Enzymatically light-controlled nitric oxide donors containing a coumarin skeleton, methods of making and uses thereof

Abstract

The application discloses an enzymatic light-controlled nitric oxide donor containing a coumarin skeleton, a preparation method and application. The enzymatic light-controlled nitric oxide donor containing the coumarin skeleton has the structure shown in the following formula: wherein R1 is H, C1-10 alkyl, C1-10 cycloalkyl, C1-4 alkoxy, phenyl or benzyl; and R2 is alkoxy, phenyl, p-tolyl, m-tolyl, o-tolyl, o-nitrophenyl, phenoxy, acetoxy or phenylfluoro. The application further provides a preparation method of the compound of the formula, and the use of the compound as the enzymatic light-controlled nitric oxide donor.

Description

TECHNICAL FIELD

[0001] The present application relates to a coumarin skeleton-containing enzymatic light-controlled nitric oxide donor, a preparation method and application thereof. BACKGROUND

[0002] Nitric oxide (NO) is endogenously synthesized by NO synthase and widely exists in mammalian tissues and cells. It is indispensable in the cardiovascular system, central nervous system and immune system. However, anything has two sides, and excessive NO can cause toxic side effects, leading to cell death. Therefore, the precise release and amount control of NO are very important for its medical and pharmacological effects. NO donors are a class of precursor drugs that can release NO in vivo, which refers to substances that do not need NOS catalysis to produce NO by themselves or with other substances. Due to its potential application value, more and more NO donors have been developed and applied to the treatment of various related diseases. However, traditional NO donors have many defects in use, such as lack of targeting and tissue specificity, too short drug action time, poor physiological stability, large toxic side effects on the human body, and difficult to grasp and control the effective dose. In recent years, a series of new NO donors have been developed and developed to solve the above problems, increasing the universality and feasibility of NO in clinical treatment, so the synthesis of nitric oxide donor compounds has become a research hotspot in recent years.

[0003] NO donors have been developed and widely used in biological research and clinical treatment. Today, the research trend of NO donors mainly focuses on the synthesis of new NO donors, the development of new application ways of traditional NO donors, the development of NO donor / drug combinations, and the research and development of tissue-specific NO donors. In these four research fields, due to the wide distribution of NO in the body, designing and synthesizing tissue-specific NO donors is the most challenging work. Due to the multiple physiological functions of NO, it is very difficult to develop a single NO donor to regulate a certain physiological function. However, in recent years, people have begun to understand this problem and have come up with some solutions, such as combining NO donors with tissue-specific ligands (especially monoclonal antibodies) to increase the targeting of drugs; in percutaneous transluminal coronary angioplasty, this drug can directly reach and act on the diseased site, while avoiding various side effects.

[0004] NO donor is a compound that releases NO after simple enzymatic or other action in vivo. It can effectively overcome the short life and short half-life of NO while storing and transporting it. It can release NO spontaneously or from specific molecular and macromolecular scaffolds under suitable stimulation (i.e. temperature, pH, light). Among them, light is a non-invasive means of regulating NO release, with the advantages of space-time control and adjustable wavelength / intensity of irradiation. Recent literature reports some light-triggered nitric oxide donors, but there are still many shortcomings in light-triggered nitric oxide donors, such as poor control effect on the position and release rate of nitric oxide. High morbidity of diabetes and vascular complications involves a series of risk factors related to oxidative stress and NO functional deficiency. Lysosomal alpha-glucosidase is a brush border membrane-bound enzyme present in the intestinal tract. Alpha-glucosidase in diabetic patients hydrolyzes oligosaccharides to release glucose, ultimately leading to postprandial hyperglycemia. Elevated blood glucose levels lead to accelerated glycosylation, resulting in advanced glycation end products (AGEs); the changes in protein structure and function caused by AGEs are considered to be the main link between diabetes and serious complications. Slowing down glucose absorption in the intestine by inhibiting the activity of alpha-glucosidase to reduce blood glucose is one of the simplest methods for treating diabetes. Long-term hyperglycemia is a characteristic of diabetes, which produces excess reactive oxygen species (ROS) such as superoxide anion, H2O2, ONOO-, NO, etc. High concentrations of NO produced by iNOS react with ROS to generate reactive nitrogen species (RNS), NO reacts with superoxide anion to generate ONOO-, and with ONOO- to generate NO2. Oxidative stress is the result of increased ROS production and decreased antioxidant systems, and oxidative stress will cause damage only when the antioxidant defense system cannot offset the excess ROS. The activation of oxidative stress produces a series of harmful metabolic processes, such as increased polyol activity, increased formation of AGEs, activation of protein kinase C, etc. In addition, clinical and experimental observations have shown that oxidative stress is the main mechanism for the occurrence of diabetes and complications. It is generally believed that during the postprandial phase, elevated blood glucose and oxidative stress can accelerate the formation of AGEs. Therefore, both hyperglycemia and AGEs are considered to be the main pathological causes of diabetic complications. Through nitric oxide photometry, we can have a clear understanding of the release kinetics, total amount and release rate of nitric oxide, which is of great significance to reveal the correlation between nitric oxide and biological reactions.

[0005] Meanwhile, the coumarin structure contained in the parent unit has certain advantages, and natural coumarin and its derivatives have been experimentally proved to have anti-diabetic activity and certain inhibitory capacity on alpha-glucosidase. Alpha-amylase and alpha-glucosidase are main target enzymes of coumarin derivatives, and the inhibitory effect can treat hyperglycemia. High-dose coumarin can produce toxicity, such as carcinogenicity and hepatotoxicity. In the existing coumarin derivative molecules, a nitroso group is introduced, NO is released under the condition of light to inhibit alpha-glucosidase, and then the biological activity is improved by the synergistic effect of the coumarin structure to reduce the toxic side effects, so that a compound with potential medicinal value is obtained. SUMMARY

[0006] In view of the fact that there are few light-controlled nitric oxide donors in the prior art, and the release position and release rate control effect are not good, the present application aims to provide an enzyme-controlled light-controlled nitric oxide donor with strong anti-interference capability, and a preparation method and application thereof.

[0007] Another object of the present application is to provide a preparation method of the above-mentioned nitric oxide donor, which is easy to obtain raw materials, simple in synthesis steps and high in yield.

[0008] Still another object of the present application is to provide a compound of formula I in the preparation of drugs for treating or preventing diabetes, hypertension-related diseases and cardiovascular diseases or related uses.

[0009] To achieve the above objects, the present application adopts the following technical solutions.

[0010] An enzyme-controlled light-controlled nitric oxide donor containing a coumarin skeleton has the following structure: In the formula, R1 is H, C1-10 alkyl, C1-10 cycloalkyl, C1-4 alkoxy, phenyl, benzyl; R2 is alkoxy, phenyl, p-tolyl, m-tolyl, o-tolyl, o-nitrophenyl, phenoxy, acetoxy, phenylfluoro.

[0011] The enzyme-controlled light-controlled nitric oxide donor containing a coumarin skeleton has the following structure:

[0012] The enzyme-controlled light-controlled nitric oxide donor containing a coumarin skeleton has the following structure:

[0013] The enzyme-controlled light-controlled nitric oxide donor containing a coumarin skeleton has the following structure:

[0014] The enzymatic light-controlled nitric oxide donor containing the coumarin skeleton has the following formula:

[0015]

[0016] The preparation method of the nitric oxide donor comprises the following steps: (1) dissolving compound 1a in an organic solvent, adding methyl iodide to participate in the reaction, adding potassium carbonate as a catalyst, heating and stirring the reaction solution, extracting the organic phase with DCM after the reaction is completed, separating, drying, and evaporating the solvent under reduced pressure to obtain a crude product, and separating and purifying the crude product by column chromatography to obtain compound 2a.

[0017]

[0018] (2) dissolving compound 2a in an organic solvent, and then adding tert-butyl nitrite and heating and stirring. After the reaction is completed, the solvent is evaporated under reduced pressure to obtain a crude product, and the crude product is separated and purified by silica gel column chromatography to obtain the required nitric oxide donor I

[0019]

[0020] The preparation method of the nitric oxide donor comprises the following steps: (3) dissolving compound 1a in an organic solvent, adding di-tert-butyl dicarbonate to participate in the reaction, adding an inorganic base as a catalyst, and heating and stirring overnight. Then quench the reaction with ice water, extract and separate with EA, dry, and evaporate the solvent under reduced pressure to obtain a crude product, which is separated and purified by column chromatography to obtain compound 3a;

[0021]

[0022] (4) dissolving compound 3a in an organic solvent, stirring the reaction solution, and then adding tert-butyl nitrite and heating and stirring. After the reaction is completed, the solvent is evaporated under reduced pressure to obtain a crude product, and the crude product is separated and purified by silica gel column chromatography to obtain the required nitric oxide donor II

[0023]

[0024] The preparation method of the nitric oxide donor comprises the following steps: (5) dissolving compound 1a in an organic solvent, adding trifluoromethanesulfonic anhydride to participate in the reaction, adding an inorganic base as a catalyst, and stirring the whole reaction system. After the reaction is completed, ice water is added to the reaction system to quench the reaction, the organic phase is extracted with DCM, and the liquid is separated. The solvent is evaporated under reduced pressure to obtain a crude product, which is separated and purified by column chromatography to obtain compound 4a

[0025]

[0026]

[0027] (6) Compound 4a is dissolved in an organic solvent, 3, 4, 5-trifluorobenzenboronic acid is added to participate in the reaction, tetraphenylphosphonium palladium is added, potassium carbonate is used as a catalyst, and heating stirring is performed. After the reaction is completed, the organic phase is extracted with DCM, and the liquid is separated. The solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography is performed to separate and purify the compound 5a

[0028]

[0029] (7) Compound 5a is dissolved in an organic solvent, the reaction solution is stirred, tert-butyl nitrite is added, and heating stirring is performed. After the reaction is completed, the solvent is evaporated under reduced pressure to obtain a crude product, and the crude product is subjected to silica gel column chromatography to obtain the desired nitric oxide donor III

[0030]

[0031] The preparation method of the nitric oxide donor comprises the following steps: (8) Compound 4a is dissolved in an organic solvent, p-methoxybenzenboronic acid is added to participate in the reaction, tetraphenylphosphonium palladium is added, potassium carbonate is used as a catalyst, and heating stirring is performed. After the reaction is completed, the organic phase is extracted with DCM, and the liquid is separated. The solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography is performed to separate and purify the compound 6a

[0032]

[0033] (9) Compound 6a is dissolved in an organic solvent, the reaction solution is stirred, tert-butyl nitrite is added, and heating stirring is performed. After the reaction is completed, the solvent is evaporated under reduced pressure to obtain a crude product, and the crude product is subjected to silica gel column chromatography to obtain the desired nitric oxide donor IV

[0034]

[0035] Any of the nitric oxide donors for use in the preparation of a medicament for treating or preventing diabetes, hypertension-related diseases, and cardiovascular diseases.

[0036] In step (1), the organic solvent of the reaction is THF

[0037] In step (1), the reaction heating temperature is 50-60°C

[0038] In step (1), the reaction time is 12-24 hours

[0039] In step (1), the eluent for column chromatography separation in the reaction is EA / PE = 1: (10-5)

[0040] In step (2), the organic solvent of the reaction is DCM

[0041] In step (2), the reaction heating temperature is 30-40°C

[0042] The reaction time in step (2) is 3-4 hours

[0043] The eluent for column chromatography separation in the reaction in step (2) is EA / PE = 1: (6-4)

[0044] The organic solvent for the reaction in step (3) is DCM

[0045] The reaction temperature in step (3) is 30-40℃

[0046] The inorganic base for the reaction in step (3) is potassium carbonate

[0047] The reaction time in step (3) is 12-24 hours

[0048] The eluent for column chromatography separation in the reaction in step (3) is EA / PE = 1: (10-8) The organic solvent for the reaction in step (4) is DCM

[0049] The heating temperature for the reaction in step (4) is 30-40℃

[0050] The reaction time in step (4) is 3-4 hours

[0051] The eluent for column chromatography separation in the reaction in step (4) is EA / PE = 1: (10-7) The organic solvent for the reaction in step (5) is DCM

[0052] The reaction temperature in step (5) is 0℃ to room temperature

[0053] The inorganic base for the reaction in step (5) is potassium carbonate

[0054] The eluent for column chromatography separation in the reaction in step (5) is EA / PE = 1: (7-5) The organic solvent for the reaction in step (6) is DMF;

[0055] The heating temperature for the reaction in step (6) is 100-110℃;

[0056] The reaction time in step (6) is 8-12 hours;

[0057] The eluent for column chromatography separation in the reaction in step (6) is EA / PE = 1: (8-5) The organic solvent for the reaction in step (7) is DCM;

[0058] The heating temperature for the reaction in step (7) is 30-40℃;

[0059] The reaction time in step (7) is 3-4 hours;

[0060] In step (7), the eluent for column chromatography separation in the reaction is EA / PE=1: (6-5) In step (8), the organic solvent for the reaction is DMF;

[0061] In step (8), the heating temperature for the reaction is 100-110℃

[0062] In step (8), the reaction time is 8-12 hours

[0063] In step (8), the eluent for column chromatography separation in the reaction is EA / PE=1: (7-5) In step (9), the organic solvent for the reaction is DCM

[0064] In step (9), the heating temperature for the reaction is 30-40℃

[0065] In step (9), the reaction time is 3-4 hours

[0066] In step (9), the eluent for column chromatography separation in the reaction is EA / PE=1: (8-6)

[0067] In order to solve the problem of slow release of NO in the dark, by adding a small amount of L-cysteine to the synthesized nitric oxide donor, dissolved in PBS buffer, the release of nitric oxide donor in the dark can be promoted

[0068] The mechanism of the enzymatic nitric oxide donor releasing nitric oxide is as follows:

[0069] The nitric oxide donor I with nitroso group, in PBS buffer, due to the reducing property of L-cysteine, can promote the release of nitric oxide with oxidizing property, and L-cysteine is a large amount of biological enzyme contained in the organism, which can coexist in a large amount in the organism. At this time, under the condition of adding L-cysteine, even in the absence of light, the amount of nitric oxide released can be increased by 50%-60%. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 is the structural formula of the enzymatic light-controlled nitric oxide donor I containing coumarin skeleton;

[0071] Figure 2 is the mechanism diagram of the release of nitric oxide of the enzymatic light-controlled nitric oxide donors I, II, III and IV containing coumarin skeleton;

[0072] Figure 3 is the nuclear magnetic hydrogen spectrum of the nitric oxide donor I;

[0073] Figure 4 is the nuclear magnetic carbon spectrum of the nitric oxide donor I;

[0074] Figure 5 NMR spectrum of nitric oxide donor II;

[0075] Figure 6 NMR spectrum of nitric oxide donor II;

[0076] Figure 7 UV absorption spectrum of nitric oxide donor I when it releases nitric oxide. Nitric oxide donor I was dissolved in neutral PBS buffer, irradiated with a 365 nm ultraviolet lamp, and the change in the UV absorption spectrum of nitric oxide donor I was recorded intermittently. It can be seen that with the continuous irradiation of the 365 nm ultraviolet lamp, the absorption peak of nitric oxide donor I gradually disappeared, and the absorption peak of the product after decomposition began to appear. And the trend gradually tends to be flat, and nitric oxide donor I is decomposed completely at about 80 s of irradiation;

[0077] Figure 8 Fluorescence spectrum of nitric oxide donor I when it releases nitric oxide. Nitric oxide donor I was dissolved in neutral PBS buffer, irradiated with a 365 nm ultraviolet lamp, and the change in the fluorescence emission spectrum of nitric oxide donor I was recorded intermittently. Since nitric oxide donor I itself has weak fluorescence, it can be seen that under the continuous irradiation of the 365 nm ultraviolet lamp, the fluorescence signal of the donor gradually increases with the increase of the irradiation time, and the release ends at 80 s;

[0078] Figure 9 UV absorption spectrum of nitric oxide donor II when it releases nitric oxide. Nitric oxide donor II was dissolved in neutral PBS buffer, irradiated with a 365 nm ultraviolet lamp, and the change in the UV absorption spectrum of nitric oxide donor II was recorded intermittently. It can be seen that with the continuous irradiation of the 365 nm ultraviolet lamp, the absorption peak of nitric oxide donor II gradually disappeared, and the absorption peak of the product after decomposition began to appear. And the trend gradually tends to be flat, and nitric oxide donor II is decomposed completely at about 80 s of irradiation;

[0079] Figure 10 Fluorescence spectrum of nitric oxide donor II when it releases nitric oxide. Nitric oxide donor II was dissolved in neutral PBS buffer, irradiated with a 365 nm ultraviolet lamp, and the change in the fluorescence emission spectrum of nitric oxide donor II was recorded intermittently. Since nitric oxide donor II itself has weak fluorescence, it can be seen that under the continuous irradiation of the 365 nm ultraviolet lamp, the fluorescence signal of the donor gradually increases with the increase of the irradiation time, and the release ends at 80 s;

[0080] Figure 11: Take the sodium nitrite solution and griess reagent in the centrifuge tube mixed evenly, while the concentration of sodium nitrite is proportional to increase, avoid light 10 min after the 3 mL with UV spectrophotometer determination 524 nm absorbance. Do three parallel experiments, take three experimental average, with the concentration of sodium nitrite on the absorbance of standard curve, get the regression equation, such as Figure 11 ;

[0081] Figure 12 Figure 1 is a scatter plot of the amount of nitric oxide released by nitric oxide donor I under five conditions in ethanol / PBS buffer solution over time. It can be seen that the maximum release efficiency of nitric oxide donor I is 38.00%, and under the condition of light, it rapidly reaches the peak value at about 100s;

[0082] Figure 13 Figure 2 is a scatter plot of the amount of nitric oxide released by nitric oxide donor I under five conditions in nanoparticle / PBS buffer solution over time. It can be seen that the maximum release efficiency of nitric oxide donor I is 36.39%, and under the condition of light, it rapidly reaches the peak value at about 10 min;

[0083] Figure 14 Figure 3 is a scatter plot of the amount of nitric oxide released by nitric oxide donor II under five conditions in ethanol / PBS buffer solution over time. It can be seen that the maximum release efficiency of nitric oxide donor II is 66.76%, and under the condition of light, it rapidly reaches the peak value at about 50s;

[0084] Figure 15 Figure 4 is a scatter plot of the amount of nitric oxide released by nitric oxide donor II under five conditions in nanoparticle / PBS buffer solution over time. It can be seen that the maximum release efficiency of nitric oxide donor II is 68.37%, and under the condition of light, it rapidly reaches the peak value at about 10 min. DETAILED DESCRIPTION

[0085] The present application will be described in detail below in conjunction with specific examples.

[0086] Example 1 Synthesis of nitric oxide donor I

[0087] (1) Compound 1a (223.23 mg, 1 mmol) was dissolved in 20 mL of THF, 283.88 mg of iodomethane, 2 mmol) was added to participate in the reaction, 345.5 mg of potassium carbonate, 2.5 mmol) was added as a catalyst, the reaction liquid was heated to 60°C and stirred, the reaction was carried out for 12 h, then 200 mL of DCM was added to extract the organic phase, the liquid was separated, dried, and the solvent was evaporated under reduced pressure to obtain a crude product, the crude product was separated by column chromatography (EA / PE = 1: (10-5)), and compound 2a (220 mg, yield 89.00%) was obtained by purification.

[0088]

[0089] (2) Compound 2a (100.00 mg, 0.43 mmol) was added to a solution of tert-butyl nitrite (133.02 mg, 1.29 mmol) in DCM (10 mL), and the reaction was stirred at 40 °C. After 4 h, the reaction was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (EA / PE = 1: (6-4)) to obtain nitric oxide donor I (89.53 mg, yield 83.88%).

[0090]

[0091] Example 2 Synthesis of nitric oxide donor II

[0092] (1) Compound 1a (223.27 mg, 1 mmol) was dissolved in 20 mL of DCM, and di-tert-butyl dicarbonate (436.50 mg, 2 mmol) was added to participate in the reaction. Potassium carbonate (345.5 mg, 2.5 mmol) was added as a catalyst, and the reaction was stirred overnight. Then, 100 mL of ice water was added to quench the reaction, and 200 mL of EA was added to extract the solution. The solvent was evaporated under reduced pressure to obtain a crude product, which was purified by column chromatography (EA / PE = 1: (10-8)) to obtain compound 3a (290.52 mg, yield 87.14%).

[0093]

[0094] (2) Compound 3a (100.00 mg, 0.30 mmol) was added to a solution of tert-butyl nitrite (123.74 mg, 1.20 mmol) in DCM (10 mL), and the reaction was stirred at 40 °C. After 4 h, the reaction was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (EA / PE = 1: (10-7)) to obtain nitric oxide donor II (87.26 mg, yield 87.00%).

[0095]

[0096] Example 3 Synthesis of nitric oxide donor III

[0097] (1) Compound 1a (893.08 mg, 4 mmol) was dissolved in 20 mL of DMF, triflic anhydride (1467.10 mg, 5.2 mmol) was added to participate in the reaction, potassium carbonate (345.5 mg, 2.5 mmol) was added as a catalyst, and the whole reaction system was cooled to 0°C and stirred. After 12 h of reaction, 100 mL of ice water was added to quench the reaction, and the organic phase was extracted with 100 mL of DCM, and separated. The solvent was evaporated under reduced pressure to obtain the crude product, which was purified by column chromatography (EA / PE = 1: (7-5)) to obtain compound 4a (1.28 g, yield 87.66%)

[0098]

[0099] (2) Compound 4a (1.83 g, 5 mmol) was dissolved in 30 mL of DMF, 3,4,5-trifluorobenzenboronic acid (1.32 g, 7.5 mmol) was added to participate in the reaction, tetrakis(triphenylphosphine)palladium (288.89 mg, 0.25 mmol) was added, potassium carbonate (1.04 g, 6 mmol) was added as a catalyst, and heated to 110°C and stirred. After 12 h of reaction, the organic phase was extracted with 100 mL of DCM, and separated. The solvent was evaporated under reduced pressure to obtain the crude product, which was purified by column chromatography (EA / PE = 1: (8-5)) to obtain compound 5a (1.45 g, yield 83.33%)

[0100]

[0101] (7) Compound 5a (100.00 mg, 0.29 mmol) was added to a solution of tert-butyl nitrite (89.71 mg, 0.87 mmol) in DCM (10 mL), and the reaction solution was heated to 40°C and stirred. After 4 h of reaction, the reaction solution was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain the crude product, which was purified by column chromatography (EA / PE = 1: (6-5)) to obtain nitric oxide donor III (78.05 mg, yield 77.48%).

[0102]

[0103] Example 4 Synthesis of Nitric Oxide Donor IV

[0104] (1) Compound 4a (2.19g, 6mmol) was dissolved in 30mL DMF, p-methoxybenzene boronic acid (1.37g, 9mmol) was added to participate in the reaction, tetrakis triphenyl phosphine palladium (277mg, 0.24mmol) was added as a catalyst, potassium carbonate (995.04mg, 7.2mmol) was heated to 110°C and stirred. After 12h of reaction, the organic phase was extracted with 100mL DCM, and the solvent was evaporated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography (EA / PE = 1: (7-5)) to obtain compound 6a (1.65g, yield 85.05%)

[0105]

[0106] (2) Compound 6a (100.00mg, 0.31mmol) was added to a solution of tert-butyl nitrite (126.84mg, 1.23mmol) in DCM (10mL), and the reaction solution was heated to 40°C and stirred. After 4h of reaction, the reaction solution was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography (EA / PE = 1: (8-6)) to obtain nitric oxide donor IV (89.35mg, yield 89.13%).

[0107]

[0108] Example 5 Detection of light-induced nitric oxide release

[0109] The process of light-induced nitric oxide release from solutions of nitric oxide donors I and II was investigated in phosphate buffer at room temperature. As shown in Figure 7 , 9, after 365nm ultraviolet light irradiation, the emission spectrum of the nitric oxide donor I and II solution changed significantly. The absorption peak of the original nitric oxide donor gradually disappeared, and the absorption peak of the product began to appear. After 70-80s of light irradiation, the ultraviolet absorption spectrum gradually tended to be consistent, indicating that the nitric oxide release from nitric oxide donor I was complete at about 80s of light irradiation. The fluorescence emission spectrum was also consistent with the predicted results, and the results are shown in Figure 8 , 10.

[0110] Example 6 Detection of whether nitric oxide is released and the amount of nitric oxide released

[0111] Next, the release of nitric oxide was detected by the Griess method, which is a well-recognized method in the industry. The most commonly used method for analyzing nitrite and nitrate is the Griess reaction. In the Griess reaction, nitrite reacts with sulfanilic acid under acidic conditions to form a diazonium ion, which then couples with α-naphthylamine to form a water-soluble red-violet azo dye with a maximum absorption wavelength at 524 nm. Therefore, the content of NO can be determined by measuring the content of nitrite by the Griess method. First, prepare the Griess reagent, and then prepare different concentrations of sodium nitrite solution and add the Griess reagent. Measure the absorbance at 524 nm, and plot the regression equation of absorbance versus sodium nitrite concentration as follows: Figure 11 .

[0112] Sample testing: Add 3.90 mL of PBS phosphate buffer, 100 μL of sample, and 100 μL of L-cysteine solution to a 10 ml centrifuge tube, respectively. After irradiation, add 1 mL of Griess reagent, and measure the absorbance value at 524 nm after 10 minutes. Substitute the measured absorbance value into the regression equation to obtain the NO release concentration. The concentration of the sample is 20 μM, and the nitric oxide release rate can be calculated. Two different solution environments (1. ethanol / PBS buffer 2. nanoparticle / PBS buffer) were used to test the sample, and the sample was prepared at a concentration of 20 μM.

[0113] Five test conditions were set: 1. Add L-cysteine, no light, room temperature; 2. 10 mW light, room temperature; 3. No light, room temperature; 4. 5 mW light, room temperature; 5. No light, 37°C

[0114] (1) Figure 12 is a scatter plot of the amount of nitric oxide released by Nitric Oxide Donor I in ethanol / PBS buffer under five conditions versus time. It can be seen that the maximum release efficiency of nitric oxide from Nitric Oxide Donor I is 38.00%, and under light conditions, it rapidly reaches a peak value at about 100 s.

[0115] (2) Figure 13 is a scatter plot of the amount of nitric oxide released by Nitric Oxide Donor I in nanoparticle / PBS buffer under five conditions versus time. It can be seen that the maximum release efficiency of nitric oxide from Nitric Oxide Donor I is 36.39%, and under light conditions, it rapidly reaches a peak value at about 10 min.

[0116] (3) Figure 14 is a scatter plot of the amount of nitric oxide released by Nitric Oxide Donor II in ethanol / PBS buffer under five conditions versus time. It can be seen that the maximum release efficiency of nitric oxide from Nitric Oxide Donor II is 66.76%, and under light conditions, it rapidly reaches a peak value at about 50 s.

[0117] (4) Figure 15 Figure 29 is a scatter plot of the amount of nitric oxide released from nitric oxide donor II over time under five conditions in nanoparticle / PBS buffer. It can be seen that the maximum nitric oxide release efficiency of nitric oxide donor II was 68.37%, and reached a peak value rapidly under light conditions at about 10 min.

[0118] It should be understood that all the modifications and variations can be made according to the above description by those of ordinary skill in the art, and all these modifications and variations shall belong to the protection scope of the appended claims of the present application.

Claims

1. An enzymatically catalytically controlled nitric oxide donor containing a coumarin backbone, having the structure shown in the following formula: ； Or it can have the structure shown in the following formula: .

2. The method for preparing the nitric oxide donor according to claim 1, characterized in that: Includes the following steps: (1) Compound 1a was dissolved in an organic solvent, iodomethane was added to participate in the reaction, potassium carbonate was added as a catalyst, the reaction solution was heated and stirred, the organic phase was extracted with DCM after the reaction was completed, the liquid was separated, dried, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound 2a. ； 1a 2a; (2) Dissolve compound 2a in an organic solvent, then add tert-butyl nitrite, heat and stir; after the reaction is complete, evaporate the solvent under reduced pressure to obtain the crude product, and then obtain the desired nitric oxide donor I by silica gel column chromatography. ； 2a Ⅰ 。 3. The method for preparing the nitric oxide donor according to claim 1, characterized in that: Includes the following steps: (1) Compound 1a was dissolved in an organic solvent, and di-tert-butyl dicarbonate was added to participate in the reaction. An inorganic base was added as a catalyst, and the mixture was heated and stirred overnight. The reaction was then quenched with ice water, and the mixture was extracted and separated by EA and dried. The solvent was evaporated under reduced pressure to obtain the crude product, which was then purified by column chromatography to obtain compound 3a. ； 1a 3a; (2) Dissolve compound 3a in an organic solvent, stir the reaction mixture, add tert-butyl nitrite, heat and stir; after the reaction is completed, evaporate the solvent under reduced pressure to obtain the crude product, and then obtain the desired nitric oxide donor II by silica gel column chromatography. ； 3a Ⅱ 。 4. Use of the nitric oxide donor of claim 1 in the preparation of medicaments for the treatment or prevention of diabetes and hypertension-related diseases.

5. Use of the nitric oxide donor of claim 1 in the preparation of medicaments for the treatment or prevention of cardiovascular diseases.

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