A pretargeted probe combination for in vivo labile iron pool imaging and preparation method and application thereof

Abstract

This invention discloses a pre-targeting probe assembly for in vivo unstable iron pool imaging, its preparation method, and its application. The pre-targeting probe assembly includes Fe... 2+ Response paramagnetic probe GdEpTz and fluorescent reporter probe 2E-Tco-Hcy, Fe 2+ Response paramagnetic probe GdEpTz with Fe 2+ Following interaction with endogenous proteins, free radicals are generated and covalent cross-linking occurs, significantly increasing the longitudinal relaxation rate (r1) by approximately 104.1%. Simultaneously, tetrazine-based Tz is efficiently enriched in the lesion region. The enriched Tz captures the fluorescent signal reporter probe 2E-Tco-Hcy via a highly efficient IEDDA bioorthogonal reaction, triggering a 155.5-fold significant enhancement of the fluorescence signal. This invention introduces a bioorthogonal pre-targeting strategy based on the reverse electron demand Diels-Alder (IEDDA) reaction, aiming to achieve Fe in deep tissues through the organic integration of "target-induced activation" and "bioorthogonal chemistry." 2+ Dynamically changing, highly selective, and high-contrast synchronous visual monitoring.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging technology, specifically relating to a pre-targeting probe assembly for imaging unstable iron pools in vivo, its preparation method, and its application. Background Technology

[0002] Iron, as an indispensable metallic trace element in life activities, possesses unique Fe... 2+ / Fe 3+ Redox potential plays a central role in regulating physiological processes such as gene transcription, DNA synthesis, oxygen transport, and electron transfer. However, this inherent redox activity is a double-edged sword; excessive Fe... 2+ It catalyzes the generation of highly reactive hydroxyl radicals via the Fenton reaction, triggering systemic oxidative stress and leading to apoptosis and tissue damage. The liver, as the body's main iron reserve and metabolic center, often suffers severe oxidative damage under pathological conditions such as hemolysis due to the accumulation of large amounts of heme released from hemoglobin degradation. Therefore, it is crucial to construct a system capable of real-time, quantitative monitoring of iron in vivo. 2+ Dynamic evolution analysis methods are of great scientific value for elucidating the molecular nature of iron balance regulation mechanisms and related pathological processes.

[0003] Although Fe 2+ While crucial for their physiological function, achieving high-fidelity monitoring in complex in vivo environments remains a technological challenge. Traditional fluorescence quenching probes are susceptible to interference from paramagnetic metals, leading to false negatives. While fluorescence enhancement probes based on chemical bond breaking excitation improve specificity, they are limited by finite light penetration depth and autofluorescence background in deep tissues. Multimodal imaging (such as FL / MR), combining the high sensitivity of optics with the high spatial resolution of magnetic resonance, often results in significant asynchrony and mismatch in the spatiotemporal distribution of imaging signals due to fundamental differences in their physical enhancement principles, detection limits, and signal activation dynamics, making it difficult to capture transient molecular events. Summary of the Invention

[0004] Regarding the iron ion (Fe) in existing technologies 2+ To address the technical bottlenecks of detection probes, such as low in vivo imaging sensitivity, poor penetration into deep tissues, and asynchronous evolution of multimodal signals, this invention provides a pre-targeting probe combination for in vivo imaging of unstable iron pools, along with its preparation method and application. This invention introduces a bioorthogonal pre-targeting strategy based on the inverse electron demand Diels-Alder (IEDDA) reaction, aiming to achieve Fe in deep tissues through the organic integration of "target-induced activation" and "bioorthogonal chemistry." 2+ Dynamically changing, highly selective, and high-contrast synchronous visual monitoring.

[0005] This invention utilizes a preferred probe to establish a lesion-residing scaffold, and then overcomes the drawback of inconsistent signal evolution between different modalities by optimizing the reaction dynamics of the secondary imaging module, thereby achieving synchronous and synergistic enhancement of MR / FL signals and high-fidelity imaging in deep tissues.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows: A pre-targeting probe assembly for in vivo imaging of unstable iron pools, the pre-targeting probe assembly comprising Fe 2+ Response paramagnetic probe GdEpTz and fluorescent signal reporter probe 2E-Tco-Hcy, Fe 2+ Response paramagnetic probe GdEpTz with Fe 2+ Following interaction with endogenous proteins, free radicals are generated and covalent cross-linking occurs, significantly increasing the longitudinal relaxation rate (r1) by approximately 104.1%. Simultaneously, the tetrazine group Tz is efficiently enriched in the lesion region. The enriched Tz captures the fluorescent signal reporter probe 2E-Tco-Hcy via a highly efficient IEDDA bioorthogonal reaction, triggering a 155.5-fold significant enhancement of the fluorescence signal. The Fe 2+ The structural formula of the responsive paramagnetic probe GdEpTz is as follows: ; The structural formula of the fluorescent signal reporting probe 2E-Tco-Hcy is as follows: .

[0007] A Fe 2+ The synthesis method of the responsive paramagnetic probe GdEpTz includes the following steps: S1. Under inert gas protection and in the presence of a base, 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane reacts with 2-(2-bromoacetamido)ethylcarbamate benzyl ester in a substitution reaction to produce compound 1, as shown in the following reaction formula: ; S2. In the presence of a catalyst, compound 1 undergoes a hydrogenation reduction reaction to produce compound 2, as shown in the following reaction equation: ; S3. In the presence of a condensing reagent and a base, Fmoc-Lys(Cbz)-OH reacts with compound 2 via an amidation reaction to generate compound 3, as shown in the following reaction equation: ; S4. In the presence of a catalyst, compound 3 undergoes a hydrogenation reduction reaction to produce compound 4, as shown in the following reaction equation: ; S5, 5-carboxy-2-adamantanone, undergoes esterification under the catalytic system generated in situ from an acyl chloride reagent and methanol, to produce compound 5, as shown in the following reaction formula: ; S6. Under alkaline conditions, compound 5 undergoes an oxime reaction with methoxyamine hydrochloride to generate compound 6, as shown in the following reaction equation: ; S7 and compound 6 undergo a cycloaddition reaction with cyclohexanone under the action of ozone to generate compound 7, as shown in the following reaction formula: ; S8. In the presence of an alkaline reagent, compound 7 undergoes a hydrolysis reaction to produce compound Ep-COOH, as shown in the following reaction equation: ; S9. In the presence of a condensing agent and a base, compound Ep-COOH undergoes an amidation reaction with compound 4 to generate compound 8, as shown in the following reaction equation: ; S10. In the presence of an alkaline reagent, compound 8 first undergoes hydrolysis, followed by chelation with a gadolinium salt to form compound Gd-Ep-NH2, as shown in the following reaction equation: ; S11. Under inert gas protection and in the presence of hydrazine hydrate and a catalyst, 4-cyanophenylacetic acid undergoes a condensation reaction with acetonitrile, followed by oxidation with sodium nitrite to produce compound Tz-COOH, as shown in the following reaction formula: ; S12. Under inert gas protection and in the presence of a condensing reagent, Tz-COOH undergoes an esterification reaction with N-hydroxysuccinimide to generate the compound Tz-NHS, as shown in the following reaction formula: ; S13. Under alkaline conditions, compound Gd-Ep-NH2 and compound Tz-NHS undergo an esterification reaction to generate the Fe... 2+ The responsive paramagnetic probe GdEpTz has the following reaction formula: .

[0008] Further, in step S1, the base is potassium carbonate, the reaction solvent is anhydrous acetonitrile, the temperature of the substitution reaction is 20~35 °C, the reaction time is 8~16 h, and the molar ratio of 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane, 2-(2-bromoacetamido)ethylcarbamate and the base is 1:1.2:2~1:2:6; In step S2, the catalyst is palladium on carbon, the reaction solvent is methanol, the reduction reaction temperature is 20~35℃, the reaction time is 12~24 h, the mass of the catalyst added is 10~15% of the mass of compound 1, and the pressure of the system after adding hydrogen is 1~1.2 atm. In step S3, the condensing agent is HATU, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is 20~40 ℃, the reaction time is 1~4 h, and the molar ratio of compound 2, Fmoc-Lys(Cbz)-OH, condensing agent and base is 1:1:1:1~1:2:2:2.

[0009] Furthermore, in step S4, the catalyst is a palladium-on-carbon catalyst, the reaction solvent is methanol, the hydrogenation reduction reaction temperature is 20~35 ℃, the reaction time is 12~24 h, the mass of the catalyst added is 10~15% of the mass of compound 3, and the pressure of the system after adding hydrogen is 1~1.2 atm; In step S5, the acyl chloride reagent is thionyl chloride, the esterification reaction temperature is 0~30 ℃, the reaction time is 6~18 h, the reaction solvent is methanol, and the molar ratio of 5-carboxy-2-adamantaneone to the acyl chloride reagent is 1:1~1:4. In step S6, both the base and the reaction solvent are pyridine, the oxime reaction temperature is 20~45 ℃, the reaction time is 2~6 h, and the molar ratio of compound 5, methoxyamine hydrochloride and base is 1:1:1~1:3:3. In step S7, the reaction solvent is anhydrous hexane, the cycloaddition reaction temperature is -20~0 ℃, the reaction time is 1~4 h, the ozone flow rate is 1.5 L / min~4 L / min, and the molar ratio of compound 6 to cyclohexanone is 1:2~1:4.

[0010] Furthermore, in step S8, the alkaline reagent is lithium hydroxide, the reaction solvent is a mixed solvent of tetrahydrofuran, methanol and water, the hydrolysis reaction temperature is 45~80 ℃, and the reaction time is 4~12 h; In step S9, the condensing agent is HATU, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is 20~40 ℃, the reaction time is 1~5 h, and the molar ratio of compound 4, compound Ep-COOH, condensing agent and base is 1:1:1:1~1:2:2:2. In step S10, the alkaline reagent is lithium hydroxide, the reaction solvent is a mixed solvent of tetrahydrofuran, methanol and water, the hydrolysis reaction temperature is 45~80 ℃, the reaction time is 4~12 h, and the gadolinium salt is selected from gadolinium trichloride.

[0011] Further, in step S11, the catalyst is 3-mercaptopropionic acid, acetonitrile is used as the solvent, the condensation reaction temperature is 20~35 ℃, the reaction time is 8~16 h, the oxidation reaction temperature is -20~0 ℃, the reaction time is 1~3 h, and the molar ratio of 4-cyanophenylacetic acid, acetonitrile, catalyst, hydrazine hydrate and sodium nitrite is 1:10:1:2:10~1:50:10:30; In step S12, the condensing agent is DIC, the reaction solvent is anhydrous chloroform, the esterification reaction temperature is -25~0℃, the reaction time is 1~4 h, and the molar ratio of Tz-COOH, N-hydroxysuccinimide and condensing agent is 1:1:1~1:3:3. In step S13, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is room temperature, the reaction time is 1~4 h, and the molar ratio of Gd-Ep-NH2, Tz-NHS and base is 1:1:1~1:5:5.

[0012] A method for synthesizing a fluorescent signal reporter probe 2E-Tco-Hcy includes the following steps: P1. Under inert gas protection and in the presence of a base, compound 9 undergoes a substitution reaction with resorcinol to form compound Hcy-OH, as shown in the following reaction equation: ; P2. Under inert gas protection and in the presence of a basic reagent, Hcy-OH, N-methyl-2-(N-methyl-Boc-amino)ethylamine, and a carbonylating agent undergo a carbamate reaction to generate compound 10, as shown in the following reaction formula: ; P3. In the presence of an acidic reagent, compound 10 undergoes a deprotection reaction to form compound 11, as shown in the following reaction equation: ; P4. In the presence of an alkaline reagent, compound 11 undergoes an amidation reaction with its non-corresponding isomer (E)-cyclooctene-succinimide carbonate to generate the fluorescent signal reporter probe 2E-Tco-Hcy, as shown in the following reaction formula: .

[0013] Furthermore, in step P1, the base is potassium carbonate, the reaction solvent is anhydrous N,N-dimethylformamide, the temperature of the substitution reaction is 20~80 °C, the reaction time is 8~16 h, and the molar ratio of compound 9, resorcinol and base is 1:1:1~1:3:5. In step P2, the basic reagent is a mixture of 4-dimethylaminopyridine and triethylamine, the reaction solvent is anhydrous dichloromethane, the carbonylating reagent is triphosgene, the temperature of the carbamate esterification reaction is 20~35 °C, the reaction time is 4~16 h, and the molar ratio of Hcy-OH, N-methyl-2-(N-methyl-Boc-amino)ethylamine, carbonylating reagent and basic reagent is 1:1:1:1~1:4:4:8; In step P3, the acidic reagent is trifluoroacetic acid, the reaction solvent is dichloromethane, the deprotection reaction temperature is -20~0 ℃, the reaction time is 0.25~0.5 h, and a mixed solvent with a TFA:DCM volume ratio of 1:2~2:1 is used as the reaction solvent. In step P4, the basic reagent is triethylamine, the reaction solvent is anhydrous dichloromethane, the amidation reaction temperature is 20~40 ℃, the reaction time is 6~16 h, and the molar ratio of compound 11, the non-corresponding isomer (E)-cyclooctene-succinimide carbonate, and the basic reagent is 1:1:3~1:5:10.

[0014] Application of a pre-targeted probe assembly for in vivo imaging of unstable iron pools in the preparation of a dual-modal contrast agent for magnetic resonance and fluorescence imaging.

[0015] Furthermore, when the pre-targeted probe combination is used as a dual-modal contrast agent, Fe is first injected into the living organism. 2+ The responsive paramagnetic probe GdEpTz solution was injected, followed by the fluorescent signal reporter probe 2E-Tco-Hcy solution after 1 hour.

[0016] This pre-targeted probe combination can serve as a high-performance magnetic resonance / fluorescence dual-modal contrast agent, enabling precise monitoring of exogenous or endogenous Fe at the in vivo level (especially in complex pathological models such as hemolytic acute liver injury). 2+ The dynamic evolution of metal ions in deep tissues. This technology breaks through the limitations of traditional detection methods, enabling high-contrast, spatiotemporally synchronized visualization analysis of metal ions, providing a powerful molecular imaging tool for the early diagnosis and pathological research of related diseases.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The pre-targeted probe combination of the present invention has high selectivity and sensitivity. Experimental results show that the paramagnetic probe GdEpTz is effective against Fe. 2+ It exhibits excellent selectivity; in the presence of proteins (HSA), Fe... 2+ Activation can reduce r1 from 7.1 mM -1 s -1 Increased to 15.13 mM -1 s -1 (Increase of 104.1%), while mediating up to 155.5 times amplification of fluorescence signal.

[0018] 2. The pre-targeted probe combination of the present invention has excellent reaction kinetics, and the system exhibits an extremely fast cascade reaction rate with a fluorescence half-activation time of only 17.1 min, which can more accurately capture transient biological events.

[0019] 3. This invention achieves spatiotemporal decoupling of molecular recognition and signal generation through a pre-targeting strategy, solving the spatiotemporal mismatch problem of multimodal signals. Utilizing a protein cross-linking scaffold constructed in situ by the initial probe overcomes the asynchronous signal evolution defect in traditional bimodal imaging. In a hemolysis-induced acute liver injury model, it achieved a 60% improvement in T1 MR contrast and a 5.0-fold improvement in fluorescence signal-to-noise ratio in the mouse liver region, ensuring consistency in spatial distribution and functional response of bimodal images.

[0020] 4. The pre-targeting probe combination of the present invention has universality and innovation. The present invention breaks through the limitation of traditional pre-targeting strategies, which are mainly limited to biological macromolecules. For the first time, it realizes the in-situ pre-targeting probe combination for unstable metal ions in vivo and achieves synergistic enhancement imaging of magnetic resonance and fluorescence dual signals. It provides a brand-new methodological framework for the design of precision medical probes and the monitoring of dynamic fluctuations of metal ions. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the imaging principle of a pre-targeted probe assembly used for imaging unstable iron pools in vivo.

[0022] Figure 2 Fe prepared in Example 1 2+ Comparison of longitudinal relaxation rate r1 of responsive paramagnetic probe GdEpTz in different systems.

[0023] Figure 3 Fe prepared in Example 1 2+ A comparison of the recognition specificity of the responsive paramagnetic probe GdEpTz for different metal ions.

[0024] Figure 4The UV-Vis absorption spectrum of GdEpTz prepared in Example 1 after undergoing a bioorthogonal reaction with 2E-Tco-Hcy is shown.

[0025] Figure 5 The fluorescence emission spectrum of GdEpTz prepared in Example 1 after undergoing a bioorthogonal reaction with 2E-Tco-Hcy is shown.

[0026] Figure 6 The diagram shows the kinetic analysis of the bioorthogonal reaction between GdEpTz prepared in Example 1 and 2E-Tco-Hcy.

[0027] Figure 7 The image shows T1 MR imaging results of the liver region in normal mice and mice with hemolysis-induced acute liver injury using GdEpTz prepared in Example 1. Figure 7 (a) shows the change of MRI images with the duration of GdEpTz. Figure 7 (b) is a graph showing the change in MRI signal intensity with the duration of GdEpTz.

[0028] Figure 8 The results of in vivo fluorescence imaging of normal mice and mice with hemolysis-induced acute liver injury using GdEpTz synergistically prepared in Example 1 are shown below. Figure 8 (a) is a graph showing the change in fluorescence imaging over time with the synergistic effect of GdEpTz and 2E-Tco-Hcy. Figure 8 (b) is a graph showing the change in fluorescence signal-to-background ratio (SBR) with the synergistic effect time of GdEpTz and 2E-Tco-Hcy. Detailed Implementation

[0029] To facilitate understanding and implementation of the present invention by those skilled in the art, the present invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] Example 1

[0031] 1. Synthesis of Compound 1: Under nitrogen protection, 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (200.0 mg, 0.5 mmol, CAS No. 114873-52-8), 2-(2-bromoacetamyl)ethylcarbamate benzyl ester (204.1 mg, 0.7 mmol, 1.3 equiv, CAS No. 1027359-36-9), and potassium carbonate (206.9 mg, 1.5 mmol) were added to anhydrous acetonitrile (15 mL), and the mixture was stirred at room temperature for 12 h. After the reaction was complete, insoluble salts were removed by filtration, and the filtrate was concentrated under reduced pressure. The concentrate was purified by rapid silica gel chromatography (eluent: dichloromethane / methanol = 100:3, v / v) to give compound 1 (280.7 mg, yield 84.4%) as a white solid.

[0032] 2. Synthesis of Compound 2: Compound 1 (1.0 g, 1.5 mmol) was dissolved in methanol, followed by the addition of palladium on carbon (Pd / C, 10 wt%, 100 mg), and then stirred overnight at room temperature under a hydrogen atmosphere (1 atm). After the reaction was complete, the catalyst was removed by filtration, and the filter cake was thoroughly washed with dichloromethane (3 × 30 mL) to obtain the washings. The filtrate and washings were combined and concentrated to dryness under reduced pressure to give compound 2 (721.3 mg, 90.4% yield) as a white solid. Compound 2 was used directly in the next reaction without further purification.

[0033] 3. Synthesis of compound 3: Fmoc-Lys(Cbz)-OH (653.3 mg, 1.3 mmol, CAS No. 105751-18-6) and HATU (494.5 mg, 1.3 mmol) were dissolved in anhydrous DMF (15 mL) to obtain a mixed solution. Then, anhydrous DMF (10 mL) containing compound 2 (530.3 mg, 1.0 mmol) and N,N-diisopropylethylamine (DIPEA, 129.2 mg, 1.0 mmol) was slowly added dropwise to the mixed solution with stirring. After the addition was complete, the reaction was continued with stirring at room temperature for 2 h. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was diluted with a mixed solvent of dichloromethane (100 mL) and water (100 mL). After separation of the layers, the aqueous phase was extracted with dichloromethane (3 × 50 mL). The organic phases were combined and washed sequentially with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100:3, v / v) to give compound 3 (451.2 mg, yield 44.5%) as a white solid.

[0034] 4. Synthesis of compound 4: Compound 3 (1.0 g, 1.0 mmol) was dissolved in methanol, followed by the addition of palladium on carbon (Pd / C, 10 wt%, 100 mg). The mixture was stirred overnight under a hydrogen atmosphere (1 atm) at room temperature. After the reaction was complete, the catalyst was removed by filtration, and the filter cake was thoroughly washed with dichloromethane (3 × 30 mL) to obtain a washing solution. The filtrate and washing solution were combined and concentrated to dryness under reduced pressure to give compound 4 (779.6 mg, yield 89.8%) as a white solid. Compound 4 was used directly in the next reaction without further purification.

[0035] 5. Synthesis of Compound 5: Thionyl chloride (0.2 mL, 2.5 mmol) was slowly added dropwise to anhydrous methanol (26.0 mL) at 0°C, with the addition rate controlled to continue for 30 min while stirring. Then, 5-carboxy-2-adamantanone (200.0 mg, 1.0 mmol) was added in portions over 10 min. The resulting reaction mixture was slowly brought to room temperature and stirred overnight at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 70:30, v / v) to give compound 5 as a white solid (150.0 mg, 70.0% yield).

[0036] 6. Synthesis of Compound 6: Compound 5 (208.1 mg, 1.0 mmol) and methoxyamine hydrochloride (91.9 mg, 1.1 mmol) were added to a round-bottom flask, followed by the addition of pyridine (2 mL). The mixture was then stirred at room temperature for 2 h. After the reaction was complete, the pH of the system was adjusted to 7 with 1.0 M HCl aqueous solution, and then ethyl acetate (300 mL) was added for extraction. The organic phase was washed with 1 M HCl aqueous solution (3 × 30 mL), and the aqueous phases were combined and back-extracted with ethyl acetate (3 × 15 mL). The organic phases from all extraction operations were combined, washed successively with saturated brine, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to give compound 6 as a solid (200.0 mg, yield 92.1%).

[0037] 7. Synthesis of Compound 7: Compound 6 (1.6 g, 6.7 mmol) and cyclohexanone (2.0 g, 20.2 mmol) were dried under vacuum and added to a 100 mL two-necked round-bottom flask, followed by the addition of anhydrous hexane. The resulting mixture was stirred at 0 °C for 10 min. Ozone (O3) was then bubbled into the mixture for 1.5 h (ozone generated by a generator, yield: 70 g / h, flow rate: 2.5 L / min). After the reaction was complete, inert nitrogen (N2) was bubbled into the two-necked round-bottom flask to remove residual ozone. The crude product was heated to room temperature and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 90:10) to give compound 7 (683.4 mg, yield 31.7%) as a colorless solid.

[0038] 1 H NMR (500 MHz, CDCl3) δ 3.64 (d, J = 8.7 Hz, 3H), 2.25-1.50 (m,23H).

[0039] 13 C NMR (126 MHz, CDCl3) δ 177.24, 177.14, 110.07, 110.04, 109.25,109.21, 77.31, 77.06, 76.80, 51.66, 39.86, 39.46, 38.20, 36.30, 36.14, 36.03,35.89, 34.70, 34.69, 33.72, 33.61, 26.59, 26.18, 24.94, 23.83, 23.81.

[0040] 8. Synthesis of compound Ep-COOH: Compound 7 (200.0 mg, 0.6 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 2 mL), followed by the addition of a methanol / water solution (2 mL, 1:1, v / v) of lithium hydroxide monohydrate (22.3 mg, 0.9 mmol). The reaction mixture was heated at 60 °C for 6 h. After the reaction was complete, the pH of the system was adjusted to approximately 5 with 1.0 M HCl aqueous solution. The organic solvent was then removed by vacuum distillation. The residue was diluted with distilled water and extracted with dichloromethane (3 × 20 mL). The combined organic phases were washed with water, dried over anhydrous sodium sulfate (Na₂SO₄), filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to give a white solid compound Ep-COOH (191.3 mg, 100% yield). Compound Ep-COOH did not require further purification and was used directly in the next reaction.

[0041] 9. Synthesis of Compound 8: Compounds Ep-COOH (400.6 mg, 1.3 mmol) and HATU (494.3 mg, 1.3 mmol) were dissolved in anhydrous DMF (15 mL). Then, an anhydrous DMF solution of compound 4 (880.5 mg, 1.0 mmol) and DIPEA (129.4 mg, 1.0 mmol) (10 mL) was slowly added dropwise to the resulting mixture. The reaction mixture was stirred at room temperature for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was diluted with a mixture of dichloromethane (100 mL) and distilled water (100 mL), and the layers were separated. The aqueous phase was extracted with dichloromethane (3 × 50 mL). The combined organic phases were washed successively with saturated brine, dried over anhydrous sodium sulfate for 30 min, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100:3, v / v) to give compound 8 as a white solid (650.2 mg, yield 55.5%).

[0042] 1 H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 7.4 Hz, 2H), 7.65 (d, J = 7.1Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 6.87 (s, 1H), 6.57 (s, 1H), 6.43 (s, 1H), 4.27 (d, J = 54.4 Hz, 10H), 3.73-1.54 (m, 59H), 1.27 (t, J = 7.3 Hz, 9H).

[0043] 13C NMR (126 MHz, CDCl3) δ 177.67, 177.58, 173.20, 172.86, 172.78,156.65, 144.11, 141.20, 136.52, 128.48, 127.96, 127.83, 127.61, 127.13,125.38, 119.83, 110.16, 109.29, 109.24, 77.31, 77.06, 76.80, 66.69, 61.29,56.45, 55.18, 53.46, 47.25, 40.52, 39.92, 39.50, 38.33, 36.41, 36.19, 36.08, 36.01, 34.69, 33.53, 31.32, 29.72, 29.70, 29.13, 26.75, 26.39, 24.94, 23.84, 22.75, 14.20, 14.16.

[0044] HRMS (ESI) Calcd for C 62 H 90 N8O 14, [M+Na] + : 1193.6474, Found: 1193.6479.

[0045] 10. Synthesis of compound Gd-Ep-NH2: Compound 8 (100.0 mg, 0.1 mmol) was dissolved in anhydrous THF (2 mL), and a methanol / water mixture of lithium hydroxide monohydrate (3.6 mg, 0.15 mmol, 1.5 equiv) (2 mL, 1:1, v / v) was added. The resulting reaction solution was heated at 60 °C for 6 h. After the reaction was completed, the pH of the system was adjusted to 6 with 1 M HCl, and then an aqueous solution of gadolinium trichloride (GdCl3, 52.7 mg, 0.2 mmol) was added. The resulting reaction solution was stirred for another 6 h to complete the chelation reaction. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The concentrate was purified by preparative reversed-phase HPLC (C18 column) to give a white solid compound Gd-Ep-NH2.

[0046] HRMS (ESI) Calcd for C 41 H 65 GdN8O 12 [M+H] + : 1020.2634, Found: 1020.2639.

[0047] 11. Synthesis of compound Tz-COOH: Under nitrogen protection, 4-cyanophenylacetic acid (100.0 mg, 0.6 mmol), acetonitrile (509.4 mg, 12.4 mmol), and 3-mercaptopropionic acid (0.1 g, 0.6 mmol, 1.0 equiv) were mixed thoroughly. The resulting mixture was cooled to 0°C, and then 80% hydrazine hydrate solution (99.5 mg, 3.1 mmol) was slowly added dropwise. After the addition was complete, the resulting reaction solution was brought to room temperature and stirred overnight. After the reaction was complete, the resulting mixture was cooled in an ice-water bath, and then pre-cooled sodium nitrite aqueous solution (856.8 mg, 12.4 mmol) was slowly added to continue the oxidation reaction for 2 h. After the reaction was complete, 1.0 M HCl aqueous solution was slowly added dropwise to neutralize the alkaline environment until no more bubbles were generated. The resulting mixture was then extracted with dichloromethane. The combined organic phases were washed successively with saturated brine and dried over anhydrous Na2SO4. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 50:50, v / v) to give a pink solid compound Tz-COOH (89.6 mg, yield 62.7%).

[0048] 12. Synthesis of compound Tz-NHS: Under nitrogen protection, compound Tz-COOH (200.0 mg, 0.9 mmol) and N-hydroxysuccinimide (NHS, 218.6 mg, 1.7 mmol) were dissolved in anhydrous chloroform (5 mL), and the resulting mixture was cooled to 0 °C. Then, an anhydrous chloroform solution of N,N-diisopropylcarbodiimide (DIC, 1.1 mmol) was slowly added dropwise to the mixture. After the addition was complete, the reaction mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was brought to room temperature and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 90:10, v / v) to give a pink solid compound Tz-NHS (195.1 mg, yield 69.0%).

[0049] 1 H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.3Hz, 2H), 4.08 (s, 2H), 3.12 (s, 3H), 2.87 (s, 4H).

[0050] 13C NMR (126 MHz, CDCl3) δ 168.84, 167.35, 166.18, 163.77, 136.03,131.42, 130.26, 128.40, 77.28, 77.02, 76.77, 37.62, 25.60, 21.19.

[0051] 13. Synthesis of compound GdEpTz: Compounds Gd-Ep-NH2 (200.0 mg, 0.2 mmol), Tz-NHS (127.6 mg, 0.4 mmol), and DIPEA (50.7 mg, 0.4 mmol) were added to anhydrous DMF (2 mL). The resulting reaction solution was stirred at room temperature for 2 h. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by preparative reversed-phase HPLC (C18 column) to obtain a pink solid compound GdEpTz, i.e., Fe. 2+ Response-type paramagnetic probe.

[0052] HRMS (ESI) Calcd for C 52 H 73 GdN 12 O 13 [M+H] + : 1232.4645, Found: 1232.4777.

[0053] 14. Synthesis of compound Hcy-OH (synthesized according to the literature JY Hyun, NR Kang, I. Shin, Org. Lett., 2018, 20, 1240-1243): Under nitrogen protection, an anhydrous DMF solution (30 mL) of compound 9 (500.0 mg, 0.7 mmol) was slowly added dropwise to an anhydrous DMF suspension (20 mL) of resorcinol (121.0 mg, 1.1 mmol) and K₂CO₃ (193.5 mg, 1.4 mmol). After the addition was complete, the resulting reaction mixture was heated to 70°C and stirred at 70°C for 12 h. After the reaction was complete, the mixture was cooled to room temperature, and the inorganic base was removed by filtration. The filtrate was concentrated under reduced pressure, and the concentrate was extracted with a mixed solvent of water (200 mL) and dichloromethane (250 mL). The aqueous phase was further extracted with dichloromethane (3 × 50 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: DCM / MeOH = 96 / 4, v / v) to give a blue solid compound Hcy-OH (175.9 mg, yield 44.6%).

[0054] 15. Synthesis of Compound 10: Triphosgene (210.7 mg, 0.7 mmol) was added to a 50 mL round-bottom flask, and the flask was evacuated for 30 min using an oil pump. This was followed by three cycles of nitrogen purging and evacuation to completely remove oxygen. Under a continuous nitrogen flow, an anhydrous dichloromethane solution (10 mL) of DMAP (216.9 mg, 1.8 mmol) was slowly added dropwise to the flask, and the mixture was stirred at room temperature for 10 min after the addition was complete. Subsequently, a dichloromethane solution (10 mL) of Hcy-OH (200.0 mg, 0.4 mmol) was added dropwise to the mixture, and the mixture was stirred at room temperature for 2 h after the addition was complete. After the stirring step was completed, a pre-prepared solution of N-methyl-2-(N-methyl-Boc-amino)ethylamine (123.8 mg, 0.7 mmol, CAS No. 121492-06-6) and TEA (179.9 mg, 1.8 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to the aforementioned precursor. After the addition was complete, the resulting reaction solution was stirred overnight at room temperature. After the reaction was completed, it was quenched, concentrated to dryness under reduced pressure to remove the solvent, and the residue was purified by rapid silica gel chromatography (eluent: DCM / MeOH = 97:3, v / v, gradient elution) to give compound 10 (121.0 mg, yield 43.8%).

[0055] 16. Synthesis of Compound 11: Compound 10 (200.0 mg, 0.3 mmol) was placed in a 50 mL flask and cooled to 0 °C in an ice bath. A mixed solution of TFA and DCM (400 µL, 1:1, v / v) was then slowly added dropwise. The resulting reaction mixture was stirred at 0 °C for 30 min, and the reaction progress was monitored by thin-layer chromatography (TLC, DCM / MeOH = 96:4, v / v). After the reaction was complete, excess TFA was removed by rotary evaporation under reduced pressure at 0 °C. The crude product was lyophilized to obtain compound 11, which was used directly in the next reaction without further purification.

[0056] 17. Synthesis of compound 2E-Tco-Hcy: Compound 11 (10.0 mg, 0.013 mmol), its axial non-corresponding isomer (E)-cyclooct-2-en-1-yl-succinimide carbonate (18.0 mg, 0.067 mmol, CAS No. 1610931-22-0), and TEA (6.8 mg, 0.067 mmol) were dissolved in anhydrous dichloromethane (1 mL). The resulting reaction solution was stirred overnight at room temperature in the dark, and the reaction was monitored by TLC (DCM / MeOH = 97 / 3, v / v). After the reaction was complete, the solvent was removed by distillation under reduced pressure to dryness. The crude product was purified by rapid silica gel chromatography (DCM / MeOH = 96 / 4, v / v) to give a blue solid compound 2E-Tco-Hcy (3 mg, yield 28.1%).

[0057] 1H NMR (500 MHz, CD3OD) ) δ 8.85 (s, 1H), 7.77 - 7.64 (m, 2H), 7.63 -7.46 (m, 3H), 7.36 (d, J = 28.0 Hz, 2H), 7.13 (d, J = 8.4 Hz, 1H), 6.72 (d, J= 15.0 Hz, 1H), 5.84 (d, J = 8.5 Hz, 1H), 5.61 (s, 1H), 5.34 (s, 1H), 4.55(s, 2H), 3.83 - 3.51 (m, 4H), 3.09 (dd, J = 20.0, 17.1 Hz, 6H), 2.80 (d, J =27.7 Hz, 4H), 2.59 (s, 1H), 2.46 (d, J = 1.8 Hz, 2H), 2.41 - 2.28 (m, 1H),2.14 (s, 2H), 1.98 (s, 3H), 1.88 (s, 6H), 1.33 (d, J = 9.9 Hz, 1H), 1.15 (s,1H), 0.88 (s, 1H)。

[0058] 13C NMR (126 MHz, CD3OD) δ 179.13, 179.07, 178.93, 160.60, 156.30,156.13, 155.99, 154.40, 154.32, 154.00, 153.83, 153.73, 153.55, 153.06,146.65, 146.50, 142.45, 142.34, 141.33, 131.99, 131.95, 131.87, 131.84,131.75, 131.45, 131.27, 131.19, 131.02, 129.74, 129.60, 128.91, 128.02, 127.56, 122.48, 119.53, 119.43, 119.29, 118.91, 118.84, 117.22, 114.76, 112.80, 109.64, 109.19, 109.06, 104.75, 82.24, 75.10, 75.04, 74.67, 70.43, 51.07, 51.00, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 43.89, 40.22,35.63, 35.43, 34.14, 33.21, 29.37, 28.84, 28.66, 28.62, 26.93, 26.86, 26.27,24.10, 23.85, 23.80, 20.06, 15.11.

[0059] HRMS (ESI) Calcd for C 44 H 52 IN3O5, [MI] + : 702.3901, Found: 702.3907.

[0060] Experiment 1: Longitudinal relaxation performance test of GdEpTz with respect to metal ions Test method: 1. The Fe prepared in Example 1 2+ The responsive paramagnetic probe GdEpTz was dissolved in ultrapure water to prepare a 10 mM probe GdEpTz stock solution.

[0061] 2. Weigh out FeSO4 and dissolve it in ultrapure water to prepare a 10 mM FeSO4 solution.

[0062] 3. Add the GdEpTz stock solution to HEPES buffer (0.1 M, pH 7.4, 25 ℃) to prepare GdEpTz solutions with final concentrations of 0.1, 0.2, 0.3, and 0.4 mM. Then, add 10 mM Fe to each GdEpTz solution sequentially. 2+ The solution and 0.6 mM human serum albumin solution were mixed to obtain a mixture system. The mixture system was incubated at room temperature in the dark for 120 min to ensure complete reaction. After incubation, the sample to be tested was obtained.

[0063] 4. T1 measurements were performed on the test samples with different GdEpTz concentrations using a 0.5 T nuclear magnetic resonance relaxation analyzer (Niumai Analytical, China). During the measurement, a reverse recovery fast spin echo sequence was used, and the entire process was conducted in a constant hot airflow to maintain a thermally stable environment of 37 °C. The longitudinal relaxation rate (r1) was calculated by the slope of a linear fitting curve of 1 / T1 versus GdEpTz concentration.

[0064] 5. Perform the experiment with the probe GdEpTz stock solution according to the method in steps 3-4, except that serum albumin solution is not added.

[0065] 6. Perform the test on the probe GdEpTz mother liquor according to the method in steps 3-4, except that FeSO4 solution is not added.

[0066] 7. Perform the experiment using the probe GdEpTz stock solution according to steps 3-4, except that serum albumin solution and Fe are not added. 2+ Solution.

[0067] 8. Prepare the probe GdEpTz mother liquor according to steps 3-4, the difference being that Fe... 2+ The solutions were changed to MgCl2, CaCl2, FeCl3, ZnSO4, KCl, NaCl, NiSO4, CoSO4, MnCl2, and CuSO4 solutions, respectively, to evaluate the specificity of the probe for different metal ions and the relaxation response in the presence of proteins.

[0068] Experimental results: Fe 2+ The longitudinal relaxation rate r1 of the responsive paramagnetic probe GdEpTz in different systems is as follows: Figure 2 As shown, by Figure 2 It can be seen that in the absence of Fe 2+ When present, the longitudinal relaxation rate (r1) of the probe is 7.4 mM. -1 s -1 Introducing Fe alone 2+Subsequently, no statistically significant difference was observed in the r1 value, indicating that simple ion activation was insufficient to induce enhanced relaxation. However, in a simulated physiological environment (containing 0.6 mMHSA), the addition of Fe... 2+ After (1.0 equiv), r1 increased from 10.2 mM -1 s -1 Significantly improved to 15.1 mM -1 s -1 The increase was as high as 48.0%, confirming Fe 2+ Mediated protein covalent coupling can significantly improve longitudinal relaxation efficiency.

[0069] Fe 2+ The responsive paramagnetic probe GdEpTz exhibits specificity for recognizing different metal ions, such as... Figure 3 As shown, by Figure 3 It can be seen that among the tested bio-related metal ions, Fe only showed activity in the presence of serum albumin. 2+ It can induce a significant upregulation of r1, while other competing ions (such as Cu) 2+ Ca 2+ Mg 2+ Fe 3+ Zn 2+ (etc.) did not cause significant fluctuations in the relaxation rate, demonstrating excellent selectivity.

[0070] Experiment 2: In vitro evaluation of the bioorthogonal reaction between GdEpTz and 2E-Tco-Hcy Test method: 1. The Fe prepared in Example 1 2+ The responsive paramagnetic probe GdEpTz was dissolved in ultrapure water to prepare a 10 mM GdEpTz stock solution.

[0071] 2. The fluorescent signal reporter probe 2E-Tco-Hcy prepared in Example 1 was dissolved in DMSO to prepare a 10 mM 2E-Tco-Hcy stock solution.

[0072] 3. Add the GdEpTz stock solution to HEPES buffer (100 mM, pH 6.5), followed by the 2E-Tco-Hcy stock solution, so that the final concentration of GdEpTz in the mixture is 30 μM and the final concentration of 2E-Tco-Hcy is 10 μM (molar ratio 3:1, containing 50% DMSO, v / v).

[0073] 4. The mixed system was incubated at 37 °C in the dark for 1 h to ensure quantitative conversion of the reaction. Subsequently, multiple indicators were characterized: the absorption spectra of each group of solutions in the range of 400-810 nm were recorded using an Evolution-220 UV-Vis spectrophotometer to monitor the shift of characteristic absorption peaks and changes in absorbance; the fluorescence emission spectra of each group of solutions were recorded using an Edinburgh FS5 fluorescence spectrophotometer under 680 nm laser excitation to evaluate the fluorescence response triggered by bioorthogonal linkages.

[0074] 5. Conduct the experiment with the 2E-Tco-Hcy mother liquor without adding GdEpTz mother liquor, following the method in steps 3-4.

[0075] Experimental results: The UV-Vis absorption spectrum of GdEpTz prepared in Example 1 after undergoing a bioorthogonal reaction with 2E-Tco-Hcy is as follows: Figure 4 As shown, by Figure 4 It can be seen that 2E-Tco-Hcy initially exhibits characteristic absorption peaks at 600 nm and 653 nm; after mixing with GdEpTz, the solution color rapidly changes from light purple to light blue, and a new characteristic absorption band appears at 700 nm.

[0076] The fluorescence emission spectrum of GdEpTz prepared in Example 1 after undergoing a bioorthogonal reaction with 2E-Tco-Hcy is as follows: Figure 5 As shown, by Figure 5 and Figure 6 It was found that the fluorescence emission intensity (λex = 680 nm) of the reaction system increased in a time-dependent manner, eventually reaching a plateau. After bioorthogonal linking 2E-Tco-Hcy with GdEpTz, its near-infrared fluorescence (NIRF) signal showed a significant "on" response at 720 nm, with the fluorescence intensity increasing by approximately 155.5 times compared to the initial state.

[0077] Kinetic analysis of the bioorthogonal reaction between GdEpTz prepared in Example 1 and 2E-Tco-Hcy is as follows: Figure 6 As shown, by Figure 6 It can be seen that the fluorescence half-activation time (t) of the GdEpTz / 2E-Tco-Hcy system is... 1 / 2 The reaction time was only 17.1 min, demonstrating fast reaction kinetics.

[0078] Experiment 3: Liver T1 MR imaging of GdEpTz in normal and hemolytic-induced acute liver injury mouse models Test method: 1. The Fe prepared in Example 1 2+The responsive paramagnetic probe GdEpTz was dissolved in ultrapure water to prepare a 10 mM GdEpTz solution.

[0079] 2. 150 μL of GdEpTz solution was intravenously injected into mice in the phenylhydrazine-treated group (+PHz) via the tail vein. Liver scans of the treated mice were performed using a 9.4 T MRI scanner (Bruker, Germany) at pre-injection (baseline) and at designated time points (0.5, 1, 2, 4, and 6 h) post-injection. During imaging, mice were anesthetized with isoflurane and placed in a prone position (tail-first). Imaging parameters were set as follows: repetition time / echo time (TR / TE) = 600 / 6 ms; matrix size = 256 × 256; slice thickness = 0.8 mm; number of slices = 12; field of view (FOV) = 35 × 35 mm; region of interest (ROI) in the liver was manually selected. Mean signal intensity (SI) values ​​were normalized using water standard samples to correct for batch-to-batch measurement differences, and the mean signal intensity of the liver at each time point was calculated using the normalized values.

[0080] 3. The healthy control group mice (-PHz) were tested according to the method in step 2.

[0081] Experimental results: GdEpTz solution was intravenously injected into normal and hemolytically induced acute liver injury mice, followed by T1 MRI imaging. The resulting MRI images and the changes in MRI signal intensity over time are shown in the figure below. Figure 7 As shown, by Figure 7 The hemolytic model mice exhibited significant enhancement of liver magnetic resonance signal, peaking at 1 hour with an increase in signal contrast enhancement (%SE) of approximately 60%, followed by a gradual decline over the next 2 hours. In contrast, healthy control mice showed only weak signal enhancement (approximately 23%). This differential response indicates that the pathologically elevated Fe in the liver of mice in the hemolytic model... 2+ It effectively activated GdEpTz and promoted its covalent binding to endogenous liver proteins. This protein anchoring effect prolonged the rotational correlation time of the paramagnetic complex, thereby improving longitudinal relaxation properties and ultimately significantly enhancing the contrast of T1 MR imaging.

[0082] Experiment 4: Fluorescence Imaging Experiment of GdEpTz in Normal and Hemolytic-Induced Acute Liver Injury Mouse Models Test method: 1. The Fe prepared in Example 1 2+ The responsive paramagnetic probe GdEpTz was dissolved in ultrapure water to prepare a 10 mM GdEpTz solution.

[0083] 2. Dissolve the fluorescent signal reporter probe 2E-Tco-Hcy prepared in Example 1 in DMSO to prepare a 0.1 mM 2E-Tco-Hcy solution.

[0084] 3. Mice in the phenylhydrazine-treated group (+PHz) were intravenously injected with 150 μL of GdEpTz solution via the tail vein. One h after GdEpTz injection, mice in the PHz-treated group were subsequently intraperitoneally injected with 200 µL of 2E-Tco-Hcy solution. Whole-body fluorescence imaging was performed at different time points (0, 0.5, 1, 1.5, 2, 3, and 4 h) following the 2E-Tco-Hcy injection using an IVIS Lumina XR III system (excitation filter: 675 nm; emission filter: 710 nm). Three groups of PHz-treated mice were included in each experiment. The fluorescence intensity in the liver was quantitatively assessed by defining regions of interest (ROIs) and analyzing total irradiance using Living Image software (PerkinElmer, USA). The total fluorescence intensity of the liver ROI is expressed as mean ± standard deviation (mean ± SD, n = 3).

[0085] 4. The mice treated with PBS (normal mice, -PHz) were tested according to the method in step 3.

[0086] Experimental results: GdEpTz solution and 2E-Tco-Hcy solution were injected sequentially into normal and hemolytically induced acute liver injury mice, followed by fluorescence imaging. The resulting fluorescence images and the changes in fluorescence signal intensity over time are shown in the figure below. Figure 8 As shown, by Figure 8 It was found that the fluorescence signal-to-background ratio (SBR) of the liver in the PHz-treated mouse group (SBR = the ratio of fluorescence intensity at different time points after 2E-Tco-Hcy injection to fluorescence intensity before 2E-Tco-Hcy injection) continuously increased over time, reaching a peak of 5.0 at 2 h after 2E-Tco-Hcy injection, and maintaining a high signal intensity for a long period. In contrast, the fluorescence signal-to-background ratio (SBR) of the liver in the PBS-treated control group reached its peak at 0.5–1 h, with a peak intensity only 1.8 times that of the baseline, and the peak response of the PHz-treated group was approximately 2.4 times that of the PBS-treated control group.

[0087] This significant signal amplification and delay reaching a peak effect indicates that pathologically elevated Fe... 2+Triggering GdEpTz to crosslink with proteins creates an in-situ "molecular anchor," effectively overcoming the drawback of the small molecule probe GdEpTz being easily eluted. Through the continuous capture of 2E-Tco-Hcy at the anchor and the occurrence of IEDDA reaction, this strategy exhibits extremely high detection sensitivity and tissue retention ability in complex pathological environments.

Claims

1. A pre-targeting probe assembly for in vivo imaging of unstable iron pools, characterized in that: The pre-targeted probe assembly includes Fe 2+ Response paramagnetic probe GdEpTz and fluorescent signal reporter probe 2E-Tco-Hcy; The Fe 2+ The structural formula of the responsive paramagnetic probe GdEpTz is as follows: ； The structural formula of the fluorescent signal reporting probe 2E-Tco-Hcy is as follows: 。 2. The Fe according to claim 1 2+ A method for synthesizing the responsive paramagnetic probe GdEpTz, characterized in that... It includes the following steps: S1. Under inert gas protection and in the presence of a base, 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane reacts with 2-(2-bromoacetamido)ethylcarbamate benzyl ester in a substitution reaction to produce compound 1, as shown in the following reaction formula: ； S2. In the presence of a catalyst, compound 1 undergoes a hydrogenation reduction reaction to produce compound 2, as shown in the following reaction equation: ； S3. In the presence of a condensing reagent and a base, Fmoc-Lys(Cbz)-OH reacts with compound 2 via an amidation reaction to generate compound 3, as shown in the following reaction equation: ； S4. In the presence of a catalyst, compound 3 undergoes a hydrogenation reduction reaction to produce compound 4, as shown in the following reaction equation: ； S5, 5-carboxy-2-adamantanone, undergoes esterification under the catalytic system generated in situ from an acyl chloride reagent and methanol, to produce compound 5, as shown in the following reaction formula: ； S6. Under alkaline conditions, compound 5 undergoes an oxime reaction with methoxyamine hydrochloride to generate compound 6, as shown in the following reaction equation: ； S7 and compound 6 undergo a cycloaddition reaction with cyclohexanone under the action of ozone to generate compound 7, as shown in the following reaction formula: ； S8. In the presence of an alkaline reagent, compound 7 undergoes a hydrolysis reaction to produce compound Ep-COOH, as shown in the following reaction equation: ； S9. In the presence of a condensing agent and a base, compound Ep-COOH undergoes an amidation reaction with compound 4 to generate compound 8, as shown in the following reaction equation: ； S10. In the presence of an alkaline reagent, compound 8 first undergoes hydrolysis, followed by chelation with a gadolinium salt to form compound Gd-Ep-NH2, as shown in the following reaction equation: ； S11. Under inert gas protection and in the presence of hydrazine hydrate and a catalyst, 4-cyanophenylacetic acid undergoes a condensation reaction with acetonitrile, followed by oxidation with sodium nitrite to produce compound Tz-COOH, as shown in the following reaction formula: ； S12. Under inert gas protection and in the presence of a condensing reagent, Tz-COOH undergoes an esterification reaction with N-hydroxysuccinimide to generate the compound Tz-NHS, as shown in the following reaction formula: ； S13. Under alkaline conditions, compound Gd-Ep-NH2 and compound Tz-NHS undergo an esterification reaction to generate the Fe... 2 + The responsive paramagnetic probe GdEpTz has the following reaction formula: 。 3. The Fe according to claim 2 2+ A method for synthesizing the responsive paramagnetic probe GdEpTz, characterized by: In step S1, the base is potassium carbonate, the reaction solvent is anhydrous acetonitrile, the temperature of the substitution reaction is 20~35 ℃, the reaction time is 8~16 h, and the molar ratio of 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane, 2-(2-bromoacetamido)ethylcarbamate and the base is 1:1.2:2~1:2:6; In step S2, the catalyst is palladium on carbon, the reaction solvent is methanol, the reduction reaction temperature is 20~35 ℃, the reaction time is 12~24 h, the mass of the catalyst added is 10~15% of the mass of compound 1, and the pressure of the system after adding hydrogen is 1~1.2 atm. In step S3, the condensing agent is HATU, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is 20~40 ℃, the reaction time is 1~4 h, and the molar ratio of compound 2, Fmoc-Lys(Cbz)-OH, condensing agent and base is 1:1:1:1~1:2:2:

2.

4. The Fe according to claim 2 2+ A method for synthesizing the responsive paramagnetic probe GdEpTz, characterized by: In step S4, the catalyst is a palladium on carbon catalyst, the reaction solvent is methanol, the hydrogenation reduction reaction temperature is 20~35 ℃, the reaction time is 12~24 h, the mass of the catalyst added is 10~15% of the mass of compound 3, and the pressure of the system after adding hydrogen is 1~1.2 atm. In step S5, the acyl chloride reagent is thionyl chloride, the esterification reaction temperature is 0~30 ℃, the reaction time is 6~18 h, the reaction solvent is methanol, and the molar ratio of 5-carboxy-2-adamantanone to the acyl chloride reagent is 1:1~1:

4. In step S6, both the base and the reaction solvent are pyridine, the oxime reaction temperature is 20~45 ℃, the reaction time is 2~6 h, and the molar ratio of compound 5, methoxyamine hydrochloride and base is 1:1:1~1:3:

3. In step S7, the reaction solvent is anhydrous hexane, the cycloaddition reaction temperature is -20~0 ℃, the reaction time is 1~4 h, the ozone flow rate is 1.5 L / min~4 L / min, and the molar ratio of compound 6 to cyclohexanone is 1:2~1:

4.

5. The Fe according to claim 2 2+ A method for synthesizing the responsive paramagnetic probe GdEpTz, characterized by: In step S8, the alkaline reagent is lithium hydroxide, the reaction solvent is a mixed solvent of tetrahydrofuran, methanol and water, the hydrolysis reaction temperature is 45~80 ℃, and the reaction time is 4~12 h. In step S9, the condensing agent is HATU, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is 20~40 ℃, the reaction time is 1~5 h, and the molar ratio of compound 4, compound Ep-COOH, condensing agent and base is 1:1:1:1~1:2:2:

2. In step S10, the alkaline reagent is lithium hydroxide, the reaction solvent is a mixed solvent of tetrahydrofuran, methanol and water, the hydrolysis reaction temperature is 45~80 ℃, the reaction time is 4~12 h, and the gadolinium salt is selected from gadolinium trichloride.

6. The Fe according to claim 2 2+ A method for synthesizing the responsive paramagnetic probe GdEpTz, characterized by: In step S11, the catalyst is 3-mercaptopropionic acid, acetonitrile is used as the solvent, the condensation reaction temperature is 20~35 ℃, the reaction time is 8~16 h, the oxidation reaction temperature is -20~0 ℃, the reaction time is 1~3 h, and the molar ratio of 4-cyanophenylacetic acid, acetonitrile, catalyst, hydrazine hydrate and sodium nitrite is 1:10:1:2:10~1:50:10:

30. In step S12, the condensing agent is DIC, the reaction solvent is anhydrous chloroform, the esterification reaction temperature is -25~0 ℃, the reaction time is 1~4 h, and the molar ratio of Tz-COOH, N-hydroxysuccinimide and condensing agent is 1:1:1~1:3:

3. In step S13, the base is DIPEA, the reaction solvent is anhydrous N,N-dimethylformamide, the amidation reaction temperature is room temperature, the reaction time is 1~4 h, and the molar ratio of Gd-Ep-NH2, Tz-NHS and base is 1:1:1~1:5:

5.

7. A method for synthesizing the fluorescent signal reporter probe 2E-Tco-Hcy as described in claim 1, characterized in that... It includes the following steps: P1. Under inert gas protection and in the presence of a base, compound 9 undergoes a substitution reaction with resorcinol to form compound Hcy-OH, as shown in the following reaction equation: ； P2. Under inert gas protection and in the presence of a basic reagent, Hcy-OH, N-methyl-2-(N-methyl-Boc-amino)ethylamine, and a carbonylating agent undergo a carbamate reaction to generate compound 10, as shown in the following reaction formula: ； P3. In the presence of an acidic reagent, compound 10 undergoes a deprotection reaction to form compound 11, as shown in the following reaction equation: ； P4. In the presence of an alkaline reagent, compound 11 undergoes an amidation reaction with its non-corresponding isomer (E)-cyclooctene-succinimide carbonate to generate the fluorescent signal reporter probe 2E-Tco-Hcy, as shown in the following reaction formula: 。 8. The method for synthesizing the fluorescent signal reporter probe 2E-Tco-Hcy according to claim 7, characterized in that: In step P1, the base is potassium carbonate, the reaction solvent is anhydrous N,N-dimethylformamide, the temperature of the substitution reaction is 20~80 ℃, the reaction time is 8~16 h, and the molar ratio of compound 9, resorcinol and base is 1:1:1~1:3:

5. In step P2, the basic reagent is a mixture of 4-dimethylaminopyridine and triethylamine, the reaction solvent is anhydrous dichloromethane, the carbonylating reagent is triphosgene, the temperature of the carbamate esterification reaction is 20~35 °C, the reaction time is 4~16 h, and the molar ratio of Hcy-OH, N-methyl-2-(N-methyl-Boc-amino)ethylamine, carbonylating reagent and basic reagent is 1:1:1:1~1:4:4:

8. In step P3, the acidic reagent is trifluoroacetic acid, the reaction solvent is dichloromethane, the deprotection reaction temperature is -20~0 ℃, the reaction time is 0.25~0.5 h, and a mixed solvent with a TFA:DCM volume ratio of 1:2~2:1 is used as the reaction solvent. In step P4, the basic reagent is triethylamine, the reaction solvent is anhydrous dichloromethane, the amidation reaction temperature is 20~40 ℃, the reaction time is 6~16 h, and the molar ratio of compound 11, the non-corresponding isomer (E)-cyclooctene-succinimide carbonate, and the basic reagent is 1:1:3~1:5:

10.

9. The use of the pre-targeting probe assembly for in vivo unstable iron pool imaging as described in claim 1 in the preparation of a dual-modal contrast agent for magnetic resonance and fluorescence imaging.

10. The application of the pre-targeting probe assembly for in vivo unstable iron pool imaging according to claim 9 in the preparation of a dual-modal contrast agent for magnetic resonance and fluorescence imaging, characterized in that: When the pre-targeted probe combination is used as a dual-modal contrast agent, Fe is first injected into the vivo body. 2+ The responsive paramagnetic probe GdEpTz solution was injected, followed by the fluorescent signal reporter probe 2E-Tco-Hcy solution after 1 hour.

Images