Preparation method and application of manganese-based micromolecular magnetic resonance imaging contrast agent

Abstract

The invention belongs to the technical field of medical materials, and discloses a preparation method and application of a manganese-based micromolecule magnetic resonance imaging contrast agent. According to the invention, a macrocyclic chelating ligand H4L is firstly prepared, then the manganese-based micromolecular complex MnL2-is prepared by using the ligand, the molecular formula of the manganese-based micromolecular complex MnL2-is C38H56MnN10O92-, and the manganese-based micromolecular complex MnL2-can be used as a magnetic resonance imaging contrast agent. Through detection, the product has very good stability, and the hydration condition q of the complex is equal to 0.78 through 17O NMR (nuclear magnetic resonance) test and relaxation rate test, which indicates that the complex has one coordination water and can provide a monitorable magnetic resonance signal. And the obtained relaxation rate r1 is equal to 2.255 mM <-1 > s <-1 >, which shows that the product has great application value.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials and relates to a magnetic resonance contrast agent, in particular to a preparation method of a manganese-based small molecule magnetic resonance imaging contrast agent and its enhanced stability and relaxation rate characteristics. Background Art

[0002] Magnetic resonance imaging (MRI) is a powerful disease diagnosis technology. Because of its great advantages in tissue penetration depth and safety, it plays a key role in improving the contrast between tumor tissue and normal tissue and shortening imaging time. In addition, because of its high soft tissue resolution, it can monitor the early diagnosis and treatment of diseases in real time, so it is often used as one of the important clinical detection methods. The physical basis of MRI is the nuclear magnetic resonance phenomenon. This imaging technology can image water molecules in organisms. MRI contrast agents can accelerate the relaxation rate of water molecules. In MRI examinations, most diagnoses can use contrast agents to enhance imaging contrast. Since the first batch of contrast agents entered the market, people have been keen on developing a large number of contrast agents to achieve higher sensitivity and contrast of magnetic resonance contrast agent imaging.

[0003] At present, commercial contrast agents are mainly gadolinium-based small molecule complexes. Although they have the characteristics of high relaxation rates, their stability can cause patients to worry about the risk of renal systemic fibrosis or brain diseases. Therefore, the development of MRI contrast agents that can replace gadolinium can not only improve the clinical MRI imaging contrast requirements and diagnostic accuracy, but also greatly reduce the toxic side effects of contrast agents on the kidneys or brain. Therefore, the design of high-performance new contrast agents is one of the research focuses in the field of MRI contrast agents. Divalent manganese, as an essential element in the body, has very low toxicity and has 5 unpaired electrons, which has a strong relaxation enhancement effect. Therefore, divalent manganese complexes can be used as excellent substitutes for gadolinium-based contrast agents. However, the thermodynamic stability and kinetic inertness of manganese complexes are lower than those of gadolinium complexes because the manganese ion has a lower charge and lacks ligand field stabilization energy (high spin d 5 Electronic configuration). Therefore, designing a special chelating ligand is a key step in developing manganese-based contrast agents. Stabilizing manganese complexes can not only prevent the conversion of divalent manganese to trivalent manganese, thereby weakening the magnetic resonance imaging ability, but also ensure sufficient thermodynamic stability and kinetic inertness to prevent excessive exposure of free manganese ions from causing "manganese poisoning". Therefore, the present invention provides a chelating ligand H of a nitrogen-containing macrocyclic carboxylic acid. 4 L and preparation of its divalent manganese complex MnL 2- Complex MnL 2-It exhibits very good stability in strong acid and other competitive metal ion environments, and has higher relaxation rate characteristics than the same type of manganese-based magnetic resonance imaging contrast agent Mn(1,4-DO2A), indicating that it is a potential excellent MRI contrast agent and is expected to be used for further clinical testing applications. Summary of the invention

[0004] The present invention aims at some deficiencies of existing magnetic resonance contrast agents and provides a novel chelating ligand H 4 L and its manganese complex MnL 2- The preparation method of MnL 2- Research on the stability characteristics and relaxation rate characteristics.

[0005] The present invention first provides a macrocyclic chelating ligand, wherein the ligand is H 4 L, where L is the first letter of the ligand, H represents the hydrogen of the carboxylic acid, and H 4 L indicates that the ligand has four ligand points that can be deprotonated to participate in coordination, and in the present invention, specifically refers to four carboxyl groups. 4 The chemical structure of L is as follows:

[0006]

[0007] The H 4 The preparation method of L comprises the following steps:

[0008] 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl) di-tert-butyl diacetate (DO2A-tBu), sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction bottle in a certain proportion, and acetonitrile (MeCN) was added as a solvent. The mixture was stirred at room temperature until dissolved and then heated to react for a certain period of time. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance. Compound H was obtained after further purification by a chromatographic column. 4 The protective group -tBu was removed by ester hydrolysis with trifluoroacetic acid (TFA) in dichloromethane (DCM) to obtain the chelating ligand H 4 L.

[0009] The H of the present invention 4 The preparation method of L uses acetonitrile with a high boiling point as a reaction solvent, and adopts a heating reflux method to obtain compound H 4 L-tBu, and then deprotected to generate ligand H 4 L. The obtained product H 4 The yield of L was increased from 47% to 91%, reducing the waste of reactants.

[0010] Furthermore, the molar ratio of DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine is 1:2:1.25.

[0011] Further, in order to control the reaction to the target product, compound H is prepared 4 The reaction temperature of L-tBu is 80°C and the reaction time is 12 hours.

[0012] Furthermore, the volume ratio of TFA and DCM used for removing the protecting group is 5:1.

[0013] The present invention also provides a preparation method of a manganese-based small molecule complex, wherein the manganese-based small molecule complex is denoted as MnL 2- , molecular formula is C 38 H 56 Mn 10 O 9 2- .

[0014] The manganese-based small molecule complex MnL 2- The chemical structure is as follows:

[0015]

[0016] The manganese-based small molecule complex MnL 2- The preparation method comprises the following steps:

[0017] The ligand H 4 L is dissolved in a reaction flask, and an aqueous solution of manganese chloride tetrahydrate is added dropwise thereto. The pH of the reaction solution is slowly adjusted to neutral using NaOH and HCl solutions until the addition of the aqueous solution of manganese chloride tetrahydrate is complete and the pH is always maintained at neutral. The reaction is carried out in two stages, that is, after a period of time at room temperature, heating is required for further reaction. After the reaction is completed, it is purified by high performance liquid chromatography (HPLC) to obtain the final product MnL 2- .

[0018] Wherein, the ligand H 4 The molar ratio of L to manganese chloride tetrahydrate is 1:1.2.

[0019] The first stage reaction temperature is 25°C and the reaction time is 4 hours;

[0020] The reaction temperature of the second stage is 65° C. and the reaction time is 4 hours.

[0021] The present invention also provides the stability of the manganese complex, 17 O NMR determination of coordinated water and its relaxation rate and other properties.

[0022] The method for testing the stability of manganese complexes comprises the following steps:

[0023] Take manganese complex MnL 2- The solution was mixed with PBS buffer in a sample tube, and 5 equivalents and 25 equivalents of Zn 2+ , 0.1M and 1.0M HCl and 5 equivalents of K + , Ca 2+ 、Na + Mg 2+ , Mn 2+ , Fe 3+ , Fe 2+ 、Co 2+ 、Ni 2+ , Cu 2+ , Cu + The stability of the complex was tested by ion detection. Under the above conditions, the product showed very good stability.

[0024] The manganese complex 17 The method for determining coordinated water by O NMR comprises the following steps:

[0025] Take manganese complex MnL 2- The solution was mixed with PBS buffer in a sample tube, and Mn-EDTA (q = 1) was used as a reference system to test the relaxation rate r of the sample at different temperatures. 2 0 [Test method refers to Gale EM, Zhu J, Caravan P. Journal of the American Chemical Society. 2013, 135(49): 18600-8.] Through calculation, the hydration condition of the product was obtained to be q=0.78, indicating that the complex has one coordinated water and can provide sufficient magnetic resonance signal.

[0026] The relaxation rate test method of the manganese complex comprises the following steps:

[0027] Take manganese complex MnL 2- The solution was mixed with PBS buffer in a sample tube, and the relaxation time T1 at different sample concentrations was tested. The relaxation rate r of the product was obtained by plotting the concentration and 1 / T1 and performing a linear fit. 1 =2.255mM -1 s -1 The test conditions are field strength 1.41T, temperature 37℃. The data show that this product is superior to the same type of Mn(1,4-DO2A)(r 1 =1.7mM -1 s -1 ) relaxation rate. [Data cited from 1 H and 17O NMR Relaxometric and ComputationalStudy on Macrocyclic Mn(II)Complexes.Inorg.Chem.2013,52,3268.]

[0028] Therefore, the manganese complex MnL 2- It can be used as a potentially safe and effective magnetic resonance imaging contrast agent.

[0029] Beneficial effects of the present invention:

[0030] (1) The present invention develops ligand H 4 Preparation conditions of L. Compound H was obtained by using acetonitrile as solvent, heating under reflux at 80°C, and adding sodium carbonate to promote the reaction. 4 L-tBu, and then deprotected to generate ligand H 4 L. The obtained product H 4 L is a tetracarboxyl macrocyclic chelating ligand, which (a) provides four carboxyl groups to participate in coordination and increase the water solubility of the sample, and (b) provides two tetranitrogen rings and two pyridine rings to participate in the stable chelation of metals and the hydrogen bonding of chelated water.

[0031] (2) The ligand H used in the present invention 4 L and manganese chloride tetrahydrate were used as starting materials, and the preparation conditions were explored in aqueous solution to obtain a manganese-based small molecule complex with good water solubility, which was recorded as MnL 2- , molecular formula is C 38 H 56 Mn 10 O 9 2- .

[0032] (3) In the manganese complex MnL 2- A series of competitive metal ions and strong acid environments were added to the PBS buffer solution, and the ultraviolet absorption of the samples was tested after 24 hours. The results showed that this product has very good stability.

[0033] (4) Through 17 O NMR test and relaxation rate test, the hydration condition of the complex was obtained to be q = 0.78, indicating that the complex has a coordinated water, which can provide a detectable magnetic resonance signal. The relaxation rate obtained is r 1 =2.255mM -1 s -1 , indicating that the product has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 For tetraazacyclic ligand H 4Chemical structure (a) and synthesis route (b) of L.

[0035] Figure 2 Ligand H 4 L(a) and manganese complex MnL 2- (b) High-resolution mass spectrum of .

[0036] Figure 3 Ligand H 4 UV absorption (a) and fluorescence spectrum (b) of L.

[0037] Figure 4 MnL 2- Chemical structure (a) and UV absorption spectrum (b).

[0038] Figure 5 MnL 2- Stability under different conditions.

[0039] Figure 6 MnL 2- of 17 O NMR graph (a) and relaxation rate graph (b). DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0041] In the embodiments of the present invention, the experimental methods without special description of specific conditions are all carried out according to the conventional methods and conditions in the art, and the materials used are all conventionally commercially available unless otherwise specified.

[0042] Reagents and instruments used in the examples of the present invention: The solvents used in the reactions were all analytically pure, and the reagents used were used directly without any special treatment unless otherwise specified.

[0043] Acetonitrile and dichloromethane: analytical grade, National Group Chemical Reagent Co., Ltd.;

[0044] Ethanol, HCl, and TFA solution: analytical grade, Shanghai Zhongshi Chemical Corporation;

[0045] Manganese chloride tetrahydrate, sodium carbonate, sodium hydroxide, anhydrous ferric chloride, ferrous chloride, cobalt nitrate, nickel chloride, zinc chloride, cupric nitrate, cuprous chloride, potassium chloride, sodium chloride, magnesium chloride, calcium chloride: analytical grade, Shanghai Chemical Reagent Co., Ltd.;

[0046] Di-tert-butyl 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetate (DO2A-tBu) and 2,6-bis(chloromethyl)pyridine: analytical grade, Anergy Company;

[0047] UV-visible spectrophotometer: Japan Shimadzu UV-2450, 800-190nm;

[0048] Nuclear magnetic resonance (NMR) spectrometer: AVANCEII 400Hz, Bruker, Switzerland;

[0049] Fluorescence spectrophotometer: Cary Eclipse, Varian Pty Ltd, Australia;

[0050] High performance liquid chromatography (HPLC): Made in Germany, Agilent Technologies.

[0051] Example 1: Ligand H 4 Preparation of L

[0052] like Figure 1 As shown in b, DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction bottle at a molar ratio of 1:2:1.25, and 20 mL of acetonitrile was added as the reaction solvent, and then the temperature was gradually raised to 80°C and heated under reflux for 12 hours. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance, which was further purified by a chromatographic column to obtain compound H 4 L-tBu. 4 In the reaction bottle of L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and the temperature was maintained at 5°C. Subsequently, 2.5 mL of TFA was added and the sample bottle was gradually returned to room temperature and reacted for 4 hours. Finally, the reaction temperature was raised to 65°C and the reaction was terminated after 6 hours. The ligand H was separated and purified by HPLC. 4 L, structural formula Figure 1 The yield of a is 91%. Figure 2 A.

[0053] Figure 3 a is the ligand H 4 The UV absorption graph of L shows that the ligand has an absorption peak at 264 nm, which comes from the π→π* transition of the pyridine ring;

[0054] Figure 3 b is ligand H 4 The fluorescence spectrum of L shows that under the excitation of 285 nm, the ligand has two strong and broad emission peaks at 332 nm and 352 nm, and a weak and broad emission peak at around 630 nm;

[0055] The product is H 4 L, whose molecular formula is C 38 H 58 N 10 O 8 , its chemical structure is:

[0056]

[0057] This embodiment is the best H 4 Preparation scheme of L.

[0058] Example 2: Ligand H 4 Preparation of L

[0059] DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction flask in a molar ratio of 1:2:1.25, and 20 mL of acetonitrile was added as the reaction solvent, and then the temperature was gradually raised to 80°C and heated under reflux for 6 hours. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance, which was further purified by a chromatographic column to obtain compound H 4 L-tBu. 4 In the reaction bottle of L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and the temperature was maintained at 5°C. Subsequently, 2.5 mL of TFA was added and the sample bottle was gradually returned to room temperature and reacted for 4 hours. Finally, the reaction temperature was raised to 65°C and the reaction was terminated after 6 hours. The ligand H was separated and purified by HPLC. 4 L, the yield is 54%. It can be seen from this comparative example that the reaction time is too short, the starting material is not completely reacted, which is not conducive to the formation of the target product.

[0060] Example 3: Ligand H 4 Preparation of L

[0061] DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction flask in a molar ratio of 1:2:1.25, and 10 mL of acetonitrile was added as the reaction solvent. The temperature was gradually raised to 80°C and refluxed for 12 hours. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance, which was further purified by a chromatographic column to obtain compound H 4 L-tBu. 4 In the reaction bottle of L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and the temperature was maintained at 5°C. Subsequently, 2.5 mL of TFA was added and the sample bottle was gradually returned to room temperature and reacted for 4 hours. Finally, the reaction temperature was raised to 65°C and the reaction was terminated after 6 hours. The ligand H was separated and purified by HPLC.4 L, the yield was 47%.

[0062] It can be seen from this comparative example that when the reaction uses less solvent, some solutes may not be completely dissolved, resulting in a lower yield of the obtained product.

[0063] Example 4: Ligand H 4 Preparation of L

[0064] DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction flask in a molar ratio of 1:2:1.5, and 20 mL of acetonitrile was added as the reaction solvent. The temperature was gradually raised to 80°C and refluxed for 12 hours. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance, which was further purified by a chromatographic column to obtain compound H 4 L-tBu. 4 In the reaction bottle of L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and the temperature was maintained at 5°C. Subsequently, 2.5 mL of TFA was added and the sample bottle was gradually returned to room temperature and reacted for 4 hours. Finally, the reaction temperature was raised to 65°C and the reaction was terminated after 6 hours. The ligand H was separated and purified by HPLC. 4 L, the yield was 74%.

[0065] It can be seen from this comparative example that an excess of the starting material 2,6-bis(chloromethyl)pyridine tends to generate other by-products, thereby resulting in a lower yield of the obtained product.

[0066] Example 5: Ligand H 4 Preparation of L

[0067] DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added to the reaction flask at a molar ratio of 1:2:1.25, and 20 mL of acetonitrile was added as the reaction solvent, and then stirred at room temperature to dissolve and react for 12 hours. After the reaction was completed, the precipitate was repeatedly washed with acetonitrile, and the supernatant was collected after standing. The supernatant was dried to obtain a light yellow oily substance, which was further purified by a chromatographic column to obtain compound H 4 L-tBu. 4 In the reaction bottle of L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and the temperature was maintained at 5 °C. Subsequently, 2.5 mL of TFA was added and the sample bottle was gradually restored to room temperature and reacted for 4 hours. Finally, the reaction temperature was raised to 65 °C and the reaction was terminated after 6 hours. The ligand H was separated and purified by HPLC. 4 L, the yield was 52%.

[0068] It can be seen from this comparative example that the reaction temperature is low and the effective collision rate between molecules is small, resulting in a low yield of the obtained product.

[0069] Example 6: Complex MnL 2- Preparation

[0070] The method of Example 1 was used to prepare H 4 L.

[0071] The ligand H 4 L (1.0mmol) was dissolved in a reaction flask and an aqueous solution of manganese chloride tetrahydrate (2.0mmol) was added dropwise thereto. The pH of the reaction solution was slowly adjusted to neutral using 1M NaOH and HCL solutions until the addition of the aqueous solution of manganese chloride tetrahydrate was complete and the pH was always maintained at neutral. The reaction was carried out in two stages, namely, after reacting at room temperature of 25°C for 4 hours, it was further heated at 65°C for 4 hours. After the reaction was completed, 32mg of the final product was obtained by HPLC purification with a yield of 78%. The mass spectrum is shown in Figure 2 b.

[0072] The product is a mononuclear manganese complex, denoted as MnL 2- , whose molecular formula is C 38 H 56 Mn 10 O 9 2- , its possible chemical structure is:

[0073]

[0074] This embodiment is a scheme for preparing the best manganese-based small molecule complex crystal material Mn. Figure 4 b is the complex MnL 2- From the ultraviolet absorption spectrum, it can be seen that there is a broad absorption peak at 265nm, which does not change much compared with the ultraviolet absorption of the ligand, indicating that pyridine does not participate in the coordination of the metal.

[0075] Comparative Example 1: Complex MnL 2- Preparation

[0076] The method of Example 1 was used to prepare H 4 L.

[0077] The ligand H 4L (1.0 mmol) was dissolved in a reaction flask and an aqueous solution of manganese chloride tetrahydrate (2.0 mmol) was added dropwise thereto. In this embodiment, no NaOH or HCL solution was added to adjust the pH value. The reaction was then carried out in two stages, i.e., after reacting at room temperature of 25°C for 4 hours, it was further heated at 65°C for 4 hours. After the reaction was completed, 7 mg of the final product was obtained by HPLC purification with a yield of 16%.

[0078] It can be seen from this comparative example that if the pH value of the reaction solution in the reaction bottle is not adjusted, some solutes will not be completely dissolved, and H 4 L cannot be completely deprotonated to participate in the coordination reaction of manganese, resulting in a low product yield.

[0079] Comparative Example 2: Complex MnL 2- Preparation

[0080] The method of Example 1 was used to prepare H 4 L.

[0081] The ligand H 4 L (1.0 mmol) was dissolved in a reaction flask and an aqueous solution of manganese chloride tetrahydrate (2.0 mmol) was added dropwise thereto. The pH of the reaction solution was slowly adjusted to neutral using 1 M NaOH and HCL solutions until the aqueous solution of manganese chloride tetrahydrate was added dropwise and the pH was always maintained at neutral. The reaction was carried out at room temperature of 25° C. for 8 hours. After the reaction was completed, 11 mg of the final product was obtained by HPLC purification with a yield of 25%.

[0082] It can be seen from this comparative example that if the temperature of the reaction flask is not raised, the reaction rate will be slow. 4 L cannot be completely deprotonated to participate in the coordination reaction of manganese, resulting in a low product yield.

[0083] Example 9: Complex MnL obtained in Example 6 2- Stability test

[0084] The stability of the complex was investigated by detecting the ultraviolet absorption of the sample. 2- PBS buffer solution was placed in a cuvette, and a UV absorption curve of the complex was measured as a blank control. Then, 5 equivalents and 25 equivalents of Zn were added to the sample solution respectively. 2+ , 5 equivalents of K + , Ca 2+ 、Na + Mg 2+ , Mn 2+ , Fe 3+ , Fe 2+ 、Co 2+ 、Ni 2+ , Cu2+ , Cu + After the addition of ions, a UV absorption curve was measured as the initial state. After 24 hours, a UV absorption curve was measured for the above mixed solution as the end state. In addition, the complex was exposed to 0.1M and 1.0M HCl to detect its UV absorption after 24 hours.

[0085] like Figure 5 As shown, by comparing the above series of UV absorption curves with the blank, the UV absorption intensity of the complex has no obvious change, showing good stability.

[0086] Example 10: Complex MnL obtained in Example 6 2- The coordinated water

[0087] The coordination water of the complex was measured by Carr-Purcell-Meiboom-Gill spin echo experiment to obtain the transverse relaxation time T2. 2 O and in a 2% HO 2 17 O was enriched in aqueous solution and the pH was adjusted to 6-7. Using the CPMG pulse sequence, H was obtained in the temperature range of T = 268 to 363 K. 2 17 The transverse relaxation time T2 of O at 9.4 T is determined by H 2 17 The line width of the O NMR signal at half height was measured 17 The transverse relaxation time of O is then used to calculate the transverse relaxation rate (r 2 0 ). Finally, through r 2 0 ~(1000 / T) is plotted and the hydration q is calculated to be 0.78. Test data and results are as follows Figure 6 As shown in a.

[0088] Example 11: Complex MnL obtained in Example 6 2- The relaxation rate

[0089] The relaxation rate of the complex was obtained by recording the T1 measurement value using a Bruker mq60 Minispec at 1.41T and 37°C. The manganese complex was diluted to different gradient concentrations with PBS solution at pH = 7.4. A series of longitudinal relaxation times T1 were measured and the experiment was repeated three times. The relaxation rate (r 1 ) is determined by the slope of the 1 / T1 vs. metal concentration curve of the five concentrations. The above operation is repeated three times in parallel, and the longitudinal relaxation rate r obtained from the three tests is 1 The average value is taken as the final result, which is 2.255mM -1 s-1 The test data and the linear fit are as follows Figure 6 As shown in b.

Claims

1. A macrocyclic chelating ligand, characterized in that: It is referred to as H4L and its chemical structure is as follows:

2. The method for preparing a macrocyclic chelating ligand according to claim 1, characterized in that: Here are the steps: Add di-tert-butyl 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetate DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine in proportion to a reaction bottle, add acetonitrile as a solvent, stir at room temperature until dissolved and then heat to react. After the reaction is completed, wash the precipitate repeatedly with acetonitrile, collect the supernatant after standing, and spin-dry the supernatant to obtain a light yellow oily substance; further purify by a chromatographic column to obtain compound H4L-tBu; The protecting group -tBu was removed by ester hydrolysis with trifluoroacetic acid (TFA) in dichloromethane (DCM) to afford the chelating ligand H4L.

3. The method for preparing a macrocyclic chelating ligand according to claim 2, characterized in that: The molar ratio of the DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine is 1:2:1.

25.

4. The method for preparing a macrocyclic chelating ligand according to claim 2, characterized in that: The reaction temperature is 80° C. and the reaction time is 12 hours.

5. The method for preparing a macrocyclic chelating ligand according to claim 2, characterized in that: When removing the protecting group -tBu, the volume ratio of TFA and DCM used was 5:

1.

6. A manganese-based small molecule complex, characterized in that: It is prepared by the macrocyclic chelate ligand according to claim 1, and has the molecular formula C 38 H 56 Mn 10 O9 2- , the structural formula is:

7. The method for preparing the manganese-based small molecule complex according to claim 6, characterized in that: Here are the steps: The ligand H4L was dissolved in a reaction flask, and an aqueous solution of manganese chloride tetrahydrate was added dropwise thereto, and the pH of the reaction solution was slowly adjusted to neutral using NaOH and HCl solutions until the addition of the aqueous solution of manganese chloride tetrahydrate was completed and the pH was always maintained at neutral; the reaction was carried out in two stages, that is, the first stage reaction was carried out at room temperature, and then heating was required for the second stage reaction. After the reaction was completed, the final product MnL was purified by high performance liquid chromatography to obtain 2- .

8. The method for preparing the manganese-based small molecule complex according to claim 7, characterized in that: The molar ratio of the ligand H4L to manganese chloride tetrahydrate is 1:1.

2.

9. The method for preparing the manganese-based small molecule complex according to claim 7, characterized in that: The temperature of the first stage reaction is 25°C and the reaction time is 4 hours; The temperature of the second stage reaction is 65° C. and the reaction time is 4 hours.

10. Use of the manganese-based small molecule complex according to claim 6 as a magnetic resonance imaging contrast agent.

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