A super inert gadolinium-dota probe coupled with non-natural dipeptide, its preparation method and application

Abstract

The application belongs to the technical field of magnetic resonance imaging contrast agents, and particularly relates to a super-inert Gd-DOTABA probe coupled with a non-natural dipeptide, a preparation method and application thereof. The probe takes super-inert Gd-DOTABA as a skeleton, covalently couples a non-natural dipeptide targeting module containing two D-4,4'-diphenylalanine residues, and can specifically bind to human serum albumin. The longitudinal relaxation efficiency r1 of the probe in pure water reaches 9.4-10.3 mM ‑1 s ‑1 , and is increased to 20.7-23.3 mM in the presence of 4.5% human serum albumin ‑1 s ‑1 . The acid dissociation half-life is 3-5 times longer than that of a clinical Gd-DOTABA. At a low dose of 0.03 mmol / kg, the probe can realize high-contrast tumor imaging and vascular imaging, has excellent biocompatibility, can be efficiently cleared through the hepatobiliary and renal pathways, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging contrast agent technology, specifically relating to an ultra-inert Gd-DOTABA probe coupled with a non-natural dipeptide, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is the cornerstone of modern diagnostic medicine. Due to its high spatial resolution and excellent soft tissue contrast, it has become an indispensable tool for clinical disease diagnosis. To improve diagnostic sensitivity, approximately 30-40% of clinical MRI procedures rely on gadolinium-based contrast agents, which accelerate proton longitudinal relaxation, thereby improving lesion visualization. However, recent clinical concerns regarding renal systemic fibrosis and gadolinium deposition in the brain (particularly in the dentate nucleus and globus pallidus) have prompted regulatory agencies and the medical community to reassess the safety of traditional gadolinium-based contrast agents. Although renal systemic fibrosis is associated with Gd in patients with renal insufficiency... 3+ While dissociation is directly related, there is also evidence that linear, less stable contrast agents are associated with higher gadolinium retention in the brain compared to more inert macrocyclic contrast agents. Therefore, there is an urgent need to develop contrast agents that possess both excellent kinetic inertness (to prevent toxic Gd) and... 3+ Next-generation gadolinium-based contrast agents with high relaxation efficiency (enabling robust imaging at significantly lower clinical doses) and high leakage.

[0003] To reduce Gd 3+ Dissociation risk has been established as a safety benchmark for macrocyclic complexes such as Gd-DOTA due to their superior thermodynamic stability and kinetic inertness compared to linear analogs. Recent advances have focused on further rigidifying the macrocyclic skeleton by introducing substituents on the ring or at the α-position of the acetate side chain to simultaneously enhance kinetic stability and proton relaxation efficiency. Within this framework, the Gd-DOTABA skeleton—with a single para-benzoic acid group at the α-position—has emerged as a particularly promising ultra-inert platform, with an acid dissociation half-life several times longer than Gd-DOTA, highlighting its strong potential as a base skeleton for developing next-generation highly safe contrast agents.

[0004] Despite their safety advantages, small-molecule macrocyclic gadolinium-based contrast agents typically exhibit low proton relaxation efficiency, ranging from 3 to 5 mM at 1.5–3.0 T. -1 s -1This is primarily attributed to its rapid molecular tumbling. An effective strategy to improve relaxation efficiency is to design probes capable of non-covalently binding to human serum albumin, the most abundant plasma protein. This binding significantly increases rotation-related time, leading to a dramatic "boom" in r1 relaxation efficiency and prolonged intravascular residence time. To this end, various albumin-binding groups have been explored, such as diphenyl, cholesterol, and trisulfonated pyrene. Based on the molecular recognition principles of bioactive peptides or proteins, it is reasonable to consider non-natural dipeptide modules as excellent and programmable platforms for targeting human serum albumin. Compared to natural peptides, non-natural peptides containing biphenylalanine and other compounds exhibit superior metabolic stability against in vivo proteases, while their modularity allows for precise regulation of the hydrophobic-hydrophilic balance to optimize human serum albumin affinity and pharmacokinetics.

[0005] Currently, there are no reports on the construction of novel magnetic resonance imaging contrast agents by coupling non-natural dipeptides based on biphenylalanine with the ultra-inert Gd-DOTABA backbone. Existing contrast agents cannot simultaneously meet the dual requirements of ultra-high kinetic inertness and ultra-high relaxation efficiency, making it difficult to achieve high-contrast tumor and vascular imaging with significantly reduced clinical doses. Furthermore, some target groups have defects such as poor in vivo metabolic stability, insufficient affinity, or poor structural tunability, which limit their further clinical translation and application. Summary of the Invention

[0006] The purpose of this invention is to provide an ultra-inert Gd-DOTABA probe coupled with a non-natural dipeptide, its preparation method and application, which can more effectively enhance the interaction between the complex and human serum albumin, and provide new ideas for the structural design of high-performance magnetic resonance imaging contrast agents.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides an ultra-inert Gd-DOTABA probe coupled with a non-natural dipeptide, the probe comprising an ultra-inert Gd-DOTABA backbone and a non-natural dipeptide targeting module covalently coupled to the Gd-DOTABA backbone; the non-natural dipeptide targeting module comprises two D-4,4'-biphenylalanine residues for specifically binding to human serum albumin.

[0008] Furthermore, the non-natural dipeptide targeting module is coupled to the benzoic acid side chain of the Gd-DOTABA backbone via a tetraethylene glycol linker arm using an amide bond, and the terminal group of the probe is a methyl ester group or a carboxyl group.

[0009] Furthermore, the probe is Gd-L1 with a terminal methyl ester group, its precise molecular weight is 1283.3930, and its molecular formula is C0.05. 60 H 67 GdN7O15 .

[0010] Furthermore, the probe is Gd-L2 with a carboxyl group at the end, its precise molecular weight is 1269.3772, and its molecular formula is C2. 59 H 65 GdN7O 15 .

[0011] This invention also provides a method for preparing the aforementioned ultrainert Gd-DOTABA probe coupled with a non-natural dipeptide, employing a convergent liquid-phase synthesis route, comprising the following steps: (1) Using D-4,4'-biphenylalanine as raw material, a non-natural dipeptide intermediate containing two biphenylalanine residues was gradually constructed through esterification, amide coupling and deprotection reactions. (2) The non-natural dipeptide intermediate and the pre-prepared Gd-DOTABA core were subjected to an amide coupling reaction under the action of a coupling agent to obtain Gd-L1 with a methyl ester group at the end. (3) Optionally, Gd-L1 can be hydrolyzed under alkaline conditions to obtain Gd-L2 with a carboxyl group at the end.

[0012] Furthermore, in both steps (1) and (2), the amide coupling reaction uses 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as the coupling agent, methanol as the reaction solvent, and room temperature as the reaction temperature; the deprotection reaction uses a trifluoroacetic acid / dichloromethane mixed solution with a volume ratio of 1:4 as the deprotection agent, and room temperature as the reaction temperature. In step (3), the hydrolysis reaction uses sodium hydroxide aqueous solution as the hydrolysant, the reaction solvent is a methanol / water mixture with a volume ratio of 1:1, and the reaction temperature is room temperature.

[0013] The present invention also provides the application of the superinert Gd-DOTABA probe coupled with the non-natural dipeptide described above in the preparation of magnetic resonance imaging contrast agents.

[0014] Furthermore, the magnetic resonance imaging contrast agent is used for tumor magnetic resonance imaging or vascular magnetic resonance imaging.

[0015] Furthermore, the dosage of the magnetic resonance imaging contrast agent is 0.03 mmol / kg, which is one-third of the clinical standard gadolinium contrast agent dosage.

[0016] Furthermore, the longitudinal relaxation efficiency r1 of the magnetic resonance imaging contrast agent in pure water at 1.4T and 37°C is 9.4-10.3 mM. -1 s -1 The longitudinal relaxation efficiency r1 in the presence of 4.5% human serum albumin was 20.7-23.3 mM. -1 s-1 Its acid dissociation half-life is 72-120 hours, which is 3-5 times longer than that of the clinical contrast agent Gd-DOTA.

[0017] The beneficial effects of this invention are as follows: This invention successfully developed a novel magnetic resonance imaging contrast agent by rationally integrating a non-natural dipeptide motif targeting human serum albumin with a kinetically ultra-inert Gd-DOTABA backbone. This agent possesses high relaxation efficiency, excellent kinetic inertness, and low-dose imaging performance, effectively solving the technical problems of insufficient relaxation efficiency, high risk of gadolinium ion leakage, and large clinical dosage associated with traditional gadolinium-based contrast agents. Compared to single-amino acid modification structures, the biphenylalanine-containing dipeptide modification strategy employed in this invention more effectively enhances the interaction between the complex and human serum albumin, providing a new approach for the structural design of high-performance magnetic resonance imaging contrast agents.

[0018] The longitudinal relaxation efficiencies r1 of the two probes Gd-L1 and Gd-L2 prepared in this invention in pure water reached 10.29 ± 0.02 mM, respectively. -1 s -1 and 9.40±0.05mM -1 s -1 It was approximately three times that of the clinical standard Gd-DOTA under the same conditions; in the presence of 4.5% human serum albumin, r1 further increased to 20.69±0.03mM. -1 s -1 and 23.30±0.02mM -1 s -1 The binding affinity was approximately six times that of Gd-DOTA, significantly superior to the control compounds Gd-L3 and Gd-L4, which contain only a single amino acid structure. At a concentration of 0.5 mM, the binding rates of the two probes to human serum albumin reached 98.7 ± 0.2% and 95.7 ± 0.2%, respectively, with dissociation constants Kd of only 2.0 μM and 2.1 μM. This high-affinity binding significantly increased the molecular rotational correlation time, resulting in a substantial leap in relaxation efficiency.

[0019] The probe of this invention also possesses excellent kinetic stability, with acid dissociation half-lives of approximately 72 hours and 120 hours, respectively, representing a 3 to 5-fold improvement in inertness compared to Gd-DOTA, and is effective even in the presence of 20 times excess Zn. 2+ It remains intact for 7 days in PBS. The large dipeptide-PEG side chain is coupled to the ultra-inert Gd-DOTABA core, in Gd... 3+ Significant steric hindrance is generated around the coordination spheres, which, combined with potential intramolecular hydrogen bonds, can effectively contain Gd. 3+ Ions are "locked" within the macrocyclic cavity to prevent toxic free Gd. 3+ The release of ions ensures the safety of contrast agents at the molecular structure level.

[0020] In vitro biocompatibility evaluation showed that, within the tested concentration range, the viability of human hepatic stellate cells and human embryonic kidney cells treated with both probes remained above 90%. Even at the highest concentration of 0.5 mM, any slight inhibitory effect observed was not statistically significant compared to the Gd-DOTA control. The in vivo imaging dosage of this invention is only 0.03 mmol / kg, which translates to an in vivo exposure concentration far lower than the highest concentration used in in vitro cytotoxicity experiments, providing a significant safety margin. In vivo acute and subacute toxicity studies further confirmed that, after single or repeated administration, the two probes did not produce detectable adverse effects on liver, kidney, or cardiac function in mice. All key serum biochemical indicators remained within the normal range, and no significant abnormalities were observed in the histomorphology of major organs. No obvious signs of lesions, necrosis, or inflammatory infiltration were observed, and the biocompatibility was comparable to the clinical benchmark Gd-DOTA.

[0021] The probes of this invention achieve high-contrast, multi-parameter magnetic resonance imaging at a low dose, only one-third of the clinical standard dose. In normal organ imaging of healthy BALB / c mice, they induced strong and persistent signal enhancement in the liver and kidneys. The normalized signal-to-noise ratio (SNR) of the liver peaked at 5 minutes post-injection, reaching 2.0 and 2.5 times the baseline, respectively, and remained at 1.5 and 1.8 times the baseline at 27 minutes. The peak SNR of the kidneys reached 2.6 and 2.3 times, respectively, far superior to the weak enhancement effect of Gd-DOTA. In the 4T1 subcutaneous tumor model, both probes provided strong and progressive tumor contrast enhancement, with the relative signal intensity continuously increasing throughout the 52-minute observation period. The clarity of tumor boundaries and internal structures improved over time, enabling effective delayed tumor imaging. In contrast, Gd-DOTA only produced weak and transient signal enhancement, failing to meet the requirements for delayed imaging.

[0022] In vascular imaging of SD rats, both probes at low doses produced robust and clear systemic vascular enhancement, far exceeding that of Gd-DOTA at the same dose and surpassing that of Gd-DOTA at the standard clinical dose. Gd-L1 achieved the clearest depiction of the systemic vascular network as early as 1 minute after injection, while Gd-L2 clearly displayed the contours of major vessels and the heart within 1-5 minutes after injection, with residual contrast still detectable in major vessels at 20 minutes, meeting the clinical requirements for high-resolution vascular imaging.

[0023] The probes of this invention also possess excellent pharmacokinetic and clearance properties, allowing them to remain in the bloodstream for an extended period after injection. Five minutes post-injection, the blood concentrations were 26.7 μg / g tissue and 34.8 μg / g tissue, respectively, approximately 1.9 times and 2.5 times that of Gd-DOTA, providing a basis for sustained vascular enhancement. Simultaneously, both probes are efficiently cleared from the body via both hepatobiliary and renal pathways, without significant accumulation in other major organs such as the heart, spleen, lungs, and brain within 24 hours, avoiding the potential risks associated with long-term in vivo residues and demonstrating promising prospects for clinical translation. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows the structure of a typical small molecule GBCA with an HSA binding group and the Gd(III) complex based on a non-natural dipeptide designed in this invention. Figure 2 The synthesis route diagram for Gd-L1 and Gd-L2; Figure 3 The figures show the acid-catalyzed dissociation kinetics and metal substitution experimental results for Gd-L1, Gd-L2, and Gd-DOTA. Figure 3 In Figure a, the acid-catalyzed dissociation kinetics of Gd-L1, Gd-L2, and Gd-DOTA at 1M HCl and 37℃ are shown. Figure 3 In the middle b, Gd-L1, Gd-L2, and Gd-DOTA are in the presence of 20 times excess Zn². + The relative relaxation time variation curve of PBS over 7 days at 37°C; Figure 4 The image shows the cell viability assay results for LX-2 cells and 293T cells. Figure 4 In the middle, a is a bar chart of cell viability of LX-2 cells after exposure to different concentrations of Gd-L1, Gd-L2 and Gd-DOTA for 24 hours; Figure 4 In the middle b, there is a bar chart showing the cell viability of 293T cells after 24 hours of exposure to different concentrations of Gd-L1, Gd-L2, and Gd-DOTA. Figure 5 Molecular docking diagrams showing the optimal binding conformations of Gd-L1 and Gd-L2 with HSA, where, Figure 5 In Figure 'a', the three-dimensional structure of the optimal binding conformation of Gd-L1 and HSA is shown. Figure 5In Figure b, the three-dimensional structure of the optimal binding conformation of Gd-L2 and HSA is shown. Figure 5 In the middle c, there is a two-dimensional schematic diagram of the key interactions in the binding pocket. HSA is represented by a white ribbon, the probe molecule is represented by a stick model, hydrogen bonds are shown as yellow dashed lines, and hydrophobic interactions are shown as red dashed circles. Figure 6 The images show magnetic resonance imaging (MRI) results of normal organs in healthy BALB / c mice. Figure 6 Image a shows a representative coronal T1-weighted liver / kidney image of a healthy BALB / c mouse after intravenous injection of 0.03 mmol / kg Gd-L1, Gd-L2, or Gd-DOTA. Figure 6 In the middle b, the normalized signal-to-noise ratio quantitative analysis curve of the liver at the corresponding time point is shown. Figure 6 In the figure, c represents the normalized signal-to-noise ratio quantitative analysis curve of the kidney at the corresponding time point; Figure 7 The image shows the tumor magnetic resonance imaging results of 4T1 tumor-bearing mice. Figure 7 In the middle section (a), a representative transverse T1-weighted tumor image of a 4T1 tumor-bearing mouse after intravenous injection of 0.03 mmol / kg Gd-L1, Gd-L2, or Gd-DOTA. Figure 7 In Figure b, the curve represents the quantitative analysis of the relative signal enhancement in the tumor region over time. Figure 8 This is a magnetic resonance imaging (MRI) image of the whole-body blood vessels of SD rats, in which... Figure 8 Image a shows a representative coronal T1-weighted image of the whole-body blood vessels in SD rats after intravenous injection of Gd-L1, Gd-L2, and Gd-DOTA. Figure 8 In Figure b, the signal intensity of the vessel of interest in the abdominal region changes over time, which is a quantitative analysis curve. Figure 8 In the middle, d represents the quantitative comparison noise ratio analysis results of key vascular structures in mice injected with Gd-L1 at different time points; Figure 8 In the middle, e represents the quantitative comparison noise ratio analysis results of key vascular structures in mice injected with Gd-L2 at different time points; Figure 9 This is a diagram showing the distribution of gadolinium in the major organs and tissues of BALB / c mice. Figure 9 In the middle section (a), there are bar charts showing the gadolinium concentration in major organs and tissues of BALB / c mice at 5 minutes, 15 minutes, and 24 hours after intravenous injection of 0.03 mmol / kg Gd-L1. Figure 9 Table b shows the gadolinium concentration in major organs and tissues of BALB / c mice at 5 minutes, 15 minutes, and 24 hours after intravenous injection of 0.03 mmol / kg Gd-L2; Figure 9The bar chart in Figure c shows the gadolinium concentration in major organs and tissues of BALB / c mice at 5 minutes, 15 minutes, and 24 hours after intravenous injection of 0.03 mmol / kg Gd-DOTA. Figure 9 The bar chart in section d shows the percentage of total uptake by the liver and kidneys at 5 and 15 minutes after injection. Figure 10 This is a graph showing the results of the in vivo safety evaluation in mice. Figure 10 Image a shows representative H&E staining images of mouse liver and kidney 24 hours after a single intravenous injection of PBS, 0.03 mmol / kg Gd-DOTA, Gd-L1, or Gd-L2 (scale bar: 50 μm). Figure 10 Figure b shows the results of serum biochemical indicators (AST, ALT, ALP, ALB) related to liver function in the acute toxicity study. Figure 10 In the acute toxicity study, c represents the analysis results of serum biochemical indicators (LDH, CK, BUN, CR) related to renal function and cardiac / skeletal muscle injury. Figure 10 In the subacute toxicity study, d represents the results of serum biochemical indicators (AST, ALT, ALP, ALB) related to liver function. Figure 10 In the subacute toxicity study, e represents the analysis results of serum biochemical indicators (LDH, CK, BUN, CR) related to renal function and cardiac / skeletal muscle injury. Figure 11 Synthetic routes for control compounds Gd-L3 and Gd-L4; Figure 12 HPLC chromatograms of Gd-L1 and Gd-L2; Figure 13 The graph shows the results of the relaxation efficiency measurements for Gd-L1 and Gd-L2. Figure 14 The graph shows the results of relaxation efficiency measurements for Gd-L3 and Gd-L4. Figure 14 In the figure, a represents the linear relationship between the relaxation rate of Gd-L3 and gadolinium concentration in the absence of 4.5% HSA at 1.4T and 37℃. Figure 14 In the figure, b represents the linear relationship between the relaxation rate of Gd-L3 and gadolinium concentration in the presence of 4.5% HSA at 1.4T and 37℃. Figure 14 The graph shows the linear relationship between the relaxation rate of Gd-L4 and gadolinium concentration in the absence of 4.5% HSA at 1.4T and 37℃. Figure 14 Linear relationship between relaxation rate and gadolinium concentration of Gd-L4 in the presence of 4.5% HSA at d=1.4T and 37℃; Figure 15 The graph shows the HSA binding affinity determination results for Gd-L1, Gd-L2, and Gd-DOTA. Figure 16Supplementary evaluation results of the kinetic inertia of Gd-L1, Gd-L2, and Gd-DOTA are shown in the figure. Figure 16 In Figure 'a', the acid-catalyzed dissociation kinetics curves of Gd-L1, Gd-L2, and Gd-DOTA in 1 mol / L hydrochloric acid solution are shown. Figure 16 In the middle b, Gd-L1, Gd-L2, and Gd-DOTA are in a solution containing 20 times the equivalent of Zn. 2+ Metal displacement kinetics curves in PBS buffer (37°C); Figure 17 Supplementary image for transverse T1-weighted magnetic resonance imaging of mouse tumors; Figure 18 H&E stained sections of major organs of a mouse; Figure 19 The ESI-MS spectrum of compound 2 is shown below. Figure 20 The image shows the ESI-MS spectrum of compound 3. Figure 21 The image shows the ESI-MS spectrum of compound 4. Figure 22 The image shows the ESI-MS spectrum of compound 5. Figure 23 The ESI-MS spectrum of compound 6 is shown below. Figure 24 ESI-HRMS spectrum of Gd-L1; Figure 25 The ESI-HRMS spectrum of Gd-L2; Figure 26 The image shows the ¹H NMR spectrum of compound 2 in CD3OD. Figure 27 Here is the ¹³C NMR spectrum of compound 2 in CD3OD; Figure 28 The ¹H NMR spectrum of compound 4 in CD3OD; Figure 29 The image shows the ¹³C NMR spectrum of compound 4 in DMSO-d6; Figure 30 The ¹H NMR spectrum of compound 5 in CD3OD; Figure 31 The ¹³C NMR spectrum of compound 5 in CD3OD; Figure 32 The ¹H NMR spectrum of compound 6 in CD3OD; Figure 33 The image shows the ¹³C NMR spectrum of compound 6 in DMSO-d6. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] General Materials and Methods Unless otherwise stated, all chemicals were analytical grade reagents, purchased from Aladdin Biotech or Maclean Biotech, and used directly without further purification. ¹H and ¹³C NMR spectra were recorded on an Oxford Quantum-1 400MHz NMR spectrometer. Chemical shifts δ are expressed in parts per million (ppm) and calibrated against residual solvent signals of D₂O, CD₃OD, or DMSO-d₆. Coupling constant J is expressed in Hertz (Hz). Coupling modes are abbreviated as follows: s (singleton), d (doublet), t (triplet), q (quartet), dd (double doublet), ddd (double double doublet), dt (double triplet), dtd (double double triplet), dq (double quartet), m (multiplet).

[0032] The purity of the complexes was analyzed using a Waters Alliance e2695 reversed-phase high-performance liquid chromatography (RP-HPLC) system, employing a Waters C18 column. For analytical HPLC, mobile phase A was an aqueous solution containing 0.05% trifluoroacetic acid, and mobile phase B was analytical grade acetonitrile. The analytical method started with 90% A and 10% B at a flow rate of 1 mL / min, with the system pressure limit set at 3000.0 psi and the flow acceleration time set at 2.00 min. Subsequently, the proportion of phase B was increased to 100% over 5.0 min, the column was washed with 100% B for 2 min, and finally, the column was equilibrated at the initial ratio for 3 min. For semi-preparative HPLC, mobile phase A was a 10 mM ammonium acetate solution, and mobile phase B was a mixture of 90% acetonitrile and 10% 10 mM ammonium acetate solution. The initial detection conditions were 70% A and 30% B at a flow rate of 1 mL / min. Then, the proportion of mobile phase B was increased to 100% over 15 min, then decreased to 30% over 2 min, and maintained at this ratio for 3 min.

[0033] Human hepatic stellate cells (LX-2), human embryonic kidney cells (293T), and 4T1 mouse breast cancer cells were purchased from iCellBioscience Inc. Female Sprague-Dawley (SD) rats and female BALB / c mice were provided by Zhejiang Experimental Animal Center. The clinical contrast agent Gd-DOTA was purchased from Hainan Shuangcheng Pharmaceutical Co., Ltd. The coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and other analytical grade chemicals were purchased from Aladdin Reagent Co., Ltd. CellCounting Kit-8 (CCK-8) was purchased from Beyotime Biotechnology. Fetal bovine serum (FBS), DMEM, and RPMI-1640 medium were purchased from Gibco.

[0034] All animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" and approved by the Animal Care and Use Committee of the Wenzhou Research Institute of the University of Chinese Academy of Sciences. In this study, PyMOL Molecular Graphics System 2.5.0 was used to generate maps of complexes binding to transport proteins; GraphPad Prism 9.5 was used for data processing and scientific drawing; MicroDicomViewer was used to process MR images; MestReNova 15 was used to process NMR, CMR, and mass spectrometry data; and Adobe Illustrator 2021 was used for image layout design. The normalized signal-to-noise ratio (SNR) was calculated using the formula SNR = SI / SDair, nSNR = SNRpost / SNRpre; tumor uptake of contrast agent was described by relative enhancement, calculated using the formula RE = (SItumor(post) - SItumor(pre)) / SDair × 100%. Experimental data are expressed as mean ± standard deviation. Statistical comparisons between multiple groups were performed using SPSS software, with a p-value less than 0.05 considered statistically significant.

[0035] Example 1: Synthesis of gadolinium-based contrast agents Gd-L1 and Gd-L2 and control compounds Gd-L3 and Gd-L4 In this embodiment, the target complexes Gd-L1 and Gd-L2 were synthesized via a convergent liquid-phase route. Their molecular structures are compared with those of typical existing HSA-bound small-molecule gadolinium-based contrast agents. Figure 1 As shown; the synthetic routes for Gd-L1 and Gd-L2 are as follows Figure 2 As shown; the synthetic routes for the control compounds Gd-L3 and Gd-L4 are as follows. Figure 11 As shown.

[0036] 1.1 Synthesis of Compound 2 Compound 1 was dissolved in 5 mL of anhydrous methanol under ice bath conditions, and 1.0 mL of thionyl chloride was slowly added dropwise. The reaction was carried out under nitrogen protection for 4 h, and the completion of the reaction was detected by reversed-phase high-performance liquid chromatography (RP-HPLC). After the reaction was completed, the solvent was removed by vacuum evaporation and the mixture was concentrated to give compound 2 (white solid, 95% yield).

[0037] The ESI-MS spectrum of compound 2 is as follows: Figure 19 As shown, the ESI-MS calculated value C 32 H 35 NO2 + [2M+H] + :511.2, measured value: 511.2; its ¹H NMR spectrum is as follows Figure 26 As shown, the ¹³ CNMR spectrum is as follows Figure 27 As shown.

[0038] 1.2 Synthesis of Compound 3 Compound 2 and Boc-D-4,4'-biphenylalanine were dissolved in 5 mL of anhydrous methanol, and DMTMM was added. The reaction was carried out under nitrogen protection for 4 h, and the completion of the reaction was detected by HPLC. After the reaction, the solvent was evaporated under reduced pressure, and the product was washed with water and filtered to obtain compound 3. Due to the poor solubility of compound 3, a precipitate formed after simple water washing, which was directly used in the next synthesis (white solid, yield 90%).

[0039] The ESI-MS spectrum of compound 3 is as follows: Figure 20 As shown, the ESI-MS calculated value C 36 H 39 N2O4 + [M+H] + :579.2, measured value:579.2.

[0040] 1.3 Synthesis of Compound 4 Compound 3 was dissolved in 4 mL of dichloromethane (DCM) solution, and 1 mL of trifluoroacetic acid (TFA) solution was added. The reaction was stirred at room temperature under nitrogen protection for 3 h, and the reaction was completed by HPLC. After the reaction, the solvent was concentrated, and the residue was washed with saturated sodium bicarbonate aqueous solution and filtered to obtain compound 4 (white solid, yield 95%).

[0041] The ESI-MS spectrum of compound 4 is as follows: Figure 21 As shown, the ESI-MS calculated value C 62 H 61 N4O6 + [2M+H] + :957.2, measured value:957.4; its ¹H NMR spectrum is as follows Figure 28 As shown, the ¹³ CNMR spectrum is as follows Figure 29 As shown.

[0042] 1.4 Synthesis of Compound 5 Compound 4 and 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid were dissolved in 5 mL of anhydrous methanol, and DMTMM was added. The reaction was stirred at room temperature under nitrogen protection for 4 h, and the reaction was completed by RP-HPLC. After the reaction was completed, the solvent was removed by vacuum evaporation, the mixture was washed with water, filtered, and dried in an oven to give compound 5 (white solid, yield 80%).

[0043] The ESI-MS spectrum of compound 5 is as follows: Figure 22 As shown, the ESI-MS calculated value C 42 H 50 N3O8 + [M+H] + 724.3, measured value: 724.3; its ¹H NMR spectrum is as follows Figure 30 As shown, the ¹³ CNMR spectrum is as follows Figure 31 As shown.

[0044] 1.5 Synthesis of Compound 6 Compound 5 was dissolved in 4 mL of LCM solution, and 1 mL of LFA solution was added. The reaction was stirred at room temperature under nitrogen protection for 3 h, and the reaction was confirmed by HPLC. After the reaction, the solvent was evaporated under reduced pressure to concentrate the mixture. The mixture was washed with saturated sodium bicarbonate aqueous solution and filtered to give compound 6 (white solid, 80% yield).

[0045] The ESI-MS spectrum of compound 6 is as follows: Figure 23 As shown, the ESI-MS calculated value C 37 H 42 N3O6 + [M+H] + :624.3, measured value:624.2; its ¹H NMR spectrum is as follows Figure 32 As shown, the ¹³ CNMR spectrum is as follows Figure 33 As shown.

[0046] 1.6 Synthesis of compound Gd-L1 Compound 6 and gadolinyl Gd-DOTABA were dissolved in 5 mL of anhydrous methanol, and DMTMM was added. The reaction was maintained under nitrogen protection for 4 h, and the reaction was detected by RP-HPLC. After the reaction, the solvent was concentrated by vacuum evaporation, and the residue was purified by semi-preparative HPLC and freeze-dried to obtain Gd-L1 (white solid, yield 80%).

[0047] The ESI-HRMS spectrum of Gd-L1 is as follows: Figure 24 As shown, the ESI-HRMS calculated value C 60 H 67 GdN7O 15 [M]: 1283.3930, Measured value: 1283.3931; its HPLC chromatogram is as follows. Figure 12 As shown, the purity meets the requirements after testing.

[0048] 1.7 Synthesis of compound Gd-L2 Gd-L1 was dissolved in 5 mL of methanol, and sodium hydroxide dissolved in 3 mL of water was added. The reaction was stirred at room temperature under nitrogen protection for 3 h, and the reaction was completed by HPLC. After the reaction was completed, the product was purified by semi-preparative HPLC and freeze-dried to obtain Gd-L2 (white solid, yield 90%).

[0049] The ESI-HRMS spectrum of Gd-L2 is as follows: Figure 25 As shown, the ESI-HRMS calculated value C 59 H 65GdN7O 15 [M]: 1269.3772, Measured value: 1269.3774; its HPLC chromatogram is as follows. Figure 12 As shown, the purity meets the requirements after testing.

[0050] 1.8 Determination of Gadolinium Content Purified samples of Gd-L1 and Gd-L2 were dissolved in deionized water to prepare stock solutions. The solutions were serially diluted with 2% nitric acid, and the concentration of gadolinium ions in the diluted solutions was determined using inductively coupled plasma mass spectrometry (ICP-MS) with three replicates. The gadolinium content in the original stock solutions was calculated based on the dilution factor, ultimately yielding the precise concentration of the gadolinium complex. The results confirmed a stoichiometric ratio of gadolinium to ligand of 1:1, confirming the successful synthesis of the target complex.

[0051] Example 2: Relaxation efficiency and HSA binding performance test 2.1 Measurement of relaxation efficiency The longitudinal relaxation time (T1) and transverse relaxation time (T2) of the contrast agent solution in pure water and 4.5% human serum albumin (HSA) were measured using a 1.4T desktop NMR analyzer at 37°C. T1 measurements were performed using a standard inversion recovery sequence, and T2 measurements were performed using a CPMG pulse sequence. The relaxation efficiency values ​​r1 and r2 were determined by the slope of a linear graph of relaxation rate versus gadolinium concentration.

[0052] The results of the relaxation efficiency tests for Gd-L1 and Gd-L2 are as follows: Figure 13 As shown, the relaxation efficiency test results for control compounds Gd-L3 and Gd-L4 are as follows: Figure 14 As shown in Table 1, the relaxation efficiency data of each complex are shown in Table 1.

[0053] Table 1. Relaxation efficiency of Gd-L1 and Gd-L2 in a 1.4T magnetic field, 37℃, water, or 4.5% HSA, and HSA binding rate at a concentration of 0.5 mgd.

[0054] In pure water, the r1 and r2 values ​​of Gd-L1 are 10.29 ± 0.02 mM, respectively. - ¹s - ¹ and 12.24±0.06mM - ¹s - ¹, the r1 and r2 values ​​of Gd-L2 are 9.40±0.05mM, respectively. - ¹s - ¹ and 11.46±0.04mM - ¹s - ¹, approximately the clinical standard Gd-DOTA under the same conditions (r1=3.21±0.11mM)- ¹s - ¹, r² = 3.33 ± 0.05 mM - ¹s - 1) 3 times.

[0055] In the presence of 4.5% HSA, the r1 of Gd-L1 increased to 20.69 ± 0.03 mM. - ¹s - ¹, r2 surged to 47.98±0.04mM - ¹s - ¹; The r1 and r2 values ​​of Gd-L2 reached 23.30 ± 0.02 mM, respectively. - ¹s - ¹ and 47.62±0.05mM - ¹s - ¹, for Gd-L1, r1 and r2 increased by 2.0 times and 3.9 times, respectively, while for Gd-L2, r1 and r2 increased by 2.5 times and 4.2 times, respectively. However, the r1 and r2 values ​​of Gd-DOTA combined with HSA were only 3.70 ± 0.05 mM. - ¹s - ¹ and 3.91±0.10mM - ¹s - ¹.

[0056] The results of the control experiment showed that the r1 of Gd-L3 in the pure water system was 7.76 mM. - ¹s - ¹, r² = 8.13 mM - ¹s - ¹, Gd-L4 has r1 = 8.80 mM - ¹s - ¹, r² = 10.36 mM - ¹s - ¹; In the 4.5% HSA system, the r1 of Gd-L3 is 14.21 mM. - ¹s - ¹, r² = 20.92 mM - ¹s - ¹, r1 of Gd-L4 is 10.59 mM - ¹s - ¹, r² = 17.48 mM - ¹s - ¹, the dipeptide-modified Gd-L1 and Gd-L2 compounds exhibit significantly better relaxation properties than the control compounds modified with a single amino acid.

[0057] 2.2 HSA binding experiment Solutions containing different concentrations of gadolinium complexes and 4.5% HSA were prepared. After incubation at 37°C for 30 min, the mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa and centrifuged at 4000 rpm for 20 min. The concentration of unbound gadolinium in the filtrate was quantified by ICP-MS. The HSA binding rate was calculated using the formula: [1 - (Gd concentration in filtrate / total Gd concentration)] × 100%.

[0058] Table 2 shows the binding rates of each complex with 4.5% w / v HSA at different concentrations. The HSA binding affinity determined by relaxation titration is shown in Table 2. Figure 15 As shown, at a concentration of 0.5 mM Gd, the HSA binding rates of Gd-L1 and Gd-L2 reached 98.7 ± 0.2% and 95.7 ± 0.2%, respectively, while Gd-DOTA showed only 2.1 ± 0.7% binding. The dissociation constants Kd of Gd-L1 and Gd-L2 were 2.0 μM and 2.1 μM, respectively, indicating that both have extremely high binding affinity to HSA.

[0059] Table 2. Binding rates of each complex with 4.5% w / v HSA at different concentrations

[0060] Example 3: Evaluation of Kinetic Inertia The stability of the complexes was evaluated under simulated ion competition and acidic conditions. The experimental results are as follows: Figure 3 and Figure 16 As shown.

[0061] 3.1 Acid dissociation experiment Gd-L1, Gd-L2, and Gd-DOTA were dissolved in 1M HCl and incubated at 37°C. Samples were taken at specified time points, and the T1 and T2 values ​​of each sample were immediately measured. The relative relaxation time change was plotted against time to monitor Gd³. + Dissociation kinetics of the complexes were studied. The results showed that clinical standard Gd-DOTA lost 50% of its structural integrity within 24 hours, while the acid dissociation half-lives of Gd-L1 and Gd-L2 were approximately 72 hours and 120 hours, respectively, representing a kinetic inertness 3 to 5 times greater than that of Gd-DOTA.

[0062] 3.2 Metal displacement experiment Gd-L1, Gd-L2, and Gd-DOTA solutions in PBS were incubated with a 20-fold molar excess of ZnCl2 at 37°C for 7 days. Samples were taken at specified time points, and T1 and T2 values ​​were measured. The results showed that all complexes remained intact within 7 days, confirming their sufficient resistance to bioassociated metal ion displacement.

[0063] Example 4: In vitro biocompatibility evaluation The cytotoxicity of Gd-L1 and Gd-L2 was assessed in LX-2 and 293T cells using the CCK-8 assay. The experimental results are as follows: Figure 4 As shown. Logarithmic growth phase LX-2 cells and 293T cells were seeded at a density of 5000 cells per well in 96-well plates. After 24 hours of incubation, the medium was replaced with serum-free medium containing different concentrations of gadolinium complexes. After another 24 hours of incubation, the medium was replaced with fresh medium containing 10% CCK-8 solution. After 4 hours of incubation, the absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage relative to untreated control cells. Each experiment was independently repeated three times.

[0064] The results showed that, within the tested concentration range, the viability of LX-2 and 293T cells treated with Gd-L1 or Gd-L2 remained above 90%; even at the highest concentration of 0.5 mM, any slight inhibitory effect observed was not statistically significant compared to the Gd-DOTA control. The dosage used for in vivo imaging in this study was 0.03 mmol / kg, which, after conversion, resulted in an in vivo exposure concentration far lower than the highest concentration used in our in vitro cytotoxicity experiments, demonstrating a significant safety margin.

[0065] Example 5: Molecular docking simulation Molecular docking simulations were performed using AutoDock Suite software to elucidate the molecular-level interactions between the novel contrast agent and HSA. The results are as follows: Figure 5 As shown.

[0066] The three-dimensional structure of the target protein HSA was obtained from the RCSB PDB database. Its binding site contains key amino acid residues such as ASP-121, GLU-119, and VAL-122. The initial structure of the target complex was constructed based on the crystal framework of a Gd-DOTA derivative. First, geometry optimization and energy minimization were performed using Avogadro software to obtain a stable conformation. Then, the protein and ligand underwent normalization preprocessing, including removing water of crystallization molecules, adding polar hydrogen atoms, calculating atomic charges using the Gasteiger method, and saving the data in PDBQT format. A docking cassette was defined based on the known active sites of the target protein. Conformation optimization and energy search were performed using the built-in algorithm of AutoDock. The conformation with the lowest binding free energy was selected as the optimal binding mode, using docking fraction and inhibition constant as evaluation metrics. PLIP was used to identify key intermolecular interactions. Finally, PyMOL software was used to visualize and render the complex structure.

[0067] The results showed that both complexes exhibited a favorable binding affinity for HSA, with negative docking fractions and low calculated inhibition constants. The docking fraction and Ki value of Gd-L1 were slightly lower than those of Gd-L2, indicating slightly stronger binding stability. Optimal binding conformation analysis revealed a conservative "dual-action" interaction mode: the carboxylate group of the Gd-DOTABA macrocycle forms hydrogen bonds with key residues of HSA as anchoring sites, while the hydrophobic biphenylalanine side chain inserts into a nearby hydrophobic pocket, stabilizing the complex through extensive hydrophobic interactions and van der Waals forces.

[0068] Example 6: Evaluation of In Vivo Magnetic Resonance Imaging Performance In vivo imaging experiments were performed using a clinical 3.0T magnetic resonance imaging scanner, with contrast agents administered via tail vein injection.

[0069] 6.1 Imaging of normal organs in healthy mice BALB / c mice were randomly divided into three groups of three, each group receiving an injection of 0.03 mmol / kg of Gd-L1, Gd-L2, or Gd-DOTA, respectively. Dynamic T1-weighted images of the abdominal region (coronal plane) were acquired before injection and at 5, 12, 20, and 27 min after injection, focusing on the liver and kidneys. Normalized signal-to-noise ratio (nSNR) was calculated to quantify the enhancement. Experimental results are shown below. Figure 6 As shown.

[0070] The results showed that Gd-L1 and Gd-L2 induced strong and sustained signal enhancement in the liver and kidneys, far exceeding the weak enhancement observed with Gd-DOTA. In liver imaging, the nSNR of Gd-L1 and Gd-L2 peaked at 5 min post-injection, reaching 2.0 and 2.5 times the baseline, respectively, and remained at 1.5 and 1.8 times the baseline at 27 min; while Gd-DOTA showed only a weak peak enhancement of 1.1 times in the liver. In kidney imaging, the kidney nSNR values ​​of Gd-L1 and Gd-L2 peaked at 5 min and remained at 2.6 and 2.3 times the baseline, respectively, at 27 min; while the kidney enhancement peak of Gd-DOTA was only 1.6 times. Signal accumulation of Gd-L1 and Gd-L2 in the intestine and gallbladder was observed during imaging, confirming the existence of the hepatobiliary excretion pathway. Both also produced significant signal enhancement in the abdominal aorta, clearly outlining the vascular contours.

[0071] 6.2 Tumor Imaging in a 4T1 Breast Cancer Model Collect 4T1 cells in the logarithmic growth phase, wash with PBS, and then... 6Cells / mL were resuspended in a 1:1 mixture of PBS and Matrigel; BALB / c mice were anesthetized, and 50 μL of the cell suspension was subcutaneously injected into the right hind limb. Tumor growth was monitored until the volume reached approximately 100-200 mm³ for imaging studies.

[0072] Mice carrying 4T1 tumors were randomly divided into three groups of three. Each group was injected with one of the three contrast agents at a dose of 0.03 mmol / kg. T1-weighted cross-sectional images of the tumors were acquired before injection and at 15, 30, 45, and 52 minutes after injection. Tumor signal enhancement was analyzed. The experimental results are as follows: Figure 7 and Figure 17 As shown.

[0073] The results showed that both Gd-L1 and Gd-L2 provided strong and progressive contrast enhancement, with the relative signal intensity continuously increasing throughout the 52-minute observation period, and the clarity of tumor boundaries and internal structures continuously improving over time. Gd-L1 provided immediate and sustained contrast enhancement, while Gd-L2 showed a significant and persistent signal increase approximately 15 minutes after injection. In contrast, Gd-DOTA produced only weak and transient signal enhancement, peaking at approximately 15 minutes and then rapidly declining to near pre-injection levels at 52 minutes, failing to achieve effective delayed tumor imaging.

[0074] 6.3 Rat vascular imaging SD rats were randomly divided into four groups of three, and injected with 0.03 mmol / kg Gd-L1, 0.03 mmol / kg Gd-L2, 0.03 mmol / kg Gd-DOTA, or 0.1 mmol / kg clinical dose Gd-DOTA, respectively. Dynamic contrast-enhanced T1-weighted angiography was performed, and whole-body vascular images were captured at 1, 5, 10, 15, and 20 min after injection. The clarity and duration of vascular enhancement were assessed. The experimental results are as follows: Figure 8 As shown.

[0075] The results showed that at a low dose of 0.03 mmol / kg, both Gd-L1 and Gd-L2 produced robust and clear vascular enhancement, far exceeding that of Gd-DOTA at the same dose and superior to Gd-DOTA at the standard clinical dose. Gd-L1 achieved the clearest depiction of the whole-body vascular network as early as 1 minute after injection, accompanied by a significant signal peak; Gd-L2 showed significant enhancement of major vessels and clear cardiac contours within 1-5 minutes after injection, and residual contrast could still be detected in major vessels at 20 minutes. In contrast, the low-dose Gd-DOTA group did not show any discernible vascular enhancement throughout the entire 20-minute observation window; even at the high clinical dose, Gd-DOTA only produced very weak vascular visualization within 1-5 minutes, with significantly lower clarity than the new reagent, and the signal decayed significantly within 5 minutes.

[0076] Example 7: In vivo biodistribution study BALB / c mice were randomly divided into three groups of three, and injected with 0.03 mmol / kg of Gd-L1, Gd-L2, or Gd-DOTA via tail vein. Mice were sacrificed at 5 min, 15 min, and 24 h post-injection. Major tissues, organs, and blood were collected, weighed, and digested with concentrated nitric acid. The gadolinium content in each digested sample was quantified using ICP-MS, and the distribution was expressed as a percentage of the injected dose per gram of tissue. The experimental results are shown below. Figure 9 As shown.

[0077] The results showed that 5 minutes after injection, Gd-L1 and Gd-L2 were mainly distributed in the kidneys, blood, and liver, with significantly higher renal and hepatic uptake than Gd-DOTA. Regarding blood retention, 5 minutes after injection, the blood concentrations of Gd-L1 and Gd-L2 were 26.7 μg / g tissue and 34.8 μg / g tissue, respectively, while Gd-DOTA was only 13.5 μg / g tissue, representing increases of approximately 1.9-fold and 2.5-fold, respectively. By 15 minutes, the gadolinium concentrations in the blood of Gd-L1 and Gd-L2 remained high, while those of Gd-DOTA had significantly decreased. Importantly, both novel reagents were efficiently cleared from the body via both hepatobiliary and renal pathways within 24 hours, without significant accumulation in other major organs.

[0078] Example 8: In vivo safety evaluation BALB / c mice were randomly divided into four groups: PBS control group, Gd-DOTA control group, Gd-L1 group, and Gd-L2 group. Each group was further divided into an acute toxicity cohort and a subacute toxicity cohort. Mice were sacrificed 24 hours after the last injection, and serum biochemical and histopathological analyses were performed. The experimental results are as follows: Figure 10 and Figure 18 As shown.

[0079] 8.1 Serum Biochemical Analysis Blood was collected from the retroorbital venous plexus, serum was separated, and key biochemical markers were analyzed, including liver function indicators (AST, ALT, ALP, ALB), kidney function indicators (BUN, CR), and cardiac / skeletal muscle injury markers (LDH, CK). Results showed that in both acute and subacute studies, compared with the PBS control group, no statistically significant changes were observed in any of the key functional indicators in the Gd-L1 and Gd-L2 groups, and all indicators remained within the normal range.

[0080] 8.2 Histopathological Analysis Major organs of mice, including heart, liver, spleen, lung, and kidney, were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. The tissue morphology was examined under a light microscope by a pathologist unaware of the treatment groups. Results showed that, compared with the PBS and Gd-DOTA control groups, the major organs of mice in the Gd-L1 and Gd-L2 groups showed no significant abnormalities in morphology, and no obvious signs of lesions, necrosis, or inflammatory infiltration were observed. Cardiac cells were uniform in size and neatly arranged; hepatocytes were regularly arranged; liver lobule structure was intact; spleen nodules were clear; the red and white medullary boundaries were distinct; alveolar structure was intact without collapse; the renal cortex-medullary boundary was clear; and the glomeruli and renal tubules had normal morphology.

[0081] 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 it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A superinert Gd-DOTABA probe coupled with a non-natural dipeptide, characterized in that, The probe comprises an ultra-inert Gd-DOTABA backbone and a non-natural dipeptide targeting module covalently coupled to the Gd-DOTABA backbone; the non-natural dipeptide targeting module comprises two D-4,4'-biphenylalanine residues for specifically binding to human serum albumin.

2. The ultrainert Gd-DOTABA probe conjugated with a non-natural dipeptide according to claim 1, characterized in that, The non-natural dipeptide targeting module is coupled to the benzoic acid side chain of the Gd-DOTABA backbone via a tetraethylene glycol linker and an amide bond. The terminal group of the probe is a methyl ester group or a carboxyl group.

3. The ultrainert Gd-DOTABA probe conjugated with a non-natural dipeptide according to claim 2, characterized in that, The probe is Gd-L1, the end group is methyl ester group, the accurate molecular weight is 1283.3930, the molecular formula is C 60 H 67 GdN7O 15 .

4. The ultrainert Gd-DOTABA probe conjugated with a non-natural dipeptide according to claim 2, characterized in that, The probe is Gd-L2 with a carboxyl group at the end; its precise molecular weight is 1269.3772, and its molecular formula is C2. 59 H 65 GdN7O 15 .

5. A method for preparing an ultrainert Gd-DOTABA probe conjugated with a non-natural dipeptide as described in any one of claims 1-4, characterized in that, The convergent liquid-phase synthesis route includes the following steps: (1) Using D-4,4'-biphenylalanine as raw material, a non-natural dipeptide intermediate containing two biphenylalanine residues was gradually constructed through esterification, amide coupling and deprotection reactions. (2) The non-natural dipeptide intermediate and the pre-prepared Gd-DOTABA core were subjected to an amide coupling reaction under the action of a coupling agent to obtain Gd-L1 with a methyl ester group at the end. (3) Optionally, Gd-L1 can be hydrolyzed under alkaline conditions to obtain Gd-L2 with a carboxyl group at the end.

6. The preparation method according to claim 5, characterized in that, In both steps (1) and (2), the amide coupling reaction used 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as the coupling agent, methanol as the reaction solvent, and room temperature as the reaction temperature; the deprotection reaction used a trifluoroacetic acid / dichloromethane mixed solution with a volume ratio of 1:4 as the deprotection agent, and room temperature as the reaction temperature. In step (3), the hydrolysis reaction uses sodium hydroxide aqueous solution as the hydrolysant, the reaction solvent is a methanol / water mixture with a volume ratio of 1:1, and the reaction temperature is room temperature.

7. The use of the ultrainert Gd-DOTABA probe coupled with a non-natural dipeptide as described in any one of claims 1-4 in the preparation of a magnetic resonance imaging contrast agent.

8. The application according to claim 7, characterized in that, The magnetic resonance imaging contrast agent is used for tumor magnetic resonance imaging or vascular magnetic resonance imaging.

9. The application according to claim 7, characterized in that, The dosage of the magnetic resonance imaging contrast agent was 0.03 mmol / kg, which is one-third of the clinical standard gadolinium contrast agent dosage.

10. The application according to claim 7, characterized in that, The longitudinal relaxation efficiency r1 of the magnetic resonance imaging contrast agent in pure water at 1.4T and 37°C is 9.4-10.3 mM. -1 s -1 The longitudinal relaxation efficiency r1 in the presence of 4.5% human serum albumin was 20.7-23.3 mM. -1 s -1 Its acid dissociation half-life is 72-120 hours, which is 3-5 times longer than that of the clinical contrast agent Gd-DOTA.

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