Monosubstituted cyclic ligand, and preparation method therefor and use thereof, and monosubstituted cyclic rare earth complex, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a monosubstituted cyclic ligand, and a preparation method therefor and a use thereof, and a monosubstituted cyclic rare earth complex, and a preparation method therefor and a use thereof, relating to the technical field of contrast agents. The monosubstituted cyclic ligand uses the ring structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as a parent ring, and an R-modified tyrosine group increases the structural rigidity of the parent ring and improves the stability of the complex. Direct introduction of benzyloxy-, ethoxy- or hydroxy-substituted benzyl substituents on the carbon atoms of the DOTA ring maintains the original eight-coordination structure, and improves the relaxation rate of the complex, in addition to increasing the stability of the complex. Compared with Gd-DOTA, both the kinetic inertia and the relaxation rate are improved. The complex formed by the ligand and Gd(III) or Eu(III) is highly stable, and has strong specificity for the liver or for inflammation, and causes no obvious damage to the heart, the liver, the spleen, the lungs or the kidneys.

Description

A monosubstituted cyclic ligand, a preparation method and application thereof, and a monosubstituted cyclic rare earth complex, a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of contrast agents, in particular to a monosubstituted cyclic ligand, a preparation method and application thereof, and a monosubstituted cyclic rare earth complex, a preparation method and application thereof. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is to obtain electromagnetic signals through the magnetic resonance phenomenon of atomic nuclei under the action of a magnetic field, and to reconstruct images through a computer. Compared with computed tomography (CT) and positron emission computed tomography (PET) imaging techniques, MRI does not cause radiation damage to the human body. However, the detection sensitivity of MRI is low, and about 40% of MRI scans need to use contrast agents to improve the detection signal, and about 40 million people worldwide use MRI contrast agents every year.

[0003] At present, the FDA publicly certified contrast agents, including the one just approved in 2022 and the one entering clinical phase I, are Gd-based contrast agents. The currently used contrast agent molecules in the clinic can be divided into two categories: linear and non-linear. Linear molecules such as Gd-DTPA, Gd-EOB-DTPA and Gd-BOPTA, most of which are non-specific magnetic resonance contrast agents, are linear contrast agents. The only two liver-specific magnetic resonance contrast agents are linear contrast agents. Currently, commercially available liver-specific MRI contrast agents are mostly linear Gd-DTPA derivatives, such as Gd-EOB-DTPA, Primovist / Eovist and Primovist / Eovist. The above-mentioned commercially available Gd-based MRI contrast agents have the problems of poor stability and poor targeting, which may cause serious health risks.

[0004]

[0005] Chinese patent CN114560821A discloses a cyclic Gd(III) complex, which uses the ring structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a mother ring, and introduces lipophilic groups R' and R'' on the alpha position and benzene ring structure of phenylacetic acid, respectively, and introduces a chiral group R at the DOTA macrocyclic position. The use of chiral group R increases the rigidity of the macrocyclic structure and improves the stability of the complex. The lipophilic groups R' and R'' can bind to the organic anion transport polypeptide of liver cells, thereby effectively improving the distribution of the cyclic Gd(III) complex as a contrast agent in the liver and gallbladder. However, the above-mentioned complex still has a non-negligible residue in the mouse liver within the considered time range, and there is a potential safety problem when used as a contrast agent. SUMMARY

[0006] Therefore, the present application aims to provide a monosubstituted cyclic ligand, a preparation method and application thereof, and a monosubstituted cyclic rare earth complex, a preparation method and application thereof.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0008] The present application provides a monosubstituted cyclic ligand having a structure shown in formula I.

[0009]

[0010] wherein R includes benzyloxy, ethoxy or hydroxyl; and the wavy line represents R configuration or S configuration.

[0011] The present application provides a preparation method of the monosubstituted cyclic ligand described in the above technical solutions, comprising the following steps: performing an ester group hydrolysis reaction on a monosubstituted cyclic ester compound under alkaline conditions to obtain the monosubstituted cyclic ligand.

[0012] The monosubstituted cyclic ester compound includes compound 7, compound 8 or compound 9.

[0013]

[0014] Preferably, the preparation method of the compound 7 comprises the following steps.

[0015] Performing a first nucleophilic substitution reaction on compound 1 and bromobenzene under alkaline conditions to obtain compound 2.

[0016] Performing an amide hydrolysis reaction on the compound 2 under acidic conditions to obtain compound 3.

[0017] Performing a second nucleophilic substitution reaction on the compound 3 and methyl bromoacetate under alkaline conditions to obtain compound 4.

[0018] Performing an ester exchange reaction on the compound 4 and diethylenetriamine to obtain compound 5.

[0019] Performing an amide reduction reaction on the compound 5 in the presence of a reducing agent to obtain compound 6.

[0020] Performing a third nucleophilic substitution reaction on the compound 6 and ethyl bromoacetate under alkaline conditions to obtain the compound 7.

[0021]

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

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

[0024] The present application provides a monosubstituted cyclic rare earth complex having a structure shown in formula II.

[0025]

[0026] wherein R comprises benzyloxy, ethoxy or hydroxyl; Ln comprises Gd(III) or Eu(III); and the wavy line represents R configuration or S configuration.

[0027] The present application provides a preparation method of the monosubstituted cyclic rare earth complex, comprising the following steps: subjecting a monosubstituted cyclic ligand 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 the above technical solution or the monosubstituted cyclic ligand prepared by the preparation method according to the above technical solution; and the metal in the rare earth metal source comprises Gd(III) or Eu(III).

[0028] The present application provides an application of the monosubstituted cyclic rare earth complex according to the above technical solution or the monosubstituted cyclic rare earth complex prepared by the preparation method according to the above technical solution in preparing a contrast agent.

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

[0030] The present application provides an application of the monosubstituted cyclic rare earth complex according to the above technical solution or the monosubstituted cyclic rare earth complex prepared by the preparation method according to the above technical solution in detecting peroxidase, wherein R of the monosubstituted cyclic rare earth complex is hydroxyl, and Ln is Gd(III).

[0031] The monosubstituted cyclic ligand with the structure shown in formula I provided by the application takes the ring structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4DOTA) as a mother ring, and the tyrosine group modified by the group R increases the rigidity of the mother ring structure, thereby improving the stability of the complex; meanwhile, the monosubstituted cyclic ligand maintains the original eight-coordination structure by introducing a benzyloxy (-OBn, denoted as ligand L1), ethoxy (-OEt, denoted as ligand L2) or hydroxyl (-OH, denoted as ligand L3) substituted benzyl group on the carbon atom of the DOTA ring, thereby increasing the stability of the complex, improving the relaxation rate of the complex, and improving the kinetic inertness and relaxation rate of the complex compared with Gd-DOTA.

[0032] The monosubstituted cyclic ligand provided by the application can form an anionic two-hydrophilic eight-coordination complex, i.e., a monosubstituted cyclic rare earth complex, with Gd(III) or Eu(III), the stability of the complex is greatly improved compared with Gd-DOTA, and fewer Gd(III) and Eu(III) residues are formed. The application can realize magnetic resonance imaging with multiple specificities by changing the R substituent group. The monosubstituted cyclic rare earth complex with the lipophilic groups -OBn and -OEt can be selectively distributed to the liver as a hepatobiliary specific contrast agent, wherein the specificity of Gd-L1 (R is -OBn, Ln is Gd(III)) to the liver is enhanced by more than 35 min, and the drug can be quickly cleared from the liver and kidney within 2 h, the safety evaluation in mice proves that the drug has no obvious damage to the heart, liver, spleen, lung and kidney, and the drug has high safety and excellent hepatobiliary diagnostic performance. In addition, the monosubstituted cyclic rare earth complex has strong responsiveness to inflammation. The monosubstituted cyclic rare earth complex Gd-L3 (R is -OH, Ln is Gd(III)) has a tyrosine residue and can participate in the peroxide cycle, and can form an oligomer of Gd-L3 in the presence of hydrogen peroxide and peroxidase, the relaxation rate of the oligomer in vitro is increased by about 3.8 times compared with that of the monomer, the oligomer can be aggregated at the lesion site of a gout mouse model in vivo, and has high contrast, and can be used for detecting the aggregation of peroxidase in vitro and in vivo, and can be used as an MPO (myeloperoxidase) responsive probe for detecting peroxidase in vitro and in vivo.

[0033] The application provides a preparation method of the monosubstituted cyclic ligand.

[0034] Further, in the preparation of the monosubstituted cyclic ester compound, the ester exchange is used to generate a cyclic structure (compound 5), so that the side chain substituent group is directly connected to the carbon atom on the ring, the eight-coordination structure of the Gd atom is retained, and the kinetic inertness and relaxation rate of the monosubstituted cyclic rare earth complex are high.

[0035] The application provides the preparation method of the single-substituted annular rare earth complex. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a synthesis route diagram of the single-substituted annular ligand and the single-substituted annular rare earth complex;

[0037] Figure 2 is an HPLC chromatogram of the ligands L1-3 at a wavelength of 254 nm;

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

[0039] Figure 4 is an HPLC chromatogram of the mixture of the complexes Gd-L1 and Eu-L1 before being separated by semi-preparative high performance liquid chromatography, and the HPLC chromatogram of the angular isomer and the edge isomer after being separated at a wavelength of 254 nm;

[0040] Figure 5 is an ESI-MS mass spectrum of the mixture of the complexes Gd-L1 and Eu-L1 before being separated by semi-preparative high performance liquid chromatography, and the ESI-MS mass spectrum of the angular isomer and the edge isomer after being separated;

[0041] Figure 6 is an ESI-MS mass spectrum of the angular isomer of the complex Eu-L1; 1 HNMR (400 MHz, D2O) spectrum;

[0042] Figure 7 is an ESI-MS mass spectrum of the edge isomer of the complex Eu-L1; 1 HNMR (400 MHz, D2O) spectrum;

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

[0044] Figure 9 is a schematic diagram of the action of Gd-L1 on OATP1B1 (a), OATP1B3 (b), OATP2B1 (c) and NTCP (d) respectively, the action of Gd-L2 on OATP1B1 (e), OATP1B3 (f), OATP2B1 (g) and NTCP (h) respectively, and the action of Gd-L3 on OATP1B1 (i), OATP1B3 (j), OATP2B1 (k) and NTCP (l) respectively;

[0045] Figure 10 is an evaluation diagram of the kinetic inertness of Gd-L 1-3Figure 3 shows the evaluation of the cytotoxicity of Gd-EOB-DTPA incubated at 37°C with LO2 (a) and 293T (b) for 24 h;

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

[0047] Figure 12 shows T1 -weighted images of the liver (a) and kidney (b) recorded at 3.0T in normal Balb / c mice after intravenous injection of Gd-L1 (0.1 mmol / kg) with (BSP) and without (no BSP) intravenous injection of sulfobromophthalein (BSP), and the 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 shows coronal and transverse images of the liver at 3.0T and the CNR (liver-to-tumor) as a function of time in coronal and transverse planes before and after intravenous injection of Gd-L1 via the tail vein;

[0049] Figure 14 shows T1 -weighted images of the liver (a) and kidney (b) recorded at 3.0T in transverse planes in normal rats before and 1 min, 10 min and 20 min after injection of Gd-EOB-DTPA and Gd-L1, respectively, and the nSNR time course of the liver (c) and kidney (d) after injection of the contrast agents;

[0050] Figure 15 shows biodistribution studies of Gd-EOB-DOTA and Gd-L1 in normal mice (a) and the total uptake of the complexes by the five organs and the brain over 5 min, 15 min and 24 h (b);

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

[0052] Figure 17 shows the results of the relaxation assay of Gd-L3 incubated with horseradish peroxidase, where (a) to (b) are the longitudinal (T1 ) and transverse (T2) relaxation rate changes of Gd-L3 with HRP / H202 ([Gd] = 0.5 mM, pH = 7.4, PBS (0.1 M), 37°C, 1.4T; HRP activity: 0, 5, 50, 500 U; H202 consumed equivalent: 0-2 eq.) plots; (c) is the plot of the longitudinal (T1 ) relaxation rate of Gd-L3 with HRP / H202 ([Gd] = 0.5 mM, HRP = 5 U, n H2O2Figure 2: Relaxivity plots of Gd-L3 incubated with (a) 0 eq, (b) 0.5 eq, (c) 1.0 eq, (d) 2.0 eq of HRP / H202; (e) T1 weighted MR image of the left leg (MSU processing); (f) T1 weighted MR image of the right leg (PBS processing, control); (g) plot of ACNR (left leg vs. right leg) as a function of time; (h) plot of nSNR of the right leg as a function of time after injection; data are expressed as mean ± standard deviation (n = 3). H2O2 Figure 2: Relaxivity plots of Gd-L3 incubated with (a) 0 eq, (b) 0.5 eq, (c) 1.0 eq, (d) 2.0 eq of HRP / H202; (e) T1 weighted MR image of the left leg (MSU processing); (f) T1 weighted MR image of the right leg (PBS processing, control); (g) plot of ACNR (left leg vs. right leg) as a function of time; (h) plot of nSNR of the right leg as a function of time after injection; data are expressed as mean ± standard deviation (n = 3). DETAILED DESCRIPTION

[0053] The present application provides a single-substituted cyclic ligand having a structure shown in Formula I:

[0054]

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

[0056] The present application provides a preparation method of the single-substituted cyclic ligand described in the above technical solution, comprising the following steps: performing ester group hydrolysis reaction on a single-substituted cyclic ester compound under alkaline conditions to obtain the single-substituted cyclic ligand; the single-substituted cyclic ester compound includes compound 7, compound 8 or compound 9:

[0057]

[0058] Unless otherwise specified, the materials and equipment used in the present application are commercially available in the art.

[0059] In the present application, the alkaline condition is preferably provided by a strong inorganic base, the strong inorganic base is preferably an alkali metal hydroxide, more preferably at least one of lithium hydroxide, sodium hydroxide and potassium hydroxide, and further preferably lithium hydroxide; the strong inorganic base is preferably used in the form of a strong inorganic base aqueous solution, the mass of the strong inorganic base to the volume of water in the strong inorganic base aqueous solution is preferably 1 g: 7-8 mL, and more preferably 1 g: 7.5 mL; the molar ratio of the single-substituted cyclic ester compound to the strong inorganic base is preferably 1: 5-6, and more preferably 1: 5.5.

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

[0061] In the present application, the temperature for the hydrolysis reaction of the ester group is preferably room temperature, and the time is preferably 10-14 h, and more preferably 11-12 h; the hydrolysis reaction of the ester group is preferably carried out under a protective atmosphere, which preferably comprises nitrogen, argon or helium, and more preferably nitrogen, and the production cost of the target product is lower when nitrogen is used.

[0062] After the hydrolysis reaction of the ester group is completed, the present application preferably further comprises: concentrating the reaction system obtained from the hydrolysis reaction of the ester group, adding an extractant to stir and then standing, solid-liquid separation, drying the obtained solid product, to obtain the monosubstituted cyclic ligand. In the present application, the concentration is preferably rotary evaporation to remove the solvent. In the present application, the extractant preferably comprises one or more of ethanol, methyl tert-butyl ether, ethyl acetate and dichloromethane. The present application does not have special limitations on the stirring, and uniform stirring is sufficient. The present application does not have special limitations on the standing time, and standing until the precipitation no longer increases is sufficient. In the present application, the solid-liquid separation preferably comprises filtration, suction filtration or centrifugal separation. In the present application, the drying temperature is preferably 40-55°C, and more preferably 45-50°C; the present application does not have special limitations on the drying time, and drying to a constant weight is sufficient.

[0063] In the present application, the preparation method of the compound 7 preferably comprises the following steps:

[0064] carrying out a first nucleophilic substitution reaction of the compound 1 with bromobenzene under alkaline conditions to obtain a compound 2;

[0065] carrying out a hydrolysis reaction of the amide of the compound 2 under acidic conditions to obtain a compound 3;

[0066] carrying out a second nucleophilic substitution reaction of the compound 3 with methyl bromoacetate under alkaline conditions to obtain a compound 4;

[0067] carrying out a trans-esterification reaction of the compound 4 with diethylenetriamine to obtain a compound 5;

[0068] carrying out a reduction reaction of the amide of the compound 5 in the presence of a reducing agent to obtain a compound 6;

[0069] carrying out a third nucleophilic substitution reaction of the compound 6 with ethyl bromoacetate under alkaline conditions to obtain the compound 7;

[0070]

[0071] In the present application, when compounds 1-4 are all S configuration, compounds 5-9, monosubstituted cyclic ligand and monosubstituted cyclic rare earth complex are all R configuration; when compounds 1-4 are all R configuration, compounds 5-9, monosubstituted cyclic ligand and monosubstituted cyclic rare earth complex are all S configuration.

[0072] In the present application, the first nucleophilic substitution reaction of compound 1 with benzyl bromide is carried out under alkaline conditions to obtain compound 2.

[0073] In the present application, the molar ratio of compound 1 to benzyl bromide is preferably 1:1.05-1.5, more preferably 1:1.1-1.15.

[0074] In the present application, the alkaline condition is preferably provided by an alkaline reagent (denoted as a first alkaline reagent), and the first alkaline reagent preferably includes a carbonate, more preferably an alkali metal carbonate, and further preferably at least one of potassium carbonate, sodium carbonate and cesium carbonate. In the present application, the molar ratio of compound 1 to the first alkaline reagent is preferably 1:2.8-4, more preferably 1:3-3.5.

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

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

[0077] After the first nucleophilic substitution reaction is completed, the present application preferably further includes: adding water to the reaction system obtained by 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 application, the extractant preferably includes ethyl acetate or dichloromethane. In the present application, the concentration method preferably includes evaporation to remove the organic solvent.

[0078] After obtaining compound 2, the present application carries out an amide hydrolysis reaction of compound 2 under acidic conditions to obtain compound 3.

[0079] In the present application, the acidic condition is 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 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 -10-10℃ (0℃). In the present application, the mass 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 the present application, the organic solvent (denoted as the second organic solvent) used in the hydrolysis reaction of the amide preferably includes dichloromethane (DCM) or tetrahydrofuran; the mass of compound 2 to the volume of the second organic solvent is preferably 1 g: 8-10 mL, more preferably 1 g: 8-9 mL.

[0081] In the present application, the temperature of the hydrolysis reaction of the amide is preferably room temperature, and the time is preferably 18-24 h, more preferably 20-22 h; 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 application 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 pumping, adding a saturated sodium bicarbonate solution until no more bubbles are generated, extraction, drying the obtained organic phase with anhydrous sodium sulfate, filtration, and concentrating the obtained liquid component to obtain compound 3. In the present application, the extractant used in the extraction preferably includes ethyl acetate or dichloromethane. In the present application, the concentration preferably includes sequentially performing rotary evaporation to remove the solvent and vacuum pumping to dry.

[0083] After obtaining compound 3, the present application carries out a second nucleophilic substitution reaction of the compound 3 with methyl bromoacetate under basic conditions to obtain compound 4.

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

[0085] In the present application, the basic condition is preferably provided by a basic reagent (denoted as the second basic reagent), which preferably includes an organic amine, more preferably at least one of N,N-diisopropylethylamine (DIPEA) and triethylamine. In the present application, the molar ratio of compound 3 to the second basic reagent is preferably 1: 1.8-2.2, more preferably 1: 1.9-2.

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

[0087] In the present application, the temperature of the second nucleophilic substitution reaction is preferably room temperature, and the time is preferably 3-10 h, more preferably 4-6 h; the second nucleophilic substitution reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably comprises nitrogen, argon or helium, more preferably nitrogen.

[0088] After the second nucleophilic substitution reaction is completed, the present application preferably further comprises: 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 the compound 4. In the present application, the extractant preferably comprises ethyl acetate or dichloromethane. In the present application, the concentration method preferably comprises sequentially evaporating the organic solvent and vacuum pumping dry.

[0089] After obtaining the compound 4, the present application carries out an ester exchange reaction of the compound 4 with diethylenetriamine to obtain the compound 5.

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

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

[0092] In the present application, the temperature of the ester exchange reaction is preferably 65-75°C, more preferably 70°C; the time is preferably 10-13 days, more preferably 11-12 days; and the second nucleophilic substitution reaction is preferably carried out under a protective atmosphere and a condensing water reflux condition, and the protective atmosphere preferably comprises nitrogen, argon or helium, more preferably nitrogen.

[0093] After the transesterification reaction is completed, the present application preferably further comprises: removing most (more than 95%, more preferably 95-99%) of the fourth organic solvent from the reaction system obtained from the transesterification reaction by rotary evaporation, filtering, washing the filter cake with ice ethanol for 2-3 times, and drying to obtain compound 5. In the present application, the temperature of the drying is preferably 45-55°C, more preferably 45-50°C; the present application does not have a special limitation on the time of the drying, and the drying can be performed until the weight is constant.

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

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

[0096] In the present application, the organic solvent (denoted as the fifth organic solvent) used in the amide reduction reaction preferably comprises tetrahydrofuran; and the concentration of the reducing agent in the fifth organic solvent is preferably 0.9-1.1M, more preferably 1M.

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

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

[0099] After obtaining compound 6, the present application carries out a third nucleophilic substitution reaction of the compound 6 with ethyl bromoacetate under basic conditions to obtain the compound 7.

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

[0101] In the present application, the basic conditions are preferably provided by a basic reagent (denoted as a third basic reagent), and the third basic reagent preferably comprises a carbonate, more preferably an alkali metal carbonate, and further preferably at least one of potassium carbonate, sodium carbonate and cesium carbonate. In the present application, the molar ratio of the compound 6 to the third basic reagent is preferably 1: 9-10, more preferably 1: 9-9.5.

[0102] In the present application, the organic solvent (denoted as the sixth organic solvent) used in the third nucleophilic substitution reaction preferably includes at least one of acetonitrile, N,N-dimethylformamide and tetrahydrofuran; the ratio of the amount of substance of the 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 the present application, 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 the third nucleophilic substitution reaction is completed, the present application preferably further includes: filtering the reaction system obtained from the third nucleophilic substitution reaction, removing the solvent from the obtained liquid component by rotary evaporation, dissolving in ethyl acetate, washing with dilute hydrochloric acid, adjusting the pH value to 8-9 (more preferably 8-8.5), extracting with dichloromethane, drying the obtained organic phase with anhydrous sodium sulfate, filtering, removing the solvent from the obtained liquid component by rotary evaporation, and drying with a vacuum pump to obtain the compound 6. In the present application, the concentration of the dilute hydrochloric acid is preferably 1-1.5 M, more preferably 1 M; the number of times of washing with dilute hydrochloric acid is preferably 2-3 times. In the present application, the number of times of dichloromethane extraction is preferably 2-3 times. In the present application, the base used for adjusting the pH value preferably includes potassium carbonate, sodium bicarbonate or a sodium hydroxide aqueous solution, and the concentration of the sodium hydroxide aqueous solution is preferably 1-1.2 M, more preferably 1-1.1 M.

[0105] In the present application, the preparation method of the compound 8 preferably includes the following step: carrying out a debenzylation reaction of the compound 7 in the presence of a catalyst and a reducing agent to obtain the compound 8.

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

[0107] In the present application, 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 the compound 8 to ammonium formate is preferably 1: 4.5-5.5, more preferably 1: 4.8-5.

[0108] In the present application, the organic solvent (denoted as the seventh organic solvent) used in the debenzylation reaction by hydrogenolysis preferably comprises an alcohol solvent, more preferably comprises ethanol and / or methanol; the ratio of the amount of substance of the compound 7 to the volume of the seventh organic solvent is preferably 1 g: 20-30 mL, more preferably 1 g: 25 mL.

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

[0110] After the completion of the debenzylation reaction by hydrogenolysis, the present application preferably further comprises: filtering the reaction system obtained from the debenzylation reaction by hydrogenolysis to remove the catalyst, and rotary evaporation of the organic solvent to obtain the compound 8.

[0111] In the present application, the preparation method of the compound 9 preferably comprises the following step: carrying out a fourth nucleophilic substitution reaction of the compound 8 with bromoethane to obtain the compound 9.

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

[0113] In the present application, 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 the present application, the fourth basic reagent preferably comprises a carbonate, more preferably comprises an alkali metal carbonate, further preferably comprises at least one of potassium carbonate, sodium carbonate and cesium carbonate; the molar ratio of the compound 8 to the fourth basic reagent is preferably 1: 5.5-6.5, more preferably 1: 6. In the present application, the eighth organic solvent preferably comprises DMF; the ratio of the mass of the compound 8 to the volume of the eighth organic solvent is preferably 1 g: 5-8 mL, more preferably 1 g: 6-7 mL.

[0114] In the present application, 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 comprises nitrogen, argon or helium, more preferably nitrogen.

[0115] After the fourth nucleophilic substitution reaction is completed, the present application preferably further comprises: 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 the compound 9. In the present application, the extractant preferably comprises ethyl acetate or dichloromethane. In the present application, the concentration preferably comprises evaporation to remove the organic solvent.

[0116] The present application provides a monosubstituted cyclic rare earth complex having a structure shown in formula II:

[0117]

[0118] wherein R comprises 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)) comprises Gd(III) or Eu(III); and the wavy line represents R configuration or S configuration.

[0119] The present application provides a preparation method of the monosubstituted cyclic rare earth complex described in the above technical solution, comprising the following steps: performing a coordination reaction on a monosubstituted cyclic ligand and a rare earth metal source to obtain the monosubstituted cyclic rare earth complex; the monosubstituted cyclic ligand is the monosubstituted cyclic ligand described in the above technical solution or the monosubstituted cyclic ligand prepared by the preparation method described in the above technical solution; and the metal in the rare earth metal source comprises Gd(III) or Eu(III).

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

[0121] In the present application, the rare earth metal source preferably comprises a Gd(III) source or a Eu(III) source; the Gd(III) source comprises gadolinium chloride and / or gadolinium nitrate, and more preferably gadolinium chloride; the Eu(III) source comprises europium chloride and / or europium nitrate, and 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 a rare earth metal source aqueous solution; the concentration of the rare earth metal source aqueous solution is preferably 1.8-2.4 g / mL, and more preferably 2-2.3 g / mL; and the adding mode of the rare earth metal source aqueous solution is preferably dropwise adding.

[0122] In the present application, the monosubstituted cyclic ligand is preferably used in the form of a monosubstituted cyclic ligand solution, the mass 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, 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 with dilute hydrochloric acid; the concentration of the dilute hydrochloric acid is preferably 1-1.2 M, more preferably 1-1.1 M.

[0123] In the present application, the temperature of the coordination reaction is preferably 100-105℃, more preferably 100-102℃; the time is preferably 18-24 h, more preferably 20-22 h; the coordination reaction is preferably carried out under the condition of refluxing condensed water; the pH value of the system during the coordination reaction is preferably neutral, the pH value is preferably adjusted with potassium carbonate, sodium bicarbonate or sodium hydroxide aqueous solution, the concentration of the sodium hydroxide aqueous solution is preferably 1-1.2 M, more preferably 1-1.1 M.

[0124] After the completion of the coordination reaction, the present application preferably further comprises: adjusting the pH value of the reaction system obtained by the coordination reaction to 10 or more, precipitating excess metal ions, centrifuging, filtering the obtained supernatant with a 0.22 μm filter head or a 0.22 μm filter membrane, freeze-drying the obtained liquid component after adjusting the pH value to 7, to obtain a monosubstituted cyclic rare earth complex. In the present application, the pH value is preferably 10-14, more preferably 10-12, further preferably 11. In the present application, the purpose of the centrifugation is to remove most of the metal precipitate; the purpose of the filter head or filter membrane filtration is to remove the remaining metal precipitate. In the present application, the adjustment of the pH value to 7 is preferably carried out with dilute hydrochloric acid, the concentration of the dilute hydrochloric acid is preferably 1-1.2 M, more preferably 1-1.1 M.

[0125] The present application also provides the use of the monosubstituted cyclic rare earth complex in the above technical solution or the monosubstituted cyclic rare earth complex prepared by the preparation method in the above technical solution in the preparation of a contrast agent. In the present application, the contrast agent preferably comprises a contrast agent for liver and gall magnetic resonance imaging and / or inflammation magnetic resonance imaging.

[0126] The present application also provides the use of the monosubstituted cyclic rare earth complex in the above technical solution or the monosubstituted cyclic rare earth complex prepared by the preparation method in the above technical solution in the detection of peroxidase, the monosubstituted cyclic rare earth complex being one in which R is a hydroxyl group and Ln is Gd(III). The exposed hydroxyl group in the monosubstituted cyclic rare earth complex can participate in peroxide cycle to realize the oligomerization of Gd-L3, and be applied as an MPO (myeloperoxidase) responsive probe for the detection of peroxidase in vivo and in vitro.

[0127] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0128] Example 1

[0129] Synthesis of the single-substituted cyclic ligand L of S configuration and R configuration according to the synthetic route shown in Fig. 1 1～3 (i.e. L1, L2 and L3) and the single-substituted cyclic rare earth complex Gd-L of S configuration and R configuration 1～3 (i.e. Gd-L1, Gd-L2 and Gd-L3) and Eu-L1-3 (i.e. Eu-L1, Eu-L2 and Eu-L3); when compounds 1-4 are all of S configuration, compounds 5-9, the single-substituted cyclic ligand and the single-substituted cyclic rare earth complex are all of R configuration; when compounds 1-4 are all of R configuration, compounds 5-9, the single-substituted cyclic ligand and the single-substituted cyclic rare earth complex are all of S configuration.

[0130] Compound 1 (3.4 mmol) was dissolved in 5 mL of DMF, and then bromobenzene (3.9 mmol) and potassium carbonate (10.2 mmol) were added successively, and stirred at room temperature under nitrogen protection for 18 h, and the reaction was monitored by TLC (PE:EA volume ratio = 2:1). 10 mL of water was added to the mixture, stirred, extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated to remove the organic solvent to obtain compound 2 (white solid, yield 96%). 1 HNMR (400 MHz, CDCl3) δ 7.46-7.28 (m, 5H), 7.04 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 5.04 (s, 2H), 4.96 (d, J = 8.4 Hz, 1H), 4.54 (q, J = 7.3, 6.7 Hz, 1H), 3.71 (s, 3H), 3.03 (tt, J = 14.2, 6.8 Hz, 2H), 1.42 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ 172.50, 157.94, 155.16, 137.02, 130.37, 128.65, 128.27, 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 DCM, 1 mL trifluoroacetic acid was added dropwise under ice bath, stirred at room temperature for 18 h under nitrogen protection, and the reaction was monitored by TLC (PE:EA = 2:1) until completion. The DCM and most of the trifluoroacetic acid (more than 95%) were removed by rotary evaporation, and a saturated solution of sodium bicarbonate was added until no more bubbles were generated. The resulting organic phase was extracted with DCM, dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the solvent and dried under vacuum to obtain compound 3 (yellow oil, yield 95%). 1 H NMR (400 MHz, CDC13) δ 7.52-7.29 (m, 5H), 7.11 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 5.04 (s, 2H), 3.71 (s, 3H), 3.03 (dd, J = 13.6, 5.2 Hz, 1H), 2.82 (dd, J = 13.6, 7.7 Hz, 1H), 1.58 (s, 2H). 13 C NMR (100 MHz, CDC13) δ 175.55, 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 DMF, DIPEA (3.5 mmol) and methyl bromoacetate (1.8 mmol) were added, and the mixture was stirred at room temperature for 3 h under nitrogen protection until the reaction was complete (TLC, PE:EA = 2:1). 10 mL of water was added to the mixture, and the organic phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the solvent and dried under vacuum to obtain compound 4 (yellow oil, yield 97%). 1 H NMR (400 MHz, CDC13) δ 7.52-7.29 (m, 5H), 7.11 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 5.04 (s, 2H), 3.71 (s, 3H), 3.03 (dd, J = 13.6, 5.2 Hz, 1H), 2.82 (dd, J = 13.6, 7.7 Hz, 1H), 1.58 (s, 2H). 13 C NMR (100 MHz, CDC13) δ 174.15, 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, then diethylenetriamine (2.8 mmol) was added dropwise, and the reaction was heated to reflux at 60 °C under nitrogen protection for 11 days. The reaction was monitored by HPLC, and the reaction limit reached about 70%. Most of the methanol was removed by rotary evaporation, and the filter cake was washed with ice ethanol for 2-3 times. After drying at 50 °C, compound 5 (white solid) was obtained in a yield of 21%. 1 HNMR (400 MHz, CDC13) δ 7.50 (s, 1H), 7.47-7.34 (m, 5H), 7.32 (t, J = 7.1 Hz, 1H), 7.14 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 5.04 (s, 2H), 3.49 (dtd, J = 37.6, 13.2, 5.6 Hz, 3H), 3.32 (dd, J = 10.2, 4.0 Hz, 1H), 3.18 (dd, 1H), 3.11 (m, 1H), 3.06-2.83 (m, 4H), 2.65 (qd, J = 10.0, 4.8 Hz, 3H), 1.92 (d, J = 7.8 Hz, 1H), 1.39 (d, J = 101.6 Hz, 1H). 13 C NMR (100 MHz, CDC13) δ 173.52, 171.78, 158.01, 136.93, 130.14, 129.18, 128.67, 128.09, 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] Compound 5 (2.5 mmol) was dissolved in borane-tetrahydrofuran (borane 1M, 60 mL) under nitrogen atmosphere, and heated to 70 °C for reflux for 24 h. Methanol was added under ice bath until no more bubbles were generated. An equal volume of 1M hydrochloric acid was added, and the reaction was heated to reflux at 70 °C for 2 h. After the temperature of the reaction solution decreased to room temperature, potassium carbonate was added to adjust the pH to 8. The reaction was extracted with dichloromethane for 3 times (30 mL each time), dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the solvent. After drying under vacuum pump, compound 6 (yellow oil) was obtained in a yield of 86%. 1HNMR (400 MHz, D20) δ 6.95 (m, 7H), 6.63 (d, J = 8.3 Hz, 2H), 4.47 (s, 2H), 3.23-2.89 (m, 10H), 2.87-2.65 (m, 6H), 2.51 (d, J = 8.4 Hz, 1H). 13 C NMR (100 MHz, D20) δ 157.32, 136.62, 130.18, 128.38, 128.29, 127.78, 127.47, 115.11, 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 acetonitrile, potassium carbonate (29.8 mmol), ethyl bromoacetate (18.0 mmol) were added successively, and the reaction was stirred at room temperature for 18 h under nitrogen atmosphere. After the reaction was completed, the filtrate was evaporated under reduced pressure to remove the solvent, dissolved in 30 mL EA, washed with 1M dilute hydrochloric acid 3 times (15 mL each time), adjusted to pH 8 with potassium carbonate, extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the solvent to obtain compound 7 (pale yellow oil). 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 ethanol, 20 wt% Pd-C (0.24 g) was added, and the air was removed, and the reaction was heated to 55°C under 2 MPa hydrogen atmosphere for 18 h. After the reaction was completed, Pd-C was removed by filtration, and the organic solvent was rotary evaporated to obtain compound 8 (yellow oil, yield 96%), without further purification. 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), then potassium carbonate (9.83 mmol) and ethyl bromide (4.90 mmol) were added under nitrogen atmosphere, and the mixture was stirred at room temperature for 20 h. 10 mL of water was added to the mixture, which was stirred uniformly, extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to remove the organic solvent to obtain compound 9 (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 aqueous lithium hydroxide solution (1 g: 7.5 mL) and ethanol (8 mL), and the mixture was stirred at room temperature for 16 h. After the reaction was completed, the solvent was removed by rotary evaporation, 7 mL of ethanol and 7 mL of methyl tert-butyl ether were added, and after stirring for 15 min, the liquid was removed by centrifugation after standing for 5 min. The obtained white solid was dried at 50°C to obtain ligand L1 with a yield of 83%.

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

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

[0141] Ligand L1 : 1 H NMR (400 MHz, D2O) δ 7.54-7.35 (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 (100 MHz, DMSO) δ 173.46, 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, D20) 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, D20) 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+ , 529.3 and 533.3; found, 270.2 and 539.4. + ; found, 270.2 and 539.4.

[0143] Ligand L3: 1 H NMR (400 MHz, D20) 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, D20) 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] + , 529.4 and 533.3; found, 517.4 [M+Li] + , 529.4 and 533.3.

[0144] Preparation of complex Gd-L1 : Ligand L1 (1 mmol) was dissolved in 15 mL weakly acidic water (pH = 6.0, 1 M hydrochloric acid was used to adjust pH) to obtain an aqueous solution of ligand L1 ; LnCl3-6H2O (1 mmol) was dissolved in 0.2 mL 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 aqueous sodium hydroxide solution. The reaction solution was refluxed at 100 °C for 20 h. After the reaction was completed, 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 freeze-dried after the pH was adjusted to 7 with 1 M hydrochloric acid to obtain complex Gd-L1.

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

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

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

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

[0149] Preparation of complex Eu-L3: The only difference between the preparation of complex Eu-L3 and the preparation of complex Eu-L1 is that ligand L1 is replaced by 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 is a HPLC chromatogram of ligand L 1～3 at a wavelength of 254 nm, it can be seen that the retention times of ligand L 1～3 are 5.886 min, 4.79 min and 2.469 min, respectively. Among them, the HPLC detection conditions of Figures 2-3 are as follows: mobile phase A is 0.05 vol% trifluoroacetic acid aqueous solution (mobile phase A), mobile phase B is acetonitrile, flow rate is 1 mL / min, and chromatographic column is Waters C18 chromatographic column (5 μm, 4.6 x 150 mm); gradient elution program: 0-10 min, the volume fraction of mobile phase B is increased from 10% to 100%, 10-12 min, the volume fraction of mobile phase B is decreased from 100% to 10%, 12-15 min, the volume fraction of mobile phase B is 10%.

[0157] Figure 3 is a HPLC chromatogram of complexes Gd-L 1～3 and Eu-L 1～3 at a wavelength of 254 nm, it can be seen that complexes Gd-L 1～3 and Eu-L 1～3 each have two absorption peaks, and the average retention times thereof are Gd-L 1～3 5.642 min, 4.408 min and 2.539 min, respectively, and Eu-L 1～3 5.642 min, 4.376 min and 2.351 min, respectively.

[0158] Figure 4 is the HPLC chromatogram of the mixture of complexes Gd-L1 and Eu-L1 before separation by semi-preparative high performance liquid chromatography, and the angular isomer and the edge isomer after separation at a wavelength of 254 nm. It can be seen that the two absorption peaks of the complex are independently separable, and there are corresponding independent peak positions on the HPLC spectrum.

[0159] Figure 5 is the ESI-MS mass spectrum of the mixture of complexes Gd-L1 and Eu-L1 before separation by semi-preparative high performance liquid chromatography, and the angular isomer and the edge isomer after separation. It can be seen that the mass-to-charge ratios of the two absorption peaks of the complex are exactly the same. In figures 4-5, the semi-preparative high performance liquid chromatography separation conditions are as follows: mobile phase A is 10 mM ammonium acetate aqueous solution, mobile phase B is a mixture of 10 mM ammonium acetate aqueous solution and acetonitrile at a volume ratio of 1:9, flow rate is 1 mL / min; the chromatographic column is a Waters C18 chromatographic column (5 μm, 4.6 x 150 mm); the gradient elution program is as follows: 0-10 min, the volume fraction of mobile phase B increases from 10% to 100%, 10-12 min, the volume fraction of mobile phase B decreases from 100% to 10%, 12-15 min, the volume fraction of mobile phase B is 10%.

[0160] Figure 6 is the ESI-MS mass spectrum of the angular isomer of complex Eu-L1. 1 Figure 7 is the HNMR (400 MHz, D2O) spectrum of the edge isomer of complex Eu-L1. 1 It can be seen from the HNMR (400 MHz, D2O) spectrum that the more hydrophilic isomer in the complex is mostly SAP (tetragonal antiprism), and the SAP and TSAP (twisted tetragonal antiprism) isomers in the more lipophilic isomer account for about half.

[0161] Test Example 1

[0162] (1) Relaxivity test

[0163] Relaxivity is a key indicator of the performance of a contrast agent. The relaxivity of a contrast agent molecule is usually affected by temperature, field strength and solutes in solution. The relaxivity (r1 and r2) of the contrast agent molecule in the present application was measured in PBS (pH = 7.4, r1) and ultrapure water (r2) using a 1.4T magnet at 37°C. As shown in Table 1, Gd-EOB-DTPA as a linear contrast agent molecule has higher relaxivity than macrocyclic complexes (Gd-L 1～3 ) with different substituents. Gd-L 1～3 has higher relaxivity than Gd-DOTA. Further test of the relaxivity in the presence of 4.5% HSA shows that Gd-L 1-3 has different degrees of binding with HSA.

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

[0165]

[0166] The results of the relaxivity parameters of Gd-DOTA at 1.4 T are shown in the literature [1] and the literature [2].

[0167] Literature [1]: Xu, W.; Ye, X.; Wu, M.; Jiang, X.; Hugo Tse, L. H.; Gu, Y.; Shu, K.; Xu, L.; Jian, Y.; Mo, G.; Xu, J.; Ding, Y.; Gao, R.; Shen, J.; Ye, F.; Yan, Z.; Dai, L., Chiral Gd-DOTA as a Versatile Platform for Hepatobiliary and Tumor Targeting MRI Contrast Agents. 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) Kinetic inertness test

[0170] Ideal MRI contrast agents require very high kinetic inertness to avoid dissociation of Gd(III) and minimize associated toxicity. There are two main mechanisms of metal dissociation: acid-assisted metal dissociation and ion or ligand competitive transmetallation. Therefore, the kinetic stability of the mono-substituted macrocyclic rare earth complexes was evaluated using relaxometry in the presence of 1 M HC1 (i.e. 0.1 M dilute HC1) or excess Zn(II) compared to Gd-DOTA and Gd-EOB-DTPA. The extent of Gd(III) dissociation was evaluated by comparing the relaxation times at each time point to the initial value. The results show that Gd-L 1～3 retained 63-79% of the complex, while Gd-DOTA lost 51% within 24 h (Fig. 8 (a) and (b)). It was reported that Gd-EOB-DTPA has a half-life of less than 5 seconds in 0.1 M HC1. To investigate the migration kinetics of the contrast agents, metal exchange experiments were performed using PBS buffer (pH = 7.4, 0.01 M) containing 1.0 mM Gd-L 1～3 and 10 mM Zn(II) incubated at 37 °C for 7 days, and no significant dissociation of Gd-L 1～3 was observed, while Gd-EOB-DTPA almost completely dissociated within 12 h (Fig. 8 (c) and (d)). Gd-L 1～3 exhibited very high stability under strongly acidic conditions and had significant Zn(II) anti-interference ability, and thus is suitable for in vivo studies.

[0171] (3) Liver uptake pathway test

[0172] The interaction between transporters (OATP1B1, OATP1B3, OATP2B1 and NTCP) and complexes was evaluated using AutoDock Site65. The results are shown in Table 2 and Figure 9, wherein Figure 9 is a schematic diagram of the interaction of Gd-L1 with OATP1B1 (a), OATP1B3 (b), OATP2B1 (c) and NTCP (d), respectively; Gd-L2 with OATP1B1 (e), OATP1B3 (f), OATP2B1 (g) and NTCP (h), respectively; Gd-L3 with OATP1B1 (i), OATP1B3 (j), OATP2B1 (k) and NTCP (l), respectively; the PDB code of OATPs is 2GFP, and the hydrophobic interaction, hydrogen bond and salt bridge between the contrast agent and the transporter are represented by gray, blue and yellow dash lines, respectively. According to Table 2 and Figure 9, it can be found that OATP1B3 has strong affinity with Gd-EOB-DTPA, with a docking score of -7.95 kal / mol, and OATP1B1 also has similar affinity with Gd-L1, with a docking score of DS = -7.74 kal / mol. On the other hand, Gd-L2 and Gd-L3 exhibit lower liver uptake properties.

[0173] This is reflected in their higher inhibition constants (DS, kal / mol) and lower docking scores (KI, μM). In the process of drug-protein interaction, both Gd-EOB-DTPA and organic anion transporting polypeptides OATP1B1 and OATP1B3 form strong hydrophobic bonds, hydrogen bonds and salt bridges. The research results highlight the significant advantage of Gd-L1 in liver uptake, which is mainly transported by OATP1B1, OATP2B1 and NTCP (sodium-taurocholate cotransporting polypeptide), which are the main pathways for liver uptake. By comprehensively understanding the interaction mechanism between these transporters and complexes, it is expected to develop more efficient and more targeted liver-specific drugs.

[0174] Table 2 Docking score (DS, kcal / mol) and inhibition constant (KI, μM) of complexes with transporters

[0175]

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

[0177] (4) CCK-8 method was used to evaluate the cytotoxicity of chelates on LO2 and 293T cells

[0178] The contrast agent is mainly absorbed by the liver and kidney after injection through the tail vein, so in this invention, CCK-8 method was used to evaluate the cytotoxicity of the chelate on LO2 (Fig. 10 (a)) and 293T (Fig. 10 (b)) cells, and the commercial contrast agent Gd-EOB-DTPA was used as a control. Cells were incubated with three different complexes Gd-L 1～3 (≤0.8 mM) at 37°C for 24 h, and then incubated with 10% CCK-8 solution for 4 h. Then the optical density was measured by a microplate reader to evaluate the cell viability, and the error bar represents the standard deviation (±SD) (n=3). The survival rate of LO2 cells treated with Gd-L1 remained above 90% at a concentration of 0.5 mM. Even at a high concentration of 0.8 mM, the three complex contrast agents Gd-L 1～3 After treatment, the cell survival rate was all above 80%. Among them, compared with Gd-EOB-DTPA, the cytotoxicity of Gd-L1 on kidney cells was significantly reduced, which indicated that it was possible to become a safer choice for patients with impaired renal function to perform magnetic resonance imaging scanning.

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

[0180] All experiments in this invention were conducted in compliance with the animal ethics guidelines of our institute and were approved by the Animal Care and Use Committee of the State Key Laboratory of Marine Medicine, Sun Yat-Sen University. The MRI was performed on a 3.0T clinical MRI scanner (Ingenia elition, Philips) equipped with a mouse-dedicated coil. The BALB / c mice (6-8 weeks, 20-25 g) were injected with the drugs (0.1 mmol / kg) via the tail vein and the T1-weighted continuous dynamic contrast-enhanced MRI was performed to observe the signal enhancement of the liver and kidney at 35 min before and after the administration. In Fig. 11, (a) is the coronal image of the liver (the mouse liver is in the yellow solid line box), (b) is the coronal image of the kidney (the mouse kidney is in the yellow solid line box), where pre, 1 min, 6 min, 12 min, 18 min, 23 min, 29 min, 35 min are the corresponding detection times before and after the administration, respectively, (c) is the normalized signal-to-noise ratio (nSNR) of the liver, (d) is the normalized signal-to-noise ratio of the kidney, (e) is the relative contrast-to-noise ratio of the liver (ACNR). After the injection of the commercial contrast agent Gd-EOB-DTPA into the body, it is rapidly distributed to the liver and kidney, and the enhancement time of the liver is more than 35 min, and it can be rapidly metabolized from the body. In the MR images obtained by injecting the complex Gd-L1 into the mouse body as an MRI contrast agent, it is observed that Gd-L1 can be selectively distributed to the liver and kidney, the enhancement time of the liver is more than 35 min, and it can be substantially cleared from the kidney within 35 min, and its hepatobiliary imaging ability is comparable to that of Gd-EOB-DTPA, and it has the potential to be used as a hepatobiliary-specific MRI contrast agent. In the MR images obtained by injecting the complex Gd-L2 into the mouse body as an MRI contrast agent, it is observed that Gd-L2 can be selectively distributed to the liver and kidney, and the contrast enhancement degree of the liver is less than that of Gd-L1. In the MR images obtained by injecting the complex Gd-L3 into the mouse body as an MRI contrast agent, almost no contrast enhancement of the kidney is observed, and it is not suitable for being used as a hepatobiliary-specific MRI contrast agent.

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

[0182] It has been demonstrated that the liver uptake of bromosulphthalein (BSP) is also mediated by OATPs. BSP was used as a competitive agent to verify the liver uptake mechanism of Gd-L1. Fig. 12 shows the T1 -weighted images of the liver (a) and kidney (b) of normal Balb / c mice recorded at 3.0T after injection of Gd-L1 (0.1 mmol / kg), and the normalized signal-to-noise ratio (nSNR) as a function of time for the liver (c) and kidney (d) within 35 min. Before the injection of Gd-L1, BSP was repeatedly injected through the tail vein to maintain a plasma concentration of 0.1 mM. The uptake of Gd-L1 by hepatocytes was significantly inhibited due to the occupation of OATP transport channels by BSP. Comparative analysis shows that the liver enhancement of the BSP(+) group within 20 min is minimal, in sharp contrast to the BSP(-) group. Given the life of the contrast agent in the body is more than 35 min, the weak enhancement of the liver also becomes apparent as BSP is rapidly metabolized in the body. The decrease in liver excretion leads to a significant increase in clearance and metabolic cycle of the kidney system. Therefore, the nSNR of the renal cortex and medulla of the BSP(+) group increases rapidly and significantly compared with the BSP(-) group. Based on these findings, it is confirmed that the expression of OATPs on the surface of hepatocyte membranes is the main pathway for the uptake of Gd-L1 by hepatocytes.

[0183] (7) Hepatobiliary imaging test

[0184] The complex Gd-L1 has hepatobiliary imaging ability comparable to Gd-EOB-DTPA, and its stability is much better than that of Gd-EOB-DTPA. To further develop its potential in hepatobiliary imaging, a mouse H22 orthotopic liver cancer model was used to study its advantages and disadvantages in detecting mouse liver cancer. After Gd-L1 was injected into the body through the tail vein, the image under 3.0T magnetic resonance was obtained, and the hepatobiliary MR image (Fig. 13) was obtained, where pre, 1 min, 6 min, 12 min, 18 min, 23 min, 29 min, 35 min, 40 min, 3 min, 9 min, 14 min, 20 min, 26 min, 32 min, 38 min, 43 min are the corresponding detection times before and after administration, respectively. In Fig. 13, the first row of images on the left is the coronal image of the liver, and the second row of images is the transverse image of the liver, and the right image is the CNR (liver to tumor) as a function of time in the coronal and transverse planes. According to the data in Fig. 13 and Table 3, Gd-L1 can significantly enhance the contrast between normal and diseased tissues in the liver, and the SI ratio of the liver parenchyma and the liver lesion tissue is significantly improved after enhancement, and the lesion boundary is clear, and the effect time is long. Table 3. Magnetic resonance signal values of Gd-L1 as a hepatobiliary specific magnetic resonance imaging contrast agent for detecting mouse orthotopic liver cancer under 3.0T magnetic resonance

[0185]

[0186] To minimize the random error in the experiment, ensure the accuracy and relevance of the imaging data obtained, further selected the weight of about ten times the mouse adult healthy rats as the experimental object, to evaluate the potential of Gd-L1 as a liver-specific contrast agent. Figure 14 is a normal rat (Wistar, 6-8 weeks) before injection of Gd-EOB-DTPA and Gd-L1 (0.1 mmol / kg), respectively, 1 min, 10 min and 20 min after injection in 3.0T capture liver (a) and kidney (b) transverse plane recorded T1 weighted image, liver (c) and kidney (d) nSNR time course after injection of contrast agent. Data are expressed as mean ± standard deviation (n = 3). In 3.0T clinical magnetic resonance imaging device using rat-specific coil, magnetic resonance scanning was performed before and after intravenous injection of Gd-L1 (0.1 mmol / kg). It is worth noting that Gd-L1 rapidly accumulates in the liver and kidney, and the pharmacokinetic characteristics of rats and mice are consistent. The enhancement effect of Gd-L1 on liver tissue is comparable to that of Gd-EOB-DTPA. This study demonstrates the potential of Gd-L1 in magnetic resonance imaging applications involving large animals, and lays the foundation for further in-depth study of its clinical application.

[0187] (8) In vivo biodistribution

[0188] To investigate the biodistribution of Gd-EOB-DTPA and Gd-L1 in vivo, the inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850) was used to quantitatively determine the Gd(III) content in blood and various organs at the designated time points after intravenous injection of each contrast agent at 0.1 mmol / kg. Figure 15 (a) is the biodistribution study of Gd-EOB-DOTA and Gd-L1 (0.1 mmol / kg) in normal mice, expressed as the percentage of Gd in each tissue, (b) is the total uptake of the complex by five internal organs and the brain within 5 min, 15 min and 24 h after injection, and the data is 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 after injection, accounting for 44.66 ± 2.98% of the administered dose. This level decreased to 32.47 ± 2.08% of the dose after 15 min. Similarly, Gd-L1 also showed rapid liver accumulation, reaching 102.32 ± 6.73 μg / g of tissue within 5 min, accounting for 40.94 ± 1.47% of the administered dose. After 15 min, the metabolism of Gd-L1 resulted in a decrease in dose to 30.98 ± 8.83%, which was statistically equivalent to the dose of Gd-EOB-DTPA. Notably, the residual amount of Gd(III) in all the tested tissues was less than 1% at 24 h after injection, reducing the potential harm to human functional organs and the immune system. In summary, these research results show that Gd-L1 has a similar excretion pathway as Gd-EOB-DTPA, with high liver enrichment and rapid clearance from the liver and kidneys. Therefore, it is expected to become a liver-specific contrast agent based on Gd(III) for magnetic resonance imaging applications.

[0189] (9) Potential toxicity test of free Gd(III) on the function of major organs

[0190] The safety issue of free Gd(III) remains the main challenge for clinical application of contrast agents. To evaluate the potential toxicity of Gd-L1, Gd-DOTA and PBS were used as a comprehensive study of the effects on the function of major organs of healthy mice. Serum biochemistry and histological examination using hematoxylin and eosin (H&E) staining were included. Acute phase mice (1 day) received one injection of drug, while sub-acute phase mice (60 days) received one injection of drug every two weeks for two months. Plasma and tissue samples were collected 24 h after the last administration. FIG. 16 is the histopathological analysis (a) and blood biochemistry analysis (b) of Gd-DOTA and Gd-L1 acute (1 day) and sub-acute (60 days) toxicity, with an equal volume of PBS as a control, ALT is alanine aminotransferase; ALB is albumin, ALKP is alkaline phosphatase, AST is aspartate aminotransferase, BUN is blood urea nitrogen, LDH is lactate dehydrogenase, CK is creatine kinase, data are expressed as mean ± standard deviation (n = 3) and there is no significant difference compared with PBS treatment. As shown in (b) of FIG. 16, no mouse death was observed and no adverse reactions were found during the whole experiment. Several important liver function indicators (ALT, AST, ALB) and kidney function indicators (BUN, CR) were measured. As shown in (a) of FIG. 16, there was no significant change in these serum biochemical indicators compared with the control group, indicating that liver and kidney function was not damaged. In addition, histological examination by H&E staining also found no obvious abnormalities compared with the control group. Heart tissue showed normal morphology, size and arrangement of myocardial cells, with no signs of inflammation or necrosis. In the liver, although mild liver congestion was observed in the central vein of the Gd-DOTA treated group, mice treated with Gd-L1 showed normal liver lobular structure, with no signs of liver damage or inflammation. Spleen tissue showed normal white and red plasma, indicating healthy spleen function. Lung tissue showed normal respiratory bronchioles, alveolar ducts, alveolar sacs and lung parenchyma. In kidney tissue, the morphology of renal tubules and glomeruli was normal, and no obvious damage or inflammation was found. In summary, Gd-L1 did not cause functional damage to the heart, liver, spleen, lungs or kidneys of mice within the specified time, showing good biocompatibility. These findings strongly demonstrate the safety and effectiveness of Gd-L1 as a potential magnetic resonance imaging contrast agent.

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

[0192] To evaluate 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) were monitored at 1.4 T under different horseradish peroxidase (HRP) activities (0 U, 5 U, 50 U, 500 U) at 37 °C, and 0.5 eq of hydrogen peroxide (H2O2, 5 mM) was added in sequence (Fig. 17 (a) longitudinal and (b) transverse). Thus, the relaxation rates were 3.16, 3.36 and 3.55 times higher than that without HRP under 5 U, 50 U and 500 U of HRP, respectively. Notably, the R2 value was also increased by 2.8 times in the presence of 5 U of HRP and 1 eq of H2O2. This indicates that Gd-L3 exhibits a very high responsiveness to HRP even at low enzyme activity and trace oxidant concentration. The relaxation rates of Gd-L3 in the presence of H2O2 / HRP (HRP, 5 U, H2O2, 2 eq) were also tested, and the results show that r1= 17.35 mM -1 s -1 , r2= 18.80 mM -1 s -1 , which is about 3.8 times higher than that of its monomer form (Fig. 17 (c)). The low affinity of Gd-L3 oligomers to HSA (Fig. 17 (d)) can facilitate their rapid clearance from the body. These results collectively indicate that the enhanced relaxation rates and responsiveness to peroxidase of Gd-L3 make it an ideal candidate for peroxidase-responsive MRI applications. Magnetic resonance imaging was performed using a 3.0 T clinical magnetic resonance (Discovery MR750, GE medical imaging system) equipped with a mouse-specific coil. The in vivo MRI signal enhancement of the left and right hind limbs of MSU-induced acute gouty Swiss mice (6-8 weeks, 20-25 g) was observed by T1-weighted continuous dynamic contrast-enhanced magnetic resonance imaging at 20 min before and after the tail vein injection of Gd-L2 and Gd-L3 (0.1 mmol / kg), respectively. Fig. 17 (e) and (f) are the liver MRI images obtained by using the complexes Gd-L2 and Gd-L3 as activatable MPO MRI contrast agents for detecting mouse liver and gallbladder, in which pre, 2 min, 5 min, 8 min, 10 min, 15 min, 20 min are the corresponding detection times before and after administration, respectively. It can be observed that both complexes produce selective contrast enhancement at the location of gout lesions in mice after being taken into the body, and Gd-L3 provides more obvious contrast enhancement than Gd-L2 (Fig. 17 (g) and (h)), which has the potential to be used as an activatable myeloperoxidase magnetic resonance imaging probe to detect myeloperoxidase enzyme in vitro and in vivo.

[0193] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A monosubstituted cyclic ligand having the structure shown in Formula I: in, R includes benzyloxy, ethoxy, 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, compound 8, or compound 9:

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. The preparation method according to claim 2, characterized in that, The preparation method of compound 9 includes the following steps: Compound 8 was subjected to a fourth nucleophilic substitution reaction with bromoethane to obtain compound 9.

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

7. The method for preparing the monosubstituted cyclic rare earth complex according to claim 6, 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 includes Gd(III) or Eu(III).

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

9. The application according to claim 8, characterized in that, The contrast agents include those used in hepatobiliary magnetic resonance imaging and / or inflammatory magnetic resonance imaging.

10. The application of the monosubstituted cyclic rare earth complex of claim 6 or the monosubstituted cyclic rare earth complex prepared by the method of claim 7 in the detection of peroxidase, wherein R of the monosubstituted cyclic rare earth complex is a hydroxyl group and Ln is Gd(III).

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