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

Abstract

The application belongs to the technical field of medical materials, and discloses a preparation method of a manganese-based small-molecule magnetic resonance imaging contrast agent and application thereof. The application first prepares a macrocyclic chelating ligand H4L, and then uses the ligand to prepare a manganese-based small-molecule complex MnL 2‑ , with a molecular formula of C 38 H 56 MnN 10 O9 2‑ , which can be used as a magnetic resonance imaging contrast agent. Through detection, the product has very good stability, and through 17 O NMR testing and relaxation rate testing, the hydration condition q=0.78 of the complex is obtained, indicating that the complex has one coordinated water and can provide a monitorable magnetic resonance signal. The obtained relaxation rate is r1=2.255mM ‑1 s ‑1 , indicating that the product has great application value.

Description

TECHNICAL FIELD

[0001] The present application 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 enhanced stability and relaxation rate characteristics thereof. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is a powerful disease diagnosis technology. It has great advantages in tissue penetration depth and safety, plays a key role in improving the contrast between tumor tissue and normal tissue, shortening the imaging time, etc. In addition, it is often used as one of the important clinical detection means because it has high soft tissue resolution and can monitor the early diagnosis and treatment of diseases in real time. The physical basis of MRI is the nuclear magnetic resonance phenomenon, and this imaging technology can image water molecules in the body. MRI contrast agents can accelerate the relaxation rate of water molecules. In MRI examination, most diagnoses can be enhanced by using contrast agents to enhance the imaging contrast. Since the first batch of contrast agents entered the market, people have been enthusiastic about developing a large number of contrast agents to achieve higher sensitivity and contrast of magnetic resonance contrast agent imaging.

[0003] At present, the commercialized contrast agent is mainly a gadolinium-based small-molecule complex. Although they have the characteristics of high relaxation rate, the stability thereof can cause patients to worry about the risk of renal fibrosis or brain disease, so 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. As a necessary element in the body, divalent manganese 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 charge of manganese ions is lower, and there is a lack of ligand field stabilization energy (high-spin d 5 electron configuration). Therefore, designing special chelating ligands is a key step in developing manganese-based contrast agents. Stabilizing manganese complexes not only prevents the transformation of divalent manganese to trivalent manganese to reduce the magnetic resonance imaging capability, but also ensures sufficient thermodynamic stability and kinetic inertness to prevent "manganese poisoning" caused by excessive exposure of free manganese ions. Therefore, the present application provides a nitrogen-containing macrocyclic carboxylic acid chelating ligand H4L and a divalent manganese complex MnL 2- 2- ​It has very good stability in strong acid and other competitive metal ion environment, and has higher relaxation rate characteristics compared with 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 applied to further clinical detection applications. SUMMARY

[0004] The present application aims at some deficiencies of existing magnetic resonance contrast agents, and provides a novel chelating ligand H4L and a preparation method of a manganese complex MnL 2- of the chelating ligand H4L. 2- The stability and relaxation rate characteristics of MnL

[0005] The present application first provides a macrocyclic chelating ligand, which is H4L, wherein L is the first letter of the ligand ligand, H represents the hydrogen of carboxylic acid, H4L indicates that the ligand has four ligand points which can be deprotonated to participate in coordination, and in the present application, the four carboxyl groups are specifically referred to. The chemical structure of the H4L is as follows:

[0006]

[0007] The preparation method of the H4L is as follows:

[0008] 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl) di-tert-butyl dicarboxylate (DO2A-tBu), sodium carbonate and 2,6-bis(chloromethyl)pyridine are added to a reaction bottle in a certain proportion, acetonitrile (MeCN) is added as a solvent, stirred at room temperature until dissolved, then heated to a certain temperature for a certain time, after the reaction is completed, the precipitate is washed repeatedly with acetonitrile, and the upper clear liquid is collected after standing. The upper clear liquid is rotary evaporated to obtain a light yellow oily substance. After further purification by chromatography column, the compound H4L-tBu is obtained. The protecting group -tBu is removed by ester hydrolysis reaction of trifluoroacetic acid (TFA) in dichloromethane (DCM) to obtain the chelating ligand H4L.

[0009] The preparation method of the H4L provided by the present application uses acetonitrile with a higher boiling point as a reaction solvent, and compound H4L-tBu is obtained by heating and refluxing, and then the protecting group is removed to generate the ligand H4L. The yield of the obtained product H4L is increased from 47% to 91%, and the waste of reactants is reduced.

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

[0011] Further, in order to control the conversion of the reaction to the target product, the reaction temperature for preparing the compound H4L-tBu is 80°C, and the reaction time is 12 hours.

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

[0013] The application also provides a preparation of a manganese-based small molecule complex, denoted as MnL 2- , and the molecular formula is C 38 H 56 MnN 10 O9 2- .

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

[0015]

[0016] The preparation method of the manganese-based small molecule complex MnL 2- is as follows:

[0017] The ligand H4L is dissolved in a reaction bottle, and an aqueous solution of manganese chloride tetrahydrate is added dropwise. The pH of the reaction solution is slowly adjusted to neutral by using NaOH and HCl solutions until the aqueous solution of manganese chloride tetrahydrate is completely added 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, further heating is required for further reaction. After the reaction is completed, the final product MnL 2- is obtained by high performance liquid chromatography (HPLC) purification.

[0018] The molar ratio of the ligand H4L and manganese chloride tetrahydrate is 1:1.2.

[0019] The first stage reaction temperature is 25℃, and the reaction time is 4 hours.

[0020] The second stage reaction temperature is 65℃, and the reaction time is 4 hours.

[0021] The application also provides a method for measuring the stability, 17 the coordinated water and its relaxation rate and other characteristics of the manganese complex.

[0022] The manganese complex stability test method comprises the following steps:

[0023] The solution of the manganese complex MnL 2- is taken in a sample tube and mixed with PBS buffer, and 5 equivalents and 25 equivalents of Zn 2+ , 0.1M and 1.0M of HCl and 5 equivalents of K + , Ca 2+ , Na + , Mg 2+ , Mn2+ Fe 3+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Cu + The stability of the complex was determined by ion testing. Under the above conditions, this product exhibited very good stability.

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

[0025] Take manganese complex MnL 2- The solution was mixed with PBS buffer in a sample tube, and the relaxation rate r2 of the sample was tested at different temperatures using Mn-EDTA (q=1) as a reference. 0 [The test method is based on Gale EM, Zhu J, Caravan P. Journal of the American Chemical Society. 2013, 135(49): 18600-8.] The hydration level of the product was calculated to be q = 0.78, indicating that the complex has a coordinating water element that can provide sufficient magnetic resonance signal.

[0026] The relaxation rate test method for the manganese complex includes 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 was tested at different sample concentrations. After plotting concentration against 1 / T1 and performing linear fitting, the relaxation rate of the product was obtained as r1 = 2.255 mM. -1 s -1 The test conditions were an electric field strength of 1.41T and a temperature of 37℃. This data indicates that this product is superior to similar Mn(1,4-DO2A) (r1=1.7mM). -1 s -1 The relaxation rate of ). [Data cited from 1 H and 17 O NMR Relaxometric and ComputationalStudy on Macrocyclic Mn(II)Complexes.Inorg.Chem.2013,52,3268.]

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

[0029] The beneficial effects of this invention are:

[0030] (1) The application develops the preparation condition of ligand H4L. By using acetonitrile solvent, adopting 80℃ heating reflux method, adding sodium carbonate to promote the reaction, compound H4L-tBu is obtained, and then deprotection generates ligand H4L. The obtained product H4L is a four-carboxyl macrocyclic chelating ligand, (a) provides four carboxyl groups to participate in coordination and increase the water solubility of the sample, (b) provides two tetraazacycles and two pyridine rings to participate in the stable chelation of metal and the hydrogen bonding of chelated water.

[0031] (2) In the application, ligand H4L and manganese chloride tetrahydrate are used as starting materials, and the preparation condition is explored in aqueous solution to obtain a manganese-based small molecule complex with good water solubility, which is recorded as MnL 2- , the molecular formula is C 38 H 56 MnN 10 O9 2- .

[0032] (3) In the PBS buffer solution of manganese complex MnL 2- , a series of competitive metal ions and strong acid environment are added respectively, and the ultraviolet absorption of the sample after 24 hours is detected, and the results show that the product has very good stability.

[0033] (4) Through 17 O NMR test and relaxation rate test, the hydration condition q=0.78 of the complex is obtained, which shows that the complex has one coordination water and can provide a monitorable magnetic resonance signal. The obtained relaxation rate is r1=2.255mM -1 s -1 , which shows that the product has great application value. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is the chemical structure (a) and synthesis path (b) of tetraazacyclic ligand H4L.

[0035] Figure 2 It is the high-resolution mass spectrum of ligand H4L (a) and manganese complex MnL 2- (b).

[0036] Figure 3 It is the ultraviolet absorption (a) and fluorescence spectrum (b) spectrum of ligand H4L.

[0037] Figure 4 It is the chemical structure (a) and ultraviolet absorption spectrum (b) of manganese complex MnL 2- .

[0038] Figure 5 It is the stability of manganese complex MnL 2- under different conditions.

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

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

[0041] In the embodiments of the present application, the experimental methods without specific conditions are carried out according to the conventional methods and conditions in the art, and the materials used are commercially available if not specially stated.

[0042] The reagents and instruments used in the embodiments of the present application: the solvents used in the reaction are all of analytical purity, and the reagents used are directly applied without any special treatment if not specially stated.

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

[0044] Ethanol, HCl, TFA solution: analytical pure, Shanghai Pilot Chemical General Company;

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

[0046] 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid di-tert-butyl ester (DO2A-tBu) and 2,6-bis(chloromethyl)pyridine: analytical pure, Anagenix Pty Ltd;

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

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

[0049] Fluorescence spectrophotometer: Australia Varian Cary Eclipse;

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

[0051] Example 1: Preparation of ligand H4L

[0052] As Figure 1 ​The reaction bottle was charged with DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine in a molar ratio of 1:2:1.25, and 20 mL of acetonitrile was added as the reaction solvent, and then gradually heated to 80°C to heat reflux, and reacted 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 spin-dried to obtain a light yellow oily substance, and compound H4L-tBu was obtained after further column purification. In the reaction bottle of H4L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and maintain the temperature 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 was heated to 65°C and reacted for 6 hours to end the reaction. Purification by HPLC separation and purification was used to obtain the ligand H4L, and the structural formula is as follows Figure 1 The mass spectrum is shown in Figure 2a, and the yield is 91%. Figure 2 The mass spectrum is shown in Figure 2a.

[0053] Figure 3 The UV absorption spectrum of the ligand H4L is shown in Figure 2a, and it can be seen from the figure that the ligand has an absorption peak at 264 nm, which is from the π→π* transition of the pyridine ring.

[0054] Figure 3 The fluorescence spectrum of the ligand H4L is shown in Figure 2b, and it can be seen from the figure that under excitation at 285 nm, the ligand has two strong broad emission peaks at 332 nm and 352 nm, and a weak broad emission peak at about 630 nm.

[0055] The product is H4L, and its molecular formula is C 38 H 58 N 10 O8, and its chemical structural formula is as follows:

[0056]

[0057] This example is the best preparation scheme of H4L.

[0058] Example 2: Preparation of ligand H4L

[0059] The DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added into the reaction bottle according to the molar ratio of 1:2:1.25, and 20 mL of acetonitrile was added as the reaction solvent, and then gradually heated to 80°C to heat reflux, and reacted 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 spin-dried to obtain a light yellow oily substance, and the compound H4L-tBu was obtained after further purification by column chromatography. In the reaction bottle of H4L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and maintain the temperature at 5°C. Subsequently, 2.5 mL of TFA was added, and the sample bottle was gradually returned to room temperature to react for 4 hours. Finally, the reaction was heated to 65°C, and the reaction was completed after 6 hours. Purification by HPLC separation was used to obtain the ligand H4L, with a yield of 54%. It can be seen from the comparative example that the reaction time is too short, and the starting material is not completely reacted, which is not conducive to the generation of the target product.

[0060] Example 3: Preparation of ligand H4L

[0061] The DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added into the reaction bottle according to the molar ratio of 1:2:1.25, and 10 mL of acetonitrile was added as the reaction solvent, and then gradually heated to 80°C to heat reflux, and reacted 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 spin-dried to obtain a light yellow oily substance, and the compound H4L-tBu was obtained after further purification by column chromatography. In the reaction bottle of H4L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and maintain the temperature at 5°C. Subsequently, 2.5 mL of TFA was added, and the sample bottle was gradually returned to room temperature to react for 4 hours. Finally, the reaction was heated to 65°C, and the reaction was completed after 6 hours. Purification by HPLC separation was used to obtain the ligand H4L, with a yield of 47%.

[0062] It can be seen from the comparative example that the solvent used in the reaction is less, which can cause incomplete dissolution of the solute, resulting in lower yield of the product.

[0063] Example 4: Preparation of ligand H4L

[0064] The DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added into the reaction bottle according to the molar ratio of 1:2:1.5, and 20 mL of acetonitrile was added as the reaction solvent, then gradually heated to 80°C to heat reflux, and reacted 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 spin-dried to obtain a light yellow oily substance, and the compound H4L-tBu was obtained after further purification by column chromatography. In the reaction bottle of H4L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and maintain the temperature at 5°C. Subsequently, 2.5 mL of TFA was added, and the sample bottle was gradually returned to room temperature to react for 4 hours. Finally, the reaction was heated to 65°C, and the reaction was completed after 6 hours of reaction. Purification by HPLC separation was used to obtain the ligand H4L, with a yield of 74%.

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

[0066] Example 5: Preparation of ligand H4L

[0067] The DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine were added into the reaction bottle according to the molar ratio of 1:2:1.5, and 20 mL of acetonitrile was added as the reaction solvent, then gradually heated to 80°C to heat reflux, and reacted 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 spin-dried to obtain a light yellow oily substance, and the compound H4L-tBu was obtained after further purification by column chromatography. In the reaction bottle of H4L-tBu, 0.5 mL of dichloromethane was added to dissolve the sample and maintain the temperature at 5°C. Subsequently, 2.5 mL of TFA was added, and the sample bottle was gradually returned to room temperature to react for 4 hours. Finally, the reaction was heated to 65°C, and the reaction was completed after 6 hours of reaction. Purification by HPLC separation was used to obtain the ligand H4L, with a yield of 74%.

[0068] As can be seen from the comparative example, the low reaction temperature results in a small effective collision rate between molecules, resulting in a lower yield of the obtained product.

[0069] Example 6: Preparation of complex MnL 2- of Example 1.

[0070] The H4L was prepared by the method of Example 1.

[0071] The ligand H4L (1.0 mmol) was dissolved in a reaction bottle and a solution of manganese chloride tetrahydrate (2.0 mmol) in water was added dropwise thereto. The pH of the reaction solution was slowly adjusted to neutral using 1M NaOH and HCL solutions until the solution of manganese chloride tetrahydrate was added dropwise and the pH was maintained at neutral. The reaction was carried out in two stages, i.e. after reacting at room temperature 25°C for 4 hours, further heating at 65°C was required for 4 hours. After the reaction was completed, the final product was obtained by HPLC purification, 32 mg, yield 78%. The mass spectrum is shown in Figure 1. Figure 2 b.

[0072] The product is a mononuclear manganese complex, denoted as MnL 2- , whose molecular formula is C 38 H 56 MnN 10 O9 2- , and its possible chemical structural formula is:

[0073]

[0074] This example is a preparation scheme of the best manganese-based small molecule complex crystal material Mn. Figure 4 b is the complex MnL 2- The ultraviolet absorption spectrum of the complex MnL

[0075] Comparative Example 1: Preparation of the complex MnL 2-

[0076] The ligand H4L was prepared by the method of Example 1.

[0077] The ligand H4L (1.0 mmol) was dissolved in a reaction bottle and a solution of manganese chloride tetrahydrate (2.0 mmol) in water was added dropwise thereto. The ligand H4L (1.0 mmol) was dissolved in a reaction bottle and a solution of manganese chloride tetrahydrate (2.0 mmol) in water was added dropwise thereto. In this example, no NaOH or HCL solution was used to adjust the pH value. Then the reaction was carried out in two stages, i.e. after reacting at room temperature 25°C for 4 hours, further heating at 65°C was required for 4 hours. After the reaction was completed, the final product was obtained by HPLC purification, 7 mg, yield 16%.

[0078] As can be seen from this comparative example, if the pH value of the reaction solution in the reaction bottle is not adjusted, the solute may not be completely dissolved, H4L cannot be completely deprotonated to participate in the coordination reaction of manganese, resulting in a low yield of the product.

[0079] Comparative Example 2: Preparation of the complex MnL 2-

[0080] The ligand H4L was prepared by the method of Example 1. ​​

[0081] The ligand H4L (1.0 mmol) was dissolved in a reaction bottle and a solution of manganese chloride tetrahydrate (2.0 mmol) in water was added dropwise thereto. The pH of the reaction solution was slowly adjusted to neutral using 1 M NaOH and HCL solutions until the solution of manganese chloride tetrahydrate was added dropwise and the pH was maintained at neutral. After 8 hours of reaction at room temperature 25 °C, the final product was obtained by HPLC purification, 11 mg, yield 25%.

[0082] As can be seen from the comparative example, without heating the reaction bottle, the reaction rate is slow, H4L cannot be fully deprotonated to participate in the coordination reaction of manganese, resulting in a low yield of the product.

[0083] Example 9: Stability test of the complex MnL 2- obtained in Example 6

[0084] The stability of the complex was explored by detecting the ultraviolet absorption of the sample. 100 μM complex MnL 2- PBS buffer solution was placed in a cuvette, and a ultraviolet absorption curve of the complex was measured as a blank control. Then, 5 equivalents and 25 equivalents of Zn 2+ , 5 equivalents of K + , Ca 2+ , Na + , Mg 2+ , Mn 2+ , Fe 3+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Cu + ions were added to the above sample solution, respectively, and a ultraviolet absorption curve was measured as the initial state. After 24 hours, the ultraviolet absorption curve of the above mixed solution was measured again as the end point state. In addition, the complex was exposed to 0.1 M and 1.0 M HCl to detect the ultraviolet absorption after 24 hours.

[0085] As shown in Figure 5 , by comparing the above series of ultraviolet absorption curves with the blank, the ultraviolet absorption intensity of the complex did not change significantly, showing good stability.

[0086] Example 10: Coordination water of the complex MnL 2- obtained in Example 6

[0087] The coordination water of the complex was measured by the Carr-Purcell-Meiboom-Gill spin echo experiment to obtain the transverse relaxation time T2. The sample was prepared in pure H2O, and the transverse relaxation time T2 was measured in 2% H2 17H2O in the presence of O, and the pH was adjusted to 6-7. Using the CPMG pulse sequence, H2 17 O transverse relaxation time T2at 9.4 T. The H2 17 O NMR signal was measured at half height 17 O transverse relaxation time, and the transverse relaxation rate (r2 0 ) was calculated. Finally, the hydration q = 0.78 was calculated by plotting r2 0 ~ (1000 / T). The test data and results are shown in Figures Figure 6 a.

[0088] Example 11: Relaxation rates of the complex MnL 2-

[0089] The relaxation rates of the complex were measured using the method for recording T1measurements at 1.41 T and 37 °C using a Bruker mq60 Minispec. The manganese complex was diluted to different concentrations in a gradient using PBS solution at pH = 7.4. The series of longitudinal relaxation times T1were measured, and the experiment was repeated three times. The relaxation rate (r1) was determined from the slope of the 1 / T1versus metal concentration curve at five concentrations. The above procedure was repeated in parallel three times, and the longitudinal relaxation rate r1from the three experiments was averaged as the final result, which was 2.255 mM -1 s -1 The test data and linear fit are shown in Figure Figure 6 b.​

Claims

1. A macrocyclic chelating ligand characterized in that, The chemical structure of H4L is as follows: 。 2. The process for the preparation of macrocyclic chelating ligands according to claim 1, characterized in that, The steps are as follows: The di-tert-butyl 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetate DO2A-tBu, sodium carbonate and 2,6-bis(chloromethyl)pyridine are added into a reaction bottle in proportion, acetonitrile is added as a solvent, and the mixture is stirred at room temperature until dissolved, and then the reaction is warmed up, after the reaction is completed, the precipitate is repeatedly washed with acetonitrile, and after standing, the supernatant is collected, and the supernatant is dried to obtain a light yellow oily substance; after further purification by a chromatographic column, the compound H4L-tBu is obtained. The protecting group -tBu is removed by ester hydrolysis reaction of trifluoroacetic acid TFA in dichloromethane DCM to obtain the chelating ligand H4L.

3. The process for the preparation of macrocyclic chelating ligands 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 process for the preparation of macrocyclic chelating ligands according to claim 2, characterized in that, The temperature of the reaction is 80 o C and the reaction time is 12 hours.

5. The process for the preparation of macrocyclic chelating ligands according to claim 2, characterized in that, When the protecting group -tBu is removed, the volume ratio of TFA and DCM used is 5:

1.

6. A manganese-based small molecule complex, characterized in that, is prepared from the macrocyclic chelate ligand of claim 1, having the formula C 38 H 56 MnN 10 O9 2- , having the formula 。 7. The method for preparing the manganese-based small molecule complex as described in claim 6, characterized in that, The steps are as follows: The ligand H4L is dissolved in a reaction bottle, and an aqueous solution of manganese chloride tetrahydrate is added dropwise thereto, and the pH of the reaction solution is slowly adjusted to neutral using NaOH and HCl solutions until the aqueous solution of manganese chloride tetrahydrate is completely added and the pH is always maintained at neutral; the reaction is carried out in two stages, i.e., the first stage reaction is carried out at room temperature 25℃ for 4 hours, and then the second stage reaction is carried out by heating, and after the reaction is completed at 65℃ for 4 hours, the final product MnL is obtained by purification through high performance liquid chromatography 2- ​ The molar ratio of the ligand H4L and manganese chloride tetrahydrate is 1:

2.

8. Use of the manganese-based small molecule complex of claim 6 as a magnetic resonance imaging contrast agent for non-disease diagnosis and treatment purposes.

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