Water-soluble signal-enhanced high-fluorine-content molecular imaging probe and preparation method thereof

Abstract

The invention discloses a water-soluble signal enhancement type high-fluorine-content molecular imaging probe and a preparation method thereof, and relates to a high-fluorine-content signal enhancement type 19F magnetic resonance molecular imaging probe of which the main structure is a 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-tricarboxylic acid (DO3A) derivative coordinated with metal ions. The probe is high in fluorine content, good in water solubility and good in biological safety, and an imaging signal with a higher signal-to-noise ratio can be obtained under the same imaging time. On the living body level, the probe has a good enrichment effect in liver-gallbladder and small intestine areas, and by combining the signal enhancement effect of the probe, a good imaging effect can be obtained, and the metabolic behavior of a living body can be traced.

Description

Technical Field

[0001] The present invention relates to the field of molecular imaging probes, and in particular to a class of water-soluble signal-enhanced high-fluorine-content molecular imaging probes and a preparation method thereof. Background Art

[0002] Molecular imaging technology is a type of non-invasive imaging technology that obtains qualitative or quantitative information by monitoring changes in the molecular level at the living body level. Among these technologies, magnetic resonance imaging technology has received widespread attention due to its advantages such as no ionizing radiation, high resolution, high penetration depth, and multi-parameter and multi-section imaging. The main magnetic resonance imaging technologies currently used in clinical practice are 1 H MRI ( 1 H MRI) is the imaging of hydrogen nuclei in human soft tissue. This is due to the fact that hydrogen nuclei have the highest nuclear magnetic resonance sensitivity among stable nuclides and are widely distributed in biological tissues (mainly H 2 O). Since the relaxation properties of hydrogen nuclei in different soft tissues are significantly different, high-resolution anatomical images can be obtained for diagnosis. However, this technology also has disadvantages such as high biological background signals and artifact interference.

[0003] Atomic nuclei with non-zero spin quantum numbers can all produce nuclear magnetic resonance in a magnetic field and can be used for magnetic resonance imaging. 19 F nuclei have attracted attention because of their nuclear magnetic resonance sensitivity and wide chemical shift distribution, which is closest to that of hydrogen nuclei. More importantly, the content of fluorine nuclei in the human body is extremely low and mainly exists in solid form (bones and teeth), which is difficult to be detected by instruments. 19 Magnetic resonance imaging of F nuclei has extremely low biological background signals and can be used as an advantageous heteronuclear MRI technique. 1 H MRI provides complementary information, thereby obtaining more information about the target area.

[0004] and 1 H MRI is different. 19 F MRI requires the use of exogenous 19 F magnetic resonance probes are used to achieve this. Currently, the more commonly used probes are liposomes wrapped in perfluorocarbons or fluorinated polymer nanoparticles. Although these probes have a high fluorine content, they are metabolized slowly in the body, affecting continuous monitoring. At the same time, further modification of these probes, such as targeted modification, is also difficult. Therefore, the development of more new high-performance 19 F magnetic resonance probe is the goal of current scientific researchers.

[0005] Enterohepatic circulation (EHC) is a substantial metabolic process regulated by negative feedback in organisms. The molecules involved in this circulation system initially accumulate in the liver, then migrate to the gallbladder and are further transported to the intestine through bile secretion. The molecules that enter the small intestine are reabsorbed in the ileum, reflux into the blood and enter the circulatory system, while the rest are excreted in the form of feces. Many endogenous fat-soluble messenger biomolecules, such as bile acids and bilirubin, can normally exert their physiological mechanisms through the EHC process. EHC is often associated with pharmacokinetic studies and can reflect liver dysfunction, abnormal bile metabolism and bile duct obstruction, as well as various digestive or circulatory system diseases, such as liver damage, inflammatory bowel disease or hepatocellular carcinoma. Therefore, visual analysis of the dynamic process of EHC is an important technical means urgently needed in the fields of biology and medicine. Summary of the invention

[0006] The purpose of the present invention is to solve the problem in the prior art 19 F magnetic resonance imaging probes generally have the above-mentioned problems of low sensitivity and poor water solubility. A type of water-soluble signal-enhanced high-fluorine-content molecular imaging probe and a preparation method thereof are provided. The probe has high fluorine content, good water solubility, and good biosafety, and can obtain an imaging signal with a stronger signal-to-noise ratio at the same imaging time. At the in vivo level, the probe has a good enrichment effect in the liver-gallbladder and small intestine regions, and combined with its signal enhancement effect, a good imaging effect can be obtained.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A class of water-soluble signal-enhanced high-fluorine-content molecular imaging probes, designed as follows:

[0009]

[0010] Among them, M includes Fe 2+ , Fe 3+ , Gd 3+ , Mn 2+ , Mn 3+ 、Dy 3+ 、Tm 3+ 、Co 2+ 、Co 3+ 、Sm 3+ 、Eu 2+ 、Eu 3+ One of the following, preferably Fe 2+ .

[0011] The main structure of the probe is a monosubstituted derivative of 1,4,7,10-tetraazacyclododecane-1,4,7-tetracarboxylic acid (DOTA) DO3A and contains 9 chemical environments with the same 19The building blocks of F atoms (perfluorotert-butyl), the two are connected by -CH 2 CONHCH 2 CH 2 The O-groups are connected to form metal ion ligands, and finally metal complexes are formed by chelating paramagnetic metal ions.

[0012] The designed metal ion complex has a high fluorine content (~25%), a single 19 F chemical shift, and the synthesis method is simple, and it can chelate a variety of paramagnetic metal ions with relaxation time shortening effect.

[0013] The present invention discloses a method for preparing a water-soluble signal-enhanced high-fluorine-content molecular imaging probe:

[0014] (1) The main starting materials in the preparation process: 1,4,7,10-tetraazacyclododecane (cyclohexane), N-Boc-ethanolamine, and perfluorotert-butyl alcohol are commercial reagents.

[0015] (2) 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate tert-butyl ester (tBu-DO3A), N-tert-butoxycarbonyl-2-(perfluorotert-butoxy)ethan-1-amine and 2-(perfluorotert-butoxy)ethan-1-amine are intermediate products synthesized in the present invention.

[0016] Specifically, the method for preparing the water-soluble signal-enhanced high-fluorine-content molecular imaging probe comprises the following steps:

[0017]

[0018] The application of a class of water-soluble signal-enhanced high-fluorine-content molecular imaging probes described in the present invention is used for dynamic real-time monitoring of the enterohepatic circulation process at the living body level.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] Among the probes of the present invention, DO3A-Fe(II)-9F has the most outstanding imaging performance. 2+ Due to the PRE effect, the fluorine nuclei in the molecule have a relatively short and suitable relaxation time, so that the probe is expected to obtain a fluorine atom magnetic resonance imaging signal with a stronger signal-to-noise ratio at the same imaging time. In addition, at the in vivo level, the probe has good signal enrichment in the liver-gallbladder, small intestine and other regions, and can participate in the enterohepatic circulation.

[0021] The molecular ligand (DO3A-9F) of the present invention has a fluorine content of 25.7%, which has significant advantages over most reported fluorine-containing small molecule magnetic resonance probes. 2+ The paramagnetic relaxation enhancement effect of paramagnetic metal ions can significantly shorten the molecular structure. 19 The relaxation time of F nuclei can be increased, thereby increasing the number of scans per unit time and significantly improving the signal-to-noise ratio of imaging. 19 It has good application prospects in F magnetic resonance imaging and has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The fluorinated building block N-tert-butoxyacyl-2-(perfluoro-tert-butoxy)ethyl-1-amine prepared in Example 1 1 HNMR spectrum (solvent: CDCl 3 ).

[0023] Figure 2 The fluorinated building block N-tert-butoxyacyl-2-(perfluoro-tert-butoxy)ethyl-1-amine prepared in Example 1 13 CNMR spectrum (solvent is CDCl 3 ).

[0024] Figure 3 The fluorinated building block N-tert-butoxyacyl-2-(perfluoro-tert-butoxy)ethyl-1-amine prepared in Example 1 19 FNMR spectrum (solvent: CDCl 3 ).

[0025] Figure 4 This is the HR-ESI-MS mass spectrum of the deprotected fluorinated building block 2-(perfluoro-tert-butoxy)ethan-1-amine prepared in Example 1.

[0026] Figure 5 tBu-DO3A prepared in Example 1 1 H NMR spectrum (solvent: CD 3 OD).

[0027] Figure 6 tBu-DO3A prepared in Example 1 13 C NMR spectrum (solvent: CD 3 OD).

[0028] Figure 7 This is the HR-ESI-MS mass spectrum of tBu-DO3A prepared in Example 1.

[0029] Figure 8This is the HR-ESI-MS mass spectrum of tBu-DO3A-COOMe prepared in Example 1.

[0030] Fig. 9 This is the HR-ESI-MS mass spectrum of tBu-DO3A-COOH prepared in Example 1.

[0031] Fig.10 This is the HR-ESI-MS mass spectrum of tBu-DO3A-9F prepared in Example 1.

[0032] Fig.11 This is the HR-ESI-MS mass spectrum of the high-fluorine-content ligand DO3A-9F prepared in Example 1.

[0033] Fig.12 The high fluorine content ligand DO3A-9F prepared in Example 1 19 F NMR spectrum (solvent: CD 3 OD).

[0034] Fig.13 The high fluorine content probe DO3A-Fe(II)-9F prepared in Example 1 19 F NMR spectrum (solvent 10% D 2 O / H 2 O), -68.51ppm is the single peak of the fluorine signal of the product (affected by Fe 2+ The impact is slightly wider).

[0035] Fig.14 This is the HR-ESI-MS mass spectrum of the high fluorine content probe DO3A-Fe(II)-9F prepared in Example 1.

[0036] Fig.15 In vitro 19 F MRI signal and 19 F concentration; (taking ligand DO3A-9F as a comparison, all materials contain 60mM 19 F; RARE sequence, T E =8.1ms, resolution MTX=32×32, slice thickness ST=20mm, fixed sampling time Accu Time=3min 12s, T R is the repetition time, from left to right: 1600, 800, 400, 200, 100, 50ms, and the sampling number NEX from left to right: 20, 40, 80, 160, 320, 640).

[0037] Fig.16 The probe DO3A-Fe(II)-9F prepared in Example 2 was detected at different repetition times TR Imaging test under (RARE sequence, T E =8.1ms, sampling number NEX=80, resolution MTX=32×32, layer thickness ST=20mm; T R They are: 50, 100, 200, 400, 800ms, and the corresponding sampling time Accu Time = 24s, 48s, 1min 36s, 3min12s, 6min24s); this experiment is to explore the full relaxation time of the probe on a 9.4T magnetic resonance imaging device.

[0038] Fig.17 The imaging test of the probe DO3A-Fe(II)-9F prepared in Example 2 under different NEX numbers (RARE sequence, T R =200ms, T E =8.1ms, resolution MTX=32×32, layer thickness ST=20mm; NEX is: 20, 40, 80, 160, 320ms, corresponding sampling time Accu Time=12s, 24s, 48s, 1min 36s, 3min 12s); This experiment is to explore the limit imaging time of the probe on a 9.4T magnetic resonance imaging machine.

[0039] Fig.18 Imaging test of probe DO3A-Fe(II)-9F prepared in Example 2 at different layer thicknesses ST (RARE sequence, T R =200ms, T E =8.1ms, NEX=320, resolution MTX=32×32. ST is 2, 5, 10, 15, 20mm, sampling time Accu Time=3min 12s); This experiment is to explore the limit imaging voxel size of the probe on the 9.4T MRI.

[0040] Fig.19 The probe DO3A-Fe(II)-9F prepared in Example 2 was detected in vitro at different resolutions. 19 F MRI signal and 19 F concentration relationship diagram (RARE sequence, T R =200ms, T E =8.1ms, ST=20mm, sampling circle number NEX=160, sampling time Accu Time=3min 12s, MTX from left to right: 32×32, 64×64, 128×128, 256×256; the imaging time from low to high resolution is 3min 12s, 6min 24s, 12min 48s, 25min 36s).

[0041] Fig. 20 Probe DO3A-Fe(II)-9F prepared in Example 2 in vivo at different resolutions 19 F MRI signal (90 minutes after injection; RARE sequence, T R =200ms, T E =8.1ms, ST=20mm, sampling circle NEX=160 / 320, sampling time Accu Time=6min 24s, MTX from left to right: 32×32, 64×64, 128×128, 256×256; the imaging time from low to high resolution is 6min 24s, 12min 48s, 25min 36s, 51min 12s).

[0042] Fig.21 The probe DO3A-Fe(II)-9F prepared in Example 2 is a live image at a resolution of 32×32. 19 F MRI signal (single-layer fluorine signal coronal imaging, time span of 4 hours; RARE sequence, T R =200ms, T E =8.1ms, MTX=32×32, ST=20mm, NEX=320, Accu Time=6min 24s, the signals in the gallbladder, small intestine and bladder areas can be clearly observed).

[0043] Fig. 22 The probe DO3A-Fe(II)-9F prepared in Example 2 is a live image at a resolution of 32×32. 19 F MRI signal (double-layer cross-sectional imaging, time span of 4 hours; RARE sequence, T R =200ms, T E =8.1ms, MTX=32×32, ST=30mm (15mm / layer, 2 layers in total), NEX=320, Accu Time=6min 24s; Epigastrium: upper abdomen, including liver-gallbladder and intestines; Hypogastrium: lower abdomen, mainly including bladder).

[0044] Fig.23 The probe DO3A-Fe(II)-9F prepared in Example 2 is a live image at a resolution of 64×64. 19 F MRI signal map (single-layer fluorine signal coronal imaging, time span of 4 hours; RARE sequence, T R =200ms, T E =8.1ms, MTX=64×64, ST=20mm, NEX=320, Accu Time=12min 48s).

[0045] Fig.24 This is the metabolic flow chart of the probe DO3A-Fe(II)-9F prepared in Example 2.

[0046] Fig.25 The MTT test results of the biosafety assessment experiment of the probe DO3A-Fe(II)-9F prepared in Example 3 (the abscissa represents the probe molecule concentration, and the ordinate represents the corresponding L02 / HepG2 cell activity).

[0047] Fig.26 Results of H&E staining experiment on the biosafety assessment experiment of the probe DO3A-Fe(II)-9F prepared in Example 3 (the tissue sections from left to right are: heart, liver, spleen, lung, and kidney; the first row of sections were taken from the control group mice injected with PBS, and the second row of sections were taken from the experimental group mice injected with the probe). DETAILED DESCRIPTION

[0048] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0049] All of this embodiment 19 F NMR measurements (including relaxation time measurements) were performed on a Bruker 600MHz (564MHz for 19 F) carried out on a nuclear magnetic resonance spectrometer, 19 F MRI test was performed on a Bruker 9.4T (376MHz for 19 F) performed on a magnetic resonance imaging device.

[0050] Example 1: Synthesis and related characterization of probe DO3A-Fe(II)-9F.

[0051] All raw materials and reagents mentioned are commercial drugs and can be used directly without further purification.

[0052]

[0053] (1) Compound 1: Synthesis of fluorinated building block N-tert-butoxyacyl-2-(perfluoro-tert-butoxy)ethan-1-amine.

[0054] This reaction is a nucleophilic substitution reaction based on the Mitsunobu reaction and needs to be carried out under anhydrous and oxygen-free conditions. N-tert-butoxyacyl-ethanolamine (1.61 g, 1.55 mL, 10.0 mmol) and triphenylphosphine (Ph 3P, 3.94g, 15.0mmol) was dissolved in 50.0mL of anhydrous tetrahydrofuran (THF), and the system was placed at 0°C. Diisopropyl azodicarboxylate (DIAD, 3.03g, 2.97mL, 15.0mmol) was added dropwise to the solution within 15 minutes and stirred vigorously. After the addition was completed, the mixture was gradually restored to room temperature, and after stirring for 5 minutes, perfluoro tert-butyl alcohol (4.72g, 2.83mL, 20.0mmol) was added. Thereafter, the system was slowly warmed to 50°C and the reaction was overnight. After the reaction was completed, silica gel column chromatography was performed using a n-hexane / ethyl acetate system (100% n-hexane-15% v / v ethyl acetate / n-hexane) to obtain 2.68g of a light yellow transparent liquid with a yield of about 68%. 1 H NMR ( Figure 1 ,600MHz,CDCl 3 ): δ4.90(1H,br s), 4.08(2H,t,J=5.4Hz), 3.40(2H,q,J=6.6Hz), 1.44(9H,s); 13 C NMR ( Figure 2 ,151MHz,CDCl 3 ): δ156.00,121.41,119.07,80.24,68.94,40.35,28.19; 19 F NMR ( Figure 3 ,564MHz,CDCl 3 ):δ-70.39(9F,s).

[0055] (2) Compound 2: Synthesis of fluorinated building block 2-(perfluoro-tert-butoxy)ethan-1-amine.

[0056] N-tert-Butoxyacyl-2-(perfluorotert-butoxy)ethane-1-amine (3.79 g, 10.0 mmol) was dissolved in 5.0 mL of dichloromethane (DCM) / trifluoroacetic acid (TFA) (1:1, v / v) system and stirred at room temperature for 3 hours. After the reaction, the solvent was removed by rotary evaporation to obtain the deprotected fluorinated building block crude product as a yellow viscous liquid. No further purification was required for this step of the reaction. HR-ESI-MS (m / z) characterization of C 6 H 7 F 9 NO[M+H] + Theoretical value: 280.0378, actual value: 280.0379, error value: 0.23ppm( Figure 4 ).

[0057] (3) Synthesis of compound 3: tBu-DO3A.

[0058] Cyclotrimethylenetetramine (10.336 g, 60.0 mmol) and anhydrous sodium acetate (14.77 g, 180.0 mmol) were dissolved in 180.0 mL of N, N-dimethylacetamide (DMA) and placed at 0 ° C. At the same time, tert-butyl bromoacetate (35.11 g, 29.1 mL, 180.0 mmol) was dissolved in 60.0 mL of DMA and dripped into the above system dropwise. Thereafter, the mixture was vigorously stirred at room temperature for 48 hours, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography using dichloromethane / methanol (100% dichloromethane-15% v / v methanol / dichloromethane) to obtain 21.87 g of a white solid with a yield of about 71%. 1 H NMR ( Figure 5 ,600MHz,CD 3 OD): δ3.41 (4H, s), 3.36 (2H, s), 3.14 (4H, t, J = 6.5 Hz), 2.98 (4H, t, J = 6.6 Hz), 2.79 (4H, t, J = 5.5 Hz), 2.69 (4H, t, J = 6.5 Hz), 1.48 (9H, s), 1.48 (18H, s), the peak of –NH– on the ring was not detected; 13 C NMR ( Figure 6 ,151MHz,CD 3 OD):δ171.18,81.20,56.23,50.37,49.09,45.62,27.03.HR-ESI-MS (m / z) characterization of C 26 H 51 N 4 O 6 [M+H] + Theoretical value: 515.3803, actual value: 515.3820, error value: 3.22ppm( Figure 7 ).

[0059] (4) Synthesis of compound 4: tBu-DO3A-COOMe.

[0060] tBu-DO3A (6.17 g, 12.5 mmol) and potassium carbonate (3.45 g, 25.0 mmol) were dissolved in 40 mL of acetonitrile, and methyl chloroacetate (2.98 g, 2.40 mL, 27.5 mmol) was added. The resulting suspension was vigorously stirred at room temperature for 12 hours. After centrifugation to remove insoluble matter, the solvent in the supernatant was removed by rotary evaporation. The crude product was separated by silica gel column chromatography using a dichloromethane / methanol system (100% dichloromethane-20% v / v methanol / dichloromethane) to obtain 6.41 g of a yellow crystalline solid with a yield of about 87%. HR-ESI-MS (m / z) characterization of C 29 H 55 N4 O 8 [M+H] + Theoretical value: 587.4014, actual value: 587.4047, error value: -1.35ppm( Figure 8 ).

[0061] (5) Synthesis of compound 5: tBu-DO3A-COOH.

[0062] tBu-DO3A-COOMe (5.864 g, 10.0 mmol) was dissolved in 45 mL of a mixed solvent of 1,4-dioxane / water (v / v=2:1) ​​containing 1.2 g of sodium hydroxide and stirred overnight at 50°C. After the reaction was completed, 1,4-dioxane was removed by rotary evaporation. The residual liquid was extracted with 180.0 mL of dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain 5.37 g of a white to light yellow foaming solid with a yield of about 90%. HR-ESI-MS (m / z) characterization of C 28 H 53 N 4 O 8 [M+H] + and C 28 H 52 N 4 O 8 Na[M+Na] + Theoretical value: 573.3858 and 595.3677, actual value: 573.3873 and 595.3699, error value: 2.69 and 3.58ppm ( Fig. 9 ).

[0063] (6) Synthesis of compound 6: tBu-DO3A-9F.

[0064] tBu-DO3A-COOH (572 mg, 1.0 mmol) was dissolved in 20.0 mL DCM, and the condensing agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 958.5 mg, 5.0 mmol), N-hydroxysuccinimide (NHS, 345 mg, 3.0 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) were added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, it was washed once with 20.0 mL of water and saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate. After that, a solution of the crude product (837 mg, 3.0 mmol) of the fluorinated building block 2-(perfluorotert-butoxy)ethyl-1-amine dissolved in 10.0 mL DCM was added to the above organic phase, and the reaction was carried out at room temperature overnight. After the reaction was completed, the solvent was removed by rotary evaporation, and the subsequent reaction was directly carried out. HR-ESI-MS (m / z) characterization of C 34 H56 F 9 N 5 O 8 Na[M+Na] + Theoretical value: 856.3877, actual value: 856.3884, error value: 0.82ppm( Fig.10 ).

[0065] (7) Compound 7: Synthesis of ligand DO3A-9F.

[0066] The crude tBu-DO3A-9F product from the previous step was dissolved in 5.0 mL TFA and stirred at 50°C overnight. After the reaction was completed, TFA was removed by rotary evaporation to obtain the crude DO3A-9F product, which was directly used for the subsequent reaction. HR-ESI-MS (m / z) characterization of C 22 H 33 F 9 N 5 O 8 [M+H] + and C 22 H 32 F 9 N 5 O 8 Na[M+Na] + Theoretical value: 666.2180 and 688.1999, actual value: 666.2181 and 688.2003, error value: 0.09 and 0.52ppm ( Fig.11 ). 19 F NMR ( Fig.12 ,564MHz,CD 3 OD):δ-71.43(9F,s).

[0067] (8) Compound 8: Synthesis of probe DO3A-Fe(II)-9F.

[0068] The crude product of the high-fluorine-containing ligand DO3A-9F from the previous step was dissolved in a v / v 50% water / methanol mixed solvent and the pH was adjusted to 3. FeCl was added 2 (254 mg, 2.0 mmol) and then 2 In an atmosphere of 45°C, the mixture was heated and stirred overnight. After the reaction, it was purified by high performance liquid chromatography (HPLC) (0-60 minutes program, eluent ratio 20% acetonitrile / 80% water-100% acetonitrile, and the components with retention time of 18.5-20.5 minutes were collected). After the solvent was removed by rotary evaporation, lyophilization was performed to obtain 2.30 g of a white to light yellow solid, with a yield of 32% from step 6. Probes based on other paramagnetic metal ions can also be synthesized by similar methods. 19 F NMR ( Fig.13,564MHz,10% D 2 O / H 2 O):δ-68.51(9F,br s); HR-ESI-MS (m / z) characterization of C 22 H 29 FeF 9 N 5 O 8 [MH] - Theoretical value: 718.1228: Actual value: 718.1215, error: -1.71ppm( Fig.14 ).

[0069] It is worth mentioning that the fluorine content of this molecular ligand (DO3A-9F) reaches 25.7%, which has significant advantages over most reported fluorine-containing small molecule magnetic resonance probes. 2+ The paramagnetic relaxation enhancement effect of paramagnetic metal ions can significantly shorten the molecular structure. 19 The relaxation time of F nuclei can be increased, thereby increasing the number of scans per unit time and greatly improving the signal-to-noise ratio of imaging.

[0070] After obtaining the probe molecule, the present invention 19 The relaxation time of F atoms was measured. 19 The relaxation time test of F is shown in Table 1 below (the solvent is 10% D 2 O / H 2 O, probe concentration 1mM, magnetic resonance center frequency set to 564MHz):

[0071] Table 1

[0072]

[0073] Note: The relaxation times of all derivatives have been measured by 600 MHz NMR spectrometer.

[0074] From the table above, we can see that the fluorine nuclei in the chelate DO3A-Fe(II)-9F have a relatively short relaxation time, which means that DO3A-Fe(II)-9F is expected to obtain a fluorine nuclear magnetic resonance signal with a stronger signal-to-noise ratio at the same imaging time. The control DO3A-Fe(III)-9F will further shorten the relaxation time of the fluorine nuclei, making it far below the detection limit of the conventional sequence, thereby reducing the fluorine nuclear magnetic resonance signal. This result lays a good foundation for subsequent imaging experiments.

[0075] Example 2: Imaging parameter testing and in vivo and in vitro imaging performance evaluation of probe DO3A-Fe(II)-9F.

[0076] DO3A-Fe(II)-9F is a molecular probe with good water solubility. In the in vitro imaging verification of this embodiment, DO3A-Fe(II)-9F was dissolved in 1×PBS and prepared to contain 10, 20, 30, 40, 50, 60 mM 19 The DO3A-Fe(II)-9F solution samples with different F concentrations were analyzed under different parameter groups (including different repetition times T R The full relaxation time of the exploration probe, the maximum sampling time of the NEX exploration probe with different numbers of circles, the maximum imaging voxel size of the ST exploration probe with different layer thicknesses, etc.) and resolution (MTX) were tested.

[0077] Fig.15 The results show that in the chelated Fe 2+ After the ionization, the probe DO3A-Fe(II)-9F has a more significant imaging signal than its ligand DO3A-9F under a shorter sampling time and corresponding parameter set, indicating that the use of Fe 2+ PRE effect regulation 19 F signaling strategy success. Fig.16 The results show that the full relaxation time of the probe is between 100 and 200 ms on the 9.4T imager. Therefore, in subsequent experiments, the repetition time T is set R =200ms. Fig.17 The results showed that the signal-enhanced probe DO3A-Fe(II)-9F can detect signals in a shorter sampling time, with a limit of 20 circles and an imaging time of 12 seconds. Fig.18 The results show that the limit detection thickness of the signal (FOV is constant) is 2mm. Fig.19 The results show that the probe still has a good linear relationship between concentration and signal intensity at high resolution. These experimental results indicate that the probe DO3A-Fe(II)-9F is expected to obtain information with higher time-space resolution.

[0078] Based on the in vitro imaging results, in the in vivo experimental group, this embodiment configured a 25mM DO3A-Fe(II)-9F (i.e., 225mM 19 F concentration) and 1% sodium ascorbate in 1× PBS solution and injected into BALB / c mice via the tail vein. 19 F Magnetic resonance imaging assessment. Figures 20-23 )

[0079] Fig. 20 is Fig.19 On this basis, we attempted high-resolution imaging of living bodies and found that the probe can also obtain high-resolution information at the living body level, among which the signals of the gallbladder and bladder are the most significant. Fig.21 The results showed that the probe was highly concentrated in the gallbladder, small intestine, and bladder. Fig. 22 This fact was further verified by the significant signals in the liver-gallbladder and small intestine regions, which strongly indicated that the probe DO3A-Fe(II)-9F was involved in the enterohepatic circulation process in the organism. Fig.23 is based on Fig. 20 As a result, high temporal and spatial resolution imaging monitoring was performed on living mice.

[0080] In summary, the probe DO3A-Fe(II)-9F 19 The F MRI signal is significantly enriched in the liver-gallbladder region and gradually moves to the small intestine, further proving that the drug is metabolized in the body through the enterohepatic circulation (liver → gallbladder → small intestine → reabsorption). The metabolic flow chart is shown in Fig.24 shown.

[0081] Example 3: Biosafety assessment of probe DO3A-Fe(II)-9F.

[0082] Cytotoxicity evaluation: DO3A-Fe(II)-9F at different concentrations was co-incubated with L02 cells and HepG2 cells, and the cytotoxicity of the probe was evaluated using the MTT test. Preliminary in vivo toxicity evaluation: 25 mM DO3A-Fe(II)-9F, i.e. 225 mM 19 The probe with a concentration of F was dissolved in a 1×PBS stock solution and injected into multiple BALB / c mice through the tail vein. At the same time, mice injected with 1×PBS solution were used as controls. All mice were killed 3 days later, and the five major organs of the heart, liver, spleen, lungs and kidneys were removed for sectioning and hematoxylin-eosin (H&E) staining experiments. The morphological changes of the tissue sections were observed and compared with the tissue sections of the control group to conduct a preliminary assessment of in vivo toxicity. From the results, it can be seen that the probe has good biocompatibility ( Fig.25 and Fig.26 ), which also lays the foundation for the clinical transformation of the probe DO3A-Fe(II)-9F as a hepatobiliary circulation tracing probe.

Claims

1. A water-soluble signal-enhanced high-fluorine-content molecular imaging probe, characterized in that: The structural formula is as follows: Among them, M includes Fe 2+ , Fe 3+ , Gd 3+ , Mn 2+ , Mn 3+ 、Dy 3+ 、Tm 3+ 、Co 2+ 、Co 3+ 、Sm 3+ 、Eu 2+ 、Eu 3+ One of them.

2. The water-soluble signal-enhanced high-fluorine-content molecular imaging probe according to claim 1, characterized in that: M is selected from Fe 2+ .

3. The method for preparing a water-soluble signal-enhanced high-fluorine-content molecular imaging probe according to claim 1, characterized in that: The following steps are involved:

4. The use of a water-soluble signal-enhanced high-fluorine-content molecular imaging probe according to claim 1, characterized in that: Used to dynamically monitor the enterohepatic circulation process in real time at the in vivo level.

Images