Fluorescent probe for targeting detection of lysosomal calcium ions and preparation method and application thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a fluorescent probe for targeted detection of lysosomal calcium ions, and a preparation method and application thereof. The lysosomal calcium ion fluorescent probe of the present application utilizes a mature calcium ion chelating group precursor, and introduces a specific lysosome-targeting fluorescent group in the structure through a triple bond. Under the action of a hydrolytic enzyme inside the lysosome, the calcium ion chelating group precursor is hydrolyzed, and then chelates with calcium ions, thereby significantly enhancing the fluorescence signal. The probe effectively avoids the interference of magnesium ions and an acidic environment, and realizes high specificity targeted imaging of lysosomal calcium ions. In vitro experiments show that the fluorescent probe can effectively anchor the lysosome, is not affected by lysosomal calcium leakage, can accurately reflect changes in the lysosomal calcium level, and is suitable for in situ detection of lysosomal calcium homeostasis. Further, the present application verifies the feasibility of the probe in detecting changes in the lysosomal calcium level in vivo through acute lung injury and pulmonary fibrosis models.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to fluorescent probes for targeted detection of lysosomal calcium ions, their preparation methods, and applications. Background Technology

[0002] Acute lung injury (ALI) and idiopathic pulmonary fibrosis (IPF) are highly fatal respiratory illnesses with a lack of effective treatments. Among them, lipopolysaccharide (LPS)-induced acute lung injury models and bleomycin (BLM)-induced pulmonary fibrosis models are classic in vivo and in vitro models for studying the pathological mechanisms of these diseases and screening for therapeutic targets.

[0003] Calcium signaling dysregulation is one of the core events in the pathological process of lung injury. Lysosomes, as one of the key intracellular calcium reservoirs, have a calcium level four orders of magnitude higher than that of the cytoplasm and are a core component of the cellular calcium signaling network. Recent studies have confirmed that lysosomal calcium homeostasis imbalance can directly lead to lysosomal dysfunction, autophagic flux blockage, and excessive activation of inflammasomes, thereby mediating alveolar epithelial cell damage and the progression of pulmonary interstitial fibrosis. It is a common core pathological driving event in pulmonary inflammation and fibrosis. Therefore, accurately observing lysosomal calcium imbalance in lung injury models is a key prerequisite for revealing new pathological mechanisms of lung injury and developing novel treatment strategies.

[0004] Currently, detection techniques for lysosomal calcium signals mainly fall into three categories: non-targeted calcium ion fluorescent probe imaging, lysosomal targeted calcium ion probe technology, and biochemical component separation and detection technology. Non-targeted calcium ion fluorescent probe imaging, using broad-spectrum calcium ion fluorescent probes such as Fluo-3 AM and Fura-2 AM, detects the total cytoplasmic calcium ion concentration after cell incubation, and is currently the mainstream approach for cellular calcium signal detection. However, these probes lack organelle targeting and cannot detect changes in lysosomal calcium levels; they only reflect the total cytoplasmic calcium level and cannot accurately characterize the lysosomal calcium homeostasis. Some reported lysosomal targeted calcium ion probes generally suffer from insufficient targeting specificity, severe interference from physiological concentrations of magnesium ions, low fluorescence response signal-to-noise ratio, poor photostability and biocompatibility, and instability under acidic pH conditions in lysosomes, making it impossible to achieve high-fidelity, long-term in-situ imaging of lysosomal calcium ion dynamics under pathophysiological conditions. Biochemical component separation and detection methods separate lysosomal components through density gradient centrifugation and then detect lysosomal calcium concentration using calcium indicators or atomic absorption spectroscopy. This method can quantitatively detect the total calcium content of lysosomes, but it requires cell disruption, cannot achieve in-situ dynamic detection, and calcium leakage is very likely to occur during sample processing, resulting in a large deviation between the detection results and the actual state in vivo.

[0005] In summary, the above detection methods have the following problems: 1. Poor detection specificity and anti-interference ability: Non-targeted fluorescent probes cannot distinguish lysosomal calcium stores from calcium signals of other organelles; existing targeted probes are easily affected by physiological concentrations of magnesium ions and fluctuations in lysosomal acidity pH, resulting in high false negative and false positive rates and failing to accurately reflect the true changes in lysosomal calcium balance. 2. Inability to achieve in situ dynamic quantitative detection: Biochemical detection methods require sample disruption, which cannot preserve physiological activity, cannot track the dynamic changes in lysosomal calcium homeostasis during the pathological process of lung injury, and sample processing is prone to calcium leakage, resulting in distorted detection results; 3. Insufficient sensitivity and spatiotemporal resolution: Existing technologies cannot capture subtle changes in lysosomal calcium homeostasis in the early stages of lung injury, nor can they establish a quantitative correlation between the degree of lysosomal calcium disorder and the pathological progression of lung injury, thus limiting the study of the early pathological mechanisms of lung injury.

[0006] Therefore, there is an urgent need to develop a fluorescent calcium ion probe that can specifically target lysosomes, has high anti-interference ability, can work stably in acidic pH environments, and can achieve in-situ dynamic quantitative detection of live cells, so as to accurately monitor lysosomal calcium imbalance in lung injury models and provide key technical support for revealing new pathological mechanisms of lung injury and screening new treatment strategies. Summary of the Invention

[0007] This invention provides a fluorescent probe for targeted detection of lysosomal calcium ions, a method for preparing the probe, and a fluorescent probe for targeted detection of lysosomal calcium ions using the present invention, comprising: The calcium ion chelating unit precursor shown in Formula I-1a;

[0008] Formula I-1a; Wherein, R1 is selected from C1~C4 hydrocarbon groups; and, the fluorescent unit of formula I-2a connected to the calcium ion chelating unit precursor;

[0009] Formula I-2a

[0010] Among them, R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -CMe2-, or -SiMe2-. The benzene ring in the compound shown in Formula I-1a is connected to the aniline benzene ring in the compound of Formula I-2a by a carbon-carbon triple bond.

[0011] CMe2, or -C(Me)2-, refers to two methyl groups (Me) attached to the same carbon atom; SiMe2, or -Si(Me)2-, refers to two methyl groups attached to the same silicon atom.

[0012] In some embodiments, the carbon at the 4-position of the benzene ring in the compound of formula I-1a is connected to the carbon at the 4-position of the aniline benzene ring in the compound of formula I-2a via a carbon-carbon triple bond, and the resulting fluorescent probe has the structure shown in formula I:

[0013] Formula I; R1, R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

[0014] The fluorescent probe of this invention contains a fluorescent group with lysosomal localization capability, which is reported for the first time. By attaching a specific lysosomal-targeting fluorescent group to a calcium ion chelating group precursor, this invention achieves highly specific targeted imaging of lysosomal calcium ions, which can accurately monitor changes in lysosomal calcium levels and avoid interference from physiological concentrations of magnesium ions and acidic pH fluctuations. Furthermore, it enables in-situ detection of lysosomal calcium homeostasis imbalance during LPS-induced acute lung injury and BLM-induced pulmonary fibrosis.

[0015] In some preferred embodiments, R1, R2, R3, R4, and R5 are independently selected from C1 to C4 alkyl groups, such as any one of methyl, ethyl, propyl, isopropyl, n-butyl, and isobutyl.

[0016] In some specific embodiments, when R2, R3, R4, and R5 in the fluorescent unit shown in Formula I-2a are all methyl groups and X is -O-, Formula I-2a is N-phenyl-9-aminopyronine, with the following structural formula: .

[0017] In some specific embodiments, the fluorescent probe (denoted as MBPY) has the following specific structure: .

[0018] The present invention also provides a method for preparing the fluorescent probe, comprising: The fluorescent probe is obtained by connecting the calcium ion chelating unit precursor shown in Formula I-1a and the fluorescent group shown in Formula I-2a through a carbon-carbon triple bond using the Sonogashira reaction.

[0019] In some embodiments, a triple bond is introduced into the calcium ion chelating unit precursor shown in Formula I-1a via a Sonogashira reaction and a deprotection step to obtain an alkynyl-modified compound of Formula I-1a, i.e., the calcium ion chelating unit, having the structure shown in I-1a' below:

[0020] Formula I-1a'; Wherein, R1 is a C1~C4 hydrocarbon group, preferably a C1~C4 alkyl group, specifically any one of methyl, ethyl, propyl, isopropyl, n-butyl, and isobutyl.

[0021] In some embodiments, the fluorescent unit represented by formula I-2a is introduced with an I atom before the Sonogashira reaction to obtain an iodinated fluorescent unit having the following formula I-2a':

[0022] Formula I-2a'; R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

[0023] In this invention, the calcium ion chelating unit precursor shown in Formula I-1a is subjected to a Sonogashira reaction and deprotection treatment to obtain a calcium ion chelating unit precursor containing a terminal alkyne group. An iodinated fluorescent unit is obtained by iodination modification of the fluorescent unit. Then, the calcium ion chelating unit precursor containing the terminal alkyne group and the iodinated fluorescent unit are subjected to a Sonogashira reaction to obtain the fluorescent probe described in this invention.

[0024] Furthermore, in a specific embodiment of the present invention, the structure of the calcium ion chelating unit precursor (compound 5) containing a terminal alkyne group is as follows: .

[0025] In this invention, the Sonogashira reaction is carried out in the presence of a catalyst. Specifically, the catalyst comprises tetrakis(triphenylphosphine)palladium and cuprous iodide.

[0026] In this invention, the solvent for the Sonogashira reaction includes tetrahydrofuran and / or triethylamine.

[0027] In this invention, the conditions for the Sonogashira reaction include: reflux stirring at 60~70°C, wherein the reaction temperature can specifically be 60°C, 65°C or 70°C.

[0028] In this invention, the calcium ion chelating unit precursor is prepared from 2-methyl-3-butyn-2-ol, 5-bromo-2-nitrophenol, and 4-(2-chloroacetyl)morpholine. In some specific embodiments, this invention also provides corresponding preparation methods: a. Sonagashira reaction: Starting with 5-bromo-2-nitrophenol and 2-methyl-3-butyn-2-ol, a coupling reaction was carried out in the presence of tetra(triphenylphosphine)palladium and cuprous iodide catalysis, with triethylamine as solvent, to prepare an alkynyl-modified intermediate; b. Deprotection reaction: The above intermediate is subjected to heating treatment with potassium hydroxide and toluene system to remove the hydroxyl protecting group, yielding the terminal alkyne intermediate; c. Introduction of acylmorpholine: The terminal alkyne intermediate and 4-(2-chloroacetyl)morpholine undergo a nucleophilic substitution reaction in acetonitrile as a solvent under potassium carbonate catalysis to introduce the acylmorpholine unit; d. Nitro reduction reaction: The above product is reacted with iron powder and ammonium chloride in a mixed solvent of ethanol and water under heating to reduce the nitro group to an amino group, yielding an aromatic amine precursor; e. N-alkylation reaction: The aromatic amine precursor and methyl bromoacetate are subjected to a substitution reaction in acetone as a solvent under the catalysis of potassium carbonate and potassium iodide to complete the modification of the calcium ion chelating unit precursor and obtain the calcium ion chelating unit of the present invention.

[0029] The present invention also provides the application of the fluorescent probe in the preparation of products for detecting lysosomal calcium ions or for preparing imaging of lysosomal calcium balance disorders.

[0030] The lysosomal calcium imbalance includes lysosomal calcium imbalance caused by lung diseases and lysosomal calcium imbalance caused by other diseases; further, the lung diseases include at least one of acute lung injury and pulmonary fibrosis; even further, the acute lung injury includes chronic obstructive pulmonary disease, hyperoxia-induced lung injury, and virus-induced lung injury. The other diseases include at least one of myocardial injury, neurodegenerative diseases, and tumors.

[0031] This invention also provides a method for detecting lysosomal calcium ions or observing lysosomal calcium imbalance, comprising: incubating the fluorescent probe of this invention as described above with test cells and then performing fluorescence imaging. This method includes detection for non-diagnostic purposes and detection for diagnostic purposes. The non-diagnostic method includes methods for detecting the content of lysosomal calcium ions in environmental samples or experimental systems and observing the distribution of calcium ions for research purposes or other non-diagnostic purposes. The diagnostic method for detecting lysosomal calcium ions involves using the fluorescent probe to image and detect lysosomal calcium ions in the test subject (in humans or animals, or samples from humans or animals) to assist in determining the lysosomal calcium imbalance related to lung diseases.

[0032] In the above method, the incubation concentration of the fluorescent probe is preferably 5–20 μM, specifically 5 μM, 10 μM, 15 μM, 20 μM or any value within the above range; the incubation time is preferably 15–60 min, specifically 15 min, 30 min, 45 min, 60 min or any value within the above range.

[0033] The fluorescent probe and detection method provided by this invention do not require cell disruption. The fluorescent probe can be directly incubated with the live cells to be tested and then subjected to fluorescence imaging, which can realize in situ dynamic quantitative detection of lysosomal calcium ion concentration, track the dynamic changes of lysosomal calcium homeostasis during pathological processes, and the detection results are more consistent with the actual state in vivo. It effectively avoids the result deviation caused by calcium leakage during sample processing, and has higher sensitivity and spatiotemporal resolution. It can promptly observe subtle changes in lysosomal calcium homeostasis in the early stage of pathology. It can be used to establish a quantitative correlation between the degree of lysosomal calcium disorder and the pathological progression of lung injury, and provide a reliable technical basis for the study of early pathological mechanisms of lung injury and the screening of new therapeutic targets.

[0034] Compared with the prior art, the present invention has advantages in at least the following aspects: 1. Significant improvement in detection specificity and anti-interference capability. The MBPY probe of this invention has precise lysosomal targeting, with a co-localization Pearson correlation coefficient of 0.93 with commercial lysosomal probes, which can completely distinguish lysosomal calcium stores from calcium signals of other organelles, solving the core defect of non-targeted probes that cannot distinguish calcium stores. At the same time, the probe uses MOPDA as the chelating unit, and its selectivity for calcium ions is much higher than that for magnesium ions. Physiological concentrations of magnesium ions have no detection interference. It is stable in the acidic pH range of lysosomes (3.0–6.0), overcoming the problems of weak anti-interference capability and poor pH stability of existing targeted probes. The fidelity of the detection results is much higher than that of existing technologies.

[0035] 2. This invention enables in-situ detection of lysosomal calcium imbalance during the pathological process of lung injury. The method of this invention does not require the disruption of cell or tissue samples; it can directly achieve in-situ imaging of lysosomal calcium signals in living cells and isolated lung tissue, fully preserving the physiological activity of the sample and fundamentally avoiding calcium leakage and result distortion caused by sample processing. 3. Significantly improved detection sensitivity and application value. The MBPY probe of this invention has an 8.6-fold fluorescence on-off factor and a high signal-to-noise ratio, enabling it to capture subtle changes in lysosomal calcium homeostasis in the early stages of lung injury, thus solving the problem of insufficient sensitivity in existing technologies. Furthermore, this invention establishes a quantitative correlation between the degree of lysosomal calcium disorder and the pathological progression of lung injury, as well as the levels of inflammatory factors, at the cellular and animal levels. This not only provides a standardized technical tool for studying the pathological mechanisms of lung injury but also offers a novel technical pathway for the early diagnosis and treatment target screening of lung injury. Its application scenarios and translational value far surpass those of existing technologies. Attached Figure Description

[0036] Figure 1 The hydrogen nuclear magnetic resonance spectrum of MBPY in Example 1; Figure 2 The image shows the carbon NMR spectrum of MBPY in Example 1. Figure 3For example, in test case 1, MBPY is used to test Ca. 2+ Fluorescence response diagram; Figure 4 For the Ca of MBPY in test example 1 2+ Anti-interference diagram of fluorescence response; Figure 5 For the Ca of MBPY in test example 1 2+ pH operating range diagram of fluorescence response; Figure 6 The graph shows the effect of MBPY on the cell viability of HeLa and BEAS 2B cells in Test Example 2. In the graph, A represents the effect on the viability of HeLa cells and B represents the effect on the viability of BEAS 2B cells. Figure 7 This is a lysosomal colocalization map of MBPY in test example 2; Figure 8 To test the lysosomal calcium imaging results of cells under exogenous stimulation using MBPY in Example 3, where A is the imaging result under stimulation with 10 μM iomycin and B is the result under stimulation with 500 μM ATP; Figure 9 To test the lysosomal calcium imaging results in the lung injury cell model in Example 4, where A is a fluorescence imaging image and B is a statistical bar chart of fluorescence intensity; Figure 10 The results of lysosomal calcium imaging in the lung injury animal model in Test Example 5 are shown in Figure A, where A is a fluorescence imaging image and B is a statistical bar chart of fluorescence intensity. Figure 11 For the detection of Ca² in lysosomes using the MBPY probe + A diagram illustrating the fluorescence response mechanism; Detailed Implementation

[0037] This invention provides a fluorescent probe for targeted detection of lysosomal calcium ions, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0039] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0040] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0041] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it means that the units for the left and right endpoints are the same. For example, "60~70℃" means that the units for the left endpoint "60" and the right endpoint "70" are both "℃".

[0042] The terms involved in this invention are explained as follows: Lysosomal calcium ions 2+ : refers to free calcium ions stored inside lysosomal organelles, and is the core second messenger regulating lysosomal functions (autophagy, phagosome maturation, membrane repair, etc.).

[0043] Lipopolysaccharide (LPS): A component of the outer wall of Gram-negative bacterial cells, used to induce the construction of a classic acute lung injury model.

[0044] Bleomycin (BLM): an antitumor antibiotic used to induce the construction of a classic pulmonary fibrosis model.

[0045] Lysosome-targeting calcium ion fluorescent probe (MBPY): A small molecule fluorescent probe with a lysosomal acidic chamber targeting group, a calcium ion specific chelating group MOPDA, and a fluorophore PY. It can specifically respond to changes in the concentration of free calcium ions in lysosomes and generate a fluorescent signal.

[0046] All test materials used in this invention are common commercially available products. The core reagents and instruments are as follows: Lysosome-targeting calcium ion fluorescent probe MBPY (in-house synthesized); human bronchial epithelial cells (BEAS-2B), human cervical cancer cells (HeLa); Dalberg modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution; Escherichia coli-derived lipopolysaccharide (LPS), bleomycin (BLM), dexamethasone (DEX), ionomycin, 5 ′– Adenosine triphosphate (ATP), probenecid, poloxamer 407 (F127); Hanks balanced salt solution (HBSS); MitoTracker Deep Red FM (mitochondrial deep red fluorescent probe), LysoTracker Deep Red FM (lysosomal deep red fluorescent probe); HEPES buffer; Cell counting kit –8 (CCK-8).

[0047] Ultraviolet-Vis absorption spectrometer (Hitachi 3900H); fluorescence spectrophotometer (Hitachi F-7000); high-resolution mass spectrometer (HRMS, Brook Dalton Autoflex III); elemental analyzer (Vario EL Cube); laser scanning confocal microscope (LSCM, Nikon C2); microplate reader; constant temperature cell culture incubator.

[0048] The abbreviation for tetra(triphenylphosphine)palladium is Pd(PPh3)4.

[0049] The lysosomal-targeting calcium ion fluorescent probe provided by this invention is abbreviated as MBPY, and its synthetic route is as follows:

[0050] MBPY was prepared via the above route, with all steps completed under standard laboratory conditions, including the following steps: a. Alkyne modification: Using the starting material (5-bromo-2-nitrophenol) and 2-methyl-3-butyn-2-ol, a first Sonogashira coupling reaction was carried out in the presence of tetra(triphenylphosphine)palladium and cuprous iodide catalysis and triethylamine as solvent to prepare an alkynyl-modified intermediate (compound 1). b. Deprotection reaction: The above intermediate was heated in a potassium hydroxide and toluene system to remove the hydroxyl protecting group, yielding a terminal alkyne intermediate (compound 2). c. Chelating group modification: The terminal alkyne intermediate and 4-(2-chloroacetyl)morpholine were subjected to a nucleophilic substitution reaction in acetonitrile under potassium carbonate catalysis to introduce an acylmorpholine unit, yielding compound 3; d. Nitro reduction reaction: Compound 3 was reacted with iron powder and ammonium chloride in a mixed solvent of ethanol and water under heating to reduce the nitro group to an amino group, yielding the MOPDA chelate unit intermediate (compound 4). e. Ester substitution reaction: The amino intermediate and methyl bromoacetate undergo a substitution reaction in acetone under the catalysis of potassium carbonate and potassium iodide to complete the modification of the chelating unit and obtain a calcium ion chelating unit precursor containing a terminal alkyne group (compound 5). f. Synthesis of iodolysosomal targeting fluorescent units: Pironine thione (compound 6) and 4-iodoaniline were coupled in acetonitrile in the presence of copper chloride and potassium chloride to prepare iodolysosomal fluorescent units (compound 7). g. Final coupling: Compounds 5 and 7 underwent a second Sonogashira coupling reaction in the presence of tetra(triphenylphosphine)palladium and cuprous iodide as catalysts, with triethylamine as the base and tetrahydrofuran as the solvent, to give the final product MBPY.

[0051] In specific embodiments of the present invention, cell experiments have confirmed that the fluorescent probe of the present invention can achieve dose-dependent quantitative detection of lysosomal calcium levels in LPS-induced acute lung injury and BLM-induced pulmonary fibrosis models in BEAS-2B cells. The fluorescence signal is always localized to lysosomal vesicles without off-target leakage, and can capture subtle changes in lysosomal calcium homeostasis in the early stage of lung injury, solving the problem that existing technologies cannot accurately detect lysosomal calcium disorders in lung injury cell models in situ. In addition to BEAS-2B cells, other lung tissue-related cells such as human alveolar type II epithelial cells (A549) and mouse lung fibroblasts can be used to construct LPS-induced acute lung injury and BLM-induced pulmonary fibrosis cell models. In animal model testing, in addition to tail vein injection, probe administration can be replaced by tracheal instillation, intraperitoneal injection, etc., by adjusting the dosage and incubation time to achieve efficient labeling of lung tissue; in addition to in vitro tissue imaging, in vivo fluorescence imaging systems can be used for real-time in vivo imaging of mouse lung tissue to achieve dynamic tracking of lysosomal calcium disorder; in addition to confocal microscopy, high-content imaging systems and two-photon fluorescence microscopy can be used for imaging detection to improve tissue penetration depth and spatiotemporal resolution.

[0052] The fluorescent probes provided by this invention can be used not only in LPS-induced acute lung injury models and BLM-induced pulmonary fibrosis models, but also for observing lysosomal calcium imbalances in other lung disease models such as hyperoxia-induced lung injury, virus infection-induced lung injury, and chronic obstructive pulmonary disease (COPD). Furthermore, they can be extended to the study and detection of mechanisms related to other lysosomal calcium signaling diseases such as myocardial injury, neurodegenerative diseases, and tumors.

[0053] The present invention will be further illustrated below with reference to the embodiments: Example 1: Synthesis of the fluorescent probe MBPY of the present invention The specific steps are as follows: Synthesis of Compound 1: 5-Bromo-2-nitrophenol (1.31 g), 2-methyl-3-butyn-2-ol (0.64 mL), Pd(PPh3)4 (25 mg), and CuI (23 mg) were dissolved in a mixed solvent of triethylamine (7.2 mL) and pyridine (3.8 mL). The reaction mixture was heated at 105 °C for 1 h. After concentration, the mixture was acidified with dilute hydrochloric acid, extracted with dichloromethane (DCM), washed with brine, and dried. The solvent was removed under reduced pressure, and the crude product was purified by rapid column chromatography, eluting with DCM. Compound 1 was given as a brownish-yellow solid (1.07 g), in 80% yield.

[0054] 1 ¹H NMR (300 MHz, CDCl₃, ppm) δ : 10.50 (s, 1H), 7.97 (d, 1H, J = 8.8Hz), 7.12 (d, 1H, J = 1.8 Hz), 6.92 (dd, 1H, J = 8.8, 1.8 Hz), 1.56 (s, 6H).

[0055] Synthesis of Compound 2: Compound 1 (200 mg) and potassium hydroxide (152 mg) were reacted in toluene (3 mL) at 110 °C for 12 h. After concentration, the reaction mixture was acidified with dilute hydrochloric acid, extracted with dichloromethane, washed with brine, and dried. The solvent was removed under reduced pressure, and the crude product was purified by rapid column chromatography, eluting with a mixture of dichloromethane and n-hexane (1:3, v / v). Compound 2 was given as a yellow-green solid (85 mg), in 58% yield.

[0056] 1 ¹H NMR (300 MHz, CDCl₃, ppm) δ : 10.50 (s, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 1.7 Hz, 1H), 7.00 (dd, J = 8.8, 1.7 Hz, 1H), 3.29 (s, 1H).

[0057] Synthesis of Compound 3: Compound 2 (200 mg), K₂CO₃ (178 mg), and 4-(2-chloroacetyl)morpholine (220 mg) were reacted in acetonitrile (5 mL) at 80 °C for 12 h. After concentration, the reaction mixture was diluted with deionized water and extracted with dichloromethane. The solvent was removed under reduced pressure, and the crude product was purified by rapid column chromatography, eluting with a mixture of n-hexane and ethyl acetate (1:2, v / v) to give compound 3 as a pale yellow solid (292 mg), in 83% yield.

[0058] 1 ¹H NMR (300 MHz, CDCl₃, ppm) δ 7.75 (d, J = 8.3 Hz, 1H), 7.20 (d, J =1.5 Hz, 1H), 7.13 (dd, J = 8.3, 1.5 Hz, 1H), 4.77 (s, 2H), 3.65-3.55 (m, 8H), 3.24 (s, 1H).

[0059] Synthesis of Compound 4: Compound 3 (550 mg) was reduced at 80 °C with an EtOH / H2O solution of iron powder (529 mg) and NH4Cl (1.01 g) (3:1, v / v). Once the reaction was complete as monitored by TLC, the mixture was filtered hot, and the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with water, dried, and concentrated. The crude product was purified by rapid column chromatography, eluting with a mixture of DCM and EA (1:1, v / v). Compound 4 was given as a yellow, viscous product (322 mg), in 65% yield.

[0060] 1 ¹H NMR (300 MHz, CDCl₃, ppm) δ 6.95 (dd, J = 8.1, 1.7 Hz, 1H), 6.84(d, J = 1.7 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 4.63 (s, 2H), 3.60 (d, J = 5.7Hz, 8H), 3.43 (d, J = 4.9 Hz, 2H), 2.88 (s, 1H).

[0061] Synthesis of Compound 5: Compound 4 (700 mg), K₂CO₃ (3.72 g), KI (669 mg), and methyl bromoacetate (1.27 mL) were reacted in acetone (10 mL) at 60 °C for 72 h. After the reaction was complete, the mixture was concentrated. The residue was dissolved in ethyl acetate, washed with saturated brine, dried, and concentrated. The crude product was purified by rapid column chromatography, eluting with a mixture of n-hexane and ethyl acetate (2:1, v / v) to give compound 5 as a pale yellow oil (697 mg), in 64% yield.

[0062] 1 H NMR (300 MHz, CDCl3, ppm), δ: 7.01 (dd, J = 8.4, 1.8 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 4.62 (s, 2H), 4.10 (s, 4H), 3.64 (s, 6H), 3.59 (m, 6H), 3.44 (m, 2H), 2.94 (s, 1H).

[0063] Synthesis of Compound 7: Compound 6, namely pyroninthion (200 mg), was dissolved in anhydrous acetonitrile (5 mL), followed by the addition of copper chloride (108 mg) and potassium chloride (250 mg). After complete consumption of pyroninthion by TLC, 4-iodoaniline (147 mg) was added. The reaction was monitored by TLC until completion, and the mixture was filtered. The solvent was removed under reduced pressure, and the crude product was purified by rapid column chromatography, eluting with a mixture of DCM and MeOH (20:1, v / v). Compound 7 (abbreviated IPY) was given as an orange solid (208 mg), in 60% yield.

[0064] 1 H NMR (300 MHz, CDCl3, ppm), δ: 7.68 (d, J = 9.2 Hz, 2H,), 7.54 (d, J= 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 6.44 (dd, J = 9.2, 2.6 Hz, 2H), 6.38 (d, J = 2.6 Hz, 2H), 2.99 (s, 12H).

[0065] Synthesis of MBPY final product: IPY (200 mg), compound 5 (186 mg), tetrakis(triphenylphosphine)palladium (23 mg), and CuI (7 mg) were dissolved in a mixed solvent of triethylamine (2 mL) and tetrahydrofuran (THF) (8 mL). The mixture was refluxed at 60 °C with stirring. After the reaction was complete, the residue was dissolved in DCM, washed with water, dried, and concentrated. The crude product was purified by rapid column chromatography, eluting with a mixture of DCM and MeOH (35:1, v / v). MBPY was given as a red solid product (153 mg), yield 50%. The 1H and 1C NMR spectra of MBPY are shown below. Figure 1 and Figure 2 .

[0066] Mp: 164.1-164.6 °C. IR (film, cm -1 ): 3436.3, 2919.2, 1743.6, 1602.1,1635.0, 1513.2, 1384.7, 1016.3, 806.6. 1 H NMR (300 MHz, CDCl3, ppm), δ : 11.03 (s, 1H), 8.02 (d, J = 9.6 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.4Hz, 2H), 7.04 (dd, J = 8.2, 1.7 Hz, 1H), 6.98 (d, J = 1.7 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.61 (dd, J = 9.6, 2.6 Hz, 2H), 6.45 (d, J = 2.6 Hz, 2H), 4.67(s, 2H), 4.13 (s, 4H), 3.66 (s, 6H), 3.62 (m, 6H), 3.47 (m, 2H), 3.08 (s,12H). 13 C NMR (75 MHz, CDCl3, ppm), δ: 170.42, 165.04, 156.18, 154.11, 148.09, 146.54, 131.78, 128.16, 125.37, 123.49, 118.11, 115.54, 109.98, 95.59, 89.29, 87.25, 66.86, 65.76, 59.70, 52.54, 50.86, 44.56, 39.27. High-resolution mass spectrometry, theoretical value: 760.3341, measured value: 760.3346. Elemental analysis theoretical values: C, 64.86; H, 5.82; N, 8.79. Measured values: C, 64.92; H, 5.61; N, 9.13.

[0067] Test Example 1: Fluorescence response of the probe to calcium ions in HBSS buffer

[0068] The specific detection method is as follows: The lysosome-targeting calcium ion fluorescent probe MBPY prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. All tests were performed in an organic sulfonate buffer containing 300 mM MOPS, 1000 mM KCl, 100 mM EGTA, and 1 U / ml esterase. The final concentration of esterase was 1 U / ml; Ca 2+ The final concentration was 3.0 mM; Mg 2+ The final concentration was 1.0 mM. The final concentration of MBPY was 10.0 μM. In fluorescence measurements, the excitation wavelength λ... ex = 488 nm. The slit width for both the excitation and emission wavelengths is set to 5 nm.

[0069] 1. Probe MBPY for Ca 2+ fluorescence response

[0070] probe MBPY for Ca 2+ The fluorescence response mechanism is shown in Figure 11 .

[0071] MBPY for Ca 2+ The detection is performed by using different concentrations of Ca 2+ Solutions (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM) were added to systems containing 10.0 µM MBPY probe and 1 U / ml esterase, respectively, to determine the effect of MBPY on Ca2+. 2+The fluorescence spectrum of the response was observed. The results showed that after chelation with calcium ions, a new fluorescence emission peak appeared at 545 nm, and the fluorescence intensity increased with increasing Ca2+. 2+ Increased concentration enhances ( Figure 3 ).

[0072] 2. Probe MBPY for Ca 2+ Selectivity of fluorescence response

[0073] In each containing Mg 2+ (1 mM), Zn 2+ (10 μM), K + (130 mM), Na + (10 mM), Ni 2+ (10 μM), Cu 2+ (10 μM), Co 2+ (10 μM) and Fe 2+ In solutions containing multiple interfering ions such as (10 μM), with or without the addition of 1.0 mM Ca 2 + Under two conditions, the fluorescence spectrum of the esterase-hydrolyzed form of MBPY (10 μM) was measured at 545 nm. The results showed that MBPY only... 2+ The fluorescence intensity is significantly enhanced in its presence and is not affected by interference from other metal ions. Figure 4 ).

[0074] 3. Probe MBPY for Ca 2+ pH range of fluorescence response

[0075] With or without 1.0 mM Ca 2+ Under the condition of pH = 3.0–10.0, the fluorescence emission intensity of the esterase hydrolysate of MBPY (10.0 μM) at 545 nm was measured. The results showed that within the pH range of 3.0–10.0, regardless of the presence of Ca... 2+ The fluorescence intensity of MBPY remained almost constant. Figure 5 ).

[0076] Test Example 2: Lysosomal localization of the probe in living cells

[0077] 1. Cytotoxicity of the probe MBPY

[0078] The effect of the probe MBPY on the cell viability of human cervical cancer cell line (HeLa) and human bronchial epithelial cell line (BEAS 2B) was determined using the CCK-8 assay. The results showed that the probe MBPY had almost no toxicity to HeLa and BEAS 2B cells in the range of 0-20 μM, indicating good biocompatibility. Figure 6(A and B in the middle).

[0079] 2. Lysosomal localization of the probe MBPY

[0080] HeLa cells were co-incubated at 37 °C with MBPY (10.0 μM) and either lysosomal red dye (0.5 μM) or mitochondrial red dye (0.2 μM) for 30 min. The probe incubation system, Hanks' Balanced Salt Solution (HBSS), contained 1 mM probenecid and 0.05% poloxamer 407. Fluorescence imaging was performed using a Nikon C2 confocal laser scanning microscope with a 40x objective lens. MBPY was excited at 488 nm and collected at 500–550 nm (green channel). The commercial dye was excited at 640 nm and collected at 650–750 nm (red channel). The results indicate that the probe MBPY has specific lysosomal targeting ability. Figure 7 ).

[0081] Test Example 3: Monitoring of dynamic changes in endogenous lysosomal calcium ions in living cells

[0082] HeLa cells were seeded in confocal culture dishes, and after adhesion, they were incubated with 10 μM MBPY for 30 min. After washing, real-time dynamic imaging was performed using LSCM. Baseline imaging: Acquire basal fluorescence images of cells in a resting state as a control; Post-stimulation imaging: Add (see...) Figure 8 (A and B) were used to track changes in lysosomal calcium ions and verify the probe's responsiveness.

[0083] Test Example 4: Observation Methods for Lysosomal Calcium Homeostasis Disorders in a Lung Injury Cell Model

[0084] (1) Cell model construction

[0085] Control group: Normally cultured human bronchial epithelial cells BEAS-2B; Dexamethasone intervention group: BEAS-2B cells were pretreated with 10, 20, and 50 μM dexamethasone for 1 h, respectively; LPS-induced acute lung injury cell model group: BEAS-2B cells were pretreated with 10 μg / mL LPS for 6 h; BLM-induced pulmonary fibrosis cell model group: BEAS-2B cells were pretreated with 50 μM BLM for 24 h.

[0086] (2) Cell imaging and detection steps

[0087] Probe incubation: Wash the cells in each group three times with PBS, add HBSS solution containing 10 μM MBPY, 1 mM probenecid, and 0.05% F127, and incubate at 37 ℃ and 5% CO2 in the dark for 30 min.

[0088] Confocal imaging: Using a Nikon C2 LSCM with a 40x objective lens and 488 nm excitation light, fluorescence images of the 500–550 nm green channel were acquired. All imaging parameters (laser power, gain, exposure time) were kept completely consistent. Results are shown in [Figure number missing]. Figure 9 A and B.

[0089] Quantitative analysis: The average fluorescence intensity of each group of images was analyzed to establish the dose-response relationship between drug concentration and lysosomal calcium level, thereby achieving quantitative detection of lysosomal calcium balance disorder.

[0090] Test Example 5: Observation Methods for Lysosomal Calcium Homeostasis in an Animal Model of Lung Injury

[0091] (1) Animal model construction

[0092] Male C57 mice were randomly divided into 3 groups, with 3 or more parallel samples in each group: Control group: Intratracheal infusion of an equal volume of sterile saline; LPS-induced acute lung injury model group: 2.5 mg / kg LPS was instilled via tracheal infusion, and the model was tested 12 h after modeling. BLM-induced pulmonary fibrosis model group: 3.5 mg / kg BLM was infused into the trachea, and the model was tested 7 days after modeling.

[0093] (2) In vivo drug delivery and tissue imaging steps

[0094] 1. Probe administration: Mice in each group were injected with MBPY probe solution via tail vein at a dose of 10 mg / kg and incubated for 30 min.

[0095] 2. Sample collection: Mice were euthanized by cervical dislocation, and lung tissue was harvested under sterile conditions. The lung tissue was washed with pre-cooled PBS or directly subjected to whole-tissue imaging. Blood was removed, and frozen sections of lung tissue were prepared.

[0096] 3. Ex vivo tissue confocal imaging: A Nikon C2 LSCM was used with 488 nm excitation light to acquire fluorescence images in the 500–550 nm green channel, ensuring complete consistency of imaging parameters for each component. Results are shown in [Figure number missing]. Figure 10 .

[0097] 4. Quantitative and correlation analysis: The average fluorescence intensity of lung tissue in each group was analyzed, and the differences between groups were statistically analyzed. Through correlation analysis, a quantitative correlation was established between the fluorescence intensity of lysosomal calcium level and the degree of pathological damage and the level of inflammatory factors in lung tissue.

[0098] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent probe for targeted detection of lysosomal calcium ions, characterized in that, include: The calcium ion chelating unit precursor shown in Formula I-1a; Formula I-1a; Wherein, R1 is selected from C1~C4 hydrocarbon groups; and, the fluorescent unit of formula I-2a connected to the calcium ion chelating unit precursor; Formula I-2a; Among them, R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-. The benzene ring in the compound shown in Formula I-1a is connected to the aniline benzene ring in the compound of Formula I-2a by a carbon-carbon triple bond.

2. The fluorescent probe according to claim 1, characterized in that, It has the structure shown in Equation I: Formula I; R1, R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

3. The fluorescent probe according to claim 1, characterized in that, R1, R2, R3, R4, and R5 are independently selected from C1 to C4 alkyl groups.

4. The fluorescent probe according to claim 1, characterized in that, It is called MBPY, and the MBPY has the following structure:

5. The method for preparing the fluorescent probe according to any one of claims 1 to 3, characterized in that, It includes: The fluorescent probe is obtained by triple bonding of the calcium ion chelating unit precursor of Formula I-1a with the fluorescent unit shown in Formula I-2a via the Sonogashira reaction.

6. The preparation method according to claim 5, characterized in that, By introducing a triple bond into the calcium ion chelating unit precursor shown in Formula I-1a through the Sonogashira reaction and deprotection step, a calcium ion chelating unit with the following structure is obtained: ； Wherein, R1 is a hydrocarbon group from C1 to C4.

7. The preparation method according to claim 5, characterized in that, The fluorescent unit shown in Formula I-2a has an I atom introduced before the Sonogashira reaction to obtain an iodofluorescent unit with the following structure: ； R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

8. The use of the fluorescent probe according to any one of claims 1 to 3 in the preparation of products for detecting lysosomal calcium ions or for imaging to monitor lysosomal calcium levels.

9. The application according to claim 8, characterized in that, The changes in lysosomal calcium levels include lysosomal calcium imbalances caused by lung diseases and lysosomal calcium imbalances caused by other diseases. The lung disease includes at least one of acute lung injury, pulmonary fibrosis, and chronic obstructive pulmonary disease; the acute lung injury includes hyperoxia-induced lung injury and / or virus infection-induced lung injury; The other diseases include at least one of myocardial injury, neurodegenerative diseases, and tumors.

10. A method for detecting lysosomal calcium ion concentration or observing lysosomal calcium disorders in lung diseases for non-diagnostic purposes, characterized in that, Fluorescence imaging was performed after incubating the fluorescent probes described in claims 1 to 3 with the cells to be tested.

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