Quinolyl fluorescent probe containing indometacin as well as preparation method and application of quinolyl fluorescent probe

Abstract

The invention discloses a quinolyl fluorescent probe containing indometacin as well as a preparation method and application of the quinolyl fluorescent probe. The structural formula of the probe is shown in the specification. According to the probe constructed by the invention, a quinoline fluorophore is coupled with a COX-2 targeting group indometacin through a variable-length carbon chain connector, so that the fluorescent probe has relatively large Stokes shift gt; and the wavelength is 160 nm. Wherein the probe ICP-1 shows excellent environmental sensitivity due to limited intramolecular rotation, selectivity and open-type fluorescence detection on COX-2 can be realized, and the probe successfully visualizes RAW 264.7 macrophages induced by lipopolysaccharide, PC12 neuronal cells stressed by glutamic acid and COX-2 up-regulation in an AD zebra fish model. Besides, ICP-1 is effectively used for screening the COX-2 inhibitor to confirm the neuroprotective effect of hararine, and it is indicated that ICP-1 is a powerful tool for real-time neuroinflammation imaging and has application value in neurodegenerative disease research.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to the preparation of a quinoline-based fluorescent probe containing indomethacin and its application in screening cyclooxygenase-2 inhibitors and neuroinflammatory imaging. Background Technology

[0002] Neuroinflammation is a pathological process characterized by chronic glial cell activation and infiltration of peripheral immune mediators, playing a central role in the progression of various neurological diseases, including Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis, and ischemic stroke. A key mediator in this inflammatory cascade is cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid into pro-inflammatory prostaglandins, particularly prostaglandin E2. E2 inhibits fibroblast proliferation, prevents their conversion to myofibroblasts, limits collagen synthesis, and promotes the degradation of newly synthesized collagen. Under physiological conditions, COX-2 is expressed at low levels in specific brain regions such as the hippocampus and cortex, participating in synaptic plasticity and cognitive function. However, COX-2 is rapidly upregulated in response to inflammatory stimuli such as cytokines, reactive oxygen species, and bacterial endotoxins, making it a key driver of neuroinflammatory responses. Furthermore, preclinical studies have shown that pharmacological inhibition of COX-2 can reduce neuroinflammation and neuronal loss, validated in models of neurodegenerative diseases and acute brain injury. For example, COX-2 inhibitors have been shown to alleviate β-amyloid-induced neurotoxicity in AD models and reduce infarct volume in experimental strokes. Therefore, real-time monitoring of COX-2 expression during neuroinflammatory processes is of great significance.

[0003] Fluorescent probes have become indispensable tools for detecting and visualizing various biomolecular interactions and pathological processes in living systems. In particular, environmentally responsive fluorescent probes enable in-situ visualization of receptor binding; their unbound state exhibits weak background fluorescence, while binding to the target significantly enhances the fluorescence signal. Typically, COX-2 inhibitors are bound to fluorophores via appropriate chemical linkages to prepare "on" probes. Despite significant efforts in developing COX-2 fluorescent probes, current sensors are still limited by small Stokes shifts and short emission wavelengths, which significantly restricts their imaging applications. Summary of the Invention

[0004] Objective of the Invention: Addressing the problems of existing technologies, this invention provides a quinoline-based fluorescent probe containing indomethacin. The quinoline-based fluorescent probe prepared by this invention couples the quinoline fluorophore to the COX-2 targeting group indomethacin (IMC) via a variable-length carbon chain connector, resulting in a large Stokes shift (>160 nm) for the fluorescent probe. Due to its restricted intramolecular rotation, the probe exhibits excellent environmental sensitivity, enabling selective and on-feed fluorescence detection of COX-2. This probe successfully visualized COX-2 upregulation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, glutamate-stressed PC12 neurons, and an AD zebrafish model. Furthermore, this probe has been effectively used to screen for COX-2 inhibitors. These findings indicate that the probe of this invention is a powerful tool for real-time neuroinflammatory imaging and has potential applications in neurodegenerative disease research.

[0005] The present invention also provides a method for preparing and applying a quinoline-based fluorescent probe containing indomethacin.

[0006] Technical solution: To achieve the above objective, the present invention provides a quinoline-based fluorescent probe containing indomethacin, the structure of which is shown below:

[0007] Where n represents the length of the carbon chain, which can be any one of 2 to 6.

[0008] Preferably, n represents the length of the carbon chain, which can be any one of 2, 4, or 6.

[0009] The fluorescent probe ICP-1-3 is obtained by using quinoline as the fluorophore and IMC as the targeting group, with the two connected by carbon chains of different lengths.

[0010] The method for preparing the indomethacin-containing quinoline-based fluorescent probe of the present invention includes the following steps:

[0011] Indomethacin and EDCI were dissolved in DMF and stirred at room temperature. Then, N-Boc-ethylenediamine (N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine), DIPEA, and HOBt were added, and the reaction mixture was stirred at room temperature. After the reaction was complete, distilled water was added to terminate the reaction, giving a white precipitate. The crude product was purified to give I-1-3.

[0012] Compound I-1-3 was dissolved in dichloromethane, followed by the slow addition of trifluoroacetic acid. The reaction mixture was stirred at room temperature. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by column chromatography to give compound C-1-3.

[0013] Cyanoacetic acid was dissolved in dichloromethane, followed by the addition of diethylamine and HATU. The mixture was stirred at room temperature. Compound C-1-3 was then added, and the reaction was stirred at room temperature. After the reaction was complete, DCM was concentrated under reduced pressure. The residue was washed successively with water, ethyl acetate, and methanol to give product IC-1-3.

[0014] Compound IC-1-3 and 6-(dimethylamino)quinoline-2-carboxaldehyde were dissolved in methanol under an argon atmosphere. A catalytic amount of piperidine was added, and the reaction mixture was stirred under heating. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by column chromatography to give ICP-1-3.

[0015] The reaction formula is shown below:

[0016] The application of the indomethacin-containing quinoline-based fluorescent probe described in this invention in the preparation of cyclooxygenase-2 (COX-2) detection reagents or tools.

[0017] The present invention relates to the application of the indomethacin-containing quinoline-based fluorescent probe in the preparation of reagents or tools for cyclooxygenase-2 (COX-2) bioimaging.

[0018] The present invention relates to the application of the indomethacin-containing quinoline-based fluorescent probe in the preparation of reagents or tools for monitoring changes in the expression level of oxidase-2 (COX-2) in cells and organisms.

[0019] The application of the indomethacin-containing quinoline-based fluorescent probe described in this invention in the preparation of reagents or tools for real-time neuroinflammatory imaging.

[0020] The present invention relates to the application of the indomethacin-containing quinoline-based fluorescent probe in the preparation of screening reagents or tools for cyclooxygenase-2 (COX-2) inhibitors.

[0021] This invention provides a COX-2-targeting fluorescent probe, ICP-1-3, using quinoline as the fluorophore and IMC as the targeting group, linked by carbon chains of varying lengths. Choosing an appropriate linker length is crucial for COX-2 probe development to avoid steric hindrance when IMC binds to the enzyme's active site. For example, ICP-1 exhibits excellent environmental sensitivity due to its restricted intramolecular rotation, enabling selective and on-feed fluorescence detection of COX-2. The probe successfully visualized COX-2 upregulation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, glutamate-stressed PC12 neurons, and an AD zebrafish model. Furthermore, ICP-1 was effectively used to screen COX-2 inhibitors, and experimental results confirmed the neuroprotective effect of halogen.

[0022] The probes of this invention enable selective and on-feed fluorescence detection of COX-2. For example, ICP-1 can be used to screen COX-2 inhibitors from natural products and applied in LPS-induced RAW 264.7 cell and zebrafish inflammation models. Furthermore, fluorescence imaging showed that ICP-1 responds to COX-2 in PC12 neurons under glutamate-induced oxidative stress. Further, ICP-1 was successfully used for COX-2 imaging in a zebrafish AD model. These results demonstrate that ICP-1 can serve as a selective COX-2 imaging probe, highlighting its potential application value in neurodegenerative diseases.

[0023] The design principle of this series of ICP fluorescent probes: The design of this series of probes (ICP-1, ICP-2, ICP-3) is based on a molecular structure-function synergistic optimization strategy. Its core lies in overcoming the limitations of traditional technologies through a triple mechanism: 6-(dimethylamino)quinoline-2-carboxaldehyde is used as a fluorescent reporter group, utilizing a push-pull electron system to induce a strong intramolecular charge transfer (ICT) effect to achieve a redshift in the emission spectrum. Simultaneously, by controlling the length of the alkyl linker arm, the intramolecular rotational freedom is restricted, significantly expanding the Stokes shift and enhancing environmental sensitivity; indomethacin (IMC) is used as a targeting module to maintain contact with CO. The specific interactions at the COX-2 active site (including Arg120 salt bridge bonding and hydrophobic cavity embedding) are key features. The short-chain variant (ICP-1) maximizes the binding efficiency of the parent ligand by using a connector arm design that precisely matches the spatial scale of the active cavity. The fluorescence activation mechanism relies on enzyme-induced conformational rigidification—the free probe undergoes non-radiative decay to quench fluorescence, while the hydrophobic microenvironment after binding to COX-2 inhibits molecular motion, triggering a significant "turn-on" response. Ultimately, by optimizing molecular size and lipid-water partitioning properties, the design meets the blood-brain barrier penetration requirements, and stable amide bond connections enable continuous imaging of neural tissue. This design, through systematic innovation in ICT-rotation-restricted synergistic photophysical optimization, target site spatial adaptation, and enzyme-responsive conformational locking, overcomes the key bottlenecks of insufficient Stokes shift, weak tissue penetration, and low activation specificity in existing probes.

[0024] This invention successfully developed a novel fluorescent probe (especially ICP-1) by combining a quinoline fluorophore with the COX-2 inhibitor indomethacin (IMC) through an optimized linker chain. Its core reaction mechanism utilizes the intramolecular rotational restriction (RIR) effect that occurs after the probe binds to the COX-2 enzyme, achieving a fluorescence "turn-on" response. This design offers significant advantages: First, the probe possesses a large Stokes shift exceeding 160 nm, effectively avoiding interference between excitation and emission light, greatly improving the signal-to-noise ratio and detection sensitivity. Second, ICP-1 exhibits extremely high selectivity and low biotoxicity, enabling successful real-time and visual monitoring of COX-2 upregulation in living cells (such as LPS-induced macrophages and glutamate-stressed neurons) and in vivo (zebrafish Alzheimer's disease model), providing a powerful tool for studying neuroinflammation. Finally, this probe was innovatively applied to high-throughput drug screening, successfully identifying COX-2 inhibitors with neuroprotective potential (such as halamine) from natural compounds, demonstrating its enormous application potential in drug development. This invention is the first to successfully introduce the intramolecular rotational restriction (RIR) mechanism into the design of COX-2 fluorescent probes. By rationally optimizing the connective arm length, a high-performance probe with an ultra-large Stokes shift (>160 nm), such as ICP-1, was created. This design effectively overcomes the signal self-absorption and background interference problems caused by the small Stokes shift of traditional probes. This study is the first to achieve specific COX-2 imaging verification of this probe in a zebrafish model of Alzheimer's disease and glutamate-stressed neurons, and pioneering its application in high-throughput functional screening of COX-2 inhibitors, successfully discovering the neuroprotective activity of natural compounds such as halamine, breaking through the technical limitations of existing COX-2 probes, which are mainly limited to tumor cell imaging.

[0025] This invention marks the first successful application of intramolecular rotational restriction (RIR) mechanisms to the construction of COX-2 fluorescent probes. By systematically optimizing the linker arm length, high-performance "turn-on" probes with ultra-large Stokes shifts (>160 nm), exemplified by ICP-1, were created. This design achieves specific fluorescence enhancement induced by molecular motion restriction after probe binding to COX-2 through precise coupling of the quinoline fluorophore to the indomethacin targeting molecule, effectively solving the signal self-absorption and background interference problems caused by the small Stokes shift of traditional probes. This innovative design not only enables specific COX-2 dynamic imaging in Alzheimer's disease zebrafish models and neuroinflammatory cells for the first time, but also expands the application of probes in high-throughput screening of COX-2 inhibitors, providing a novel molecular tool for neuroinflammation research and anti-inflammatory drug discovery.

[0026] Furthermore, this invention employs an intramolecular rotationally restricted (RIR) mechanism and precisely optimizes the core parameter of the linker arm length. Through systematic synthesis of probes with only different linker arm lengths (ICP-1 C2, ICP-2 C4, ICP-3 C6) and parallel comparative experiments, it is conclusively demonstrated that ICP-1, with only the short chain (C2) linker, can achieve specific recognition of COX-2—its fluorescence is significantly quenched in inhibitor competition experiments. In contrast, ICP-2 / 3, due to steric hindrance caused by excessively long linker arms, results in non-specific binding and fails to respond to competition, highlighting the precision and necessity of the structural design of this invention.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] This invention prepares a quinolinyl fluorescent probe containing indomethacin, which significantly overcomes the core limitations of existing COX-2 fluorescent probes by optimizing the fluorophore structure and intramolecular rotational confinement mechanism. Compared with traditional probes (such as the BODIPY-IMC system), its technological breakthroughs are mainly reflected in:

[0029] Existing probes are generally limited by narrow Stokes shifts (typically 50-80 nm) and short-wavelength emission (<600 nm), leading to overlapping excitation / emission spectra and interference from autofluorescence in biological tissues. The probes prepared in this invention, such as ICP-1, utilize the push-pull electron effect of quinoline derivatives to achieve a 176 nm Stokes shift (a 120% increase in the maximum reported in the literature) and 648 nm near-infrared emission, resulting in complete separation of the excitation / emission peaks and a 5.3-fold improvement in signal-to-noise ratio compared to conventional probes (validated in a RAW 264.7 cell model). The intramolecular rotation-restricted design endows it with excellent environmental sensitivity, showing a >20-fold increase in fluorescence intensity in a glycerol / PBS mixture and a 12.8-fold increase in fluorescence quantum yield upon binding with COX-2.

[0030] Unlike single-function imaging probes, this invention's probe is the first to integrate in-situ imaging and high-throughput inhibitor screening. Its flow cytometry-based screening platform can complete the screening of a natural compound library within 30 minutes, successfully identifying halamine, a COX-2 inhibitor with superior inhibitory efficacy compared to indomethacin (increased inhibition rate by 38.7%). This probe further enables dynamic efficacy assessment of the inhibitor; in LPS-induced macrophage and AD zebrafish models, fluorescence intensity decay was linearly correlated with COX-2 activity inhibition.

[0031] Addressing the challenge of blood-brain barrier penetration in neurological research, ICP-1's molecular weight (649 Da) and lipid-water partition coefficient (calculated LogP = 3.52) meet the requirements for central nervous system penetration, successfully enabling dynamic COX-2 imaging in AlCl3-induced AD zebrafish brains. It maintains fluorescence stability for >12 hours in vivo, supporting longitudinal monitoring of neuroinflammatory processes.

[0032] This invention systematically solves key technical bottlenecks such as narrow Stokes displacement, weak tissue penetration, and limited functionality through the innovative design of quinoline fluorophores. Validation data in various pathological models (LPS-macrophages, glutamate-neurons, AD-zebrafish) demonstrate that this probe not only provides a highly sensitive tool for studying neuroinflammation mechanisms but also establishes an efficient screening platform for the development of drugs against neurodegenerative diseases.

[0033] This invention employs a simple and efficient one-step condensation reaction to achieve the efficient linkage of quinoline aldehyde derivatives and indomethacin derivatives under mild conditions (methanol solvent, argon protection, and a catalytic amount of piperidine). A series of probes are obtained by systematically adjusting the linker arm lengths (C2 / C4 / C6). The preparation process requires no complex purification techniques (conventional column chromatography is sufficient for separation), and the structures of all probes have been fully confirmed by 1H / C NMR spectroscopy and high-resolution mass spectrometry. This demonstrates that the synthetic route has advantages such as simple operation, reliable yield, and good reproducibility, laying a solid foundation for large-scale preparation for subsequent biological applications.

[0034] The probe of this invention exhibits superior detection performance: based on the intramolecular rotationally restricted (RIR) mechanism, the ICP-1 probe achieves a highly selective and sensitive "turn-on" fluorescence response to COX-2, and its ultra-large Stokes shift (>160 nm) effectively overcomes the problem of autofluorescence interference in bioimaging. At the application level, this probe has successfully achieved multi-level detection from cells to living organisms: it not only clearly traces COX-2 upregulation in LPS-induced macrophages and glutamate-stressed neurons, but also, for the first time, achieves dynamic visualization of neuroinflammation in a zebrafish model of Alzheimer's disease; simultaneously, it has been successfully applied to high-throughput screening of COX-2 inhibitors, discovering the anti-neuroinflammatory activity of natural compounds such as hamamipine, highlighting its important tool value in disease mechanism research and drug development. Attached Figure Description

[0035] Figure 1 The chemical structures and fluorescence excitation / emission spectra of fluorescent probes ICP-1, ICP-2, and ICP-3 in 10 mM PBS buffer (pH 7.4, containing 1% dimethyl sulfoxide) are shown.

[0036] Figure 2The fluorescence emission spectra of the three probes (10 μM) in a PBS / glycerol (Gly) mixture are shown. The top image shows the corresponding fluorescence captured under UV light irradiation at an excitation wavelength of 365 nm.

[0037] Figure 3 The logarithm of the probes (ICP-1, ICP-2, and ICP-3) is (Log( The linear relationship between viscosity logarithm (η) and viscosity logarithm (η).

[0038] Figure 4 The fluorescence spectra of probe ICP-1 (10 μM) in 1,4-dioxane-water mixtures at different ratios (1,4-dioxane: 0%~100%) are shown.

[0039] Figure 5 The fluorescence spectra of ICP-1 (10 μM) in solvents of different polarities are shown.

[0040] Figure 6 Cell viability of ICP-1, ICP-2, and ICP-3 in RAW 264.7 cells after 24 hours was assessed using the standard MTT assay.

[0041] Figure 7 Figure A shows a fluorescence microscope image of HeLa cells stained with probes ICP-1, ICP-2, or ICP-3 (10 μM), some of which were pretreated with celecoxib (50 μM) for 24 hours before staining. Red channel: excitation wavelength λex = 488 nm, emission wavelength λem = 570 nm. Figure B shows a quantitative analysis of the fluorescence intensity of the experiments shown in Figure A.

[0042] Figure 8 Fluorescence images and corresponding intensity analysis of RAW264.7 cells after pretreatment with LPS (0, 250, or 500 ng / mL, 12 h) followed by incubation with ICP-1 (1 μM, 30 min). Red channel: excitation wavelength (λ). ex = 488 nm, emission wavelength (λ) em = 570-616 nm. Scale bar: 20 μm. Data are expressed as mean ± standard error (SEM). .

[0043] Figure 9 RAW 264.7 cells were analyzed for flow cytometry thermography. Cells were first stimulated with LPS (500 ng / mL, 24 h), then treated with a variety of natural products (20 μM, 12 h), and finally co-incubated with ICP-1 (2 μM, 30 min).

[0044] Figure 10 The images show fluorescence images of RAW 264.7 cells after stimulation with LPS (500 ng / mL, 24 h), treatment with indomethacin, trihydroxyphenol acetone, or halogen (20 μM each, 24 h), and subsequent incubation with an ICP-1 probe (1 μM, 30 min).

[0045] Figure 11 The image shows the fluorescence of zebrafish stimulated by LPS. The zebrafish were first exposed to LPS (500 ng / mL, 24 h), then treated with phloretin or harmin (20 μM each, 24 h), and finally incubated with the ICP-1 probe (1 μM, 30 min).

[0046] Figure 12 Fluorescence intensity analysis of LPS-stimulated zebrafish was performed. Zebrafish were first exposed to LPS (500 ng / mL, 24 h), followed by treatment with phloretin or harmin (20 μM each, 24 h), and finally incubated with an ICP-1 probe (1 μM, 30 min). Data are expressed as mean ± standard error (SEM). .

[0047] Figure 13 Imaging of neuroinflammation induced by glutamate in PC12 cells. PC12 cells were first incubated with glutamate (5 mM, 24 h), then treated with or without canine (20 μM, 12 h), and finally incubated with ICP-1 (1 μM, 30 min). Red channel: excitation wavelength (λex) = 488 nm, emission wavelength (λem) = 570-616 nm.

[0048] Figure 14 Fluorescence images and corresponding intensity analysis of the probe ICP-1 in an AlCl3-induced AD zebrafish model. Zebrafish were exposed to 100 μg / L AlCl3 for 12 h, followed by treatment with harmin or indomethacin (20 μM) for an additional 24 h, and finally incubated with ICP-1 (1 μM) for 30 min. Scale bar: 20 μm.

[0049] Figure 15 RAW264.7 cells were incubated for 24 h with different probe concentrations (0, 0.5, 1, 2, 4, 8 μM) of (a) ICP-1, (b) ICP-2, and (c) ICP-3. Error bars are ±SD (n=3).

[0050] Figure 16 The image shows a heatmap of flow cytometry results. RAW264.7 was stimulated with 500 ng / mL LPS for 24 h, then incubated with different natural products (20 μM) for 12 h, and then incubated with ICP-1 (2 μM) for 30 min. Error bars are ±SD (n=3). Detailed Implementation

[0051] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0052] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0053] Example 1

[0054] A series of methods for preparing the probe ICP-1-3 are described below:

[0055] Indomethacin (500 mg, 1.5 mmol) and EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 573 mg, 3.0 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 30 minutes. Then, N-Boc-ethylenediamine (2.25 mmol) or N-Boc-1,4-butanediamine (2.25 mmol) or N-Boc-1,6-hexanediamine (2.25 mmol), DIPEA (N,N-diisopropylethylamine, 387 mg, 3.0 mmol) and HOBt (1-hydroxybenzotriazole, 608 mg, 4.5 mmol) were added, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, distilled water (30 mL) was added to terminate the reaction, resulting in a white precipitate. The crude product was purified by column chromatography (PE / EA = 100:1, volume ratio) to obtain compound I-1-3 (i.e., I-1, I-2, or I-3) (wherein, the reaction mixture was reacted with N-Boc-ethylenediamine to obtain I-1, N-Boc-1 and 4-butanediamine to obtain I-2, and N-Boc-1 and 6-hexanediamine to obtain I-3), which was a light yellow solid.

[0056] Compounds I-1, I-2, or I-3 (1.63 mmol) were dissolved in dichloromethane (10 mL), followed by the slow addition of trifluoroacetic acid (3 mL) at 0°C. The reaction mixture was stirred at room temperature for 4 hours (the reaction progress was monitored by thin-layer chromatography). After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (DCM / MeOH = 10:1, v / v) to give compounds C-1, C-2, or C-3 as white solids.

[0057] Cyanoacetic acid (148 mg, 1.75 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of diethylamine (505 mg, 5 mmol) and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 950 mg, 2.5 mmol). The mixture was stirred at room temperature for 1 hour. Then, compounds C-1, C-2, or C-3 (1.25 mmol) were added, and the reaction was stirred at room temperature for 6 hours. After the reaction was complete, the DCM was concentrated under reduced pressure. The residue was washed successively with water, ethyl acetate, and methanol to give products IC-1, IC-2, or IC-3.

[0058] Compounds IC-1, IC-2, or IC-3 (0.32 mmol) and 6-(dimethylamino)quinoline-2-carboxaldehyde (64 mg, 0.32 mmol) were dissolved in 3 mL of methanol under an argon atmosphere. A catalytic amount of piperidine (1.4 μL, 16 μmol) was added, and the reaction mixture was stirred at 60°C for 6 hours. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by column chromatography (DCM-methanol = 60:1, v / v) to give ICP-1, ICP-2, or ICP-3 as a deep red solid.

[0059] ICP-1, dark red solid (42 mg, yield 21%) 1H NMR (400 MHz, CDCl3) δ 8.09 (s,1H), 7.99 (d, J = 9.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.76-7.71 (m, 2H),7.56 (d, J = 8.4 Hz, 1H), 7.47-7.42 (m, 2H), 7.38 (dd, J = 9.4, 2.8 Hz, 1H), 6.95 (t, J = 5.6 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.83 (d, J = 9.0 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.56-6.49 (m, 2H), 3.75 (d, J = 0.8 Hz, 3H), 3.64 (s, 2H), 3.52 (td, J = 6.9, 6.2, 4.0 Hz, 2H), 3.46 (dd, J = 7.0, 4.4 Hz, 2H), 3.15 (s, 6H), 2.37 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 170.9, 168.4, 162.1,156.2, 151.4, 145.0, 144.8, 142.2, 139.3, 136.6, 133.9, 133.7, 131.3 (2C),131.2, 131.0, 130.9, 130.4, 129.2 (2C), 123.9, 120.1, 117.2, 115.1, 112.6,112.2, 104.2, 103.6, 100.7, 55.7, 41.0, 40.5, 39.9, 32.1, 13.4. HR-MS (ESI): m / z calcd for C 36 H 33 ClN6O4 [M+H] + : 649.2325, found: 649.2314.

[0060] ICP-2, dark red solid (53 mg, yield 25%) 1H NMR (400 MHz, CDCl3) δ 8.32 (s,1H), 7.98 (dd, J = 12.2, 9.0 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.61 (d, J =8.5 Hz, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.38 (dd, J = 9.4, 2.8 Hz, 1H), 6.90(d, J = 2.6 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.68(dd, J = 9.0, 2.4 Hz, 1H), 6.63 (t, J = 5.8 Hz, 1H), 5.88-5.84 (m, 1H), 3.81(s, 3H), 3.65 (s, 2H), 3.32-3.28 (m, 4H), 3.15 (s, 6H), 2.39 (s, 3H), 1.74(s, 2H), 0.89-0.81 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 170.0, 168.3, 160.7,156.2, 151.2, 149.9, 145.0, 142.2, 139.5, 136.4, 133.8, 133.6, 131.2 (2C),131.2, 130.9, 130.8, 130.4, 129.2 (2C), 123.7, 120.0, 117.5, 115.2, 112.9,112.3, 105.0, 103.6, 100.9, 55.8, 40.5, 39.9, 39.2, 32.3, 26.9, 26.7, 13.4.HR-MS (ESI): m / z calcd for C 38 H 37 ClN6O4 [M+H] + : 677.2638, found: 677.2613.

[0061] ICP-3, dark red solid (40 mg, yield 23%) 1H NMR (400 MHz, CDCl3) δ 8.34 (s,1H), 8.03 (d, J = 9.4 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.67-7.63 (m, 2H),7.50-7.42 (m, 2H), 7.39 (dd, J = 9.5, 2.8 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 6.74 (d, J = 2.8 Hz, 1H), 6.69 (dd, J = 9.1, 2.5Hz, 1H), 6.55 (t, J = 5.9 Hz, 1H), 5.76 (t, J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.64 (s, 2H), 3.28-3.18 (m, 4H), 3.15 (s, 6H), 2.39 (s, 3H), 1.47-1.40 (m, 4H), 1.36-1.25 (m, 4H). 13 C NMR (101 MHz, CDCl3) δ 169.8, 168.3, 160.5, 156.3,149.9, 139.5, 136.3, 133.9, 133.6, 131.2 (2C), 131.0, 130.9, 130.8, 130.4,129.2 (2C), 123.6, 120.1, 117.5, 115.1, 113.0, 112.3, 103.6, 100.9, 55.8,40.5 (2C), 40.2, 39.3, 32.3, 29.3, 29.3, 26.2 (2C), 13.3. HR-MS (ESI): m / zcalcd for C 40 H 41 ClN6O4 [M+H] + : 705.2951, found: 705.2950.

[0062] Example 2

[0063] Fluorescence spectra of ICP-1, ICP-2, and ICP-3

[0064] The excitation and emission spectra of ICP-1, ICP-2, and ICP-3 (10 μM) in PBS buffer solution (10 mM, pH 7.4, 1% DMSO) were measured using a fluorescence spectrophotometer, and signals in the wavelength range of 300–800 nm were collected. Figure 1 As shown, the maximum excitation wavelengths of compounds ICP-1, ICP-2, and ICP-3 in PBS (10 mM, pH 7.4, 1% DMSO) buffer were 470 nm, 467 nm, and 465 nm, respectively; the maximum emission wavelengths were 651 nm, 647 nm, and 643 nm, respectively; and the Stokes shifts were 181 nm, 180 nm, and 178 nm, respectively. This indicates that the above probes have strong near-infrared fluorescence emission and large Stokes shifts.

[0065] Example 3

[0066] Spectroscopic testing of viscosity, polarity and pH response of ICP-1

[0067] This invention first investigated the relationship between the fluorescence intensity and viscosity of ICP-1 by adding 0%–99% glycerol to a PBS buffer solution (10 mM, pH 7.4, 1% DMSO) to change the solution viscosity. As the glycerol content increased, the solution viscosity increased, and the fluorescence intensity of ICP-1 increased, as shown below. Figure 2 As shown. Furthermore, by fitting the Förster–Hoffmann equation, a good linear relationship was found between the logarithm of fluorescence intensity (Log I651) and the logarithm of viscosity (Log η), indicating that the probe ICP-1 is sensitive to viscosity, such as... Figure 3 As shown in the figure. The correlation between the fluorescence intensity and polarity of the probe ICP-1 was then further evaluated. The changes in fluorescence intensity of ICP-1 (10 μM) in 1,4-dioxane-water systems with different ratios were tested. With increasing 1,4-dioxane ratio (0%–100%), the solution polarity decreased, the fluorescence signal increased, and a blue shift occurred, as shown in the figure. Figure 4 As shown, when the Δf of the solution increased from 0.2050 to 0.3200, the fluorescence intensity of ICP-1 decreased by approximately 13-fold, and the maximum fluorescence signal (Imax) showed a good linear relationship with Δf. Simultaneously, the fluorescence signal changes of ICP-1 in solvents of different polarities (ordered from highest to lowest polarity: water, dimethyl sulfoxide, methanol, N,N-dimethylformamide, acetonitrile, acetone, 1,4-dioxane, chloroform, ethyl acetate, tetrahydrofuran, dichloromethane, toluene) were tested. The normalized spectrum of the probe (…) Figure 5The results showed that the emission wavelength of ICP-1 generally exhibited a redshift trend with increasing solvent polarity. These results all indicate that the probe ICP-1 is sensitive to polarity. After confirming the environmental sensitivity of the probe ICP-1, we further investigated the effects of other physiological environmental factors (such as pH) on the fluorescence signal intensity. The results showed that the fluorescence intensity of ICP-1 did not change significantly in PBS at pH (3–11), demonstrating good pH stability.

[0068] Example 4

[0069] Cytotoxicity tests for ICP-1, ICP-2, and ICP-3

[0070] The MTT assay was used to detect the toxicity of ICP-1, ICP-2, and ICP-3 to HeLa and RAW264.7 cells. This experiment included a PBS group (outer wells for interference prevention), a blank control group (DMEM complete medium only), a cell control group (0 μM probe concentration, cells in DMEM complete medium only), and experimental groups (containing 5 probe concentrations). Cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 ~1×10 4 Cells were incubated overnight at 37 ℃ in a 5% CO2 incubator. After cell attachment, culture media containing different concentrations of the compound were added (concentration gradients of 0.5, 1, 2, 4, 8 μM for HeLa cells and 0.5, 1, 2, 4, 8 μM for RAW264.7 cells), with 5 replicates for each concentration. After 24 h of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well. Incubation continued for another 4 h in the dark. After incubation, the original culture medium was discarded, and 150 μL of DMSO solution was added to each well. The wells were shaken for 10 min to fully dissolve the formazan crystals, and the absorbance of each well was measured (at 490 nm) using a microplate reader.

[0071] Before performing flow cytometry and confocal imaging, the toxicity of ICP-1, ICP-2, and ICP-3 to HeLa cells and RAW264.7 cells was assessed using the MTT assay. At a concentration of 2 μM, the survival rates of probes ICP-1, ICP-2, and ICP-3 in HeLa cells were all greater than 80%. Figure 6 As shown, the survival rate of the above probes was greater than 80% in RAW264.7 cells at a concentration of 2 μM. Figure 15As shown, ICP-1, ICP-2, and ICP-3 exhibited low cytotoxicity at a concentration of 2 μM. Based on the results of the toxicity tests, a concentration of 2 μM was used for subsequent flow cytometry detection, and a concentration of 1 μM was used for cell confocal imaging.

[0072] Example 5

[0073] Flow cytometry detection of the binding affinity of ICP-1, ICP-2, ICP-3 to COX-2

[0074] HeLa cells were seeded in 6-well plates and incubated overnight. After cell attachment, different concentrations of celecoxib solution (0, 20, 40, 80 μM) were added and incubated at 37 °C for 30 min. After incubation, the original culture medium was discarded, and the cells were washed three times with PBS (10 mM, pH 7.4). Then, they were co-incubated with 2 μM ICP-1, ICP-2, and ICP-3 at 37 °C for 30 min, respectively. After washing three times with PBS, the fluorescence intensity of cells in the PerCP-A channel was detected by flow cytometry.

[0075] ICP-1 exhibited high fluorescence intensity in HeLa cells with high COX-2 expression. However, with the addition of celecoxib, a selective inhibitor of COX-2, the fluorescence intensity decreased sharply, and further decreased with increasing celecoxib concentration. Figure 7 As shown. However, ICP-2 and ICP-3 did not show a significant decrease in fluorescence intensity due to celecoxib competing with it for the target, indicating that ICP-1 has the best targeting ability for COX-2 among the three. Therefore, ICP-1 was selected for further research in subsequent cell imaging and other experiments.

[0076] Example 6

[0077] Confocal imaging of RAW264.7 stimulated by ICP-1 and LPS

[0078] RAW264.7 cells were seeded on confocal discs and cultured overnight. After cell attachment, different concentrations of LPS (0, 250, 500 ng / mL) were added for stimulation for 12 h. After washing three times with PBS, ICP-1 (1 μM) was added and incubated for 30 min. After incubation, the cells were washed three times with PBS and confocal imaging was performed (red channel, λex=488 nm, λem=570-616 nm).

[0079] RAW264.7 cells were induced to polarize with LPS, thereby increasing COX-2 levels. Figure 8As shown, compared with the control group, LPS-stimulated cells showed higher fluorescence intensity under a confocal microscope after incubation with ICP-1, and the fluorescence intensity changed more significantly with increasing stimulation concentration.

[0080] Example 7

[0081] ICP-1 screening of natural products with anti-inflammatory activity

[0082] RAW264.7 cells were seeded in 12-well plates and incubated overnight. After cell attachment, 500 ng / mL LPS was added and incubated for 24 h. Cells were then washed three times with PBS, and 20 μM of different natural products were added. Figure 16 (Incubate for 12 h, using indomethacin as a control drug. After incubation, wash with PBS, add ICP-1 (2 μM) and incubate for 30 min. After washing 3 times with PBS, detect the fluorescence intensity of cells in the PerCP-A channel using flow cytometry. Verify the screening results using confocal imaging. Seed RAW264.7 cells on confocal dishes and culture overnight. Treat cells with the selected natural products according to the above procedure. After incubating with ICP-1 (1 μM) for 30 min, wash 3 times with PBS and perform confocal imaging (red channel, λex=488nm, λem=570-616 nm).)

[0083] Using an LPS-induced inflammatory cell model, natural drugs with anti-inflammatory activity were screened based on flow cytometry fluorescence intensity in the cells. Among these drugs, those incubated with phlorizin, halogenated alkaloids, and betulin showed significantly reduced fluorescence intensity. Figure 9 As shown. Next, the effect of confocal imaging convective fluorescence detection was verified. The intracellular fluorescence intensity of cells incubated with the selected natural drugs was significantly lower than that of cells stimulated with only LPS or with the positive control drug indomethacin. Figure 10 As shown in the figure. Among the drugs screened, halalkaloid showed the best effect, therefore halalkaloid was selected as a control for subsequent experiments.

[0084] Example 8

[0085] ICP-1 confocal imaging of a glutamate-induced PC12 cell neurodegenerative disease model

[0086] PC12 cells were seeded on confocal dishes and incubated overnight. After cell attachment, they were incubated with glutamate (5 mM) for 12 h or with a mixture of glutamate (5 mM) and halogen (20 μM) for 12 h. After washing, ICP-1 (1 μM) was added and incubation continued for 30 min. After incubation, the cells were washed three times with PBS and then subjected to confocal imaging (red channel, λex=488 nm, λem=570-616 nm).

[0087] First, a cell model of neurodegenerative disease was established by inducing cytotoxic damage in PC12 cells stimulated with glutamate. After glutamate stimulation, the intracellular fluorescence intensity was significantly upregulated compared to the control group. Figure 13 As shown, the intracellular fluorescence intensity of cells co-incubated with halamine, a natural drug with anti-inflammatory activity, was lower than that stimulated with glutamate alone. This demonstrates that ICP-1 can monitor changes in COX-2 levels in nerve cells.

[0088] Example 9

[0089] ICP-1 confocal imaging of an AlCl3-induced AD zebrafish model

[0090] The hatched zebrafish were randomly grouped and placed in six-well plates, with approximately 6-8 zebrafish (3-7 day old juveniles) per well, and 3 replicates per group. Each well contained 2 mL of culture medium for incubation. The specific groupings were as follows: (a): 2 μM ICP-1 for 30 min; (b): 100 μg / L AlCl3 for 12 h + 2 μM ICP-1 for 30 min; (c): 100 μg / L AlCl3 for 12 h + 20 μM harmine for 24 h + 2 μM ICP-1 for 30 min; (d): 100 μg / L AlCl3 for 12 h + 20 μM domethacin for 24 h + 2 μM ICP-1 for 30 min. After incubation, the zebrafish were anesthetized, washed, and subjected to confocal imaging (red channel, λ). ex =488 nm, λ em =570 - 616 nm).

[0091] First, an AlCl3-induced Alzheimer's disease (AD) zebrafish model was established. Confocal imaging results showed that the fluorescence level was significantly increased in the AlCl3-stimulated disease model group. Figure 11 and Figure 12As shown, compared with the control group, LPS-treated zebrafish exhibited a significantly enhanced red fluorescence signal. However, when LPS-stimulated zebrafish were further treated with trihydroxyphenol acetone or halamine, the fluorescence intensity decreased significantly, indicating a decrease in COX-2 levels. Furthermore, the fluorescence intensity decreased upon the addition of the COX-2 inhibitor indomethacin and the anti-inflammatory drug halamine. Figure 14 As shown, this demonstrates that ICP-1 is capable of COX-2 bioimaging.

Claims

1. A quinolinyl fluorescent probe containing indomethacin, characterized in that, The structure of the fluorescent probe is shown below: ； Where n represents the number of carbon chains, which can be any number from 2 to 6.

2. The quinoline-based fluorescent probe containing indomethacin according to claim 1, characterized in that, The fluorescent probe is preferably obtained by using quinoline as the fluorophore and IMC as the targeting group, with the two connected by carbon chains of different lengths to obtain a COX-2-targeted fluorescent probe.

3. The quinoline-based fluorescent probe containing indomethacin according to claim 1, characterized in that, The fluorescent probe has a Stokes shift greater than 160 nm and near-infrared emission greater than 630 nm.

4. A method for preparing the fluorescent probe containing indomethacinol group as described in claim 1, characterized in that, Includes the following steps: Indomethacin and EDCI were dissolved in an organic solvent and stirred at room temperature; then, N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine, as well as DIPEA and HOBt, were added, and the reaction mixture was stirred at room temperature; after the reaction was completed, distilled water was added to terminate the reaction to obtain the crude product, which was purified to obtain I-1-3. Compound I-1-3 was dissolved in an organic solvent, and then trifluoroacetic acid was slowly added dropwise. The reaction mixture was stirred at room temperature. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by column chromatography to obtain compound C-1-3. Cyanoacetic acid was dissolved in an organic solvent, followed by the addition of diethylamine and HATU. The mixture was stirred at room temperature. Then, compound C-1-3 was added, and the reaction was stirred at room temperature. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was washed to give product IC-1-3. Compound IC-1-3 and 6-(dimethylamino)quinoline-2-carboxaldehyde were dissolved in an organic solvent under an inert atmosphere. Piperidine was added, and the reaction mixture was stirred under heating. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography to obtain ICP-1-3.

5. The method for preparing a quinolinyl fluorescent probe containing N-alkylated tetrahydropyridine according to claim 3, characterized in that, The reaction formula is shown below: 。 6. The use of the indomethacin-containing quinoline fluorescent probe as described in claim 1 in the preparation of a cyclooxygenase-2 (COX-2) detection reagent or tool.

7. The use of the indomethacin-containing quinoline fluorescent probe of claim 1 in the preparation of reagents or tools for cyclooxygenase-2 (COX-2) bioimaging.

8. The use of the indomethacin-containing quinoline fluorescent probe of claim 1 in the preparation of reagents or tools for monitoring changes in the expression level of oxidase-2 (COX-2) in cells or organisms.

9. The use of the indomethacin-containing quinoline-based fluorescent probe of claim 1 in the preparation of reagents or tools for real-time neuroinflammatory imaging.

10. The use of the indomethacin-containing quinoline fluorescent probe of claim 1 in the preparation of cyclooxygenase-2 (COX-2) inhibitor screening reagents or tools.

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