Compounds, chelates, and methods of making and using the same in the preparation of magnetic resonance imaging contrast agents

Abstract

The application discloses a compound, a chelate, a preparation method of the compound and the chelate and application of the compound and the chelate in preparation of a magnetic resonance imaging contrast agent, and belongs to the technical field of medical imaging. The application specifically discloses a compound as shown in the formula (I) or a stereoisomer, a pharmaceutically acceptable salt or a mixture thereof, which can significantly improve plasma exposure, has high bioavailability, can prolong an in-vivo imaging time window, has long-acting and low-dose imaging advantages, and has good clearance characteristics and low tissue residue risk, thereby reducing free Gd 3+ release hidden dangers, and providing a long-acting, low-dose and high-safety magnetic resonance contrast solution for clinical use.

Description

Technical Field

[0001] This invention belongs to the field of medical imaging technology, specifically relating to a compound, a chelate, its preparation method, and its application in the preparation of magnetic resonance imaging contrast agents. Background Technology

[0002] Gadolinium (III)-based contrast agents (GBCAs) are widely used clinically in magnetic resonance imaging (MRI). Once in the body, gadolinium-containing chelates significantly shorten the relaxation time of protons in tissues, thereby enhancing the clarity and contrast of MRI images and improving lesion detection rates. They provide more diagnostically valuable information than plain scans for the localization and characterization of lesions in the brain, spinal cord, and central nervous system. They are also used for MRI of the abdomen, chest, pelvis, limbs, and other human tissues, as well as for renal function assessment. Compared to other contrast agents, gadolinium contrast agents offer high tissue resolution and good safety.

[0003] Currently available gadolinium contrast agents all contain a nine-coordinate gadolinium ion [Gd(III)], which can coordinate with an octardate polyaminocarboxylic acid ligand and a water molecule. According to the ligand structure, they can be divided into two types: linear and macrocyclic. There are six linear gadolinium contrast agents: gadopentetate diglucamine (Magnevist), gadodiamine (Omniscan), gadofosylamine, gadoxetate disodium (Primovist), gadobutrol diglucamine (MultiHance), and gadofosyltetrate trisodium. There are three macrocyclic gadolinium contrast agents: gadoteric acid dimeglumine (Dotarem), gadoteryl alcohol (ProHance), and gadobutrol (Gadovist). Most GBCAs are passively distributed extracellularly in vivo and are mainly excreted through the kidneys, while some contrast agents (such as disodium gadoxetate and gadobutrazol) also have hepatocyte uptake properties and can be used for liver-specific imaging.

[0004] To improve the imaging performance of gadolinium contrast agents, existing technologies mainly employ two strategies: one is to increase the number of inner-layer water molecules (q), i.e., to increase the number of water molecules directly coordinated with Gd(III), thereby significantly enhancing relaxation efficiency; the other is to slow down the rotational diffusion of the molecule, by increasing the molecular volume or introducing protein-binding groups to reduce the rotational-dependent time of the molecule, thereby improving the relaxation rate. Furthermore, the stability of the chelate is also a key factor in the design of GBCAs, because free Gd... 3+ Ions have potential toxicity, and their safety is particularly prominent for patients with impaired kidney function.

[0005] Although existing technologies such as patents CN107667096B (Comparative Example 2) and CN110035996B (Comparative Example 3) have disclosed gadolinium-based polymeric contrast agents with different configurations, attempting to improve relaxation performance by increasing molecular volume, these polymeric structures based on traditional macrocyclic skeletons, while pursuing high performance, still generally suffer from the following irreconcilable drawbacks: Due to the increased complexity of the molecular structure and the increase in steric hindrance, these polymers often exhibit a high tendency for intermolecular aggregation in solution. This may not only make it difficult to maintain the stability of the injection solution in the long term, but may also interfere with the coordination environment between water molecules and metal centers, thus limiting the actual improvement in relaxation rate; more importantly, a significant increase in molecular weight is often accompanied by the deterioration of pharmacokinetic behavior, and its plasma half-life in vivo may exhibit unexpected changes. It is difficult to achieve the ideal "long-acting" imaging window, and it is easy to cause retention in non-specific tissues, making it difficult to achieve the best balance between "high relaxation rate" and "ideal in vivo clearance and safety". This disconnect between complex structures, high preparation costs, and actual clinical performance (such as stability and pharmacokinetic parameters) is a pain point that existing technologies urgently need to address, and it is also the key to the breakthrough that this invention is committed to.

[0006] Therefore, there is still an urgent need in this field to develop a new type of gadolinium chelate that not only has a significantly higher relaxation rate to support efficient imaging at lower doses, but also has better pharmacokinetic characteristics. At the same time, it should maintain or even improve chelation stability and in vivo clearance efficiency, so as to truly achieve a three-in-one clinical solution for magnetic resonance imaging that is "long-acting, low-dose, and highly safe". Summary of the Invention

[0007] The purpose of this invention is to provide a compound, a chelate, a method for preparing the same, and its application in the preparation of magnetic resonance imaging contrast agents. This novel gadolinium chelate can significantly increase plasma exposure and prolong the imaging time window at equivalent or lower doses, thereby achieving long-acting, low-dose magnetic resonance imaging while reducing tissue residue and free Gd. 3+ To mitigate risks and overcome the shortcomings of existing contrast agents, such as short imaging time, strong dose dependence, and insufficient safety.

[0008] This application is achieved through the following technical solution: In a first aspect, the present invention provides a chelate or a stereoisomer thereof, a pharmaceutically acceptable salt, or a mixture thereof, as shown in formula (I): ; in: R1 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4Alkoxy, hydroxy, carboxyl, ester, amide, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 1-4 Mercaptoalkyl, mercaptoether, ether, C 3-6 cycloalkyl or phenyl; R2 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 3-6 Cycloalkyl or phenyl.

[0009] Furthermore, R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Halogenated alkyl, hydroxyl, carboxyl, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 1-4 Mercaptoalkyl, mercaptoether, C 3-6 cycloalkyl or phenyl; R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl or C 3-6 Cycloalkyl.

[0010] Furthermore, R1 is selected from hydrogen, hydroxyl, carboxyl, C 1-4 Hydroxyalkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Carboxyalkyl, C 1-4 Mercaptoalkyl, mercaptoether, phenyl; R2 is selected from hydrogen, C 1-4 Alkyl, C 3-6 Cycloalkyl.

[0011] Furthermore, R1 is selected from methyl, deuterated methyl, halomethyl, carboxyl, C 1-2 Hydroxyalkyl, C 1-2 mercaptoalkyl, C 1-2 Carboxyalkyl, phenyl; R2 is selected from hydrogen, isobutyl, or cyclopropyl.

[0012] Furthermore, the above chelates are selected from: , , , , , , , , , , , , , , or .

[0013] In a second aspect, the present invention provides a compound of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a mixture thereof: ; R1 and R2 are defined in the same way as any of the terms in the first aspect.

[0014] Furthermore, the above compounds are selected from: , , , , , , , , , , , , , , or .

[0015] Furthermore, the hydrogen in any of the above-mentioned compound structures can be replaced by one or more deuterium atoms.

[0016] Thirdly, the present invention provides a method for preparing a compound as shown in formula (II), comprising: Compound d and compound h were condensed to obtain compound i, and compound i was further deprotected to obtain compound (II); R1 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, hydroxy, carboxyl, ester, amide, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 1-4 Mercaptoalkyl, mercaptoether, ether, C 3-6 cycloalkyl or phenyl; R2 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 3-6 cycloalkyl or phenyl; R1' is the protection form of R1 or R1; R3 is selected from methyl, tert-butyl, or benzyl.

[0017] Fourthly, the present invention provides a method for preparing a chelate as shown in formula (I), comprising: Compound of formula (I) was prepared by chelation reaction of the compound shown in formula (II); The definitions of R1 and R2 are the same as the corresponding definitions of any item in the first aspect.

[0018] Fifthly, the present invention provides the use of a chelate or stereoisomer of formula (I), a pharmaceutically acceptable salt or a mixture thereof, or a compound or stereoisomer of formula (II), a pharmaceutically acceptable salt or a mixture thereof, in the preparation of a magnetic resonance imaging contrast agent.

[0019] Furthermore, preferably, the contrast agent is used for enhanced imaging of blood vessels.

[0020] Furthermore, the aforementioned contrast agent is used for contrast enhancement in magnetic resonance imaging (MRI) of systemic lesions in mammals, preferably for contrast enhancement in MRI of lesions of the central nervous system. Further, the mammal includes humans; the systemic lesions include, but are not limited to, the central nervous system, abdomen, chest, blood vessels, bones, and muscles; the central nervous system includes, but is not limited to, the brain and spinal cord. The blood vessels include, but are not limited to, cerebral blood vessels. The abdomen includes, but is not limited to, the liver and kidneys.

[0021] Preferably, the pharmaceutical composition is in the form of a lyophilized powder or a ready-to-use injection.

[0022] The compound names corresponding to the English abbreviations mentioned in this invention are: Pd / C: Palladium on carbon.

[0023] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate.

[0024] TFA: Trifluoroacetic acid.

[0025] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The gadolinium chelate and its salts of the present invention exhibit significantly higher relaxation (r1) in in vitro pure water and plasma than the comparative compounds, demonstrating greater potential for MRI contrast enhancement and providing a basis for achieving stronger signals or reduced-dose equivalent imaging at the same dose.

[0027] 2. The pharmacokinetic AUC and C0.05 of the gadolinium chelate and its salt in vivo according to the present invention. max It is higher, especially at equimolar doses, plasma exposure is about 8-10 times higher than the commercially available control, and it has higher bioavailability; in addition, it can maintain a higher peak signal even when the dose is at least halved, achieving "reduced dose without reduced effect", and the signal persistence window is extended by 1 time, with rapid elution within 2 hours and tissue residue close to the background, combining the advantages of long-lasting imaging and low exudation safety.

[0028] 3. The gadolinium chelates and their salts of the present invention also have good scavenging properties and low risk of tissue residue, thereby reducing free Gd. 3+ This approach aims to eliminate potential risks and provide a long-lasting, low-dose, and highly safe magnetic resonance imaging (MRI) contrast solution for clinical use. Attached Figure Description

[0029] Figure 1 MRI images of cerebral blood vessels in mice before and after administration of compounds 1-3 and compound 8; Figure 2 The graph shows the Δ signal intensity-time curves of cerebral blood vessels in mice after administration of compounds 1-3 and compound 8. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] The inventive concept of this application: In order to overcome the shortcomings of existing gadolinium contrast agents (such as structures similar to Comparative Examples 2 and 3) in that it is difficult to simultaneously achieve optimal relaxation rate, pharmacokinetics, and safety, the inventors constructed a specific tetraazacyclododecane tetramer core framework and purposefully introduced substituents (R1 / R2) of specific polarity and volume at its bridging or terminal positions. They discovered that such structures can synergistically achieve the following: (a) Due to the moderate increase in molecular volume and the interaction of specific groups, molecular rotation is slowed down, and relaxation rate is increased; (b) Due to the influence of specific groups (such as hydroxyl and carboxyl groups), the protein binding or distribution characteristics are altered, thereby significantly increasing plasma exposure and prolonging the imaging window; (c) Maintain good water solubility and renal clearance pathway for rapid clearance.

[0032] Based on the above inventive concept, this application provides a chelate or its stereoisomer, a pharmaceutically acceptable salt, or a mixture thereof as shown in formula (I): ; in: R1 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, hydroxy, carboxyl, ester, amide, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 1-4 Mercaptoalkyl, mercaptoether, ether, C 3-6 cycloalkyl or phenyl; R2 is selected from hydrogen, deuterium, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Carboxyalkyl, C 3-6 Cycloalkyl or phenyl.

[0033] The core inventive point of this application lies in the construction of a novel tetraazacyclododecane-1-yl]acetic acid tetrameric gadolinium chelate through specific structural design. Its superior comprehensive properties, such as high relaxivity, high plasma exposure, and rapid clearance, are not simply a superposition of existing polymeric structures, but rather stem from a sophisticated, non-obvious "structure-performance synergy" mechanism. Specifically: 1. Core skeletal structure: Precisely regulating the balance between molecular rotation and hydrophilicity. The compound of this application (as shown in Formula I) is a highly symmetrical and compact tetramer formed by coupling four DOTA derivative units through specific short-chain / rigid linkers. Unlike the polymers in prior art CN107667096B (Comparative Example 2) and CN110035996B (Comparative Example 3), the structure of this application avoids excessive molecular weight increase and structural rigidity. This "moderate increase" design can effectively slow down the overall rotational correlation time (τR) of the molecule, which is a key kinetic factor for improving the longitudinal relaxation rate (r1) (as shown in Experimental Example 1, compound 8 has an r1 as high as 10.90 mM). -1 s -1 On the other hand, the compact structure prevents molecules from randomly coiling or unfavorably aggregating in solution, ensuring unobstructed water channels inside the molecules, which is conducive to the efficient exchange of water molecules with Gd³⁺ centers.

[0034] 2. Targeted functionalization of substituents (R1, R2): synergistic optimization of pharmacokinetic behavior The key innovation of this application lies in the introduction of selected functional groups with specific physicochemical properties at specific positions (R1, R2) in the core tetramer backbone. This is fundamentally different from the relatively simple linking arms or substituents in the comparative examples.

[0035] Hydrophilic / functional groups at the R1 site (such as hydroxyl, carboxyl, mercapto, carboxyalkyl, etc.): The introduction of these groups is not merely a matter of presence or absence. They significantly enhance the overall hydrophilicity and biocompatibility of the entire macromolecular ligand. More importantly, groups like hydroxyl and carboxyl can engage in controlled, reversible, weak interactions with plasma components (such as albumin). This interaction does not lead to irreversible binding and accumulation, but moderately slows down the renal filtration rate of the molecule. This is the structural basis for the compound in this application to maintain rapid clearance (T1 / 2 of only about 3.5 hours) while significantly increasing plasma exposure (AUC 8-10 times that of the control, see Experimental Example 2) and extending the imaging window. It cleverly balances the contradiction between long-term effectiveness and rapid clearance.

[0036] Small, rigid groups (such as hydrogen, isobutyl, cyclopropyl, etc.) at the R2 site: These groups are mainly used to fine-tune the steric hindrance and electronic environment of the core linkage region. They can stabilize the core linkage conformation, prevent adverse conformational changes from affecting chelation stability, and avoid introducing excessively large hydrophobic fragments that could lead to non-specific tissue adsorption, thereby helping to reduce the risk of tissue residue.

[0037] In summary, the inventiveness of this application does not stem from a change in a single structural feature, but rather from the synergistic effect between the core tetrameric backbone and specific functionalized substituents: the compact tetrameric backbone is the physical basis for achieving ultra-high relaxation rates; the carefully selected R1 / R2 groups enable precise regulation of the compound's distribution, circulation, and clearance behavior in vivo, translating the potential of high relaxation rates into practical long-acting, low-dose imaging capabilities. The entire molecule maintains good water solubility and a moderate molecular size, ensuring that it is still primarily cleared rapidly via the renal pathway. Its compact structure and highly stable DOTA derivative units also maximize the chelation stability of Gd³⁺, reducing the risk of free gadolinium release at the source.

[0038] Existing technologies (Comparative Examples 2 and 3) only focus on increasing the degree of polymerization to improve relaxation rate, failing to systematically address the resulting pharmacokinetic degradation and safety risks. This application, however, by revealing and utilizing the aforementioned synergistic structure-activity relationship of the "skeleton-substituent" relationship, designs a class of novel compounds with predictable performance, achieving optimized integration of relaxation efficiency, in vivo behavior, and safety at the molecular level. This constitutes the prominent substantive feature and significant progress of this invention, and is therefore non-obvious.

[0039] The structure of the compound in the embodiments of this invention is determined by nuclear magnetic resonance (NMR). 1 Determined by ¹H NMR or LC-MS.

[0040] In this invention, "room temperature" refers to 10–35°C.

[0041] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0042] The synthesis methods described in Examples 1-8 below are exemplary. Those skilled in the art can prepare other compounds within the scope of the claims of this application by replacing the corresponding starting materials (i.e., "compound 1a" or similar intermediates with different R1 and R2) and using the same or similar synthesis strategies, based on the synthetic route.

[0043] Example 1: Preparation of Compound 1 [4,10-bis(carboxymethyl)-7-{1-oxoylide-1-[(2-{4,7,10-tris[2-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]propionyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]prop-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 1) The synthesis route is as follows: Step 1: Synthesis of compound 1c 3.68 g (15.13 mmol, 1.00 eq.) of compound 1a and 7.79 g (15.13 mmol, 1.00 eq.) of compound 1b were dissolved in 400 mL of acetonitrile. Then, 6.27 g (45.39 mmol, 3.00 eq.) of potassium carbonate and 0.23 g (1.51 mmol, 0.10 eq.) of sodium iodide were added. The reaction system was stirred and heated to 60 °C under nitrogen protection and stirred overnight. After the reaction was completed by TLC monitoring, the mixture was filtered. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain 5.12 g (50%, 7.57 mmol) of intermediate compound 1c.

[0044] LC-MS(ESI+): m / z=677.5(M+H) + .

[0045] Step 2: Synthesis of compound 1d 5.12 g (7.57 mmol, 1.00 eq.) of compound 1c, 3.58 g (1.51 mol, 0.20 eq.) of 10% Pd / C and 230 ml of isopropanol were added to a hydrogenation reactor. The reactor was purged with hydrogen three times, pressurized to P=1.00 MPa, heated to 100℃ and reacted for 8 h. Heating was stopped, the reactor was cooled, the gas was vented, the reactor was opened, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography to obtain 3.56 g (80%, 6.07 mmol) of intermediate compound 1d.

[0046] LC-MS (ESI-): m / z = 585.4 (MH) - .

[0047] Step 3: Synthesis of 1g of compound 3.45 g (20.00 mmol, 1.00 eq.) of compound 1e and 19.72 g (88.00 mmol, 4.40 eq.) of compound 1f were dissolved in 800 mL of acetonitrile. Then, 16.59 g (120.00 mmol, 6.00 eq.) of potassium carbonate and 1.32 g (8.80 mmol, 0.44 eq.) of sodium iodide were added. The reaction system was stirred and heated to 60 °C under nitrogen protection and stirred overnight. After the reaction was completed by TLC monitoring, the mixture was filtered. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain 5.07 g (34%, 6.80 mmol) of intermediate compound 1 g.

[0048] LC-MS(ESI+): m / z=745.7(M+H) + .

[0049] Step 4: Synthesis of compound 1h 5.07 g (6.80 mmol, 1.00 eq.) of 1 g of compound was added to 200 mL of cyclopentyl methyl ether (200 mL), cooled to 0 °C, and cyclopentyl methyl ether hydrochloric acid solution (136.00 mmol, 20.00 eq.) was added dropwise. The reaction mixture was stirred at 0 °C for 1 h and then stirred overnight at room temperature. Dioxane (50 mL) and another batch of cyclopentyl methyl ether hydrochloric acid solution (408.00 mol, 60.00 eq.) were then added, and stirring continued at room temperature. The resulting suspension was concentrated under reduced pressure to give compound 1 h (4.33 g), without further purification.

[0050] Step 5: Synthesis of Compound 1i 1.76 g (3.00 mmol, 12.00 eq.) of compound 1d, 1.33 g (3.50 mmol, 14.00 eq.) of HATU and 220 ml of N,N-dimethylacetamide were added to a 500 mL three-necked flask. The reaction system was stirred and cooled to 0 °C under nitrogen protection. Then, 0.97 g (7.50 mmol, 30.00 eq.) of N,N-diisopropylethylamine dissolved in 90 ml of N,N-dimethylacetamide was added. The resulting reaction mixture was stirred at 0-5°C for 30 min, then 159.1 mg (0.25 mmol, 1.00 eq.) of the compound was added for 1 h, and the reaction was continued to be stirred at room temperature for 3 h. The reaction solution was added to 3 L of purified water, and stirring was continued. 2 L of dichloromethane was added for extraction, and the organic phase was collected. The organic phase was washed three times with 2 L of saturated sodium chloride water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 1i. This crude product did not require further characterization and was directly used in the next chemical step.

[0051] LC-MS(ESI+): m / z(z=2)=1281.9(M+2H) 2+ m / z (z = 3) = 854.9 (M + 3H) 3+ m / z (z = 4) = 641.5 (M + 4H) 4+ .

[0052] Step Six: Synthesis of Compound 1j The crude product 1i obtained in the previous step was treated with 120 mL of TFA and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to obtain an oil, which was separated by reversed-phase preparative chromatography to give 155.7 mg (overall yield of 30% of the two-step chemical reaction, 0.08 mmol) of intermediate compound 1j.

[0053] LC-MS(ESI+): m / z(z=2)=973.6 (M+2H) 2+ m / z (z=3)=649.4 (M+3H) 3+ m / z (z = 4) = 487.3 (M + 4H) 4+ .

[0054] Step 7: Synthesis of Compound 1 97.3 mg (0.05 mmol, 1.00 eq.) of compound 1j was dissolved in 100 mL of purified water. After adding 36.3 mg (0.10 mmol, 2.00 eq.) of gadolinium oxide, the reaction mixture was heated to 90-100 °C and stirred for 5 h. The reaction solution was then cooled to room temperature and lyophilized. The crude product was separated by reverse preparative chromatography to obtain 89.7 mg (70%, 0.035 mmol) of compound 1 with a purity of 98.6%.

[0055] LC-MS(ESI+): m / z(z=2)=1283.4(M+2H) 2+ m / z (z = 3) = 855.9 (M + 3H) 3+ m / z (z = 4) = 642.2 (M + 4H) 4+ .

[0056] Example 2: Preparation of Compound 2 [7,10-bis(carboxymethyl)-4-{3-hydroxy-1-oxoylide-1-[(2-{4,7,10-tris[2-({3-hydroxy-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]propionyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]propyl-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 2) The preparation method is the same as that in Example 1, except that 1a in Example 1 is replaced with intermediate 2a to obtain compound 2. The synthesis yield in step seven is 76% and the purity is 98.9%.

[0057] LC-MS(ESI+): m / z(z=2)=1315.4(M+2H) 2+ m / z (z = 3) = 877.2 (M + 3H) 3+ m / z (z = 4) = 658.2 (M + 4H) 4+ .

[0058] The synthetic route for compound 2b is as follows: Step 1: Synthesis of compound 2a-1 5.67 g (35.17 mmol, 1.00 eq.) O-tert-butyl-serine, 14.65 g (123.10 mmol, 3.50 eq.) KBr and 47.43 g (281.36 mmol, 8.00 eq.) HBr (48%) were added to a 250 ml three-necked flask containing 57 ml of drinking water. The reaction system was stirred and cooled to 0 °C.

[0059] 2.55 g (36.91 mmol, 1.05 eq.) of NaNO2 was dissolved in 26 ml of drinking water and added dropwise to the reaction system. The internal temperature was controlled at 0±5℃ during the dropwise addition. After the dropwise addition was completed, the mixture was stirred at 0±5℃ for 4 h and monitored by TLC. After the reaction was completed, the mixture was extracted with 113 ml of ethyl acetate, washed with 113 ml of water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 6.81 g (86%, 30.24 mmol) of intermediate compound 2a-1.

[0060] LC-MS (ESI-): m / z = 223.1 (MH) - .

[0061] Step 2: Synthesis of compound 2a 4.79 g (21.30 mmol, 1.00 eq.) of compound 2a-1 and 4.42 g (31.95 mmol, 1.50 eq.) of potassium carbonate were added to a 500 mL three-necked flask containing 100 mL of N,N-dimethylformamide. The reaction mixture was stirred and cooled to 0 °C under nitrogen protection. Then, 4.37 g (25.56 mmol, 1.2 eq.) of benzyl bromide was added dropwise. The reaction mixture was brought to room temperature and stirred overnight. After the reaction was completed by TLC, 3 L of ethyl acetate and 1 L of water were added to the reaction mixture for washing and extraction twice. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography to give 4.84 g (72%, 15.35 mmol) of intermediate compound 2a.

[0062] LC-MS(ESI+): m / z=258.1(Mt-Bu+H) + .

[0063] Example 3: Preparation of Compound 3 [7,10-bis(carboxymethyl)-4-{1-carboxy-2-oxoylide-2-[(2-{4,7,10-tris[2-({2-carboxy-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]ethyl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 3) The preparation method is the same as that in Example 1, except that 1a in Example 1 is replaced with intermediate 3a to obtain compound 3. The synthesis yield in step seven is 65% and the purity is 98.3%.

[0064] LC-MS(ESI+): m / z(z=2)=1343.3(M+2H) 2+ m / z (z = 3) = 895.9 (M + 3H) 3+ m / z (z = 4) = 672.2 (M + 4H) 4+ .

[0065] The synthetic route for compound 3a is as follows: Step 1: Synthesis of compound 3a 12.55 g (50.14 mmol, 1.00 eq.) of compound 3a-1, 8.40 g (55.15 mmol, 1.10 eq.) of DBU and 154 mL of tetrahydrofuran were added to the reaction flask and stirred until the temperature was reduced to 0±5℃.

[0066] 17.46 g (52.65 mmol, 1.05 eq.) of carbon tetrabromide was dissolved in 200 mL of tetrahydrofuran and added dropwise to the reaction system. The internal temperature was controlled at 0±5℃ during the dropwise addition. After the dropwise addition was completed, the mixture was stirred at 0±5℃ for 2 h and monitored by TLC. After the reaction was completed, 250 mL of saturated ammonium chloride solution was added to quench the reaction. The mixture was stirred and allowed to rise naturally to room temperature. 500 mL of dichloromethane was added for extraction and separation. The aqueous phase was extracted again with 250 mL of dichloromethane. The organic phases were combined and washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain 7.92 g (48%, 24.06 mmol) of intermediate compound 3a.

[0067] LC-MS(ESI+): m / z=272.1(Mt-Bu+H) + .

[0068] Example 4: Preparation of Compound 4 [7,10-bis(carboxymethyl)-4-{1-oxoylide-3-mercapto-1-[(2-{4,7,10-tris[2-({3-mercapto-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]propionyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]prop-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 4) The preparation method is the same as that in Example 2, except that O-tert-butyl-serine in Example 2 is replaced with S-tert-butyl-DL-cysteine ​​hydrochloride to obtain compound 4. The synthesis yield in step seven is 64%, and the purity is 97.8%.

[0069] LC-MS(ESI+): m / z(z=2)=1347.3(M+2H) 2+ m / z (z = 3) = 898.5 (M + 3H) 3+ m / z (z = 4) = 674.2 (M + 4H) 4+ .

[0070] Example 5: Preparation of Compound 5 [7,10-bis(carboxymethyl)-4-[6-oxoylide-9-(4,7,10-tris{6-oxoylide-5-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]-7-aza-2-thionon-9-yl}-1,4,7,10-tetraazacyclododecane-1-yl)-7-aza-2-thionon-5-yl]-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 5) The preparation method is the same as that in Example 2, except that O-tert-butyl-serine in Example 2 is replaced with DL-methionine to obtain compound 5. The synthesis yield in step seven is 74%, and the purity is 98.2%.

[0071] LC-MS(ESI+): m / z(z=2)=1403.4(M+2H) 2+ m / z (z = 3) = 935.9 (M + 3H) 3+ m / z (z = 4) = 702.2 (M + 4H) 4+ .

[0072] Example 6: Preparation of Compound 6 [7,10-bis(carboxymethyl)-4-{3-hydroxy-1-oxylidene-1-[(2-{4,7,10-tris[2-({3-hydroxy-1-oxylidene-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]butyl-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 6) The preparation method is the same as that in Example 2, except that O-tert-butyl-serine in Example 2 is replaced with O-tert-butyl-DL-threonine to obtain compound 6. The synthesis yield in step seven is 77% and the purity is 98.7%.

[0073] LC-MS(ESI+): m / z(z=2)=1343.4M+2H) 2+ m / z (z = 3) = 895.9 (M + 3H) 3+ m / z (z = 4) = 672.2 (M + 4H) 4+ .

[0074] Example 7: Preparation of Compound 7 [7,10-bis(carboxymethyl)-4-{3-carboxy-1-oxoylide-1-[(2-{4,7,10-tris[2-({3-carboxy-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]propionyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]propyl-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 7) The preparation method is the same as that in Example 2, except that O-tert-butyl-serine in Example 2 is replaced with 2-amino-4-[(2-methylprop-2-yl)oxy]-4-oxonylbutyric acid to obtain compound 7. The synthesis yield of step seven is 61%, and the purity is 98.1%.

[0075] The structure of 2-amino-4-[(2-methylprop-2-yl)oxy]-4-oxonylbutyric acid is: .

[0076] LC-MS(ESI+): m / z(z=2)=1371.3M+2H) 2+ m / z (z = 3) = 914.6 (M + 3H) 3+ m / z (z = 4) = 686.2 (M + 4H) 4+ .

[0077] Example 8: Preparation of Compound 8 [4,7-bis(carboxymethyl)-10-{4-carboxyl-1-oxylidene-1-[(2-{4,7,10-tris[2-({4-carboxyl-1-oxylidene-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butyl}amino)ethyl]-1,4,7,10-tetraazacyclododecane-1-yl}ethyl)amino]butyl-2-yl}-1,4,7,10-tetraazacyclododecane-1-yl]tetragadolinium acetate (compound 8) The preparation method is the same as that in Example 2, except that O-tert-butyl-serine in Example 2 is replaced with DL-glutamic acid-5-tert-butyl ester to obtain compound 8. The synthesis yield in step seven is 60%, and the purity is 98.4%.

[0078] LC-MS(ESI+): m / z(z=2)=1399.4(M+2H) 2+ m / z (z = 3) = 933.3 (M + 3H) 3+ m / z (z = 4) = 700.2(M + 4H) 4+ .

[0079] The intermediates and their mass spectrometry data included in the above embodiments are shown in the table below: Comparative Example 1: Gadovist (purchased from Anaiji Chemical, purity over 98%) .

[0080] Comparative Example 2: .

[0081] The compound of Example 7 in patent CN107667096B was prepared according to the preparation method of Example 7 and is used as Comparative Example 2 of this application. Finally, it was purified by reversed-phase preparative chromatography and dried to obtain the target Comparative Example 2 compound with a purity of 98.5%. The structure was consistent with the analysis, and the LC-MS (ESI+) of the main product was: m / z (z=2) = 1311.7 (M+2H). 2+ m / z (z=3) = 873.1 (M+3H) 3+ The chemical structure of the product is the same as that of Comparative Example 2 above.

[0082] Comparative Example 3: .

[0083] The compound of Example 6 was prepared according to the preparation method in Example 6 of patent CN110035996B, and is used as Comparative Example 3 of this application. Finally, it was purified by reversed-phase preparative chromatography and dried to obtain the target Comparative Example 3 compound with a purity of 98.1%. The structure was consistent with the analysis, and the LC-MS (ESI+) of the main product was: m / z (z=2) = 1373.4 (M+2H). 2+ m / z (z=3) = 916.0 (M+3H) 3+ The chemical structure of the product is the same as that of Comparative Example 3 above.

[0084] Experimental Example 1: Relaxation Measurement 1. Instruments Meishi Medical 7.0T Small Animal Magnetic Resonance Imaging System.

[0085] 2. Preparation of test samples At room temperature, using pure water and human blood plasma as matrices, the compounds of the examples and comparative examples were prepared into test samples with concentrations of 1.0, 0.75, 0.6, 0.5, 0.4, and 0.25 mmol Gd / L, respectively. The samples were sonicated for 2 min to remove air bubbles, centrifuged to ensure the liquid level was consistent, sealed to prevent evaporation, and placed in a honeycomb array sample holder in order of concentration for later use.

[0086] 3. Test methods Before the formal scan, after the gradient and radio frequency tests pass, the sample is placed at the center of the coil, and local shimming (based on the water peak) is performed until the linewidth is <30–50 Hz. A rapid localization image is acquired to ensure that the single-layer slice covers all samples. After confirmation, localization / tuning / matching / field modulation are performed, and the geometry of the coil and sample is recorded. T1 arrays of all concentration samples are acquired sequentially. The sequence parameters (IR-SE sequence) are as follows: Field of view (FOV) 80×80 mm²; matrix 128×128; thickness 3 mm; repetition time (TR) 3000 ms; echo time (TE) 7.5 ms; 8 TI arrays with times of 10, 300, 580, 860, 1150, 1400, 1700, and 2000 ms respectively; accumulation count 1; number of layers 1.

[0087] 4. Data Processing and Result Analysis Data processing: The average signal is taken within the circular ROI (avoiding the boundary by 1–2 pixels) for each sample, and T1 (m) is fitted. T1 is then converted to R1 = 1 / T1 (unit: s⁻¹; T1 needs to be converted to seconds). A univariate linear regression is performed on (R1, [C]): R1 = r1[C] + R10, and the resulting slope r1 is the relaxation rate of the compound (unit: mM^-1 s^-1).

[0088] The relaxation properties of each compound measured in pure water and human plasma are shown in the table below: Table 1. Relaxability in water and human plasma (mM^-1 s^-1) As shown in Table 1, the compounds of this application exhibit high relaxation rates (r1) in both water and human plasma. Compound 8 shows the highest relaxation rate, with r1 values ​​exceeding 10 in both water and human plasma, significantly superior to the comparative compounds, demonstrating its superior relaxation performance. These results indicate that the compounds of this application possess excellent relaxation performance and greater potential for MRI contrast enhancement, providing a basis for achieving stronger signals at the same dose or equivalent imaging with reduced dose.

[0089] Experimental Example 2: Pharmacokinetic Experiment in SD Rats 1. Experimental animals SPF-grade male SD rats, 6-8 weeks old, weighing 180-250g, with 6 rats in each group, were randomly assigned to groups according to their weight and had free access to food and water.

[0090] 2. Administration Test samples: compounds from each example and comparative example; Administration method: Weigh the patient before administration. Calculate the dosage based on body weight at 0.1 mmol Gd / kg. Prepare the injection solution using 0.9% sodium chloride as the solvent. Prepare and use immediately. Administer intravenously.

[0091] 3. Sample collection and processing After blood was collected via the jugular vein, the blood samples were placed on ice. At least 150 µL of blood was collected per time point. The samples were temporarily placed on ice before centrifugation and the plasma was separated by centrifugation within one hour. The collected whole blood samples were placed in blood collection tubes containing anticoagulant (EDTA-K2) and centrifuged at 6800 g at 2-8ºC for 6 min. At least 75 µL of plasma was collected and stored in a -80ºC freezer for testing.

[0092] 4. Data Processing and Result Analysis Bioanalysis: An ICP-MS standard curve was established to detect the plasma concentration of gadolinium, with an upper limit of quantification of 20,000 ng / mL and a lower limit of quantification of 0.1 ng / mL. The accuracy of quality control samples was evaluated simultaneously with the analysis of the samples. The accuracy of more than 66% of the quality control samples was between 80-120%.

[0093] Data processing: Pharmacokinetic parameters, such as AUC, were calculated using WinNonlin based on blood drug concentration data at different time points. all AUC inf T 1 / 2 C max and T max When plotting plasma drug concentration-time curves, BLQ is always recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; C max Previous BLQs (including "No peak") are calculated as 0; C max Subsequent BLQs (including "No peak") will not be included in the calculation.

[0094] The main pharmacokinetic parameters (Mean ± SD) of each compound in rats are shown in the table below: Table 2. Pharmacokinetic parameters of gadolinium in plasma (Mean ± SD) As shown in Table 2, at equimolar gadolinium doses, the AUC of the compounds in the embodiments of this application in rats was... all and AUC inf All were significantly higher than the comparative compounds, approximately 8-10 times higher, indicating a significantly increased overall in vivo exposure level of the compounds in this application. Meanwhile, the C of the compounds in this application... max The value is approximately twice that of the comparative example, indicating that it can rapidly reach a high peak blood drug concentration after administration, which is beneficial for quickly producing a significant imaging enhancement effect. Furthermore, the half-life (T5) of the compound in this application... 1 / 2 The concentration of the compound was significantly lower than that of the comparative compound, indicating that it has a faster clearance rate in vivo.

[0095] The above results indicate that the compound of this application achieves high exposure and high peak value while possessing good clearance properties and low risk of tissue residue, thereby reducing free Gd. 3+ Eliminate potential hazards and enhance safety.

[0096] Experimental Example 3: Evaluation of Magnetic Resonance Imaging (MRI) Results This study aims to evaluate the magnetic resonance efficacy of the compound in the examples and the comparative compound in mice, including the magnetic resonance enhancement effects on cerebral blood vessels, liver, kidneys and muscles, and their pharmacodynamic characteristics, through a single imaging test and pharmacodynamic test.

[0097] 1. Test materials Instrument: MedSci 7.0T Small Animal Magnetic Resonance Imaging System.

[0098] Animals: Balb / c mice, 7-12 weeks old; single imaging test, males, 3 mice per group; time-dependent test, half males and half females, 2 mice per group.

[0099] Test samples: Take each compound and prepare injection solutions using 0.9% sodium chloride as solvent. Prepare and use immediately.

[0100] 2. Dosing regimen Single imaging test: All test samples were administered at an equivalent dose of 0.1 mmol Gd / kg.

[0101] Pharmacokinetic studies (time-dependent): Compound 1 was administered at 0.1 mmol Gd / kg; Compound 8 and Compounds 2 and 3 were administered at a low dose of 0.04 mmol Gd / kg.

[0102] 3. Test methods Mice were fasted overnight before the examination and anesthetized with 1%-1.5% isoflurane gas at a ventilation rate of 0.8-1.0 L / minO2. After the mice were anesthetized, a tail vein access was established.

[0103] Cerebral angiography: Mice were fixed in a prone position in a magnetic resonance imaging (MRI) scanner, using a special head-mounted coil. After tuning and shimming, cerebral vascular MRA data were acquired before drug administration. Subsequently, the test substance was injected via the tail vein, and data were acquired after drug administration. Single-shot imaging test: Images were acquired before drug administration (pre) and immediately after drug administration. Pharmacokinetic test (time-dependent): Images were acquired before drug administration (pre) and at multiple time points, including 0.5 min, 5 min, 15 min, and 30 min after drug administration.

[0104] 4. Data Processing MRI angiography was performed using 3D CE-MRA sequences. The MRI images were analyzed and processed using ImageJ and MatLab software. The signal intensity (SI) of the target region (blood vessel) was measured, and the imaging effect was expressed as MRI Δ signal intensity. The calculation formula is as follows: MRI signal intensity = (signal intensity after drug administration / signal intensity before drug administration) - 1.

[0105] 5. Results Analysis 5.1 Single Imaging Table 3. Δ signal intensity of cerebral blood vessels before and after drug administration (Mean±SD) Figure 1The images of cerebral blood vessels in mice before and after administration of compounds 1-3 and compound 8 are shown in Table 3. As can be seen from the data, under the same dosage, compound 8 of this application achieved a significantly better cerebral vascular enhancement effect than the comparative compounds after administration. Its imaging effect had the highest brightness, sharp vascular edges, and no signal spillover, which was significantly better than the comparative compounds.

[0106] 5.2 Pharmacokinetics 5.2.1 Cerebrovascular Pharmacokinetics Table 4. Δ signal intensity of cerebral blood vessels on MRI (Mean ± SD) Figure 2 The image shows the Δ signal intensity-time curve of mouse brain blood vessels on MRI. Figure 2 As shown in the trend and the data in Table 4, at a lower dose (0.04 mmol Gd / kg), compound 8 of this application achieved a signal enhancement peak of approximately 9.3 times within 0.5 min after administration, significantly higher than the comparative compound; and maintained a high signal intensity at 15 min, indicating a longer effective imaging time window. Simultaneously, its signal intensity decreased to near the level of the comparative compound at 30 min, indicating rapid clearance in vivo without significant delayed accumulation. These results demonstrate that the compound of this application possesses the combined advantages of "high peak value, longer effective window, and rapid clearance," consistent with its pharmacokinetic experimental results.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chelate or a pharmaceutically acceptable salt thereof, or a mixture thereof, characterized in that, The chelate is selected from: , , , , , , , .

2. A compound or a pharmaceutically acceptable salt thereof, or a mixture thereof, characterized in that, The compound is selected from: , , , , , , ,or .

3. A method for preparing the compound as described in claim 2, characterized in that, Compound d and compound h were condensed to obtain compound i, and compound i was further deprotected to obtain compound (II); ； in, R1 and R2 correspond to the groups of the compound described in claim 2; R1' is the protection form of R1 or R1; R3 is selected from methyl, tert-butyl, or benzyl.

4. A method for preparing the chelate as described in claim 1, characterized in that, It includes: Compound d and compound h were condensed to obtain compound i, and compound i was further deprotected to obtain compound (II); The compound of formula (II) was prepared by chelation reaction to obtain the compound of formula (I); in, R1 and R2 correspond to the groups of the chelate described in claim 1; R1' is the protection form of R1 or R1; R3 is selected from methyl, tert-butyl, or benzyl.

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