Gadolinium-free magnetic resonance contrast agent and preparation method thereof

Abstract

The invention relates to a non-gadolinium magnetic resonance contrast agent and a preparation method thereof, and belongs to the field of contrast agents. The invention aims to provide a non-gadolinium contrast agent with high biological safety aiming at the risks of renal fibrosis and cerebral deposition existing in a gadolinium-based magnetic resonance contrast agent. The invention provides a phosphate group modified non-gadolinium magnetic resonance contrast agent. The phosphate group modified non-gadolinium magnetic resonance contrast agent consists of a ligand of a phosphate group substituted trans-cyclohexanediamine tetraacetic acid derivative (tmPCDTA or tqPCDTA) and metal ions Fe < 3 + > or Mn < 2 + >, the contrast agent disclosed by the invention is high in biological safety: endogenous Fe < 3 + > / Mn < 2 + > is used for replacing gadolinium, and cell experiments prove that the survival rate of 4T1 cells is not influenced at the concentration of 500 mu M; clinical applicability is high, kidney excretion is rapid, bladder signals are enhanced 3-5 minutes after mouse intravenous injection, and liver retention is avoided.

Description

Technical Field

[0001] This invention belongs to the field of contrast agent technology, and relates to non-gadolinium magnetic resonance contrast agents and their preparation methods. Background Technology

[0002] Magnetic resonance imaging (MRI) is a widely used biomedical diagnostic tool. In clinical diagnosis, contrast agents are frequently used to improve the contrast between diseased and normal tissues. It has been reported that the usage rate of contrast agents in clinical MRI examinations is as high as 30-40%. Early research on T1 MRI contrast agents mainly focused on gadolinium (Gd) complexes. Currently, Gd(III)-based MRI contrast agents are widely used in clinical practice. However, Gd is an exogenous metal, and repeated use of Gd-based contrast agents may lead to renal fibrosis and brain deposition. Based on this, in recent years, endogenous manganese (Mn) or iron (Fe)-based contrast agents have received some attention as alternatives to gadolinium. Compared to Gd(III), Fe(III) and Mn(II), as metal ions stored and circulating in the human body, play a crucial role and exhibit higher biocompatibility (in the human body: Mn 20mg vs. Gd 0mg vs. Fe 5000mg). Therefore, it is of great significance to develop Fe(III) or Mn(II)-based contrast agents and to develop Gd(III)-based contrast agents to replace clinically used ones.

[0003] As MRI contrast agents, the high paramagnetism of the central metal Fe(III) or Mn(II) is essential for their effectiveness. However, oxides in the human body, such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, as well as reducing species such as glutathione, can cause redox reactions in Fe(III) or Mn(II), which can not only damage biological tissues but also potentially convert highly paramagnetic Fe(III) or Mn(II) into less paramagnetic Fe(II) or Mn(III), rendering the contrast agent ineffective. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide a non-gadolinium magnetic resonance contrast agent, and another objective is to provide a method for preparing a non-gadolinium magnetic resonance contrast agent.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention utilizes tCDTA as the core framework, replacing one carboxylic acid structure in tCDTA with a phosphate group to synthesize tmPCDTA, which, after coordination with iron or manganese, yields Fe-tmPCDTA / Mn-tmPCDTA. Furthermore, tetraphosphate-substituted tqPCDTA is synthesized, and after coordination with iron or manganese, Fe-tqPCDTA / Mn-tqPCDTA is synthesized.

[0007] This invention provides a non-gadolinium magnetic resonance imaging (MRI) contrast agent, which is composed of trans-cyclohexanediaminetetraacetic acid (TGA) coordinated with a metal ion, wherein one to four carboxyl groups of the trans-cyclohexanediaminetetraacetic acid are replaced by phosphate groups, and the metal ion is Fe. 3+ or Mn 2+ ;

[0008] Preferably, the structural formula of the non-gadolinium magnetic resonance contrast agent is as follows:

[0009]

[0010] Furthermore, the preparation method of the non-gadolinium magnetic resonance contrast agent includes the following steps:

[0011] S1: Synthesize phosphate-modified trans-cyclohexanediaminetetraacetic acid derivative ligands tmPCDTA or tqPCDTA;

[0012] S2: The ligand is reacted with metal ions Fe... 3+ or Mn 2+ They react in aqueous solution to form a complex;

[0013] S3: Purify and freeze-dry to obtain the non-gadolinium magnetic resonance contrast agent;

[0014] Preferably, the preparation method of the tmPCDTA is as follows:

[0015] (a) Trans-cyclohexanediamine and benzyl bromide were reacted in acetonitrile at a molar ratio of 10:1 for 24 hours. The mixture was then concentrated, washed with saturated Na2CO3 solution, extracted with DCM, and dried to obtain compound 1.

[0016] (b) Compound 1 was dissolved in a DMF solution containing diisopropylethylamine and potassium iodide, and ethyl bromide of tervastatin was added and reacted for 6 hours. After concentration and saturation with N2... a Compound 2 was obtained by washing with 2CO3 solution, DCM extraction, and column chromatography purification; the molar ratio of compound 1 to diisopropylethylamine, potassium iodide, and ethyl bromide tervastatin was 1:6:6:5.

[0017] (c) Compound 2 was reacted with Pd / C in methanol solution under H2 atmosphere for 24 hours by stirring, and then filtered and concentrated to obtain compound 3; the mass fraction of Pd / C in the methanol solution was 5%;

[0018] (d) Compound 3 was reacted with 4 mmol of paraformaldehyde in a tetrahydrofuran solution containing 4 mmol of diethyl phosphite at 95 °C for 24 hours. The mixture was then concentrated, washed with saturated Na2CO3 solution, extracted with DCM, and purified by column chromatography to obtain compound 4.

[0019] (e) Compound 4 was hydrolyzed in 6M HCl at 105°C for 24 hours, purified by preparative HPLC, and lyophilized to obtain the tmPCDTA;

[0020] Preferably, the preparation method of the tqPCDTA is as follows:

[0021] Trans-cyclohexanediaminetetraacetic acid and phosphorous acid were dissolved in p-xylene, phosphorus trichloride was added dropwise, the mixture was refluxed for 6 hours, dried under vacuum, and then crystallized in a water / ethanol mixed solvent to obtain the tqPCDTA.

[0022] Preferably, the ligand is related to the metal ion Fe. 3+ The method for preparing non-gadolinium magnetic resonance contrast agents is as follows:

[0023] The ligands tmPCDTA or tqPCDTA were mixed with FeCl3·6H2O in an aqueous solution at an equal molar concentration. The pH was adjusted to 2.5 and the reaction was carried out for 24 hours. After adjusting the pH to neutral, the mixture was desalted by Sephadex G-25, centrifuged, and lyophilized to obtain the non-gadolinium magnetic resonance contrast agent.

[0024] Preferably, the ligand is related to the metal ion Mn. 2+ The method for preparing non-gadolinium magnetic resonance contrast agents is as follows:

[0025] The ligands tmPCDTA or tqPCDTA were mixed with MnCl2·4H2O in an aqueous solution at an equal molar concentration. The pH was adjusted to 6.5 and the reaction was carried out for 12 hours. Then the pH was adjusted to neutral, and the mixture was purified by RP-HPLC (C18 column) and lyophilized to obtain the non-gadolinium magnetic resonance contrast agent.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. Biosafety

[0028] Using endogenous metal ions (Fe) 3+ / Mn 2+ It can replace exogenous gadolinium (Gd) and fundamentally avoid the risks of kidney fibrosis and brain deposition caused by gadolinium-based contrast agents.

[0029] Cytotoxicity experiments confirmed that Fe-tmPCDTA and Fe-tqPCDTA, at concentrations up to 500 μM, had no statistically significant effect on the survival rate of 4T1 cells (comparable to Gd-DTPA), demonstrating their excellent biocompatibility.

[0030] 2. Stability

[0031] Resistance to metal ion competition: Under conditions of 37℃ and pH 7.4, with an equimolar amount of Zn 2+After 72 hours of incubation, the absorbance change rate at 260 nm was ≤5%, which is significantly better than that of traditional carboxylic acid ligand contrast agents.

[0032] Antioxidant and reducing properties: After incubation with 25 μM sodium ascorbate for 20 minutes, the absorbance at 265 nm did not decrease, thus avoiding imaging failure caused by changes in metal valence state.

[0033] 3. Clinical applicability

[0034] Pharmacokinetic optimization: In vivo mouse experiments showed that it is rapidly excreted by the kidneys after intravenous injection, avoiding liver retention and reducing the risk of long-term toxicity.

[0035] Phosphate-modified ligands (tmPCDTA / tqPCDTA) significantly improve the thermodynamic stability and water molecule interaction efficiency of complexes through high negative charge and steric hindrance effect, overcoming the limitations of traditional carboxylic acid ligands.

[0036] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0038] Figure 1 To investigate the kinetic inertness of iron(III) complexes using UV-Vis spectroscopy: (a), (b): Fe-tmPCDTA or Fe-tqPCDTA (150 μM) was reacted with 150 μM Zn under the conditions of pH 7.4 and 37 °C (in 50 mM tris buffer). 2+ (c) and (d): Fe-tmPCDTA or Fe-tqPCDTA (150 μM) was incubated in 30 mM phosphate-buffered saline (PBS) containing 37.5 mM sodium bicarbonate (NaHCO3) and 0.75 mM disodium hydrogen phosphate (Na2HPO4); (e) and (f): Fe-tmPCDTA or Fe-tqPCDTA (150 μM) was incubated in 150 mM hydrochloric acid (HCl); (g) and (h): Fe-tmPCDTA or Fe-tqPCDTA (7.5 μM) was incubated in aqueous solution with 25 μM sodium ascorbate.

[0039] Figure 2To assess the effects of different concentrations of Fe-PCDTA (including Fe-tqPCDTA and Fe-tmPCDTA) and Gd-DTPA on 4T1 cells at 37°C for 24 hours using the CCK-8 assay kit, the study aimed to evaluate the cytotoxicity of these cells. (The experiment was repeated n=3 times, and data are presented as mean ± standard deviation.)

[0040] Figure 3 Magnetic resonance imaging (MRI) of Fe-PCDTA: (a) Representative coronal T1-weighted MR images of mice acquired before intravenous injection of Fe-PCDTA (0.2 mmol / kg) and at 0.5, 3, 15, and 30 minutes after injection. (b) Curves showing the change in normalized signal-to-noise ratio (nSNR) over time in the heart and kidneys. (Number of experiments repeated n = 3; data are presented as mean ± standard deviation). Detailed Implementation

[0041] 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 be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0042] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0043] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0044] Example 1: Synthesis of tmPCDTA ligands

[0045] Step 1: At room temperature, trans-cyclohexanediamine (28.55 g, 250 mmol) was added to 150 mL of acetonitrile. Benzyl bromide (4.28 g, 25 mmol) was dissolved in 40 mL of acetonitrile and slowly added dropwise to the above solution over 1 hour. After 24 hours, the reaction solution was concentrated, saturated Na₂CO₃ solution was added, and extraction was performed using DCM. The organic phase was washed several times with NaCl, dried over anhydrous Na₂SO₄, and concentrated to give product 1 (yellow oil, 4.93 g, 97%).

[0046] Characterization data: ¹H NMR (400 MHz, CDCl₃) δ 7.20–7.40 (m, 5H, HAr), 3.94 (d, J = 13.2 Hz, 1H, CH₂), 3.68 (d, J = 13.2 Hz, 1H, CH₂), 2.37–2.43 (m, 1H, CH), 2.08–2.18 (m, 2H, CH₂), 1.87–1.91 (m, 1H, CH), 1.47–1.79 (m, 5H, CH₂, NH), 0.95–1.36 (m, 4H, CH₂). ESI-MS: m / z = 205.05 [M+H]⁺; calcd: 205.17.

[0047] Step 2: At room temperature (RT), ethyl bromide tervastatin (9.75 g, 50 mmol) was added to 10 mL of N,N-dimethylformamide (DMF) solution containing compound 1 (2.04 g, 10 mmol), diisopropylethylamine (7.74 g, 60 mmol), and potassium iodide (1.70 g, 63 mmol). After reacting for 6 hours, the reaction solution was concentrated, saturated sodium carbonate (Na₂CO₃) solution was added, and extraction was performed with dichloromethane (DCM). The organic phase was washed three times with saturated sodium carbonate solution, then dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated to give the crude product. After purification by rapid column chromatography, pure compound 2 (3.0 g, 55% yield, yellow oil) was obtained.

[0048] Characterization data: 1H NMR (400MHz, CDCl3) δ 7.44 (d, J = 7.2Hz, 2H, HAr), 7.28 (t, J = 7.2Hz, 2H, HAr), 7.21 (t, J = 7.2Hz, 1H, HAr), 3.99 (d, J = 13.2Hz, 1H, CH2), 3.66 (d, J = 13.2Hz, 1H, CH2), 3.44 (d, J = 2.8Hz, 4H, CH2), 3.28 (d, J = 16.8Hz, 1H, CH2), 3. 25(d,J=16.8Hz,1H,CH2),2.69(td,J1=10.4Hz,J2=3.6Hz,1H,CH),2.51(td,J1=10.8Hz,J2=3.6Hz,1H,CH),1. 97-2.10(m,2H,CH2),1.62-1.76(m,2H,CH2),1.45(s,18H,tBu),1.44(s,9H,tBu),1.10-1.31(m,4H,CH2).13C NMR (101MHz, CDCl3) δ172.1(C=O,1C),171.7(C=O,2C),140.1(CAr,1C),129.3(CAr,2C),128.0(CAr,2C),126.7(CAr,1C),80.2(quaternarycarbon of tBu,3C),63.4(CH,1C),60.7(CH,1C),54.7(NCH2,1C),53.2(NCH2,2C),52.6(NCH2,1C),29.6(CH2,1C),2 8.1(CH3,9C),28.0(CH2,1C),25.9(CH2,1C),25.7(CH2,1C).ESI-MS: m / z=547.75[M+H]+; calcd.:547.37.

[0049] Step 3: Compound 2 (3.0 g, 5.49 mmol) was reacted with palladium / carbon (Pd / C, 400 mg, 5% by mass) in methanol (MeOH) under a hydrogen (H2) atmosphere at 1 atm with stirring. After 24 hours, the palladium / carbon catalyst was removed by filtration, and the remaining reaction solution was concentrated to give compound 3 (2.27 g, 4.98 mmol, 91% yield), as a colorless oil.

[0050] Characterization data: ¹H NMR (400 MHz, CDCl₃) δ 3.24–3.90 (m, 7H, CH₂, NH), 2.36–2.38 (m, 2H, CH), 1.92–2.00 (m, 2H, CH₂), 1.60–1.79 (m, 2H, CH₂), 1.45 (s, 18H, tBu), 1.44 (s, 9H, tBu), 1.02–1.28 (m, 4H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ 171.7 (C=O, 2C), 171.0 (C=O, 1C), 80.9 (quaternary carbon of tBu, 1C), 80.7 (quaternary carbon of tBu, 1C). tBu,2C),66.7(CH,1C),57.6(NCH2,2C),52.7(NCH2,1C),48.1(CH,1C),30.7(CH2,1C),28.1(CH3 of tBu,9C),26.9(CH2,1C),25.5(CH2,1C),24.3(CH2,1C).ESI-MS: m / z=457.25[M+H]+; calcd.:457.33

[0051] Step 4: At room temperature (RT), paraformaldehyde (0.11 g, 4 mmol) was added to 20 mL of tetrahydrofuran (THF) solution containing compound 3 (1.32 g, 3.0 mmol) and diethyl phosphite (0.39 g, 4.0 mmol). After reacting at 95 °C for 24 hours, the reaction solution was concentrated, saturated sodium carbonate (Na₂CO₃) solution was added, and extraction was performed with dichloromethane (DCM). Subsequently, the organic phase was dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated to obtain the crude product. After purification by rapid column chromatography, pure compound 4 (0.9 g, 49% yield, colorless oil) was obtained.

[0052] Characterization data: 1H NMR (400 MHz, CDCl3) δ 4.04 - 4.22 (m, 4H, CH2), 3.39 - 3.72 (m, 7H, CH2), 3.10 - 3.18 (m, 1H, CH), 2.73 (td, J1 = 10.0 Hz, J2 = 3.6 Hz, 1H, CH), 2.57 (td, J1 = 10.4 Hz, J2 = 3.2 Hz, 1H, CH), 2.00 - 2.05 (m, 2H, CH2), 1.62 - 1.71 (m, 2H, CH2), 1.45 (s, 27H, tBu), 1.30 (td, J1 = 6.8 Hz, J2 = 2.4 Hz, 6H, CH3), 1.00 - 1.22 (m, 4H, CH2). 13C NMR (101 MHz, CDCl3) δ 171.8 (d, J(P - C) = 1.0 Hz, C = O, 1C), 171.5 (C = O, 2C), 80.5 (quaternary carbon of tBu, 2C), 80.3 (quaternary carbon of tBu, 1C), 63.9 (NCH2, 1C), 62.21 (d, J(P - C) = 5.6 Hz, CH, 1C), 62.15 (d, J(P - C) = 4.3 Hz, CH, 1C), 61.6 (d, J(P - C) = 6.9 Hz, POCH2, 1C), 54.4 (d, J(P - C) = 5.8 Hz, POCH2, 1C), 53.1 (NCH2, 2C), 45.8 (d, J(P - C) = 163.4 Hz, PCH2N, 1C), 30.2 (CH2, 1C), 28.8 (CH2, 1C), 28.12 (CH3 of tBu, 6C), 28.14 (CH3 of tBu, 3C), 25.6 (CH2, 2C), 16.52 (d, J(P - C) = 5.0 Hz, CH3 of POCH2CH3, 1C), 16.46 (d, J(P - C) = 4.8 Hz, CH3 of POCH2CH3, 1C). ESI-MS: m / z = 607.30 [M + H]+; calcd.: 607.37

[0053] Step 5: Compound 4 (0.9 g, 1.48 mmol) was stirred in 20 mL of hydrochloric acid (6 M) solution. After reacting at 105 °C for 24 hours, the solution was concentrated and purified by preparative high performance liquid chromatography (HPLC), then concentrated again and lyophilized to give the product tmPCDTA·HCl (0.35 g, yield 62%, white solid). Characterization data: ¹H NMR (400 MHz, CDCl₃) δ 2.53–3.81 (m, 10H, CH, CH₂), 1.45–1.77 (m, 2H, CH₂), 1.14–1.32 (m, 2H, CH₂), 0.62–0.89 (m, 2H, CH₂). ¹H NMR (400 MHz, D₂O) δ 2.78–4.05 (m, 10H, NCH₂, CH), 1.95–2.19 (m, 2H, CH₂, Cyclo), 1.66–1.82 (m, 2H, CH₂, Cyclo), 1.06–1.37 (m, 4H, CH₂, Cyclo). ¹³C NMR(101MHz,D2O)δ172.0(C=O,2C),171.4(C=O,1C),63.6(CH,2C),62.8(NCH2,1C),62.2(NCH2,2C),62.1(NCH2,1C),24.1(CH2,Cyclo,1C),23.7 (CH2, Cyclo, 1C). 23.6 (CH2, Cyclo, 1C), 23.4 (CH2, Cyclo, 1C). ESI-MS: m / z=383.00[M+H]+; calcd.: 383.12, m / z=381.05[MH]-; calcd.: 381.11.

[0054] Example 2: Synthesis of tqPCDTA ligands

[0055] Phosphorous acid (1.82 g, 22.0 mmol) was added to a solution of trans-cyclohexane-1,2-diaminetetraacetic acid (trans-CDTA, 1.73 g, 5.0 mmol) dissolved in 30 mL of p-xylene. Under reflux, 1.92 mL of phosphorus trichloride (3.0 g, 22 mmol) was added dropwise to this reflux mixture. After reacting for 6 hours, the p-xylene was collected and dried under vacuum. Subsequently, deionized water and ethanol were added to the mixture. The purified product, tqPCDTA, precipitated in the solvent mixture (white solid, 75% yield).

[0056] Characterization data: 1 H NMR(400MHz,D2O)δ2.69–3.35(m,10H,NCH 2,CH),1.86–1.89(m,2H,CH2,Cyclo),1.53–1.55(m,2H,CH2,Cyclo),0.94–1.14(m,4H,CH2,Cyclo). 13 C NMR (101 MHz, D₂O) δ 63.3 (d, J P-C =11.2Hz,2C,CH),48.4(d,J) P-C =146.5Hz,2C CH2,),45.5(dd,J P-C(1) =146.0Hz, J P-C(2) =12.7Hz,2C,CH2),23.6(CH2,Cyclo,2C),22.4(CH2,Cyclo,2C).

[0057]

[0058] Ligand synthesis steps

[0059] Example 3: Synthesis of iron-based and manganese-based contrast agents

[0060] Ferric chloride hexahydrate (FeCl3·6H2O, 0.30 g, 1.1 mmol) was added to 5 mL of an aqueous solution containing 1 mmol of the ligand tmPCDTA·HCl. The pH of the solution was adjusted to 2.5, and the reaction was carried out at room temperature for 24 hours. After the reaction was complete, the pH was adjusted to neutral to promote the precipitation of free iron ions, and the reaction mixture was then desalted using a Sephadex G-25 column. Finally, the solution was centrifuged, filtered, and freeze-dried to give a pale yellow solid product (Fe-tmPCDTA, yield 49%). Fe-tqPCDTA was synthesized using the same method (yield: 53%, yellow solid). Characterization data: Fe-tmPCDTA.ESI-MS: [M+H] - m / z=433.95,calcd.:434.02,[M+Na] - m / z=455.95,calcd.:456.00.[M] 2- m / z=216.50,calcd.:216.51.Fe-tmPCDTA.ESI-MS:[M+3H] 2- m / z = 270.45, calcd: 270.47.

[0061] By using Mn 2+The precise ligand concentration was determined by titrating the equal fractions of the solution. The ligand (0.525 mmol, 1.05 eq) and MnCl₂·4H₂O (98.95 mg, 0.5 mmol) were mixed in 5 mL of H₂O, and the pH was adjusted to 6.5. After 12 hours, the pH was adjusted to neutral. The mixture was purified by RP-HPLC using a C18 column. The solution was lyophilized to give Mn-tmPCDTA (yield: 55%, gray powder) and Mn-tqPCDTA gray powder (yield: 63%, red solid). Characterization data: Mn-tmPCDTA.ESI-MS: [M+H+Na] - m / z=455.95,calcd.:456.01.[M+H] 2- m / z=216.50,calcd.:216.51.Mn-tqPCDTA ESI-MS:[M+4H] 2- m / z=270.45,calcd.:270.48.[M+3H+Na] 2- m / z=281.45,calcd.:281.47.[M+2H+3Na] - m / z=607.85,calcd.:607.91.[M+4H+Na] - m / z = 563.90, calcd: 563.94.

[0062]

[0063] Example 4: Contrast Agent Performance Test

[0064] The kinetic stability of paramagnetic complexes is crucial for their practical application in organisms, as it directly affects their persistence in the biological environment. In this embodiment, taking Fe-tqPCDTA and Fe-tmPCDTA as examples, the kinetic inertness of iron(III) complexes under different conditions was analyzed, including their interactions with bio-related metal ions, anions, and sodium ascorbate. Their stability under acidic conditions was also evaluated.

[0065] The kinetic stability of the contrast agent was assessed by monitoring changes in absorbance at 260 nm in the UV-Vis spectrum under different conditions. This was taken into account the presence of highly coordinating Zn in the human body. 2+ Perform Zn 2+ Metal exchange experiment; at 37°C, with an equimolar amount of Zn 2+ After co-incubation for 72 hours and monitoring of absorbance changes at 260 nm, the results showed that the contrast agent Fe-tqPCDTA exhibited superior stability. Figure 1(a and 1b). The human body also contains abundant phosphate species, and its stability was assessed by co-incubating the contrast agent with a large excess of free phosphate. Figure 1 c and 1d show that after co-incubation at 37°C for 72 hours, their absorption band at 260 nm changed very little, demonstrating their stability, especially for Fe-tqPCDTA. Figure 1 As shown in e and 1f, the contrast agent (150 mM) was co-incubated with 150 mM hydrochloric acid. The results showed that Fe-tmPCDTA gradually degraded within 72 hours at 37 °C, while Fe-tqPCDTA exhibited significant stability with almost no change in absorbance at 260 nm.

[0066] Fe 2+ and Fe 3+ The redox cycle of iron can induce oxidative toxicity in vivo; therefore, the redox stability of iron-based contrast agents is crucial. The redox stability of iron(III) complexes was assessed using a sodium ascorbate assay, where 7.5 μM of the contrast agent was co-incubated with 25 μM sodium ascorbate for 20 minutes. No change in the absorbance peak of ascorbic acid at 265 nm indicated the redox stability of the contrast agent (the absorbance of Fe-tCDTA at the corresponding position decreased by more than 50% within 20 minutes). Figure 1 g and 1h).

[0067] In magnetic resonance imaging (MRI), relaxation rate refers to the ability of a contrast agent to shorten the longitudinal or transverse relaxation time of proton spins in neighboring water molecules. This is typically assessed using spin inversion recovery experiments and is the first step in characterizing contrast agent efficacy. Longitudinal relaxation rate (T1 relaxation rate, r1) and transverse relaxation rate (T2 relaxation rate, r2) are used to quantify the effect of a unit concentration of contrast agent on relaxation time. (For Fe-tmPCDTA, the r1 value is 1.40 mM compared to Fe-tCDTA.) - 1 s -1 and 2.27mM -1 s -1 (Compared to Fe-tCDTA, the r1 value of Fe-tqPCDTA is 1.97 mM). -1 s -1 and 2.27mM -1 s -1 ).

[0068] To assess the potential cytotoxicity of Fe(III)-PCDTA complexes (Fe-tqPCDT and Fe-tmPCDTA), we used a CCK-8 assay to determine cell viability in 4T1 cells, with Gd-DTPA as a reference control. Figure 3As shown, both Fe(III) complexes exhibited negligible cytotoxicity, with biocompatibility characteristics similar to Gd-DTPA. Even after treatment with high concentrations (500 μM) following 24 hours of incubation, cell viability remained statistically undifferentiated compared to the control group. These results demonstrate that Fe(III)-PCDTA complexes possess excellent biocompatibility and extremely low cytotoxicity, highlighting their advantages as safe and clinically promising candidates for MRI contrast agents.

[0069] Taking advantage of the favorable physicochemical properties of Fe(III)-PCDTA complexes—including kinetic inertness, redox stability, moderate relaxation rate, and low cytotoxicity—we evaluated their applicability in in vivo T1-weighted magnetic resonance imaging (MRI). Balb / c mice aged 6–8 weeks were selected and intravenously injected with contrast agent (0.2 mmol / kg). Dynamic contrast-enhanced MRI showed rapid blood clearance and significant T1 signal enhancement in the heart and kidneys. Figure 3 A), which is similar to the previously reported performance of Gd-DTPA. Pharmacokinetic analysis was performed using normalized signal-to-noise ratio (nSNR) time curves within the ventricular cavity. Figure 3 B), the results showed a transient increase in signal in the kidneys, with Fe-tqPCDTA eliciting a more significant response compared to Fe-tmPCDTA. Figure 3 C). Notably, the contrast changes in the liver or gallbladder were minimal, while the rapid enhancement of the bladder signal (3–5 minutes post-injection) highlighted renal excretion as the primary elimination pathway. This contrasts with the prolonged renal retention of the hydrophilic phosphonate analog Gd-HP-DO3P, which is attributed to ion-pair-mediated plasma protein binding. Despite its high negative charge, Fe-PCDTA's rapid clearance likely stems from its reduced protein binding affinity. Overall, the similar nSNR curves of Fe-PCDTA and Gd-DTPA suggest similar biodistribution, pharmacokinetics, and extracellular fluid distribution characteristics, making Fe-PCDTA a potential biocompatible alternative for clinical MRI.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended 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 present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A non-gadolinium magnetic resonance imaging contrast agent, characterized in that: The non-gadolinium magnetic resonance imaging agent is composed of trans-cyclohexanediaminetetraacetic acid (TCA) coordinated with a metal ion, wherein one to four carboxyl groups of the trans-cyclohexanediaminetetraacetic acid are replaced by phosphate groups, and the metal ion is Fe. 3+ or Mn 2+ .

2. The non-gadolinium magnetic resonance contrast agent according to claim 1, characterized in that: The structural formula of the non-gadolinium magnetic resonance contrast agent is as follows:

3. The method for preparing the non-gadolinium magnetic resonance contrast agent according to claim 1, characterized in that, Includes the following steps: S1: Synthesize phosphate-modified trans-cyclohexanediaminetetraacetic acid derivative ligands tmPCDTA or tqPCDTA; S2: The ligand is reacted with metal ions Fe 3+ or Mn 2+ They react in aqueous solution to form a complex; S3: Purify and freeze-dry to obtain the non-gadolinium magnetic resonance contrast agent.

4. The preparation method according to claim 3, characterized in that, The preparation method of the tmPCDTA is as follows: (a) Trans-cyclohexanediamine and benzyl bromide were reacted in acetonitrile at a molar ratio of 10:1 for 24 hours. The mixture was then concentrated, washed with saturated Na2CO3 solution, extracted with DCM, and dried to obtain compound 1. (b) Compound 1 was dissolved in a DMF solution containing diisopropylethylamine and potassium iodide, and ethyl bromide of pivalate was added and reacted for 6 hours. After concentration, washing with saturated Na2CO3 solution, DCM extraction, and purification by column chromatography, compound 2 was obtained. The molar ratio of compound 1 to diisopropylethylamine, potassium iodide, and ethyl bromide of pivalate was 1:6:6:

5. (c) Compound 2 was reacted with Pd / C in methanol solution under H2 atmosphere for 24 hours by stirring, and then filtered and concentrated to obtain compound 3; the mass fraction of Pd / C in the methanol solution was 5%; (d) Compound 3 was reacted with 4 mmol of paraformaldehyde in a tetrahydrofuran solution containing 4 mmol of diethyl phosphite at 95 °C for 24 hours. The mixture was then concentrated, washed with saturated Na2CO3 solution, extracted with DCM, and purified by column chromatography to obtain compound 4. (e) Compound 4 was hydrolyzed in 6M HCl at 105°C for 24 hours, purified by preparative HPLC, and lyophilized to obtain the tmPCDTA.

5. The preparation method according to claim 3, characterized in that, The preparation method of the tqPCDTA is as follows: Trans-cyclohexanediaminetetraacetic acid and phosphorous acid were dissolved in p-xylene, phosphorus trichloride was added dropwise, the mixture was refluxed for 6 hours, dried under vacuum, and then crystallized in a water / ethanol mixed solvent to obtain the tqPCDTA.

6. The preparation method according to claim 3, characterized in that, The ligand and the metal ion Fe 3+ The method for preparing non-gadolinium magnetic resonance contrast agents is as follows: The ligands tmPCDTA or tqPCDTA were mixed with FeCl3·6H2O in an aqueous solution at an equal molar concentration. The pH was adjusted to 2.5 and the reaction was carried out for 24 hours. After adjusting the pH to neutral, the mixture was desalted using Sephadex G-25, centrifuged, and lyophilized to obtain the non-gadolinium magnetic resonance contrast agent.

7. The preparation method according to claim 3, characterized in that, The ligand and the metal ion Mn 2+ The method for preparing non-gadolinium magnetic resonance contrast agents is as follows: The ligands tmPCDTA or tqPCDTA were mixed with MnCl2·4H2O in an aqueous solution at an equal molar concentration. The pH was adjusted to 6.5 and the reaction was carried out for 12 hours. Then the pH was adjusted to neutral, and the mixture was purified by RP-HPLC and lyophilized to obtain the non-gadolinium magnetic resonance contrast agent.

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