A type of water-soluble signal-enhanced high-fluorine-content molecular imaging probe and its preparation method

Abstract

The application relates to a water-soluble signal-enhanced high-fluorine-content molecular imaging probe and a preparation method thereof, and relates to a high-fluorine-content signal-enhanced molecular imaging probe with a main structure of 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid (DO3A) derivative and metal ion coordination 19 The F magnetic resonance molecular imaging probe has high fluorine content, good water solubility and good biological safety, can obtain imaging signals with stronger signal-to-noise ratio under the same imaging time, has good enrichment effect in the liver-bile and small intestine regions on the living body level, can obtain good imaging effect in combination with the signal enhancement effect, and can track the living body metabolism behavior.

Description

TECHNICAL FIELD

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

[0002] Molecular imaging technology is a kind of non-invasive imaging technology, which obtains qualitative or quantitative information by monitoring the molecular level changes at the level of living body. Among these technologies, magnetic resonance imaging technology has received extensive attention due to its advantages of no ionizing radiation, high resolution, high penetration depth, multi-parameter and multi-profile imaging, etc. At present, the magnetic resonance imaging technology mainly used in clinical is 1 H magnetic resonance imaging 1 (H MRI), that is, 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 nuclei and are widely distributed in biological tissues (mainly in the form of H2O). Because 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 the disadvantages of high biological background signal and the presence of artifacts.

[0003] Nuclei with spin quantum number not equal to 0 can produce nuclear magnetic resonance phenomenon in a magnetic field and can be used for magnetic resonance imaging. Among stable nuclei, 19 F nuclei are of interest due to their close nuclear magnetic resonance sensitivity to hydrogen nuclei and wide chemical shift distribution. 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, so 19 F nuclear magnetic resonance imaging has extremely low biological background signal and can be used as a quite advantageous heteronuclear MRI technology to provide supplementary information for 1 H MRI, thereby obtaining more information about the target region.

[0004] Unlike 1 H magnetic resonance imaging, 19 F magnetic resonance imaging needs to rely on exogenous 19 F magnetic resonance probes. The commonly used probes are liposomes wrapped with perfluorocarbon or fluorine-containing polymer nanoparticles. Although the fluorine content of this kind of probe is very high, the metabolism of this kind of probe in the body is relatively slow, which affects the continuous monitoring. At the same time, further modification of these probes, such as targeting modification, also has certain difficulties. Therefore, developing more new high-performance 19 F magnetic resonance probes is the goal of current researchers.

[0005] The enterohepatic circulation (EHC) is a substantial metabolic process regulated by negative feedback in organisms. The molecules involved in the circulation system are initially accumulated in the liver, then migrated to the gallbladder, and further transported to the intestine through bile secretion. The molecules entering the small intestine are reabsorbed in the ileum, return to the blood into the circulatory system, and the remaining part is excreted in the form of feces. Many endogenous lipid-soluble messenger biological molecules, such as bile acids and bilirubin, can normally play their physiological mechanisms through the EHC process. The EHC is often related to pharmacokinetic studies, which can reflect liver dysfunction, abnormal bile metabolism and bile duct obstruction, and various digestive system or circulatory system diseases, such as liver damage, inflammatory bowel disease or hepatocellular carcinoma. Therefore, visualizing the dynamic process of EHC is an important technical means urgently needed in the fields of biology and medicine. SUMMARY

[0006] The present application aims to solve the above problems in the prior art 19 The F magnetic resonance imaging probe has the above problems of low sensitivity and poor water solubility, and provides a water-soluble signal-enhanced high-fluorine-content molecular imaging probe and a preparation method thereof. The probe has high fluorine content, good water solubility and good biological safety, and can obtain imaging signals with stronger signal-to-noise ratio in the same imaging time. In the aspect of living body, the probe has good enrichment effect in the liver-gallbladder and small intestine regions, and combined with its signal enhancement effect, good imaging effect can be obtained.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A water-soluble signal-enhanced high-fluorine-content molecular imaging probe is designed as follows:

[0009]

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

[0011] The main structure of the probe is a monosubstituted derivative DO3A of 1,4,7,10-tetraazacyclododecane-1,4,7-tetracarboxylic acid (DOTA) and a 9-chemical-environment-same 19The building blocks of F atom (perfluorinated t-butyl), which are connected by-CH2CONHCH2CH2O- group to form metal ion ligand, and finally form metal complex by chelating paramagnetic metal ion.

[0012] The designed metal ion complex has high fluorine content (about 25%), single 19 F chemical shift, and the synthesis method is simple, and various paramagnetic metal ions with relaxation time shortening effect are chelated.

[0013] The application discloses a preparation method of a water-soluble signal enhancement type high fluorine content molecular imaging probe.

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

[0015] (2) 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,4,7-tri-tert-butyl acetate (tBu-DO3A), N-tert-butoxycarbonyl-2-(perfluorinated t-butoxy) ethan-1-amine and 2-(perfluorinated t-butoxy) ethan-1-amine are intermediate products synthesized in the application.

[0016] Specifically, the preparation method of the water-soluble signal enhancement type high fluorine content molecular imaging probe comprises the following steps.

[0017]

[0018] The application of the water-soluble signal enhancement type high fluorine content molecular imaging probe can be used for dynamic real-time monitoring of a liver-intestine circulation process in a living body.

[0019] Compared with the prior art, the technical scheme of the application has the following beneficial effects:

[0020] In the probe, the imaging performance of DO3A-Fe (II)-9F is the most outstanding. 2+ Due to the PRE effect of Fe, the fluorine nuclei in the molecule have a relatively short and suitable relaxation time, so that the probe can obtain a fluorine nuclear magnetic resonance imaging signal with a stronger signal-to-noise ratio under the same imaging time.

[0021] The fluorine content of the molecular ligand (DO3A-9F) reaches 25.7%, which has a significant advantage over most reported fluorine-containing small molecular 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 effectively 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-(perfluorotert-butoxy)ethyl-1-amine prepared in Example 1 1 HNMR spectrum (solvent: CDCl3).

[0023] Figure 2 The fluorinated building block N-tert-butoxyacyl-2-(perfluorotert-butoxy)ethyl-1-amine prepared in Example 1 13 CNMR spectrum (solvent is CDCl3).

[0024] Figure 3 The fluorinated building block N-tert-butoxyacyl-2-(perfluorotert-butoxy)ethyl-1-amine prepared in Example 1 19 FNMR spectrum (solvent: CDCl3).

[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: CD3OD).

[0027] Figure 6 tBu-DO3A prepared in Example 1 13 C NMR spectrum (solvent: CD3OD).

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

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

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

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

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

[0033] Figure 12 The high fluorine content ligand DO3A-9F prepared in Example 1 19 F NMR spectrum (solvent: CD3OD).

[0034] Figure 13 The high fluorine content probe DO3A-Fe(II)-9F prepared in Example 1 19 F NMR spectrum (solvent is 10% D2O / H2O), -68.51ppm is the single peak of fluorine signal of the product (affected by Fe 2+ The impact is slightly broadened).

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

[0036] Figure 15 The probe DO3A-Fe(II)-9F prepared in Example 2 was 19 F MRI signal and 19 F concentration relationship diagram; (taking ligand DO3A-9F as a comparison, the materials all 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 cycle NEX from left to right: 20, 40, 80, 160, 320, 640).

[0037] Figure 16 The probe DO3A-Fe(II)-9F prepared in Example 2 was detected at different repetition times T R Imaging test under (RARE sequence, T E =8.1ms, sampling number NEX=80, resolution MTX=32×32, slice thickness ST=20mm; T R= 200 ms, T = 8.1 ms, resolution MTX = 32 x 32, slice thickness ST = 20 mm; NEX in turn: 50, 100, 200, 400, 800 ms, corresponding to the sampling time Accu Time = 24 s, 48 s, 1 min 36 s, 3 min 12 s, 6 min 24 s); this experiment was to explore the full relaxation time of the probe on the 9.4T MRI.

[0038] Figure 17 Imaging test of the probe DO3A-Fe(II)-9F prepared for Example 2 at different NEX (RARE sequence, T R = 200 ms, T E = 8.1 ms, resolution MTX = 32 x 32, slice thickness ST = 20 mm; NEX in turn: 50, 100, 200, 400, 800 ms, corresponding to the sampling time Accu Time = 24 s, 48 s, 1 min 36 s, 3 min 12 s, 6 min 24 s); this experiment was to explore the full relaxation time of the probe on the 9.4T MRI.

[0039] Figure 18 Imaging test of the probe DO3A-Fe(II)-9F prepared for Example 2 at different slice thickness ST (RARE sequence, T R = 200 ms, T E = 8.1 ms, NEX = 320, resolution MTX = 32 x 32. ST in turn: 2, 5, 10, 15, 20 mm, sampling time Accu Time = 3 min 12 s); this experiment was to explore the limit imaging pixel size of the probe on the 9.4T MRI.

[0040] Figure 19 In vitro imaging of the probe DO3A-Fe(II)-9F prepared for Example 2 at different resolutions 19 F MRI signal vs 19 F concentration graph (RARE sequence, T R = 200 ms, T E = 8.1 ms, ST = 20 mm, sampling number NEX = 160, sampling time Accu Time = 3 min 12 s, MTX from left to right in turn: 32 x 32, 64 x 64, 128 x 128, 256 x 256; imaging time from low to high resolution in turn: 3 min 12 s, 6 min 24 s, 12 min 48 s, 25 min 36 s).

[0041] Figure 20 In vivo imaging of the probe DO3A-Fe(II)-9F prepared for Example 2 at different resolutions (90 min post-injection time point; RARE sequence, T 19 F MRI signal (90 min post-injection time point; RARE sequence, TR = 200 ms, T E = 8.1 ms, ST = 20 mm, number of sampling turns NEX = 160 / 320, sampling time Accu Time = 6 min 24 s, MTX from left to right: 32x32, 64x64, 128x128, 256x256; imaging times from low to high: 6 min 24 s, 12 min 48 s, 25 min 36 s, 51 min 12 s).

[0042] Figure 21 In vivo DO3A-Fe(II)-9F prepared as in Example 2 at 32x32 resolution 19 F MRI signal (single layer fluorine signal coronal imaging, time span of 4 hours; RARE sequence, T R = 200 ms, T E = 8.1 ms, MTX = 32x32, ST = 20 mm, NEX = 320, Accu Time = 6 min 24 s, signal of gallbladder, small intestine and urinary bladder regions clearly visible).

[0043] Figure 22 In vivo DO3A-Fe(II)-9F prepared as in Example 2 at 32x32 resolution 19 F MRI signal (double layer transversal imaging, time span of 4 hours; RARE sequence, T R = 200 ms, T E = 8.1 ms, MTX = 32x32, ST = 30 mm (15 mm / layer, 2 layers), NEX = 320, Accu Time = 6 min 24 s; Epigastrium: upper abdomen, containing liver-biliary and intestinal tracts; Hypogastrium: lower abdomen, mainly containing urinary bladder).

[0044] Figure 23 In vivo DO3A-Fe(II)-9F prepared as in Example 2 at 64x64 resolution 19 F MRI signal (single layer fluorine signal coronal imaging, time span of 4 hours; RARE sequence, T R = 200 ms, T E = 8.1 ms, MTX = 64x64, ST = 20 mm, NEX = 320, Accu Time = 12 min 48 s).

[0045] Figure 24 Metabolic scheme of probe DO3A-Fe(II)-9F prepared as in Example 2.

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

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

[0048] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clear, specific embodiments are described in detail below.

[0049] All the 19 F NMR tests (including relaxation time tests) were performed on a Bruker 600MHz (564MHz for 19 F) nuclear magnetic resonance spectrometer, 19 F MRI tests were performed on a Bruker 9.4T (376MHz for 19 F) magnetic resonance imaging device.

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

[0051] The raw reagents mentioned are commercial drugs and do not need to be further purified and can be directly used.

[0052]

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

[0054] The reaction is a nucleophilic substitution reaction based on Mitsunobu reaction, which needs to be carried out under anhydrous and anaerobic conditions. N-tert-butoxyacyl-ethanolamine (1.61 g, 1.55 mL, 10.0 mmol) and triphenylphosphine (Ph3P, 3.94 g, 15.0 mmol) were dissolved in 50.0 mL of anhydrous tetrahydrofuran (THF), and the system was placed in 0°C. Diisopropyl azodicarboxylate (DIAD, 3.03 g, 2.97 mL, 15.0 mmol) was added dropwise to the solution within 15 minutes, and stirred vigorously. After the end of the dropwise addition, the mixture was gradually returned to room temperature, and after stirring for 5 minutes, perfluoro-tert-butyl alcohol (4.72 g, 2.83 mL, 20.0 mmol) was added. Thereafter, the system was slowly warmed to 50°C, and the reaction was carried out overnight. After the reaction was completed, silica gel column chromatography was carried out using a n-hexane / ethyl acetate system (100% n-hexane-15% v / v ethyl acetate / n-hexane) to obtain 2.68 g of a light yellow transparent liquid, with a yield of about 68%. 1 H NMR Figure 1 , 600 MHz, CDC13): δ 4.90 (1H, br s), 4.08 (2H, t, J = 5.4 Hz), 3.40 (2H, q, J = 6.6 Hz), 1.44 (9H, s); 13 C NMR Figure 2 , 151 MHz, CDC13): δ 156.00, 121.41, 119.07, 80.24, 68.94, 40.35, 28.19; 19 F NMR Figure 3 , 564 MHz, CDC13): δ -70.39 (9F, s).

[0055] (2) Synthesis of compound 2: fluoro-block 2-(perfluoro-tert-butoxy)ethan-1-amine.

[0056] N-tert-butoxyacyl-2-(perfluoro-tert-butoxy)ethan-1-amine (3.79 g, 10.0 mmol) was dissolved in a 5.0 mL 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 crude deprotected fluoro-block as a yellow viscous liquid, which was used without further purification. HR-ESI-MS (m / z) of C6H7F9NO [M+H] + Theoretical value: 280.0378, Actual value: 280.0379, Error value: 0.23 ppm 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. Simultaneously, tert-butyl bromoacetate (35.11 g, 29.1 mL, 180.0 mmol) was dissolved in 60.0 mL of DMA and dripped dropwise into the above system. 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 give 21.87 g of a white solid with a yield of about 71%. 1 H NMR ( Figure 5 , 600MHz,CD3OD):δ3.41(4H,s),3.36(2H,s),3.14(4H,t,J=6.5Hz),2.98(4H,t,J=6.6Hz),2.79(4H,t,J=5.5Hz),2.69(4H,t,J=6.5Hz),1.48(9H,s),1.48(18H,s), the peak of –NH– on the ring was not detected; 13 C NMR ( Figure 6 ,151MHz,CD3OD):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 N4O6[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 N4O8[M+H] +Theoretical value: 587.4014, actual value: 587.4047, error value: -1.35 ppm 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 at 50 °C overnight. 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 white to light yellow foaming solid with a yield of about 90%. HR-ESI-MS (m / z) characterization of C 28 H 53 N4O8[M+H] + and C 28 H 52 N4O8Na[M+Na] + Theoretical value: 573.3858 and 595.3677, actual value: 573.3873 and 595.3699, error value: 2.69 and 3.58 ppm Figure 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 of DCM, and a 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, each was washed once with 20.0 mL of water and saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate. Then, a solution of the crude product of fluoro building block 2-(perfluoro-t-butoxy)ethan-1-amine (837 mg, 3.0 mmol) dissolved in 10.0 mL of DCM was added to the above-mentioned 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 H 56 F9N5O8Na[M+Na] + Theoretical value: 856.3877, actual value: 856.3884, error value: 0.82 ppm Figure 10 ).

[0065] (7) Synthesis of compound 7: Ligand DO3A-9F.

[0066] The crude tBu-DO3A-9F from 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 give the crude DO3A-9F product, which was used directly for the following reaction. HR-ESI-MS (m / z) for C 22 H 33 F9N5O8[M+H] + and C 22 H 32 F9N5O8Na[M+Na] + Theoretical: 666.2180 and 688.1999, Actual: 666.2181 and 688.2003, Error: 0.09 and 0.52 ppm Figure 11 ). 19 F NMR Figure 12 , 564 MHz, CD3OD): δ -71.43 (9F, s).

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

[0068] The crude high fluorine content ligand DO3A-9F from previous step was dissolved in v / v 50% water / methanol mixed solvent and adjusted to pH ~ 3. After the addition of FeCl2(254 mg, 2.0 mmol), it was heated and stirred at 45 °C overnight under N2atmosphere. After the reaction was completed, it was purified by high performance liquid chromatography (HPLC) (0-60 min time program, eluent ratio 20% acetonitrile / 80% water - 100% acetonitrile, fractions collected at retention time 18.5-20.5 min). After the removal of solvent by rotary evaporation, it was freeze-dried to give 2.30 g of white to light yellow solid with 32% yield from step 6. Probes based on other paramagnetic metal ions can also be synthesized in a similar way. 19 F NMR Figure 13 , 564 MHz, 10% D2O / H2O): δ -68.51 (9F, br s); HR-ESI-MS (m / z) for C 22 H 29 FeF9N5O8[M-H] - Theoretical: 718.1228: Actual: 718.1215, Error: -1.71 ppm Figure 14 ).

[0069] It is worth mentioning that the content of fluorine in the molecular ligand (DO3A-9F) reaches 25.7%, which is significantly advantageous compared to most reported fluorine-containing small molecular magnetic resonance probes. The paramagnetic relaxation enhancement effect of the paramagnetic metal ions such as Fe 2+ can significantly shorten the relaxation time of F atomic nucleus in the molecular structure, thereby increasing the number of scans per unit time and effectively improving the signal-to-noise ratio of imaging. 19

[0070] After obtaining the probe molecule, the present application tests the relaxation time of F atomic nucleus. The relaxation time of F in the probe is tested as shown in Table 1 (the solvent is 10% D2O / H2O, the probe concentration is 1mM, and the magnetic resonance center frequency is set to 564MHz): 19 19

[0071] Table 1

[0072]

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

[0074] From the above table, it can be observed that the fluorine atomic nucleus in the chelate DO3A-Fe(II)-9F has a relatively short relaxation time, which means that DO3A-Fe(II)-9F is expected to obtain a fluorine atomic nucleus magnetic resonance signal with a stronger signal-to-noise ratio under the same imaging time. The control DO3A-Fe(III)-9F will further shorten the relaxation time of the fluorine atomic nucleus, making it much lower than the detection limit of the conventional sequence, and thus reducing the fluorine atomic nucleus magnetic resonance signal. This result lays a good foundation for subsequent imaging experiments.

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

[0076] DO3A-Fe(II)-9F is a molecular probe with good water solubility. In this embodiment, the in vitro imaging verification is carried out by dissolving DO3A-Fe(II)-9F in 1×PBS to prepare DO3A-Fe(II)-9F solution samples containing 10, 20, 30, 40, 50 and 60mM 19 F respectively. The relaxation time of F atomic nucleus in the probe is tested under different parameter groups (including different repetition times T R The probe limit sampling time is explored under different numbers of NEX, the probe limit imaging voxel size is explored under different slice thicknesses ST, and the resolution (MTX) is tested.

[0077] Figure 15 ​​​The results show that after chelating Fe 2+ ions, the probe DO3A-Fe(II)-9F has more significant imaging signal than its ligand DO3A-9F at shorter sampling time and corresponding parameter set, indicating that the PRE effect of Fe 2+ can be used to regulate 19 the F signal strategy successfully. Figure 16 The results show that the total relaxation time of the probe is between 100-200 ms on a 9.4T imager, so in the subsequent experiments, the repetition time T R = 200 ms is set. Figure 17 The results show that the signal-enhanced probe DO3A-Fe(II)-9F can detect signal at a shorter sampling time, with a limit of 20 circles and an imaging time of 12 s. Figure 18 The results show that the limit of signal detection thickness (FOV is constant) is 2 mm. Figure 19 The results show that the probe still has a good concentration-signal intensity linear relationship at high resolution. These experimental results indicate that the probe DO3A-Fe(II)-9F is expected to obtain higher time-spatial resolution information.

[0078] Based on the in vitro imaging results, in the in vivo experimental group, this embodiment configures a 1×PBS solution containing 25mM DO3A-Fe(II)-9F (i.e. 225mM 19 F concentration) and 1% sodium ascorbate, and injects it into the BALB / c mouse body through the tail vein. Immediately after injection, the mouse is evaluated by 19 F magnetic resonance imaging. Figures 20 to 23 )

[0079] Figure 20 Based on Figure 19 , high-resolution imaging of living bodies is attempted, and it is found that the probe can also obtain high-resolution information at the level of living bodies, with the signals of the gallbladder and bladder being the most significant. Figure 21 The results show that the probe has a larger enrichment concentration in the gallbladder, small intestine, and bladder regions, Figure 22 further verifying this fact. The significant signals in the liver-gallbladder and small intestine regions strongly indicate that the probe DO3A-Fe(II)-9F participates in the hepatobiliary circulation process in the organism. Figure 23 Based on Figure 20 the results, high-time-spatial resolution imaging monitoring of the living mouse is performed.

[0080] In summary, the 19The F MRI signal is significantly enriched in the liver-gallbladder region and gradually moves toward the small intestine, further proving that the drug is metabolized in vivo through the enterohepatic circulation (liver → gallbladder → small intestine → reabsorption). The metabolic flow chart is shown in Figure 2. Figure 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: 25mM DO3A-Fe(II)-9F was prepared, i.e., 225mM 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. After 3 days, all mice were killed, 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 the in vivo toxicity. It can be seen from the results that the probe has good biocompatibility ( Figure 25 and Figure 26 ), which also lays the foundation for the clinical transformation of the probe DO3A-Fe(II)-9F as a hepatoenterocirculation tracing probe.

Claims

1. A water-soluble signal-enhancing high-fluorine-content molecular imaging probe, characterized in that: The structural formula is as follows: Where M is Fe 2+ .

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

3. The use of the water-soluble signal-enhancing high-fluorine-content molecular imaging probe according to claim 1, characterized in that: Used to prepare reagents for dynamic real-time monitoring of enterohepatic circulation processes at the in vivo level.

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