A monosubstituted cyclic ligand, its preparation method and application; a monosubstituted cyclic rare earth complex, its preparation method and application.

Abstract

This invention provides a monosubstituted cyclic ligand, its preparation method, and its application, as well as a monosubstituted cyclic rare earth complex, its preparation method, and its application, relating to the field of contrast agent technology. The monosubstituted cyclic ligand provided by this invention uses a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ring structure as the parent ring. The R-modified tyrosine group increases the rigidity of the parent ring structure and improves the stability of the complex. Benzyl substituents, ethoxy, or hydroxyl-substituted benzyl groups, are directly introduced onto the carbon atoms of the DOTA ring, maintaining the original eight-coordinate structure. This increases the stability of the complex while also improving its relaxation rate; both kinetic inertness and relaxation rate are improved compared to Gd-DOTA. The complexes formed by this ligand with Gd(III) or Eu(III) exhibit high stability and strong specificity for liver or inflammation, without significant damage to the heart, liver, spleen, lungs, or kidneys.

Description

Technical Field

[0001] This invention relates to the field of contrast agent technology, specifically to a monosubstituted cyclic ligand and its preparation method and application, and a monosubstituted cyclic rare earth complex and its preparation method and application. Background Technology

[0002] Magnetic resonance imaging (MRI) obtains electromagnetic signals through the magnetic resonance phenomenon of atomic nuclei under the influence of a magnetic field, and then reconstructs images using a computer. Compared to imaging techniques such as computed tomography (CT) and positron emission tomography (PET), MRI poses no radiation risk to the human body. However, MRI has relatively low detection sensitivity, and approximately 40% of MRI scans require the use of contrast agents to enhance the detection signal. Globally, approximately 40 million people use MRI contrast agents annually.

[0003] Currently, all contrast agents publicly approved by the FDA, including those approved in 2022 and those entering Phase I clinical trials, are Gd-based. Clinically used contrast agent molecules can be broadly classified into linear and non-linear categories. Linear molecules, such as Gd-DTPA, Gd-EOB-DTPA, and Gd-BOPTA, are mostly non-specific magnetic resonance imaging (MRI) contrast agents. The only two hepatobiliary-specific MRI contrast agents are linear. Currently, most commercially available hepatobiliary-specific MRI contrast agents are linear Gd-DTPA derivatives, such as Gd-EOB-DTPA, Primovist / Eovist, and Primovist. These commercially available Gd-based MRI contrast agents suffer from poor stability, resulting in significant gadolinium residue, and poor targeting, posing serious health risks.

[0004]

[0005] Chinese patent CN114560821A discloses a cyclic Gd(III) complex using the ring structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as the parent ring. Lipophilic groups R' and R'' are introduced at the α-position of phenylacetic acid and the benzene ring structure, respectively, and a chiral group R is introduced at the macrocyclic position of DOTA. The chiral group R increases the rigidity of the macrocyclic structure, improving the stability of the complex. The lipophilic groups R' and R'' can bind to hepatocyte organic anion transport peptides, thereby effectively improving the distribution of the cyclic Gd(III) complex in the liver and gallbladder when used as a contrast agent. However, the above complex still has significant residues in mouse livers within the considered time frame, posing potential safety concerns for its use as a contrast agent. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a monosubstituted cyclic ligand, its preparation method and application, and a monosubstituted cyclic rare earth complex, its preparation method and application. The monosubstituted cyclic ligand provided by this invention forms complexes with Gd(III) or Eu(III) with high stability, strong specificity for the liver and inflammation, and can be rapidly cleared from the liver and kidneys in a short time, exhibiting high safety.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a monosubstituted cyclic ligand having the structure shown in Formula I:

[0009]

[0010] R includes benzyloxy, ethoxy, or hydroxyl groups; the wavy line indicates the R configuration or S configuration.

[0011] This invention provides a method for preparing the monosubstituted cyclic ligand described above, comprising the following steps: hydrolyzing the ester group of a monosubstituted cyclic ester compound under alkaline conditions to obtain the monosubstituted cyclic ligand;

[0012] The monosubstituted cyclic ester compounds include compound 7, compound 8, or compound 9:

[0013]

[0014] Preferably, the preparation method of compound 7 includes the following steps:

[0015] Compound 1 was reacted with benzyl bromide under basic conditions to undergo a first nucleophilic substitution reaction to give compound 2;

[0016] Compound 2 was subjected to an amide hydrolysis reaction under acidic conditions to obtain compound 3;

[0017] Compound 3 was reacted with methyl bromoacetate under alkaline conditions to undergo a second nucleophilic substitution reaction to obtain compound 4;

[0018] Compound 4 was subjected to an ester exchange reaction with diethylenetriamine to obtain compound 5;

[0019] Compound 5 was subjected to an amide reduction reaction in the presence of a reducing agent to obtain compound 6;

[0020] Compound 6 was reacted with ethyl bromoacetate under alkaline conditions to undergo a third nucleophilic substitution reaction to obtain compound 7;

[0021]

[0022] Preferably, the preparation method of compound 8 includes the following steps: subjecting compound 7 to a hydrogen debenzylation reaction in the presence of a catalyst and a reducing agent to obtain compound 8.

[0023] Preferably, the preparation method of compound 9 includes the following steps: reacting compound 8 with bromoethane in a fourth nucleophilic substitution reaction to obtain compound 9.

[0024] This invention provides a monosubstituted cyclic rare earth complex having the structure shown in Formula II:

[0025]

[0026] Wherein, R includes benzyloxy, ethoxy or hydroxyl; Ln includes Gd(Ⅲ) or Eu(Ⅲ); the wavy line indicates the R configuration or S configuration.

[0027] This invention provides a method for preparing the monosubstituted cyclic rare earth complex described in the above technical solution, comprising the following steps: performing a coordination reaction between a monosubstituted cyclic ligand and a rare earth metal source to obtain the monosubstituted cyclic rare earth complex; wherein the monosubstituted cyclic ligand is the monosubstituted cyclic ligand described in the above technical solution or the monosubstituted cyclic ligand prepared by the above technical solution; and wherein the metal in the rare earth metal source includes Gd(Ⅲ) or Eu(Ⅲ).

[0028] This invention provides the application of the monosubstituted cyclic rare earth complexes described in the above technical solutions or the monosubstituted cyclic rare earth complexes prepared by the above technical solutions in the preparation of contrast agents.

[0029] Preferably, the contrast agent includes a contrast agent for hepatobiliary magnetic resonance imaging and / or inflammatory magnetic resonance imaging.

[0030] This invention provides the application of the monosubstituted cyclic rare earth complexes described in the above technical solution or the monosubstituted cyclic rare earth complexes prepared by the above technical solution in the detection of peroxidase, wherein R of the monosubstituted cyclic rare earth complex is a hydroxyl group and Ln is Gd(Ⅲ).

[0031] The monosubstituted cyclic ligand with the structure shown in Formula I provided by this invention uses the cyclic structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4DOTA) as the parent ring. The tyrosine group modified by group R increases the rigidity of the parent ring structure, thereby improving the stability of the complex. At the same time, this invention maintains the original eight-coordinate structure by directly introducing benzyl groups substituted with benzyloxy (-OBn, denoted as ligand L1), ethoxy (-OEt, denoted as ligand L2), or hydroxyl (-OH, denoted as ligand L3) onto the carbon atom of the DOTA ring, thereby increasing the stability of the complex and improving its relaxation rate. Both the kinetic inertness and relaxation rate are improved compared to Gd-DOTA.

[0032] The monosubstituted cyclic ligands provided by this invention can form anionic amphiphilic eight-coordinate complexes with Gd(III) or Eu(III)—monosubstituted cyclic rare earth complexes. These complexes exhibit significantly improved stability compared to Gd-DOTA, without forming substantial Gd(III) and Eu(III) residues. This invention enables various specific magnetic resonance imaging techniques by changing the R substituent. Monosubstituted cyclic rare earth complexes with lipophilic groups -OBn and -OEt can selectively distribute to the liver as hepatobiliary-specific contrast agents. Specifically, Gd-L1 (R = -OBn, Ln = Gd(III)) shows enhanced liver specificity for over 35 minutes and is rapidly cleared from the liver and kidneys within 2 hours. Safety assessments in mice demonstrate that this drug causes no significant damage to the heart, liver, spleen, lungs, or kidneys, exhibiting high safety and excellent hepatobiliary diagnostic performance. Furthermore, the monosubstituted cyclic rare earth complexes demonstrate strong responsiveness to inflammation. The monosubstituted cyclic rare earth complex Gd-L3 (R is -OH, Ln is Gd(III)) contains tyrosine residues and can participate in the peroxide cycle. In the presence of hydrogen peroxide and peroxidase, it forms an oligomer of Gd-L3. Its relaxation rate in vitro is about 3.8 times higher than that of its monomer. In vivo, it can accumulate at the lesion site in a mouse model of gout, showing high contrast. It can be used to detect peroxidase accumulation in vitro and in vivo and can be used as an MPO (myeloperoxidase) responsive probe for the detection of peroxidase in vitro and in vivo.

[0033] This invention provides a method for preparing the monosubstituted cyclic ligands described in the above technical solution. The preparation method provided by this invention is simple in process, easy to operate, environmentally friendly, low in production cost, and suitable for industrial production.

[0034] Furthermore, in the process of preparing monosubstituted cyclic ester compounds, the present invention uses transesterification to generate a cyclic structure (to obtain compound 5), so that the side chain substituents are directly connected to the carbon atoms on the ring, retaining the stable eight-coordinate structure of the Gd atom. The monosubstituted cyclic rare earth complex has high kinetic inertness and relaxation rate.

[0035] This invention provides a method for preparing the monosubstituted cyclic rare earth complexes described above. The preparation method provided by this invention is simple in process, easy to operate, environmentally friendly, low in production cost, and suitable for industrial production. Attached Figure Description

[0036] Figure 1 Synthetic routes for monosubstituted cyclic ligands and monosubstituted cyclic rare earth complexes;

[0037] Figure 2 The HPLC chromatograms of ligands L1-3 at a wavelength of 254 nm are shown.

[0038] Figure 3 For the complex Gd-L 1～3 and Eu-L 1～3 HPLC chromatogram at a wavelength of 254 nm;

[0039] Figure 4 The image shows the mixture of complexes Gd-L1 and Eu-L1 before semi-preparative high-performance liquid chromatography separation, and the HPLC chromatograms of the separated angular and edge isomers at a wavelength of 254 nm.

[0040] Figure 5 The image shows the ESI-MS mass spectra of the mixture of complexes Gd-L1 and Eu-L1 before separation by semi-preparative high-performance liquid chromatography, and the separated angular and edge isomers.

[0041] Figure 6 An angular isomer of the complex Eu-L1 1 HNMR (400MHz, D2O) spectrum;

[0042] Figure 7 The edge isomer of the complex Eu-L1 1 HNMR (400MHz, D2O) spectrum;

[0043] Figure 8 Gd-L 1～3 Gd-EOB-DTPA in 1M HCl (a and b) and 10mM Zn 2+ Graphs assessing the kinetic inertness of PBS solutions (c and d);

[0044] Figure 9Schematic diagrams illustrating the interactions of Gd-L1 with OATP1B1(a), OATP1B3(b), OATP2B1(c), and NTCP(d); Schematic diagrams illustrating the interactions of Gd-L2 with OATP1B1(e), OATP1B3(f), OATP2B1(g), and NTCP(h); Schematic diagrams illustrating the interactions of Gd-L3 with OATP1B1(i), OATP1B3(j), OATP2B1(k), and NTCP(l).

[0045] Figure 10 Gd-L 1-3 Graphs evaluating the cytotoxicity of Gd-EOB-DTPA after incubation at 37°C with LO2(a) and 293T(b) for 24 h, respectively;

[0046] Figure 11 Coronal images of the liver (a) and kidney (b), normalized signal-to-noise ratio (nSNR) of the liver (c) and kidney (d), and contrast-to-noise ratio of the liver relative to muscle (e);

[0047] Figure 12 T1-weighted images of the liver (a) and kidney (b) of normal Balb / c mice after ingestion of Gd-L1 (0.1 mmol / kg) at 3.0T in vivo, without and with sulfobromophthalein (BSP), and normalized signal-to-noise ratio (nSNR) of the liver (c) and kidney (d) as a function of time over 35 min; data are expressed as mean ± standard deviation (n = 3).

[0048] Figure 13 The images show coronal and transverse sections of the liver under 3.0T MRI before and after Gd-L1 injection via tail vein, as well as the relationship between CNR (liver to tumor) and time in the coronal and transverse sections.

[0049] Figure 14 T1-weighted images of the liver (a) and kidney (b) captured at 3.0T transverse plane before injection of Gd-EOB-DTPA and Gd-L1, and at 1 min, 10 min and 20 min after injection. The nSNR time progression of the liver (c) and kidney (d) after injection of contrast agent.

[0050] Figure 15 Figure (a) shows the biodistribution of Gd-EOB-DOTA and Gd-L1 in normal mice and the total uptake of the complex in the five internal organs and brain at 5 min, 15 min and 24 h after injection (b).

[0051] Figure 16Histopathological analysis (a) and blood biochemical analysis (b) for acute (1 day) and subacute (60 days) poisoning of Gd-DOTA and Gd-L1;

[0052] Figure 17 The results of the relaxation assay of Gd-L3 incubated with horseradish peroxidase are shown in the figure. (a)–(b) show the longitudinal (T1) and transverse (T2) relaxation rate changes of Gd-L3 with HRP / H2O2 ([Gd] = 0.5 mM, pH = 7.4, PBS (0.1 M), 37℃, 1.4T; HRP activity: 0 U, 5 U, 50 U, 500 U; H2O2 equivalents consumed: 0–2 eq.); (c) shows the relaxation rate changes of Gd-L3 with HRP / H2O2 ([Gd] = 0.5 mM, HRP = 5 U, n) without HSA. H2O2 =2.0eq) relaxation rate plot, (d) is the relaxation rate plot of Gd-L3 and HRP / H2O2 in the presence of 4.5% HSA ([Gd] = 0.5mM, HRP = 5U, n H2O2 (e) T1-weighted MR image of the left foot (MSU treatment); (f) T1-weighted MR image of the right foot (PBS treatment, control group); (g) ΔCNR (left foot relative to right foot) as a function of time; (h) nSNR of the right foot after injection as a function of time; data are expressed as mean ± standard deviation (n = 3). Detailed Implementation

[0053] This invention provides a monosubstituted cyclic ligand having the structure shown in Formula I:

[0054]

[0055] R includes benzyloxy (-OBn, denoted as ligand L1), ethoxy (-OEt, denoted as ligand L2), or hydroxyl (-OH, denoted as ligand L3); the wavy line indicates the R configuration or S configuration.

[0056] This invention provides a method for preparing the monosubstituted cyclic ligand described above, comprising the following steps: hydrolyzing the ester group of a monosubstituted cyclic ester compound under alkaline conditions to obtain the monosubstituted cyclic ligand; wherein the monosubstituted cyclic ester compound includes compound 7, compound 8, or compound 9.

[0057]

[0058] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0059] In this invention, the alkaline conditions are preferably provided by an inorganic strong base, which is preferably an alkali metal hydroxide, more preferably including at least one of lithium hydroxide, sodium hydroxide, and potassium hydroxide, and even more preferably lithium hydroxide; the inorganic strong base is preferably used in the form of an aqueous solution of the inorganic strong base, and the mass ratio of the inorganic strong base to the volume of water in the aqueous solution of the inorganic strong base is preferably 1 g: 7-8 mL, more preferably 1 g: 7.5 mL; the molar ratio of the monosubstituted cyclic ester compound to the inorganic strong base is preferably 1: 5-6, more preferably 1: 5.5.

[0060] In this invention, the solvent used for the hydrolysis reaction of the ester group is preferably a polar organic solvent; the polar organic solvent preferably includes at least one of ethanol, methanol and tetrahydrofuran, more preferably ethanol; the molar ratio of the monosubstituted cyclic ester compound to the volume of the polar organic solvent is preferably 1g:10-15mL, more preferably 1g:12-13mL.

[0061] In this invention, the hydrolysis reaction of the ester group is preferably carried out at room temperature and for a time of 10-14 hours, more preferably 11-12 hours. The hydrolysis reaction of the ester group is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen. When nitrogen is used, the production cost of the target product is lower.

[0062] After completing the hydrolysis reaction of the ester group, the present invention preferably further includes: concentrating the reaction system obtained from the hydrolysis reaction of the ester group, adding an extractant, stirring, allowing it to stand, separating the solid and liquid components, and drying the obtained solid product to obtain the monosubstituted cyclic ligand. In the present invention, the concentration is preferably achieved by rotary evaporation to remove the solvent. In the present invention, the extractant preferably includes one or more of ethanol, methyl tert-butyl ether, ethyl acetate, and dichloromethane. The present invention does not have a special limitation on the stirring; stirring until uniform is achieved is sufficient. The present invention does not have a special limitation on the standing time; standing until precipitation no longer increases is sufficient. In the present invention, the solid-liquid separation preferably includes filtration, vacuum filtration, or centrifugation. In the present invention, the drying temperature is preferably 40–55°C, more preferably 45–50°C; the present invention does not have a special limitation on the drying time; drying to constant weight is sufficient.

[0063] In this invention, the preparation method of compound 7 preferably includes the following steps:

[0064] Compound 1 was reacted with benzyl bromide under basic conditions to undergo a first nucleophilic substitution reaction to give compound 2;

[0065] Compound 2 was subjected to an amide hydrolysis reaction under acidic conditions to obtain compound 3;

[0066] Compound 3 was reacted with methyl bromoacetate under alkaline conditions to undergo a second nucleophilic substitution reaction to obtain compound 4;

[0067] Compound 4 was subjected to an ester exchange reaction with diethylenetriamine to obtain compound 5;

[0068] Compound 5 was subjected to an amide reduction reaction in the presence of a reducing agent to obtain compound 6;

[0069] Compound 6 was reacted with ethyl bromoacetate under alkaline conditions to undergo a third nucleophilic substitution reaction to obtain compound 7;

[0070]

[0071] In this invention, when compounds 1 to 4 are all in the S configuration, compounds 5 to 9, the monosubstituted cyclic ligand, and the monosubstituted cyclic rare earth complex are all in the R configuration; when compounds 1 to 4 are all in the R configuration, compounds 5 to 9, the monosubstituted cyclic ligand, and the monosubstituted cyclic rare earth complex are all in the S configuration.

[0072] In this invention, compound 1 is reacted with benzyl bromo under alkaline conditions to undergo a first nucleophilic substitution reaction to obtain compound 2.

[0073] In this invention, the molar ratio of compound 1 to benzyl bromo is preferably 1:1.05 to 1.5, more preferably 1:1.1 to 1.15.

[0074] In this invention, the alkaline conditions are preferably provided by an alkaline reagent (denoted as the first alkaline reagent), which preferably includes a carbonate, more preferably an alkali metal carbonate, and even more preferably at least one selected from potassium carbonate, sodium carbonate, and cesium carbonate. In this invention, the molar ratio of compound 1 to the first alkaline reagent is preferably 1:2.8 to 4, more preferably 1:3 to 3.5.

[0075] In this invention, the organic solvent used in the first nucleophilic substitution reaction (denoted as the first organic solvent) preferably includes at least one of N,N-dimethylformamide (DMF), tetrahydrofuran, and acetonitrile; the molar ratio of compound 1 to the volume of the first organic solvent is preferably 1 g: 5-8 mL, more preferably 1 g: 5-6 mL.

[0076] In this invention, the temperature of the first nucleophilic substitution reaction is preferably room temperature, and the time is preferably 18-24 hours, more preferably 20-22 hours; the first nucleophilic substitution reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0077] After completing the first nucleophilic substitution reaction, the present invention preferably further includes: adding water to the reaction system obtained from the first nucleophilic substitution reaction, adding an extractant for extraction, drying the obtained organic phase with anhydrous sodium sulfate, filtering, and concentrating the obtained liquid component to constant weight to obtain compound 2. In the present invention, the extractant preferably includes ethyl acetate or dichloromethane. In the present invention, the concentration method preferably includes evaporation to remove the organic solvent.

[0078] After obtaining compound 2, the present invention performs an amide hydrolysis reaction on compound 2 under acidic conditions to obtain compound 3.

[0079] In this invention, the acidic conditions are preferably provided by an acidic reagent, which preferably includes trifluoroacetic acid or hydrochloric acid-dioxane; the concentration of hydrochloric acid in the hydrochloric acid-dioxane mixture is preferably 0.8–1.5 M (i.e., mol / L), more preferably 1–1.2 M; the trifluoroacetic acid is preferably added dropwise to the mixture of compound 2 and the second organic solvent at -10 to 10°C (0°C). In this invention, the mass ratio of compound 2 to the volume of the acidic reagent is preferably 1 g: 2–2.5 mL, more preferably 1 g: 2–2.2 mL.

[0080] In this invention, the organic solvent used for the hydrolysis reaction of the amide (denoted as the second organic solvent) preferably includes dichloromethane (DCM) or tetrahydrofuran; the mass ratio of compound 2 to the volume of the second organic solvent is preferably 1g:8-10mL, more preferably 1g:8-9mL.

[0081] In this invention, the hydrolysis reaction of the amide is preferably carried out at room temperature and for a time of 18-24 hours, more preferably 20-22 hours; the hydrolysis reaction of the amide is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0082] After the hydrolysis reaction of the amide is completed, the present invention preferably further includes: removing the second organic solvent from the reaction system obtained from the hydrolysis reaction of the amide by rotary evaporation, removing more than 95% (more preferably 95-99%) of the acidic reagent by vacuum pump, adding a saturated sodium bicarbonate solution until no more bubbling occurs, extracting, drying the obtained organic phase with anhydrous sodium sulfate, filtering, and concentrating the obtained liquid component to obtain compound 3. In the present invention, the extractant used for extraction preferably includes ethyl acetate or dichloromethane. In the present invention, the concentration preferably includes sequentially removing the solvent by rotary evaporation and drying by vacuum pump.

[0083] After obtaining compound 3, the present invention reacts compound 3 with methyl bromoacetate under alkaline conditions to carry out a second nucleophilic substitution reaction to obtain compound 4.

[0084] In this invention, the molar ratio of compound 3 to methyl bromoacetate is preferably 1:1 to 1.2, more preferably 1:1 to 1.1.

[0085] In this invention, the alkaline conditions are preferably provided by an alkaline reagent (denoted as the second alkaline reagent), which preferably includes an organic amine, more preferably at least one selected from N,N-diisopropylethylamine (DIPEA) and triethylamine. In this invention, the molar ratio of compound 3 to the second alkaline reagent is preferably 1:1.8 to 2.2, more preferably 1:1.9 to 2.

[0086] In this invention, the organic solvent used for the second nucleophilic substitution reaction (denoted as the third organic solvent) preferably includes at least one of N,N-dimethylformamide, tetrahydrofuran, and acetonitrile; the molar ratio of the compound 3 to the volume of the third organic solvent is preferably 1 g: 5 to 10 mL, more preferably 1 g: 8 to 10 mL.

[0087] In this invention, the temperature of the second nucleophilic substitution reaction is preferably room temperature, and the time is preferably 3 to 10 hours, more preferably 4 to 6 hours; the second nucleophilic substitution reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0088] After completing the second nucleophilic substitution reaction, the present invention preferably further includes: adding water to the reaction system obtained from the second nucleophilic substitution reaction, adding an extractant for extraction, drying the obtained organic phase with anhydrous sodium sulfate, filtering, and concentrating the obtained liquid component to obtain compound 4. In the present invention, the extractant preferably includes ethyl acetate or dichloromethane. In the present invention, the concentration method preferably includes sequentially evaporating to remove the organic solvent and then drying with a vacuum pump.

[0089] After obtaining compound 4, the present invention performs an ester exchange reaction between compound 4 and diethylenetriamine to obtain compound 5.

[0090] In this invention, the molar ratio of compound 4 to diethylenetriamine is preferably 1:1 to 1.2, more preferably 1:1 to 1.05; the diethylenetriamine is preferably added dropwise.

[0091] In this invention, the organic solvent used in the transesterification reaction (denoted as the fourth organic solvent) preferably includes at least one of methanol (MeOH), ethanol and acetonitrile; the volume ratio of compound 4 to the fourth organic solvent is preferably 1g:50-60mL, more preferably 1g:55-58mL.

[0092] In this invention, the temperature of the transesterification reaction is preferably 65-75°C, more preferably 70°C; the time is preferably 10-13 days, more preferably 11-12 days; the second nucleophilic substitution reaction is preferably carried out under a protective atmosphere and reflux of condensate, wherein the protective atmosphere preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0093] After completing the transesterification reaction, the present invention preferably further includes: rotary evaporating the reaction system obtained from the transesterification reaction to remove most (more than 95%, more preferably 95-99%) of the fourth organic solvent, filtering, washing the filter cake with ice-cold ethanol 2-3 times, and drying to obtain compound 5. In the present invention, the drying temperature is preferably 45-55°C, more preferably 45-50°C; the present invention does not have a special limitation on the drying time, drying to constant weight is sufficient.

[0094] After obtaining compound 5, the present invention performs an amide reduction reaction on compound 5 in the presence of a reducing agent to obtain compound 6.

[0095] In this invention, the molar ratio of compound 5 to reducing agent is preferably 1:20 to 30, more preferably 1:22 to 25.

[0096] In this invention, the organic solvent used for the reduction reaction of the amide (denoted as the fifth organic solvent) preferably includes tetrahydrofuran; the concentration of the reducing agent in the fifth organic solvent is preferably 0.9 to 1.1 M, more preferably 1 M.

[0097] In this invention, the temperature of the reduction reaction of the amide is preferably 65-75°C, more preferably 70°C; the time is preferably 18-27 h, more preferably 20-24 h.

[0098] After completing the reduction reaction of the amide, the present invention preferably further includes: cooling the reaction system obtained from the reduction reaction of the amide to room temperature, quenching the reaction, adjusting the pH value to 8-9 (more preferably 8-8.5), extracting, drying the obtained organic phase with anhydrous sodium sulfate, filtering, and concentrating the obtained liquid component to obtain compound 6. In the present invention, the quenching agent used in the quenching reaction preferably includes a mixed solution of methanol and dilute hydrochloric acid or a mixed solution of methanol and water; the concentration of the dilute hydrochloric acid is preferably 1-1.5M, more preferably 1-1.2M; the mass ratio of compound 5 to the volume of dilute hydrochloric acid is preferably 1g:20-30mL, more preferably 1g:20-25mL; the mass ratio of compound 5 to the volume of water is preferably 1g:20-30mL, more preferably 1g:20-25mL; the temperature of the quenching reaction is preferably -10 to 10℃. The preferred temperature is -5 to 5°C; the preferred time is 1.5 to 2.2 hours, more preferably 2 hours; the quenching reaction is preferably carried out under reflux conditions; taking a mixed solution of methanol and dilute hydrochloric acid as the quenching agent as an example, the preferred quenching reaction is: adding methanol dropwise at -10 to 10°C (more preferably -5 to 5°C) until no more bubbling occurs, then adding dilute hydrochloric acid, and carrying out the quenching reaction under reflux conditions; both the reduction reaction and the quenching reaction of the amide are preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon, or helium, more preferably nitrogen. In this invention, the alkali used to adjust the pH value preferably includes an aqueous solution of potassium carbonate, sodium bicarbonate, or sodium hydroxide, and the concentration of the aqueous solution of sodium hydroxide is preferably 1 to 1.2 M, more preferably 1 to 1.1 M. In this invention, the concentration preferably includes sequentially removing the solvent by rotary evaporation and drying by a vacuum pump.

[0099] After obtaining compound 6, the present invention reacts compound 6 with ethyl bromoacetate under alkaline conditions with a third nucleophilic substitution reaction to obtain compound 7.

[0100] In this invention, the molar ratio of compound 6 to ethyl bromoacetate is preferably 1:5.5 to 6.5, more preferably 1:5.5 to 6.

[0101] In this invention, the alkaline conditions are preferably provided by an alkaline reagent (denoted as the third alkaline reagent), which preferably includes a carbonate, more preferably an alkali metal carbonate, and even more preferably at least one selected from potassium carbonate, sodium carbonate, and cesium carbonate. In this invention, the molar ratio of compound 6 to the third alkaline reagent is preferably 1:9 to 10, more preferably 1:9 to 9.5.

[0102] In this invention, the organic solvent used in the third nucleophilic substitution reaction (denoted as the sixth organic solvent) preferably includes at least one of acetonitrile, N,N-dimethylformamide and tetrahydrofuran; the molar ratio of compound 6 to the volume of the sixth organic solvent is preferably 1 g: 10-20 mL, more preferably 1 g: 10-15 mL.

[0103] In this invention, the temperature of the third nucleophilic substitution reaction is preferably room temperature, and the time is preferably 18-27 h, more preferably 18-20 h; the third nucleophilic substitution reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0104] After completing the third nucleophilic substitution reaction, the present invention preferably further includes: filtering the reaction system obtained from the third nucleophilic substitution reaction, removing the solvent by rotary evaporation of the resulting liquid component, dissolving it in ethyl acetate, washing with dilute hydrochloric acid, adjusting the pH to 8-9 (more preferably 8-8.5), extracting with dichloromethane, drying the resulting organic phase with anhydrous sodium sulfate, filtering, removing the solvent by rotary evaporation of the resulting liquid component, and drying under vacuum to obtain compound 6. In the present invention, the concentration of the dilute hydrochloric acid is preferably 1-1.5M, more preferably 1M; the number of times the dilute hydrochloric acid is washed is preferably 2-3 times. In the present invention, the number of times the dichloromethane is extracted is preferably 2-3 times. In the present invention, the alkali used to adjust the pH preferably includes an aqueous solution of potassium carbonate, sodium bicarbonate, or sodium hydroxide, and the concentration of the aqueous solution of sodium hydroxide is preferably 1-1.2M, more preferably 1-1.1M.

[0105] In this invention, the preparation method of compound 8 preferably includes the following steps: subjecting compound 7 to a hydrogen debenzylation reaction in the presence of a catalyst and a reducing agent to obtain compound 8.

[0106] In this invention, the catalyst preferably comprises palladium on carbon and / or palladium hydroxide; the mass fraction of palladium on carbon is preferably 10-20%, more preferably 15-20%. In this invention, the mass ratio of compound 7 to the catalyst is preferably 1:0.2-0.3, more preferably 1:0.2-0.25.

[0107] In this invention, the reducing agent preferably includes hydrogen or ammonium formate; the pressure of the hydrogen is preferably 2-3 MPa, more preferably 2-2.5 MPa; the molar ratio of compound 8 to ammonium formate is preferably 1:4.5-5.5, more preferably 1:4.8-5.

[0108] In this invention, the organic solvent used in the hydrogen debenzylation reaction (referred to as the seventh organic solvent) preferably includes an alcohol solvent, more preferably ethanol and / or methanol; the molar ratio of compound 7 to the volume of the seventh organic solvent is preferably 1g:20-30mL, more preferably 1g:25mL.

[0109] In this invention, the temperature of the hydrogen debenzylation reaction is preferably 52-58°C, more preferably 55°C; the time is preferably 18-36 h, more preferably 18-25 h; the hydrogen debenzylation reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0110] After completing the hydrogen debenzylation reaction, the present invention preferably further includes: filtering the reaction system obtained from the hydrogen debenzylation reaction to remove the catalyst, and evaporating the organic solvent to obtain compound 8.

[0111] In this invention, the preparation method of compound 9 preferably includes the following steps: reacting compound 8 with bromoethane in a fourth nucleophilic substitution reaction to obtain compound 9.

[0112] In this invention, the molar ratio of compound 8 to bromoethane is preferably 1:3 to 5, more preferably 1:3 to 3.5.

[0113] In this invention, the fourth nucleophilic substitution reaction is preferably carried out in the presence of a basic reagent (denoted as the fourth basic reagent) and an organic solvent (denoted as the eighth organic solvent). In this invention, the fourth basic reagent preferably comprises a carbonate, more preferably an alkali metal carbonate, and even more preferably at least one selected from potassium carbonate, sodium carbonate, and cesium carbonate; the molar ratio of compound 8 to the fourth basic reagent is preferably 1:5.5 to 6.5, more preferably 1:6. In this invention, the eighth organic solvent preferably comprises DMF; the mass ratio of compound 8 to the volume ratio of the eighth organic solvent is preferably 1 g:5 to 8 mL, more preferably 1 g:6 to 7 mL.

[0114] In this invention, the temperature of the fourth nucleophilic substitution reaction is preferably room temperature, and the time is preferably 18-27 h, more preferably 20-24 h; the fourth nucleophilic substitution reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium, more preferably nitrogen.

[0115] After completing the fourth nucleophilic substitution reaction, the present invention preferably further includes: adding water to the reaction system obtained from the fourth nucleophilic substitution reaction, adding an extractant for extraction, drying the obtained organic phase with anhydrous sodium sulfate, filtering, and concentrating the obtained liquid component to obtain compound 9. In the present invention, the extractant preferably includes ethyl acetate or dichloromethane. In the present invention, the concentration method preferably includes evaporation to remove the organic solvent.

[0116] This invention provides a monosubstituted cyclic rare earth complex having the structure shown in Formula II:

[0117]

[0118] Wherein, R includes benzyloxy (Gd-L1 or Eu-L1), ethoxy (Gd-L2 or Eu-L2) or hydroxyl (Gd-L3 or Eu-L3); Ln (trivalent, denoted as Ln(Ⅲ)) includes Gd(Ⅲ) or Eu(Ⅲ); the wavy line indicates the R configuration or S configuration.

[0119] This invention provides a method for preparing the monosubstituted cyclic rare earth complex described in the above technical solution, comprising the following steps: performing a coordination reaction between a monosubstituted cyclic ligand and a rare earth metal source to obtain the monosubstituted cyclic rare earth complex; wherein the monosubstituted cyclic ligand is the monosubstituted cyclic ligand described in the above technical solution or the monosubstituted cyclic ligand prepared by the above technical solution; and wherein the metal in the rare earth metal source includes Gd(Ⅲ) or Eu(Ⅲ).

[0120] In this invention, the molar ratio of the monosubstituted cyclic ligand to the metal in the rare earth metal source is preferably 1:1 to 1.1, more preferably 1:1 to 1.05.

[0121] In this invention, the rare earth metal source preferably includes a Gd(III) source or an Eu(III) source; the Gd(III) source includes gadolinium chloride and / or gadolinium nitrate, more preferably gadolinium chloride; the Eu(III) source includes europium chloride and / or europium nitrate, more preferably europium chloride; the rare earth metal source is preferably a water-soluble rare earth metal source, and is used in the form of an aqueous solution of the rare earth metal source; the concentration of the aqueous solution of the rare earth metal source is preferably 1.8-2.4 g / mL, more preferably 2-2.3 g / mL; the aqueous solution of the rare earth metal source is preferably added dropwise.

[0122] In this invention, the monosubstituted cyclic ligand is preferably used in the form of a monosubstituted cyclic ligand solution. The mass ratio of the monosubstituted cyclic ligand to the volume of the solvent in the monosubstituted cyclic ligand solution is preferably 1 g: 20-30 mL, more preferably 1 g: 20-25 mL. The solvent is preferably weakly acidic water, and the pH value of the weakly acidic water is preferably 4-6, more preferably 5-6. The weakly acidic water is preferably obtained by adjusting the pH value of water to weakly acidic using dilute hydrochloric acid. The concentration of the dilute hydrochloric acid is preferably 1-1.2 M, more preferably 1-1.1 M.

[0123] In this invention, the temperature of the coordination reaction is preferably 100–105°C, more preferably 100–102°C; the time is preferably 18–24 h, more preferably 20–22 h; the coordination reaction is preferably carried out under reflux conditions of condensate; the pH value of the system during the coordination reaction is preferably neutral, and the pH value is preferably adjusted by an aqueous solution of potassium carbonate, sodium bicarbonate, or sodium hydroxide, and the concentration of the aqueous solution of sodium hydroxide is preferably 1–1.2 M, more preferably 1–1.1 M.

[0124] After completing the coordination reaction, the present invention preferably further includes: adjusting the pH of the reaction system obtained by the coordination reaction to above 10, precipitating excess metal ions, centrifuging, filtering the resulting supernatant with a 0.22 μm filter or a 0.22 μm filter membrane, adjusting the pH of the resulting liquid component to 7, and then freeze-drying to obtain a monosubstituted cyclic rare earth complex. In the present invention, the pH value is preferably 10-14, more preferably 10-12, and even more preferably 11. In the present invention, the purpose of centrifugation is to remove most of the metal precipitate; the purpose of filtration with a filter or filter membrane is to remove the remaining metal precipitate. In the present invention, adjusting the pH value to 7 is preferably done using dilute hydrochloric acid, and the concentration of the dilute hydrochloric acid is preferably 1-1.2 M, more preferably 1-1.1 M.

[0125] This invention also provides the application of the monosubstituted cyclic rare earth complexes described in the above-described technical solutions, or the monosubstituted cyclic rare earth complexes prepared by the above-described preparation methods, in the preparation of contrast agents. In this invention, the contrast agent preferably includes contrast agents for hepatobiliary magnetic resonance imaging and / or inflammatory magnetic resonance imaging.

[0126] This invention also provides the application of the monosubstituted cyclic rare earth complexes described in the above-described technical solutions, or the monosubstituted cyclic rare earth complexes prepared by the above-described technical solutions, in the detection of peroxidase, wherein R in the monosubstituted cyclic rare earth complexes is a hydroxyl group and Ln is Gd(III). The exposed hydroxyl group in the monosubstituted cyclic rare earth complexes can participate in the peroxide cycle to achieve Gd-L3 oligomerization, and can be used as an MPO (myeloperoxidase) responsive probe for the detection of peroxidase in vivo and in vitro.

[0127] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0128] Example 1

[0129] according to Figure 1 The synthetic route shown synthesizes monosubstituted cyclic ligands L in S and R configurations. 1～3 (i.e., L1, L2, and L3) as well as the S- and R-configurations of monosubstituted cyclic rare earth complexes Gd-L 1～3 (i.e., Gd-L1, Gd-L2 and Gd-L3) and Eu-L1 to 3 (i.e., Eu-L1, Eu-L2 and Eu-L3); when compounds 1 to 4 are all in the S configuration, compounds 5 to 9, the monosubstituted cyclic ligands and the monosubstituted cyclic rare earth complexes are all in the R configuration; when compounds 1 to 4 are all in the R configuration, compounds 5 to 9, the monosubstituted cyclic ligands and the monosubstituted cyclic rare earth complexes are all in the S configuration.

[0130] Compound 1 (3.4 mmol) was dissolved in 5 mL of LMF, and benzyl bromide (3.9 mmol) and potassium carbonate (10.2 mmol) were added sequentially. The mixture was stirred at room temperature under nitrogen protection for 18 h, and the reaction was monitored for completion by TLC (PE:EA volume ratio = 2:1). 10 mL of water was added to the mixture, and the mixture was stirred. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to remove the organic solvent, yielding compound 2 (white solid, yield 96%). 1 HNMR (400MHz, CDCl3) δ7.46-7.28(m,5H),7.04(d,J=8.5Hz,2H),6.90(d,J=8.6Hz,2H),5.04(s,2H),4. 96(d,J=8.4Hz,1H),4.54(q,J=7.3,6.7Hz,1H),3.71(s,3H),3.03(tt,J=14.2,6.8Hz,2H),1.42(s,9H). 13 CNMR(100MHz,CDCl3)δ172.50,157.94,155.16,137.02,130.37,128.65,1 28.27,128.03,127.54,114.96,79.98,70.05,54.57,52.27,37.53,28.36.

[0131] Compound 2 (1.3 mmol) was dissolved in 4 mL of DCM, and 1 mL of trifluoroacetic acid was added dropwise under ice bath conditions. The mixture was stirred for 18 h under nitrogen protection at room temperature, and the reaction was monitored for completeness by TLC (PE:EA = 2:1). DCM and most (over 95%) of the trifluoroacetic acid were removed by rotary evaporation. A saturated sodium bicarbonate solution was added until no more bubbles were observed, and the mixture was extracted with DCM. Anhydrous sodium sulfate was added to the resulting organic phase, and the mixture was dried. The solution was filtered, and the solvent was removed by rotary evaporation of the filtrate. The solution was then dried under vacuum to give compound 3 (a yellow oil, 95% yield). 1 HNMR (400MHz, CDCl3) δ7.52-7.29(m,5H),7.11(d,J=8.6Hz,2H),6.92(d,J=8.6Hz,2H),5.04( s,2H),3.71(s,3H),3.03(dd,J=13.6,5.2Hz,1H),2.82(dd,J=13.6,7.7Hz,1H),1.58(s,2H). 13 C NMR (100MHz, CDCl3) δ175.55,157.82,137.08,130.35,129.47,128.65,128.02,127.53,115.00,70.06,55.97,52.04,40.22.

[0132] Compound 3 (1.8 mmol) was dissolved in 5 mL of DMF, and DIPEA (3.5 mmol) and methyl bromoacetate (1.8 mmol) were added. The reaction was carried out under nitrogen protection and stirred at room temperature for 3 h until complete reaction (TLC, PE:EA = 2:1). 10 mL of water was added to the mixture, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation of the filtrate. The filtrate was then dried under vacuum to give compound 4 (a yellow oil, yield 97%). 1 H NMR (400MHz, CDCl3) δ7.47-7.28(m,5H),7.13(d,J=8.6Hz,2H),6.92(d,J=8.6Hz,2H),5.04(s ,2H),3.68(d,J=6.5Hz,6H),3.58(t,J=6.7Hz,1H),3.40(q,J=17.2Hz,2H),3.02-2.90(m,2H). 13 C NMR (100MHz, CDCl3) δ174.15,172.11,157.78,137.10,130.26,129.17,128.60,127.97,127.53,114.91,69.98,62.25,51.86,49.05,38.66.

[0133] Compound 4 (2.8 mmol) was dissolved in 56 mL of methanol, followed by the dropwise addition of diethylenetriamine (2.8 mmol). The mixture was heated under nitrogen protection and refluxed in an oil bath at 60 °C for 11 days. The reaction was monitored by HPLC, and the reaction limit was approximately 70%. Most of the methanol was removed by rotary evaporation, and the mixture was filtered. The filter cake was washed 2–3 times with ice-cold ethanol and dried at 50 °C to obtain compound 5 (white solid, yield 21%). 1 HNMR (400MHz, CDCl3) δ7.50 (s, 1H), 7.47-7.34 (m, 5H), 7.32 (t, J = 7.1Hz, 1H), 7.1 4(d,J=8.2Hz,2H),6.93(d,J=8.2Hz,2H),5.04(s,2H),3.49(dtd,J=37.6,13.2,5. 6Hz,3H),3.32(dd,J=10.2,4.0Hz,1H),3.18(dd,1H),3.11(m,1H),3.06-2.83(m, 4H), 2.65 (qd, J=10.0, 4.8Hz, 3H), 1.92 (d, J=7.8Hz, 1H), 1.39 (d, J=101.6Hz, 1H). 13 C NMR (100MHz, CDCl3) δ173.52,171.78,158.01,136.93,130.14,129.18,128.67,128.0 9,127.54,115.35,70.11,67.55,53.69,45.25,44.96,39.04,38.22,37.48.(m / z)for C 22 H 28 N4O3:Calcd, 419.2 [M+Na] + ;found, 419.2[M+Na] + .

[0134] In a nitrogen atmosphere and under ice bath conditions, compound 5 (2.5 mmol) was dissolved in borane-tetrahydrofuran (1 M borane, 60 mL), heated to 70 °C and refluxed for 24 h. After cooling to room temperature, methanol was added under ice bath conditions until no more bubbling occurred. An equal volume of 1 M hydrochloric acid was then added, and the mixture was refluxed at 70 °C for 2 h. Once the reaction solution cooled to room temperature, potassium carbonate was added to adjust the pH to 8. The mixture was extracted three times with dichloromethane (30 mL each time), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated by rotary evaporation to remove the solvent. The filtrate was then dried under vacuum to obtain compound 6 (a yellow oil, 86% yield). 1HNMR (400MHz, D2O) δ6.95 (m, 7H), 6.63 (d, J = 8.3Hz, 2H), 4.47 (s, 2H), 3.23-2.89 (m, 10H), 2.87-2.65 (m, 6H), 2.51 (d, J = 8.4Hz, 1H). 13 C NMR(100MHz,D2O)δ157.32,136.62,130.18,128.38,128.29,127.78,127.47,1 15.11,69.53,55.24,47.22,44.08,43.72,43.43,43.08,42.58,40.97,35.54.

[0135] Compound 6 (3.0 mmol) was dissolved in 10 mL of acetonitrile under a nitrogen atmosphere. Potassium carbonate (29.8 mmol) and ethyl bromoacetate (18.0 mmol) were added sequentially, and the mixture was stirred at room temperature for 18 h. After the reaction was complete, the mixture was filtered, and the solvent was removed by evaporation under reduced pressure. The filtrate was dissolved in 30 mL of EA, washed three times with 1 M dilute hydrochloric acid (15 mL each time), and the pH was adjusted to 8 with potassium carbonate. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation of the filtrate to give compound 7 (a pale yellow oil). (m / z) for C 33 H 54 N4O9:Calcd, 713.4 [M+H] + and 735.4 [M+Na] + ;found, 713.4 and 735.4.

[0136] Compound 7 (1.7 mmol) was dissolved in 30 mL of ethanol, and 20 wt% Pd-C (0.24 g) was added. After removing air, the mixture was heated to 55 °C and refluxed for 18 h under a hydrogen atmosphere of 2 MPa. After the reaction was complete, Pd-C was removed by filtration, and the organic solvent was evaporated to dryness to give compound 8 (yellow oil, 96% yield), which required no further purification. (m / z) for C 31 H 50 N4O9:Calcd, 623.4 [M+H] + and 645.3 [M+Na] + ; found, 623.4 and 645.5.

[0137] Compound 8 (1.64 mmol) was dissolved in DMF (5 mL), followed by the addition of potassium carbonate (9.83 mmol) and bromoethane (4.90 mmol). The mixture was reacted at room temperature for 20 h under a nitrogen atmosphere. 10 mL of water was added to the mixture, and the mixture was stirred until homogeneous. The mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, and the organic solvent was removed by rotary evaporation of the filtrate to give compound 9 (a yellow oil, yield 66%). (m / z) for C 33 H 54 N₄O₉:Calcd, 673.4 [M+Na] + ;found,673.5.

[0138] Synthesis of ligand L1: Compound 7 (1 mmol) was dissolved in a mixed solution containing 5.5 mmol of lithium hydroxide aqueous solution (1 g: 7.5 mL) and ethanol (8 mL), and reacted at room temperature for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and 7 mL of ethanol and 7 mL of methyl tert-butyl ether were added. The mixture was stirred for 15 min, allowed to stand for 5 min, and centrifuged to remove the liquid. The resulting white solid was dried at 50 °C to obtain ligand L1, with a yield of 83%.

[0139] Synthesis of ligand L2: The only difference from the synthesis of ligand L1 was that compound 7 was replaced with compound 8, with a yield of 72%.

[0140] Synthesis of ligand L3: The only difference from the synthesis of ligand L1 was that compound 7 was replaced with compound 9, with a yield of 74%.

[0141] Ligand L1: 1 H NMR(400MHz,D2O)δ7.54-7.35(m,5H),7.28-7.16(m,2H),7.04(s,2H),5.16(s,2H),4.21-2.46(m,27H). 13 C NMR (100MHz, DMSO) δ173.46,173.43,172.01,171.83,157.15,142.73,137.26,130.40,129.68,128.58,127.99,127.87 ,114.91,69.31,64.29,54.48,54.36,54.32,53.95,53.87,53.79,53.74,53.65,53.10,53.05,51.20,31.01.(m / z)for C 30 H 40 N4O9:Calcd, 301.1 [M+2H] 2+ / 2and 601.3[M+H] +; found, 301.2 and 601.3.

[0142] Ligand L2: 1 H NMR (400 MHz, D2O) δ 7.22 (t, J = 7.9 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 4.20 (s, 1H), 4.09 (q, J = 7.0 Hz, 3H), 4.02 - 2.49 (m, 24H), 1.35 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, D2O) δ 169.37, 169.08, 168.94, 168.75, 157.09, 130.46, 129.14, 115.25, 64.41, 55.90, 55.68, 54.67, 54.31, 53.13, 51.90, 51.64, 49.86, 49.21, 47.38, 46.79, 46.13, 30.73, 13.92. (m / z) for C 25 H 38 N4O9: Calcd, 270.1 [M + 2H] 2+ / 2 and 539.3 [M + H] + ; found, 270.2 and 539.4.

[0143] Ligand L3: 1 H NMR (400 MHz, D²O) δ 7.18 (d, J = 7.7 Hz, 2H), 6.88 (d, J = 5.6 Hz, 2H), 3.80 - 2.90 (m, 2³H), 2.62 (d, J = 11.6 Hz, 1H). 13 C NMR (100 MHz, D2O) δ 176.88, 175.31, 170.89, 170.13, 154.53, 130.14, 128.55, + 115.84, 59.58, 57.94, 56.63, 55.84, 52.29, 51.16, 49.7², 49.55, 46.41, 46.16, 45.88, 45.74, 30.59. (m / z) for C 23 H 34 N4O9: Calcd, 517.2 [M + Li] + , 529.2 and 533.2; found, 517.4 [M + Li] + , 529.4 and 533.3.

[0144] Note: In the translation of the NMR data, for better understanding in the English context, it might be more common to write something like "1H at 4.20 (singlet)", "3H at 4.09 (quartet, J = 7.0 Hz)" etc. But according to the strict rules, the original format is maintained. Also, the chemical shift values and coupling constants are kept as they are in the original text. And the chemical formula and mass spectrometry data are translated as accurately as possible while keeping the original notations. The -

[0144] tags are left unchanged as required.Preparation of complex Gd-L1: Ligand L1 (1 mmol) was dissolved in 15 mL of weakly acidic water at pH 6.0 (adjusted with 1 M dilute hydrochloric acid) to obtain an aqueous solution of ligand L1; LnCl3·6H2O (1 mmol) was dissolved in 0.2 mL of water to obtain an aqueous solution of LnCl3. The aqueous solution of LnCl3 was added dropwise to the aqueous solution of ligand L1, and then the pH of the reaction solution was adjusted to neutral with 1 M sodium hydroxide solution. The mixture was refluxed at 100 °C for 20 h. After the reaction was complete, the pH was adjusted to 11 with 1 M sodium hydroxide to precipitate excess metal ions. The reaction solution was centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was adjusted to pH 7 with 1 M hydrochloric acid and then lyophilized to obtain complex Gd-L1.

[0145] Preparation of complex Gd-L2: The only difference from the preparation of complex Gd-L1 is that ligand L1 is replaced with ligand L2.

[0146] Preparation of complex Gd-L3: The only difference from the preparation of complex Gd-L1 is that ligand L1 is replaced with ligand L3.

[0147] Preparation of complex Eu-L1: The only difference from the preparation of complex Gd-L1 is that LnCl3·6H2O is replaced with EuCl3·6H2O.

[0148] Preparation of complex Eu-L2: The only difference from the preparation of complex Eu-L1 is that ligand L1 is replaced with ligand L2.

[0149] Preparation of complex Eu-L3: The only difference from the preparation of complex Eu-L1 is that ligand L1 is replaced with ligand L3.

[0150] Complex Gd-L1: High resolution of ESI-MS (m / z) for C 30 H 36 GdN4O9:Calcd, 754.1723 [M] － ;found,754.1726.

[0151] Complex Gd-L2: High resolution of ESI-MS (m / z) for C 25 H 34 GdN4O9:Calcd, 692.1567 [M] － ;found,692.1579.

[0152] Complex Gd-L3: High resolution of ESI-MS (m / z) for C 23 H 30GdN4O9:Calcd, 664.1254 [M] － ;found,664.1262.

[0153] Complex Eu-L1: High resolution of ESI-MS (m / z) for C30H36EuN4O9: Calcd, 749.1695 [M] － ;found,749.1693.

[0154] Complex Eu-L2: High resolution of ESI-MS (m / z) for C25H34EuN4O9: Calcd, 687.1538 [M] － ;found,687.1541.

[0155] Complex Eu-L3: High resolution of ESI-MS (m / z) for C23H30EuN4O9: Calcd, 659.1225 [M] － ;found,659.1238.

[0156] Figure 2 For ligand L 1～3 The HPLC chromatogram at a wavelength of 254 nm shows that ligand L... 1～3 The retention times were 5.886 min, 4.79 min, and 2.469 min, respectively. Figures 2-3 HPLC detection conditions: Mobile phase A was 0.05 vol% trifluoroacetic acid aqueous solution (mobile phase A), mobile phase B was acetonitrile, flow rate was 1 mL / min, and the chromatographic column was a Waters C18 column (5 μm, 4.6 × 150 mm); gradient elution program: 0–10 min, the volume fraction of mobile phase B increased from 10% to 100%; 10–12 min, the volume fraction of mobile phase B decreased from 100% to 10%; 12–15 min, the volume fraction of mobile phase B was 10%.

[0157] Figure 3 For the complex Gd-L 1～3 and Eu-L 1～3 The HPLC chromatogram at a wavelength of 254 nm shows that the complex Gd-L 1～3 and Eu-L 1～3 Both exhibit two absorption peaks, with average retention times of Gd-L, respectively. 1～3 The retention times were 5.642 min, 4.408 min, and 2.539 min, respectively, for Eu-L. 1～3The retention times were 5.642 min, 4.376 min, and 2.351 min, respectively.

[0158] Figure 4 The HPLC chromatograms of the complexes Gd-L1 and Eu-L1 before semi-preparative high-performance liquid chromatography (HPLC) separation, and the angular and edge isomers after separation, at a wavelength of 254 nm, show that the two absorption peaks of the complexes are independent and separable, with corresponding independent peak positions on the HPLC chromatogram.

[0159] Figure 5 The images show the ESI-MS mass spectra of the mixture of complexes Gd-L1 and Eu-L1 before semi-preparative high-performance liquid chromatography (HPLC) separation, and the separated angular and edge isomers. It is evident that the mass-to-charge ratios of the two absorption peaks in the complex are identical. Figures 4-5 The semi-preparative high-performance liquid chromatography (HPLC) separation conditions were as follows: mobile phase A was a 10 mM ammonium acetate aqueous solution, mobile phase B was a mixture of 10 mM ammonium acetate aqueous solution and acetonitrile at a volume ratio of 1:9, and the flow rate was 1 mL / min; the chromatographic column was a Waters C18 column (5 μm, 4.6 × 150 mm); the gradient elution program was as follows: 0–10 min, the volume fraction of mobile phase B increased from 10% to 100%; 10–12 min, the volume fraction of mobile phase B decreased from 100% to 10%; 12–15 min, the volume fraction of mobile phase B was 10%.

[0160] Figure 6 An angular isomer of the complex Eu-L1 1 HNMR (400MHz, D2O) spectrum, Figure 7 The edge isomer of the complex Eu-L1 1 The HNMR (400MHz, D2O) spectrum shows that most of the hydrophilic isomers in the complex are SAP (tetragonal antiprism), while the hydrophilic isomers, SAP and TSAP (twisted tetragonal antiprism), are equally represented.

[0161] Test Example 1

[0162] (1) Relaxation rate test

[0163] Relaxation rate is a key indicator for evaluating the performance of contrast agents. The relaxation rate of contrast agent molecules is typically affected by temperature, electric field strength, and the solute in the solution. The relaxation rates (r1 and r2) of the contrast agent molecules in this invention were measured using a 1.4T magnet at a constant temperature of 37°C in PBS (pH = 7.4, r1) and ultrapure water (r2), respectively. As shown in Table 1, Gd-EOB-DTPA, as a linear contrast agent molecule, compared to macrocyclic complexes (Gd-L... 1～3 It has a higher relaxation rate. Gd-L1～3 Due to the addition of different substituents, it exhibits a higher relaxation rate compared to Gd-DOTA. Further testing of the relaxation rate in the presence of 4.5% HSA showed that Gd-L... 1-3 It has varying degrees of integration with HSA.

[0164] Table 1 Gd-L 1～3 Relaxation rate parameters (mM) at 1.4T -1 s -1 )

[0165]

[0166] The relaxation rate parameters of Gd-DOTA at 1.4T are shown in references [1] and [2].

[0167] Literature [1]: Xu, W.; Ye, X.; Wu, M.; Jiang, X.; Hugo Tse, LH; Gu, Y.; Shu, K.; Xu, L.; Jian, Y.; Mo, G.; a Versatile Platform for Hepatobiliary and Tumor Targeting MRIContrastAgents.J.Med.Chem.2023,66(21),14669-14682.

[0168] Literature [2]: Xu, W.; Lu, Y.; Xu, J.; Li, H.; Lan, R.; Gao, R.; Ding, Y.; Ye, X.; Shu, K.; Ye, F.; Yan, Z.; Dai, L., Rational Design of Gd-DOTA-Type Contrast Agents for Hepatobiliary Magnetic Resonance Imaging.J.Med.Chem.2023,66(13),8993-9005.

[0169] (2) Dynamic inertia test

[0170] Ideal magnetic resonance imaging contrast agents require extremely high kinetic inertness to avoid Gd(III) dissociation and minimize associated toxicity. Metal dissociation occurs through two main mechanisms: acid-assisted metal dissociation and ion- or ligand-competitive transmetalation. Therefore, the kinetic stability of monosubstituted cyclic rare earth complexes was assessed using relaxation methods in the presence of 1M HCl (i.e., 0.1M dilute hydrochloric acid) or excess Zn(II), with Gd-DOTA and Gd-EOB-DTPA as controls. The degree of Gd(III) dissociation was assessed by comparing the relaxation time at each time point with the initial values. The results showed that after 7 days of incubation at room temperature and 1M HCl, Gd-L... 1～3 63-79% of the complex was retained, while Gd-DOTA lost 51% within 24 hours. Figure 8 (a) and (b)). It has been reported that Gd-EOB-DTPA has a half-life of less than 5 seconds in 0.1 M HCl. To study the migration kinetics of the contrast agent, a mixture containing 1.0 mM Gd-L... 1～3 Metal transfer experiments were performed by incubating the sample with 10 mM Zn(II) in PBS buffer (pH = 7.4, 0.01 M) at 37°C for 7 days. No Gd-L was observed. 1～3 There was significant dissociation, while Gd-EOB-DTPA almost completely dissociated within 12 hours. Figure 8 (c) and (d)). Gd-L 1～3 It exhibits extremely high stability under strongly acidic conditions and has significant Zn(II) anti-interference ability, making it suitable for in vivo studies.

[0171] (3) Liver uptake pathway test

[0172] The interactions between transport proteins (OATP1B1, OATP1B3, OATP2B1, and NTCP) and their complexes were assessed using AutoDock Site65. The results are shown in Table 2 and... Figure 9 As shown, where, Figure 9 Schematic diagrams illustrating the interactions of Gd-L1 with OATP1B1(a), OATP1B3(b), OATP2B1(c), and NTCP(d); Schematic diagrams illustrating the interactions of Gd-L2 with OATP1B1(e), OATP1B3(f), OATP2B1(g), and NTCP(h); Schematic diagrams illustrating the interactions of Gd-L3 with OATP1B1(i), OATP1B3(j), OATP2B1(k), and NTCP(l); The PDB code for OATPs is 2GFP. Hydrophobic interactions, hydrogen bonds, and salt bridges between the contrast agent and the transporter are represented by gray, blue, and yellow dashes, respectively. (Based on Table 2 and...) Figure 9It was found that OATP1B3 has a strong affinity for Gd-EOB-DTPA, with a docking score of -7.95 kcal / mol, while OATP1B1 has a similar affinity for Gd-L1, with a docking score of DS = -7.74 kcal / mol. On the other hand, Gd-L2 and Gd-L3 exhibited low hepatic uptake characteristics.

[0173] This is reflected in their high inhibition constant (DS, kcal / mol) and low docking fraction (KI, μM). During drug-protein interactions, Gd-EOB-DTPA forms strong hydrophobic bonds, hydrogen bonds, and salt bridges with organic anion transport peptides OATP1B1 and OATP1B3. The results highlight the significant advantage of Gd-L1 in hepatic uptake, primarily via OATP1B1, OATP2B1, and NTCP (sodium-taurocholate cotransporter), which are the main pathways for hepatic uptake. A comprehensive understanding of the interaction mechanisms between these transporters and their complexes holds promise for developing more efficient and targeted liver-specific drugs.

[0174] Table 2. Docking fraction (DS, kcal / mol) and inhibition constant (KI, μM) of complexes to transport proteins

[0175]

[0176] Reference [3]: Journal of Medicinal Chemistry, 2023, 66, 14669-14682.

[0177] (4) The CCK-8 assay was used to evaluate the cytotoxicity of the chelate to LO2 and 293T cells.

[0178] Contrast agents are primarily absorbed by the liver and kidneys after intravenous injection via the tail vein. Therefore, in this invention, the CCK-8 assay was used to evaluate the effect of the chelate on LO2 (… Figure 10 (a) and 293T ( Figure 10 (b) Cell cytotoxicity was assessed, with the commercial contrast agent Gd-EOB-DTPA used as a control. Cells were compared with three different Gd-L complexes. 1～3 (≤0.8mM) was incubated at 37℃ for 24h, followed by incubation with 10% CCK-8 solution for 4h. The optical density was then measured using a microplate reader to assess cell viability. Error bars represent standard deviation (±SD) (n=3). The survival rate of LO2 cells treated with Gd-L1 remained above 90% at a concentration of 0.5mM. Even at a high concentration of 0.8mM, the three complex contrast agents Gd-L1... 1～3After treatment, cell viability remained above 80%. Among these, Gd-L1 showed significantly reduced cytotoxicity to kidney cells compared to Gd-EOB-DTPA, suggesting it may be a safer option for magnetic resonance imaging (MRI) scans in patients with impaired renal function.

[0179] (5) In vivo MRI studies of normal mice

[0180] All experiments in this invention adhered to the animal ethics guidelines of our institution and were approved by the Animal Care and Use Committee of the Wenzhou Institute of the Chinese Academy of Sciences. Magnetic resonance imaging (MRI) was performed using a 3.0T clinical MRI scanner (Ingeniaelition, Philips) equipped with a mouse-specific coil. BALB / c mice (6–8 weeks old, 20–25g) were administered the drug via tail vein injection (0.1 mmol / kg). T1-weighted continuous dynamic contrast-enhanced MRI was performed before and 35 minutes after drug administration to observe signal enhancement in the liver and kidneys. Figure 11 (a) shows a coronal image of the liver (the mouse liver is within the yellow solid box), and (b) shows a coronal image of the kidney (the mouse kidney is within the yellow solid box). Pre, 1 min, 6 min, 12 min, 18 min, 23 min, 29 min, and 35 min represent the detection times before and after drug administration, respectively. (c) shows the normalized liver signal-to-noise ratio (nSNR), (d) shows the normalized kidney signal-to-noise ratio, and (e) shows the contrast-to-noise ratio of the liver relative to muscle (ΔCNR). The commercial contrast agent Gd-EOB-DTPA, after injection, rapidly distributes to the liver and kidneys, with enhancement time in the liver exceeding 35 min, and is rapidly metabolized within the body. In MR images obtained by injecting the complex Gd-L1 as an MRI contrast agent into mice, Gd-L1 was selectively distributed to the liver and kidneys. The contrast enhancement time in the liver exceeded 35 minutes, and it was largely cleared from the kidneys within 35 minutes. Its hepatobiliary imaging capability was comparable to Gd-EOB-DTPA, showing potential as a hepatobiliary-specific MRI contrast agent. In MR images obtained by injecting the complex Gd-L2 as an MRI contrast agent into mice, Gd-L2 was selectively distributed to the liver and kidneys, but the contrast enhancement in the liver was less than that of Gd-L1. In MR images obtained by injecting the complex Gd-L3 as an MRI contrast agent into mice, almost no contrast enhancement in the kidneys was observed, making it unsuitable as a hepatobiliary-specific MRI contrast agent.

[0181] (6) Hepatic absorption mechanism of Gd-L1

[0182] Previous studies have confirmed that hepatic absorption of bromosulfonphthalein (BSP) is also mediated by OATPs. BSP was used as a competitive reagent to verify the hepatic absorption mechanism of Gd-L1. Figure 12T1-weighted images of the liver (a) and kidney (b) of normal Balb / c mice after injection of Gd-L1 (0.1 mmol / kg) at 3.0T are shown. The normalized signal-to-noise ratio (nSNR) of the liver (c) and kidney (d) over 35 min is shown as a function of time. Prior to Gd-L1 injection, BSP was repeatedly injected via the tail vein to maintain a plasma concentration of 0.1 mM. Because BSP occupies the OATP transport channel, hepatocyte uptake of Gd-L1 was significantly inhibited. Comparative analysis showed that the BSP(+) group exhibited the least liver enhancement within 20 min, in stark contrast to the BSP(-) group. Given that the contrast agent has a lifetime in vivo exceeding 35 min, even the slight liver enhancement became apparent due to the rapid metabolism of BSP. Reduced hepatic excretion led to a significant increase in clearance and renal metabolic cycle. Therefore, compared with the BSP(-) group, the nuclear magnetic resonance imaging signal (nSNR) of both the renal cortex and medulla increased rapidly and significantly in the BSP(+) group. Based on these findings, it is confirmed that the expression of OATPs on the hepatocyte membrane surface is the main pathway for hepatocyte uptake of Gd-L1.

[0183] (7) Hepatobiliary imaging test

[0184] The complex Gd-L1 possesses hepatobiliary imaging capabilities comparable to Gd-EOB-DTPA, but with significantly superior stability. To further explore its potential in hepatobiliary imaging, a mouse H22 orthotopic hepatocellular carcinoma model was used to investigate its advantages and disadvantages in detecting mouse hepatocellular carcinoma. Images of Gd-L1 injected via the tail vein and then visualized under 3.0T MRI were obtained, along with hepatobiliary MR images. Figure 13 ), where pre, 1min, 6min, 12min, 18min, 23min, 29min, 35min, 40min, 3min, 9min, 14min, 20min, 26min, 32min, 38min, and 43min are the corresponding detection times before and after drug administration, respectively. Figure 13 In the left image, the first row shows the coronal plane of the liver, the second row shows the transverse plane, and the right image shows the CNR (liver to tumor) versus time in both the coronal and transverse planes. According to... Figure 13 As shown in Table 3, Gd-L1 significantly enhances the contrast between normal and diseased liver tissues. After enhancement, the SI ratio between liver parenchyma and diseased tissues is significantly increased, and the lesion boundaries are clearly defined, with a long duration of action. Table 3: Magnetic resonance signal values ​​of Gd-L1 as a hepatobiliary-specific magnetic resonance imaging contrast agent for detecting orthotopic hepatocellular carcinoma in mice under 3.0T MRI.

[0185]

[0186] To minimize random errors in the experiment and ensure the accuracy and relevance of the obtained imaging data, adult healthy rats weighing approximately ten times that of mice were selected as experimental subjects to evaluate the potential of Gd-L1 as a liver-specific contrast agent. Figure 14 T1-weighted images of the liver (a) and kidney (b) in a transverse plane were captured at 3.0T in normal rats (Wistar, 6–8 weeks old) before injection of Gd-EOB-DTPA and Gd-L1 (0.1 mmol / kg), and at 1 min, 10 min, and 20 min after injection. The nSNR time progression of the liver (c) and kidney (d) after contrast agent injection was also recorded. Data are expressed as mean ± standard deviation (n = 3). MRI scans were performed before and after intravenous injection of Gd-L1 (0.1 mmol / kg) using a rat-specific coil in a 3.0T clinical MRI apparatus. Notably, Gd-L1 accumulated rapidly in the liver and kidneys, with consistent pharmacokinetic characteristics in rats and mice. The enhancing effect of Gd-L1 on liver tissue was comparable to that of Gd-EOB-DTPA. This study demonstrates the potential of Gd-L1 in MRI applications involving large animals and lays the foundation for further in-depth research into its clinical applications.

[0187] (8) Biodistribution within the body

[0188] To investigate the biodistribution of Gd-EOB-DTPA and Gd-L1 in vivo, inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850) was used to quantitatively determine the Gd(III) content in blood and various organs at specified time points after intravenous injection of each contrast agent at 0.1 mmol / kg. Figure 15(a) shows the biodistribution of Gd-EOB-DOTA and Gd-L1 (0.1 mmol / kg) in normal mice, expressed as the percentage of Gd in each tissue. (b) shows the total uptake of the complex in the five viscera and brain at 5 min, 15 min, and 24 h post-injection. Data are expressed as mean ± standard deviation (n = 3). ICP-MS analysis showed that Gd-EOB-DTPA accumulated rapidly in the liver, reaching 129.12 ± 16.94 μg / g within 5 min post-injection, accounting for 44.66 ± 2.98% of the administered dose. After 15 min, this level decreased to 32.47 ± 2.08% of the dose. Similarly, Gd-L1 also showed rapid hepatic accumulation, reaching 102.32 ± 6.73 μg / g tissue within 5 min, accounting for 40.94 ± 1.47% of the administered dose. After 15 minutes, the metabolism of Gd-L1 reduced the dose to 30.98 ± 8.83%, which is statistically comparable to the dose of Gd-EOB-DTPA. Notably, 24 hours after injection, the residual Gd(III) levels in all tested tissues were less than 1%, reducing potential harm to human functional organs and the immune system. In summary, these results indicate that the excretion pathway of Gd-L1 is similar to that of Gd-EOB-DTPA, with high liver accumulation and rapid clearance by the liver and kidneys. Therefore, it holds promise as a Gd(III)-based liver-specific contrast agent for magnetic resonance imaging applications.

[0189] (9) Potential toxicity test of free Gd(III) to major organ functions

[0190] Safety concerns regarding free Gd(III) remain a major challenge for the clinical application of contrast agents. To assess the potential toxicity of Gd-L1, a comprehensive study was conducted on the effects of Gd-DOTA and PBS on the major organ functions of healthy mice. This included serum biochemical assays and histological examination using hematoxylin and eosin (H&E) staining. Acute-phase mice received a single injection (1 day), while subacute-phase mice (60 days) received injections every two weeks for two consecutive months. Plasma and tissue samples were collected 24 hours after the last administration. Figure 16 Histopathological analysis (a) and blood biochemical analysis (b) of acute (1 day) and subacute (60 days) Gd-DOTA and Gd-L1 poisoning were performed, with an equal volume of PBS as a control. ALT was alanine aminotransferase; ALB was albumin; ALKP was alkaline phosphatase; AST was aspartate aminotransferase; BUN was blood urea nitrogen; LDH was lactate dehydrogenase; and CK was creatine kinase. Data are expressed as mean ± standard deviation (n = 3), and there was no significant difference from PBS treatment. Figure 16As shown in Figure (b), no mouse deaths or adverse reactions were observed throughout the experiment. Several important liver function indicators (ALT, AST, ALB) and kidney function indicators (BUN, CR) were measured. Figure 16 As shown in (a), these serum biochemical indicators did not change significantly compared to the control group, indicating no impairment of liver and kidney function. Furthermore, histological examination by H&E staining revealed no significant abnormalities compared to the control group. Cardiac tissue showed normal morphology, size, and arrangement of cardiomyocytes, with no signs of inflammation or necrosis. In the liver, although mild hepatic congestion was observed in the central vein of the Gd-DOTA-treated group, Gd-L1-treated mice showed normal hepatic lobular structure, with no signs of liver damage or inflammation. Spleen tissue showed normal white and red serous fluid, indicating healthy spleen function. Lung tissue showed normal respiratory bronchi, alveolar ducts, alveolar sacs, and lung parenchyma. In kidney tissue, renal tubules and glomeruli showed normal morphology, with no obvious damage or inflammation. In conclusion, Gd-L1 did not cause functional damage to the heart, liver, spleen, lungs, or kidneys of mice within the prescribed time, demonstrating good biocompatibility. These findings strongly demonstrate the safety and efficacy of Gd-L1 as a potential contrast agent for magnetic resonance imaging.

[0191] (10) As an activatable myeloperoxidase magnetic resonance imaging probe

[0192] To assess the sensitivity of Gd-L3 to peroxidase, the longitudinal (T1) and transverse (T2) relaxation rates of Gd-L3 (0.5 mM) solution (PBS, pH = 7.4, 0.1 M) at different horseradish peroxidase (HRP) activities (0 U, 5 U, 50 U, 500 U) were monitored at 37 °C and 1.4 T. Hydrogen peroxide (H2O2, 5 mM) was added sequentially. Figure 17 (a) longitudinal and (b) transverse). Therefore, the relaxation rates at 5 U, 50 U, and 500 U of HRP were 3.16, 3.36, and 3.55 times higher than those without HRP, respectively. Notably, even in the presence of only 5 U HRP and 1 eq of H2O2, the R2 value increased by 2.8 times. This indicates that Gd-L3 exhibits extremely high responsiveness to HRP, even at low enzyme activity and trace oxidant concentrations. The relaxation rate of Gd-L3 in the presence of H2O2 / HRP (HRP, 5 U, H2O2, 2 eq) was also tested, showing r1 = 17.35 mM. -1 s -1 r2 = 18.80 mM -1 s -1 It is about 3.8 times higher than its monomeric form. Figure 17(c)). Gd-L3 oligomers have a low affinity for HSA. Figure 17 This may promote its rapid clearance from the body. These results collectively indicate that the enhanced relaxation rate of Gd-L3 and its responsiveness to peroxidase make it an ideal candidate for peroxidase-responsive MRI applications. Magnetic resonance imaging was performed using a 3.0T clinical MRI (Discovery MR750, GE MEDI contrast system) with a mouse-specific coil. In an MSU-induced acute gout Swiss mouse model (6–8 weeks, 20–25 g), Gd-L2 and Gd-L3 (0.1 mmol / kg) were injected intravenously via the tail vein. T1-weighted continuous dynamic contrast-enhanced magnetic resonance imaging was performed before and 20 min after administration to observe the MRI signal enhancement in the left and right hind limbs. Figure 17 Images (e) and (f) show hepatobiliary MRI images obtained from mice using complexes Gd-L2 and Gd-L3 as MRI contrast agents for activating MPO. Pre, 2 min, 5 min, 8 min, 10 min, 15 min, and 20 min represent the detection times before and after drug administration, respectively. It was observed that both complexes produced selective contrast enhancement at the site of gout lesions in mice after ingestion, with Gd-L3 providing a more pronounced contrast enhancement compared to Gd-L2. Figure 17 (g) and (h) have the potential to serve as activatable myeloperoxidase magnetic resonance imaging probes for detecting myeloperoxidase aggregation in vivo and in vitro.

[0193] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monosubstituted cyclic ligand having the structure shown in Formula I: Formula I; in, R represents benzyloxy or hydroxyl; the wavy line indicates the R configuration or S configuration.

2. The method for preparing the monosubstituted cyclic ligand according to claim 1, comprising the following steps: The monosubstituted cyclic ester compound was subjected to ester group hydrolysis under alkaline conditions to obtain the monosubstituted cyclic ligand. The monosubstituted cyclic ester compounds include compound 7 or compound 8: 。 3. The preparation method according to claim 2, characterized in that, The preparation method of compound 7 includes the following steps: Compound 1 was reacted with benzyl bromide under basic conditions to undergo a first nucleophilic substitution reaction to give compound 2; Compound 2 was subjected to an amide hydrolysis reaction under acidic conditions to obtain compound 3; Compound 3 was reacted with methyl bromoacetate under alkaline conditions to undergo a second nucleophilic substitution reaction to obtain compound 4; Compound 4 was subjected to an ester exchange reaction with diethylenetriamine to obtain compound 5; Compound 5 was subjected to an amide reduction reaction in the presence of a reducing agent to obtain compound 6; Compound 6 was reacted with ethyl bromoacetate under alkaline conditions to undergo a third nucleophilic substitution reaction to obtain compound 7; 。 4. The preparation method according to claim 2, characterized in that, The preparation method of compound 8 includes the following steps: Compound 7 was subjected to a hydrogen debenzylation reaction in the presence of a catalyst and a reducing agent to obtain compound 8.

5. A monosubstituted cyclic rare earth complex having the structure shown in Formula II: Formula II; in, R represents benzyloxy or hydroxyl; Ln represents Gd(Ⅲ); the wavy line indicates the R configuration or S configuration.

6. The method for preparing the monosubstituted cyclic rare earth complex according to claim 5, comprising the following steps: The monosubstituted cyclic ligand is subjected to a coordination reaction with a rare earth metal source to obtain the monosubstituted cyclic rare earth complex; the monosubstituted cyclic ligand is the monosubstituted cyclic ligand according to claim 1 or the monosubstituted cyclic ligand prepared by the preparation method according to any one of claims 2 to 5. The rare earth metal source is Gd(Ⅲ).

7. The application of the monosubstituted cyclic rare earth complex of claim 5 or the monosubstituted cyclic rare earth complex prepared by the preparation method of claim 6 in the preparation of contrast agents.

8. The application according to claim 7, characterized in that, The contrast agent is a contrast agent for hepatobiliary magnetic resonance imaging and / or inflammatory magnetic resonance imaging.

9. The application of the monosubstituted cyclic rare earth complex of claim 5 or the monosubstituted cyclic rare earth complex prepared by the method of claim 6 in the detection of peroxidase in the diagnosis and treatment of non-diseases, wherein R of the monosubstituted cyclic rare earth complex is a hydroxyl group and Ln is Gd(III).

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