1.1 g, 77%). 1H NMR (300MHz, CDCl3) δ7.16(d,J=2.4Hz,1H),6.72(d,J=8.6Hz,1H),6.62(s,1H),6.54(dd,J=8.6,2.8Hz,1H),3.74(s,3H),3.28(s,2H),1.51(s,9H). 13 C NMR(101MHz, CDCl3)δ154.3,153.4,130.6,128.1,120.0,110.8,107.9,80.6,55.8,28.4.HRMS calcd for C 12 H 19 N2O3 + [M+H] + 239.1396, found 239.1389.

[0057] 7-Hydroxy-4-methylcoumarin carboxylic acid chloride (238 mg, 1 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.03 mL, 0.2 mmol), DMAP (12 mg, 0.1 mmol), and compound 3-2 (47 mg, 0.2 mmol) were added in sequence, and the reaction was stirred at room temperature for 4 h. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3 × 10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 2) to obtain a white solid WNO1-Boc (55 mg, 62%). 1 H NMR (400MHz, d 6 -DMSO) δ11.06(s,1H),10.55(s,1H),7.61(d,J=8.7Hz,1H),7.29(d,J=2.5Hz,1H),6.91(d,J=8.5Hz,1H),6.82(d d,J=8.7,2.4Hz,1H),6.80–6.70(m,2H),6.15(t,J=1.3Hz,1H),3.75(s,3H),2.38(d,J=1.2Hz,3H),1.60(s,9H). 13 CNMR(101MHz,d 6-DMSO)δ161.7,160.8,155.3,155.0,154.1,151.5,148.8,127.9,127.2,122.6 ,113.4,112.5,110.8,109.9,109.9,102.7,101.3,84.2,56.0,28.2,18.6.HRMS calcd for C 23 H 24 N2NaO7 + [M+Na] + 463.1481, found 463.1476.

[0058] The compound WNO1-Boc (40 mg, 1 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1 mL) was added. The reaction was stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO1 (22 mg, 72%). 1 H NMR (400MHz, d 6 -DMSO) δ9.28(s,1H),7.84(d,J=8.6Hz,1H),7.38(d,J=2.3Hz,1H),7.30(dd,J=8.6,2.3Hz,1H),7.10(d,J=8.7Hz ,1H),6.41(dd,J=3.8,2.2Hz,2H),6.28(dd,J=8.6,2.8Hz,1H),4.15(s,2H),3.70(s,3H),2.47(d,J=1.3Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.3,158.7,156.9,154.1,153.6,152.9,139.1,128.0,126.7,118.9,117.4,116.8,115.7,114.4,113.9,110.4,55.5,18.7.HRMS calcd for C 18 H 17 N2O5 + [M+H] + 341.1137, found 341.1136.

[0059] Synthesis of compound WNO2:

[0060]

[0061] Compound 3-2 (1 g, 4.2 mmol) was dissolved in dimethyl sulfoxide (10 mL), potassium carbonate (1.73 g, 12.6 mmol) was added, and iodomethane (0.26 mL, 4.2 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, 1N dilute hydrochloric acid (100 mL) was added to quench the reaction. Ethyl acetate (3×20 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-3 (254 mg, 24%). 1 HNMR (300MHz, CDCl3) δ7.25–7.21(m,1H),6.74(d,J=8.7Hz,1H),6.64(dd,J=8.7,2.9Hz,1H),6.54(s,1H),3.76(s,3H),2.79(s,3H),1.51(s,9H). 13 C NMR(101MHz, CDCl3)δ153.6,134.8,128.0,115.2,110.5,108.4,80.5,55.8,32.5,28.4.HRMS calcd for C 13 H 21 N2O3 + [M+H] + 253.1552, found 253.1547.

[0062] 7-Hydroxy-4-methylcoumarin carboxylic acid chloride (1.12 g, 4.71 mmol) was dissolved in anhydrous dichloromethane (20 mL), and DIPEA (0.26 mL, 1.57 mmol), DMAP (95 mg, 0.78 mmol), and compound 3-3 (398 mg, 1.57 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (40 mL) was added to quench the reaction. Dichloromethane (3 × 20 mL) was added to extract, and the organic phases were combined and washed with saturated brine (40 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 2) to obtain a white solid WNO2-Boc (559 mg, 78%). 1 H NMR (400MHz, d 6-DMSO)δ9.15(s,1H),7.78(d,J=8.7Hz,0.3H),7.61(d,J=8.7Hz,0.6H),7.41–7.32(m,1H),7. 24(d,J=2.9Hz,1H),7.19(d,J=2.2Hz,1H),7.11(dd,J=8.6,5.2Hz,1H),6.88–6.79(m,1H),6. 79–6.69(m,1H),6.37(d,J=1.4Hz,0.3H),6.15(d,J=1.5Hz,0.6H),3.77(d,J=3.0Hz,3H),3.2 8(s,2H),3.16(s,1H),2.44(d,J=1.3Hz,1H),2.38(d,J=1.2Hz,2H),1.61(s,6H),1.49(s,3H). 13 CNMR(101MHz,d 6 -DMSO)δ161.7,160.8,158.8,155.4,154.1,150.8,148.7,136.3,129.3,127.2,126.6,124.3,118.9,117.5,113.9, 113.4,112.5,110.7,110.5,109.8,109.1,102.7,101.3,84.5,79.9,56.1,55.8,28.6,28.1,27.4,18.7,18.6.HRMS calcd forC 24 H 26 N2NaO7 + [M+Na] + 477.1638, found 477.1639.

[0063] The compound WNO2-Boc (200 mg, 1 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (2 mL) was added. The reaction was stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO2 (154 mg, 98%). 1 H NMR (400MHz, d 6-DMSO) δ7.83(d,J=8.6Hz,0.2H),7.77(d,J=8.7Hz,0.8H),7.38(d,J=2.3Hz,0.2H),7 .32(dd,J=8.7,2.3Hz,0.2H),7.21(d,J=2.3Hz,0.8H),7.15–7.04(m,1.6H),6.99(d, J=8.6Hz,0.2H),6.38(dd,J=8.0,1.4Hz,1H),6.32(t,J=2.5Hz,1H),6.15(dd,J=8.6, 2.8Hz,1H),3.68(s,3H),3.25(s,0.7H),3.14(s,2.3H),2.45(dd,J=9.9,1.2Hz,3H). 13 CNMR(101MHz,d 6 -DMSO)δ160.3,159.8,154.7,153.9,153.8,153.6,145.9,129.2,126.6,120.9,119.0,117.4,113.9,110.5,102.6,100.5,55.3,37.5,18.7.HRMS calcd forC 19 H 19 N2O5 + [M+H] + 355.1294, found 355.1285.

[0064] Synthesis of compound WNO3:

[0065]

[0066] 7-Amino-4-methylcoumarin carboxylic acid chloride (170 mg, 0.72 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.04 mL, 0.24 mmol), DMAP (15 mg, 0.12 mmol), and compound 3-2 (57 mg, 0.24 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and a white solid WNO3-Boc (50 mg, 47%) was obtained by rapid column chromatography separation (ethyl acetate / petroleum ether=1 / 2). 1 H NMR (300MHz, d 6-DMSO)δ9.52(s,1H),8.58(s,1H),7.94(s,1H),7.68(s,1H),7.65–7.59(m,1H),7.43(d,J=8.8Hz,1H),7.31(dd,J=8 .7,2.1Hz,1H),7.06(s,1H),6.71(dd,J=8.9,2.9Hz,1H),6.27–6.16(m,1H),3.72(s,3H),2.39(s,3H),1.45(s,9H). 13 CNMR(75MHz,d 6 -DMSO)δ160.8,156.5,154.6,153.9,153.9,153.6,144.3,132.6,126.5,126.3 ,124.3,114.7,114.2,111.9,110.4,109.8,104.6,80.1,55.8,28.6,18.6.HRMS calcd for C 23 H 25 N3NaO6 + [M+Na] + 462.1641, found 462.1635.

[0067] The compound WNO3-Boc (20 mg, 0.05 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.2 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO3 (12 mg, 70%). 1 H NMR (400MHz, CDCl3) δ9.44(s,1H),8.11(s,1H),7.71–7.60(m,2H),7.31(dd,J=8.7,2.1Hz,1H),7.14(d,J=8.7H z,1H),6.54(d,J=2.8Hz,1H),6.47(d,J=8.7Hz,1H),6.19(d,J=1.3Hz,1H),3.69(s,3H),2.37(d,J=1.2Hz,3H). 13 C NMR (101MHz, CDCl3) δ165.5,162.7,159.3,158.7,158.6,149.1,132.5,131. 2,124.8,122.5,119.6,118.9,116.7,111.5,109.5,109.2,60.5,23.3.HRMS calcd for C 18 H18 N3O4 + [M+H] + 340.1297, found 340.1300.

[0068] Synthesis of compound WNO4:

[0069]

[0070] 7-Amino-4-methylcoumarin carboxylic acid chloride (561 mg, 2.37 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.13 mL, 0.79 mmol), DMAP (47 mg, 0.39 mmol), and compound 3-3 (200 mg, 0.79 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO4-Boc (138 mg, 39%). 1 H NMR(300MHz, CDCl3)δ7.92(d,J=2.9Hz,1H),7.51–7.39(m,2H),7.23–7.11(m,2H),6.80(s,1H),6.68(dd,J=8.7 ,2.9Hz,1H),6.54(s,1H),6.15(d,J=1.3Hz,1H),3.88(s,3H),3.25(s,3H),2.38(d,J=1.3Hz,3H),1.50(s,9H). 13 C NMR (75MHz, CDCl3) δ161.3,160.8,154.8,154.5,152.5,152.3,142.2,137.2,129.0,125 .0,121.8,115.3,115.2,113.1,110.5,106.5,105.0,81.8,55.8,36.8,28.4,18.7.HRMS calcd for C 24 H 27 N3NaO6 + [M+Na] + 476.1798, found 476.1794.

[0071] The compound WNO4-Boc (52 mg, 0.05 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.5 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO4 (33 mg, 85%). 1 H NMR (400MHz, d 6 -DMSO)δ8.16(s,1H),7.72(d,J=2.0Hz,1H),7.61(d,J=8.7Hz,1H),7.46(dd,J=8.8,2.1Hz,1H),7.03(d,J=8.6Hz,1H),6.49(d, J=2.8Hz,1H),6.36(dd,J=8.6,2.8Hz,1H),6.20(d,J=1.3Hz,1H),4.61(s,2H),3.74(s,3H),3.11(s,3H),2.39(d,J=1.2Hz,3H). 13 CNMR(101MHz,d 6 -DMSO)δ160.8,159.9,159.0,158.6,155.0,154.1,153.8,144.6,130.1,125.7,116.1,114.4,114.2,111.9,106.0,102.6,55.5,36.9,18.5.HRMS calcd forC 19 H 20 N3O4 + [M+H] + 354.1454, found 354.1449.

[0072] Synthesis of compound WNO5:

[0073]

[0074] 7-Methylamino-4-methylcoumarin carboxylic acid chloride (333 mg, 1.32 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.07 mL, 0.44 mmol), DMAP (26 mg, 0.22 mmol), and compound 3-2 (104 mg, 0.44 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO5-Boc (109 mg, 55%). 1 H NMR(300MHz, CDCl3)δ7.63(d,J=8.4Hz,1H),7.40–7.31(m,1H),7.21(d,J=8.8Hz,1H),7.08(s,1H),7.05–6 .93(m,2H),6.64(dd,J=8.9,2.9Hz,1H),6.25(s,1H),3.76(s,3H),3.40(s,3H),2.43(s,3H),1.38(s,9H). 13 C NMR (75MHz, CDCl3) δ160.5,157.7,155.5,154.4,154.0,152.0,146.7,133.0,127.2,125 .9,123.0,122.6,118.4,114.9,114.4,110.9,108.5,80.8,55.6,37.5,28.3,18.8.HRMS calcdfor C 24 H 27 N3NaO6 + [M+H] + 476.1798, found 476.1794.

[0075] The compound WNO5-Boc (80 mg, 0.05 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.8 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO5 (49 mg, 77%). 1H NMR(300MHz,CD3OD)δ7.83(d,J=8.3Hz,1H),7.46(d,J=8.5Hz,2H),7.25–7.14(m,1H),6.9 1(d,J=8.1Hz,2H),6.31(d,J=1.4Hz,1H),3.81(s,3H),3.45(s,3H),2.48(d,J=1.3Hz,3H). 13 CNMR(75MHz,CD3OD)δ161.4,158.6,156.4,154.0,153.6,146.5,129.5,128.2, 126.1,124.4,122.5,118.4,114.5,113.8,113.4,108.5,55.0,36.9,17.3.HRMS calcd for C 19 H 20 N3O4 + [M+H] + 354.1454, found 354.1446.

[0076] Synthesis of compound WNO6:

[0077]

[0078] 7-Methylamino-4-methylcoumarin carboxylic acid chloride (406 mg, 1.62 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.09 mL, 0.54 mmol), DMAP (33 mg, 0.27 mmol), and compound 3-3 (272 mg, 1.07 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO6-Boc (193 mg, 62%). 1 H NMR (300MHz, CDCl3) δ7.28(d,J=6.7Hz,1H),7.24(d,J=2.9Hz,1H),6.80–6.71(m,3H),6.39(dd,J=8.7,2.9Hz,1H ),6.35(s,1H),6.19(d,J=1.4Hz,1H),3.66(s,3H),3.16(s,3H),3.02(s,3H),2.35(d,J=1.3Hz,3H),1.51(s,9H). 13C NMR (75MHz, CDCl3) δ161.3,160.5,159.4,153.9,152.0,151.9,147.6,135.3,127.7,125.8 ,124.7,121.8,117.6,114.4,113.8,109.3,103.6,81.1,55.6,39.8,38.9,28.4,18.7.HRMS calcd for C 25 H 29 N3NaO6 + [M+Na] + 490.1954, found 490.1949.

[0079] The compound WNO6-Boc (120 mg, 0.26 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1.2 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO6 (87 mg, 72%). 1 H NMR (300MHz, CD3OD) δ7.56(d,J=8.5Hz,1H),6.98(dd,J=8.5,2.2Hz,1H),6.93(d,J=2.1Hz,1H),6.80(d,J=8.7Hz,1H),6.33(dd ,J=8.7,2.8Hz,1H),6.25(q,J=1.3Hz,1H),6.15(d,J=2.8Hz,1H),3.63(s,3H),3.19(s,3H),3.03(s,3H),2.42(d,J=1.3Hz,3H). 13 C NMR (75MHz, CD3OD) δ161.6,161.4,159.7,153.7,148.2,139.4,128.5,126.2,125 .3,125.3,121.0,117.0,113.0,112.1,106.9,102.9,54.6,38.4,37.0,17.3.HRMS calcd for C 20 H 22 N3O4 + [M+H] + 368.1610, found 368.1605.

[0080] Synthesis of compound WNO7:

[0081]

[0082] Dissolve o-phenylenediamine (2.16 g, 20 mmol) in dichloromethane (100 mL), then cool the reaction solution to 0 ° C, and slowly add Boc2O (872 mg, 4 mmol). Then warm the reaction solution to room temperature and stir for 4 hours. After the reaction is complete, add water (400 mL) to dilute the reaction solution, then add dichloromethane (3×50 mL) to extract, and then combine the organic phases and wash with saturated brine (50 mL), dry over anhydrous sodium sulfate solid and filter. After filtration, the filtrate is concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-4 (760 mg, 91%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0083] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-4 (104mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO7-Boc (136mg, 66%). 1 H NMR (400MHz, d 6 -DMSO)δ9.77(s,1H),8.61(s,1H),8.23(s,1H),7.78(d,J=8.1Hz,1H),7.70(d,J=8.7Hz,1H),7.66(d,J=2.1Hz,1H), 7.39–7.30(m,2H),7.20–7.11(m,1H),7.11–7.02(m,1H),6.24(d,J=1.3Hz,1H),2.41(d,J=1.3Hz,3H),1.47(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.5,154.2,153.8,153.0,144.1,130.2,129.7,126.5,125 .9,125.6,123.8,123.1,114.7,114.3,111.9,104.6,79.8,28.6,18.5.HRMS calcd for C 22 H23 N3NaO5 + [M+Na] + 432.1535, found 432.1532.

[0084] The compound WNO7-Boc (40 mg, 0.09 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.4 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO7 (13 mg, 47%). 1 H NMR (400MHz, d 6 -DMSO)δ9.59(s,1H),8.40(s,1H),7.77–7.62(m,2H),7.43–7.34(m,2H),7.13–7.00(m,2H ),6.94(t,J=7.6Hz,1H),6.25(dd,J=9.3,1.4Hz,1H),3.64(s,2H),2.42(d,J=1.2Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,159.1,158.7,154.5,153.8,153.5,144.1,127.7,126.5,125.9,125.5,119.7,114.8,114.3,112.0,104.8,18.5.HRMS calcd for C 17 H 16 N3O3 + [M+H] + 310.1192, found 310.1186.

[0085] Synthesis of compound WNO8:

[0086]

[0087] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and aniline (0.04mL, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and white solid WNO8 (30mg, 20%) was obtained by rapid column chromatography separation (ethyl acetate / petroleum ether=1 / 2). 1 H NMR (400MHz, d 6 -DMSO)δ9.24(s,1H),8.89(s,1H),7.70(dd,J=8.7,6.1Hz,1H),7.64(d,J=2.1Hz,1H),7.55–7.44 (m,2H),7.40–7.28(m,3H),7.07–6.99(m,1H),6.24(dd,J=7.7,1.4Hz,1H),2.41(d,J=1.3Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.5,153.8,152.7,143.9,139.7,129.4,126.5,122.8,119.0,114.8,114.3,112.0,104.7,18.5.HRMS calcd for C 17 H 15 N2O3 + [M+H] + 295.1083, found 295.1077.

[0088] Synthesis of compound WNO9:

[0089]

[0090] 3-Nitro-4-aminophenol (4g, 25.95mmol) was dissolved in N,N-dimethylformamide (200mL), and iodomethane (1.62mL, 25.95mmol) and NaH (1.04g, 25.95mmol, dispersed in mineral oil, mass fraction 60%) were added in sequence. The reaction was stirred at room temperature overnight. After the reaction was complete, water (400mL) was added to the reaction solution to quench the reaction. Ethyl acetate (3×50mL) was added to extract, and the organic phases were combined and washed with saturated brine (50mL), dried over anhydrous sodium sulfate solid and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a yellow solid 3-5 (3.04g, 68%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0091] Compound 3-5 (3.04 g, 17.91 mmol) was dissolved in dichloromethane (20 mL), and Boc2O (3.91 g, 17.91 mmol) and DMAP (218 mg, 1.79 mmol) were added in sequence. Finally, the reaction was stirred at room temperature overnight. After the reaction was completed, 1N dilute hydrochloric acid (50 mL) was added to quench the reaction. Ethyl acetate (3×20 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate solid and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-6 (1.23 g, 26%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0092] Compound 3-6 (759 mg, 2.83 mmol) was dissolved in ethyl acetate (60 mL) and pyridine (10 mL), and dihydrated stannous chloride (3.19 g, 14.15 mmol) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate (3×10 mL), and then the obtained filtrate was diluted with water (200 mL), and then ethyl acetate (3×40 mL) was added for extraction, and the organic phases were combined and washed with saturated brine (40 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-7 (384 mg, 57%). 1 H NMR (300MHz, CDCl3) δ7.12–7.01(m,1H),6.38–6.28(m,2H),6.00(s,1H),3.76(s,3H),1.51(s,9H).13 C NMR(101MHz, CDCl3)δ158.7,154.5,142.7,127.3,117.2,104.5,102.5,80.4,55.4,28.4.HRMS calcd for C 12 H 19 N2O3 + [M+H] + 239.1396, found 239.1390.

[0093] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-7 (119mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO9-Boc (83mg, 38%). 1 H NMR (500MHz, d 6 -DMSO) δ9.79(s,1H),8.36(s,1H),8.15(s,1H),7.66(d,J=8.7Hz,1H),7.61(d,J=2.1Hz,1H),7.56(s,1H),7.29(dd ,J=8.7,2.1Hz,1H),7.10(s,1H),6.64–6.51(m,1H),6.21(d,J=9.0Hz,1H),3.72(s,3H),2.38(s,3H),1.43(s,9H). 13 C NMR (126MHz, d 6 -DMSO)δ160.6,157.5,154.5,153.7,152.7,143.9,143.4,128.0,126.5,121.4 ,115.0,114.7,114.3,112.2,112.0,105.1,104.6,79.5,55.7,28.6,18.5.HRMS calcd for C 23 H 25 N3NaO6 + [M+Na] + 462.1641, found 462.1638.

[0094] The compound WNO9-Boc (28 mg, 0.26 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.3 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO9 (16 mg, 79%). 1 H NMR (400MHz, d 6 -DMSO) δ9.74(s,1H),8.86(s,1H),7.80–7.69(m,2H),7.40(dd,J=8.7,2.1Hz,1H),7.35(d,J=8.8Hz,1H),7. 03(d,J=2.8Hz,1H),6.92(dd,J=8.8,2.8Hz,1H),6.27(d,J=1.4Hz,1H),3.80(s,3H),2.43(d,J=1.3Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.6,159.4,154.4,153.8,153.5,143.5,133.4,126.5,125.6,119.2,115.1,114.7,112.3,112.0,111.3,105.3,56.1,18.5.HRMS calcd for C 18 H 18 N3O4 + [M+H] + 340.1297, found 340.1292.

[0095] Synthesis of compound WNO10:

[0096]

[0097] 5-Methyl-2-nitroaniline (1.5 g, 10 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL), and Boc2O (2.18 g, 10 mmol) and NaH (400 mg, 10 mmol, dispersed in mineral oil, mass fraction 60%) were added in sequence. The reaction was refluxed overnight. After the reaction was complete, the reaction solution was cooled to room temperature, and then water (200 mL) was added to quench the reaction. Ethyl acetate (3×25 mL) was added to extract, and the organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate solid and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-8 (0.9 g, 36%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0098] Compound 3-8 (0.9 g, 3.6 mmol) was dissolved in ethyl acetate (60 mL) and pyridine (10 mL), and stannous chloride dihydrate (4.06 g, 18 mmol) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate (3×10 mL), and then the obtained filtrate was diluted with water (200 mL), and then ethyl acetate (3×40 mL) was added for extraction, and the organic phases were combined and washed with saturated brine (40 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-9 (381 mg, 43%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0099] 7-Amino-4-methylcoumarin carboxylic acid chloride (355 mg, 1.5 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.08 mL, 0.5 mmol), DMAP (30 mg, 0.25 mmol), and compound 3-9 (111 mg, 0.5 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO10-Boc (154 mg, 60%). 1H NMR(400MHz,d6-DMSO)δ9.64(s,1H),8.55(s,1H),8.08(s,1H),7.69(d,J=8.7Hz,1H),7.67–7.56(m,2H),7.33(dd,J=8.7,2.1Hz,1H),7 .19(d,J=6.9Hz,1H),6.96(dd,J=8.4,2.0Hz,1H),6.24(dd,J=10.8,1.3Hz,1H),2.41(dd,J=3.1,1.2Hz,3H),2.28(s,3H),1.47(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.5,154.2,153.8,153.1,144.1,133.2,129.9,129.4,128.7 ,126.5,126.0,125.8,114.7,114.2,111.9,104.5,79.7,28.6,21.0,18.5.HRMS calcd for C 23 H 25 N3NaO5 + [M+Na] + 446.1692, found 446.1686.

[0100] Compound WNO10-Boc (50 mg, 0.12 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (0.5 mL) was then added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO10 (32 mg, 83%). 1 H NMR (400MHz, d 6 -DMSO)δ9.82(s,1H),8.80(s,1H),7.77–7.66(m,2H),7.44–7.35(m,1H),7.30–7.20(m,1H) ),7.03(d,J=7.1Hz,2H),6.25(dd,J=6.0,1.4Hz,1H),2.42(d,J=1.3Hz,3H),2.31(s,3H). 13 C NMR (101MHz,d 6-DMSO)δ160.7,159.2,158.9,154.5,153.8,153.7,144.0,135.9,127.6,126.5,126.2,122.6,114.9,114.4,112.0,105.0,21.0,18.5.HRMS calcd forC 18 H 18 N3O3 + [M+H] + 324.1348, found 324.1347.

[0101] Synthesis of compound WNO11:

[0102]

[0103] Dissolve 1,2-dimethoxy-4,5-dinitrobenzene (2g, 8.77mmol) in ethanol (44mL), and add dihydrate stannous chloride (19.79g, 87.7mmol) and concentrated hydrochloric acid (35mL) in sequence. Heat the reaction to 88°C and stir overnight. After the reaction is complete, cool the reaction solution to room temperature, then add 4N sodium hydroxide (150mL) to adjust the reaction solution to pH = 10, and a large amount of white precipitate is produced. Filter the precipitate, and wash the filter cake with dichloromethane (3×15mL). Add dichloromethane (3×25mL) to the filtrate for extraction, and then combine the organic phases and wash with saturated brine (50mL), dry over anhydrous sodium sulfate solid and filter. After filtration, the filtrate is concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 1) to obtain a yellow solid 3-10 (1.32g, 90%). .This compound is a known compound, 1 H NMR analysis was consistent with that reported in the literature.

[0104] Compound 3-10 (1.32 g, 7.88 mmol) was dissolved in dichloromethane (50 mL), and then the reaction solution was cooled to 0 ° C, and then Boc2O (872 mg, 4 mmol) was slowly added. Then the reaction solution was warmed to room temperature and stirred for 4 h. After the reaction was complete, water (400 mL) was added to dilute the reaction solution, and then dichloromethane (3×50 mL) was added for extraction, and the organic phases were combined and washed with saturated brine (50 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a black solid 3-11 (420 mg, 60%). This compound is a known compound, 1 H NMR analysis was consistent with that reported in the literature.

[0105] 7-Amino-4-methylcoumarin carboxylic acid chloride (355 mg, 1.5 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.08 mL, 0.5 mmol), DMAP (30 mg, 0.25 mmol), and compound 3-11 (134 mg, 0.5 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO11-Boc (173 mg, 73%). 1 H NMR (400MHz, d 6 -DMSO) δ9.66(s,1H),8.47(s,1H),8.00(s,1H),7.70(s,1H),7.64(d,J=2.1Hz,1H),7.34(dd,J=8.7,2. 1Hz, 2H), 6.93 (s, 1H), 6.23 (d, J = 1.4Hz, 1H), 3.74 (d, J = 6.7Hz, 6H), 2.41 (d, J = 1.3Hz, 3H), 1.47 (s, 9H). 13 CNMR(101MHz,d 6 -DMSO)δ161.1,160.7,154.5,154.3,153.8,153.2,146.9,146.4,145.7,144.2,135. 9,126.5,114.7,114.2,111.9,110.2,104.5,79.6,56.3,56.2,28.6,18.5.HRMScalcd for C 24 H 27 N3NaO7 + [M+Na] + 492.1747, found 492.1742.

[0106] Compound WNO11-Boc (50 mg, 0.12 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.5 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO11 (39 mg, 96%). 1 H NMR (400MHz, d 6-DMSO) δ9.84(s,1H),8.79(s,1H),7.72(dd,J=5.4,3.3Hz,2H),7.39(dd,J=8.7,2.1Hz,1H),6.99(s ,1H),6.92(s,1H),6.25(d,J=1.4Hz,1H),4.18(s,2H),3.79(d,J=2.5Hz,7H),2.42(d,J=1.2Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,159.1,158.8,154.5,153.8,148.0,147.2,143.9,126.5,124.2,115.0,114.5,112.1,110.6,107.2,105.1,56.4,18.5.HRMS calcd for C 19 H 20 N3O5 + [M+H] + 370.1403, found 370.1403.

[0107] Synthesis of compound WNO12:

[0108]

[0109] Dissolve 4-fluoro-1,2-phenylenediamine (1.26g, 10mmol) in acetonitrile (100mL), add potassium bicarbonate (2g, 20mmol), then cool the reaction solution to 0°C, and slowly add Boc2O (872mg, 4mmol). Then warm the reaction solution to room temperature and stir overnight. After the reaction is completed, add water (200mL) to dilute the reaction solution, then add ethyl acetate (3×50mL) for extraction, and combine the organic phases and wash with saturated brine (50mL), dry over anhydrous sodium sulfate solid and filter. After filtration, the filtrate is concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a white solid 3-12 (1.53g, 68%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0110] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-12 (113mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO12-Boc (40mg, 18%). 1 H NMR (400MHz, d 6 -DMSO)δ9.87(s,0.8H),9.57(s,0.2H),8.78(s,0.2H),8.57(s,0.8H),8.35(s,0. 8H),8.11(s,0.2H),7.85(dd,J=11.5,3.0Hz,0.8H),7.71(d,J=8.7Hz,1H),7.64(t ,J=2.3Hz,1H),7.33(dd,J=8.7,2.1Hz,1H),7.26(s,0.8H),7.05–6.94(m,0.2H), 6.93–6.80(m,0.8H),6.36–6.09(m,1H),2.41(d,J=1.2Hz,3H),1.59–1.36(m,9H). 19 F NMR (376MHz, d 6 -DMSO)δ-115.5,-117.9. 13 C NMR (101MHz,d 6 -DMSO) δ161.2,160.7,160.6,158.8,154.5,153.8,153.7,153.3,152.6,144.1,143.6,135.9,128.5,126.6,126.5,12 4.4,114.8,114.7,114.5,114.2,112.1,111.9,109.5,109.2,107.7,104.7,104.6,80.4,79.8,28.6,28.5,18.5.HRMS calcd for C 22 H 22 FN3NaO5 + [M+Na] + 450.1441, found 450.1438.

[0111] The compound WNO12-Boc (40 mg, 0.12 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.4 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO12 (14 mg, 54%). 1 H NMR (400MHz, d 6 -DMSO)δ9.90(s,0.9H),9.44(s,0.1H),8.82(s,0.9H),8.01(s,0.1H),7.78 –7.63(m,2H),7.48–7.30(m,1.9H),7.23(dd,J=8.7,6.2Hz,0.1H),7.12(dd, J=8.8,5.7Hz,0.9H),7.04–6.89(m,0.9H),6.58(dd,J=11.0,2.9Hz,0.1H), 6.46–6.38(m,0.1H),6.29–6.18(m,1H),5.05(s,2H),2.41(d,J=1.2Hz,3H). 19 F NMR (376MHz, d 6 -DMSO)δ-117.5,-119.8. 13 C NMR (101MHz,d 6 -DMSO)δ160.7,157.1,154.5,153.8,153.1,143.8,130.6,129.4,126.5,121.8,1 17.9,114.9,114.5,112.1,111.8,111.5,111.2,110.9,105.0,104.5,18.5.HRMS calcdfor C 17 H 15 FN3O3 + [M+H] + 328.1097, found 328.1091.

[0112] Synthesis of compound WNO13:

[0113]

[0114] Dissolve 2-nitro-5-chloroaniline (4g, 23.18mmol) in N,N-dimethylformamide (50mL), and add dimethylamine aqueous solution (8.8mL, 57.95mmol, mass fraction 33%) and potassium carbonate (5.76g, 41.72mmol) in sequence. Heat the reaction to 150°C and stir for 12h. After the reaction is complete, cool the reaction solution to room temperature, and then pour the reaction solution into ice water to produce a large amount of yellow precipitate. Filter the precipitate and wash the filter cake with water (3×15mL). The filter cake is then concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 1) to obtain a yellow solid 3-13 (1.25g, 30%). This compound is a known compound, 1 H NMR analysis was consistent with literature reports.

[0115] Compound 3-13 (1.25 g, 6.9 mmol) was dissolved in dichloromethane (20 mL), and Boc2O (2.26 g, 10.35 mmol) and DMAP (40 mg, 0.32 mmol) were added in sequence. The reaction solution was stirred at room temperature overnight. After the reaction was completed, water (200 mL) was added to dilute the reaction solution, and dichloromethane (3×50 mL) was added for extraction. The organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate solid, and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 3) to obtain a yellow solid 3-14 (236 mg, 12%). 1 H NMR (300MHz, CDCl3) δ8.13(dd,J=9.4,1.1Hz,1H),6.57(dd,J=9.5,2.8Hz,1H),6.39(d,J=2.8Hz,1H),3.09(d,J=1.2Hz,6H),1.40(s,9H). 13 C NMR (101MHz, CDCl3) δ155.3,152.7,138.9,128.7,125.2,105.7,99.2,81.3,40.3,28.3.HRMS calcd for C 13 H 20 N3O4 + [M+H] + 282.1454, found 282.1449.

[0116] Compound 3-14 (154 mg, 0.55 mmol) was dissolved in methanol (15 mL), 10% Pd / C (15 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred at room temperature overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 3) to obtain a white solid 3-15 (129 mg, 93%). 1 H NMR (300MHz, CDCl3) δ7.05(d,J=2.8Hz,1H),6.75(d,J=8.6Hz,1H),6.66(s,1H),6.47(dd,J=8.6,2.8Hz,1H),3.22(s,2H),2.86(s,6H),1.53(s,9H). 13 C NMR (101MHz, CDCl3) δ153.5,146.4,128.4,128.2,120.4,110.8,108.4,80.3,41.8,28.4.HRMS calcd for C 13 H 22 N3O2 + [M+H] + 252.1712, found 252.1720.

[0117] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-15 (129mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and a white solid WNO13-Boc (100mg, 44%) was obtained by rapid column chromatography separation (ethyl acetate / petroleum ether=1 / 2). 1 H NMR (400MHz, d 6-DMSO) δ9.49(s,1H),8.46(s,1H),7.81(s,1H),7.68(d,J=8.7Hz,1H),7.63(d,J=2.1Hz,1H),7.37–7.27(m,2H),6.8 9–6.80(m,1H),6.55(dd,J=8.9,2.9Hz,1H),6.22(d,J=1.4Hz,1H),2.88(s,6H),2.41(d,J=1.3Hz,3H),1.47(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.5,153.9,153.8,153.7,148.5,144.5,137.4,132.6,126.4 ,114.6,114.0,111.7,109.6,108.3,104.4,102.1,79.7,40.9,28.6,18.5.HRMS calcd for C 24 H 28 N4NaO5 + [M+Na] + 475.1957, found 475.1955.

[0118] The compound WNO13-Boc (50 mg, 0.11 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.5 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO13 (24 mg, 62%). 1 H NMR (400MHz, d 6 -DMSO)δ9.99(s,1H),8.71(s,1H),7.77–7.61(m,2H),7.35(dd,J=8.7,2.1Hz,1H),7.17(d,J=8.6Hz,1H),6. 56(d,J=2.7Hz,1H),6.50(dd,J=8.8,2.7Hz,1H),6.27–6.16(m,1H),4.02(s,2H),2.94(s,6H),2.39(s,3H). 13 CNMR(101MHz,d 6-DMSO)δ160.8,154.5,154.0,153.9,147.0,144.6,138.4,127.0,126.4,119.2,115.0,114.7,114.0,111.7,107.3,104.5,42.3,18.5.HRMS calcd for C 19 H 21 N4O3 + [M+H] + 353.1614, found 353.1607.

[0119] Synthesis of compound WNO14:

[0120]

[0121] Dissolve 2-nitroaniline (2.76 g, 20 mmol) in glacial acetic acid (8 mL), heat the reaction solution to 50 ° C, and then slowly add NBS (3.56 g, 20 mmol). Stir the reaction solution at 50 ° C for 3 h. After the reaction is complete, cool the reaction solution to room temperature, and then pour the reaction solution into ice water to produce a large amount of yellow precipitate. Filter the precipitate and wash the filter cake with water (3×15 mL). The filter cake is then concentrated under reduced pressure by a vacuum pump and recrystallized from ethanol to obtain a yellow solid 3-16 (3.3 g, 76%). This compound is a known compound, 1 H NMR analysis was consistent with that reported in the literature.

[0122] Compound 3-16 (3.3 g, 15.34 mmol) was dissolved in anhydrous tetrahydrofuran (200 mL), and Boc2O (3.34 g, 15.34 mmol) and NaH (1.22 g, 30.68 mmol, dispersed in mineral oil, mass fraction 60%) were added in sequence. The reaction was refluxed overnight. After the reaction was complete, the reaction solution was cooled to room temperature, and then water (400 mL) was added to quench the reaction. Ethyl acetate (3×50 mL) was added to extract, and the organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate solid and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-17 (4.75 g, 98%). This compound is a known compound, 1 H NMR analysis was consistent with that reported in the literature.

[0123] Palladium acetate (45 mg, 0.2 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (187 mg, 0.3 mmol) and sodium tert-butoxide (134 mg, 1.4 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (316 mg, 1 mmol) was dissolved in anhydrous toluene (10 mL), and dimethylamine (0.9 mL, 1.8 mmol, 2 M solution of tetrahydrofuran) was added, and the solution was added to the sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-18 (136 mg, 44%). 1 H NMR (400MHz, CDCl3) δ9.21 (s, 1H), 8.32 (d, J = 9.4Hz, 1H), 7.42 (d, J = 3.0Hz, 1H), 7.07 (dd, J = 9.4, 3.0Hz, 1H), 3.01 (s, 6H). 13 C NMR(101MHz, CDCl3)δ152.8,145.8,137.4,125.6,122.5,120.6,107.1,81.1,40.6,28.3.HRMS calcd for C 13 H 19 N3NaO4 + [M+Na] + 304.1273, found 304.1267.

[0124] Compound 3-18 (136 mg, 0.44 mmol) was dissolved in methanol (10 mL), 10% Pd / C (14 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump to obtain a colorless transparent oily liquid 3-19 (95 mg, 86%). 1 H NMR (400MHz, CDCl3) δ7.01(d,J=8.7Hz,1H),6.22(dd,J=8.7,2.7Hz,1H),6.16(d,J=2.7Hz,1H),5.98(s,1H),2.92(s,6H),1.51(s,9H). 13C NMR(101MHz, CDCl3)δ150.2,142.4,129.9,127.3,114.0,104.5,101.3,80.1,40.9,28.4.HRMS calcd for C 13 H 22 N3O2 + [M+H] + 252.1712, found 252.1705.

[0125] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-19 (125mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO14-Boc (162mg, 72%). 1 H NMR (400MHz, d 6 -DMSO)δ9.79(s,1H),8.22(s,1H),8.08(s,1H),7.74–7.62(m,2H),7.31(dd,J=8.8,2.0Hz,2H) ,7.02(s,1H),6.51–6.37(m,1H),6.22(d,J=1.4Hz,1H),2.88(s,6H),2.40(s,3H),1.45(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.8,155.0,154.5,153.9,152.9,149.2,144.0,134.8,127.8,126.7 ,126.5,118.2,114.7,114.2,111.9,107.9,104.6,79.3,40.9,28.6,18.5.HRMS calcd forC 24 H 28 N4NaO5 + [M+Na] + 475.1957, found 475.1954.

[0126] The compound WNO14-Boc (66 mg, 0.11 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.6 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO14 (34 mg, 64%). 1 H NMR (400MHz, d 6 -DMSO) δ9.88(s,1H),8.76(s,1H),7.71(dd,J=5.4,3.3Hz,2H),7.38(dd,J=8.7,2.1Hz,1 H),7.00(s,2H),6.70(d,J=9.2Hz,1H),6.24(d,J=1.5Hz,1H),2.91(s,6H),2.42(s,3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.5,153.8,153.5,144.1,142.8,140.8,138.8,133.9,132.7,126.5,126.2,114.9,114.3,112.0,104.8,42.3,18.5.HRMS calcd forC 19 H 21 N4O3 + [M+H] + 353.1614, found 353.1610.

[0127] Synthesis of compound WNO15:

[0128]

[0129] The compound WNO3-Boc (174 mg, 0.4 mmol) was dissolved in dichloromethane (10 mL), and the reaction solution was cooled to -45 ° C, and then boron tribromide (2 mL, 2 mmol, 1 M dichloromethane solution) was added. The reaction solution was warmed to room temperature and stirred for 1 h. After the reaction was completed, ice water (50 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO15 (73 mg, 52%). 1 H NMR (400MHz, d 6-DMSO)δ9.20(s,1H),9.00(s,1H),7.69–7.63(m,2H),7.32(dd,J=8.7,2.1Hz,1H),6.94(d,J= 8.4Hz, 1H), 6.22 (dd, J=11.0, 2.1Hz, 2H), 6.06 (dd, J=8.4, 2.7Hz, 1H), 2.41 (d, J=1.2Hz, 3H). 13 C NMR (101MHz,d 6 -DMSO)δ160.8,156.1,154.6,153.9,153.9,144.6,143.6,127.7,126.4,116.0,114.6,113.9,111.7,104.9,104.3,103.0,18.5.HRMS calcd for C 17 H 16 N3O4 + [M+H] + 326.1141, found 326.1144.

[0130] Synthesis of compound WNO16:

[0131]

[0132] The compound WNO9-Boc (100 mg, 0.23 mmol) was dissolved in dichloromethane (10 mL), and the reaction solution was cooled to -45 ° C, and then boron tribromide (2.3 mL, 2.3 mmol, 1 M dichloromethane solution) was added. The reaction solution was warmed to room temperature and stirred for 1 h. After the reaction was completed, ice water (50 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO16 (60 mg, 80%). 1 H NMR (400MHz, d 6 -DMSO)δ9.75(s,1H),9.57(s,2H),8.89(s,1H),7.79–7.68(m,2H),7.38(dd,J=8.7,2.1Hz,1H),7.25(d, J=8.7Hz,1H),6.85(d,J=2.6Hz,1H),6.73(dd,J=8.7,2.7Hz,1H),6.26(s,1H),4.88(s,2H),2.42(s,3H). 13 C NMR (101MHz,d 6-DMSO)δ160.6,158.0,154.4,153.8,153.5,143.5,138.0,133.3,126.5,125.8,117.0,115.0,114.7,113.4,112.3,105.2,18.5.HRMS calcdfor C 17 H 16 N3O4 + [M+H] + 326.1141, found 326.1135.

[0133] Synthesis of compound WNO17:

[0134]

[0135] Palladium acetate (45 mg, 0.2 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (187 mg, 0.3 mmol) and sodium tert-butoxide (134 mg, 1.4 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (316 mg, 1 mmol) was dissolved in anhydrous toluene (10 mL), and diethylamine (0.19 mL, 1.8 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-20 (136 mg, 43%). 1 HNMR (400MHz, CDCl3) δ9.11 (s, 1H), 8.24 (d, J = 9.4Hz, 1H), 7.37 (d, J = 3.1Hz, 1H), 7. 00(dd,J=9.4,3.2Hz,1H),3.39(q,J=7.1Hz,4H),1.56(s,9H),1.19(t,J=7.0Hz,6H). 13 C NMR(101MHz, CDCl3)δ152.9,143.3,138.0,124.4,122.9,120.0,106.5,81.0,44.5,28.3,12.4.HRMS calcd for C 15 H 23 N3NaO4 + [M+Na] + 332.1586, found 332.1580.

[0136] Compound 3-20 (136 mg, 0.43 mmol) was dissolved in methanol (10 mL), 10% Pd / C (14 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump to obtain a colorless transparent oily liquid 3-21 (82 mg, 67%). 1 H NMR (400MHz, CDCl3) δ6.96 (d, J=8.7Hz, 1H), 6.15 (dd, J=8.7, 2.8Hz, 1H), 6.09 (d, J=2.8Hz ,1H),5.96(s,1H),3.78(s,2H),3.32(q,J=7.1Hz,4H),1.53(s,9H),1.16(t,J=7.1Hz,6H). 13 C NMR (101MHz, CDCl3) δ154.9,147.4,142.8,127.7,112.7,103.7,80.0,44.5,28.4,12.7.HRMS calcd for C 15 H 26 N3O2 + [M+H] + 280.2025, found 280.2019.

[0137] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (10mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-21 (139mg, 0.5mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO17-Boc (50mg, 72%). 1 H NMR (400MHz, d 6-DMSO)δ9.79(s,1H),8.19(s,1H),8.05(s,1H),7.77–7.61(m,2H),7.31(dd,J=9.0,2.3Hz,2H),7.06–6.90(m,1H),6.36(dd ,J=8.7,2.9Hz,1H),6.23(d,J=1.4Hz,1H),3.32(q,J=6.9Hz,4H),2.41(d,J=1.2Hz,3H),1.47(s,9H),1.11(t,J=6.9Hz,6H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,155.1,154.5,153.8,152.9,146.2,144.1,135.2,130.2,128.1,12 6.5,117.0,114.7,114.2,111.9,107.2,104.6,79.2,44.5,28.7,18.5,13.0.HRMS calcd for C 26 H 33 N4O5 + [M+H] + 481.2451, found 481.2449.

[0138] Compound WNO17-Boc (49 mg, 0.10 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.4 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO17 (29 mg, 76%). 1 H NMR (400MHz, d 6 -DMSO)δ9.36(s,1H),7.87(s,1H),7.61–7.50(m,2H),7.21(dd,J=8.6,2.2Hz,1H),6.93(s,1H),6.58(d,J=8.6Hz ,1H),6.26(d,J=8.7Hz,1H),6.09(s,1H),4.18(s,2H),3.06(q,J=7.1Hz,4H),2.28(s,3H),0.92(t,J=7.0Hz,6H). 13 C NMR (101MHz,d 6-DMSO)δ160.8,154.5,153.8,153.1,144.4,141.2,131.2,130.1,127.1,126.4,118.5,114.6,114.0,111.7,110.3,104.4,45.3,18.5,12.9.HRMS calcd for C 21 H 25 N4O3 + [M+H] + 381.1927, found 381.1920.

[0139] Synthesis of compound WNO18:

[0140]

[0141] Palladium acetate (45 mg, 0.2 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (187 mg, 0.3 mmol) and sodium tert-butoxide (134 mg, 1.4 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (316 mg, 1 mmol) was dissolved in anhydrous toluene (10 mL), piperidine (153 mg, 1.8 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-22 (106 mg, 33%). 1 HNMR (400MHz, CDCl3) δ9.35 (s, 1H), 8.36 (d, J = 9.3Hz, 1H), 7.64 (d, J = 3.0Hz, 1H), 7.2 9–7.25(m,1H),3.24–3.13(m,4H),1.78–1.72(m,4H),1.66–1.62(m,2H),1.56(s,9H). 13 C NMR(101MHz, CDCl3)δ147.1,141.4,127.8,125.1,122.0,119.0,111.1,81.3,50.5,28.3,25.6,24.0.HRMS calcd for C 16 H 24 N3O4 + [M+H] + 322.1767, found 322.1762.

[0142] Compound 3-22 (106 mg, 0.33 mmol) was dissolved in methanol (10 mL), 10% Pd / C (11 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump to obtain a colorless transparent oily liquid 3-23 (77 mg, 80%). 1 H NMR(400MHz,CDC l3) δ7.04(d,J=8.6Hz,1H),6.41(dd,J=8.6,2.7Hz,1H),6.37(d,J=2.6Hz,1H),6.04(s, 1H),3.79(s,2H),3.16–3.06(m,4H),1.77–1.65(m,6H),1.62–1.56(m,2H),1.53(s,9H). 13 C NMR (101MHz, CDCl3) δ154.5,151.6,141.9,126.7,116.1,108.3,105.3,80.3,51.0,28.4,25.8,24.4.HRMS calcd forC 16 H 26 N3O2 + [M+H] + 292.2025, found 292.2017.

[0143] 7-Amino-4-methylcoumarin carboxylic acid chloride (128 mg, 0.54 mmol) was dissolved in anhydrous dichloromethane (5 mL), and DIPEA (0.03 mL, 0.18 mmol), DMAP (11 mg, 0.09 mmol), and compound 3-23 (53 mg, 0.18 mmol) were added in sequence, and the reaction was stirred at room temperature for 30 min. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO18-Boc (37 mg, 24%). 1 H NMR (300MHz, d 6-DMSO)δ9.74(s,1H),8.27(s,1H),8.04(s,1H),7.68(s,1H),7.65–7.59( m,1H),7.47(s,1H),7.29(dd,J=8.7,2.1Hz,1H),7.01(d,J=8.7Hz,1H),6 .59(dd,J=8.9,2.6Hz,1H),6.20(d,J=1.3Hz,1H),3.07(t,J=5.3Hz,4H), 2.38(d,J=1.2Hz,3H),1.65–1.55(m,4H),1.55–1.48(m,2H),1.42(s,9H). 13 CNMR(75MHz,d 6 -DMSO)δ160.7,154.9,154.6,153.8,153.0,150.2,144.1,132.3,127.5,126.6,120. 3,114.8,114.3,112.0,111.5,109.7,104.7,79.4,50.5,28.7,25.8,24.5,18.6.HRMS calcd for C 27 H 33 N4O5 + [M+H] + 493.2451, found 493.2442.

[0144] Compound WNO18-Boc (20 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was then added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO18 (12 mg, 77%). 1 H NMR (500MHz, d 6 -DMSO)δ9.39(s,1H),7.89(s,1H),7.68–7.58(m,2H),7.31(dd,J=8.7,2.1Hz,1H),7.21–7.09(m,1H),6.66(d,J=8.6Hz,1H),6. 53(t,J=5.0Hz,1H),6.19(q,J=1.2Hz,1H),2.90(t,J=5.5Hz,4H),2.38(d,J=1.3Hz,3H),1.69–1.56(m,4H),1.50–1.43(m,2H). 13 C NMR (126MHz, d 6-DMSO)δ160.7,154.5,153.7,153.1,144.3,133.8,126.4,126.1,118.0,11 7.6,114.6,114.5,114.0,113.3,111.8,104.4,52.3,26.1,24.3,18.5.HRMS calcd forC 22 H 25 N4O3 + [M+H] + 393.1927, found 393.1921.

[0145] Synthesis of compound WNO19:

[0146]

[0147] Palladium acetate (45 mg, 0.2 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (187 mg, 0.3 mmol) and sodium tert-butoxide (134 mg, 1.4 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (316 mg, 1 mmol) was dissolved in anhydrous toluene (10 mL), and N-methylbenzylamine (218 mg, 1.8 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-24 (109 mg, 30%). 1 H NMR (400MHz, CDCl3) δ9.19 (s, 1H), 8.27 (d, J = 9.4Hz, 1H), 7.46 (d, J = 3.1Hz, 1H), 7.39–7.32 (m, 2H), 7.28 (d,J=7.4Hz,1H),7.25–7.20(m,2H),7.07(dd,J=9.4,3.1Hz,1H),4.58(s,2H),3.09(s,3H),1.55(s,9H). 13 HRMS calcd for C 19 H 24 N3O4 + [M+H]+ 358.1767, found 358.1762.

[0148] Compound 3-24 (109 mg, 0.31 mmol) was dissolved in ethanol (5 mL) and tetrahydrofuran (2.5 mL), and iron powder (174 mg, 3.1 mmol) and ammonium chloride (10 mg, 0.18 mmol) were added in sequence. The reaction system was then heated to reflux and stirred overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth to remove excess iron powder, and the obtained filtrate was extracted with ethyl acetate (3×10 mL). The filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a brown transparent oily liquid 3-25 (65 mg, 64%). 1 H NMR (500MHz, CDCl3) δ7.33–7.28(m,2H),7.28–7.18(m,3H),6.95(d,J=8.7Hz,1H),6.19(dd,J=8.6 ,2.8Hz,1H),6.11(t,J=2.1Hz,1H),5.96(s,1H),4.48(s,2H),2.96(d,J=1.6Hz,3H),1.49(s,9H). 13 HRMS calcd for C 19 H 26 N3O2 + [M+H] + 328.2025, found 328.2021.

[0149] 7-Amino-4-methylcoumarin carboxylic acid chloride (384 mg, 1.62 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.09 mL, 0.54 mmol), DMAP (33 mg, 0.27 mmol), and compound 3-25 (175 mg, 0.54 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO19-Boc (195 mg, 68%). 1H NMR (400MHz, d 6 -DMSO)δ9.74(s,1H),8.17(s,1H),8.03(s,1H),7.65(d,J=8.6Hz,1H),7.60(d,J=2.1Hz,1H),7.36(s,1H),7.29(q,J=6.3,4.4H z,3H),7.20(d,J=8.3Hz,3H),6.95(s,1H),6.47–6.34(m,1H),6.19(s,1H),4.52(s,2H),2.96(s,3H),2.37(s,3H),1.42(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,155.0,154.6,153.8,153.0,148.0,144.1,139.5,130.2,129.0,128.0,127.5,127. 3,126.5,118.1,114.8,114.3,112.0,107.8,105.7,104.7,79.3,56.1,39.2,28.7,18.6.HRMScalcd for C30H31N4O5 - [MH] - 527.2294, found 527.2297.

[0150] The compound WNO19-Boc (195 mg, 0.37 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1.9 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO19 (98 mg, 62%). 1 H NMR (400MHz, d 6 -DMSO)δ9.92(s,1H),9.07(s,1H),7.79–7.67(m,2H),7.43–7.30(m,3H),7.30–7.19(m,3H),7.12(d,J =8.5Hz,1H),6.76(s,1H),6.66(d,J=8.8Hz,1H),6.25(s,1H),4.60(s,2H),3.04(s,3H),2.41(s,3H). 13 C NMR (101MHz,d 6-DMSO)δ160.7,154.4,153.8,153.7,145.1,144.6,144.1,143.8,138.7,129.0,1 27.7,127.5,126.5,122.3,117.2,115.2,115.0,114.5,112.1,105.1,18.5.HRMS calcd for C 25 H 25 N4O3 + [M+H] + 429.1927, found 429.1922.

[0151] Synthesis of compound WNO20:

[0152]

[0153] Palladium acetate (403 mg, 1.8 mmol) was dissolved in anhydrous toluene (10 mL), BINAP (1.68 g, 2.7 mmol) and sodium tert-butoxide (672 mg, 7 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous toluene (10 mL), azocyclobutane (513 mg, 9 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3 × 20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and yellow solid 3-26 (400 mg, 23%) was obtained by rapid column chromatography separation (ethyl acetate / petroleum ether = 1 / 10). 1 HNMR(500MHz, CDCl3)δ9.19(s,1H),8.26(d,J=9.1Hz,1H),7.10(d,J=2.8Hz,1H),6 .72(dd,J=9.1,2.8Hz,1H),3.89(t,J=7.2Hz,4H),2.54–2.33(m,2H),1.52(s,9H). 13 C NMR (126MHz, CDCl3) δ152.7,147.3,137.4,126.2,122.5,119.5,106.2,81.2,52.7,28.3,16.9.HRMS calcd for C 14 H 19 N3NaO4[M+Na] + 316.1273, found 316.1268.

[0154] Compound 3-26 (200 mg, 0.83 mmol) was dissolved in methanol (10 mL), 10% Pd / C (20 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred at room temperature overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a yellow solid 3-27 (123 mg, 56%). 1 H NMR (500MHz, CDCl3) δ6.94 (d, J=8.4Hz, 1H), 5.95 (s, 1H), 5.88 (dd, J=8.4, 2.5Hz, 1H),5.82(d,J=2.5Hz,1H),3.79(t,J=7.3Hz,4H),2.36–2.45(m,2H),1.49(s,9H). 13 C NMR(126MHz, CDCl3)δ151.9,142.5,129.9,127.4,114.4,102.9,99.7,80.1,52.5,28.4,16.8.HRMScalcd for C 14 H 22 N3O2 + [M+H] + 264.1712, found 264.1716.

[0155] 7-Amino-4-methylcoumarin carboxylic acid chloride (140 mg, 0.59 mmol) was dissolved in anhydrous dichloromethane (5 mL), and DIPEA (0.29 mL, 1.77 mmol), DMAP (36 mg, 0.3 mmol), and compound 3-27 (156 mg, 0.1 mmol) were added in sequence, and the reaction was stirred at room temperature for 30 min. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO20-Boc (132 mg, 48%). 1 H NMR (500MHz, d 6-DMSO)δ9.77(s,1H),8.20(s,1H),8.04(s,1H),7.66(d,J=8.7Hz,1H),7.60(d,J=2.1Hz,1H),7.29(dd,J=8.7,2.1Hz,1H),7.01(s,1H), 6.95(s,1H),6.20(d,J=1.3Hz,1H),6.06(d,J=8.6Hz,1H),3.75(t,J=7.1Hz,4H),2.38(d,J=1.2Hz,3H),2.34–2.22(m,2H),1.43(s,9H). 13 CNMR(126MHz,d 6 -DMSO)δ160.7,155.2,154.6,153.7,152.9,150.9,144.1,130.3,127.9,126.6,11 8.6,114.7,114.3,112.0,108.6,106.4,104.7,79.2,52.6,28.6,18.5,16.8.HRMS calcd forC 25 H 28 N4NaO5 + [M+Na] + 487.1957, found 487.1950.

[0156] The compound WNO20-Boc (132 mg, 0.28 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1.3 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO20 (78 mg, 77%). 1 H NMR (500MHz, d 6 -DMSO)δ9.78(s,1H),8.58(s,1H),7.68–7.64(m,2H),7.33(dd,J=8.7,2.2Hz,1H),6.88(d,J=8.4Hz,1H), 6.60(d,J=2.5Hz,1H),6.24–6.14(m,2H),3.74(t,J=7.2Hz,4H),2.38(d,J=1.3Hz,3H),2.30–2.20(m,2H). 13 C NMR (126MHz, d 6-DMSO)δ160.6,154.5,153.7,153.3,148.8,144.1,140.0,126.4,121.0,114.8,114.2,111.9,108.9,108.5,107.5,104.7,53.0,31.2,18.5.HRMS calcd for C 20 H 21 N4O3 + [M+H] + 365.1614, found 365.1611.

[0157] Synthesis of compound WNO21:

[0158]

[0159] Palladium acetate (403 mg, 1.8 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (1.68 g, 2.7 mmol) and sodium tert-butoxide (672 mg, 7 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous toluene (10 mL), and tetrahydropyrrole (639 mg, 9 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-28 (488 mg, 32%). 1 HNMR(500MHz, CDCl3)δ9.09(s,1H),8.23(d,J=9.2Hz,1H),7.19(d,J=3.0Hz,1H) ,6.84(dd,J=9.2,3.0Hz,1H),3.38–3.17(m,4H),2.13–1.96(m,4H),1.52(s,9H). 13 C NMR(126MHz, CDCl3)δ152.8,143.4,137.8,124.4,122.7,119.6,105.9,80.9,47.9,28.3,25.5.HRMScalcd for C 15 H 21 N3NaO4 + [M+Na] + 330.1430, found 330.1424.

[0160] Compound 3-28 (488 mg, 1.59 mmol) was dissolved in methanol (10 mL), 10% Pd / C (49 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred at room temperature overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a yellow solid 3-29 (354 mg, 80%). 1 H NMR (400MHz, CDCl3) δ6.94 (d, J=8.5Hz, 1H), 6.00 (dd, J=8.5, 2.6Hz, 1H), 5.94 (d ,J=2.6Hz,1H),3.76(s,2H),3.37–3.10(m,4H),2.10–1.88(m,4H),1.49(s,9H). 13 C NMR(101MHz, CDCl3)δ154.9,147.7,142.9,127.8,112.9,103.4,99.8,80.0,47.8,28.5,25.6.HRMScalcd for C 15 H 24 N3O2 + [M+H] + 278.1869, found 278.1860.

[0161] 7-Amino-4-methylcoumarin carboxylic acid chloride (355mg, 1.5mmol) was dissolved in anhydrous dichloromethane (5mL), and DIPEA (0.08mL, 0.5mmol), DMAP (30mg, 0.25mmol), and compound 3-29 (354mg, 1.27mmol) were added in sequence, and the reaction was stirred at room temperature for 30min. After the reaction was completed, water (20mL) was added to quench the reaction. Dichloromethane (3×10mL) was added to extract, and the organic phases were combined and washed with saturated brine (20mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO21-Boc (218mg, 36%). 1 H NMR (500MHz, d 6-DMSO)δ9.75(s,1H),8.15(s,1H),8.02(s,1H),7.70–7.59(m,2H),7.34–7.25(m,1H),7.15(s,1H),7.03–6.90 (m,1H),6.19(d,J=10.7Hz,2H),3.19(d,J=5.7Hz,4H),2.36(d,J=3.0Hz,3H),2.00–1.87(m,4H),1.43(s,9H). 13 C NMR (126MHz, d 6 -DMSO) δ160.6,155.1,154.5,153.7,152.8,146.6,144.1,135.1,130.1,128.1,126. 4,117.1,114.6,114.2,111.9,107.1,104.5,79.7,79.1,48.0,28.6,25.5,18.5.HRMS calcd for C 26 H 31 N4O5 + [M+H] + 479.2294, found 479.2288.

[0162] The compound WNO21-Boc (218 mg, 0.46 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (2 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO21 (131 mg, 75%). 1 H NMR (500MHz, d 6 -DMSO)δ9.46(s,1H),7.97(s,1H),7.71–7.58(m,2H),7.31(dd,J=8.7,2.1Hz,1H),6.85(s,1H), 6.69(d,J=8.5Hz,1H),6.26–6.12(m,2H),3.10(s,4H),2.38(d,J=1.2Hz,3H),2.01–1.87(m,4H). 13 C NMR (126MHz, d 6 -DMSO)δ160.7,154.5,153.8,153.0,144.3,141.6,136.9,130.1,126.4,119.6,119.2,114.6,114.0,111.8,107.3,104.4,48.4,25.3,18.5.HRMS calcd for C21 H 23 N4O3 + [M+H] + 379.1770, found 379.1766.

[0163] Synthesis of compound WNO22:

[0164]

[0165] Palladium acetate (403 mg, 1.8 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (1.68 g, 2.7 mmol) and sodium tert-butoxide (672 mg, 7 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous toluene (10 mL), morpholine (783 mg, 9 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-30 (355 mg, 22%). 1 HNMR (500MHz, CDCl3) δ9.57 (s, 1H), 8.62 (d, J = 9.3Hz, 1H), 7.83 (d, J = 3.0Hz, 1H), 7.45 (dd ,J=9.3,3.0Hz,1H),4.09(dd,J=5.6,3.9Hz,4H),3.38(dd,J=5.8,3.8Hz,4H),1.76(s,9H). 13 C NMR (101MHz, CDCl3) δ152.6,146.1,136.8,128.7,124.2,122.1,110.7,81.5,66.7,49.2,28.3.HRMS calcd for C 15 H 21 N3NaO5 + [M+Na] + 346.1379, found 346.1374.

[0166] Compound 3-30 (335 mg, 1.04 mmol) was dissolved in methanol (10 mL), 10% Pd / C (34 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a white solid 3-31 (224 mg, 73%). 1 H NMR (500MHz, CDCl3) δ7.03 (d, J=8.6Hz, 1H), 6.33 (dd, J=8.7, 2.7Hz, 1H), 6.28 (d ,J=2.7Hz,1H),6.11(s,1H),3.89–3.76(m,4H),3.14–2.98(m,4H),1.49(s,9H). 13 C NMR(126MHz, CDCl3)δ154.4,150.5,142.1,126.9,116.9,107.2,104.4,80.3,66.9,49.6,28.4.HRMScalcd for C 15 H 24 N3O3 + [M+H] + 294.1818, found 294.1816.

[0167] 7-Amino-4-methylcoumarin carboxylic acid chloride (547 mg, 2.31 mmol) was dissolved in anhydrous dichloromethane (5 mL), and DIPEA (0.13 mL, 0.77 mmol), DMAP (48 mg, 0.39 mmol), and compound 3-31 (224 mg, 0.77 mmol) were added in sequence, and the reaction was stirred at room temperature for 30 min. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO22-Boc (236 mg, 62%). 1 H NMR (400MHz, d 6-DMSO)δ9.78(s,1H),8.34(s,1H),8.12(s,1H),7.75–7.59(m,2H),7.53(s,1H),7.31(d,J=8.7Hz,1H),7.10(d,J=8. 7Hz,1H),6.65(d,J=8.9Hz,1H),6.21(s,1H),3.76(t,J=4.5Hz,4H),3.08(t,J=3.8Hz,4H),2.40(s,3H),1.45(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.8,154.5,153.8,152.9,149.4,144.0,134.5,127.4,126.5,12 1.0,114.7,114.3,111.9,110.8,109.0,104.6,79.5,66.6,49.3,28.6,18.5.HRMS calcd for C 26 H 30 N4NaO6 + [M+Na] + 517.2063, found 517.2057.

[0168] The compound WNO22-Boc (140 mg, 0.28 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1.4 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO22 (78 mg, 70%). 1 H NMR (500MHz, d 6 -DMSO)δ10.01(s,1H),8.97(s,1H),7.72–7.63(m,2H),7.35(dd,J=8.7,2.1Hz,1H),7.09–6.97(m,2H),6.78(dd,J =8.8, 2.8Hz, 1H), 6.20 (d, J = 1.4Hz, 1H), 3.72 (dd, J = 6.0, 3.5Hz, 4H), 3.06 (t, J = 4.8Hz, 4H), 2.38 (d, J = 1.3Hz, 3H). 13 C NMR (126MHz, d 6-DMSO)δ160.6,154.5,153.7,153.5,152.7,144.0,130.4,130.1,126.4,122.2,114.9,114.3,113.2,112.1,112.0,104.9,66.4,49.6,18.5.HRMS calcd for C 21 H 23 N4O4 + [M+H] + 395.1719, found 395.1712.

[0169] Synthesis of compound WNO23:

[0170]

[0171] Palladium acetate (224 mg, 1 mmol) was dissolved in anhydrous toluene (10 mL), and BINAP (933 mg, 1.5 mmol) and sodium tert-butoxide (672 mg, 7 mmol) were added in sequence, and the reaction solution was placed in a sealed tube and stirred at room temperature for 30 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous toluene (10 mL), and N-methylaniline (5.35 g, 50 mmol) was added, and the solution was added to a sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered through diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-32 (144 mg, 8%). 1 H NMR(400MHz, CDCl3) δ9.34(s,1H),8.30(d,J=9.3Hz,1H),7.69(d,J=2.9Hz,1H),7.36– 7.29(m,2H),7.22(dd,J=9.3,2.9Hz,1H),7.10–7.02(m,3H),3.33(s,3H),1.53(s,9H). 13 CNMR(101MHz, CDCl3)δ152.7,148.0,143.9,137.1,129.8,128.8,126.9,123.4,122.4,122.1,113.4,81.6,40.5,28.4.HRMS calcd for C 18 H 22 N3O4 + [M+H] + 344.1610, found 344.1606.

[0172] Compound 3-32 (119 mg, 0.35 mmol) was dissolved in ethanol (5 mL) and tetrahydrofuran (2.5 mL), and iron powder (192 mg, 3.5 mmol) and ammonium chloride (93 mg, 1.75 mmol) were added in sequence. The reaction system was then heated to reflux and stirred overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth to remove excess iron powder, and the obtained filtrate was extracted with ethyl acetate (3×10 mL). The filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a white solid 3-33 (78 mg, 71%). 1 H NMR (500MHz, CDCl3) δ7.27–7.22(m,2H),7.08(d,J=8.4Hz,1H),7.05–6.97(m,2H),6.9 5–6.86(m,1H),6.49–6.40(m,2H),6.07(s,1H),3.77(s,2H),3.25(s,3H),1.51(s,9H). 13 HRMS calcd for C 18 H 24 N3O2 + [M+H] + 314.1869, found 314.1859.

[0173] 7-Amino-4-methylcoumarin carboxylic acid chloride (467 mg, 2.01 mmol) was dissolved in anhydrous dichloromethane (5 mL), and DIPEA (0.33 mL, 2.01 mmol), DMAP (41 mg, 0.34 mmol), and compound 3-33 (211 mg, 0.67 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO23-Boc (189 mg, 55%). 1 H NMR (400MHz, d 6-DMSO)δ9.72(s,1H),8.42(s,1H),8.14(s,1H),7.65(d,J=8.7Hz,1H),7.62–7.54(m,2H),7.33–7.19(m,3H),7.15(d,J=8.7Hz,1H),7.0 0–6.94(m,2H),6.94–6.85(m,1H),6.70(dd,J=8.8,2.6Hz,1H),6.19(d,J=1.4Hz,1H),3.23(s,3H),2.37(d,J=1.3Hz,3H),1.44(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ160.7,154.7,154.6,153.8,152.9,149.2,146.5,144.0,134.5,129.7,127.4,126.6 ,123.2,121.0,119.6,116.4,115.1,114.8,114.4,112.0,104.7,79.7,40.7,28.7,18.6.HRMS calcd for C 29 H 31 N4O5 + [M+H] + 515.2294, found 515.2206.

[0174] The compound WNO23-Boc (189 mg, 0.37 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1.8 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO23 (112 mg, 73%). 1 H NMR (500MHz, d 6 -DMSO) δ9.93(s,1H),9.18(s,1H),7.67(dd,J=5.4,3.3Hz,2H),7.39–7.30(m,3H),7.19(d,J=8.8Hz,1H),7.11(d,J=7.9Hz,1H),7. 06(t,J=7.4Hz,1H),6.86(d,J=2.7Hz,1H),6.76(dd,J=8.8,2.7Hz,1H),6.20(d,J=1.5Hz,1H),3.25(s,3H),2.37(d,J=1.3Hz,3H). 13 C NMR (126MHz, d 6-DMSO)δ160.6,154.4,153.7,153.6,148.3,143.7,132.5,130.0,126.4,124.7,123 .5,123.0,117.4,115.3,115.0,114.5,114.2,112.1,108.0,105.2,40.6,18.5.HRMS calcd for C 24 H 23 N4O3 + [M+H] + 415.1770, found 415.1761.

[0175] Synthesis of compound WNO24:

[0176]

[0177] Palladium acetate (224 mg, 1 mmol) was dissolved in anhydrous 1,4-dioxane (15 mL), and Xantphos (867 mg, 1.5 mmol) and cesium carbonate (2.43 g, 7.5 mmol) were added in sequence, and then the reaction solution was placed in a sealed tube and stirred at 60 ° C for 15 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous 1,4-dioxane (15 mL), formamide (270 mg, 6 mmol) was added, and then the solution was added to the sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered with diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-34 (614 mg, 43%). 1 H NMR (400MHz, d 6 -DMSO) δ10.62–10.48(m,0.8H),10.35(d,J=10.8Hz,0.2H),9.50(s,0.8H),9.47(s,0.2H),8.84(d,J=10.8Hz,0.2H),8.37(d,J=2.4Hz,0.8 H),8.34(d,J=1.8Hz,0.8H),7.77(s,0.2H),7.74(dd,J=8.9,2.5Hz,0.8H),7.60(d,J=8.9Hz,0.8H),7.53(d,J=1.5Hz,0.4H),1.45(s,9H). 13 C NMR (101MHz,d 6-DMSO)δ160.6,153.0,141.5,134.8,128.5,125.6,125.0,115.5,80.8,28.4.HRMS calcd for C 12 H 15 N3NaO5 + [M+Na] + 304.0909, found 304.0904.

[0178] Compound 3-34 (205 mg, 0.73 mmol) was dissolved in methanol (10 mL), 10% Pd / C (21 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred at room temperature overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a white solid 3-35 (154 mg, 84%). 1 H NMR (400MHz, d 6 -DMSO)δ9.90(s,0.3H),9.88–9.81(m,0.7H),8.57(dd,J=11.1,1.8Hz,0.3H),8.19(s,0.7H),8.14(d,J=2.0Hz,1H),7.14 –6.90(m,1.7H),6.67–6.41(m,0.7H),6.44(t,J=2.2Hz,0.3H),6.35(dd,J=8.5,2.4Hz,0.3H),4.96(s,2H),1.43(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ162.8,159.6,154.4,143.0,142.2,136.0,125.7,120.5,120.2,108.2,107.0,106.4,105.4,79.2,79.1,28.8.HRMS calcd for C 12 H 17 N3NaO3 + [M+Na] + 274.1168, found 274.1161.

[0179] 7-Amino-4-methylcoumarin carboxylic acid chloride (521 mg, 2.2 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.12 mL, 0.73 mmol), DMAP (45 mg, 0.37 mmol), and compound 3-35 (184 mg, 0.73 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO24-Boc (68 mg, 18%). 1 H NMR (500MHz, d 6 -DMSO) δ10.28–10.08(m,1H),9.75(d,J=13.8Hz,1H),8.48(s,1H),8.23(d,J=1.9Hz,1H),8.17(s,1H),8.05(s,1H),7.68(d,J=8.7Hz,1H),7.63( d,J=2.1Hz,1H),7.35(dd,J=8.7,2.4Hz,1H),7.30(dd,J=8.7,2.1Hz,1H),7.19(s,1H),6.21(d,J=1.3Hz,1H),2.39(d,J=1.2Hz,3H),1.43(s,9H). 13 C NMR (126MHz, d 6 -DMSO)δ162.9,160.6,159.9,154.8,154.5,154.4,153.7,152.8,143.9,136.0, 126.7,126.5,115.1,114.7,114.3,113.5,112.0,104.6,79.7,28.6,18.5.HRMS calcd for C 23 H 23 N4O6 - [MH] - 451.1618, found 451.1626.

[0180] The compound WNO24-Boc (68 mg, 0.15 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.6 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO24 (27 mg, 33%). 1H NMR (500MHz, d 6 -DMSO)δ10.24(s,1H),9.76(d,J=10.5Hz,1H),8.84(s,1H),8.24(d,J=1.9Hz,1H),7.73–7.62(m,3 H),7.42–7.30(m,2H),7.09(dd,J=20.6,8.6Hz,1H),6.22(d,J=1.4Hz,1H),2.39(d,J=1.3Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ163.6,163.1,160.7,160.2,159.1,158.8,154.4,153.9,153.5,143.8,130.6,126.7,126 .5,122.8,118.0,117.0,116.3,115.1,114.5,112.1,108.0,105.8,105.1,102.3,99.0,18.5.HRMS calcd for C 18 H 16 N4NaO4 + [M+Na] + 375.1069, found 375.1061.

[0181] Synthesis of compound WNO25:

[0182]

[0183] Palladium acetate (224 mg, 1 mmol) was dissolved in anhydrous 1,4-dioxane (15 mL), and Xantphos (867 mg, 1.5 mmol) and cesium carbonate (2.43 g, 7.5 mmol) were added in sequence, and then the reaction solution was placed in a sealed tube and stirred at 60 ° C for 15 min. Compound 3-17 (1.58 g, 5 mmol) was dissolved in anhydrous 1,4-dioxane (15 mL), N-methylformamide (354 mg, 6 mmol) was added, and then the solution was added to the sealed tube, heated to 100 ° C and stirred for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and filtered with diatomaceous earth. Ethyl acetate (3×20 mL) was added to wash the filter cake, and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 10) to obtain a yellow solid 3-36 (369 mg, 25%). 1H NMR(500MHz,CDC l3)δ9.60(s,1H),8.66(d,J=9.2Hz,0.88H),8.60(d,J=9.3Hz,0.12H),8.49(s,0.88H),8.38(s,0.12H),8.30(d,J=2.7Hz,0.12H),8.0 1(d,J=2.7Hz,0.88H),7.82(dd,J=9.3,2.7Hz,0.12H),7.46(dd,J=9.2,2.7Hz,0.88H),3.39(s,0.36H),3.33(s,2.64H),1.56(s,9H). 13 C NMR (126MHz, CDCl3) δ161.7,152.1,136.1,136.0,134.3,129.5,122.3,119.0,82.4,32.1,28.2.HRMS calcd forC 13 H 17 N3NaO5 + [M+Na] + 318.1066, found 318.1061.

[0184] Compound 3-36 (369 mg, 0.73 mmol) was dissolved in methanol (10 mL), 10% Pd / C (37 mg, 10% wt) was added, the air in the reaction system was removed, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol (3×10 mL), and then the obtained filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 4) to obtain a white solid 3-37 (266 mg, 80%). 1 H NMR (400MHz, CDCl3) δ8.40 (s, 1H), 7.27–7.24 (m, 1H), 6.57 (d, J = 8.1Hz, 2H), 6.19 (s, 1H), 3.92 (s, 2H), 3.25 (s, 3H), 1.51 (s, 9H). 13 C NMR(101MHz, CDCl3)δ162.4,154.0,141.6,140.5,126.3,122.8,113.4,111.3,81.1,77.5,32.3,28.4.HRMS calcd for C 13 H 19 N3NaO3 + [M+Na] + 288.1324, found 288.1318.

[0185] 7-Amino-4-methylcoumarin carboxylic acid chloride (711 mg, 3 mmol) was dissolved in anhydrous dichloromethane (10 mL), and DIPEA (0.17 mL, 1 mmol), DMAP (61 mg, 0.5 mmol), and compound 3-37 (266 mg, 1 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a white solid WNO25-Boc (277 mg, 59%). 1 H NMR (400MHz, d 6 -DMSO)δ9.75(s,1H),8.63(s,1H),8.53(s,1H),8.33(d,J=20.7Hz,1H),7.87(d,J=2.7Hz,1H),7.68–7.58(m,2H),7.37(d,J=8.5Hz,1H),7.30 (dd,J=8.7,2.3Hz,1H),7.02(dd,J=8.6,2.7Hz,1H),6.17(d,J=2.7Hz,1H),3.45(s,0.5H),3.23(s,2.5H),2.37(d,J=2.5Hz,3H),1.48(s,9H). 13 C NMR (101MHz,d 6 -DMSO)δ162.4,160.6,154.5,154.3,153.5,152.9,152.8,143.7,139.6,134.2,12 7.0,126.8,126.3,116.7,114.6,114.3,112.0,104.7,79.9,31.8,28.6,18.4.HRMS calcd forC 24 H 25 N4O6 - [MH] - 465.1774, found 465.1780.

[0186] The compound WNO25-Boc (277 mg, 0.15 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (0.6 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with dichloromethane and petroleum ether to obtain a white solid WNO25 (182 mg, 63%).1 H NMR (400MHz, d 6 -DMSO)δ9.66(s,1H),8.40(d,J=19.4Hz,2H),7.76–7.64(m,2H),7.45–7.34(m,2H),6.99(d, J=2.2Hz,2H),6.23(d,J=1.4Hz,1H),3.76–3.75(m,2H),3.17(s,3H),2.41(d,J=1.3Hz,3H). 13 C NMR (101MHz,d 6 -DMSO)δ162.4,160.7,154.5,153.8,153.3,144.0,135.4,135.1,127.4, 126.5,119.8,119.1,119.0,114.8,114.3,112.0,104.8,32.2,18.5.HRMS calcd for C 19 H 19 N4O4 + [M+H] + 367.1406, found 367.1405.

[0187] Example 2 Preparation of fluorescent probe WNO26, such as Figure 2 The reaction flow chart of the fluorescent probe WNO26 is shown:

[0188] Synthesis of compound WNO26:

[0189]

[0190] Dissolve 4-nitro-1,8-naphthalene dicarboxylic anhydride (5 g, 20.57 mmol) in ethanol (100 mL), add 4-aminobutyric acid (2.33 g, 22.63 mmol), and then heat the reaction solution to reflux and stir for 8 h. After the reaction is complete, cool the reaction solution to room temperature, and then pour the reaction solution into ice water to produce a large amount of yellow precipitate. Filter the precipitate and wash the filter cake with water (3×15 mL). The filter cake is then concentrated under reduced pressure by a vacuum pump and recrystallized from ethanol to obtain a yellow solid 3-38 (3.23 g, 48%). 1 H NMR (400MHz, d 6-DMSO) δ12.05(s,1H),9.43(d,J=2.3Hz,1H),8.89(d,J=2.3Hz,1H),8.74(d,J=8.3Hz,1H),8.64(d,J =7.3Hz,1H),8.04(t,J=7.8Hz,1H),4.10(t,J=6.9Hz,2H),2.35(t,J=7.3Hz,2H),2.01–1.84(m,2H).

[0191] Compound 3-38 (3.23 g, 9.85 mmol) was dissolved in methanol (58 mL), concentrated sulfuric acid (2.3 mL) was added dropwise, and then the reaction solution was heated to reflux and stirred overnight. After the reaction was complete, the reaction solution was cooled to room temperature, and then poured into ice water to produce a large amount of yellow precipitate. The precipitate was filtered and the filter cake was washed with water (3×20 mL). The filter cake was then concentrated under reduced pressure by a vacuum pump to obtain a yellow solid 3-39 (3.03 g, 90%). 1 H NMR (500MHz, d 6 -DMSO) δ8.73–8.68(m,1H),8.64–8.61(m,1H),8.60(d,J=8.0Hz,1H),8.55(d,J=8.0Hz,1H),8.09(d d,J=8.7,7.3Hz,1H),4.09(t,J=6.9Hz,2H),3.51(s,3H),2.42(t,J=7.3Hz,2H),1.98–1.89(m,2H).

[0192] Compound 3-39 (1.2 g, 2.22 mmol) was dissolved in methanol (25 mL) and tetrahydrofuran (25 mL), 10% Pd / C (120 mg, 10% wt) was added, and then the air in the reaction system was removed, and finally the reaction system was stirred overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, and the obtained filtrate was concentrated under reduced pressure by a vacuum pump to obtain a yellow solid 3-40 (358 mg, 49%). 1 H NMR (400MHz, d 6 -DMSO) δ8.62(dd,J=8.5,1.2Hz,1H),8.44(dd,J=7.3,1.1Hz,1H),8.20(d,J=8.4Hz,1H),7.67(dd,J=8.4,7.3Hz,1H ),7.46(s,2H),6.86(d,J=8.4Hz,1H),4.06(t,J=6.9Hz,2H),3.52(s,3H),2.37(t,J=7.4Hz,2H),1.97–1.84(m,2H).

[0193] Compound 3-40 (60 mg, 0.19 mmol) was dissolved in anhydrous dichloromethane (5 mL), and triethylamine (0.03 mL, 0.19 mmol) was added. Triphosgene (28 mg, 0.095 mmol) was then dissolved in anhydrous dichloromethane (5 mL), and then added to the dichloromethane solution of compound 3-40 and triethylamine. The reaction was stirred at room temperature for 2 h, and then the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was vacuumed on an oil pump for 2 h to remove excess triphosgene and phosgene produced as a byproduct of the reaction, and the main component of the remaining solid was compound 3-41. The obtained compound 3-41 was dissolved in anhydrous dichloromethane (10 mL), triethylamine (0.05 mL, 0.38 mmol) and compound 3-31 (67 mg, 0.23 mmol) were added in sequence, and finally the reaction was stirred at room temperature overnight. After the reaction was completed, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added for extraction, and the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate solid and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether=1 / 2) to obtain a yellow solid WNO26-Boc (27 mg, 22%). 1 H NMR (400MHz, d 6 -DMSO)δ9.91(s,1H),8.71(d,J=8.6Hz,2H),8.57(dd,J=7.9,4.9Hz,2H),8.48(d,J=8 .4Hz,1H),7.96(dd,J=8.5,7.3Hz,1H),7.60(d,J=4.9Hz,1H),7.13(s,1H),6.69(dd,J =8.8,2.8Hz,1H),4.11(t,J=6.8Hz,2H),3.77(dd,J=6.1,3.5Hz,4H),3.53(s,3H),3.1 0(dd,J=5.8,3.6Hz,4H),2.42(t,J=7.3Hz,2H),2.02–1.89(m,2H),1.58–1.38(m,9H). 13 C NMR (101MHz,d 6 -DMSO) δ173.4,164.2,163.6,161.7,160.6,154.7,152.9,149.4,142.2,132.9,131.3,130.5,129. 1,128.5,127.4,126.7,123.1,123.0,121.2,115.7,79.4,66.6,51.7,49.4,31.5,28.6,23.5.HRMS calcdfor C33 H 37 N5NaO8 + [M+Na] + 654.2540, found 654.2536.

[0194] The compound WNO26-Boc (20 mg, 0.03 mmol) was dissolved in dichloromethane (5 mL), and then hydrochloric acid (0.03 mL, 0.12 mmol, 4 M solution of 1,4-dioxane) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with methanol and ether to obtain a yellow solid WNO26 (12 mg, 75%). 1 H NMR(400MHz, CDCl3)δ9.75(s,1H),8.79(d,J=1.1Hz,1H),8.78–8.69(m,1H),8.57(d,J=8.5Hz, 1H),8.50(dd,J=7.3,0.9Hz,1H),8.41(d,J=8.5Hz,1H),7.88(dd,J=8.6,7.3Hz,1H),7.27(d,J =2.7Hz,1H),6.72(d,J=8.6Hz,1H),6.57(dd,J=8.6,2.7Hz,1H),4.66(s,1H),4.07(t,J=6.9Hz ,2H),3.80–3.66(m,4H),3.51(s,3H),2.99–2.88(m,4H),2.45–2.36(m,2H),1.97–1.87(m,2H). 13 C NMR (101MHz, CDCl3) δ178.2,169.0,168.4,157.7,148.5,147.4,138.9,137.7,136.0,134.9,133.8,13 3.3,131.2,130.6,127.7,127.4,122.1,120.0,119.8,118.3,116.9,71.6,56.5,55.6,36.2,28.2.HRMS calcdfor C 28 H 30 N5O6 + [M+H] + 532.2196, found 532.2193.

[0195] Example 3 Preparation of fluorescent probe WNO27, such as Figure 3 The reaction flow chart of the fluorescent probe WNO27 is shown below:

[0196] 2,3,3-Trimethyl-3H-indole (5 g, 31.4 mmol) was dissolved in acetonitrile (200 mL), 1-iodopropane (15.2 mL, 157 mmol) was added, and the reaction solution was heated to reflux and stirred for 15 h. After the reaction was complete, the reaction solution was cooled to room temperature, the precipitate was filtered, and the filter cake was washed with ether (3×15 mL). The filter cake was recrystallized from acetone to obtain a purple solid 3-42 (9.33 g, 90%). 1 H NMR (400MHz, d 6 -DMSO)δ8.09–7.98(m,1H),7.92–7.82(m,1H),7.70–7.60(m,2H),4.55–4.41 (m,2H),2.88(s,3H),1.99–1.83(m,2H),1.56(s,6H),1.01(t,J=7.4Hz,3H).

[0197] Compound 3-42 (1.09 g, 3.32 mmol) was dissolved in ethanol (50 mL), 4-aminobenzaldehyde (481 mg, 3.98 mmol) was added, and the reaction solution was heated to reflux and stirred overnight. After the reaction was complete, the reaction solution was cooled to room temperature, and then the reaction solution was added dropwise to ether (200 mL), and a large amount of yellow precipitate was precipitated. The precipitate was filtered and the filter cake was washed with ether (3×20 mL). The filter cake was then concentrated under reduced pressure by a vacuum pump to obtain a yellow solid 3-43 (1.35 g, 94%). 1 H NMR (400MHz, d 6 -DMSO) δ8.25(d,J=15.5Hz,1H),8.00(d,J=8.4Hz,2H),7.72(dd,J=16.5,7.6Hz,2H),7.54–7.39(m,2H),7.20(d,J=15.5Hz ,1H),6.75(d,J=8.5Hz,2H),5.44(s,2H),4.45(t,J=7.2Hz,2H),1.77(q,J=7.2Hz,2H),1.70(s,6H),0.94(t,J=7.3Hz,3H).

[0198] Compound 3-43 (96 mg, 0.31 mmol) was dissolved in anhydrous dichloromethane (8 mL), pyridine (40 μL, 0.49 mmol) was added, and the reaction solution was cooled to 0 ° C. p-Nitrophenyl chloroformate (200 mg, 0.99 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL), and then slowly added to the dichloromethane solution of compound 3-43 and pyridine. The reaction solution was first stirred at 0 ° C for 0.5 h, and then the reaction solution temperature was raised to room temperature and stirred for 3 h. The main component of the reaction solution is compound 3-44, which can be directly used in the next step. Compound 3-31 (363 mg, 1.24 mmol) was dissolved in anhydrous tetrahydrofuran (6 mL), and then pyridine (2 mL, 24.71 mmol) was added. The reaction solution whose main component is compound 3-44 was extracted with a syringe with a long needle, and slowly added to the tetrahydrofuran solution of compound 3-31 and pyridine under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 12 hours. After the reaction was complete, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added to extract, and the organic phases were combined and washed with saturated brine (20 mL), and the anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump, and separated by rapid column chromatography (methanol / dichloromethane=1 / 10) to obtain a yellow solid WNO27-Boc (164 mg, 70%). 1 H NMR (400MHz, d 6 -DMSO)δ10.03(s,1H),8.46(d,J=16.0Hz,1H),8.36(s,1H),8.33–8.19(m,3H),7.97 –7.87(m,2H),7.70(d,J=8.8Hz,2H),7.66–7.59(m,2H),7.55(d,J=13.5Hz,1H),7.16 –7.07(m,1H),6.67(dd,J=9.0,2.7Hz,1H),4.74–4.59(m,2H),3.84–3.71(m,4H),3. 16–3.02(m,4H),1.96–1.87(m,2H),1.82(s,6H),1.47(s,9H),1.03(t,J=7.4Hz,3H). 13 C NMR (101MHz,d 6-DMSO)δ182.0,154.8,154.6,152.8,149.5,146.1,144.1,141.4,134.5,133.2,130.2,129.6,129.5,128 .3,127.4,123.6,120.9,118.2,115.5,110.1,79.4,66.6,52.5,49.3,47.8,28.6,26.5,22.2,11.3.HRMS calcd forC 37 H 46 N5O4 + [M] + 624.3544, found 624.3537.

[0199] The compound WNO27-Boc (54 mg, 0.07 mmol) was dissolved in dichloromethane (5 mL), and then hydrochloric acid (0.07 mL, 0.28 mmol, 4 M solution of 1,4-dioxane) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with methanol and ether to obtain a yellow solid WNO27 (21 mg, 46%). 1 H NMR (400MHz, CD3OD) δ8.44(d,J=16.1Hz,2H),8.15–8.04(m,3H),7.88–7.75(m,5H),7.67–7.62(m,2H),7.56(d,J=16.1Hz,2H),7.32–7.22(m,2H) ),7.02(d,J=8.1Hz,2H),4.64(t,J=7.4Hz,2H),3.90–3.85(m,4H),3.29 –3.26(m,4H),2.02(d,J=7.1Hz,2H),1.86(s,6H),1.11(t,J=7.4Hz,3H). 13 C NMR (101MHz, CD3OD) δ182.3,179.2,178.1,166.4,154.8,154.0,145.1,143.7,141.0,132.1,129.5,129.3,1 28.8,123.6,122.8,118.8,114.7,112.0,109.7,101.1,66.1,52.5,49.4,48.4,25.6,21.9,10.0.HRMScalcd for C 32 H 38 N5O2 + [M] + 524.3020, found 524.3018.

[0200] Example 4 Preparation of fluorescent probe WNO28, such as Figure 4 The reaction flow chart of the fluorescent probe WNO28 is shown below:

[0201] 3-Nitrophenol (521 mg, 3.75 mmol) was dissolved in acetonitrile (10 mL), potassium carbonate (519 mg, 3.75 mmol) was added, and the reaction solution was stirred at room temperature for 15 min. 1.5 mmol of 1 g IR-780 [according to the literature report (Tian, ​​X.; Li, Z.; Sun, Y.; Wang, P.; Ma, H., Near-Infrared Fluorescent Probes for Hypoxia Detection Via Joint Regulated Enzymes: Design, Synthesis, and Application in Living Cells and Mice. Anal. Chem. 2018, 90, 13759-13766.), IR-780 was first reacted with m-nitrophenol to synthesize compound 3-45] was dissolved in acetonitrile (5 mL),

[0202] The structural formula of IR-780 is as follows:

[0203]

[0204] Add it to the reaction solution, heat the reaction solution to 50°C and stir for 4h. After the reaction is complete, wait for the reaction solution to cool to room temperature, and concentrate the reaction solution under reduced pressure with a vacuum pump. The solid obtained after concentration is dissolved in dichloromethane (100mL), and the organic phase is washed with water (3×50mL), and then washed with saturated brine (20mL). After washing, the organic phase is dried with anhydrous sodium sulfate solid and filtered. After filtration, the filtrate is concentrated under reduced pressure with a vacuum pump to obtain a purple viscous substance. The purple viscous substance is dissolved in methanol (50mL), and then anhydrous stannous chloride (2.84g, 15mmol) and concentrated hydrochloric acid (3.2mL) are added in sequence. The reaction solution is heated to 70°C and stirred overnight. After the reaction is complete, wait for the reaction solution to cool to room temperature, add saturated sodium carbonate solution (200mL) to quench the reaction, then add dichloromethane (3×20mL) to extract, and after the organic phases are combined, they are washed with saturated brine (40mL), and the anhydrous sodium sulfate solid is dried and filtered. After filtration, the filtrate was concentrated under reduced pressure using a vacuum pump and separated by flash column chromatography (dichloromethane / methanol=1 / 10) to give a purple solid 3-45 (521 mg, 65%). 1H NMR (400MHz, CD3OD) δ8.66 (d, J=14.3Hz, 1H), 7.65–7.54 (m, 2H), 7.55–7.45 (m,1H),7.43–7.30(m,3H),6.80(dd,J=8.6,2.1Hz,1H),6.74(d,J=2.1Hz,1 H), 6.29 (d, J = 14.3Hz, 1H), 4.21 (t, J = 7.3Hz, 2H), 2.81 (t, J = 6.1Hz, 2H), 2. 74(t,J=6.2Hz,2H),2.05–1.87(m,4H),1.82(s,6H),1.10(t,J=7.4Hz,3H).

[0205] Compound 3-45 (80 mg, 0.15 mmol) was dissolved in anhydrous dichloromethane (4 mL), pyridine (20 μL, 0.25 mmol) was added, and the reaction solution was cooled to 0 ° C. p-Nitrophenyl chloroformate (90 mg, 0.45 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL), and then slowly added to the dichloromethane solution of compound 3-45 and pyridine. The reaction solution was first stirred at 0 ° C for 0.5 h, and then the temperature of the reaction solution was raised to room temperature and stirred for 3 h. The main component of the reaction solution is compound 3-46, which can be directly used for the next step. Compound 3-31 (142 mg, 0.6 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), and then pyridine (1 mL, 12.36 mmol) was added. The reaction solution with the main component of compound 3-46 was extracted with a syringe with a long needle, and slowly added to the tetrahydrofuran solution of compound 3-31 and pyridine under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 12 h. After the reaction was complete, water (20 mL) was added to quench the reaction. Dichloromethane (3×10 mL) was added for extraction, and the organic phases were combined and washed with saturated brine (20 mL). The anhydrous sodium sulfate solid was dried and filtered. After filtration, the filtrate was concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane=1 / 10) to obtain a purple solid WNO28-Boc (29 mg, 24%). 1H NMR (400MHz, CD3OD) δ8.71(d,J=14.9Hz,1H),8.01(d,J=2.0Hz,1H),7.85(d,J=2.7Hz,1H),7.66(d,J =7.2Hz,1H),7.58–7.44(m,3H),7.40(dd,J=8.7,4.9Hz,2H),7.27(s,1H),7.11(dd,J=8.4,2.1Hz,1H) ,7.07–6.97(m,1H),6.52(d,J=14.9Hz,1H),4.18(t,J=7.5Hz,2H),3.88(t,J=4.7Hz,4H),2.81–2.65( m,4H),1.93(d,J=6.1Hz,2H),1.89(d,J=7.8Hz,2H),1.85(s,6H),1.54(s,9H),1.04(t,J=7.4Hz,3H). 13 CNMR(101MHz,CD3OD)δ177.8,161.6,155.4,154.0,153.1,145.7,143.5,142.2,141.8,134.0,133.5,129.0,128.2,127.7,127.1,122.5,117.0 ,115.8,114.4,113.1,112.7,111.6,109.5,108.1,105.1,104.1,103.7 ,80.3,65.6,51.7,50.7,46.1,28.9,27.4,23.9,20.9,20.3,10.3.HRMS calcd for C 44 H 52 N5O5 + [M] + 730.3963,found730.3966.

[0206] The compound WNO28-Boc (100 mg, 0.12 mmol) was dissolved in dichloromethane (5 mL), and then hydrochloric acid (0.12 mL, 0.48 mmol, 4 M solution of 1,4-dioxane) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure by a vacuum pump. The solid obtained after concentration was recrystallized with methanol and ether to obtain a purple solid WNO28 (83 mg, 91%). 1H NMR (400MHz, CD3OD) δ8.78(d,J=14.9Hz,1H),7.93(d,J=2.0Hz,1H),7.68(d,J=7.4Hz,1H),7.57(t, J=5.0Hz,2H),7.54–7.45(m,2H),7.45–7.32(m,3H),7.23(d,J=2.7Hz,1H),7.14(dd,J=8.9,2.7Hz,1 H),6.56(d,J=15.0Hz,1H),4.36(t,J=7.4Hz,2H),3.95(t,J=4.8Hz,4H),3.40–3.35(m,4H),2.82(t, J=6.0Hz,2H),2.75(d,J=6.2Hz,2H),1.97(q,J=7.3,5.7Hz,4H),1.85(s,6H),1.11(t,J=7.3Hz,3H). 13 C NMR (101MHz, CD3OD) δ177.9,161.5,153.9,153.8,148.5,145.7,143.1,142.1,141.7,133.4,131.7,128.9,128.1,128.0,127.5,127.1,12 3.7,122.5,117.2,116.2,114.4,114.3,112.9,112.7,104.5,103.7,65.8,50.7,50.0,46.3,28.8,27.1,23.7,20.9,20.3,10.3.HRMScalcd for C 39 H 44 N5O3 + [M] + 630.3439, found 630.3439.

[0207] 2. In vitro detection and characterization of fluorescent probes

[0208] 1. Experimental Methods

[0209] The absorption and fluorescence spectra of the fluorescent probe were measured using a fluorescence spectrophotometer Hitachi F-4600 and an enzyme reader BIOTEK CYTATION 5. When the fluorescence intensity was detected in a 96-well plate, the enzyme reader BIOTEK CYTATION 5 was used for determination. Unless otherwise specified, when detecting the reaction, selectivity and detection limit of the probe with the enzyme or small molecule, all probes were configured into a 1mM DMSO stock solution. When detecting the sensitivity of the probe to the enzyme or small molecule, 1μL or 2μL of the probe stock solution was taken and diluted to 200μL with PBS buffer in a 96-well plate, and the final concentration of the probe was 5μM or 10μM. Unless otherwise specified, the equivalent ratio of the probe to the enzyme or small molecule used in this application refers to the ratio of the amount of substance. NO donor (DEA NONOate) was purchased from Sigma, HDAC6 was purchased from Cayman Chemical, and the above reagents were configured into corresponding stock solutions according to the method in the instruction manual. Unless otherwise specified, the reactions were carried out in a constant temperature box at 37°C.

[0210] like Figure 1 As shown, the fluorescence response degree of NO fluorescent probe molecular library reacting with NO. (A) The fluorescence response degree of NO fluorescent probe WNO1-WNO6 with different linkers reacting with NO. (B) Reaction conditions: PBS buffer solution (10mM, pH=7.4); WNO1-WNO25 (10μM); NO donor (1mM); incubation time 60min; T=37℃; λex=360nm; λem=450nm. Data are shown as mean ± standard deviation (n=3).

[0211] The present invention relates to the connection structure of o-phenylenediamine and fluorophore ( Figure 1 A) and o-phenylenediamine structures ( Figure 1 The structure-activity relationship between B, 1C) and NO responsiveness was systematically studied, and the optimal connection structure and reactive group structure were identified. Figure 1 A results verify the feasibility of the design strategy of NO fluorescent probe in this application, that is, the coumarin core fluorophore can be connected to the o-phenylenediamine structure through a carbamate or carbonamide linker to achieve a fluorescent response to NO. More importantly, these results also show that when 7-amino-4-methylcoumarin and o-phenylenediamine are connected with carbonamide as a linker, the probe WNO3 exhibits stability in aqueous solution and high reactivity to NO. Therefore, this linker structure is a more suitable linking group between the fluorophore and o-phenylenediamine. Figure 1B The results show that subtle structural differences in the o-phenylenediamine reactive groups have an important influence on the NO fluorescence response of the probe. Among them, the o-phenylenediamine structure with a dimethylamino substituent at the amino position, i.e., the reactive group in probe WNO14, shows the best NO reactivity. At the same time, these results also suggest that by replacing different amino substituents in the probe WNO14 structure, it is expected to further optimize the reactivity and fluorescence response of the probe. In order to further improve the reactivity and fluorescence responsiveness of the probe to NO, the present invention designed nine fluorescent probes WNO17-WNO25 by introducing different amino substituents at the amino position of o-phenylenediamine, and characterized their photophysical properties for reactions with NO. Figure 1 As shown in Figure C, except for WNO17 and WNO23, which have a certain background fluorescence, the background fluorescence of the other probes before reacting with NO is weak. After reacting with NO, WNO17-WNO25 all have different degrees of fluorescence enhancement response, which shows that the para position of the amino group of o-phenylenediamine is a suitable position to introduce different amino substituents. Among them, the fluorescence intensity of WNO22 before and after the reaction with NO is enhanced by about 30 times, which is the probe with the strongest fluorescence response multiple. Compared with the above-mentioned probes WNO3 and WNO14 with better performance, the fluorescence response multiple of WNO22 before and after the reaction with NO is larger. These results show that the introduction of morpholine substituents in the para position of the free amino group of the o-phenylenediamine structure further improves the reactivity and fluorescence response of the probe to NO. Therefore, the o-phenylenediamine structure with morpholine substituents is the optimal reaction group screened by this application, and its mechanism may be to improve the nitrosation efficiency of the free amino group and / or the formation and hydrolysis efficiency of benzotriazole, but the specific mechanism remains to be studied.

[0212] like Figure 5 As shown, the fluorescence spectrum of the probe WNO26 (10 μM) after reacting with NO donor (0-2 mM) at 37 ° C. Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37 ° C; λex = 440 nm. As expected, due to the acylation of the naphthylimide fluorophore, WNO26 itself exhibits weak fluorescence before reacting with NO; with the reaction with NO, the fluorescence intensity of WNO26 shows a significant enhancement, and its fluorescence intensity at the maximum emission wavelength of about 554 nm gradually increases with the increase of NO concentration.

[0213] like Figure 6As shown in the figure, the fluorescence spectrum of the probe WNO27 (10 μM) after reacting with NO donor (0-2 mM) at 37°C. Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37°C; λex = 492 nm. As expected, due to the acylation of the merocyanine fluorophore, WNO27 itself exhibited weak fluorescence before reacting with NO, as shown in Figure 2. Figure 6 As shown; with the reaction with NO, the fluorescence intensity of WNO27 showed a significant enhancement, and its fluorescence intensity at the maximum emission wavelength of about 559nm gradually increased with the increase of NO concentration.

[0214] like Figure 7 Figure 2 shows the fluorescence spectrum of the probe WNO28 (10 μM) after reacting with NO donor (0-2 mM) at 37°C. Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37°C; λex = 674 nm. As expected, due to the acylation of the hemicyanine fluorophore, WNO28 itself exhibited weak fluorescence before reacting with NO. Figure 4 As shown; with the reaction with NO, the fluorescence intensity of WNO28 showed a significant enhancement, and its fluorescence intensity at the maximum emission wavelength of about 700nm gradually increased with the increase of NO concentration.

[0215] 2. Detection Limit and Selectivity

[0216] In order to determine the detection limit, the fluorescent probe was prepared with DMSO to a 1mM DMSO stock solution. According to the experimental requirements, the 1mM stock solution was diluted to 5μM or 10μM. The enzyme or NO donor to be tested was first prepared into a stock solution with an equivalent ratio of 0.01-0.2, and then the probe was incubated with different concentrations of the enzyme or NO donor to be tested in a constant temperature box at 37°C for 30min or 1h, and the fluorescence intensity after the reaction was measured.

[0217] For selective determination, different probes were used with different potential interfering substances. If not otherwise specified, the potential interfering substances were obtained from the reagent company and prepared into corresponding stock solutions according to the instructions.

[0218] like Figure 8 As shown, the kinetics and selectivity of probe WNO26 in detecting NO. (A) Fluorescence response kinetics of probe WNO26 (10 μM) to NO, NO donor (DEA NONOate, 1 mM). (B) Fluorescence response intensity of probe WNO26 (10 μM) to NO and other potential interferents. NaClO (500 μM), NaNO2 (500 μM), H2O2 (500 μM), KO2 (500 μM), 1O2(500μM),·OH(500μM),Cys(500μM),GSH(500μM),MGO(1mM),DHA(1mM),AA(1mM),ONOO － (100 μM), NO donor (DEA NONOate, 500 μM). Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37°C; λ ex =440nm; em =554 nm. Data are shown as mean ± standard deviation (n=3).

[0219] The present application analyzes the kinetics of the reaction between probe WNO26 and NO. The results show that after adding NO donor (DEANONOate, 1 mM), the fluorescence signal of WNO26 increases with time and reaches the maximum fluorescence signal plateau at about 20 minutes. Figure 8 As shown in A. In contrast, in the absence of NO, the fluorescence signal of WNO26 remains basically unchanged, indicating that the probe remains stable in aqueous solution. Next, the present application studied the selectivity of WNO26 for NO detection, that is, the response of WNO26 to various potential interferents, including sodium hypochlorite, sodium nitrite, hydrogen peroxide, potassium superoxide, singlet oxygen, hydroxyl radicals, cysteine, glutathione, methylglyoxal, ascorbic acid, dehydroascorbic acid and peroxynitrite, etc. The results are shown in Figure 8 As shown in B. The results showed that WNO26 only showed a strong fluorescence enhancement in response to NO, but had basically no fluorescence response to the potential interferents tested, thus confirming the selectivity of the probe WNO26.

[0220] like Fig. 9 As shown, the characterization of probe WNO26 for detecting NO in vitro. (A) The linear relationship between the fluorescence intensity at 554 nm and the NO donor concentration after the reaction of probe WNO26 (10 μM) with NO donor (0-2.0 μM). Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37 ° C; λ ex =440nm; em =554 nm. Data are shown as mean ± SD (n = 3). (B) Fluorescence response of probe WNO26 (10 μM) to NO donor (1 mM) at different pH values.

[0221] This application studies the detection limit of WNO26 ( Fig. 9 A) and the effects of different pH conditions on WNO26 detection of NO ( Fig. 9B). The results showed that under low NO donor concentration conditions (0-2 μM), there was a good linear correlation between the fluorescence intensity of WNO26 and the NO donor concentration (R 2 =0.9742). Based on LOD=3×S / N, the detection limit of WNO26 for NO is estimated to be about 0.25μM. The background fluorescence signal of WNO26 has almost no change in the range of pH=4-10; when NO donor is added, there is a significant fluorescence response in the range of pH=4-10, which indicates that WNO26 can be used for the detection of NO under different pH conditions.

[0222] like Fig.10 As shown, the kinetics and selectivity of probe WNO27 for detecting NO. (A) The fluorescence response kinetics of probe WNO27 (10 μM) to NO donor (1 mM), NO donor (DEA NONOate, 1 mM). (B) The fluorescence response intensity of probe WNO27 (10 μM) to NO and other potential interferents. NaClO (500 μM), NaNO2 (500 μM), H2O2 (500 μM), KO2 (500 μM), 1 O2(500μM),·OH(500μM),Cys(500μM),GSH(500μM),MGO(1mM),DHA(1mM),AA(1mM),ONOO － (500 μM), NO donor (DEA NONOate, 500 μM). Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37°C; λex = 492 nm; λem = 559 nm. Data are shown as mean ± standard deviation (n = 3).

[0223] The present application analyzes the kinetics of the reaction between probe WNO27 and NO. The results show that after adding NO donor (DEANONOate, 1 mM), the fluorescence signal of WNO27 increases with time and reaches the maximum fluorescence signal plateau at about 5 minutes. Fig.10 As shown in A. In contrast, in the absence of NO, the fluorescence signal of WNO27 remains basically unchanged, indicating that the probe remains stable in aqueous solution. Next, the present application studied the selectivity of WNO27 for NO detection, that is, the response of WNO27 to various potential interferents, including sodium hypochlorite, sodium nitrite, hydrogen peroxide, potassium superoxide, singlet oxygen, hydroxyl radicals, cysteine, glutathione, methylglyoxal, ascorbic acid, dehydroascorbic acid and peroxynitrous acid, etc. The results are shown in Fig.10 As shown in B. The results showed that WNO27 only showed a strong fluorescence enhancement in response to NO, but had basically no fluorescence response to the potential interferents tested, thus confirming the selectivity of the probe WNO27.

[0224] like Fig.11 As shown, the characterization of probe WNO27 for detecting NO in vitro. Figure (A) Linear relationship between the fluorescence intensity at 559nm of probe WNO27 (10μM) after reaction with NO donor (0-2.0μM) and NO donor concentration. Reaction conditions: PBS buffer solution (10mM, pH=7.4); incubation time 60min; T=37℃; λex=492nm; λem=559nm. Data are shown as mean±standard deviation (n=3). Figure (B) Fluorescence response of probe WNO27 (10μM) to NO donor (1mM) at different pH. The results show that under the condition of low NO donor concentration (0-2μM), there is a good linear correlation between WNO27 fluorescence intensity and NO donor concentration (R2=0.9758). Based on LOD=3×S / N, the detection limit of WNO27 for NO is calculated to be approximately 0.16μM. The background fluorescence signal of WNO27 hardly changes in the range of pH = 4-10; when NO donor is added, there is a significant fluorescence response in the range of pH = 4-10, which indicates that WNO27 can be used for the detection of NO under different pH conditions.

[0225] like Fig.12 As shown, Figure 7 Characterization of the kinetics and selectivity of probe WNO28 for NO detection. (A) Fluorescence response kinetics of probe WNO28 (10 μM) to NO donor (1 mM), NO donor (DEA NONOate, 1 mM). (B) Fluorescence response intensity of probe WNO28 (10 μM) to NO and other potential interferents. NaClO (100 μM), NaNO2 (100 μM), H2O2 (100 μM), KO2 (100 μM), 1 O2(100μM),·OH(100μM),Cys(100μM),GSH(100μM),MGO(100μM),DHA(100μM),AA(100μM),ONOO － (100μM), NO donor (DEA NONOate, 500μM). Reaction conditions: PBS buffer solution (10mM, pH=7.4); incubation time 60min; T=37℃; λex=674nm; λem=700nm. Data are shown as mean ± standard deviation (n=3). The present application analyzes the kinetics of the reaction between probe WNO28 and NO. The results show that after the addition of NO donor (DEA NONOate, 1mM), the fluorescence signal of WNO28 increases with time and reaches the maximum fluorescence signal plateau at around 30 minutes. Fig.12As shown in A. In contrast, in the absence of NO, the fluorescence signal of WNO28 remains basically unchanged, indicating that the probe remains stable in aqueous solution. Next, the present application studied the selectivity of WNO28 for NO detection, that is, the response of WNO28 to various potential interferents, including sodium hypochlorite, sodium nitrite, hydrogen peroxide, potassium superoxide, singlet oxygen, hydroxyl radicals, cysteine, glutathione, methylglyoxal, ascorbic acid, dehydroascorbic acid and peroxynitrous acid, etc. The results are shown in Fig.12 As shown in B. The results showed that WNO28 only showed a strong fluorescence enhancement in response to NO, but had basically no fluorescence response to the potential interferents tested, thus confirming the selectivity of the probe WNO28.

[0226] like Fig.13 The following is the characterization of the probe WNO28 for detecting NO in vitro. (A) The linear relationship between the fluorescence intensity at 700 nm and the NO donor concentration after the probe WNO28 (10 μM) reacted with the NO donor (0-1.2 μM). Reaction conditions: PBS buffer solution (10 mM, pH = 7.4); incubation time 60 min; T = 37 ° C; λex = 674 nm; λem = 700 nm. Data are shown as mean ± standard deviation (n = 3). (B) Fluorescence response of the probe WNO28 (10 μM) to the NO donor (1 mM) at different pH.

[0227] At the same time, this application evaluated the detection limit of WNO28 ( Fig.13 A) and the effect of different pH on WNO28 detection of NO ( Fig.13 B). The results showed that under low NO donor concentration conditions (0-1.2 μM), there was a good linear correlation between the fluorescence intensity of WNO28 and the NO donor concentration (R2=0.9729). Based on LOD=3×S / N, the detection limit of WNO28 for NO was calculated to be approximately 0.32 μM. The background fluorescence signal of WNO28 hardly changed in the range of pH=4-10; when the NO donor was added, there was an optimal fluorescence response at around pH=7.4, but the fluorescence response was significantly weakened at pH=4-6 and pH=9-10, indicating that WNO28 is not very suitable for detecting NO under extreme pH conditions, but has a good response to NO near the pH related to physiological conditions.

[0228] 3. Live Cell Fluorescence Imaging Study of Nitric Oxide Fluorescent Probe

[0229] Intracellular detection and imaging experiments of exogenous NO by WNO27 and WNO28:

[0230] HeLa cells were incubated in DEME medium containing 10% fetal bovine serum and cultured at 37°C with a CO2 content of 5%. Before cell imaging, HeLa cells were seeded in a confocal dish and allowed to adhere overnight. The next day, they were washed twice with PBS, and the cells were incubated with probes WNO27 (5μM) or WNO28 (2μM) for 60 minutes. Then, nitric oxide donors with a final concentration of 0-500μM were added to the cells and incubated in an incubator for 30 minutes. The cells were then washed three times with PBS and confocal fluorescence imaging was performed.

[0231] Imaging parameters: Cells were imaged using a Nikon A1R laser confocal microscope. The fluorescence signal of WNO27 was excited at 400 nm and collected at 510-590 nm. The fluorescence signal of probe WNO28 was excited at 640 nm and collected at 663-738 nm. Image fluorescence intensity was quantified using Image J, and statistical analysis was performed using one-way ANOVA in GraphPad Prism.

[0232] Fig.14 Confocal fluorescence imaging of exogenous NO by probe WNO27 in HeLa cells. (A) Fluorescence imaging of probe WNO27 (5 μM) and HeLa cells under NO donor (0-500 μM) treatment conditions. The scale bar represents 50 μm. (B) Fluorescence quantitative analysis of Figure A. The ordinate represents the fluorescence intensity of cells relative to the condition without NO donor. The data are shown as the mean relative fluorescence intensity ± standard deviation (n=3). The excitation wavelength of the fluorescence signal is 400 nm, and the collection band is 510-590 nm.

[0233] Fig.15 Confocal fluorescence imaging of exogenous NO by probe WNO28 in HeLa cells. (A) Fluorescence imaging of probe WNO28 (2 μM) and HeLa cells under NO donor (0-500 μM) treatment conditions. The scale bar represents 50 μm. (B) Fluorescence quantitative analysis of Figure A. The ordinate represents the fluorescence intensity of cells relative to the condition without NO donor. The data are shown as the mean relative fluorescence intensity ± standard deviation (n=3). The excitation wavelength of the fluorescence signal is 640 nm, and the collection band is 663-738 nm.

[0234] Results Analysis

[0235] HeLa cells were first incubated with the probe WNO27 (5 μM) for 60 min, then NO donor (0-500 μM) was added and incubated for another 30 min, and finally imaged under a confocal fluorescence microscope. The results are shown in Figure 2. Fig.14As shown in the figure, it can be seen that the background fluorescence intensity of probe WNO27 is weak when no NO donor is added. However, after adding NO donor, the fluorescence signal of probe WNO27 is significantly enhanced. Fig.14 The quantitative analysis results of B show that the fluorescence response of probe WNO27 gradually increases with the increase of NO donor concentration, which indicates that probe WNO27 can detect and image NO in living cells.

[0236] Similarly, HeLa cells were first incubated with the probe WNO28 (2 μM) for 60 min, then NO donors (0-500 μM) were added and incubated for another 30 min, and finally imaged under a confocal fluorescence microscope. The results are shown in Figure 2. Fig.15 As shown in the figure, it can be seen that without the addition of NO donor, the background fluorescence intensity of probe WNO28 is very weak. After adding NO donor, the fluorescence signal of probe WNO28 is significantly enhanced. Fig.15 The quantitative analysis results of B show that the fluorescence response of probe WNO28 gradually increases with the increase of NO donor concentration, which indicates that probe WNO28 can detect and image NO in living cells.

[0237] In summary, the present invention designs a modular NO fluorescent probe construction strategy based on the reaction between o-phenylenediamine and NO, that is, the fluorophore is connected to the o-phenylenediamine structure through its phenolic hydroxyl group or aromatic amine group with a carbamate or carbonamide linker to construct a NO fluorescent probe. The present invention envisions that NO undergoes a nitrosylation reaction with the amine group on o-phenylenediamine, followed by intramolecular cyclization to form a benzotriazole structure, and then hydrolyzes to remove the benzotriazole and generate a free fluorophore, accompanied by significant fluorescence enhancement to achieve NO detection. In order to verify this strategy, the present invention uses coumarin as a fluorophore, and first designs and synthesizes a first group of coumarin fluorescent probes with different carbamate or carbonamide linkers, namely WNO1-WNO6, which not only confirms that NO can indeed cause the fluorescence response of the probe, but also finds that when coumarin is connected to o-phenylenediamine with a carbonamide structure, the reaction activity and fluorescence response to NO are higher. Based on this, the present invention then designs and synthesizes a second group of coumarin fluorescent probes, namely WNO7-WNO16, which have substituents of different types and positions on the o-phenylenediamine structure. The screening results of the second group of fluorescent probes showed that the introduction of a dimethylamino substituent at the position of the free amino group in the o-phenylenediamine structure would increase the NO reactivity and fluorescence response of the probe. Immediately afterwards, the third group of coumarin fluorescent probes, namely WNO17-WNO25, was designed and synthesized to screen and optimize the o-phenylenediamine reaction group. The third group of coumarin fluorescent probes has different amino substituents at the position of the free amino group in the o-phenylenediamine structure. The screening results show that the introduction of a morpholine substituent at the position of the free amino group will lead to an enhanced fluorescence response of the probe to NO, which is better than other substituents. Therefore, through the above-mentioned screening of coumarin fluorescent probes WNO1-WNO25, the present application found that carbonamide is the optimal linker structure between the amino fluorophore and o-phenylenediamine, and o-phenylenediamine with a morpholine substituent is the optimal NO reaction group.

[0238] Based on this, in order to verify the modularity and universality of the NO fluorescent probe design strategy, the present application then connected the optimal o-phenylenediamine reactive group to fluorophores of different wavelengths through the optimal linker structure, including naphthylimide, merocyanine and hemicyanine dye fluorophores, etc., and designed and synthesized NO fluorescent probes WNO26-WNO28 of different wavelengths. The experimental results show that the probes WNO26-WNO28 can all be used for the detection of NO in PBS buffer solution, and have high selectivity. At the same time, the present application used HeLa cells for fluorescence imaging experiments and found that WNO27 and WNO28 can be used for the detection and imaging of exogenous NO in living cells. Therefore, the modularity and universality of the NO fluorescent probe construction strategy designed by this application have been preliminarily verified. In the future, the o-phenylenediamine reactive group can be connected to more fluorophores of different wavelengths and optical properties through a carbonamide structure, so as to conveniently and quickly develop a series of NO fluorescent probes.

[0239] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A class of small molecule fluorescent probes for detecting nitric oxide, characterized in that: The fluorescent probe comprises one of the following structures: 。 2. Use of a class of small molecule fluorescent probes for detecting nitric oxide according to claim 1 in detecting nitric oxide.

3. Application of a class of small molecule fluorescent probes for detecting nitric oxide according to claim 1 in the detection and imaging of nitric oxide in solutions, living cells and living animals.

4. Use of a small molecule fluorescent probe for detecting nitric oxide according to claim 3 in preparing a nitric oxide detection kit or test paper.

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