Highly inert Gd-DOTA-based contrast agent with hydrophobic side chain as well as preparation method and application of highly inert Gd-DOTA-based contrast agent

Abstract

The invention discloses a highly inert Gd-DOTA-based contrast agent with a hydrophobic side chain as well as a preparation method and application of the highly inert Gd-DOTA-based contrast agent, and two novel Gd-DOTA-based contrast agents Gd-L1 and Gd-L2 are successfully developed by respectively connecting hydrophobic 3-phenyl propylamine and 3, 3-diphenyl propylamine to Gd-DOTABA. The two novel Gd-DOTA-based contrast agents remarkably improve the chemical inertness of the complex, the dissociation half-life period of the complex in a 1 M HCl solution at 37 DEG C is about 5 times that of Gd-DOTA, and a powerful guarantee is provided for clinical safety; the MRI (Magnetic Resonance Imaging) performance of the liver in a mouse shows that Gd-L1 is equivalent to commercially available Gd-BOPTA and is suitable for imaging of a liver and gall system; after the Gd-L2 is combined with the human serum albumin, the longitudinal relaxation rate is obviously increased (r1 = 14.8 mM <-1 > s <-1 >, 1.4 T, 37 DEG C), the blood elimination half-life period in a rabbit model is about 18 minutes, and the Gd-L2 is suitable for vascular MRI (Magnetic Resonance Imaging). The two contrast agents avoid the spleen residual problem, the organ accumulation possibility is reduced, and a safer and more efficient MRI diagnostic tool is provided for clinic.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a highly inert Gd-DOTA-based contrast agent with hydrophobic side chains, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is an effective and non-invasive clinical diagnostic tool, and the use of contrast agents (CAs) is crucial for better distinguishing lesions from normal tissue. For nearly 40 years, gadolinium ions (Gd) have been widely used... 3+ Paramagnetic complexes of Gd have been used in longitudinal (T1)-weighted MRI, but free Gd from less inert complexes... 3+ It may cause serious patient side effects, such as renal systemic fibrosis (NSF) and Gd. 3+ Accumulation in tissues and organs. To avoid unacceptable health risks, medical regulatory agencies have strictly limited the use of less inert GBCAs such as Gd-DTPA, Gd-DTPA-BMA, and Gd-DTPA-BMEA. However, public concerns have not been alleviated due to the lack of clinical alternatives to specific GBCAs, such as Gd-BOPTA and Gd-EOB-DTPA for hepatobiliary MRI.

[0003] DOTA is considered a chelator for Gd 3+ As the gold standard, Gd-DOTA is the most stable and inert of clinically available GBCAs, but its application is limited by rapid renal excretion. Functional modifications of Gd-DOTA by linking additional groups (such as peptides, responsive agents, and carriers) have been extensively explored to reveal its potential as a precise MRI lesion probe. Since linking a biomolecule to Gd-DOTA via one arm reduces the stability of the complex by 2 to 3 orders of magnitude, it is preferred to immobilize the linker at the α-position, resulting in analogs such as Gd-DOTAGA, Gd-DOTASA, and Gd-DOTABA. In our previous study, Gd-DOTABA was considered more inert than Gd-DOTA, possibly due to limited conversion between its stereoisomers. Using a similar strategy, hydrophobic p-benzyloxyphenyl / benzyl groups were immobilized on the pendulous arms or macrocycles of Gd-DOTA (i.e.,...). Figure 1 The addition of Gd-BP-DOTA and Gd-BB-DOTA to macrocycles yields more inert GBCAs with liver-targeting properties, facilitating the diagnostic MRI of hepatocellular carcinoma (HCC). The inertness of Gd-DOTABA can be further enhanced by introducing chiral groups onto the macrocycle, enabling the development of liver or tumor-targeting agents. For example, Figure 1 Gd-NP-Et4DOTA was screened as a liver-specific GBCA, and no Gd was observed after incubation in 1 M HCl solution at room temperature for more than 100 days.3+ leakage.

[0004] Gd-DOTABA, as a representative of highly inert GBCAs, exhibits significantly better chelating stability than traditional GBCAs (such as Gd-DTPA) and performs excellently in liver-targeted MRI. However, it suffers from spleen residue issues in practical applications. The spleen is one of the main organs for gadolinium deposition, and long-term residue may lead to trace amounts of Gd due to local microenvironmental factors (such as low pH or enzymatic activity). 3+ Release. Free Gd 3+ Contrast agents may diffuse into other tissues via the bloodstream, increasing the risk of renal systemic fibrosis or organ accumulation. Residual contrast agents in the spleen may cause signal interference, especially in hepatobiliary-specific imaging, potentially leading to false-negative results (such as missing small liver metastases). Furthermore, residual signals may mask pathological changes in the spleen itself (such as splenic infarction or lymphoma); additionally, the state of the spleen may affect the efficacy of immunotherapy in patients with hepatocellular carcinoma. Patients who underwent splenectomy and received targeted therapy combined with immunotherapy showed significantly prolonged progression-free survival and overall survival, suggesting that the splenic microenvironment may be involved in immune regulation. Recent research has focused on developing macromolecules and nanomaterials for magnetic resonance angiography (MRA) as contrast agents, relying on their large size to prolong circulation time in the blood. For example, albumin, polyacrylic acid, nanoclusters, and cross-linked ligands loaded with paramagnetic metals have been used as MRA contrast agents, all due to the increased rotation-related time (τ) of the macromolecules. R This maintains a high longitudinal relaxation rate (r1). Another strategy is to develop small molecule contrast agents that have a high affinity for albumin, which is abundant in blood, thus limiting its distribution within blood vessels. For example, the discontinued MS-325 is equipped with a biphenylcyclohexyl group to bind albumin, while B22956 / 1, a combination of Gd-DTPA and a deoxycholic acid analog, is also in clinical trials. Figure 1 Small molecule contrast agents (CAs), such as patient-used Gd-DTPA and Gd-DOTA, are rapidly excreted via the kidneys, resulting in a relatively short window for diagnostic magnetic resonance imaging (MRI). However, protein-binding groups, such as phenyl derivatives, can endow Gd-DTPA with specific organ targeting. For example, Gd-BOPTA and Gd-EOB-DTPA can achieve significant hepatocyte uptake by binding to transmembrane transport proteins such as organic anion transport peptides (OATPs). On the other hand, the tight binding of contrast agents to proteins restricts their rotation, thereby significantly increasing relaxation rate and enhancing imaging results.

[0005] Therefore, there is an urgent need for a Gd-DOTA-based contrast agent that is not prone to residue in the spleen, does not affect the accuracy of detection, and has a stable structure. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a highly inert Gd-DOTA-based contrast agent with hydrophobic side chains, its preparation method and application, to solve the problem of spleen residue in the practical application of existing Gd-DOTABA.

[0007] To achieve the above and other related objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a highly inert Gd-DOTA-based contrast agent having a hydrophobic side chain, having the structural formula shown in Formula I: , Formula I

[0009] In Equation I, R is H or -Ph.

[0010] Secondly, the present invention provides a method for preparing a highly inert Gd-DOTA-based contrast agent, the method comprising the following steps:

[0011] (a) Methyl 4-(1-bromo-2-methoxy-2-oxoethyl) benzoate was dissolved in methanol and then mixed with an aqueous solution of potassium carbonate for hydrolysis. The product was concentrated, acidified, extracted and dried. The residue was mixed with tetrahydrofuran and tert-butyl trichloroacetate for transesterification. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain compound 2.

[0012] (b) Compound 2 was dissolved in acetonitrile and subjected to a nucleophilic substitution reaction with cyclotrenin under nitrogen protection. After the reaction was completed, the solvent was evaporated and purified by silica gel column chromatography to obtain compound 3.

[0013] (c) Compound 3 was dissolved in acetonitrile and subjected to an intramolecular nucleophilic addition reaction with tert-butyl bromoacetate under alkaline conditions to introduce a tert-butyl ester group at the nucleophilic site. After the reaction was completed, the solvent was evaporated, and the compound was subjected to acid extraction, drying and concentration. The compound was then purified by silica gel column chromatography to obtain compound 4.

[0014] (d) Compound 4, tetrahydrofuran, and methanol were mixed and then subjected to hydrolysis reaction by adding lithium hydroxide aqueous solution. After neutralization, the reaction solution was concentrated, extracted, and dried. The product was dissolved in N,N-dimethylformamide and under the action of a condensing agent, it underwent an amidation reaction with amine compounds. The reaction solution was extracted, washed with water, concentrated and dried, and then purified by silica gel column chromatography to obtain compound 5 or compound 6.

[0015] (e) Hydrolyze compound 5 or compound 6 under acidic conditions at 60°C, evaporate the solvent to obtain ligand L1 or L2.

[0016] (f) The ligand L1 or L2 is heated to boiling under neutral conditions to carry out a coordination reaction with an aqueous solution of gadolinium chloride hexahydrate. The target complex is separated by a semi-preparative high performance liquid chromatography system and then freeze-dried to obtain compound Gd-L1 or Gd-L2, which is the highly inert Gd-DOTA-based contrast agent.

[0017] The highly inert Gd-DOTA-based contrast agent of this invention is a lipophilically modified Gd-DOTA analog. Its construction begins with methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate (compound 1), the synthesis of which is based on previous reports. The methyl acetate of compound 1 was treated with K₂CO₃ and tert-butyltrichloroacetylimide, and then replaced with tert-butyl acetate to give compound 2. Cyclotane was conjugated with compound 2 and tert-butylbromoacetate in a two-step process to give compound 4. Its methyl ester was then hydrolyzed with LiOH to allow for a subsequent reaction with 3-phenylpropylamine or 3,3-diphenylpropylamine in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole (HoBt). Compounds 5 and 6 were then purified using a silica gel column and treated with 3 M hydrochloric acid to give ligands L1 and L2, respectively, for coordinating Gd. 3+ The final complexes Gd-L1 and Gd-L2 were further purified using a semi-preparative high-performance liquid chromatography (HPLC) system. Intermediates and ligands were then processed... 1 H, 13 Gd was characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). 3+ The complex was confirmed by HPLC-MS.

[0018] .

[0019] Preferably, step (a) includes:

[0020] (a1) Hydrolysis reaction: Methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate was dissolved in methanol to prepare solution A; potassium carbonate was dissolved in water to prepare solution B; then solution B was mixed with solution A and stirred overnight at room temperature to obtain the reaction mixture;

[0021] (a2) Post-treatment: The reaction mixture was concentrated under vacuum, and then the pH of the system was adjusted to acidic with concentrated hydrochloric acid. The mixture was extracted multiple times with dichloromethane, and all the dichloromethane extracts were combined and washed with water. Then anhydrous sodium sulfate was added to the organic phase for drying, filtered, and the filtrate was dried under vacuum to obtain the residue.

[0022] (a3) Esterification reaction: Under nitrogen protection, the above residue was mixed with anhydrous tetrahydrofuran and tert-butyl trichloroacetate and stirred at room temperature for 12 h to obtain an intermediate reaction solution;

[0023] (a4) Purification: The intermediate reaction solution obtained in step (a3) ​​was concentrated, and the crude product was purified by silica gel column chromatography to obtain compound 2.

[0024] Preferably, in step (a), the molar ratio of methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate to tert-butyl trichloroacetate is 1:1.5~2. Preferably, in step (a), the molar ratio of methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate to tert-butyl trichloroacetate is 1:2.

[0025] Preferably, in step (b), the molar ratio of compound 2 to cyclohexane is 1:0.8~1.

[0026] More preferably, in step (b), the molar ratio of compound 2 to cyclohexane is 1:1.

[0027] Preferably, step (c) includes:

[0028] (c1) Esterification reaction: Compound 3 was dissolved in acetonitrile and potassium carbonate was added. Tert-butyl bromide ester was added under stirring to obtain a mixture. The mixture was stirred overnight at room temperature. After the reaction was completed, the solvent was evaporated to obtain the residue.

[0029] (c2) Acid washing: Dissolve the residue obtained in step (c1) in ethyl acetate and extract it multiple times with hydrochloric acid solution. Combine the aqueous phases and add potassium carbonate to the aqueous phase to adjust the pH of the system to neutral.

[0030] (c3) Purification: The neutralized aqueous phase was extracted multiple times with dichloromethane, all dichloromethane phases were combined and dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain product 4.

[0031] Preferably, in step (c), the molar ratio of compound 3 to tert-butylbromoacetic acid ester is 1:3~6.

[0032] More preferably, in step (c), the molar ratio of compound 3 to tert-butylbromoacetic acid ester is 1:4~5.

[0033] Preferably, step (d) includes:

[0034] (d1) Hydrolysis reaction: Compound 4 was dissolved in a mixed solution of tetrahydrofuran and methanol, and an aqueous solution of lithium hydroxide was added under stirring. The reaction was stirred at room temperature for 6 hours.

[0035] (d2) Neutralization and extraction: The mixture obtained in step (d1) was neutralized with hydrochloric acid solution, and the residue after vacuum concentration was dissolved in ethyl acetate. Then, it was washed with water several times and the organic phase was collected. Anhydrous sodium sulfate was added to the organic phase for dehydration treatment, filtered, and the product was obtained by vacuum drying.

[0036] (d3) Amide reaction: The product obtained in step (d2) was dissolved in N,N-dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole were added. The mixture was stirred at room temperature for 0.5 h, and then 3-phenyl-1-propane or 3,3-diphenylpropane and N,N-diisopropylethylamine were added in sequence. The mixture was stirred for another 6 h to obtain the reaction solution.

[0037] (d4) Purification: The reaction solution obtained in step (d3) was mixed with ethyl acetate and then washed with water. The organic phase was concentrated under vacuum and dried with anhydrous sodium sulfate. The filtered product was purified by silica gel column chromatography to obtain compound 5 or compound 6.

[0038] Preferably, in step (d), the molar ratio of compound 4 to N,N-diisopropylethylamine and 3-phenyl-1-propane or 3,3-diphenylpropane is 1:1.5~2.2:1~1.5.

[0039] More preferably, in step (d), the molar ratio of compound 4 to N,N-diisopropylethylamine and 3-phenyl-1-propane or 3,3-diphenylpropane is 1:2:1.1.

[0040] Preferably, in step (e): the condensing agent is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole in a molar ratio of 1:1; the amine compound is 3-phenylpropylamine or 3,3-diphenylpropylamine, wherein when the amine compound is 3-phenylpropylamine, compound 5 is synthesized; and when the amine compound is 3,3-diphenylpropylamine, compound 6 is synthesized.

[0041] Preferably, step (e) includes: mixing compound 5 or 6 with a 3 M hydrochloric acid solution, heating the mixture to 60°C and stirring overnight, cooling to room temperature, and then evaporating the solution under vacuum to obtain ligand L1 or L2.

[0042] Preferably, step (f) includes: dissolving ligand L1 or L2 in water, then adding an aqueous solution of gadolinium chloride hexahydrate, adjusting the pH of the system to neutral by adding sodium hydroxide solution dropwise at 80°C, stirring and boiling overnight, and forming Gd through coordination bonds. 3+The complex is a central ion with L1 or L2 as ligands; then the complex is separated using a semi-preparative high-performance liquid chromatography system, and the collected portion is freeze-dried to obtain Gd-L1 and Gd-L2 powders, which are the highly inert Gd-DOTA-based contrast agents.

[0043] Preferably, in step (f), the molar ratio of ligand L1 or L2 to gadolinium chloride hexahydrate is 1:1 to 1.2.

[0044] More preferably, in step (f), the molar ratio of ligand L1 or L2 to gadolinium chloride hexahydrate is 1:1.

[0045] Thirdly, the present invention provides the application of the highly inert Gd-DOTA-based contrast agent described above or the highly inert Gd-DOTA-based contrast agent prepared by the preparation method described above in the preparation of contrast agents for hepatobiliary or vascular MRI.

[0046] Preferably, the highly inert Gd-DOTA-based contrast agents are Gd-L1 and Gd-L2, wherein Gd-L1 is suitable for hepatobiliary imaging and Gd-L2 is suitable for vascular MRI.

[0047] The present invention has the following beneficial effects:

[0048] (1) Innovative molecular design: This invention successfully developed two novel Gd-DOTA-based contrast agents, Gd-L1 and Gd-L2, by introducing an HSA-binding group similar to MS-325 (i.e., 3,3-diphenylpropylamine) and its less hydrophobic counterpart, 3-phenylpropylamine. This design significantly improves the chemical inertness of the complexes, with a shorter dissociation half-life (t0.05) in 1 M HCl solution at 37 °C. 1 / 2 It is approximately 5 times more potent than Gd-DOTA, providing strong assurance for clinical safety;

[0049] (2) Precise targeting characteristics: In vivo studies using systematic mouse, rat, and rabbit models revealed that Gd-L2, after binding to human serum albumin (HSA), exhibited a significantly increased longitudinal relaxation rate (r1 = 14.8 mM). -1 s -1 (1.4 T, 37℃), and with a blood elimination half-life of about 18 minutes in rabbit models, it has become an effective candidate drug for vascular MRI, suitable for vascular MRI angiography, and can clearly show the microcirculation structure; while Gd-L1, due to its different affinity for hepatocytes and OATP, shows excellent targeting in hepatobiliary MRI, avoiding non-specific distribution, and has become a potential candidate contrast agent for the diagnosis of hepatobiliary diseases.

[0050] (3) Both contrast agents were confirmed through systemic metabolism studies to be designed to effectively avoid the problem of traditional GBCAs residues in the spleen and significantly reduce Gd. 3+ This mitigates risks and the possibility of organ accumulation, providing a safe basis for long-term clinical application.

[0051] (4) Gd-L1 and Gd-L2 target the hepatobiliary system and the vascular system respectively, forming a complementary scheme to meet the precise imaging needs of different clinical scenarios. Attached Figure Description

[0052] Figure 1 This is a design diagram for the synthesis of the highly inert Gd-DOTA-based contrast agent described in this invention.

[0053] Figure 2 A comparative graph evaluating the binding properties of GBCAs with HSA; where (A) the longitudinal relaxation rate (r1) of Gd-DOTA, Gd-BOPTA, Gd-L1, and Gd-L2 in the absence / with 4.5% (w / v) HSA under a magnetic field of 1.4 T and 37 °C; (B) the dissociation constant (K) of Gd-BOPTA, Gd-L1, and Gd-L2 with HSA. d (c) Fitting results; (d) The binding percentage of 0.5 mM GBCAs with 4.5% (w / v) HSA after incubation at pH 7.4 and 37°C for 30 minutes; (e) Schematic diagram of the HSA binding pocket containing Gd-L2 (HSA crystal structure sourced from the Protein Database, PDB number 1AO6).

[0054] Figure 3 T1-weighted MRI images of mice after intravenous injection of GBCAs; (A) MRI images of the liver region recorded over 30 minutes after injection of Gd-BOPTA, Gd-L1, and Gd-L2 at doses of 0.1 mmol / kg, respectively; (B) Images of the kidney region after treatment with Gd-BOPTA (a), Gd-L1 (b), and Gd-L2 (c); (C) and (D) show the signal changes in the liver and kidney, respectively, expressed as relative enhancement values; data are presented as mean ± standard deviation (n=3).

[0055] Figure 4 The diagram shows a simulated interaction between GBCAs (Gd-L1 and Gd-L2) and transport proteins (OATP1B1 and OATP1B3); where (A) is the docking conformation of Gd-L1 with OATP1B1; (B) is the docking conformation of Gd-L2 with OATP1B1; (C) is the docking conformation of Gd-L1 with OATP1B3; and (D) is the docking conformation of Gd-L2 with OATP1B3.

[0056] Figure 5 Comparative images of vascular MRI performance in rat models (Gd-BOPTA, Gd-L1, and Gd-L2); (A) Whole-body MRI images of rats acquired using 3D DCE sequence after intravenous bolus injection of Gd-BOPTA (0.1 mmol / kg), Gd-L1 (0.05 mmol / kg), and Gd-L2 (0.05 mmol / kg), respectively; (B) Coronal and sagittal MRI images of rats 2 minutes after Gd-L2 injection; (C)-(E) Changes in vascular-muscle contrast-to-noise ratio (CNR) of specific vessels after injection of Gd-BOPTA, Gd-L1, and Gd-L2, respectively; NA indicates data unavailable. Data are expressed as mean ± standard deviation (n=3; ns indicates no significant difference; P<0.05; P<0.01; P<0.001; ****P<0.0001; independent samples t-test).

[0057] Figure 6 This study presents an MRA study of a rabbit model under 3.0 T MRI. (A) Three-dimensional reconstruction results of MR images acquired using TOF sequences; (B) 3D DCE image acquired 1 minute after Gd-L2 injection; (C) magnified views of specific regions in the coronal and sagittal images of the rabbit; (D) Changes in the vessel-muscle contrast-to-noise ratio (CNR) of specific vessels within 5 minutes after Gd-L2 injection; NA indicates data unavailable. Data are expressed as mean ± standard deviation (n=3; ns indicates no significant difference; P<0.05; P<0.01; P<0.001; independent samples t-test).

[0058] Figure 7 The biodistribution characteristics of GBCAs are shown; (AC) the gadolinium content in various tissues and organs of mice at 5 minutes, 30 minutes and 24 hours after injection of Gd-BOPTA, Gd-L1 and Gd-L2 (0.05 mmol / kg dose); (D) the time-dependent elimination curve of Gd-L2 in blood in rabbit model (24-hour dynamic monitoring).

[0059] Figure 8The biosafety of Gd-L1 and Gd-L2 was evaluated (with Gd-BOPTA as a control); (A) cytotoxicity of Gd-BOPTA, Gd-L1, and Gd-L2 to LX-2 cells (test concentration gradients: 0.05, 0.1, 0.2, 0.3, 0.5, and 1.0 mM); (B) erythrocyte hemolysis rate (inset corresponding to hemolysis in the Gd-L2 group); (C) levels of enzyme indicators in mouse serum (ALT, AST, ALP, CK, LDH); (D) levels of biomarkers in mouse serum (ALB, BUN, CR); (E) H&E staining histological examination of major organs (heart, liver, spleen, lung, and kidney); data are expressed as mean ± standard deviation (n=3).

[0060] Figure 9 , Figure 10 respectively ligand L1 1 H NMR spectrum, 13 1000 C NMR spectrum (solvent: CDCl3)

[0061] Figure 11 , Figure 12 , Figure 13 They are ligands L2 and L2 respectively. 1 H NMR spectrum, 13 C10 NMR spectrum (solvent: CDCl3) and ESI-MS plot.

[0062] Figure 14 , Figure 15 ESI-MS plots for Gd-L1 and Gd-L2, respectively.

[0063] Figure 16 The graph shows the change of 1 / T2 of Gd-BT-DO3A, Gd-DOTA, Gd-L1 and Gd-L2 with incubation time in 1 M HCl solution at 37 °C. Detailed Implementation

[0064] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0065] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0066] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0067] In this invention, methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate (compound 1) is prepared according to the literature Henig, J.; Toth, E.; Engelmann, J.; Gottschalk, S.; Mayer, HA, MacrocyclicGd 3+ Chelates Attached to A Silsesquioxane Core as Potential MagneticResonance Imaging Contrast Agents: Synthesis, PhysicochemicalCharacterization, and Stability Studies. Inorg. Chem. 2010, 49 (13), 6124-6138.

[0068] In this invention, Gd 3+ Quantitative analysis: Gd in the sample was analyzed using an Agilent 7850 inductively coupled plasma mass spectrometer (ICP-MS). 3+ Quantitative analysis was performed. A calibration curve was established before each measurement, including standards containing gadolinium (Gd) diluted with 2% nitric acid solution. 3+ The stock solution (1000 ppm, GSB Testing & Certification Co., Ltd., Beijing, China) yielded 5, 10, 30, 50, 100, and 200 ppb solutions. The samples were diluted with 2% nitric acid to the appropriate Gd concentration. 3+ The concentration was determined and filtered through a 0.22 μm membrane before injection. During measurement, 1 ppb of terbium (Tb) was simultaneously injected. 3+ The solution was used as an internal standard.

[0069] Example 1

[0070] This embodiment provides a method for preparing highly inert Gd-DOTA-based contrast agents Gd-L1 and Gd-L2, including the following steps:

[0071] (a) Methyl 4-(1-bromo-2-methoxy-2-oxoethyl) benzoate was dissolved in methanol and then mixed with an aqueous solution of potassium carbonate for hydrolysis. The product was concentrated, acidified, extracted and dried. The residue was mixed with tetrahydrofuran and tert-butyl trichloroacetate for transesterification. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain compound 2.

[0072] Compound 2 was prepared by the following method:

[0073] (a1) Hydrolysis reaction: Methyl 4-(1-bromo-2-methoxy-2-oxoethyl) benzoate (4.0 g, 13.9 mmol) was dissolved in 20 mL of methanol (MeOH) to prepare solution A; potassium carbonate (K2CO3, 2.9 g, 20.9 mmol) was dissolved in 20 mL of water to prepare solution B. Then solution B was mixed with solution A and stirred overnight at room temperature to obtain the reaction mixture.

[0074] (a2) Post-treatment: The reaction mixture was concentrated under vacuum, and then the pH of the system was adjusted to acidic with concentrated hydrochloric acid (HCl). The mixture was extracted with dichloromethane (DCM, 30 mL × 3), and all the dichloromethane extracts were combined and washed with water (30 mL × 1). Anhydrous sodium sulfate (Na2SO4) was then added to the collected organic phase for drying. The mixture was filtered, and the filtrate was dried under vacuum to obtain the residue. The collected residue was used directly in the next reaction without further purification.

[0075] (a3) Esterification reaction: Under nitrogen protection, the above residue was mixed with anhydrous tetrahydrofuran (THF, 40 mL) and tert-butyl trichloroacetate (6.1 g, 27.8 mmol), and stirred at room temperature for 12 h to obtain an intermediate reaction solution;

[0076] (a4) Purification: The intermediate reaction solution obtained in step (a3) ​​was concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1) to obtain compound 2 (3.1 g, yield 67%).

[0077] 1 1H nuclear magnetic resonance (NMR) spectra (400 MHz, DMSO-d6, δ ppm): 1.40 (single peak, 9H), 3.88 (single peak, 3H), 5.92 (single peak, 1H), 7.64 (double peak, 2H, J = 8.44 Hz), 8.02 (double peak, 2H, J = 8.40 Hz). 13C NMR spectrum (100 MHz, DMSO-d6, δ ppm): 27.83, 52.79, 52.82, 59.11, 83.57, 128.81, 130.14, 130.58, 141.94, 166.20, 166.96.

[0078] (b) Compound 2 was dissolved in acetonitrile and subjected to a nucleophilic substitution reaction with cyclotrenin under nitrogen protection. After the reaction was completed, the solvent was evaporated and purified by silica gel column chromatography to obtain compound 3.

[0079] Compound 3 was prepared by the following method:

[0080] Compound 2 (3.0 g, 9.1 mmol) was dissolved in 30 mL of acetonitrile (ACN), and cyclen (1.5 g, 9.1 mmol) was added. The mixture was then stirred for 6 hours at room temperature under a nitrogen atmosphere. After drying the solvent, the residue was purified by silica gel column chromatography (ethyl acetate / methanol = 10 / 1) to give product compound 3 (2.2 g, 69% yield).

[0081] 1 1H NMR spectra (400 MHz, CDCl3, δ ppm): 1.47 (single peak, 9H), 2.57 (multiple peak, 2H), 2.75 (multiple peak, 2H), 2.80 (multiple peak, 2H), 2.88 (multiple peak, 2H), 2.98 (multiple peak, 8H), 3.93 (single peak, 3H), 4.64 (single peak, 1H), 7.47 (double peak, 2H, J = 8.24 Hz), 8.03 (double peak, 2H, J = 8.36 Hz). 13 C10 NMR spectra (100 MHz, CDCl3, δ ppm): 28.20, 45.42, 47.40, 48.79, 49.88, 52.36, 69.96, 82.72, 129.33, 129.94, 142.03, 166.68, 170.97. Electrospray ionization mass spectrometry (ESI-MS) m / z: [M+H] + C 22 H 37 The calculated value of N4O4 is 421.3; the measured value is 421.2.

[0082] (c) Compound 3 was dissolved in acetonitrile and subjected to an intramolecular nucleophilic addition reaction with tert-butyl bromoacetate under alkaline conditions to introduce a tert-butyl ester group at the nucleophilic site. After the reaction was completed, the solvent was evaporated, and the compound was subjected to acid extraction, drying and concentration. The compound was then purified by silica gel column chromatography to obtain compound 4.

[0083] Compound 4 was prepared by the following method:

[0084] (c1) Esterification reaction: Compound 3 (2.0 g, 7.1 mmol) was dissolved in 30 mL of acetonitrile, and potassium carbonate (K2CO3, 2.9 g, 21.4 mmol) was added. Tert-butyl bromide ester (5.6 g, 28.5 mmol) was added under stirring to obtain a mixture. The mixture was stirred overnight at room temperature. After the reaction was completed, the solvent was evaporated to obtain the residue.

[0085] (c2) Acid washing: Dissolve the residue obtained in step (c1) in 50 mL of ethyl acetate and extract with 1 M hydrochloric acid solution (30 mL × 3). Combine the aqueous phases and add potassium carbonate to the aqueous phase to adjust the pH of the system to neutral.

[0086] (c3) Purification: The neutralized aqueous phase was extracted with dichloromethane (30 mL × 3), all dichloromethane phases were combined and dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give compound 4 (3.1 g, yield 86%).

[0087] 1 1H NMR spectra (400 MHz, CDCl3, δ ppm): 1.34 (multiplet, 13H), 1.39 (multiplet, 23H), 2.14 (multiplet, 1H), 2.30 (multiplet, 1H), 2.70 (multiplet, 15H), 3.06 (multiplet, 3H), 3.24 (single, 1H), 3.42 (multiplet, 1H), 3.83 (single, 2H), 3.86 (single, 1H), 4.43 (single, 0.6H), 4.61 (single, 0.4H), 7.03 (doublet, 1H, J = 8.04 Hz), 7.46 (doublet, 1H, J = 8.12 Hz), 7.93 (multiplet, 2H). 13 C NMR spectrum (100 MHz, CDCl3, δ ppm): 26.73, 26.90, 26.95, 27.14, 27.17, 27.23, 43.66, 47.10, 47.23, 47.73, 4 8.45, 51.03, 51.17, 51.29, 51.51, 51.78, 54.58, 54.86, 55.09, 55.30, 64.42, 6 8.51, 79.69, 80.60, 81.06, 81.26, 81.91, 128.04, 128.35, 128.40, 128.82, 129 .13, 136.19, 165.58, 165.92, 169.97, 170.14, 170.25, 172.00, 172.13, 172.48. ESI-MS m / z: [M+H] + C 40H 67 N4O 10 The calculated value is 763.5; the measured value is 763.4.

[0088] (d) Compound 4, tetrahydrofuran, and methanol were mixed and then hydrolyzed in an aqueous lithium hydroxide solution. After neutralization, the reaction solution was concentrated, extracted, and dried. The product was dissolved in N,N-dimethylformamide and reacted with amine compounds under the action of a condensing agent. The reaction solution was extracted, washed with water, concentrated, and dried. Then it was purified by silica gel column chromatography to obtain compound 5 or 6.

[0089] Compound 5 was prepared by the following method:

[0090] (d1) Hydrolysis reaction: Compound 4 (3.0 g, 3.9 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (20 mL each), and lithium hydroxide (LiOH, 0.19 g, 7.8 mmol) dissolved in 5 mL of water was added while stirring. The reaction was stirred at room temperature for 6 hours.

[0091] (d2) Neutralization and extraction: The mixture obtained in step (d1) was neutralized with 1 M hydrochloric acid solution. The residue after vacuum concentration was dissolved in 50 mL of ethyl acetate, then washed with water (20 mL × 3), and the organic phase was collected. Anhydrous sodium sulfate was added to the organic phase for dehydration, filtered, and the product was obtained by vacuum drying.

[0092] (d3) Amide reaction: 2 g of the product obtained in step (d2) (2.6 mmol) was dissolved in 30 mL of N,N-dimethylformamide (DMF), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1.0 g, 5.3 mmol) and 1-hydroxybenzotriazole (HoBt, 0.7 g, 5.3 mmol) were added. The mixture was stirred at room temperature for 0.5 h, and then 3-phenyl-1-propylamine (0.4 g, 3.1 mmol) and N,N-diisopropylethylamine (DIPEA, 0.7 g, 5.3 mmol) were added sequentially. The mixture was stirred for another 6 h to obtain the reaction solution.

[0093] (d4) Purification: The reaction solution obtained in step (d3) was mixed with 80 mL of ethyl acetate and then washed with water (30 mL × 3). The organic phase was concentrated under vacuum and dried with anhydrous sodium sulfate. The filtered product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 as eluent) to obtain compound 5.

[0094] When preparing compound 6, simply replace 3-phenyl-1-propane (0.4 g, 3.1 mmol) with 3,3-diphenylpropane (0.6 g, 3.1 mmol), and the remaining process conditions are exactly the same as those for preparing compound 5.

[0095] Compound 5: 1 1H NMR spectra (400 MHz, CDCl3, δ ppm): 1.33 (single, 9H), 1.42 (single, 10H), 1.52 (single, 17H), 1.95 (triple, 2H, J = 7.52 Hz), 2.69 (triple, 2H, J = 7.72 Hz), 3.46 (broad peak, 23H), 5.11 (single, 1H), 7.20 (multiple, 6H), 7.62 (single, 1H), 7.82 (single, 2H); 13 C NMR spectrum (100 MHz, CDCl3, δ ppm): 27.71, 27.91, 28.00, 31.02, 33.38, 39.92, 50.26, 52.46, 54.07, 55.22, 63.67, 82.20, 82.76 , 85.09, 125.87, 127.77, 128.44, 128.50, 130.06, 135.15, 141.84, 165.67, 167.41, 170.87; ESI-MS m / z:[M+H] + C 48 H 76 The calculated value of N5O9 is 866.6; the measured value is 866.5.

[0096] Compound 6: 1 1H NMR spectra (400 MHz, CDCl3, δ ppm): 1.32 (single, 9H), 1.42 (single, 10H), 1.50 (single, 16H), 2.39 (quartet, 2H, J = 7.44 Hz), 3.37 (broad peak, 23H), 4.02 (triple, 2H, J = 7.72 Hz), 5.07 (single, 1H), 7.13 (multiple, 3H), 7.26 (multiple, 8H), 7.45 (single, 1H), 7.74 (doublet, 2H, J = 7.68 Hz); 13C10 NMR spectra (100 MHz, CDCl3, δ ppm): 27.75, 27.94, 28.04, 35.05, 38.76, 39.25, 41.60, 45.89, 49.37, 50.42, 51.43, 52.49, 54.19, 55.15, 82.25, 82.77, 84.99, 126.32, 127.90, 128.56, 128.63, 130.03, 144.57, 165.77, 167.24, 171.07; ESI-MS m / z: [M+H] + C 54 H 80 The calculated value of N5O9 is 942.6; the measured value is 942.5.

[0097] (e) Hydrolyze compound 5 or compound 6 under acidic conditions at 60°C, evaporate the solvent to obtain ligand L1 or L2.

[0098] Ligand L1 and ligand L2 were prepared according to the following method:

[0099] Compound 5 or 6 (3.0 mmol) was mixed with 3 M hydrochloric acid (HCl) solution (30 mL), and the mixture was heated to 60 °C and stirred overnight. After cooling to room temperature, the solution was evaporated under vacuum to give ligand 1 or 2 (L1 or L2).

[0100] like Figures 9-10 As shown, L1: 1 1H NMR spectra (400 MHz, D2O, δ ppm): 1.59 (single peak, 2H), 2.32 (single peak, 2H), 3.09 (broad peak, 24H), 5.26 (single peak, 1H), 6.88 (multiple peaks, 5H), 7.23 (single peak, 2H), 7.49 (single peak, 2H); 13 C NMR spectrum (100 MHz, D2O, δ ppm): 30.17, 32.65, 39.63, 43.22, 45.57, 51.48, 53.29, 54.07, 55.24, 63.59, 125.8 4, 127.84, 128.32, 128.40, 130.24, 134.42, 141.75, 168.87, 173.31, 174.54; ESI-MS m / z:[M+H] + C 32 H 44 The calculated value of N5O9 is 642.3; the measured value is also 642.3.

[0101] like Figures 11-13 As shown, L2: 11H NMR spectrum (400 MHz, D2O, δ ppm): 2.33 (single peak, 3H), 3.28 (broad peak, 25H), 5.41 (single peak, 1H), 7.22 (multiple peaks, 10H), 7.46 (single peak, 2H), 7.76 (single peak, 2H). 13 C NMR spectrum (100 MHz, D2O / CD3CN, δ ppm): 31.64, 33.69, 37.86, 38.37, 43.10, 45.27, 47.69, 48.33, 51.24, 52.77, 54.70, 62.93, 125.94 , 127.09, 127.19, 127.38, 128.22, 128.37, 130.02, 134.02, 144.26, 167.71, 168.55, 172.63; ESI-MS m / z:[M+H] + C 38 H 48 The calculated value of N5O9 is 718.3; the measured value is also 718.3.

[0102] (f) The ligand L1 or L2 is heated to boiling under neutral conditions to carry out a coordination reaction with an aqueous solution of gadolinium chloride hexahydrate. The target complex is separated by a semi-preparative high performance liquid chromatography system and then freeze-dried to obtain compound Gd-L1 or Gd-L2, which is the highly inert Gd-DOTA-based contrast agent.

[0103] Gd-L1 and Gd-L2 were prepared as follows: Ligand L1 or L2 (3.0 mmol) was dissolved in 30 mL of water, and then gadolinium chloride hexahydrate (GdCl3·6H2O, 1.1 g, 3.0 mmol) dissolved in 5 mL of water was added. The solution was heated to 80 °C, and the pH was adjusted to neutral by adding 1 M sodium hydroxide (NaOH) solution dropwise while stirring continuously. The solution was then boiled overnight. Finally, the complexes were separated using a semi-preparative high-performance liquid chromatography (HPLC) system, and the collected fraction was freeze-dried to obtain Gd-L1 and Gd-L2 powders. The complexes were characterized by ESI-MS, and their purity was checked by HPLC.

[0104] like Figure 14 , 15 As shown, Gd-L1: purity, 98.08%. ESI-MS m / z: [M]⁻, C 32 H 39 Calculated GdN₅O₉: 795.2; Measured: 795.1. Gd-L₂: Purity: 98.50%. ESI-MS m / z: [M] - C 38 H 43Calculated value of GdN5O9: 871.2; Measured value: 871.2.

[0105] Example 2: Performance testing of compounds Gd-L1 and Gd-L2 prepared in Example 1.

[0106] (1) Lipid solubility assessment:

[0107] The lipophilicity of Gd-L1 and Gd-L2 was determined by HPLC, and their log P values ​​and binding affinity to HSA were evaluated. The results showed that both Gd-L1 and Gd-L2 exhibited high binding affinity to HSA, indicating their high lipophilicity. The r1 and r2 values ​​of Gd-L1 and Gd-L2 in PBS were significantly higher than those of Gd-DOTA, suggesting that their binding affinity to HSA enhanced their water solubility.

[0108] (2) HSA binding characteristics:

[0109] Relaxation rate is an important parameter for measuring the efficiency of paramagnetic complexes in improving the relaxation rate in aqueous media. After small molecule contrast agents bind to relatively large proteins, their rotational correlation time (τ) is... R The same as the carrier significantly improves relaxation rate and reduces the amount of contrast agent required for effective scanning. This invention investigated the enhancement of relaxation rate induced by Gd-L1 and Gd-L2 binding in phosphate buffered saline (PBS, pH 7.4) using a 1.4 T magnet (37 °C), while also testing two commercially available gadolinium chelates, Gd-DOTA and Gd-BOPTA.

[0110] like Figure 2 As shown in Figure A, the r1 values ​​of the gadolinium chelates, as monohydrate complexes, increased proportionally with increasing molecular weight, in the order of Gd-DOTA, Gd-BOPTA, Gd-L1, and Gd-L2, ranging from 3.2 to 5.5 mM. -1 s -1 Between these values, Gd-DOTA and Gd-BOPTA were considered highly hydrophilic, with log P values ​​of -2.87 and -2.33, respectively, while the log P values ​​of Gd-L1 and Gd-L2, estimated using HPLC, were much higher, at 1.34 and 1.67, respectively. A positive correlation was subsequently observed between their log P values ​​and the corresponding increase in relaxivity after the addition of 4.5% HSA. The r1 value of Gd-DOTA remained essentially unchanged, while the r1 value of Gd-BOPTA increased by 32%, determined to be 3.6 and 5.7 mM, respectively. -1 s -1In contrast, with 4.5% HSA present, the r1 values ​​of Gd-L1 and Gd-L2 reached 1.8 times and 2.6 times their initial values, respectively, from 4.9 and 5.5 mM. -1 s -1 Jumping to 8.8 and 14.5 m M -1 s -1 Gd-L2 showed the best performance in terms of binding to enhance relaxation rate, indicating that it has a higher affinity for HSA.

[0111] Further, by titrating an increase in HSA into the 0.1 mM complex and recording the longitudinal relaxation time (T1) of the mixture, the equilibrium dissociation constant (K) was obtained by fitting the result to the corresponding HSA concentration. d , Figure 2 (B) The binding of Gd-BOPTA, Gd-L1, and Gd-L2 to HSA was investigated. It was predicted that, under equilibrium conditions, 0.1 mM Gd-L2 would achieve half-maximal binding with 0.17 mM HSA, while the simulated binding of HSA to Gd-L1 was 0.24 mM. Gd-BOPTA showed a significantly lower affinity for HSA, with its K0... d The fitted value was as high as 8.81 mM. For example... Figure 2 As shown in Figure C, after incubating 0.5 mM Gd-L2 with 4.5% HSA for 30 minutes, approximately 61.6% of Gd-L2 bound to HSA, while the binding values ​​for Gd-BOPTA and Gd-L1 were only 4.3% and 16.5%, respectively. In contrast, due to the lack of aromatic groups, the binding of Gd-DOTA to HSA was negligible.

[0112] The binding sites of Gd-L1 and Gd-L2 with HSA were simulated using molecular docking techniques. The molecular structures of Gd-L1 and Gd-L2 were optimized based on the crystal structure of Gd-DOTA, and then the AutoDock suite was used for docking studies with HSA. Figure 2 As shown in Figure D, Gd-L1 and Gd-L2 are housed in the same cavity of the HSA through interactions including salt bridges, hydrophobic interactions, hydrogen bonds, and π-stacking. Amino acid residues, including GLU-244, ASP-249, and GLU-252, interact with the tertiary amine of the Gd-L1 macrocycle via salt bridge formation. The phenyl groups at the side chain ends are expected to interact hydrophobically with HIS-247, GLN-204, LEU-203, and THR-243. Furthermore, the HIS-247 residues induce π-stacking interactions and hydrogen bonds with the p-carboxylphenyl groups attached to the arm. In contrast, due to the diphenyl group at the lipophilic end of Gd-L2, additional hydrophobic interactions with LEU-103 are estimated, and additional salt bridges are also expected between the amine group and the carboxyl group of LYS-240.

[0113] (3) Inertia assessment

[0114] Gadolinium ions (Gd) in acyclic gadolinium chelators (GBCAs) 3+ The leakage is mainly due to the interaction with endogenous zinc ions (Zn). 2 + ) and / or copper ions (Cu 2+ The process is caused by a metal substitution reaction, while in cyclic GBCAs, this process mainly occurs through acid catalysis. In our previous study, gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd-DOTA) GBCAs were subjected to a reaction containing 10 mM Zn. 2+ Incubation in phosphate-buffered saline (PBS, pH 7.4) at 50°C for 7 days showed no detectable Gd. 3+ The leakage occurred, while the linear gadoxetate disodium (Gd-EOB-DTPA) underwent a metal displacement reaction instantaneously. Acyclic GBCAs are extremely fragile in acidic environments; their half-life (t) in 0.1 M hydrochloric acid (HCl) solution has been reported to be very short. 1 / 2 (Less than 5 seconds.) Since gadolinium-L1 (Gd-L1) and gadolinium-L2 (Gd-L2) are expected to be more inert than Gd-DOTA, the inertness of Gd-L1 and Gd-L2 was evaluated using a solution containing 1 M HCl at 37 °C. Meanwhile, gadotate dimeglumine (Gd-DO3A-butrol, Gd-BT-DO3A) with a cyclic structure was selected for comparative studies with Gd-DOTA.

[0115] like Figure 16 As shown, the transverse relaxation time (T2) of GBCAs was continuously recorded in 1M HCl solution for up to one week, and a nonlinear fit was performed on 1 / T2 and the incubation time to obtain the pseudo-first-order rate constant (k). n ) and half-life (t 1 / 2 According to the results in Table 1, Gd-DOTA's t 1 / 2 It takes approximately 3 hours, and due to the lack of an acetic acid arm, the t of Gd-BT-DO3A... 1 / 2 Only 1 hour. The t of Gd-L1 and Gd-L2 1 / 2The observational durations were 13.5 hours and 16.5 hours, respectively, approximately five times that of Gd-DOTA. Our previous study found that the TSAP configuration was present in a higher proportion (TSAP:SAP = 0.8:1) of Gd-DOTA analogs with a phenyl group introduced at the α-position of one arm compared to unmodified Gd-DOTA (twisted square antiprism configuration, TSAP, to square prism configuration, SAP, in a 1:4 ratio). This suggests that the phenyl group on the arm may limit the dynamic transformation of Gd-DOTA stereoisomers, thereby significantly improving the inertness of Gd-L1 and Gd-L2. Therefore, this design strategy minimizes Gd-DOTA in vivo. 3+ The possibility of leakage may alleviate patients' risk of renal systemic fibrosis (NSF) and other free Gd. 3+ The burden caused by induced side effects.

[0116] Table 1. Dissociation rate constants and half-lives of Gd-L1 and Gd-L2 in 1 M HCl solution at 37 °C (with Gd-BT-DO3A and Gd-DOTA as controls) .

[0117] (4) In vivo MRI studies in mice:

[0118] The excretion of gadolinium chelates was primarily observed in vivo using T1-weighted MRI in normal mice. Under anesthesia, mice were injected with Gd-BOPTA, Gd-L1, and Gd-L2, respectively, and images were acquired for 30 minutes using a 3.0 T clinical magnetic field (Philips, Ingenia Elition). Figure 3 As shown in Figure A, both Gd-BOPTA and Gd-L1 exhibited a visually progressively increasing hepatic uptake, with similar relative enhancement values, the highest being approximately 160%. Figure 3 C). Therefore, Gd-L1 holds promise as a gadolinium chelate for hepatobiliary MRI. In contrast, Gd-L2 exhibits significantly lower hepatic uptake, resulting in limited relative enhancement of liver signal, but shows a steady increasing trend during recording, reaching its peak at approximately 105% 30 minutes post-injection. Figure 3 C). For example Figure 3 As shown in Figure B, all three gadolinium chelates immediately filled the kidneys after injection. By 30 minutes post-injection, the gadolinium chelates in the renal cortex were largely cleared, but those in the renal medulla remained clearly visible. In particular, Gd-L2 showed a relatively longer retention time in the renal medulla, likely due to its higher lipophilicity.

[0119] (5) Computer simulation study of hepatocyte transport

[0120] The differences in hepatocyte uptake of Gd-BOPTA, Gd-L1, and Gd-L2 were further explored through computer simulation (in silico) studies. Since hepatic uptake of drugs is mediated by transporters, organic anion transport peptides (OATPs) and sodium-dependent taurocholic acid cotransport peptides (NTCPs) are two major types of transporters expressed on the surface of hepatocyte sinusoids. Therefore, this invention uses them as carriers to accommodate Gd-BOPTA, Gd-L1, and Gd-L2, respectively. The docking results of Gd-L1 and Gd-L2 with the two most abundant OATP isoforms, namely OATP1B1 and OATP1B3, are shown below. Figure 4 As shown.

[0121] Table 2. Binding energies (kcal / mol) of Gd-BOPTA, Gd-L1, and Gd-L2 to OATP and NTCP transporters .

[0122] As shown in Table 2, the docking scores of Gd-BOPTA, Gd-L1, and Gd-L2 after being contained within OATP1B1 did not differ significantly. The calculated value for Gd-L1 was slightly lower at -5.45 kcal / mol, while the values ​​for Gd-BOPTA and Gd-L2 were -6.45 and -6.54 kcal / mol, respectively. Compared to OATP1B1, NTCP and OATP2B1 appear to have higher affinity for these three gadolinium contrast agents (GBCAs) because more energy is expected to be released during docking (see Table 2). Gd-L2 showed similar affinity for NTCP and OATP2B1, with docking scores of -10.50 and -10.86 kcal / mol, respectively. Similar energy release was also observed when Gd-BOPTA interacted with NTCP and OATP2B1, at -8.55 and -8.28 kcal / mol, respectively. When Gd-L1 was contained within NTCP, the recorded docking score was as high as -11.84 kcal / mol, while its docking score with OATP2B1 was slightly lower at -9.50 kcal / mol.

[0123] When Gd-L2 is incorporated into OATP1B3, its docking score is extremely low, estimated at -3.77 kcal / mol, which is only about half the docking score of Gd-BOPTA (see Table 2). Figure 4As shown in Figure D, the lipophilic side chain of Gd-L2 undergoes extensive hydrophobic interactions with the aromatic and hydrophobic residues of OATP1B3 (such as PHE-73, LEU-294, and ILE-69). Simultaneously, salt bridges and hydrogen bonds are formed between the chelating atoms (i.e., O and N) and acid- or amine-containing residues (such as HIS-169, LYS-41, and ASP-453). In contrast, due to the stronger hydrophilicity of Gd-BOPTA, the hydrogen bonds and salt bridges formed between the chelating agent and polar groups, including amino and hydroxyl groups, play a more significant role in peptide-mediated transport (see Figure D). Figure 4 Considering the similarity of the interactions between Gd-L1 and OATP1B3 (see...), Figure 4 While Gd-L2 has a higher energy output (i.e., -6.56 kcal / mol), the biphenyl tail of Gd-L2 may create significant steric hindrance, hindering the transport of OATP1B3. In contrast, the surface of human serum albumin (HSA) provides an open cavity, allowing Gd-L2 to be accommodated more readily through hydrophobic interactions. Therefore, the Gd-L2 binding to HSA is more stable, but its flexibility in being accommodated by hepatocyte transporters is lower, potentially resulting in slower hepatic uptake compared to Gd-BOPTA and Gd-L1.

[0124] (6) In vivo vascular magnetic resonance imaging (MRI) study in rats

[0125] Because Gd-L2 is found to bind tightly to human serum albumin (HSA) and is slowly excreted by the liver, it is considered an ideal contrast agent for angiogenesis-enhanced magnetic resonance imaging (MRA) due to its expected longer circulation time. This invention uses a rat model to evaluate the in vivo efficacy of the studied gadolinium-based contrast agents (GBCAs) for angiogenesis imaging. As a commonly used contrast agent in clinical magnetic resonance angiography (MRA), Gd-BOPTA was first injected at the recommended dose of 0.1 mmol / kg. Images were then continuously recorded using a T1 dynamic contrast-enhanced (DCE) sequence on a Philips 3.0 T clinical magnet (Ingenia elition) within 1 to 30 minutes after injection for comparative studies. Figure 5 As shown in Figure A, cardiovascular vessels and their branches can be clearly identified one minute after injection; however, as... Figure 5 The contrast-to-noise ratio (CNR) of blood vessels versus muscle, shown in Figure C, indicates that 2 minutes post-injection, Gd-BOPTA levels in blood vessels are significantly reduced due to rapid hepatic uptake and renal excretion. Five minutes post-injection, signal levels in most blood vessels are almost identical to those in muscle. Figure 5As can be seen, the vascular imaging window is similar when half-dose Gd-BOPTA is injected, but the background noise is relatively strong. After injection of 0.05 mmol / kg Gd-L1, the CNR of rat blood vessels is similar to that after Gd-BOPTA injection (…). Figure 4 D). Due to the interference of immediate signal enhancement in the liver parenchyma, the inferior vena cava and aorta in rats injected with Gd-BOPTA or Gd-L1 were difficult to distinguish. Figure 5 A). One to two minutes after Gd-L1 injection, the decrease in CNR of vessels such as the carotid artery and aortic arch was not as significant as that of Gd-BOPTA, which may be due to the higher affinity of Gd-L1 for albumin.

[0126] Gd-L2 was intravenously injected into rats at a dose of 0.05 mmol / kg, and images with enhanced vascularization could be recorded 1 minute after injection. Figure 5 A). Images of the rat's entire vascular system 2 minutes after injection, as shown below. Figure 4 As shown in Figure B, major arteries and veins are marked in both coronal and sagittal planes. The carotid artery, jugular vein, and subclavian vein, as branches of the cardiovascular system, are clearly visible. In particular, venous branches diffusing from the liver tissue can be identified, and the complete connection from the aortic arch to the abdominal aorta is clearly visible in the sagittal plane. The inferior vena cava closely accompanies the abdominal aorta, both extending from the pelvis to the leg. Compared to Gd-BOPTA and Gd-L1, the vascular CNR decrease in Gd-L2 is more gradual, with the most significant decrease occurring between 5 and 10 minutes post-injection, but with a slight delay, indicating a longer effective imaging time. Figure 5 As shown in E. After Gd-L2 injection, the inferior vena cava and aorta were not observable until more than 10 minutes after injection due to increased noise in the liver and subsequently the intestines (following the Gd-L2 excretion pathway).

[0127] (7) In vivo MRI study of blood vessels in rabbits

[0128] In clinical practice, time-of-flight (TOF) sequences are widely used to provide vascular contrast for diagnosing arterial diseases. Their principle relies primarily on blood flow velocity relative to static tissue, rather than the use of exogenous contrast agents (CA). However, signal generation is limited by saturation effects; in magnetic resonance angiography (MRA) practice, signal loss occurs in turbulent vessels, vessels with excessively slow flow velocities, and vessels where blood flow is parallel to the imaging plane. This invention evaluates the clinical application potential of Gd-L2 as a vascular MRI contrast agent in a rabbit model and compares its performance with that of TOF sequences. Figure 6As shown in Figure A, in coronary and sagittal angiography images acquired using TOF sequences, cardiovascular vessels (such as the aortic arch and heart) are barely visible, with only a few vessels highlighted in the head and neck region. In contrast, after a bolus injection of Gd-L2 at a dose of 0.05 mmol / kg, cardiovascular vessels and vessels connecting to the head are clearly visible. Figure 6 B). The carotid artery can be clearly distinguished from the adjacent jugular vein in the neck, and branches branching from the subclavian vessels connect (approximately 4.2 mm in width). Figure 6 C) It can also be clearly traced after spreading within the organization. For example... Figure 6 As shown in Figure C, the diffusion of peripheral blood vessels with a width of approximately 0.7 mm can also be well represented.

[0129] from Figure 6 In the sagittal image of rabbit B, the aorta originates from the aortic arch and runs parallel to the inferior vena cava in the abdomen, clearly visible throughout, with an initial width of approximately 4.3 mm. Figure 6 C). The TOF sequence only showed the aorta, which was difficult to distinguish below the aortic arch, and could not differentiate it from the closely adjacent inferior vena cava. However, in rabbits injected with Gd-BOPTA and Gd-L1 at doses of 0.1 mmol / kg and 0.05 mmol / kg respectively, neither of these important vessels was clearly visualized, and the cardiovascular imaging window for both groups of dynamic contrast-enhanced (DCE) imaging was less than 2 minutes, while the imaging window for the Gd-L2 group was approximately 5 minutes. Figure 6 D shows the changes in contrast-to-noise ratio (CNR) of rabbit blood vessels after Gd-L2 injection. Within an imaging window of up to 5 minutes, the CNR of blood vessels and muscles decreased as vascular visibility decreased. Two minutes after injection, the aorta and inferior vena cava were almost invisible due to increased abdominal noise. Intuitively, Gd-L2 tends to provide consistent contrast enhancement for limb vessels (such as those in the popliteal fossa), which is helpful for effective diagnosis. Figure 6 C).

[0130] (8) Distribution of organisms

[0131] Because mice used for magnetic resonance (MR) scanning are pre-anesthetized, the hepatic dynamics of gadolinium carbonate contrast agents (GBCAs) are easily affected by hypothermia induced by anesthesia, leading to reduced initial uptake and slower elution rates of GBCAs in the mouse liver. Therefore, this invention further employed inductively coupled plasma mass spectrometry (ICP-MS) to study the precise biodistribution of GBCAs in freely moving mice. Mice were sacrificed at 5 minutes, 30 minutes, and 24 hours post-injection, and major organs and tissues were sent for gadolinium ion (Gd) analysis. 3+ Quantitative analysis. For example... Figure 6As shown in Tables 3 and 4, 5 minutes after injection, the Gd-L1 content in liver tissue was approximately 40 micrograms of Gd per gram of tissue. 3+ This accounts for 25% of the injected dose, while the content of Gd-BOPTA is similar, at 31 micrograms of Gd per gram of tissue. 3+ (Approximately 20% of the injected dose). Subsequently, 30 minutes after injection, the Gd-L1 content in liver tissue rapidly decreased to 14 micrograms of Gd per gram of tissue. 3+ This indicates a relatively rapid rate of liver excretion, with the Gd-BOPTA content 30 minutes after injection being almost identical to that 5 minutes after injection. Similarly, the Gd-L2 content in liver tissue remained similar between 5 and 30 minutes after injection, approximately 25 micrograms of Gd per gram of tissue. 3+ This accounts for 16% of the injected dose.

[0132] Five minutes after Gd-L2 injection, the level of Gd in the blood of mice was... 3+ The concentration is approximately 20 micrograms per gram of blood, more than twice the concentrations of Gd-L1 and Gd-BOPTA. Thirty minutes after injection, approximately 7 micrograms of Gd are still present per gram of blood. 3+ This indicates that Gd-L2 has a relatively long retention time in blood vessels. Five minutes after injection, Gd levels in the kidneys... 3+ The content was low, but Gd levels in the intestines increased 30 minutes after injection. 3+ The high levels indicate that Gd-L2 is primarily excreted via the liver. After 24 hours, very low levels of Gd-BOPTA, Gd-L1, and Gd-L2 residues were observed in organs and tissues, suggesting that they were effectively cleared from the body.

[0133] Table 3. Content of Gd(III) in various organs and tissues of mice after intravenous injection of Gd-L1, Gd-L2 and Gd-BOPTA (μg Gd / g) .

[0134] Table 4. Uptake of Gd-L1, Gd-L2 and Gd-BOPTA by mouse liver and kidney (expressed as a percentage of the injected dose). .

[0135] By detecting Gd in rabbit blood 3+ The decrease in Gd-L2 levels was further evaluated to assess its hemophagocytic pharmacokinetics. Figure 7 As shown in Figure D, after bolus injection via the ear vein, Gd-L2 levels decreased rapidly, and very few complexes remained in the blood approximately one hour post-injection. A double-exponential formula was used to analyze Gd levels in blood samples collected at typical time points. 3+ By fitting the data at different levels, the blood distribution half-life (t) can be estimated. 1 / 2 , α) and blood elimination half-life (t1 / 2 , β) are approximately 1.9 minutes and 18 minutes respectively.

[0136] (9) Biosafety assessment

[0137] This invention systematically studied the biosafety of Gd-L1 and Gd-L2 by sequentially detecting cytotoxicity, hemolysis, serum biomarker levels, and organ histological state, and compared them with commercially available Gd-BOPTA. Since the liver is the main organ for the excretion of Gd-L1, Gd-L2, and Gd-BOPTA, this invention used the human hepatic stellate cell line LX-2 to evaluate their cytotoxicity. Figure 8 As shown in Figure A, even at concentrations as high as 1 mM, the inhibitory effects of Gd-BOPTA and Gd-L1 on LX-2 cells were negligible, indicating good cell tolerance. Gd-L2, however, exhibited significant toxicity at a concentration of 0.5 mM, with a cell viability of approximately 74%; at 1.0 mM, cell viability approached half-maximal inhibition. The unexpected toxicity of Gd-L2 to cells is thought to be related to its hydrophobic biphenyl group, which may promote its interaction with specific biomolecules. Considering that the practical application dose of Gd-L2 is as low as 0.05 mmol / kg, its toxicity in vivo should be relatively limited. Subsequently, Gd-L1 and Gd-L2 were applied to rat erythrocytes, and the hemolysis rate was calculated after incubation at 37°C for 2 hours. Figure 8 As shown in Figure B, red blood cells in pure water served as a positive control, and their hemolysis rate was defined as 100% due to the cell membrane rupture effect caused by osmotic pressure loss; while phosphate-buffered saline (PBS) served as a negative control. Subsequently, the red blood cell suspension in PBS was incubated with Gd-L1 or Gd-L2 at concentrations ranging from 0.05 to 1.0 mM. Even at a concentration of 1.0 mM, the hemolysis rate was the same as that in the PBS group, indicating that they have good biocompatibility in blood. Figure 7 B).

[0138] This invention further evaluated the in vivo biosafety of Gd-L1 and Gd-L2 in ICR mice. Blood biochemical parameters and tissue characteristics were analyzed after four weeks of continuous administration, and compared with mice treated with Gd-BOPTA. The following parameters related to liver function were detected using a kit: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and albumin (ALB); and parameters related to kidney function were: blood urea nitrogen (BUN) and creatinine (CR). Myocardial function marker creatine kinase (CK) was also measured. The obtained enzyme activities and the levels of biomolecules in mouse serum are as follows: Figure 7As shown in C and 7D, these biomarkers showed good consistency regardless of whether Gd-L1, Gd-L2, or Gd-BOPTA was used. The histological condition of major mouse organs (including heart, liver, spleen, lungs, and kidneys) was further examined under a microscope using hematoxylin and eosin (H&E) staining. Figure 7 As shown in Figure E, no obvious pathological changes were observed in any of the examined organs. Specifically, no abnormalities such as inflammatory infiltration were found in the liver sections of all three groups of mice; no fibrosis or hyperplasia was observed in the lungs and spleen; and magnified images of the kidney tissues of all three groups of mice showed clear and abundant glomeruli. In conclusion, cell viability, hemolysis assays, and in vivo experiments all demonstrate that Gd-L1 and Gd-L2 possess good biocompatibility.

[0139] In summary, macrocyclic gadolinium-based contrast agents (GBCAs), such as disodium gadoxetate (Gd-DOTA), are highly inert, therefore gadolinium ions (Gd...)... 3+ The risk of leakage is low, and it is expected to be safe in clinical applications. In this invention, Gd-DOTA analogs (i.e., Gd-L1 and Gd-L2) suitable for use as contrast agents in liver and vascular magnetic resonance imaging (MRI) were developed by modifying the side chains with lipid-soluble groups, including phenylpropylamine and biphenylpropylamine. The phenyl analogs on the side arms further inhibit acid-induced Gd in hydrochloric acid solution and at physiological temperatures. 3+ Leakage and inhibition effects are significantly enhanced. In vivo liver MRI imaging shows that Gd-L1 is comparable to commercially available Gd-BOPTA. After binding to human serum albumin (HSA), Gd-L2 significantly increases the longitudinal relaxation rate and prolongs blood circulation time. Studies in rats and rabbits have shown that it provides effective vascular MRI imaging at relatively low concentrations. Systemic metabolic studies confirm that its design effectively avoids the problem of traditional GBCAs residue in the spleen, significantly reducing Gd... 3+ By mitigating risks and the potential for organ accumulation, a safe foundation is provided for long-term clinical application. Gd-L1 and Gd-L2 target the hepatobiliary and vascular systems respectively, forming a complementary approach to meet the precise imaging needs of various clinical scenarios. In summary, modifying one arm of Gd-DOTA with a phenyl analogue has become an effective design method to improve the inertness of gadolinium-based contrast agents. Furthermore, by combining with appropriate chemical groups, Gd-DOTA analogues with specific applications can be developed, making it an ideal strategy for providing safe MRI diagnostic reagents for a variety of clinical scenarios.

[0140] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A highly inert Gd-DOTA-based contrast agent with hydrophobic side chains, characterized in that, It has the structural formula shown in Equation I: ， Formula I In Equation I, R is H or -Ph.

2. A method for preparing the highly inert Gd-DOTA-based contrast agent according to claim 1, characterized in that, The preparation method includes the following steps: (a) Methyl 4-(1-bromo-2-methoxy-2-oxoethyl) benzoate was dissolved in methanol and then mixed with an aqueous solution of potassium carbonate for hydrolysis. The product was concentrated, acidified, extracted and dried. The residue was mixed with tetrahydrofuran and tert-butyl trichloroacetate for transesterification. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain compound 2. (b) Compound 2 was dissolved in acetonitrile and subjected to a nucleophilic substitution reaction with cyclotrenin under nitrogen protection. After the reaction was completed, the solvent was evaporated and purified by silica gel column chromatography to obtain compound 3. (c) Compound 3 was dissolved in acetonitrile and subjected to an intramolecular nucleophilic addition reaction with tert-butyl bromoacetate under alkaline conditions to introduce a tert-butyl ester group at the nucleophilic site. After the reaction was completed, the solvent was evaporated, and the compound was subjected to acid extraction, drying and concentration. The compound was then purified by silica gel column chromatography to obtain compound 4. (d) Compound 4, tetrahydrofuran, and methanol were mixed and then hydrolyzed in an aqueous lithium hydroxide solution. After neutralization, the reaction solution was concentrated, extracted, and dried. The product was dissolved in N,N-dimethylformamide and reacted with amine compounds under the action of a condensing agent. The reaction solution was extracted, washed with water, concentrated, and dried. Then it was purified by silica gel column chromatography to obtain compound 5 or 6. (e) Hydrolyze compound 5 or compound 6 under acidic conditions at 60°C, evaporate the solvent to obtain ligand L1 or L2. (f) The ligand L1 or L2 is heated to boiling under neutral conditions to carry out a coordination reaction with an aqueous solution of gadolinium chloride hexahydrate. The target complex is separated by a semi-preparative high performance liquid chromatography system and then freeze-dried to obtain compound Gd-L1 or Gd-L2, which is the highly inert Gd-DOTA-based contrast agent.

3. The preparation method according to claim 2, characterized in that, In step (a), the molar ratio of methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate to tert-butyl trichloroacetate is 1:1.5~2.

4. The preparation method according to claim 2, characterized in that, In step (b), the molar ratio of compound 2 to cyclohexane is 1:0.8~1.

5. The preparation method according to claim 2, characterized in that, In step (c), the molar ratio of compound 3 to tert-butylbromoacetic acid ester is 1:3~6.

6. The preparation method according to claim 2, characterized in that, In step (d), the molar ratio of compound 4 to N,N-diisopropylethylamine and amine compounds is 1:1.5~2.2:1~1.5; in step (f), the molar ratio of ligand L1 or L2 to gadolinium chloride hexahydrate is 1:1~1.

2.

7. The preparation method according to claim 2, characterized in that, In step (e), the condensing agent is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole in a molar ratio of 1:

1.

8. The preparation method according to claim 2, characterized in that, In step (e), the amine compound is 3-phenylpropylamine or 3,3-diphenylpropylamine.

9. The preparation method according to claim 2, characterized in that, In step (a), the molar ratio of methyl 4-(1-bromo-2-methoxy-2-oxoethyl)benzoate to tert-butyl trichloroacetate is 1:2; in step (b), the molar ratio of compound 2 to cyclohexanetin is 1:1; in step (c), the molar ratio of compound 3 to tert-butylbromoacetate is 1:4~5; in step (d), the molar ratio of compound 4 to N,N-diisopropylethylamine and 3-phenyl-1-propylamine or 3,3-diphenylpropylamine is 1:1.5~2.2:1~1.5; in step (f), the molar ratio of ligand L1 or L2 to gadolinium chloride hexahydrate is 1:1~1.

2.

10. The use of a highly inert Gd-DOTA-based contrast agent according to claim 1 or a highly inert Gd-DOTA-based contrast agent prepared by any one of claims 2 to 9 in the preparation of contrast agents for hepatobiliary or vascular MRI.

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