A light-controlled nitric oxide donor with good water solubility, preparation and application thereof

Abstract

The present application discloses a water-soluble light-controlled nitric oxide donor, its preparation and application. Specifically, the present application provides a compound with the structure shown in formula I, which has the advantages of good water solubility, stable properties and the like. The present application also provides a preparation method of the compound of formula I, and the compound of formula I is used for preparing a medicament for treating diseases benefiting from the release of nitric oxide, such as nervous system diseases and cardiovascular diseases. The nitric oxide donor of formula I is a water-soluble light-controlled nitric oxide donor, and can realize the light-controlled release of nitric oxide in zebrafish.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nitric oxide donors, and particularly relates to a nitric oxide donor, preparation and application thereof. BACKGROUND

[0002] Nitric oxide can dominate a variety of signal pathways and participate in the regulation of a variety of physiological and pathological processes, including the conduction of neural signals, the dynamic balance of microvessels and large vessels, the regulation of immune inflammation, the inhibition of tumor occurrence and metastasis, apoptosis, infection immune response, angiogenesis, etc., but the prerequisite is that the concentration of nitric oxide needs to be in a suitable range. High concentration of nitric oxide in vivo can mediate neurotoxicity, and can induce nerve death through oxidative stress, production of active oxygen intermediates and damage to antioxidant systems, and high concentration of nitric oxide can affect various biological effects in vivo through non-enzymatic reactions with superoxide anions (O 2− ) and can cause irreversible damage to lipids, proteins, DNA, etc. and deplete glutathione in vivo. Nitric oxide is also a gas transmitter involved in vasodilation, neuronal signal transmission and immune response, and can inhibit platelet aggregation and fungal growth in vivo, so its application is very extensive. The synthesis of nitric oxide in vivo is limited by many factors, and the half-life of nitric oxide is short, only a few seconds, but the balance of nitric oxide level in vivo 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 and nitrosamine compounds. However, these donors have certain limitations, such as spontaneous release after being added to various biological matrices, poor stability in biological media, etc. Recent literature reports some light-controlled nitric oxide donors. Light-sensitive nitric oxide donors with light as a stimulating signal have the advantage of overcoming uncontrollable release dose. However, light-controlled nitric oxide donors still have deficiencies, such as instability, poor water solubility, and the need for a cosolvent for solubilization, and too much cosolvent can cause great harm 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 wavelength, high quantum efficiency, and good water solubility. Based on this, a new light-controlled nitric oxide donor of formula I is designed and synthesized using rhodamine 6G as a fluorescent group. SUMMARY

[0004] In view of the problems of few light-controlled nitric oxide donors and poor water solubility in the prior art, the present application aims to provide a light-controlled nitric oxide donor with good water solubility.

[0005] 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.

[0006] Another object of the present invention is to provide Formula I for preparing medicaments for treating or preventing neurological and cardiovascular diseases, or for related uses.

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

[0008] A photosensitive nitric oxide donor with good water solubility has the structure shown in Formula I.

[0009]

[0010] A method for synthesizing a readily water-soluble, light-controlled nitric oxide donor includes the following steps:

[0011] (1) Dissolve R6G in an organic acid and stir in a water bath, then add an aqueous solution of sodium nitrite. Monitor the reaction by thin-layer chromatography. After the reaction is complete, extract the reaction solution and evaporate to dryness to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain the desired nitric oxide donor formula I.

[0012]

[0013] In step (1), the organic acid in the reaction is glacial acetic acid.

[0014] In step (1), the molar ratio of sodium nitrite and formula I in the reaction is 0.5-20.

[0015] In step (1), the reaction time is 0.5 to 24 hours.

[0016] In step (1), the eluent for the silica gel column chromatography separation in the reaction is dichloromethane:methanol = 10:0.5-5.

[0017] One of the above-mentioned water-soluble nitric oxide donors is used to release nitric oxide via photoradiation in aqueous solutions, cells, and zebrafish.

[0018] The mechanism by which the water-soluble, light-controlled nitric oxide donor of this invention releases nitric oxide is as follows:

[0019] The N-nitroso bond in the nitric oxide donor molecule is unstable. After irradiation with a 365 nm ultraviolet lamp, the N-nitroso bond breaks, releasing one molecule of nitric oxide and R6G, thereby restoring fluorescence.

[0020] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects:

[0021] Upon illumination, the donor releases a single nitric oxide moiety, with its fluorescence intensity gradually increasing with irradiation time. Compared to existing nitric oxide donors, its main advantages are: firstly, donor formulation I releases nitric oxide upon irradiation with a 365 nm ultraviolet lamp, and its fluorescence intensity gradually increases with irradiation time; while its fluorescence intensity remains essentially unchanged in the absence of light. This facilitates precise light-controlled nitric oxide release, enabling research into the mechanisms by which nitric oxide participates in pathological processes and its application in the treatment of related diseases; secondly, the donor compound has a simple synthesis procedure, high yield, and good stability and water solubility. It is anticipated that this donor will have a broader application prospect in the biomedical field in the future. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of posture I.

[0023] Figure 2 This is a diagram illustrating the mechanism of nitric oxide release by donor type I under light irradiation.

[0024] Figure 3 This is the hydrogen NMR spectrum of donor formula I.

[0025] Figure 4 This is the mass spectrum of donor formula I.

[0026] Figure 5 The emission spectrum of a 2.0 μM solution of Formula I was obtained after irradiation with a 365 nm UV lamp. Formula I was dissolved in a neutral phosphate buffer solution and irradiated with a 365 nm UV lamp, and the changes in the fluorescence emission spectrum of Formula I were recorded intermittently (excitation wavelength 480 nm). The figure shows that the fluorescence signal of the donor gradually increased with increasing irradiation time, and the release ended after 150 minutes.

[0027] Figure 6 This is the fluorescence spectrum of nitric oxide released during the decomposition of Formula I by DAN. Formula I and DAN of the same concentration were dissolved in neutral phosphate buffer, irradiated with 365 nm ultraviolet light, and the changes in fluorescence emission spectrum were recorded intermittently.

[0028] Figure 7 This involves zebrafish imaging. After co-culturing zebrafish with Formula I for 30 min, the zebrafish were washed three times with neutral phosphate buffer solution and subjected to fluorescence microscopy. They were then irradiated with a 365 nm ultraviolet lamp, and the fluorescence microscopy of the zebrafish was recorded intermittently until the fluorescence intensity no longer changed. Implementation

[0029] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0030] Example 1 Synthesis of Nitric Oxide Donor Formula I

[0031] R6G (0.1198 g, 0.25 mmol) was dissolved in glacial acetic acid (3 mL) and stirred in an ice-water bath. Then, an aqueous solution of NaNO2 (0.0735 g, 1.07 mmol) was added. The reaction was monitored, and after completion, the mixture was extracted with DCM and evaporated to dryness under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain pure compound I.

[0032]

[0033] Example 2 Detection of Nitric Oxide Release

[0034] Formula I (2 μM) and DAN (2 μM) were simultaneously added 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 Furthermore, the fluorescence signal gradually increased with prolonged irradiation time, reaching a near-equilibrium level at 50 minutes. This demonstrates that Formula I successfully released nitric oxide upon irradiation, as shown in the results. Figure 6 .

[0035]

[0036] Example 3: Photocontrolled release of nitric oxide in zebrafish

[0037] In zebrafish imaging experiments, donor I was co-cultured with zebrafish for 30 min. The zebrafish were then washed three times with neutral phosphate buffer and fixed onto a glass slide using a fixative. Subsequently, confocal fluorescence imaging was performed. Figure 7 As shown, a) is an image of zebrafish under bright-field conditions; b) is an image of live zebrafish initially under dark-field conditions, where only a very weak fluorescence signal was observed. Subsequently, the zebrafish were irradiated with a 365 nm UV lamp, and fluorescence microscopy imaging was intermittently recorded until the fluorescence intensity no longer changed. The results indicate that donor I can achieve photocontrolled release of nitric oxide within 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 water-soluble, light-controlled nitric oxide donor as described in claim 1, characterized in that, Includes the following steps: R6G was dissolved in acid and stirred at a certain temperature. Then, an aqueous solution of inorganic salt was added, and the reaction was monitored. After the reaction was completed, the product was extracted with an organic solvent and evaporated to dryness to obtain a crude product. The crude product was 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: The dissolution in this reaction is carried out under acidic conditions, and the acid selected is glacial acetic acid; The inorganic salt solution mentioned is sodium nitrite. The reaction time for the test is 0.5-24 hours.

4. Use of the water-soluble, light-controlled nitric oxide donor of claim 1 in the preparation of medicaments for diseases benefiting from nitric oxide release.

5. The use of the water-soluble, light-controlled nitric oxide donor as described in claim 4, characterized in that, The diseases that benefit from nitric oxide release are selected from neurological diseases and cardiovascular diseases.

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