Polychromatic fluorine magnetic resonance imaging probes based on metallo-organic cages

Abstract

This invention discloses a multicolor fluorine magnetic resonance imaging probe based on a metal-organic cage. First, a rhodium-based metal-organic cage is formed by the self-assembly of a fluorinated isophthalic acid ligand and a rhodium salt, covalently introducing the first characteristic fluorine signal. Then, leveraging the coordination ability of the rhodium nodes in the rhodium-based metal-organic cage, it coordinates with a fluorinated pyridine derivative, introducing a second characteristic fluorine signal. This results in a multicolor probe containing both fluorine signals within the metal-organic cage, ensuring that all signals exhibit completely consistent pharmacokinetic behavior in vivo. This achieves true multi-channel synchronous imaging, solving the fundamental problem of spatiotemporal asynchrony of mixed probe signals. The probe system of this invention features precise structure, strong design flexibility, high imaging sensitivity, and multicolor imaging capabilities, providing a new solution for existing fluorine probes with single-channel imaging or complex structures. It also provides a new tool for in vivo multi-target biological imaging and dynamic monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biomedical imaging technology and relates to a multicolor fluorine magnetic resonance imaging probe based on a metal-organic cage. Specifically, it relates to a molecular probe system that utilizes the controllable coordination of a metal-organic cage to precisely integrate multiple fluorine signals with different chemical shifts to achieve multi-channel fluorine magnetic resonance imaging. Background Technology

[0002] Magnetic Resonance Imaging (MRI), as a non-invasive, radiation-free core technology in medical imaging, has established an irreplaceable position in clinical diagnosis and life science research. Its imaging basis primarily relies on the abundance of water protons within living organisms (…). 1 The nuclear magnetic resonance signal emitted by H) is detected by... 1 The differences in relaxation properties of H in different tissue environments can generate high-resolution anatomical images with excellent soft tissue contrast. However, traditional 1 1H MRI has inherent limitations in functional imaging at the molecular and cellular levels. It primarily reflects the physical and chemical environment of water molecule distribution, lacking inherent specific signal contrast for particular biomolecules, metabolic pathways, or cellular activities. Therefore, developing targeted molecular probes is crucial for expanding the capabilities of MRI in order to visualize specific physiological or pathological processes, such as the tumor microenvironment, inflammatory activity, or enzyme activity.

[0003] Fluorine magnetic resonance imaging (FMRI) 19 fMRI is a promising molecular imaging technique that has emerged against this backdrop. 19 The F nucleus has 100% natural abundance and a gyrometry ratio of approximately 83% that of protons, thus possessing excellent magnetic resonance detection sensitivity. More importantly, the content of endogenous fluorine-containing substances in living organisms is extremely low, which means... 19 fMRI achieves true "dark-field" imaging because there is almost no background signal interference in living organisms. This characteristic makes... 19 fMRI can directly, quantitatively, and specifically detect and image introduced fluorine-containing probes, and the signal intensity has a good linear relationship with the probe concentration. 19 fMRI has shown great potential in fields such as drug metabolism tracking, cell tracing, and inflammation and tumor-targeted imaging, providing researchers with a powerful and quantifiable molecular window.

[0004] although 19 fMRI has significant advantages in principle, but its technological development bottlenecks are highly concentrated in the design and performance of the probe itself. An ideal 19 fMRI probes need to meet several stringent conditions: First, they must carry a sufficient number of equivalent [missing information].19 F atoms (typically ≥10 per molecule) are needed to generate a detectable signal intensity, overcoming relatively low sensitivity; secondly, the probe's... 19 The F nucleus should have a short longitudinal relaxation time (T1) to enable rapid signal acquisition; furthermore, for biomedical applications, the probe must have good biocompatibility, appropriate in vivo stability, and ideal pharmacokinetic behavior; finally, in order to expand imaging capabilities, the probe should ideally be able to respond to specific biological stimuli (such as pH, enzymes, metal ions) or be able to report multiple parameters simultaneously.

[0005] Currently reported 19 fMRI probe systems can be mainly categorized into the following types, but all of them focus on generating a single signal:

[0006] 1. Perfluorinated carbon nanoemulsions (PFC Nanoemulsions): This is the most classic and widely used type. 19 fMRI probes. They encapsulate thousands of perfluorinated carbon molecules (such as perfluoro-15-crown-5-ether, PFCE; perfluorooctyl bromide, PFOB) within phospholipid or surfactant-stabilized nanodroplets. Each droplet contains a massive amount of equivalent... 19 F atoms can produce extremely strong single... 19 F signals are well-suited for angiography, cell labeling (such as macrophage tracking), and tumor targeting studies. However, their limitations include: a) their particle size is typically large (>100 nm), which may affect tissue penetration and clearance; b) the signal originates from a large collection of molecules, making precise molecular-level design and functional modification difficult; and c) they primarily provide a single "switch" signal with limited functionality.

[0007] 2. Fluorine-labeled polymers and dendritic macromolecules: Fluorine-containing groups (such as trifluoromethyl) are covalently attached to the polymer backbone or the periphery of dendritic macromolecules through chemical synthesis. These probes allow for the control of the degree of polymerization or generation to regulate the number of fluorine atoms and are easily surface-functionalized (e.g., by attaching targeted ligands). However, the challenges include: a) potentially complex synthetic steps and non-uniform molecular weight distribution; b) 19 The relaxation time of the F nucleus may be prolonged due to restricted molecular motion, affecting imaging efficiency; c) Similarly, usually only one primary nucleus can be generated. 19 F signal peak.

[0008] 3. Probes based on small molecule-metal complexes: Design small molecules containing multiple trifluoromethyl or perfluoroalkyl chains, or utilize paramagnetic metal ions (such as GdO2) 3+ Fe 3+ It coordinates with fluorine-containing ligands and shortens the time by utilizing the paramagnetic effect. 19The T1 relaxation time of fluorine (F) is also relevant. These probes have well-defined structures, but each molecule carries a limited number of fluorine atoms, often requiring high concentrations to obtain sufficient signals, and their metabolism in vivo may be rapid. Paramagnetic probes are even more complex to design, requiring precise control of the metal-fluorine atom distance to optimize relaxation enhancement.

[0009] Clearly, the aforementioned monochromatic probe system has promoted... 19 While fMRI has been developed, it falls short in its ability to handle complex biological systems. Life processes are often the result of the synergistic effects of multiple factors and targets. For example, simultaneously monitoring different cell populations within a tumor (such as tumor cells and immune cells), tracking multiple biomarkers (such as the activity of two enzymes), or differentiating different pathological stages in an inflammatory region all require imaging tools capable of providing multiple independent pieces of information. Therefore, the development of multicolor or multichannel imaging technologies is crucial. 19 fMRI probes, that is, a probe that can simultaneously detect two or more resolvable signals. 19 F-signals have become a cutting-edge pursuit in this field.

[0010] In existing technologies, multicolor is achieved 19 Initial attempts at fMRI primarily involved physical hybridization strategies, namely, combining two or more known fMRI techniques with different physical characteristics. 19 Monochromatic probes with F chemical shifts (such as two different perfluorinated carbon droplets, or one droplet and one fluorinated small molecule) are simply co-injected into the organism. However, this method has fundamental drawbacks: a) Pharmacokinetic mismatch: Different probes have different sizes, surface properties, and hydrophilicity / hydrophobicity, leading to significant differences in their distribution, metabolism, and clearance rates in vivo. Their signals cannot be precisely correlated in time and space, and cannot reliably reflect two events in the same microenvironment or the same cell, rendering multicolor imaging meaningless for accurate correlation. b) Spectral resolution challenges: The chemical shift range of small molecule fluorinated compounds is limited (usually concentrated between -70 and -120 ppm). If the chemical shifts of the two selected fluorine sources are too close, their signal peaks are prone to overlap, broadening, or shift drift under the complex magnetic field inhomogeneity and metabolite interference in vivo, making it difficult to achieve clear, stable resolution and quantification. c) Complex dosage and toxicity control: The dosage and safety of each component need to be optimized and validated separately, increasing the complexity of the application.

[0011] Therefore, researchers have realized that ideal multicolor... 19 fMRI probes should be a single, structurally well-defined entity that integrates multiple reporter units, each contributing a unique and stable signal. 19F chemical shift signals. This design ensures that all signals exhibit completely consistent pharmacokinetic behavior, achieving true spatiotemporal synchronization, and allows for maximizing the chemical shift difference between signals through molecular design, avoiding mutual interference. However, how to controllably integrate multiple fluorine signals with different chemical environments on a stable nano or molecular support while maintaining their signal independence is a significant challenge in synthetic chemistry and materials science.

[0012] In recent years, metal-organic cages (MOCs) have emerged as a novel supramolecular platform, attracting the attention of molecular imaging probe designers. MOCs are discrete, three-dimensional structures with cavities, formed by the self-assembly of multiple organic ligands with metal ions or metal clusters via coordination bonds. Unlike continuous metal-organic frameworks, MOCs are discrete, soluble molecular entities. They possess the following unique advantages: 1. Definite structure: Their composition, size, and shape are atomically precise and can be precisely designed and predicted through the selection of ligands and metals. 2. Tunable cavities: The size of the internal cavities is adjustable, allowing for the encapsulation of guest molecules. 3. Rich surface chemistry: The outer surfaces are easily functionalized. 4. Dynamics and reversibility: The coordination bonds of some MOCs exhibit a degree of dynamic reversibility, allowing for the reversible binding and exchange of guest molecules.

[0013] In the field of biomedical imaging, molecular structures (MOCs) have shown potential as ideal probe platforms: their rigid framework allows for atomically precise spatial arrangement of paramagnetic metal nodes and high-density fluorine-containing reporter units; their well-defined molecular structure ensures batch-to-batch homogeneity and optimizable structure-performance relationships; and their ultra-small nanoscale size may lead to unique in vivo distribution behavior. More importantly, the modularity and programmability of MOCs make it possible to integrate multiple functional modules on the same molecular framework, providing an ideal carrier for constructing precisely tunable multicolor imaging probes.

[0014] In summary, the existing 19 fMRI probe technology, in its transition from "monochromatic" to practical "multicolor" imaging, has encountered bottlenecks such as inconsistencies in pharmacokinetics and difficulties in signal resolution. Metal-organic cages (MOCs) with precise structures and modifiability offer a novel platform approach to overcome these challenges. Summary of the Invention

[0015] In response to the existing 19Most fluorine magnetic resonance imaging (FMRI) probes are single-signal carriers, and there are technical problems such as pharmacokinetic mismatch and difficulty in reliably distinguishing signals when achieving multi-signal imaging by physically mixing multiple probes. This invention aims to provide a multicolor fluorine magnetic resonance imaging probe based on a metal-organic cage (MOC). The probe of this invention uses a rhodium-based MOC as its core structure, introducing multiple fluorine signals through both covalent and coordination bonds, thereby achieving multicolor fluorine magnetic resonance imaging. Specifically, this invention first forms a fluorine-containing rhodium-based MOC by coordinating a fluorinated isophthalic acid ligand with a rhodium salt, thereby introducing the first characteristic fluorine signal through covalent bonds. Then, a second fluorine signal is integrated into the fluorine-containing rhodium-based MOC through reversible and directional coordination between the rhodium metal nodes in the rhodium-based MOC and a fluorinated pyridine derivative. Furthermore, the fluorine signal introduced by coordination has strong adjustability and reversibility, and multiple fluorinated pyridine derivatives with different fluorine NMR chemical shifts can be designed to achieve on-demand design of multicolor fluorine probes.

[0016] The objective of this invention can be achieved through the following methods: In a first aspect, the present invention provides a method for preparing a multicolor fluorine magnetic resonance imaging probe based on a metal-organic cage, comprising the following steps: S1. The dihalogen compound is mixed and dissolved with potassium carbonate, and dimethyl 5-hydroxyisophthalate is added under inert conditions to produce product 1. S2. Mix and dissolve product 1 with potassium carbonate, and add a fluorinated phenol compound under an inert atmosphere to react and generate product 2. S3. Mix product 2 with sodium hydroxide solution to react and generate fluorinated isophthalic acid ligand; S4. Fluorinated isophthalic acid ligand, rhodium salt, and sodium carbonate are mixed and dissolved to react and generate a fluorinated rhodium-based metal organic cage. S5. Mix and dissolve the diol compound, the fluorinated carboxylic acid compound, EDCl and DMAP to react and generate product 3; S6. Mix product 3, pyridine carboxylic acid compounds, EDCl and DMAP and sonicate to dissolve them, reacting to generate fluorinated pyridine derivatives. S7. Mix and dissolve the fluorine-containing rhodium-based metal organic cage from step S4 and the fluorine-containing pyridine derivative from step S6 to obtain the multicolor fluorine magnetic resonance imaging probe.

[0017] As one embodiment of the present invention, in step S1, the dihalogen compound includes one or more of 1,8-dibromooctane, 1,2-di(2-chloroethoxy)ethane, and bis[2-(2-chloroethoxy)ethyl] ether.

[0018] In one embodiment of the present invention, in step S2, the fluorinated phenol compound includes 4-(trifluoromethoxy)phenol.

[0019] In one embodiment of the present invention, in step S4, the rhodium salt includes rhodium acetate.

[0020] In one embodiment of the present invention, in step S5, the diol compound includes one or more of 1,2-dodecanediol, diethylene glycol, triethylene glycol, 3,6,9,12,15,18-hexaoxaecosane-1,20-diol, and 3,6,9,12,15,18,21,24-octaoxaecosane-1,26-diol; the fluorinated carboxylic acid compound includes one or more of 3,5-difluorophenylacetic acid, 4-(difluoromethoxy)benzoic acid, and 3,5-bis(trifluoromethylbenzoic acid).

[0021] As one embodiment of the present invention, in step S6, the pyridine carboxylic acid compound includes one or more of 3,5-pyridinedicarboxylic acid and 4-pyridinecarboxylic acid.

[0022] In one embodiment of the present invention, in steps S1, S2, S3 and S4, the reaction temperature is 70-110°C and the time is 3-48 h.

[0023] In one embodiment of the present invention, in steps S5 and S6, the reaction temperature is 18-35°C and the time is 15-25h.

[0024] In one embodiment of the present invention, the two types of fluorine in the multicolor fluorine magnetic resonance imaging probe have a nuclear magnetic resonance chemical shift difference of more than 5 ppm. This provides sufficient signal differentiation, and the synthesis of these fluorine-containing compounds employs relatively mature chemical reactions, followed by purification and structural confirmation.

[0025] Secondly, the present invention provides a multicolor fluorine magnetic resonance imaging probe prepared by the method described above.

[0026] Thirdly, the present invention provides an application of the multicolor fluorine magnetic resonance imaging probe in magnetic resonance imaging.

[0027] As one embodiment of the present invention, the application is: applying a multicolor fluorine magnetic resonance imaging probe to a sample or object to be tested, and performing fluorine magnetic resonance imaging scanning. The present invention provides this multicolor... 19 Application of the F-probe in magnetic resonance imaging. The probe can be prepared into solutions of different concentrations for use in... 19 F-type magnetic resonance imaging technology can simultaneously acquire signal channel images corresponding to different chemical shifts, with good imaging results, clear and uniform images, and a linear relationship between signal intensity and concentration.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. In this invention, a fluorinated isophthalic acid ligand and a rhodium salt are self-assembled to form a rhodium-based metal-organic cage, thereby covalently introducing the first characteristic fluorine signal. Then, by utilizing the coordination ability of the rhodium nodes in the rhodium-based metal-organic cage, it is coordinated with a fluorinated pyridine derivative to coordinate and introduce the second characteristic fluorine signal, thus obtaining a metal-organic cage multicolor probe containing two fluorine signals, thereby realizing multicolor fluorine magnetic resonance imaging.

[0029] 2. This invention utilizes supramolecular assembly to integrate multicolor... 19 The F signal is integrated into a single molecular entity, ensuring that all signals have completely consistent pharmacokinetic behavior in vivo, realizing true multi-channel synchronous imaging, and solving the fundamental problem of spatiotemporal asynchrony of mixed probe signals.

[0030] 3. The present invention adopts a modular design, which can design fluorinated isophthalic acid ligands and fluorinated pyridine derivatives with corresponding fluorine chemical shifts as needed. The two can be flexibly combined to achieve high-resolution signal separation.

[0031] 4. The probe system of this invention has the characteristics of precise structure, strong design, high imaging sensitivity, and multicolor imaging, providing a new solution for existing fluorine probes with single-channel imaging or complex structures, and also providing a new tool for multi-target bioimaging and dynamic monitoring at the in vivo level; at the same time, the metal-organic cage platform has the potential for further functionalization, laying the foundation for the development of new diagnostic and therapeutic agents that integrate multicolor imaging, targeting, and therapy. Attached Figure Description

[0032] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is the process for constructing the multicolor probe of the present invention; Figure 2 The fluorinated isophthalic acid ligand LF and the fluorinated rhodium-based metal organic cage in Example 2 1 H NMR spectrum; Figure 3 The fluorinated isophthalic acid ligand LF and the fluorinated rhodium-based metal organic cage in Example 2 19 F NMR spectrum; Figure 4 The infrared spectrum of the fluorinated isophthalic acid ligand LF and the fluorinated rhodium-based metal-organic cage in Example 2; Figure 5 This is a gel permeation chromatogram of the fluorinated isophthalic acid ligand LF and the fluorinated rhodium-based metal organic cage in Example 2; Figure 6 The DLS spectrum of the fluorine-containing rhodium-based metal-organic cage in Example 2; Figure 7The AFM spectrum of the fluorine-containing rhodium-based metal-organic cage in Example 2; Figure 8 For Probe 1 and Probe 2 1 H MRI and multicolor 19 F MRI results. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0034] Example 1 This embodiment provides a method for preparing fluorinated isophthalic acid ligands, the steps of which are as follows: S1. 1,8-Dibromooctane (1.00 g, 3.68 mmol) and potassium carbonate (0.56 g, 4.05 mmol) were dispersed in 12 mL of acetonitrile. Dimethyl 5-hydroxyisophthalate (0.77 g, 3.68 mmol) was dissolved in a mixture of 3 mL DMF and 6 mL acetonitrile, and added dropwise to the above solution under nitrogen atmosphere. The mixture was refluxed at 100 °C and stirred for 12 h. After cooling, the reaction solution was concentrated and then extracted three times with ethyl acetate (50 mL) and saturated sodium bicarbonate solution. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography.

[0035] S2. The product obtained in step S1, 5-((8-bromooctyl)oxy)dimethyl isophthalate (0.62 g, 1.55 mmol), and potassium carbonate (0.24 g, 1.70 mmol) were dispersed in 6 mL of acetonitrile. 4-(trifluoromethoxy)phenol (0.28 g, 1.55 mmol) was dissolved in a mixture of 1.5 mL DMF and 3 mL acetonitrile, and added dropwise to the above solution under nitrogen atmosphere. The mixture was refluxed at 100 °C and stirred for 12 h. After cooling, the reaction solution was concentrated and then extracted three times with ethyl acetate (25 mL) and saturated sodium bicarbonate solution. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography.

[0036] S3. Dissolve the product obtained in step S2, 5-((8-(4-(trifluoromethoxy)phenoxy)octyl)oxy)dimethyl isophthalate (0.50 g, 1.00 mmol), in 10 mL of tetrahydrofuran, and dissolve sodium hydroxide (0.12 g, 3.00 mmol) in water. Mix the two solutions, reflux at 80 °C, and stir for 3 h. After the reaction is complete, adjust the pH to 1 with dilute hydrochloric acid, concentrate the reaction solution, and then extract with dichloromethane. Collect the organic phase, dry it with anhydrous MgSO4, filter, concentrate, and vacuum dry to obtain a light yellow solid, which is the fluorinated isophthalic acid ligand LF.

[0037] Example 2 This embodiment provides a method for preparing a fluorine-containing rhodium-based metal organic cage, referring to the literature "Phase Transfer of Rhodium(II)-Based Metal". The method described in "Organic Polyhedra Bearing Coordinatively Bound Cargo Enables Molecular Separation. J. Am. Chem. Soc. 2019, 141, 18349-18355" is as follows: S4. The product obtained in step S3 (100 mg, 0.21 mmol), Rh2(OAc)4·2MeOH (50 mg, 0.11 mmol), and sodium carbonate (22 mg) were dispersed in 3 mL of DMA and sonicated for 5 minutes to mix thoroughly. The mixture was heated to 100 °C and kept at that temperature for 48 h. After the reaction was completed, the mixture was cooled to room temperature, centrifuged, and the supernatant was added dropwise to 50 mL of methanol to precipitate. The precipitate was then centrifuged, the solid was collected, washed with methanol, and dried to obtain a green solid containing a fluorinated rhodium-based metal organic cage.

[0038] S5. The obtained fluorinated isophthalic acid ligand LF is reacted with a fluorinated rhodium-based metal-organic cage. 1 H NMR ( Figure 2 ), 19 FNMR ( Figure 3 ), infrared spectrum ( Figure 4 Gel permeation chromatography (GPC) Figure 5 The structure was tested and verified.

[0039] 1 1H NMR: Compared to the ligand, the proton signal of the fluorine-containing rhodium-based metal-organic cage is broadened and shifted to a lower field overall (e.g., aromatic hydrogen shifts from 8.06 / 7.63 ppm to 8.28 / 7.47 ppm), confirming the coordination and cage structure formation.

[0040] 19F NMR: The fluorine-containing rhodium-based metal-organic cage shows only a single sharp peak at -58.4 ppm, proving that the chemical environment of the fluorine atom is completely equivalent and the structure is highly symmetrical; the signal broadening and shift further corroborate the influence of the metal.

[0041] Infrared spectroscopy: After coordination, the C=O peak of the carboxyl group decreased from 1595.6 cm⁻¹. -1 It split and shifted to 1574.3 cm. -1 and 1365.5 cm -1 This conforms to the characteristics of carboxylate bridging / chelation coordination.

[0042] Gel permeation chromatography: The elution time of the fluorine-rhodium-based metal organic cage was significantly reduced (14.22 min vs. 18.01 min), the apparent molecular weight (10402 g / mol) was basically consistent with the theoretical value, and the polydispersity index (1.13) was very low, indicating that the structure was uniform and stable.

[0043] S6. Particle size analysis of fluorine-containing rhodium-based metal organic cages ( Figure 6 ) and atomic force microscope ( Figure 7 )test.

[0044] Dynamic light scattering: The hydrodynamic diameter in tetrahydrofuran is about 5.6 nm, with a narrow particle size distribution (PDI = 0.23), indicating good dispersibility and uniform size.

[0045] Atomic force microscopy: The morphology shows independently dispersed nanoparticles with no obvious aggregation; the average height is about 3.6 nm, which is consistent with the theoretical scale of its cubic octahedral cage.

[0046] Example 3 This embodiment provides a method for preparing the fluoropyridine derivative Py1, the steps of which are as follows: S7. 1,2-Dodecanediol (1.00 g, 4.94 mmol), 3,5-difluorophenylacetic acid (0.85 g, 4.94 mmol), EDCl (1.04 g, 5.43 mmol), and DMAP (0.12 g, 0.99 mmol) were added to 10 mL of DMF and sonicated for 5 minutes to dissolve. The reaction was allowed to proceed at room temperature for 18 h. After the reaction was confirmed to be complete by TLC, 20 mL of water was added to quench the reaction, followed by extraction with 100 mL of ethyl acetate. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography.

[0047] S8. The product obtained in step S7, 150 mg (0.42 mmol) of 2-(3,5-difluorophenyl)acetic acid 12-hydroxydodecyl ester, 3,5-pyridinedicarboxylic acid (32 mg, 0.19 mmol), EDCl (81 mg, 0.42 mmol), and DMAP (9 mg, 0.08 mmol), were added to 3 mL of DMF and sonicated for 5 minutes to dissolve. The reaction was allowed to proceed at room temperature for 18 h. After the reaction was confirmed to be complete by TLC, 6 mL of water was added to quench the reaction, followed by extraction with 30 mL of ethyl acetate. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography. The NMR chemical shift of Py1 was approximately -110.0 ppm.

[0048] Example 4 This embodiment provides a method for preparing the fluoropyridine derivative Py2, the steps of which are as follows: S9. 1,2-Dodecanediol (320 mg, 1.58 mmol), 4-(difluoromethoxy)benzoic acid (300 mg, 1.58 mmol), EDCl (330 mg, 1.74 mmol), and DMAP (40 mg, 0.32 mmol) were added to 3 mL of DMF and sonicated for 3 minutes to dissolve. The reaction was allowed to proceed at room temperature for 18 h. After the reaction was confirmed to be complete by TLC, 6 mL of water was added to quench the reaction, followed by extraction with 30 mL of ethyl acetate. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography.

[0049] S10. The product obtained in step S9, 147 mg (0.39 mmol) of 12-hydroxydodecyl 4-(difluoromethoxy)benzoic acid, 3,5-pyridinedicarboxylic acid (30 mg, 0.18 mmol), EDCl (76 mg, 0.39 mmol), and DMAP (9 mg, 0.07 mmol), were added to 3 mL of DMF and sonicated for 5 minutes to dissolve. The reaction was allowed to proceed at room temperature for 18 h. After the reaction was confirmed to be complete by TLC, 6 mL of water was added to quench the reaction, followed by extraction with 30 mL of ethyl acetate. The organic phase was collected, dried over anhydrous MgSO4, filtered, and concentrated. Finally, it was purified by silica gel column chromatography. The NMR chemical shift of Py2 was approximately -81.7 ppm.

[0050] Example 5 This embodiment provides a method for preparing the multicolor probe Probe 1, refer to... Figure 1 The path and steps are as follows: S11. The fluorine-containing rhodium-based metal organic cage (30 mg, 0.002 mmol) obtained in step S4 and the product Py1 (46 mg, 0.05 mmol) obtained in step S8 were completely dissolved in 2 mL of dichloromethane and stirred for 5 minutes. The solvent was then removed by evaporation under vacuum. The resulting solid was washed with methanol and dried under vacuum to obtain the multicolor probe Probe 1.

[0051] Example 6 This embodiment provides a method for preparing the multicolor probe Probe 2, refer to... Figure 1 The path and steps are as follows: S12. The fluorine-containing rhodium-based metal organic cage (30 mg, 0.002 mmol) obtained in step S4 and the product Py2 (48 mg, 0.05 mmol) obtained in step S10 were completely dissolved in 2 mL of dichloromethane and stirred for 5 minutes. The solvent was then removed by evaporation under vacuum. The resulting solid was washed with methanol and dried under vacuum to obtain the multicolor probe Probe 2.

[0052] S13. Perform relaxation time tests on Probe1 and Probe2 (results are shown in Table 1 and Table 2) to verify their relaxation performance.

[0053] ideal 19 f MRI probes need to possess both a short T1 (allowing for rapid repeated acquisitions and improving the signal-to-noise ratio) and a long T2 (slow signal attenuation and high intensity per acquisition). T1 was determined using inversion recovery sequences, and T2 was determined using Carr-Purcell-Meiboom-Gill sequences. In Tables 1 and 2, the T2 / T1 ratios for probes 1 and 2 are greater than 0.4; this range is generally considered beneficial for achieving high 19f MRI imaging efficiency.

[0054] Table 1

[0055] Table 2

[0056] Due to the high efficiency, stability, and universality of pyridine coordination with rhodium metal nodes, this single-molecule-level probe has a precise and highly adjustable structure, capable of outputting two or more independently detectable chemical shifts. 19 F nuclear magnetic resonance signal. To better demonstrate the feasibility of the design, [the following was synthesized]. 19 The chemical shift of F is approximately -58.4 ppm for fluorinated isophthalic acid ligands; [The following is a separate, unrelated sentence:] A synthesis of fluorinated isophthalic acid ligands was performed. 19Two fluorinated pyridine derivatives with chemical shifts of approximately -110.0 ppm and -81.7 ppm respectively constituted multicolor probes Probe 1 and Probe 2 with different signal combinations.

[0057] Performance test examples Imaging effects of the multicolor fluorine magnetic resonance imaging probes Probe 1 and Probe 2 prepared in Examples 5 and 6 ( Figure 8 (To be tested)

[0058] S1, Probe 1 19 fMRI performance testing.

[0059] Probe 1 was prepared into solutions of different concentrations (2 M, 1 M, 0.5 M, 0.25 M, 0.125 M) and placed in NMR tubes for imaging on a small animal magnetic resonance imaging system. 19 MRI scans showed fluoride signals at -58.4 ppm (marked in red) and -110.0 ppm (marked in blue).

[0060] S2, Probe 2 19 fMRI performance testing.

[0061] Probe 2 was prepared into solutions of different concentrations (2 M, 1 M, 0.5 M, 0.25 M, 0.125 M) and placed in NMR tubes for imaging on a small animal magnetic resonance imaging system. 19 MRI scans showed fluoride signals at -58.4 ppm (marked in red) and -81.7 ppm (marked in green).

[0062] The results are as follows Figure 8 As shown, within an extremely short acquisition time of only 3.2 minutes, almost all concentration points of both probes could be clearly imaged, and the image signal-to-noise ratio showed a regular increase with increasing probe concentration. This directly confirms that both probes possess efficient and sensitive multicolor imaging capabilities in solution systems. 19 fMRI capability.

[0063] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for fabricating a multicolor fluorine magnetic resonance imaging probe based on a metal-organic cage, characterized in that, Includes the following steps: S1. The dihalogen compound is mixed and dissolved with potassium carbonate, and dimethyl 5-hydroxyisophthalate is added under inert conditions to produce product 1. S2. Mix and dissolve product 1 with potassium carbonate, and add a fluorinated phenol compound under an inert atmosphere to react and generate product 2. S3. Mix product 2 with sodium hydroxide solution to react and generate fluorinated isophthalic acid ligand; S4. Fluorinated isophthalic acid ligand, rhodium salt, and sodium carbonate are mixed and dissolved to react and generate a fluorinated rhodium-based metal organic cage. S5. Mix and dissolve the diol compound, the fluorinated carboxylic acid compound, EDCl and DMAP to react and generate product 3; S6. Mix product 3, pyridine carboxylic acid compounds, EDCl and DMAP and sonicate to dissolve them, reacting to generate fluorinated pyridine derivatives. S7. Mix and dissolve the fluorine-containing rhodium-based metal organic cage from step S4 and the fluorine-containing pyridine derivative from step S6 to obtain the multicolor fluorine magnetic resonance imaging probe.

2. The preparation method according to claim 1, characterized in that, In step S1, the dihalogen compound includes one or more of 1,8-dibromooctane, 1,2-di(2-chloroethoxy)ethane, and bis[2-(2-chloroethoxy)ethyl] ether.

3. The preparation method according to claim 1, characterized in that, In step S2, the fluorinated phenol compound includes 4-(trifluoromethoxy)phenol.

4. The preparation method according to claim 1, characterized in that, In step S4, the rhodium salt includes rhodium acetate.

5. The preparation method according to claim 1, characterized in that, In step S5, the diol compound includes one or more of 1,2-dodecanediol, diethylene glycol, triethylene glycol, 3,6,9,12,15,18-hexaoxaecosane-1,20-diol, and 3,6,9,12,15,18,21,24-octaoxaecosane-1,26-diol; the fluorinated carboxylic acid compound includes one or more of 3,5-difluorophenylacetic acid, 4-(difluoromethoxy)benzoic acid, and 3,5-bis(trifluoromethyl)benzoic acid.

6. The preparation method according to claim 1, characterized in that, In step S6, the pyridine carboxylic acid compound includes one or more of 3,5-pyridinedicarboxylic acid and 4-pyridinecarboxylic acid.

7. The preparation method according to claim 1, characterized in that, The two types of fluorine in the multicolor fluorine magnetic resonance imaging probe have a nuclear magnetic resonance chemical shift difference of more than 5 ppm.

8. A multicolor fluorine magnetic resonance imaging probe obtained by the preparation method according to any one of claims 1-7.

9. The application of the multicolor fluorine magnetic resonance imaging probe as described in claim 8 in magnetic resonance imaging.

10. The application according to claim 9, characterized in that, The application is as follows: applying a multicolor fluorine magnetic resonance imaging probe to the sample or object to be tested to perform fluorine magnetic resonance imaging scanning.

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