Light-operated nitric oxide donor with good water solubility as well as preparation and application of light-operated nitric oxide donor

Abstract

The invention relates to a light-operated nitric oxide donor with good water solubility as well as preparation and application of the light-operated nitric oxide donor. Specifically, the invention provides a compound with a structure as shown in a formula I. The compound has the advantages of good water solubility, stable properties and the like. The invention further provides a preparation method of the compound shown in the formula I, the compound is used for preparing drugs beneficial to diseases of nitric oxide release, and the diseases are selected from nervous system diseases and cardiovascular diseases. The nitric oxide donor represented by the formula I is used as a light-operated nitric oxide donor with good water solubility, and can realize light-operated release of nitric oxide in a zebra fish body.

Description

Technical Field

[0001] The invention belongs to the field of nitric oxide donors, and in particular relates to nitric oxide donors, preparation and application thereof. Background Art

[0002] Nitric oxide can dominate multiple signaling pathways and participate in regulating a variety of physiological and pathological processes, including the conduction of neural signals, the dynamic balance of microvessels and macrovessels, the regulation of immune inflammation, the inhibition of tumor occurrence and metastasis, cell apoptosis, immune response to infection, angiogenesis, etc. However, the prerequisite is that the concentration of nitric oxide must be within an appropriate range. Excessive concentration of nitric oxide in the body can mediate neurotoxicity and induce neuronal death through oxidative stress, the production of reactive oxygen intermediates, and damage to the antioxidant system. In addition, high concentrations of nitric oxide can react with superoxide anions (O 2− ) reaction affects various biological functions in the body, and also causes irreversible damage to lipids, proteins, DNA, etc. and depletes glutathione in the body. Nitric oxide is also a gaseous transmitter involved in vasodilation, neuronal signal transduction and immune response, and can inhibit platelet aggregation and fungal growth in organisms, so it is widely used. The synthesis of nitric oxide in the body is subject to many restrictions, and nitric oxide has a short life span, with a half-life of only a few seconds, but the balance of nitric oxide levels in the body is crucial to the health of the body, so nitric oxide donors are very necessary for the prevention of human diseases.

[0003] The nitric oxide donors reported in the literature include metal nitroso compounds, nitrosamine compounds, etc. However, these donors have certain limitations, such as spontaneous release once the donors are added to various biological matrices; poor stability in biological media, etc. Recently, some light-controlled nitric oxide donors have been reported in the literature. Light-sensitive nitric oxide donors with light as the stimulation signal have the advantage of overcoming the uncontrollable release dose. However, there are still shortcomings in light-controlled nitric oxide donors. For example, some of them are unstable in nature and have poor water solubility, requiring solubilization with co-solvents, and more co-solvents are more harmful to the human body. Therefore, it is necessary to develop a light-controlled nitric oxide donor with good water solubility and stable properties. Rhodamine derivatives have the advantages of long excitation and emission wavelengths, high quantum efficiency, and good water solubility. Based on this, the present invention designs and synthesizes a new type of light-controlled nitric oxide donor formula I with rhodamine 6G as a fluorophore. Summary of the invention

[0004] In view of the problem that the light-controlled nitric oxide donors in the prior art are relatively small and have poor water solubility, the present invention aims to provide a light-controlled nitric oxide donor with good water solubility.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned nitric oxide donor, which has readily available raw materials, simple synthesis steps and high yield.

[0006] Another object of the present invention is to provide formula I for preparing drugs for treating or preventing nervous system diseases and cardiovascular diseases or related uses.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A light-controlled nitric oxide donor with good water solubility, whose structure is shown in formula I.

[0009]

[0010] A method for synthesizing a light-controlled nitric oxide donor with good water solubility comprises the following steps: (1) R6G is dissolved in an organic acid, stirred in a water bath, and then an aqueous solution of sodium nitrite is added. The reaction is monitored by thin layer chromatography. After the reaction is completed, the reaction solution is extracted and dried to obtain a crude product. The crude product is purified by silica gel column chromatography to obtain the desired nitric oxide donor Formula I.

[0011]

[0012] In step (1), the organic acid in the reaction is glacial acetic acid.

[0013] In step (1), the molar ratio of sodium nitrite to formula I in the reaction is 0.5-20.

[0014] In step (1), the reaction time is 0.5 to 24 hours.

[0015] In step (1), the eluent for the silica gel column chromatography separation in the reaction is dichloromethane:methanol=10:0.5-5.

[0016] A well-water-soluble nitric oxide donor is used to release nitric oxide in aqueous solution, cells and zebrafish by light irradiation.

[0017] The mechanism of releasing nitric oxide by the light-controlled nitric oxide donor with good water solubility of the present invention is as follows: The N-nitroso bond in the molecular structure of the nitric oxide donor is unstable. After irradiation with 365 nm ultraviolet light, the N-nitroso bond breaks, releasing a portion of nitric oxide and R6G, thereby restoring fluorescence.

[0018] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: After the donor is irradiated with light, it can release a part of nitric oxide, and its fluorescence intensity gradually increases with the increase of illumination time. Compared with the existing nitric oxide donors, its main advantages are: first, the donor formula I releases nitric oxide after irradiation with 365 nm ultraviolet light, and the fluorescence intensity gradually increases with the illumination time; while its fluorescence intensity remains basically unchanged without light, which is conducive to the precise light-controlled release of nitric oxide, and is convenient for studying the mechanism of nitric oxide's participation in pathological processes and the treatment of related diseases; second, the donor compound has a simple synthesis step, high yield, good stability and water solubility. It is estimated that this donor will have a broader application prospect in the biomedical field in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of Form I.

[0020] Figure 2 This is a diagram of the mechanism of release of nitric oxide by donor formula I under light irradiation.

[0021] Figure 3 This is the H NMR spectrum of donor formula Ⅰ.

[0022] Figure 4 is the mass spectrum of donor formula Ⅰ.

[0023] Figure 5 After irradiation with a 365 nm UV lamp, the emission spectrum of a 2.0 μM solution of Formula I was obtained. Formula I was dissolved in a neutral phosphate buffer solution, irradiated with a 365 nm UV lamp, and the changes in the fluorescence emission spectrum of Formula I were recorded intermittently (excitation wavelength was 480 nm). As can be seen from the figure, the fluorescence signal of the donor gradually increased with the increase of illumination time, and the release ended at 150 minutes.

[0024] Figure 6 This is the fluorescence spectrum of nitric oxide released when DAN tests the decomposition of Formula I. The same concentration of Formula I and DAN are dissolved in neutral phosphate buffer, illuminated with 365 nm ultraviolet light, and the changes in the fluorescence emission spectrum are recorded intermittently.

[0025] Figure 7 The zebrafish was imaged. Formula I was co-cultured with zebrafish for 30 min, and then washed three times with neutral phosphate buffer solution, and then fluorescence microscopy was performed. Then, it was irradiated with a 365 nm ultraviolet lamp, and the fluorescence microscopy imaging of the zebrafish was recorded intermittently until the fluorescence intensity no longer changed. Implementation

[0026] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0027] Example 1 Synthesis of Nitric Oxide Donor Formula I R6G (0.1198 g, 0.25 mmol) was dissolved in glacial acetic acid (3 mL), stirred in an ice-water bath, and then an aqueous solution of NaNO2 (0.0735 g, 1.07 mmol) was added. The reaction was monitored, and after completion, it was extracted with DCM and dried under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain pure compound I.

[0028]

[0029] Example 2 Detection of Nitric Oxide Release Formula I (2 μM) and DAN (2 μM) were added simultaneously to a phosphate buffer solution at pH = 7.4. The solution was irradiated with a 365 nm UV lamp, and its fluorescence emission spectrum was recorded intermittently. The results showed that after irradiation with a 365 nm UV lamp, a new fluorescence emission peak appeared at 410 nm ( Figure 6 ), and the fluorescence signal gradually increased with the extension of irradiation time, and basically reached equilibrium at 50 min. This proves that formula I successfully released nitric oxide after irradiation. Figure 6 .

[0030]

[0031] Example 3 Light-controlled release of nitric oxide in zebrafish In the zebrafish imaging experiment, donor I was co-cultured with zebrafish for 30 min. The zebrafish was washed three times with neutral phosphate buffer solution and fixed on a slide with fixative. Subsequently, confocal fluorescence imaging was performed. Figure 7 As shown in the figure, a) is the zebrafish imaging under bright field conditions; b) is the live zebrafish imaging under dark field conditions at the beginning, and only a very weak fluorescence signal is observed. Then, the zebrafish is irradiated with a 365 nm ultraviolet lamp and the fluorescence microscopic imaging of the zebrafish is recorded intermittently until the fluorescence intensity no longer changes. The results show that donor I can achieve light-controlled release of nitric oxide in zebrafish.

Claims

1. A light-controlled nitric oxide donor with good water solubility, the structural formula of which is shown in Formula I: 。 2. The method for preparing a light-controlled nitric oxide donor with good water solubility according to claim 1, characterized in that: The following steps are involved: R6G is dissolved in an acid, stirred at a certain temperature, and then an aqueous solution of an inorganic salt is added, and the reaction is monitored. After the reaction is completed, it is extracted with an organic solvent and dried to obtain a crude product. The crude product is purified to obtain a pure compound of formula I; 。 3. The preparation method according to claim 2, characterized in that: The preparation method has the following characteristics: In this reaction, the dissolution is carried out under acidic conditions, and the acid selected is glacial acetic acid; The inorganic salt solution mentioned, the selected inorganic salt is sodium nitrite; The detection reaction time is: 0.5-24 hours.

4. Use of the water-soluble light-controlled nitric oxide donor according to claim 1 in the preparation of drugs for diseases that benefit from the release of nitric oxide.

5. The use of a light-controlled nitric oxide donor with good water solubility as claimed in claim 4, characterized in that: The disease that benefits from the release of nitric oxide is selected from the group consisting of a nervous system disease and a cardiovascular disease.

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