A magnetic resonance imaging contrast agent Hyp-DOTA-Gd and its preparation method and application

Abstract

The invention discloses a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd and a preparation method and application thereof. The method comprises: reacting DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in anhydrous DMF, and then purifying to obtain the compound Hyp-DOTA; dissolving the compound Hyp-DOTA in a PBS solution, and then adding gadolinium chloride for chelation reaction to obtain the nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd; the contrast agent of the invention is a ligand Hyp-DOTA chelated with Gd 3+ The contrast agent sequentially exerts three functions: glomerular filtration, imaging of ROS-activated renal enrichment, and enzymatic renal clearance. It is expected to achieve in vivo imaging of renal ROS content and dynamic distribution while avoiding renal damage.

Description

Technical Field

[0001] The present invention relates to multidisciplinary cross-fields such as material science, nephrology, and medical imaging, and in particular to a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd and a preparation method and application thereof. Background Art

[0002] Studies have found that renal reactive oxygen species (ROS) are key markers for the early diagnosis of acute kidney injury and play an important role in the occurrence and development of acute kidney injury. Optical detection methods for renal ROS have been widely used in the early diagnosis of acute kidney injury, but the penetration depth of light in tissues is limited and difficult to apply clinically. Magnetic resonance imaging (MRI) has the characteristics of non-invasiveness, unlimited penetration depth, and high spatial resolution, but it is difficult to accurately image the content and dynamic distribution of renal ROS. Most of the reported ROS-responsive MRI nanocontrast agents have a hydrated particle size (20-200nm) far exceeding the glomerular filtration membrane clearance threshold (6-8nm). They are difficult to pass through glomerular filtration in the early stage of renal injury, and cannot be enriched in the kidney and specifically image renal ROS in the early stage of acute kidney injury. In addition, they are affected by urination, and their retention time in the kidney is short and the enrichment rate is low, resulting in the fluctuation of the MRI signal in the kidney with urination, which cannot accurately reflect the physiological and pathological changes of renal ROS.

[0003] Therefore, it is necessary to develop a magnetic resonance imaging contrast agent Hyp-DOTA-Gd that can solve the above technical problems. Summary of the invention

[0004] The purpose of the present invention is to provide a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd and its preparation method and application. The nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd integrates three functions: glomerular filtration, renal enrichment of ROS activation, and enzymatic renal clearance, and can play a role in sequence. It is expected to achieve quantitative imaging of renal ROS in the early stage of acute kidney injury, while avoiding kidney damage. Based on this, an early non-invasive imaging diagnosis method for acute kidney injury is developed, and a scientific prevention and treatment plan is adopted in advance for high-risk patients, which has important clinical significance for the treatment and recovery of kidney injury.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a magnetic resonance imaging contrast agent Hyp-DOTA-Gd is provided, and the structural formula of the contrast agent is as follows:

[0007]

[0008] In a second aspect of the present invention, a method for preparing a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd is provided, the method comprising:

[0009] DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are reacted in anhydrous DMF, and then purified to obtain compound Hyp-DOTA;

[0010] The compound Hyp-DOTA is dissolved in a PBS solution, and then gadolinium chloride is added for chelation reaction to obtain a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd. The reaction formula is as follows:

[0011]

[0012] Furthermore, the molar ratio of the compound Hyp-DOTA to the gadolinium chloride is 1:1.

[0013] Furthermore, the pH of the PBS solution is 6.8.

[0014] Preferably, the chelating reaction is carried out under stirring conditions.

[0015] Furthermore, the usage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and anhydrous DMF is (0.1-0.25) g: (0.06-0.1) g: (0.01-0.02) g: (17-18) μl: (0.2-0.4) ml.

[0016] Furthermore, the usage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and anhydrous DMF is 0.197g:0.08g:0.012g:17.5μl:0.3ml.

[0017] Furthermore, in the reaction for preparing the compound Hyp-DOTA, the reaction temperature is 20 to 30° C. and the reaction time is 8 to 15 hours.

[0018] In a second aspect of the present invention, a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd prepared by the method is provided.

[0019] In the third aspect of the present invention, there is provided a use of the magnetic resonance imaging contrast agent Hyp-DOTA-Gd in the preparation of an imaging tool for acute kidney injury.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] 1. A magnetic resonance imaging contrast agent Hyp-DOTA-Gd of the present invention comprises H 2 O 2 and ClO - The boronate ester bond is highly sensitive to ROS and has an inactive ester bond with low sensitivity to esterase. 2 O 2 or ClO - The fast reaction can form highly reactive quinone methyl groups that can react and bind with protein nucleophilic groups to enhance T1WI signals and achieve H 2 O 2 and ClO - Enrichment and imaging; the inactive ester bond is slowly hydrolyzed under the action of intracellular esterase to achieve renal clearance of the contrast agent. The three functions of the contrast agent, glomerular filtration, ROS-activated renal enrichment imaging, and enzymatic renal clearance, play a role in sequence. The present invention utilizes ① the property that the highly reactive quinone methyl group can react and bind with protein nucleophilic groups (such as amino groups); ② the property that the inactive ester bond can be slowly hydrolyzed under the action of esterase, and innovatively designs a programmed self-fixation and renal clearance sub-component magnetic resonance imaging contrast agent Hyp-DOTA-Gd, which integrates the three functions of glomerular filtration, ROS-activated renal enrichment, and enzymatic renal clearance, and can play a role in sequence, and non-invasively obtain ROS (H) in the kidney area in vivo. 2 O 2 On this basis, we developed a specific quantitative imaging method for renal ROS, selectively enhanced T1WI signals in the kidney, dynamically acquired the content and spatial distribution of ROS in the kidney, and established a magnetic resonance imaging method for renal ROS, which made up for the shortcomings of current magnetic resonance imaging in selectively imaging renal ROS. It provides a new strategy for the early non-invasive imaging diagnosis of acute kidney injury, and also provides an imaging tool for systematically studying the mechanism of action of dynamic changes of ROS in acute kidney injury.

[0022] 2. The preparation method of the magnetic resonance imaging contrast agent Hyp-DOTA-Gd of the present invention introduces Hyp with oxidation response. Under the stimulation of oxidation response, Hyp can generate compounds with active free radicals, which can specifically bind to proteins in the renal injury site, so that the contrast agent is enriched in the kidney and T1 imaging is enhanced. The inactive ester bond is broken under the action of intracellular esterase, and the contrast agent accumulated in the kidney is slowly excreted from the body after imaging, avoiding damage to the kidney by the contrast agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are result data of some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 It is the synthetic route of Hyp-DOTA-Gd;

[0025] Figure 2 This is the H NMR spectrum of the prepared contrast agent Hyp-DOTA-Gd;

[0026] Figure 3 Gd in different groups of HK-2 cells 3+ concentration and T1WI signals of cell dispersions;

[0027] Figure 4 Confocal images of the ROS production results of HK-2 cells in different groups (A) and H 2 O 2 Relative content graph (B);

[0028] Figure 5 are the serum creatinine and urea nitrogen concentrations of mice at different time points after cisplatin injection;

[0029] Figure 6 It is the confocal image of ROS in mouse kidney at different time points after injection of cisplatin;

[0030] Figure 7 is the H level in the kidney of mice at different time points after injection of cisplatin 2 O 2 Relative concentration diagram of

[0031] Figure 8 is the H&E staining of mouse kidneys at different time points after cisplatin injection;

[0032] Fig. 9 These are the imaging effects and signal intensity diagrams of different contrast agents at different time points after the injection of cisplatin. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0036] According to a typical implementation of an embodiment of the present invention, a magnetic resonance imaging contrast agent Hyp-DOTA-Gd is provided, and the structural formula of the contrast agent is as follows:

[0037]

[0038] According to another typical implementation of the present invention, a method for preparing a magnetic resonance imaging contrast agent Hyp-DOTA-Gd is provided, the method comprising:

[0039] DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are reacted in anhydrous DMF, and then purified to obtain compound Hyp-DOTA;

[0040] The compound Hyp-DOTA is dissolved in a PBS solution, and then gadolinium chloride is added for chelation reaction to obtain a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd. The reaction formula is as follows:

[0041]

[0042] In the above technical solution, the compound 5 can be directly purchased or synthesized.

[0043] As an optional embodiment, the preparation method of compound 5 comprises:

[0044] S1, adding methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide and 2,2-azobisisobutyronitrile to an organic solution for reaction, and then filtering, rotary evaporating and purifying to obtain compound 1;

[0045] S2, dissolving the compound 1 in a dioxane aqueous solution containing silver sulfate for reaction, and then filtering and extracting, collecting the organic phase solution and spinning it to dryness, to obtain compound 2;

[0046] S3, reacting the compound 2 with diethylaminosulfur trifluoride in an organic solvent, and then washing and purifying to obtain compound 3;

[0047] S4, reacting the compound 3, bis(pinacol)diboron, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate in a DMF solution, then adding [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride to continue the reaction, adding ether to the reaction solution after the reaction, and then washing, collecting the organic layer solvent and drying to obtain compound 4;

[0048] S5, dissolving the compound 4 in THF / H 2 O(3 / (1) was then added with LiOH for reaction, followed by extraction, collection of the organic layer and rotary evaporation to obtain compound 5;

[0049] The reaction formula for preparing the compound 5 is as follows:

[0050]

[0051] In the above technical solution, the DOTA-OH can be directly purchased or synthesized.

[0052] As an optional embodiment, the preparation method of DOTA-OH comprises:

[0053] 1,4,7,10-tetraazacyclododecane and tert-butyl bromoacetate were reacted in anhydrous acetonitrile, and then cesium carbonate was added to continue the reaction. After the reaction was completed, the filtrate was collected, spin-dried and purified to obtain compound 6;

[0054] The compound 6 and 2-bromoethanol are reacted in anhydrous acetonitrile, and then cesium carbonate is added to continue the reaction. After the reaction is completed, the filtrate is filtered, collected, spin-dried and purified to obtain the compound DOTA-OH.

[0055] As an optional specific embodiment of the present invention, the present invention provides a method for preparing a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd, comprising the following steps:

[0056]

[0057] S1, adding methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide and 2,2-azobisisobutyronitrile to a carbon tetrachloride solution, reacting for a period of time, filtering and rotary evaporating the reaction solution, and purifying to obtain compound 1;

[0058] S2, dissolving compound 1 in dioxane / pure water (1 / 1) containing silver sulfate, filtering the reaction solution after a period of reaction, extracting, collecting the organic phase solution and drying it to obtain compound 2;

[0059] S3, adding compound 2 and diethylaminosulfur trifluoride to anhydrous dichloromethane and reacting for a period of time, washing the reaction solution, and purifying to obtain compound 3;

[0060] S4. Compound 3, bis(pinacol)diboron, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate are added to the DMF solution in sequence to react for a period of time, and [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride is added again to continue the reaction. After the reaction is completed, ether is added to the reaction solution for washing. The organic layer solvent is collected and dried to obtain compound 4;

[0061] S5, compound 4 was dissolved in THF / H 2 O (3 / 1) and then LiOH was added to react for a period of time, the solution was diluted, extracted, the organic layer was collected, and the compound 5 was obtained by rotary evaporation;

[0062] S6, 1,4,7,10-tetraazacyclododecane, and tert-butyl bromoacetate were added to anhydrous acetonitrile in sequence, and cesium carbonate was added after a period of reaction to continue the reaction. After the reaction was completed, the filtrate was collected, dried by spin drying, and purified to obtain compound 6;

[0063] S7, compound 6 and 2-bromoethanol were added to anhydrous acetonitrile in sequence, and cesium carbonate was added after a period of reaction to continue the reaction. After the reaction was completed, the filtrate was collected and spin-dried. The crude product was dissolved in DCM / TFA (1 / 1), reacted in an ice bath for a period of time, and then spin-dried to obtain the compound DOTA-OH after purification;

[0064] S8, DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine were added to anhydrous DMF in sequence. After reacting at room temperature for a period of time, the compound Hyp-DOTA was purified. An equimolar amount of gadolinium chloride was added to a PBS (pH=6.8) solution containing the compound Hyp-DOTA, and stirred overnight to obtain the contrast agent Hyp-DOTA-Gd.

[0065] Steps S1, S2, S3, S4, S5, S6, S7, and S8 in the embodiment of the present invention do not represent a strict sequential relationship and the order can be changed according to the situation. For example, S6 and S7 are steps for preparing DOTA-OH and can also be used as S1 and S2.

[0066] As one of the preferred schemes of the specific implementation scheme, the reaction in step S1 is refluxed for 12 hours under a nitrogen atmosphere, and the purification is separation and purification by a silica gel column (hexane / ethyl acetate=20 / 1);

[0067] Preferably, the usage ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile and carbon tetrachloride is 2.3 g: 1.9 g: 0.164 g: 25 ml.

[0068] As one of the preferred schemes of the specific implementation scheme, the reaction in step S2 is refluxed under light-proof conditions, and the reaction time is 1 hour; the extraction is ethyl acetate extraction three times;

[0069] Preferably, the usage ratio of silver sulfate and dioxane / pure water (1 / 1) is 0.499 g:20 ml.

[0070] As one of the preferred schemes of the specific implementation scheme, the reaction in step S3 is carried out under a nitrogen atmosphere at 0°C for 12 hours; the washing is carried out three times with pure water and brine; the purification is carried out by separation and purification with a silica gel column (hexane / ethyl acetate = 20 / 1);

[0071] Preferably, the usage ratio of anhydrous dichloromethane, compound 2 and diethylaminosulfur trifluoride is 20 ml: 0.235 g: 0.0464 g.

[0072] As one of the preferred schemes of the specific implementation scheme, the reaction in step S4 is carried out under a nitrogen atmosphere, heated to 100°C for 3 hours; the continued reaction time is 4 hours; washing is performed three times with pure water and salt water; drying is performed with anhydrous sodium sulfate;

[0073] Preferably, the usage ratio of the DMF solution, compound 3, bis(pinacol)diboron, the initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, the added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate and ether is 5ml:0.185g:0.228g:0.306g:0.306g:0.221g:50ml.

[0074] As one of the preferred schemes of the specific implementation scheme, the reaction in the step S5 is carried out at 0°C for 12 hours; the extraction is performed by ethyl acetate extraction three times;

[0075] Preferably, the compound 4, THF / H 2 The usage ratio of O(3 / 1) and LiOH is 0.0151 g:0.8 ml:150 μl.

[0076] As one of the preferred schemes of the specific implementation scheme, the reaction in step S6 is heated to reflux at 100° C. for 1 hour; the continued reaction time is 24 hours; and the purification is performed by HPLC;

[0077] Preferably, the usage ratio of 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, anhydrous acetonitrile and cesium carbonate is 0.172 g: 0.585 g: 10 ml: 0.977 g.

[0078] As one of the preferred schemes of the specific implementation scheme, in the step S7, the reaction is heated to reflux at 100° C. for 1 hour; the continued reaction time is 24 hours; the reaction is carried out in an ice bath for 12 hours, and the purification is carried out by HPLC;

[0079] Preferably, the usage ratio of the compound 6, 2-bromoethanol, anhydrous acetonitrile, cesium carbonate and DCM / TFA (1 / 1) is 0.307 g: 0.248 g: 10 ml: 0.664 g: 10 ml.

[0080] As one of the preferred schemes of the specific implementation scheme, the reaction time in the step S8 is 12 hours; the purification is carried out by HPLC;

[0081] Preferably, the usage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, anhydrous DMF and gadolinium chloride is 0.197g:0.08g:0.012g:17.5μl:0.3ml:0.072g.

[0082] The present invention also provides a contrast agent prepared by the above-mentioned method for preparing a self-fixing and renal-clearing magnetic resonance imaging contrast agent, wherein the contrast agent contains H 2 O 2 The boronate esters are highly sensitive to esterases and ClO-(ROS) and the inactive ester bonds are less sensitive to esterases.

[0083] The following will describe in detail a magnetic resonance imaging contrast agent Hyp-DOTA-Gd and its preparation method and application in combination with embodiments, comparative examples and experimental data.

[0084] Example 1: An oxidation-responsive self-fixing and renal-cleared magnetic resonance imaging contrast agent and its preparation method

[0085] 1. First, prepare programmed ROS (H 2 O 2 and ClO - ) The ligand Hyp-DOTA, which responds to self-immobilization and enzymatic renal clearance of contrast agents, was characterized by H NMR spectroscopy. Figure 2 The preparation method of the Hyp-DOTA is as follows:

[0086] (1) Dissolve methyl 4-bromo-3-methylbenzoate (2.3 g, 10 mmol), N-bromosuccinimide (1.9 g, 11 mmol), and 2,2-azobisisobutyronitrile (0.164 g, 1 mmol) in 25 mL of carbon tetrachloride solution and reflux for 12 h under N2 protection. After the reaction is completed, the reaction solution is filtered and rotary evaporated. The crude product is separated and purified by silica gel column chromatography (hexane / ethyl acetate = 20 /

[0087] (1) obtaining compound 1;

[0088] (2) Compound 1 was dissolved in 20 mL of dioxane / pure water (1 / (1)) containing silver sulfate (0.499 g, 1.6 mmol), and refluxed for 1 h in the dark. After the reaction was completed, the reaction solution was filtered and extracted three times with ethyl acetate. The organic phase solution was collected and dried to obtain compound 2.

[0089] (3) Compound 2 (0.235 mg, 0.96 mmol) and diethylaminosulfur trifluoride (0.0464 g, 2.88 mmol) were added to 20 mL of anhydrous dichloromethane and reacted at 0°C for 12 h under N2 protection. After the reaction, the reaction solution was washed three times with pure water and brine respectively. The crude product was separated and purified by silica gel column chromatography (hexane / ethyl acetate = 20 / (1)) to obtain compound 3.

[0090] (4) Compound 3 (0.185 g, 0.75 mmol), bis(pinacolato)diboron (0.228 g, 0.9 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (0.306 g, 0.0375 mmol) and potassium acetate (0.221 g, 2.25 mmol) were added to 5 mL of DMF solution in sequence, and heated to 100°C under N2 protection. After reacting for 3 h, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (0.306 g, 0.0375 mmol) was added again, and the reaction was continued for 4 h. After the reaction was completed, 50 mL of ether was added to the reaction solution, and the solution was washed three times with water and brine in sequence. The organic layer solvent was collected, and anhydrous sodium sulfate was added to dry to obtain compound 4.

[0091] (5) 0.8 mL THF / H 2 O(3 / (1) LiOH (150 μL, 0.15 mmol) was added. The above solution was reacted at 0°C for 12 h, and then 1 mL of pure water was added to dilute it. Finally, it was extracted three times with ethyl acetate, the organic layer was collected, and the ethyl acetate was removed by rotary evaporation to obtain compound 5.

[0092] (6) 1,4,7,10-Tetraazacyclododecane (0.172 g, 1 mmol) and tert-butyl bromoacetate (0.585 g, 3 mmol) were added to 10 mL of anhydrous acetonitrile. After heating under reflux at 100°C for 1 h, cesium carbonate (0.977 g, 3 mmol) was added and the reaction was continued for 24 h. After the reaction was completed, the filtrate was collected and dried, and purified by HPLC to obtain compound 6.

[0093] (7) Compound 6 (0.307 g, 1 mmol) and 2-bromoethanol (0.248 g, 2 mmol) were added to 10 mL of anhydrous acetonitrile in sequence. After heating under reflux at 100°C for 1 h, cesium carbonate (0.664 g, 2 mmol) was added and the reaction was continued for 24 h. After the reaction was completed, the filtrate was collected and dried by rotation. The crude product was dissolved in 10 mL of DCM / TFA (1 / (1), reacted in an ice bath for 12 h, dried by rotation, and purified by HPLC to obtain compound DOTA-OH.

[0094] (8) DOTA-OH (0.197 g, 0.5 mmol), compound 5 (0.08 g, 0.275 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.012 g, 0.0325 mmol), and N,N-diisopropylethylamine (17.5 μL, 0.1 mmol) were added to 0.3 mL of anhydrous DMF in sequence. After reacting at room temperature for 12 h, the compound Hyp-DOTA was separated and purified by HPLC.

[0095] 2. Add an equimolar amount of GdCl to the PBS (pH = 6.8) solution containing the ligand Hyp-DOTA. 3 , through ligand chelation of Gd 3+ The contrast agent is Hyp-DOTA-Gd; specifically:

[0096] Gadolinium chloride (0.072 g, 0.275 mmol)) was added to a PBS solution (pH=6.(8)) containing the compound Hyp-DOTA, and the mixture was stirred overnight to obtain a contrast agent Hyp-DOTA-Gd.

[0097] Example 2: A self-fixing and renal-cleared MRI contrast agent Hyp-DOTA-Gd and its preparation method

[0098] Embodiment 2 is substantially the same as Embodiment 1, except that:

[0099] In step (1), the reflux reaction is carried out under a nitrogen atmosphere for 18 hours; the amount ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile and carbon tetrachloride is 3.45 g: 2.59 g: 0.246 g: 35 ml.

[0100] In step (2), reflux for 2 hours under light-proof conditions; the amount ratio of silver sulfate and dioxane / pure water (1 / (1)) is 0.749 g:30 ml.

[0101] In step (3), the reaction was carried out at 0°C under a nitrogen atmosphere for 24 hours; the usage ratio of anhydrous dichloromethane, compound 2, and diethylaminosulfur trifluoride was 30 ml: 0.3525 g: 0.0696 g.

[0102] In step (4), the reaction is heated to 100°C under a nitrogen atmosphere for 5 hours; the dosage ratio of DMF solution, compound 3, bis(pinacol)diboron, initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and ether is 8ml:0.2775g:0.342g:0.459g:0.459g:0.332g:75ml.

[0103] In step (5), the solution is reacted at 0°C for 24 hours; compound 4, THF / H 2 The usage ratio of O(3 / (1) and LiOH is 0.0226 g:1.2 ml:225 μl.

[0104] In step (6), the reaction is heated under reflux at 100° C. for 2 hours; the dosage ratio of 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, anhydrous acetonitrile and cesium carbonate is 0.258 g: 0.8775 g: 15 ml: 1.4655 g.

[0105] In step (7), the reaction is heated under reflux at 100° C. for 2 hours; the amount ratio of compound 6, 2-bromoethanol, anhydrous acetonitrile, cesium carbonate, and DCM / TFA (1 / (1)) is 0.46 g: 0.372 g: 15 ml: 0.996 g: 10 ml.

[0106] In step (8), the reaction time at room temperature is 24 hours; the dosage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, anhydrous DMF, and gadolinium chloride is 0.295 g: 0.12 g: 0.015 g: 20 μl: 0.4 ml: 0.108 g.

[0107] Example 3: A self-fixing and renal-cleared MRI contrast agent Hyp-DOTA-Gd and its preparation method

[0108] Embodiment 3 is substantially the same as Embodiment 1, except that:

[0109] In step (1), the reflux reaction is carried out under a nitrogen atmosphere for 12 hours; the amount ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile and carbon tetrachloride is 3.85 g: 2.59 g: 0.395 g: 30 ml.

[0110] In step (2), reflux for 1 hour under light-proof conditions; the amount ratio of silver sulfate to dioxane / pure water (1 / (1)) is 0.849 g:30 ml.

[0111] In step (3), the reaction was carried out at 0°C for 12 hours under a nitrogen atmosphere; the usage ratio of anhydrous dichloromethane, compound 2, and diethylaminosulfur trifluoride was 30 ml: 0.368 g: 0.0776 g.

[0112] In step (4), the reaction is heated to 100°C under a nitrogen atmosphere for 5 hours; the dosage ratio of DMF solution, compound 3, bis(pinacol)diboron, initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and ether is 8ml:0.2775g:0.342g:0.585g:0.585g:0.332g:75ml.

[0113] In step (5), the solution is reacted at 0°C for 12 hours; compound 4, THF / H 2 The usage ratio of O(3 / (1) and LiOH is 0.0226 g:1.5 ml:250 μl.

[0114] In step (6), the reaction is heated under reflux at 100° C. for 1 hour; the dosage ratio of 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, anhydrous acetonitrile and cesium carbonate is 0.313 g:1.05 g:15 ml:1.775 g.

[0115] In step (7), the reaction is heated under reflux at 100° C. for 1 hour; the amount ratio of compound 6, 2-bromoethanol, anhydrous acetonitrile, cesium carbonate, and DCM / TFA (1 / (1)) is 0.521 g: 0.372 g: 15 ml: 0.996 g: 10 ml.

[0116] In step (8), the reaction time at room temperature is 12 hours; the dosage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, anhydrous DMF, and gadolinium chloride is 0.295 g: 0.144 g: 0.015 g: 20 μl: 0.4 ml: 0.13 g.

[0117] Example 4: A self-fixing and renal-cleared MRI contrast agent Hyp-DOTA-Gd and its preparation method

[0118] Embodiment 4 is substantially the same as Embodiment 1, except that:

[0119] In step (1), the reflux reaction is carried out under a nitrogen atmosphere for 18 hours; the amount ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile and carbon tetrachloride is 3.945 g: 2.89 g: 0.395 g: 40 ml.

[0120] In step (2), reflux for 3 hours under light-proof conditions; the amount ratio of silver sulfate and dioxane / pure water (1 / (1)) is 0.996 g:30 ml.

[0121] In step (3), the reaction was carried out at 0°C for 18 hours under a nitrogen atmosphere; the usage ratio of anhydrous dichloromethane, compound 2, and diethylaminosulfur trifluoride was 30 ml: 0.468 g: 0.177 g.

[0122] In step (4), the reaction is heated to 100°C under a nitrogen atmosphere for 5 hours; the dosage ratio of DMF solution, compound 3, bis(pinacol)diboron, initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and ether is 10ml: 0.3445g: 0.368g: 0.674g: 0.674g: 0.388g: 75ml.

[0123] In step (5), the solution is reacted at 0°C for 18 hours; compound 4, THF / H 2 The usage ratio of O(3 / (1) and LiOH is 0.0336 g:1.5 ml:275 μl.

[0124] In step (6), the reaction is heated under reflux at 100° C. for 1 hour; the dosage ratio of 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, anhydrous acetonitrile and cesium carbonate is 0.387 g:1.55 g:15 ml:1.975 g.

[0125] In step (7), the reaction is heated under reflux at 100° C. for 1 hour; the amount ratio of compound 6, 2-bromoethanol, anhydrous acetonitrile, cesium carbonate, and DCM / TFA (1 / (1)) is 0.661 g: 0.572 g: 15 ml: 0.996 g: 10 ml.

[0126] In step (8), the reaction time at room temperature is 12 hours; the dosage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, anhydrous DMF, and gadolinium chloride is 0.295 g: 0.189 g: 0.015 g: 20 μl: 0.4 ml: 0.17 g.

[0127] Example 5: A self-fixing and renal-cleared MRI contrast agent Hyp-DOTA-Gd and its preparation method

[0128] Embodiment 5 is substantially the same as Embodiment 1, except that:

[0129] In step (1), the reflux reaction is carried out under a nitrogen atmosphere for 18 hours; the dosage ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile and carbon tetrachloride is 4.045 g: 2.97 g: 0.395 g: 40 ml.

[0130] In step (2), reflux for 3 hours under light-proof conditions; the amount ratio of silver sulfate and dioxane / pure water (1 / (1)) is 0.996 g:35 ml.

[0131] In step (3), the reaction was carried out at 0°C under a nitrogen atmosphere for 24 hours; the usage ratio of anhydrous dichloromethane, compound 2, and diethylaminosulfur trifluoride was 30 ml: 0.668 g: 0.279 g.

[0132] In step (4), the reaction is heated to 100°C under a nitrogen atmosphere for 5 hours; the dosage ratio of DMF solution, compound 3, bis(pinacol)diboron, initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and ether is 15ml:0.377g:0.398g:0.694g:0.694g:0.428g:75ml.

[0133] In step (5), the solution is reacted at 0°C for 24 hours; compound 4, THF / H 2 The usage ratio of O(3 / (1) and LiOH is 0.0536 g:1.5 ml:275 μl.

[0134] In step (6), the reaction is heated under reflux at 100° C. for 1 hour; the dosage ratio of 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, anhydrous acetonitrile and cesium carbonate is 0.387 g:1.55 g:18 ml:2.575 g.

[0135] In step (7), the reaction is heated under reflux at 100° C. for 1 hour; the amount ratio of compound 6, 2-bromoethanol, anhydrous acetonitrile, cesium carbonate, and DCM / TFA (1 / (1)) is 0.696 g: 0.572 g: 15 ml: 0.996 g: 10 ml.

[0136] In step (8), the reaction time at room temperature is 12 hours; the dosage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, anhydrous DMF, and gadolinium chloride is 0.375 g: 0.239 g: 0.045 g: 20 μl: 0.4 ml: 0.216 g.

[0137] Experimental Example 1

[0138] The contrast agent of Example 1 was co-cultured with human renal cortical proximal tubule epithelial cells (HK-2), and then H 2 O 2 The final concentration was 200 μM and the culture was continued for 4 h. HK-2 cells were collected and divided into two parts. One part of the cells was measured for protein content using the BCA protein kit. The second part of the cells was nitrified and dissolved, and then analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). 2 O 2 Gd in treated cells 3+ The concentration (125.3 μg mg-1 protein) was significantly higher than that of the control group 2 O 2 Gd in treated cells 3+ Concentration (75.6μg mg-1protein), reference Figure 3 (Left). At the same time, cells were co-cultured with contrast agents and then added with different concentrations of H 2 O 2 Magnetic resonance imaging showed that the signal intensity of T1WI increased with the increase of H 2 O 2 The concentration of H 2 O 2 The treated cells showed lower T1WI signals, referring to Figure 3 (right). This proves that the contrast agent of the present invention can inhibit H 2 O 2 Enrichment and imaging.

[0139] Experimental Example 2: ROS content determination

[0140] 1. Cisplatin treatment group: 10 μmol / L cisplatin solution was co-cultured with HK-2 cells for 24 h. The total ROS (H 2 O 2 、ClO-、·OH、O 2-) content. The results are as follows Figure 4 As shown in A, HK-2 cells treated with cisplatin will produce ROS, and the intracellular ROS content is positively correlated with the concentration of cisplatin, while ROS is almost undetectable in cells not treated with cisplatin.

[0141] 2. Use H 2 O 2 Detection reagent, detect intracellular H 2 O 2 The results are as follows Figure 4 B shows that H 2 O 2 The content of H in HK-2 cells was significantly higher than that in cells not treated with cisplatin. 2 O 2 The content was positively correlated with the concentration of cisplatin.

[0142] 3. Mice were injected with 20 mg kg-1 cisplatin via the abdomen, and blood was collected at 12, 24, 48, 72, and 96 hours after cisplatin injection, and then the mice were killed (cervical dislocation method). Analysis of BUN and Scr in the blood of mice showed that at 12, 24, and 48 hours after administration, the BUN and Scr of the mice remained in the normal range, while at 72 hours after administration, the BUN and Scr of the mice were abnormally increased. Figure 5 Subsequently, ROS and H in the mouse kidneys were detected by confocal laser scanning microscopy (CLSM) and microplate reader, respectively. 2 O content, reference Figure 6 and Figure 7 Cisplatin accumulates in kidney tissue and produces a large amount of H 2 O 2 , the generated H 2 O 2 The amount of cisplatin was positively correlated with the administration time.

[0143] 4. H&E staining analysis of mouse kidney sections revealed the following results: Figure 8 As shown in the figure, 72 hours after administration, the renal tubules underwent obvious damage, such as cell necrosis and disappearance of the brush border, and a large number of inflammatory cells infiltrated into the renal tissue, while there was no obvious damage to the renal tissue at other earlier administration time points.

[0144] The above results prove that after cisplatin is enriched in the kidney, it first induces cells and tissues to produce a large amount of ROS, which further causes renal tubular necrosis or apoptosis, leading to acute kidney injury. Based on this, it can be inferred that renal ROS is an early marker of cisplatin-induced acute kidney injury, and the content of renal ROS is related to the degree of renal injury.

[0145] Experimental Example 3: Comparison between the contrast agent Hyp-DOTA-Gd of Example 1 and the commercial contrast agent DOTA-Gd

[0146] 48 hours after the administration of cisplatin (20 mg / kg) (it has been shown that no renal tubular damage has occurred, but a large amount of ROS is produced in the renal tissue), the contrast agent Hyp-DOTA-Gd prepared by the present invention and the commercial contrast agent DOTA-Gd (0.1 mmol / kg Gd) were injected intravenously, and magnetic resonance imaging images and T1WI signal values ​​of the kidney were collected at different injection time points.

[0147] The results are as follows Fig. 9 As shown, the T1WI signal in the kidney of mice reached the highest value 10 minutes after the injection of Hyp-DOTA-Gd, and its signal value was 1.25 times higher than that of mice injected with DOTA-Gd. Half an hour after administration, the signal values ​​in the kidney of the two groups of mice gradually decreased. This shows that Hyp-DOTA-Gd can react quickly with ROS in the kidney, selectively enrich in the kidney, and enhance the T1WI signal. At the same time, the contrast agent will be slowly cleared from the kidney, and it is expected to avoid its long-term enrichment in the kidney and cause renal damage. This is because the contrast agent Hyp-DOTA-Gd of the present invention can generate compounds with active free radicals under the stimulation of oxidative response, which can specifically bind to proteins in the renal injury site, so that the contrast agent is enriched in the kidney and enhances T1 imaging. The inactive ester bond is broken under the action of intracellular esterase, and the contrast agent accumulated in the kidney is slowly excreted from the body after imaging, avoiding damage to the kidney by the contrast agent.

[0148] Therefore, it is feasible to construct a magnetic resonance imaging method for renal ROS using this contrast agent, which is expected to enable early diagnosis of acute kidney injury induced by ischemia / reperfusion, sepsis, and nephrotoxin, and provide an imaging tool for the study of the injury mechanism of acute kidney injury.

[0149] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or apparatus.

[0150] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0151] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A magnetic resonance imaging contrast agent Hyp-DOTA-Gd, characterized in that: The structural formula of the contrast agent is shown below: 。 2. A method for preparing the magnetic resonance imaging contrast agent Hyp-DOTA-Gd according to claim 1, characterized in that: The method comprises: DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are reacted in anhydrous DMF, and then purified to obtain compound Hyp-DOTA; The compound Hyp-DOTA is dissolved in a PBS solution, and then gadolinium chloride is added for chelation reaction to obtain a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd. The reaction formula is as follows: 。 3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound Hyp-DOTA to the gadolinium chloride is 1:

1.

4. The preparation method according to claim 2, characterized in that: The pH of the PBS solution is 6.

8.

5. The preparation method according to claim 2, characterized in that: The usage ratio of DOTA-OH, compound 5, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and anhydrous DMF is (0.1-0.25) g: (0.06-0.1) g: (0.01-0.02) g: (17-18) μl: (0.2-0.4) ml.

6. The preparation method according to claim 2, characterized in that: In the reaction for preparing the compound Hyp-DOTA, the reaction temperature is 20 to 30° C. and the reaction time is 8 to 15 hours.

7. The preparation method according to claim 2, characterized in that: The chelating reaction is carried out under stirring conditions.

8. The method for preparing a nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd according to claim 2, characterized in that: The preparation method of compound 5 comprises: S1, adding methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide and 2,2-azobisisobutyronitrile to an organic solution for reaction, and then filtering, rotary evaporating and purifying to obtain compound 1; S2, dissolving the compound 1 in a dioxane aqueous solution containing silver sulfate for reaction, and then filtering and extracting, collecting the organic phase solution and spinning it to dryness, to obtain compound 2; S3, reacting the compound 2 with diethylaminosulfur trifluoride in an organic solvent, and then washing and purifying to obtain compound 3; S4, reacting the compound 3, bis(pinacol)diboron, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium acetate in a DMF solution, then adding [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride to continue the reaction, adding ether to the reaction solution after the reaction, and then washing, collecting the organic layer solvent and drying to obtain compound 4; S5, dissolving the compound 4 in THF / H2O with a volume ratio of 3:1, adding LiOH to react, and then extracting, collecting the organic layer and rotary evaporating to obtain compound 5; The reaction formula for preparing the compound 5 is as follows: 。 9. The preparation method according to claim 8, characterized in that: In S1, the dosage ratio of methyl 4-bromo-3-methylbenzoate, N-bromosuccinimide, 2,2-azobisisobutyronitrile, and organic solution is 2.3 g: 1.9 g: 0.164 g: 25 ml, and the organic solution is selected from carbon tetrachloride, toluene, dimethyl sulfoxide, and dioxane; In S2, the dosage ratio of silver sulfate and dioxane / pure water is 0.499 g:20 ml, and the volume ratio of dioxane to pure water in the dioxane aqueous solution is 1:1; In S3, the usage ratio of anhydrous dichloromethane, compound 2, and diethylaminosulfur trifluoride is 20ml:0.235g:0.0464g, and the organic solvent is selected from anhydrous dichloromethane, chloroform, acetonitrile, and dioxane; In S4, the usage ratio of the DMF solution, compound 3, bis(pinacol)diboron, initial amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, added amount of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, potassium acetate, and ether is 5ml:0.185g:0.228g:0.306g:0.306g:0.221g:50ml; In the S5, the usage ratio of the compound 4, THF / H2O with a volume ratio of 3:1, and LiOH is 0.0151 g: 0.8 ml: 150 μl.

10. Use of the nuclear magnetic resonance imaging contrast agent Hyp-DOTA-Gd according to claim 1 in the preparation of an imaging agent for nuclear magnetic resonance imaging analysis of acute kidney injury.

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