Strong fluorescence recovery light-operated nitric oxide donor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemistry, in particular to a light-operated nitric oxide donor with strong fluorescence recovery as well as a preparation method and application of the light-operated nitric oxide donor. According to the light-operated nitric oxide donor with strong fluorescence recovery and the preparation method of the light-operated nitric oxide donor, the donor has good light sensitivity, light stability and anti-interference capacity under ultraviolet irradiation, and accurate control over nitric oxide released by the donor in time and space can be achieved. Meanwhile, the donor is subjected to fluorescence quenching, but the fluorescence intensity is greatly improved by about 110 times after ultraviolet irradiation, accurate synchronization of NO release and detection is achieved, NO with extremely low concentration can also be detected, the method is suitable for real-time monitoring of trace NO in organisms, and the method is low in raw material cost, simple in synthesis step and high in yield. The compound can be used for preparing medicines or related medicines for treating cardiovascular diseases, Alzheimer's disease, inflammation, tumors and neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and particularly to a photo-controlled nitric oxide donor with strong fluorescence recovery, a preparation method thereof, and an application thereof. Background Art

[0002] Nitric oxide (NO) is an important endogenous molecule with multiple uses and plays an important role in signal regulation in multiple physiological and pathological reactions such as the nervous system, immune system, and cardiovascular regulation, which has great significance for the prevention and treatment of diseases. However, NO has a short half-life and is extremely easy to diffuse in tissues, and insufficient generation of NO in the body will cause a series of pathological conditions. Nitric oxide donors are an important way to provide exogenous NO, but traditional spontaneously dissociating NO donors have limitations in detecting the release site, release dose, and release rate of NO, etc.

[0003] The progress of a photochemical reaction can be controlled by the exposure time and position. Photo-sensitive NO donors using light as a stimulation signal precisely control the release of NO through an external light source, providing precise release in terms of time and space. The release of NO by photo-induced nitric oxide donors is a powerful solution for providing exogenous nitric oxide. The change amplitude of the fluorescence signal of the photo-controlled nitric oxide donor after illumination is small, which will directly affect the detection sensitivity. Low sensitivity may make it difficult to accurately detect the release of NO in a low-concentration NO environment. In contrast, a photo-controlled nitric oxide donor with strong fluorescence recovery can still ensure detection reliability when reducing the donor usage dose, effectively reducing the risk of biological toxicity. This breakthrough design not only provides a high-resolution dynamic monitoring tool for the research of NO-related mechanisms in the fields of cardiovascular diseases, neural regulation, and tumor treatment, but also opens up a new way for the optimization of drug delivery systems and precision medicine applications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a photo-controlled nitric oxide donor with strong fluorescence recovery, a preparation method thereof, and an application thereof in view of the deficiencies of the above-mentioned prior art. The fluorescence intensity of the donor can increase by 110 times under ultraviolet light irradiation. The donor has fluorescence quenching in the unilluminated state, with low background fluorescence. After ultraviolet light irradiation, the fluorescence intensity is greatly restored, with a high signal-to-background contrast and enhanced detection accuracy. Ultraviolet light irradiation synchronously releases NO and activates fluorescence, realizing high-resolution monitoring of the release site and kinetics, and is used in the preparation of drugs for treating cardiovascular diseases, Alzheimer's disease, inflammation, tumors, and neurodegenerative diseases or related uses.

[0005] The present invention provides a photo-controlled nitric oxide donor with strong fluorescence recovery, and the chemical structural formula of the donor is: .

[0006] The present invention provides a method for preparing the above-mentioned light-controlled nitric oxide donor with strong fluorescence recovery, comprising the following steps: S1. Take 3-morpholinophenol, 3-bromo-N-methylaniline, and cesium carbonate and dissolve them in dimethyl sulfoxide. First, introduce an inert gas, then add cuprous bromide and picolinic acid to obtain a reaction solution. Introduce the inert gas again, heat and stir the reaction solution. After the reaction is completed, cool it to room temperature, and obtain compound 1 through extraction and column chromatography separation and purification; S2. Take compound 1 prepared in S1 and ninhydrin and dissolve them in acetonitrile. Then add glacial acetic acid and a strong acid to obtain a reaction solution. Heat and stir the reaction solution. After the reaction is completed, cool it to room temperature, and obtain compound 2 through extraction and column chromatography separation and purification; S3. Take compound 2 prepared in S2 and dissolve it in a weak acid. Stir it in an ice-water bath, and dropwise add an aqueous solution of sodium nitrite to obtain a reaction solution. After the reaction is completed, filter the reaction solution by suction, and obtain the light-controlled nitric oxide donor after washing.

[0007] According to the preparation method provided by the present invention, in S1, the molar volume ratio of 3-morpholinophenol, 3-bromo-N-methylaniline, cesium carbonate, dimethyl sulfoxide, cuprous bromide, and picolinic acid is 4 mmol: 1.21 mL: 8 mmol: 10 mL: 1.5 mmol: 1.2 mmol.

[0008] According to the preparation method provided by the present invention, in S1, the inert gas is argon. The time for the first introduction of the inert gas is 20 min, the time for the second introduction of the inert gas is 10 min, the temperature for the heating and stirring reaction is 120 °C, the time for the heating and stirring reaction is 15 h, and the extraction agent for extraction is ethyl acetate.

[0009] According to the preparation method provided by the present invention, in S2, the strong acid is concentrated sulfuric acid. The molar volume ratio of compound 1, ninhydrin, acetonitrile, glacial acetic acid, and concentrated sulfuric acid is 1.5 mmol: 2.1 mmol: 5 mL: 5 mL: 0.5 mL.

[0010] According to the preparation method provided by the present invention, in S2, the temperature for the heating and stirring reaction is 75 °C, the time for the heating and stirring reaction is 12 h, and the extraction agent for extraction is chloroform.

[0011] According to the preparation method provided by the present invention, in S3, the weak acid is glacial acetic acid. The molar volume ratio of compound 2, glacial acetic acid, and the aqueous solution of sodium nitrite is 0.6 mmol: 5 mL: 0.6 mmol.

[0012] According to the preparation method provided by the present invention, in S3, the temperature of the ice-water bath is 0 °C, and the time of the ice-water bath is 15 min.

[0013] The present invention also provides an application of the above-mentioned photo-controlled nitric oxide donor with strong fluorescence recovery. The photo-controlled nitric oxide donor with strong fluorescence recovery can synchronously release nitric oxide and activate fluorescence under ultraviolet light irradiation, realizing high-resolution monitoring of the release site and kinetics.

[0014] The present invention has the following advantages compared with the prior art: The present invention provides a photo-controlled nitric oxide donor with strong fluorescence recovery and a preparation method thereof. Under ultraviolet light irradiation, the donor has good photosensitivity, photostability, and anti-interference ability, and can realize precise control of the release of nitric oxide by the donor in terms of time and space. At the same time, the donor is fluorescence quenched, but the fluorescence intensity increases significantly by about 110 times after ultraviolet light irradiation, realizing precise synchronization of NO release and detection. Even very low concentrations of NO can be detected, which is suitable for real-time monitoring of trace amounts of NO in vivo. This method uses cheap raw materials, has simple synthesis steps and high yields.

[0015] The photo-controlled nitric oxide donor with strong fluorescence recovery provided by the present invention is a photo-controlled nitric oxide donor that can be rapidly released under photo-controlled conditions, and is used in the preparation of drugs for treating cardiovascular diseases, Alzheimer's disease, inflammation, tumors, and neurodegenerative diseases or related uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 is the 1H NMR spectrum of Compound 1 in Example 1; Figure 2 is the 1H NMR spectrum of Compound 2 in Example 1; Figure 3 is the 1H NMR spectrum of the donor in Example 1; Figure 4 is the high-resolution mass spectrum of the photolyzed donor; Figure 5 is the fluorescence emission spectrum of the donor releasing nitric oxide upon photoirradiation; Figure 6 is the ultraviolet absorption spectrum of the donor releasing nitric oxide upon photoirradiation; Figure 7 is the graph of the change in fluorescence intensity of the donor solution upon photoirradiation over time in the presence of interfering substances; Figure 8 is the photosensitivity test graph of the donor; Figure 9 It is a graph of the photostability test of the donor and Compound 2 after irradiation with a 500 nm xenon lamp; Figure 10 It is a graph of the photo-controlled release of nitric oxide by the donor in A549 cells. Detailed implementation manners

[0018] Example 1

[0019] This example provides a preparation method of a photo-controlled nitric oxide donor with strong fluorescence recovery, and the specific steps are as follows: S1. Take 4 mmol of 3-morpholinophenol, 1.21 mL of 3-bromo-N-methylaniline, and 8 mmol of cesium carbonate and dissolve them in 10 mL of dimethyl sulfoxide. Pass argon for 20 min, then add 1.5 mmol of copper(I) bromide and 1.2 mmol of picolinic acid to obtain a reaction solution. Pass argon again for 10 min, heat and stir the reaction solution to 120 °C and react for 15 h. After the reaction is completed, cool to room temperature, extract with ethyl acetate to obtain a crude product, and purify the crude product by column chromatography. The column chromatography eluent is petroleum ether:ethyl acetate = 7:3 to obtain a milky white Compound 1; The structural formula of Compound 1 is: ; S2. Take 1.5 mmol of Compound 1 prepared in S1 and 2.1 mmol of ninhydrin and dissolve them in 5 mL of acetonitrile. Then add 5 mL of glacial acetic acid and 0.5 mL of concentrated sulfuric acid to obtain a reaction solution. Heat and stir the reaction solution to 75 °C for reflux condensation for 12 h. After the reaction is completed, cool to room temperature, extract with chloroform to obtain a crude product, and purify the crude product by column chromatography to obtain a purple Compound 2; The structural formula of Compound 2 is: ; S3. Take 0.6 mmol of Compound 2 prepared in S2 and dissolve it in 5 mL of glacial acetic acid. Stir on an ice bath at 0 °C for 15 min. Take 0.6 mmol of sodium nitrite and dissolve it in 2 mL of distilled water, and dropwise add it to the S2 solution. Detect the reaction by thin layer chromatography. After the reaction is completed, filter the reaction solution by suction, and wash it repeatedly with water to obtain a purple donor, which is the photo-controlled nitric oxide donor.

[0020] The structural formula of the donor is: .

[0021] Example 2 This example provides a detection of the photo-induced nitric oxide release of the donor prepared in Example 1.

[0022] The process of photoinduced release of nitric oxide from the donor solution was investigated in a neutral phosphate buffer solution at 25 °C.

[0023] The mechanism of nitric oxide release from the donor is as follows:

[0024] As Figure 5 shown, the fluorescence intensity of the donor solution at 560 nm after irradiation with 365 nm ultraviolet light gradually increased with the increase of irradiation time and gradually stabilized after 70 min.

[0025] As Figure 6 shown, the absorbance at 530 nm increased with the increase of ultraviolet light irradiation time.

[0026] The experimental results of the fluorescence emission spectrum and ultraviolet-visible absorption spectrum showed that the donor successfully released nitric oxide after irradiation with 365 nm ultraviolet light.

[0027] Example 3

[0028] This example provides the selectivity of the donor prepared in Example 1.

[0029] As Figure 7 shown, the anti-interference performance of the donor was tested.

[0030] In a neutral phosphate buffer solution, the donor concentration was 10 μmol / L and the interfering ion concentration was 2 mmol / L, and the fluorescence emission spectrum was measured.

[0031] The interfering ions from 1 - 9 in the figure were, in turn: none, H2O2, ascorbic acid, BSA, HSA, CYS, tryptophan, GSH, and NaHS; When the interfering ions were present, the time required to reach fluorescence saturation was slightly reduced, indicating that this fluorescence correction method is not interfered by other media for detecting the release amount of nitric oxide.

[0032] Example 4

[0033] This example explored the light sensitivity of the donor prepared in Example 1.

[0034] The donor was dissolved in a neutral phosphate buffer solution, and after irradiating the solution with 365 nm light for 5 min, turning off the light source for 5 min was taken as one cycle.

[0035] As Figure 8 shown, the fluorescence intensity did not change during the 5 min in the dark, and the fluorescence intensity increased significantly after 5 min of light irradiation, indicating that the donor has good light sensitivity.

[0036] Example 5

[0037] This example explores the photostability of the donor prepared in Example 1 and Compound 2 after irradiation with a 500 nm xenon lamp.

[0038] The donor and Compound 2 were separately dissolved in neutral phosphate buffer solution, and the fluorescence emission spectrum was measured once every 5 min under irradiation with a 500 nm xenon lamp for 40 min continuously.

[0039] As Figure 9 shown, after continuous irradiation with a 500 nm xenon lamp for 40 min, the fluorescence intensity basically did not change, indicating that both the donor and Compound 2 have good photostability under continuous irradiation with a 500 nm xenon lamp.

[0040] Example 6

[0041] This example explores the intracellular imaging of the donor prepared in Example 1.

[0042] The donor and A549 cells were co-cultured for 15 min and then cultured in an incubator at 37 °C containing 5% CO2. After the cells adhered to the wall, they were cultured in two groups: the first group was the blank group; the second group was the ultraviolet irradiation group. Cell imaging was performed on the cells without irradiation and irradiated for 5 min, 10 min, 15 min, and 20 min respectively.

[0043] As Figure 10 shown, as the irradiation time increased, the fluorescence signal became stronger and stronger, and the fluorescence intensity was positively correlated with the irradiation time.

[0044] The results showed that the donor can achieve visible light-controlled release of nitric oxide in cells and has uses in the preparation of drugs for treating cardiovascular diseases, Alzheimer's disease, inflammation, tumors, and neurodegenerative diseases or related applications.

[0045] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A light-controlled nitric oxide donor with strong fluorescence recovery, characterized in that: The chemical structure of the donor is: 。 2. A method for preparing a light-controlled nitric oxide donor with strong fluorescence recovery as claimed in claim 1, characterized in that: The following steps are involved: S1, dissolving 3-morpholinephenol, 3-bromo-N-methylaniline and cesium carbonate in dimethyl sulfoxide, introducing an inert gas once, then adding cuprous bromide and 2-pyridinecarboxylic acid to obtain a reaction solution, introducing an inert gas again, heating and stirring the reaction solution, cooling to room temperature after the reaction is completed, and obtaining compound 1 through extraction and column chromatography separation and purification; S2, dissolving the compound 1 prepared in S1 and ninhydrin hydrate in acetonitrile, adding glacial acetic acid and a strong acid to obtain a reaction solution, heating and stirring the reaction solution, cooling to room temperature after the reaction is completed, and extracting and separating and purifying by column chromatography to obtain compound 2; S3. Dissolve the compound 2 prepared in S2 in a weak acid, stir in an ice-water bath, add sodium nitrite aqueous solution dropwise to obtain a reaction solution, and after the reaction is completed, filter the reaction solution and wash it to obtain a light-controlled nitric oxide donor.

3. The preparation method according to claim 2, characterized in that: The molar volume ratio of 3-morpholinephenol, 3-bromo-N-methylaniline, cesium carbonate, dimethyl sulfoxide, cuprous bromide and 2-picolinic acid described in S1 is 4 mmol:1.21 mL:8 mmol:10 mL:1.5 mmol:1.2 mmol.

4. The preparation method according to claim 2, characterized in that: The inert gas in S1 is argon, the time for passing the inert gas once is 20 min, the time for passing the inert gas again is 10 min, the temperature of the heating and stirring reaction is 120°C, the time for the heating and stirring reaction is 15 h, and the extraction agent is ethyl acetate.

5. The preparation method according to claim 2, characterized in that: The strong acid in S2 is concentrated sulfuric acid, and the molar volume ratio of the compound 1, ninhydrin, acetonitrile, glacial acetic acid and concentrated sulfuric acid is 1.5 mmol:2.1 mmol:5 mL:5 mL:0.5 mL.

6. The preparation method according to claim 2, characterized in that: The temperature of the heating and stirring reaction in S2 is 75° C., the time of the heating and stirring reaction is 12 h, and the extraction solvent is chloroform.

7. The preparation method according to claim 2, characterized in that: The weak acid in S3 is glacial acetic acid, and the molar volume ratio of the compound 2, glacial acetic acid and sodium nitrite aqueous solution is 0.6 mmol:5 mL:0.6 mmol.

8. The preparation method according to claim 2, characterized in that: The temperature of the ice water bath in S3 is 0°C, and the duration of the ice water bath is 15 min.

9. An application of the light-controlled nitric oxide donor with strong fluorescence recovery as claimed in claim 1, characterized in that: The light-controlled nitric oxide donor with strong fluorescence recovery can synchronously release nitric oxide and activate fluorescence under ultraviolet light irradiation, thereby achieving high-resolution monitoring of release sites and dynamics.

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