A kind of lysosome and mitochondrion double-targeted light-controlled nitric oxide donor, its preparation method and application

Abstract

This invention discloses a photosensitive nitric oxide donor that targets both lysosomes and mitochondria, its preparation method, and its applications, belonging to the fields of biomedical materials and medicinal chemistry. The nitric oxide donor has a structure as shown in formula DNOD560, which simultaneously includes a morpholino group for lysosome targeting and a rhodamine fluorophore for mitochondrial targeting, and uses an N-nitroso bond as the photosensitive nitric oxide release unit. The donor undergoes fluorescence quenching under physiological conditions and can undergo N-N bond cleavage under specific wavelength (e.g., 365 nm) illumination, quantitatively releasing nitric oxide and simultaneously generating the strongly fluorescent product DLMF560, enabling self-reporting and visualization of the release process. This donor has advantages such as high release efficiency (95% release efficiency within 70 minutes) and good photostability. Crucially, co-localization experiments have confirmed that it can simultaneously target lysosomes and mitochondria in cells, achieving in-situ, photosensitive release of nitric oxide at the subcellular organelle level, and has been successfully applied to live-cell (A549) imaging, providing a novel tool for studying the function of nitric oxide in subcellular organelles and developing precision treatment strategies.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials, drug delivery, and molecular imaging, specifically to a light-controlled nitric oxide (NO) donor capable of simultaneously targeting lysosomes and mitochondria, its preparation method, and its application in biomedical research and disease treatment. Background Technology

[0002] Nitric oxide (NO) is an endogenous gaseous signaling molecule that plays a complex dual role in tumor biology, with its effects exhibiting concentration- and spatial distribution dependence. High concentrations of NO can induce mitochondrial dysfunction and lysosomal membrane permeability, thereby strongly inducing tumor cell apoptosis. However, NO's extremely short half-life makes it difficult to accumulate to effective therapeutic concentrations in specific subcellular organelles, severely limiting its clinical application. Photocontrolled NO donors, enabling precise spatiotemporal control of NO release, represent an ideal strategy to address these challenges. Subcellular organelle-targeted NO donors are crucial for elucidating the subcellular functions of NO and improving therapeutic specificity. Lysosomes and mitochondria are two key organelles regulating cell survival and death, and are also potential targets for tumor therapy.

[0003] Currently, several NO donors targeting single organelles (such as lysosomes or mitochondria) have been reported. For example, He et al. reported a lysosome-targeting NO donor, but its NO release was too rapid to maintain a sustained effect. However, to date, no NO donors capable of simultaneously targeting lysosomes and mitochondria have been reported.

[0004] Developing a novel donor that combines lysosomal and mitochondrial dual-targeting capabilities and enables controlled NO release and real-time monitoring is of great significance for a deeper understanding of the subcellular mechanism of NO action and for developing efficient dual-target anticancer strategies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel dual-targeted, light-controlled nitric oxide donor for lysosomes and mitochondria. This donor enables precise NO release and fluorescence self-reporting in both organelles under light irradiation.

[0006] Another objective of this invention is to provide a method for preparing the aforementioned NO donor, which has a clear route and can efficiently prepare the target product.

[0007] Another object of the present invention is to provide the use of the above-mentioned NO donor in the preparation of medicaments and tools for the treatment of diseases (such as tumors) or for subcellular biological research.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this invention provides a lysosome and mitochondrial dual-targeting photocontrolled nitric oxide donor, namely the compound DNOD560 having the structure shown in Formula I. The innovative design of this molecule lies in the simultaneous introduction of a morpholine group as a lysosome-targeting unit and a rhodamine derivative (DLMF560) as a mitochondrial-targeting and fluorescent reporter unit, linked by a photosensitive N-nitrosamine bond (-N(NO)-). In the absence of light, the donor exists as a non-fluorescent spironolactone; upon irradiation with a specific wavelength (e.g., 365 nm), the N-N bond undergoes homolytic cleavage, simultaneously releasing one molecule of NO and the strongly fluorescent open-ring form of DLMF560. The released DLMF560 targets mitochondria due to its cationic properties, while the retained morpholine group ensures simultaneous targeting of the lysosome, thus achieving co-targeting of two organelles by a single molecule.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned NO donor. This method uses 3-morpholinephenol and 3-bromo-N-methylaniline as starting materials, constructs the intermediate DL via a copper-catalyzed coupling reaction, then condenses and cyclizes it with hydrated ninhydrin to obtain the fluorophore DLMF560, and finally connects it to a NO-releasing unit via a nitrosation reaction to obtain the target product DNOD560.

[0010] Thirdly, this invention provides applications for the aforementioned NO donor. This donor can be used to prepare drugs that release NO via light control, and is particularly suitable for therapeutic applications requiring simultaneous intervention of lysosomal and mitochondrial functions. Furthermore, it is also a unique tool for studying the role of NO in physiological and pathological processes such as lysosomal-mitochondrial crosstalk.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The invented NO donor possesses dual targeting capabilities of lysosomes and mitochondria, breaking the limitation of targeting a single organelle. Its fluorescence enhancement during NO release is up to 110 times, and the release amount can be quantitatively correlated. The release efficiency reaches 95% after 70 minutes of illumination, enabling real-time quantitative and visual monitoring. Moreover, the release rate is moderate and the effect duration is better. The molecule has a clear structure, is simple to synthesize and easy to prepare, and has already been validated in A549 lung cancer cells with photocontrolled drug release and localization imaging. It has good biocompatibility and promising application prospects. Attached Figure Description

[0012] Figure 1 This is a structural diagram of the compound DNOD560 (Formula I) of the present invention.

[0013] Figure 2 This is a schematic diagram illustrating the mechanism of NO release and fluorophore generation by photolysis of the donor DNOD560 in this invention.

[0014] Figure 3This is the ¹H NMR spectrum of the intermediate DL.

[0015] Figure 4 This is the ¹H NMR spectrum of the fluorophore DLMF560.

[0016] Figure 5 This is the ¹H NMR spectrum of the target donor DNOD560.

[0017] Figure 6 This is a high-resolution mass spectrum (HRMS) image of the photodecomposition products of DNOD560.

[0018] Figure 7 This is a comparison chart of high performance liquid chromatography (HPLC) analysis of DNOD560 and DLMF560.

[0019] Figure 8 This is a comparison of the UV-Vis absorption and fluorescence emission spectra of DLMF560 and DNOD560 under and without 365 nm illumination.

[0020] Figure 9 The graph shows the changes in the ultraviolet absorption spectrum (A) and fluorescence emission spectrum (B) of DNOD560 solution under continuous illumination at 365 nm, as well as the fluorescence standard curve of DLMF560 (C, D).

[0021] Figure 10 The spectrum of NO emission from DNOD560 under illumination was confirmed using electron paramagnetic resonance (EPR) spectroscopy and the PTIO capture method.

[0022] Figure 11 These are fluorescence confocal microscopy images and quantitative fluorescence intensity analysis diagrams of DNOD560 in A549 cells under different illumination times.

[0023] Figure 12 This is a co-localization imaging image of DNOD560 with commercial lysosomal probe Lyso-Tracker Green and mitochondrial probe Mito-Tracker Deep Red FM in A549 cells, demonstrating its dual targeting of lysosomes and mitochondria. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following embodiments.

[0025] (1) Synthesis of intermediate DL: Under argon protection, 3-morpholinephenol (0.7163 g, 4 mmol), 3-bromo-N-methylaniline (approximately 1.21 mL), cesium carbonate (2.6068 g, 8 mmol), and 10 mL of dimethyl sulfoxide were added to a reaction flask. After purging with argon for 20 minutes, cuprous bromide (0.2151 g, 1.5 mmol) and 2-pyridinecarboxylic acid (0.1477 g, 1.2 mmol) were added, and argon was purged again for 10 minutes. The mixture was heated to 120 °C and stirred for 15 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 7:3, v / v) to give a white solid DL (0.7021 g, yield 62%).

[0026]

[0027] (2) Synthesis of the fluorophore DLMF560: DL (0.426 g, 1.5 mmol) and ninhydrin hydrate (0.356 g, 2.1 mmol) were dissolved in 7 mL of acetonitrile, and 5 mL of glacial acetic acid and 0.5 mL of concentrated sulfuric acid were added. The mixture was heated to 100 °C and refluxed for 24 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to give a purple solid DLMF560 (0.1245 g, yield 20%).

[0028] Structural characterization data: 1 H NMR (600 MHz, DMSO- d 6) δ 7.97 (d, J =7.7 Hz, 1H), 7.77(t, J =7.6 Hz, 1H), 7.70 (t, J =7.7 Hz, 1H), 7.23 (d, J =7.7 Hz, 1H), 6.77 (s,1H), 6.72 (d, J =9.0 Hz, 1H), 6.51 (d, J =8.8 Hz, 1H), 6.40 (d, J =8.4 Hz, 1H), 6.33 (d, J =10.2 Hz, 2H), 6.24 (d, J=5.7 Hz, 1H), 3.75-3.69 (m, 4H), 3.17 (t, J =4.7 Hz, 4H), 2.69 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 168.84, 152.65,152.41, 152.03, 151.98, 135.37, 129.88, 128.30, 128.23, 126.52, 124.45,123.98, 111.16, 105.16, 100.94, 96.28, 84.25, 65.89, 47.53, 29.41. HRMS (ESI+): m / z calcd for C 25 H 23 N2O4 + [M] + : 415.1653; found: 415.1632.

[0029]

[0030] (3) Synthesis of the target donor DNOD560: DLMF560 (0.0828 g, 0.2 mmol) was dissolved in 5 mL of glacial acetic acid under ice-water bath cooling and stirring. A solution of sodium nitrite (0.0207 g, 0.3 mmol) dissolved in 1 mL of water was slowly added dropwise. After the addition was complete, the reaction was stirred at 0 °C for 30 minutes. After the reaction was completed, the mixture was filtered, and the solid was washed three times with water to obtain purple powder DNOD560 (0.063 g, yield 71%).

[0031] Structural characterization data: 1 H NMR (600 MHz, Chloroform- d ) δ 8.05 (d, J =5.9 Hz, 1H), 7.66 (d, J =25.6 Hz, 2H), 7.46 (s, 1H), 7.27 (s, 1H), 7.18 (d, J =5.6 Hz, 1H), 6.90 (d, J =6.8 Hz, 1H), 6.75-6.68 (m, 2H), 6.63 (d, J =7.5 Hz, 1H), 3.85 (s,4H), 3.44 (s, 3H), 3.23 (s, 4H). 13C NMR (150 MHz, DMSO- d 6) δ 170.19, 153.54,153.39, 153.05, 152.93, 151.49, 135.87, 130.68, 129.07, 128.98, 127.76,125.56, 124.71, 112.04, 110.93, 109.70, 106.28, 101.24, 96.92, 66.42, 47.95,30.01. HRMS (ESI + ): m / z calcd for C 25 H 21 N3O5 [M+H] + : 444.1559; found: 444.2041.

[0032] The above data confirms the successful synthesis of the target molecule DNOD560.

[0033]

[0034] Example 2: Mechanism and Performance of Photocontrolled NO Release by DNOD560 (1) Mechanism verification: The solution of DNOD560 after irradiation at 365 nm was analyzed by HRMS and HPLC. HRMS showed a strong peak at m / z = 415.1974, which is consistent with the theoretical molecular weight of the fluorophore DLMF560. Figure 6 HPLC analysis showed that after light irradiation, the DNOD560 solution exhibited a new peak at the same retention time (3.7 min) as the DLMF560 standard. Figure 7 These results confirm that light exposure leads to the quantitative conversion of DNOD560 into DLMF560.

[0035] (2) Spectral properties: such as Figure 8 As shown, DNOD560 itself exhibits weak absorption at 530 nm and almost no fluorescence at 560 nm. In contrast, DLMF560 shows strong absorption and fluorescence emission at both 530 nm and 560 nm. After irradiation with 365 nm light for 50 minutes, the spectrum of the DNOD560 solution was almost identical to that of DLMF560, confirming the photoconversion.

[0036] (3) Release kinetics and efficiency: such as Figure 9 As shown, under 365 nm illumination, the absorbance at 530 nm and the fluorescence intensity at 560 nm of the DNOD560 solution increased synchronously over time, reaching a plateau after 70 minutes. This was confirmed by the fluorescence standard curve of DLMF560. Figure 9According to calculations (C, D), 10 μM DNOD560 can release approximately 9.5 μM NO after 70 minutes of illumination, with a release efficiency as high as 95%, indicating that the donor has an extremely high NO release capacity.

[0037] (4) EPR experiment: such as Figure 10 As shown, a mixed solution of DNOD560 and the NO scavenger PTIO exhibits the characteristic EPR signal of PTIO before illumination. After 10 minutes of illumination, this signal decays and the characteristic signal of PTI appears, directly proving that NO is generated during the illumination process.

[0038] Example 3: Dual-targeting and NO release imaging of DNOD560 in living cells A549 cells were co-incubated with 10 μM DNOD560, followed by a light experiment. Figure 11 As shown, unilluminated cells exhibit weak fluorescence. After irradiation with 365 nm light, the yellow fluorescence of the cells significantly increased, and the intensity increased with irradiation time (5, 10, 20 minutes) (increasing 6.5-fold after 20 minutes of irradiation), confirming the intracellular light-controlled NO release and fluorescence activation. Key co-localization experiments are shown below. Figure 12 As shown. A group of cells were simultaneously stained with DNOD560 and the lysosomal probe Lyso-Tracker Green. After light exposure, the yellow fluorescence of DNOD560 (…) Figure 12 A1) and the green fluorescence of Lyso-Tracker Green ( Figure 12 A2) High overlap ( Figure 12 A3), with a Pearson correlation coefficient of 0.99 ( Figure 12 A5). Another group of cells was simultaneously stained with DNOD560 and the mitochondrial probe Mito-Tracker Deep Red FM. After light exposure, the yellow fluorescence of DNOD560 ( Figure 12 B1) and the red fluorescence of Mito-Tracker DeepRed FM ( Figure 12 B2) also showed significant overlap ( Figure 12 B3), Pearson correlation coefficient is 0.90 ( Figure 12 (B5). This result demonstrates that DNOD560 possesses the unique ability to simultaneously target lysosomes and mitochondria.

[0039] In summary, this invention successfully designed and synthesized the first light-controlled nitric oxide donor, DNOD560, which simultaneously targets lysosomes and mitochondria. This donor innovatively integrates dual-targeting groups, a fluorescent self-reporter unit, and a light-controlled NO release unit into a single molecule. Under illumination, it can efficiently and quantitatively release NO, and the release process can be visualized. Cell experiments confirmed its precise localization to lysosomes and mitochondria, achieving in situ NO release at the subcellular organelle level. This invention provides an unprecedented molecular tool for studying the cross-functional interaction of NO between subcellular organelles and its role in disease, and demonstrates great application potential in the field of dual-target precision anticancer therapy.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A light-controlled nitric oxide donor that targets both lysosomes and mitochondria, characterized in that, It is a compound having the structure shown in formula DNOD560: Or, or a pharmaceutically acceptable salt thereof.

2. A method for preparing the lysosomal and mitochondrial dual-targeting light-controlled nitric oxide donor as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of intermediate DL: 3-morpholine phenol and 3-bromo-N-methylaniline were heated and reacted in dimethyl sulfoxide in the presence of base and copper catalyst. After the reaction was completed, the mixture was extracted, dried and purified by column chromatography to obtain white solid DL. ； (2) Synthesis of fluorophore DLMF560: The DL obtained in step (1) was heated and refluxed with hydrated ninhydrin under acidic conditions. After the reaction was completed, the DL was extracted, dried and purified by column chromatography to obtain purple solid DLMF560. ； (3) Synthesis of the target donor DNOD560: The DLMF560 obtained in step (2) was dissolved in glacial acetic acid and reacted with sodium nitrite aqueous solution at low temperature. After the reaction was completed, the purple solid DNOD560 was obtained by filtration and washing. 。 3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of 3-morpholinephenol to 3-bromo-N-methylaniline is 1:(1-1.5); the base is cesium carbonate; the copper catalyst is cuprous bromide; the reaction temperature is 110-130℃, and the reaction time is 14-16 hours; the eluent used for column chromatography purification is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of (6:1)-(8:1); In step (2), the molar ratio of DL to ninhydrin hydrate is 1:(1.3-1.5); the acidic conditions are provided by glacial acetic acid and concentrated sulfuric acid; the reaction temperature is 95-105℃ and the reaction time is 22-26 hours; the eluent used for column chromatography purification is a mixed solvent of dichloromethane and methanol with a volume ratio of (18:1)-(22:1); In step (3), the molar ratio of DLMF560 to sodium nitrite is 1: (1.4-1.6); the reaction temperature is -5 to 5℃, and the reaction time is 25 to 35 minutes.

4. The use of the lysosome and mitochondrial dual-targeting light-controlled nitric oxide donor as described in claim 1 in the preparation of drugs or research tools for light-controlled release of nitric oxide.

5. The application according to claim 4, characterized in that, The light-controlled release can simultaneously target the lysosomes and mitochondria of the cell.

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