Enzyme-targeted response type benzyl fluorine bridged glucoside gadolinium compound and application thereof as magnetic resonance contrast agent

Abstract

The invention discloses an enzyme targeted response type benzyl fluorine bridged glucoside gadolinium compound and application thereof as a magnetic resonance contrast agent. According to the invention, a high-stability macrocyclic gadolinium contrast agent is subjected to structural modification, a glycoside group and a benzyl fluorine bridging group with specific biological enzyme responsiveness are introduced, and the benzyl fluorine bridging group triggered by enzyme response forms a quinone methylation active intermediate; the quinone intermediate can react with a nucleophilic biomacromolecule functional group to form a covalent bond to obtain a gadolinium biomacromolecule compound, so that the magnetic resonance relaxation rate of gadolinium molecules can be remarkably improved, the enhancement of an MRI signal is realized, and the real-time specific enzyme tracking function of MRI is realized. Compared with the existing non-targeted responsive gadolinium contrast agent, the contrast agent provided by the invention has specific biological enzyme targeted responsiveness, can be used for real-time and non-invasive monitoring of diagnosis and treatment of enzyme function abnormality diseases, and has important significance in the aspects of precision medical treatment and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of magnetic resonance imaging, and particularly relates to an enzyme-targeting response type glycoside gadolinium compound bridged by benzyl fluoride and application thereof as a magnetic resonance contrast agent. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is a non-invasive, non-radioactive, high-biological penetration, high-resolution real-time 3D medical imaging technology, which is widely used for imaging of internal structures of human body. MRI uses a strong magnetic field and radio frequency waves to track the magnetic resonance relaxation process of water molecules in the body to generate real-time images of the internal structure of the body, and has irreplaceable advantages in clinical diagnosis of soft tissue, brain, spinal cord, joints and the like. The strength of the magnetic resonance signal depends on the relaxation rate of the protons of the water molecules in the local environment. Since the water protons have only one single electron, the magnetic dipole moment is weak, so the key limitation of MRI technology is its low imaging sensitivity.

[0003] In order to improve the imaging sensitivity of MRI technology, about 30%-40% of MRI scans in clinical diagnosis need to use MRI contrast agents for magnetic resonance enhancement. Since gadolinium metal ions have 7 unpaired single electrons, they are the best elements for magnetic resonance enhancement, and gadolinium contrast agents are the only type of MRI contrast agents that have been clinically approved and widely used. At present, about 8 gadolinium magnetic resonance contrast agents approved for use in Europe and the United States, such as Magnevist (Gd-DTPA), Dotarem (Gd-DTOA) and the like, are all MRI signal enhancement type contrast agents, but they do not have biological targeting responsiveness, cannot track specific life processes or disease types, and have low magnetic resonance relaxation rate (4-5 mM -1 s -1 ). In addition, about 4 early approved linear gadolinium contrast agents have low stability and safety problems of releasing biotoxic gadolinium ions, leading to problems of kidney-derived systemic fibrosis and brain gadolinium deposition, and have been restricted in use in Europe and the United States. At present, only about 4 macrocyclic gadolinium contrast agents are available for safe use in clinical practice. Therefore, the development of new types of gadolinium MRI contrast agents with biological targeting responsiveness, high relaxation rate and high stability is an important field of international frontier research and competition. SUMMARY

[0004] In view of the problems that the gadolinium magnetic resonance contrast agents approved for use in clinical practice do not have biological targeting responsiveness, have low relaxation rate, and cannot track specific life functions and disease states, the application provides an enzyme-targeting response type glycoside gadolinium compound bridged by benzyl fluoride and application thereof as a magnetic resonance contrast agent.

[0005] The structural formula of the enzyme-targeting response type glycoside gadolinium compound bridged by benzyl fluoride is as follows: , Wherein, R1 is a biological enzyme response group, and is specifically selected from one of glucoside, galactoside, aminohexoside, glucuronide, mannose, lactoside, cellulose glycoside, chitosan glycoside, and phosphate group; R2 is a group of enzymatic reaction and biological macromolecule reaction, and is specifically selected from one of F, CHF2, Br, Cl, and I; R3 is a gadolinium chelate MRI active group, and is specifically selected from one of gadolinium chelate of diethylene triamine pentaacetic acid (Gd-DTPA), gadolinium chelate of 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid (Gd-DO3A), gadolinium chelate of 2,2',2''-[3,6,9-triazine-1(2,6)-pyridine cyclododecane-3,6,9-triyl]tris{5-[(2,3-dihydroxypropyl)amino]-5-oxopentanoic acid} (Gadopiclenol), and gadolinium chelate of 4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazine-13-acid (Gd-BOPTA).

[0006] The preparation method of the enzyme-targeting response type glycoside gadolinium compound bridged by benzyl fluoride is as follows: (1) compound 1 and fluorination reagent are added into a dry organic solvent at a molar ratio of 1:1-5, and stirred for 0.5-5 hours at-20 o C-25 o C under nitrogen or inert gas atmosphere; after the reaction is completed, saturated sodium bicarbonate, ammonium chloride or sodium carbonate solution is added, and dichloromethane, ethyl acetate or chloroform is used for extraction, the organic phase is collected and concentrated, and finally, silica gel column chromatography is used for separation and purification; (2) the product prepared in step (1), compound 4 and carbonate are added into a dry acetonitrile, methanol or tetrahydrofuran solvent at a molar ratio of 1:1:1-2:1:5, and stirred for 8-24 hours at 0 o C - 25 o C under nitrogen or inert gas atmosphere; the supernatant is collected by centrifugation and concentrated by rotary evaporation, and the concentrated mixture is separated and purified by silica gel column chromatography, and then dissolved in THF, methanol, water or ethanol solvent; 10-50 molar amounts of lithium hydroxide or sodium hydroxide solution are added at 0 o C - 25 o C, and stirred for 1-10 hours; after that, an acidic reagent is used to adjust the pH to 5-7, and the solvent is removed by concentration; distilled water or deionized water is added, an acidic reagent or an alkaline reagent is used to adjust the pH value to 5-7, and 1-3 molar amounts of gadolinium reagent are added, and the temperature is adjusted to 100 oC stirring overnight, pH maintained at 5.5-7; centrifugal collection of supernatant was concentrated, redissolved in 0-0.1% ammonia solution, purified by filter membrane filtration, freeze-dried to obtain the enzyme-targeting responsive benzyl fluoride bridged glycoside gadolinium compound; The compound 1 is selected from one of glucoside-substituted bromoacetylamino benzyl alcohol compound, galactoside-substituted bromoacetylamino benzyl alcohol compound, aminohexoside-substituted bromoacetylamino benzyl alcohol compound, glucuronic acid-substituted bromoacetylamino benzyl alcohol compound, mannose-substituted bromoacetylamino benzyl alcohol compound, lactoside-substituted bromoacetylamino benzyl alcohol compound, cellulose glycoside-substituted bromoacetylamino benzyl alcohol compound, chitosan glycoside-substituted bromoacetylamino benzyl alcohol compound or phosphoric acid-substituted bromoacetylamino benzyl alcohol compound. The compound 4 is selected from one of diethylenetriamine pentaacetic acid chelate (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid chelate (DO3A), 2,2',2"-[3,6,9-triaza-1(2,6)-pyridinocyclododecane-3,6,9-triyl]tris{5-[(2,3-dihydroxypropyl)amino]-5-oxopentanoic acid} chelate, 4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid chelate (BOPTA).

[0007] The fluorination reagent is selected from one of diethylamino sulfur trifluoride, sulfur hexafluoride, bis(2-methoxyethyl)aminosulfur trifluoride.

[0008] The organic solvent is dichloromethane, trichloromethane, ethyl acetate or tetrahydrofuran; the carbonate is sodium carbonate or potassium carbonate.

[0009] The acidic reagent is hydrochloric acid, acetic acid or acetic acid; the basic reagent is sodium hydroxide, potassium hydroxide or lithium hydroxide solution.

[0010] The application of the above-mentioned enzyme-targeting responsive benzyl fluoride bridged glycoside gadolinium compound as a magnetic resonance contrast agent.

[0011] This invention modifies the structure of a highly stable macrocyclic gadolinium contrast agent by introducing specific enzyme-responsive glycosidic groups and benzylfluoro bridging groups. The enzyme-responsive benzylfluoro bridging groups form a quinone methylation active intermediate. This quinone intermediate can react with nucleophilic biomolecular functional groups to form covalent bonds, yielding a gadolinium biomolecular complex. This significantly improves the magnetic resonance relaxation rate of gadolinium molecules, enhancing MRI signals and enabling real-time, specific tracking of enzyme function in MRI. Compared to existing non-targeting gadolinium contrast agents, the contrast agent of this invention possesses specific enzyme-targeting responsiveness, making it suitable for real-time, non-invasive monitoring of the diagnosis and treatment of enzyme dysfunction diseases, and holding significant importance in precision medicine. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the galactosidase response mechanism of the GalFGd (6) molecule prepared in Example 1 to changes in magnetic resonance signals.

[0013] Figure 2 The graph shows the change in the magnetic resonance relaxation rate of the galactosidase response of the GalFGd (6) molecule prepared in Example 1.

[0014] Figure 3 The graph shows the variation of the galactosidase response magnetic resonance relaxation rate of the GalFGd (6) molecule prepared in Example 1 over a wide magnetic field range.

[0015] Figure 4 The GalFGd (6) molecule prepared in Example 1 was used for MRI imaging to track gene therapy in GM1 mice.

[0016] Figure 5 The high-resolution mass spectrum of the GalFGd (6) molecule prepared in Example 1. Detailed Implementation

[0017] Example 1: Gadolinium compounds responsive to galactosidase and fluorobridged The synthesis reaction formula is: , (1) Synthesis of compound 3: As shown in the above synthetic reaction formula, compound 1 and compound 2 were added to dry dichloromethane in a reaction equivalent ratio of 1:1.2, and the mixture was heated at 0°C. o The mixture was stirred for 0.5 h under a nitrogen atmosphere (C). A saturated NaHCO3 solution was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL × 3). The dichloromethane organic phase was collected and concentrated. The concentrated mixture was purified by silica gel column chromatography (eluent: 100 / 30 v / v dichloromethane / ethyl acetate) to give the white target product in 57% yield.

[0018] 1 H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.47(s, 1H), 7.07 (d, J = 8.85 Hz, 1H), 5.47-5.53 (m, 2H), 5.20-5.40 (m, 2H),4.99-5.12 (m, 2H), 4.04-4.25 (m, 2H),4.06-4.09 (m, 1H), 4.02 (s, 2H), 2.19(s, 3H), 2.07 (s, 6H), 2.02 (s, 3H). 13 C NMR (125 MHz, CDCl3) δ 170.34,170.19, 170.07, 169.52, 163.39, 151.65, 151.61, 132.41, 127.09, 126.96,121.98, 121.96, 121.39, 121.33, 115.88, 100.15, 80.18, 78.86, 71.21, 70.63,68.27, 66.83, 61.40, 53.43, 29.35, 20.68, 20.66, 20.58. HR-MS of 3C23H27BrFNO11(M+Na) + cal. 614.0649, Found. 614.0645. (2) Synthesis of GalFGd (6) molecule: Compound 3, Compound 4 and K2CO3 were added in dry acetonitrile solvent at a reaction equivalent of 1.2:1:3, the mixed solution was stirred at room temperature under nitrogen atmosphere for 18 hours. The supernatant was collected by centrifugation and concentrated by rotary evaporation, and the concentrated mixture was separated and purified by silica gel column chromatography (eluent: 95 / 5 v / v dichloromethane / methanol) to prepare Compound 5. Compound 5 was dissolved in THF (10 mL) and treated with 2-bromo-2-methylpropionyl bromide (0.5 mL, 4.5 mmol) at 0 oC condition, 20 equivalents of LiOH solution was added and stirred for 5 hours. The pH was adjusted to 6.5 using 0.5 M HC1 and concentrated to remove THF. 10 mL of deionized water was added to the concentrated solution, the pH was adjusted to 5.8-6.1 using a 0.5 M HC1 / 0.5 M NaOH system, 1.2 equivalents of GdCl3·6H2O was added, and it was stirred at room temperature overnight while maintaining the pH at 5.8-6.1. The supernatant was collected by centrifugation, concentrated, dissolved in 0.1% ammonia water, filtered through a 0.2 pm PVDF syringe filter, and freeze-dried to obtain the target gadolinium molecule, the benzyl fluoride bridged gadolinium compound GalFGd (6), with a total yield of about 16% for the three steps. The HR-MS of GalFGd (6) is shown in Figure 5 The target gadolinium molecule has a specific gadolinium isotope molecule, which matches the predicted molecular weight, proving the correctness of the structure of the target molecule.

[0019] 1.41T, 37 o C condition, the benzyl fluoride bridged gadolinium compound prepared above was subjected to the action of galactosidase, as shown in Figure 2 The magnetic resonance relaxation rate of the compound decreased with incubation time, and the MRI signal weakened in the PBS buffer system; while in the presence of serum protein (HSA), the magnetic resonance relaxation rate gradually increased, and the MRI signal was enhanced. This fully demonstrates that the compound forms a quinone methylation active intermediate in the enzymatic reaction, which can bond in situ with biological macromolecules such as serum proteins, etc., increase the size of the gadolinium molecule, and increase the relaxation rate and MRI signal.

[0020] As shown in Figure 3 In the 0.01 -HSA 100 MHz (2.3 * 10 -4 - 2.3 T) magnetic field range, the magnetic resonance relaxation rate of the compound in the PBS+HSA+galactosidase condition is higher than that of the control group without HSA or galactosidase in the whole test magnetic field strength, further confirming the bonding properties of the compound with biological macromolecules such as serum proteins in the enzymatic reaction, significantly improving the relaxation rate and MRI signal.

[0021] The benzyl fluoride bridged gadolinium compound prepared above was used as an MRI contrast agent to track GM1 gangliosidosis and gene diagnosis and treatment in real time: as Figure 4As shown, by means of intravenous injection, the GM1 mice with gene diagnosis and treatment have obvious MRI signal enhancement, and have high tissue resolution, the magnetic resonance strength gradually increases and is stable in the high MRI signal state, which proves that the in-situ bonding mode of the benzyl fluoride bridged gadolinium compound can effectively slow down the clearance rate of the molecule, prolong the MRI imaging window, and improve the imaging quality. For the control group, the MRI signal of the GM1 mice has no obvious enhancement, and the MRI signal gradually decreases with time, which is significantly different from the GM1 mice with gene diagnosis and treatment. The mouse experiment fully proves that the prepared benzyl fluoride bridged gadolinium compound can realize real-time and non-invasive MRI tracking of the gene diagnosis and treatment effect of the mice, which has important significance for promoting clinical conversion research.

Claims

1. An enzyme-targeted, responsive, benzyl fluoride-bridged, glycosyl gadolinium compound, characterized in that, The structural formula is as follows: ， Wherein, R1 is a biological enzyme response group, R2 is a group of enzymatic reaction and biological macromolecular reaction, R3 is a gadolinium chelate MRI active group.

2. The enzyme-targeted responsive benzyl fluoride-bridged glycosyl Gd-based compound according to claim 1, characterized in that, R1 is specifically selected from one of glucoside, galactoside, aminohexoside, glucuronide, mannose, lactoside, cellulose sugar, chitosan sugar and phosphoric acid group.

3. The enzyme-targeted responsive benzyl fluoride-bridged glycosyl Gd-based compound according to claim 1, characterized in that, R2 is specifically selected from one of F, CHF2, Br, Cl and I.

4. The enzyme-targeted responsive benzyl fluoride-bridged glycosyl Gd-based compound of claim 1, wherein, R3 is specifically selected from one of gadolinium diethylene triamine pentaacetic acid chelate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid gadolinium chelate, 2,2',2''-[3,6,9-triazine-1(2,6)-pyridine ring ten-3,6,9-triyl] tri{5-[(2,3-dihydroxypropyl) amino]-5-oxopentanoic acid} gadolinium chelate, 4-carboxy-5,8,11-tri(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazine-13- acid gadolinium chelate.

5. A process for the preparation of an enzyme-targeted, responsive, benzyl fluoride-bridged glycosyl gadolinium complex, characterized by, The specific steps of the preparation method are: (1) adding compound 1 and fluorination reagent in a molar ratio of 1:1-5 into a dry organic solvent, stirring the reaction at -20 o C-25 o C temperature under nitrogen or inert gas atmosphere for 0.5-5 hours; after the reaction is completed, adding saturated sodium bicarbonate, ammonium chloride or sodium carbonate solution, and extracting with dichloromethane, ethyl acetate or chloroform, collecting the organic phase and concentrating, and finally separating and purifying by silica gel column chromatography; (2) adding the product prepared in step (1), compound 4 and carbonate in a molar ratio of 1:1:1-2:1:5 into a dry acetonitrile, methanol or tetrahydrofuran solvent, stirring the reaction at 0 o C - 25 o C temperature under nitrogen or inert gas atmosphere for 8-24 hours; centrifuging to collect the supernatant and rotary evaporating to concentrate, separating and purifying the concentrated mixture by silica gel column chromatography, and then dissolving in THF, methanol, water or ethanol solvent; adding 10-50 molar amounts of lithium hydroxide or sodium hydroxide solution at 0 o C - 25 o C temperature, stirring the reaction for 1-10 hours, then adjusting the pH to 5-7 using an acidic reagent, and concentrating to remove the solvent; adding distilled water or deionized water, adjusting the pH to 5-7 using an acidic reagent or a basic reagent, and then adding 1-3 molar amounts of gadolinium reagent at room temperature to 100 o C overnight, keeping the pH at 5.5-7; centrifuging to collect the supernatant and concentrate, and then dissolving in 0-0.1% ammonia water solution, filtering and purifying through a filter membrane, and freeze-drying to obtain the enzyme-targeting responsive benzyl fluoride bridged glycoside gadolinium compound. The compound 1 is selected from one of glucoside-substituted bromoacetylamino benzyl alcohol compound, galactoside-substituted bromoacetylamino benzyl alcohol compound, aminohexoside-substituted bromoacetylamino benzyl alcohol compound, glucuronide-substituted bromoacetylamino benzyl alcohol compound, mannose-substituted bromoacetylamino benzyl alcohol compound, lactoside-substituted bromoacetylamino benzyl alcohol compound, cellulose sugar-substituted bromoacetylamino benzyl alcohol compound, chitosan sugar-substituted bromoacetylamino benzyl alcohol compound or phosphoric acid group-substituted bromoacetylamino benzyl alcohol compound. The compound 4 is selected from one of diethylene triamine pentaacetic acid chelate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid chelate, 2,2',2''-[3,6,9-triazine-1(2,6)-pyridine ring ten-3,6,9-triyl] tri{5-[(2,3-dihydroxypropyl) amino]-5-oxopentanoic acid} chelate, 4-carboxy-5,8,11-tri(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazine-13- acid chelate.

6. The preparation method according to claim 5, characterized in that, The fluorination reagent is selected from one of diethylamino sulfur trifluoride, sulfur hexafluoride and bis(2-methoxyethyl)amino sulfur trifluoride.

7. The preparation method according to claim 5, characterized in that, The organic solvent is dichloromethane, trichloromethane, ethyl acetate or tetrahydrofuran; the carbonate is sodium carbonate or potassium carbonate.

8. The preparation method according to claim 5, characterized in that, The acidic reagent is hydrochloric acid, acetic acid or acetic acid; the basic reagent is sodium hydroxide, potassium hydroxide or lithium hydroxide solution.

9. The preparation method according to claim 5, characterized in that, The gadolinium reagent is gadolinium trichloride, gadolinium oxide or gadolinium nitrate.

10. The use of the enzyme-targeting response benzyl fluoride bridged glycoside gadolinium compound according to any one of claims 1-4 as a magnetic resonance contrast agent.

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