CN117777174A - Contrast agent and application thereof - Google Patents

Contrast agent and application thereof Download PDF

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CN117777174A
CN117777174A CN202311789988.XA CN202311789988A CN117777174A CN 117777174 A CN117777174 A CN 117777174A CN 202311789988 A CN202311789988 A CN 202311789988A CN 117777174 A CN117777174 A CN 117777174A
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contrast agent
protein
compound
phenylboronic acid
bound
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刘世勇
潘文浩
岑杰
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University of Science and Technology of China USTC
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University of Science and Technology of China USTC
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Abstract

The invention provides a contrast agent, which has a structure shown in any one of formulas A-H. The contrast agent structure provided by the invention contains aldehyde group, o-aldehyde phenylboronic acid or o-acetyl phenylboronic acid functional elements, and has higher metal binding constant; the O-aldehyde phenylboronic acid and O-acetyl phenylboronic acid functional motifs can realize rapid protein coupling under physiological conditions, so that higher relaxation rate is generated, meanwhile, the circulation time of the contrast agent is effectively prolonged, the enrichment degree of the magnetic resonance contrast agent in a tumor area is enhanced, and the signal to noise ratio of the tumor area is obviously enhanced. The amide bond linkage is favorable for improving the complexing efficiency and the complexing stability of the macrocyclic ring to metal, so that the contrast agent has higher ion binding stability and can be quickly coupled with protein under physiological conditions to form the protein binding contrast agent.

Description

Contrast agent and application thereof
Technical Field
The invention relates to the technical field of materials, in particular to a contrast agent and application thereof.
Background
Magnetic Resonance Imaging (MRI) is the first modality to evaluate many diseases. Due to its adjustable soft tissue contrast, high spatial and temporal resolution, and the lack of ionizing radiation properties, it is a major tool in clinical radiology diagnostics. The use of paramagnetic Gd (III) -based magnetic resonance contrast agents (GBCAs) can further enhance anatomical features and improve diagnostic accuracy. Currently, clinically approved GBCAs are limited to providing anatomical or functional information and cannot provide molecular data.
In order to extend the functionality of MRI to molecular imaging systems, a special series of contrast agents, called bio-reactive GBCAs, have been developed in recent years by researchers. These contrast agents may alter the magnetic resonance signal to conditionally respond to specific biochemical events by selectively binding (i.e., targeting GBCA) or performing a bi-orthogonal chemical transformation (i.e., activating GBCA). Traditionally, the term "bioreactive GBCAs" refers only to activatable GBCAs, and not targeted contrast agents. However, a bioresponsive agent is defined as a "material that is sensitive to, interacts with or is driven by biological signals or pathologies", and a targeted magnetic resonance contrast agent interacts with molecular biomarkers through selective binding. Thus, "bioreactive GBCAs" may be used to designate targeted and activatable GBCAs.
Targeting GBCAs involves coupling a targeting ligand to Gd (III) complexes. This allows GBCA to selectively bind to target biomarkers, typically a protein that is more abundant in the disease state than in the healthy state, since biomacromolecule-bound GBCA is retained, unbound GBCA is rapidly cleared, and the magnetic resonance signal can be increased by differential accumulation of GBCAs. Furthermore, receptor-bound GBCA is generally due to τ compared to unbound GBCA R Increase r by increase of r 1 The magnetic resonance signals are further enhanced. Encouraging, many advances have recently been made in the study of targeted GBCAs that will find application in future clinical magnetic resonance diagnostics, providing a promising solution for this field.
Since the number of people suffering from tumor diseases is increasing in recent years, development of contrast agents with tumor diagnosis functions is a medical problem to be solved urgently. On the other hand, in practical application, the contrast agent often has the problems of poor stability of complex metal and biotoxicity caused by free metal, so that a novel contrast agent with a higher metal binding constant is needed to realize good biocompatibility.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a contrast agent and application thereof, wherein the contrast agent can bind to proteins under physiological conditions, and the original activity and structure of the proteins are maintained.
To achieve the above object, the present invention provides a contrast agent having a structure represented by any one of the following formulas a to H:
wherein M is Gd, ga, cu or Zr;
m is an integer of 0 to 400;
n is an integer of 1 to 11;
R 1 is NH or O;
R 2 is any one of the following structures:
R 3 is H or methyl;
indicating the connection location.
The invention provides a series of novel contrast agents, which contain aldehyde group, o-aldehyde phenylboronic acid and o-acetyl phenylboronic acid functional units and are connected by adopting different connecting sections to obtain the contrast agents, wherein the contrast agents containing the aldehyde group functional units have a relaxation rate r slightly higher than DOTA-Gd molecules 1 The method comprises the steps of carrying out a first treatment on the surface of the Contrast agent containing o-aldehyde phenylboronic acid and o-acetyl phenylboronic acid functional units connected by urea bonds has slightly higher relaxation rate r compared with DOTA-Gd molecules 1 And can be physiologically compatible with proteinsUnder the condition of rapid coupling to form protein-binding contrast agent; GBCAs containing o-aldehyde phenylboronic acid and o-acetyl phenylboronic acid functional units and connected by adopting amide bonds have a slightly higher relaxation rate r compared with DOTA-Gd molecules 1 Can be quickly coupled with protein under physiological conditions to form protein-bound GBCAs, and the amide bond is helpful for improving the macrocyclic pair Gd 3+ Is a complex efficiency and a complex stability.
In another aspect, the present invention provides a method of preparing the above-described contrast agent.
The preparation method of the compound shown in the formula A, E-H is similar, taking the compound shown in the formula A as an example, and the reaction equation of the preparation process is as follows:
specifically, the method comprises the following steps:
s1) mixing a compound shown in a formula 3 with DO3A for reaction to obtain a compound shown in a formula 4;
s2) hydrogenating the compound shown in the formula 4 and palladium carbon to remove benzyloxycarbonyl protection to obtain a compound shown in the formula 5;
s3) mixing a compound shown in a formula 5 with a compound shown in a formula 6 for reaction to obtain a compound shown in a formula 7;
s4) removing tert-butyl ester protection of the compound shown in the formula 7 and complexing the compound with a metal element to obtain the compound shown in the formula A.
The solvent for the reaction in step S1) is preferably acetonitrile or DMF, the reaction temperature is preferably 25 ℃, and the reaction time is preferably 12h.
The preparation process reaction equation of the compound shown in the formula B is as follows:
s1) mixing a compound shown in a formula 1 with formic acid for reaction to obtain a compound shown in a formula 2;
s2) mixing a compound shown in a formula 2 with diphenyl azide phosphate and triethylamine for reaction to obtain a compound shown in a formula 3;
s3) mixing a compound shown in a formula 3 with DO3A for reaction to obtain a compound shown in a formula 4;
s4) mixing a compound shown in a formula 4 with ethylene glycol amine for reaction to obtain a compound shown in a formula 5;
s5) removing tert-butyl ester protection of the compound shown in the formula 5 and complexing the compound with a metal element to obtain the compound shown in the formula B.
The preparation process reaction equation of the compound shown in the formula C is as follows:
the preparation process reaction equation of the compound shown in the formula D is as follows:
the contrast agent provided by the invention can be well dissolved in aqueous solution, has higher metal binding constant, can be quickly combined with protein under physiological conditions to enhance contrast performance, further generates higher relaxation rate, effectively prolongs the circulation time of the contrast agent, enhances the enrichment degree of the magnetic resonance contrast agent in a tumor area, and realizes remarkable enhancement of the signal-to-noise ratio in the tumor area. Specifically, the contrast agent containing FPBA or APBA functional motifs can react with amino groups on proteins rapidly to form protein-bound contrast agent, capture of an MPS system is avoided under the action of the proteins, long circulation time in blood is achieved, tumor cell uptake is promoted under the mediation of SPARC proteins with high expression in tumor regions, and the imaging signal to noise ratio of the tumor regions is remarkably enhanced. The specific reaction scheme is shown in figure 1.
In view of the above, the present invention provides a protein-binding type contrast agent, which is obtained by reacting and binding the above-mentioned contrast agent and protein in a physiological environment.
The protein-bound contrast agent can be directly injected for use.
The method for preparing the protein-bound contrast agent is not particularly limited, and preferably comprises the following steps:
mixing the contrast agent and the protein in a physiological environment, and reacting to obtain the protein-binding contrast agent.
Preferably, the physiological environment is PBS buffer or sodium chloride solution.
Preferably, the pH of the PBS buffer is between 5.0 and 11.0, preferably 7.4.
Preferably, the temperature of the reaction is 25-37.5 ℃, and the time of the reaction is 0.5-1 h.
Preferably, the molar ratio of the contrast agent to the protein is 1:1 to 100:1, preferably 1:1.
In another aspect, the present invention provides a protein-bound contrast agent composition comprising the above-described contrast agent or the above-described protein-bound contrast agent.
In another aspect, the present invention provides the use of the above-described contrast agent, the above-described protein-bound contrast agent or the above-described protein-bound contrast agent composition for the preparation of a contrast agent for imaging diagnosis.
In some embodiments, the concentration of the contrast agent at the final injection into the living body is 0.07-0.12mg/kg.
In some embodiments, the contrast agent can be used for specific imaging diagnosis of lesions such as tumors, and particularly can effectively prolong the circulation time of the contrast agent during imaging diagnosis, enhance the enrichment degree of the contrast agent in a tumor area and realize remarkable enhancement of the signal-to-noise ratio in the tumor area. The method can be particularly applied to imaging diagnosis such as magnetic resonance, positron Emission Tomography (PET), CT and the like.
Compared with the prior art, the invention provides a contrast agent, which has a structure shown in any one of formulas A-H. The contrast agent structure provided by the invention contains aldehyde group, o-aldehyde phenylboronic acid or o-acetyl phenylboronic acid functional elements, and can generate higher relaxation rate; the O-aldehyde phenylboronic acid and O-acetyl phenylboronic acid functional motifs can realize rapid protein coupling under physiological conditions, so that the circulation time of a contrast agent is effectively prolonged, the enrichment degree of a magnetic resonance contrast agent in a tumor area is enhanced, and the signal-to-noise ratio of the tumor area is obviously enhanced. The amide bond linkage is favorable for improving the complexing efficiency and the complexing stability of the macrocyclic ring to metal, so that the contrast agent has higher ion binding stability and can be quickly coupled with protein under physiological conditions to form the protein binding contrast agent.
Drawings
FIG. 1 is a schematic illustration of the reaction of a contrast agent and a protein;
FIG. 2 is a nuclear magnetic hydrogen spectrum of compound Cbz-NH-DO 3A;
FIG. 3 is a high resolution mass spectrum of compound Cbz-NH-DO 3A;
FIG. 4 is a compound NH 2 -nuclear magnetic hydrogen spectrum of DO 3A;
FIG. 5 is a compound NH 2 -high resolution mass spectrometry of DO 3A;
FIG. 6 is a nuclear magnetic resonance hydrogen spectrum of the compound 4-APBAP-AM-DOTA;
FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of the compound 4-APBA-AM-DOTA;
FIG. 8 is a high resolution mass spectrum of the compound 4-APBA-AM-DOTA-Gd;
FIG. 9 is an in vitro test r at 9.4T for a series of GBCAs according to the invention 1 Results;
FIG. 10 is an in vitro test r at 9.4T and 1.5T for a series of GBCAs according to the invention 1 Comparing the results;
FIG. 11 is a series of GBCAs mice in vivo T according to the invention 1 Magnetic resonance imaging results;
FIG. 12 is a series of GBCAs mice in vivo T according to the invention 1 A magnetic resonance imaging liver signal change curve;
FIG. 13 shows T in vivo in a series of GBCAs mice according to the invention 1 Magnetic resonance imaging kidney signal change curve;
FIG. 14 shows T in vivo in a series of GBCAs mice according to the invention 1 Magnetic resonance imaging tumor signal profile.
FIG. 15 is compound 3-FPBAP-CON 3 Nuclear magnetic hydrogen spectrum;
FIG. 16 is Compound 3-FPBAP-CON 3 Nuclear magnetic carbon spectrum;
FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of compound 3-FPBA-DO 3A;
FIG. 18 is a nuclear magnetic resonance hydrogen spectrum of compound 3-FPBA-DOTA;
FIG. 19 is a high resolution mass spectrum of the compound 3-FPBA-DOTA-Gd.
Detailed Description
In order to further illustrate the present invention, the contrast agent and the preparation thereof provided by the present invention are described in detail below with reference to examples.
The raw materials used in the invention are described as follows:
the protein of Murine Serum (MSA) was purchased from aladine and used as such. Anhydrous N, N-Diisopropylethylamine (DIPEA), pentafluorophenol (PFp-OH), 4-Dimethylaminopyridine (DMAP), 2-chloropyridine, potassium tert-butoxide, 4-acetylbenzoic acid, boron trifluoride etherate, bisboronic acid pinacol ester, [ Ir (COD) (OMe) ]] 2 Triphenylarsine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), pentafluorophenol, hexamethylenediamine, bromoacetyl bromide, N-Diisopropylethylamine (DIPEA), tri-tert-butyl 1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetate (DO 3A), diethanolamine, 4- (aminomethyl) benzoic acid, purchased from Anaglycone chemical, and used as received. Sodium carbonate (Na) 2 CO 3 ) Sodium bicarbonate (NaHCO) 3 ) Sodium hydroxide (NaOH), sodium sulfate (Na) 2 SO 4 ) Furan, N-methylmorpholine (NMM), tetrahydrofuran (THF), dichloromethane (DCM), petroleum Ether (PE), ethyl Acetate (EA), diethyl ether (Et) 2 O), dimethyl sulfoxide (DMSO) was purchased from national drug control chemical company, ltd, and used directly without prior treatment. The water was Deionized (DI) using a Milli-Q SP reagent water system (Millipore) to achieve a resistivity of 18.4 M.OMEGA.cm. Unless otherwise indicated, all other reagents were purchased from national pharmaceutical group chemical company, ltd, and used as received.
The following examples will further illustrate the invention A, E-H, wherein m=0; n=5; m=gd; r is R 1 ＝NH；
For example, it is only for better understanding of the present invention, and not to limit the scope of the present invention.
Example 1
In the first step, compound 8 is synthesized:
cbz protection reaction on hexamethylenediamine single-ended amino group:
the preparation method comprises the following steps: hexamethylenediamine (15.00 g,129.31 mmol) was added to a DCM (120 mL) and the mixture was ice-washed. Benzyl chloroformate (4.40 g,25.80 mmol) dissolved in DCM (40 mL) was then slowly added dropwise to the above system, after which the reaction was resumed at room temperature for 4 hours. 100mL of DCM was added to the system to dilute, and the system was washed with 100mL of saturated sodium chloride solution, anhydrous Na 2 SO 4 The dried organic phase was added, filtered, and the organic solvent was removed by rotary evaporation, and the mixture was further separated and purified by column chromatography on silica gel using DCM/methanol (20/1, v/v) as eluent to give a white solid (5.80 g, yield: 90.1%).
Second step, compound 9 is synthesized:
preparing an active ester intermediate of a connecting section:
the preparation method comprises the following steps: bromoacetyl bromide (4.38 g,21.70 mmol) was dissolved in DCM (30 mL) and then ice-bath, DIPEA (2.97 g,22.99 mmol), pentafluorophenol (3.99 g,21.70 mmol) were dissolved in DCM (30 mL), the above system was added dropwise, and then the reaction was continued at room temperature for 1 hour. Washed with water (3X 30 mL), anhydrous Na was added 2 SO 4 The dried organic phase was added, filtered, and after spin-drying the solvent using a rotary evaporator, the mixture was further separated and purified by silica gel column chromatography using DCM as an eluent to give a white solid (5.67 g, yield: 86.6%).
And a third step of: synthesis of Compound 10:
the preparation method comprises the following steps: compound 9 (2.81 g,9.23 mmol) was dissolved in DMF (20 mL) and then ice-bath, compound 8 (2.10 g,8.39 mmol) and DIPEA (1.30 g,10.06 mmol) dissolved in DMF (10 mL) were added dropwise, the reaction was continued for 5 hours at room temperature, DCM (50 mL) was added for dilution, water (3X 80 mL) was used for washing, dried over anhydrous sodium sulfate, filtered, the organic solvent was removed by rotary evaporation, and the crude product was further isolated and purified by silica gel column chromatography using PE/EA (4/1, v/v) as eluent to give a white solid (2.56 g, yield: 82.2%).
Fourth step: synthesis of Compound Cbz-NH-DO3A:
the preparation method comprises the following steps: compound 10 (2.25 g,6.05 mmol) and potassium carbonate (1.59 g,9.80 mmol) were mixed and added to anhydrous acetonitrile (60 mL) followed by ice bath, then DO3A (2.97 g,5.76 mmol) solution in anhydrous acetonitrile (25 mL) was added dropwise and the reaction was continued at room temperature for 12 hours. The filtrate was collected by filtration, the solvent was removed by rotary evaporation, diluted with DCM (100 mL) added, washed with water (2X 100 mL), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation, and the crude product was further isolated and purified by silica gel column chromatography using DCM/MeOH (20/1, v/v) as eluent to give a white solid (3.85 g, yield: 83.3%).
Fifth step: synthesis of Compound NH 2 -DO3A：
The preparation method comprises the following steps: compound Cbz-NH-DO3A (1.00 g,0.12 mmol) was dissolved in methanol (50 mL) and 0.1g of 10% palladium on carbon powder was added to the system. The reaction flask was evacuated/purged with nitrogen three times to purge residual air. Next, H2 was added to the reaction mixture. The reaction was stopped by stirring at room temperature for 8 hours. The reaction was filtered through celite filter aid and the residue was washed with MeOH (3×30 mL). Mixing the filtrates, and evaporating to dryness; after further drying in a vacuum oven, compound 3a was obtained as a white powder (0.80 g, yield: 96.0%).
Sixth step: synthesis of Compound 4-APBAP-AM-DO3A:
the preparation method comprises the following steps: 4-APBAP-PFp (1.32 g,1.97 mmol) was dissolved in anhydrous DMF (20 mL) followed by an ice bath and then NH was dissolved in anhydrous DMF (10 mL) 2 A solution of DO3A (1.10 g,1.64 mmol) and DIPEA (0.42 g,0.33 mmol) was added dropwise to the above system and the reaction was continued at room temperature for 7 hours. DCM (50 mL) was added for dilution, washed with deionized water (2X 60 mL), and anhydrous Na was added to the system 2 SO 4 Drying, filtration, spin-removal of the organic solvent using a rotary evaporator, and further separation and purification of the mixture by silica gel column chromatography using DCM/MeOH (20/1, v/v) as eluent gave a white solid (1.25 g, yield: 81.0%).
Seventh step: synthesis of Compound 4-APBAP-AM-DOTA:
the preparation method comprises the following steps: 4-APBAP-AM-DO3A (0.50 g,0.53 mmol) was added to dioxane hydrochloride solution (10 mL), reacted at room temperature for 6 hours, the resulting solid was dissolved in water by filtration, washed with diethyl ether (2X 30 mL), and lyophilized to give the product (0.39 g, yield: 95%).
Eighth step: synthesis of the compound 4-APBA-AM-DOTA-Gd:
the preparation method comprises the following steps: 4-APBA-AM-DOTA (0.050 g,0.072 mmol) was dissolved in deionized water (2 mL) and GdCl was added dropwise 3 ·6H 2 O (0.040 g,0.11 mmol), aqueous NaOH (1.0M) was slowly added to adjust the pH of the solution to 6.5, stirred at room temperature for 24 hours, and free Gd was removed by ion exchange resin 3+ Lyophilization gives the desired product (0.060 g, yield: 98%).
Example 2: preparation of protein-bound GBCAs
The 4-APBA-AM-DOTA-Gd magnetic resonance contrast agent prepared in the example 1 and protein are reacted in 10mM pH 7.4PBS buffer solution in physiological environment at the temperature of 25 ℃, and the molar ratio of the contrast agent to the protein is 1:1 are mixed and stirred for reaction for 1 hour, and the directly injectable protein-binding contrast agent is prepared.
Example 3:1.5T and 9.4T in vitro MRI experiments
MRI studies were performed using a volumetric radio frequency coil on a 9.4T/400mm wide bore scanner (agilent technologies, santa Clara, CA, USA). For in vitro and in vivo phantom MRI experiments, the longitudinal relaxation time (T 1 ) The parameter is echo Time (TE) =16.18 ms,8 repetitions (TR): 843. 1117.378, 1141.050, 1835.969, 2342.620, 3050.310, 4233.124 and 9000ms, field of view (FOV) =32 mm×32mm, matrix size=128×128, bw 50khz,5 slices, slice thickness 1mm. Select T when tr=843 ms 1 The MR phantom images are weighted. Scanning was performed using a GE Signal Horizon 1.5T MR scanner with an axial 3D FGRE sequence. MRI intensity was compared to untreated controls (PBS mixture). The test results at 1.5T showed a significant increase in relaxation rate of GBCAs to nearly 5-fold after albumin binding. And the relaxation rate of GBCAs after albumin is combined in the test result at 9.4T is slightly enhanced.
Example 4:9.4T in vivo MRI experiments
In vivo MRI studies, isoflurane (3.5% anesthetic stunned, 1.0% -1.5% maintained) was used in air/O during the scan 2 (2:1) anesthetizing a nude mouse with a tumor. The animals were placed on a specially designed bassinet and inserted into magnets. Throughout the experiment, a physiological monitoring unit (model 1030; SA Instruments, inc., stony Brook, NY) was used. During the experiment, the body temperature of the animals was maintained at 36.5℃to allow for the followingWith a heating pad. Longitudinal relaxation time (T) 1 ) With a fast acquired relaxation enhancement sequence, the parameter is echo Time (TE) =15.3 ms,6 repetitions (TR): 650. 1047.3, 1557.9, 2273.4, 3478.7 and 9000ms, field of view (FOV) =32 mm×32mm, matrix size=128×128, slice thickness=1 mm (12 pieces, gap=0), BW at 50kHz, 1 average. Select T when tr=650 ms 1 The coronal MR image is weighted. During the scan, accurate measurements and markers were used to ensure consistent location of the mouse tumor in the animal stent and the mouse tumor in the magnet. MRI acquisitions are performed using respiratory triggers to reduce respiratory motion induced artifacts. The imaging experiment result shows that the GBCAs combined with albumin obviously enhances the aggregation in the tumor area, reduces the accumulation in liver and kidney organs, and further reduces the toxic and side effects of the medicine.
The following examples will further illustrate invention B, wherein m=gd;
for example, it is only for better understanding of the present invention, and not to limit the scope of the present invention.
Example 5
First, compound 2 is synthesized:
bromoboric acid pinacol ester reaction:
the preparation method comprises the following steps: solid starting materials such as: compound 1 (15.00 g,52.61 mmol), pinacol biborate (14.83 g,58.39 mmol), catalyst PdCl 2 (dppf) (1.27 g,1.74 mmol) and potassium acetate (15.64 g,159.40 mmol) were added to the system, and the vacuum was replaced with argon atmosphere and repeated three times; then 200mL of anhydrous dioxane is added, and the mixture is vacuumized and replaced to argon atmosphere for three times; heating to 100 ℃ under argon atmosphere to react for about 3 hours; the system was cooled to room temperature, diluted with DCM and washed with waterAnhydrous Na 2 SO 4 The dried organic phase was added, and after filtration, the organic solvent was removed by rotary evaporation, and the mixture was further separated and purified by silica gel column chromatography using PE/EA (20/1, v/v) as an eluent to give a white solid (14.91 g, yield: 85.3%).
Second step, compound 3 is synthesized:
removing tert-butyl ester protection:
the preparation method comprises the following steps: placing HCOOH at 10 ℃ for precooling; 100mL of HCOOH was added to compound 2 (10.00 g,30.10 mmol), the reaction was stirred at 10deg.C, TLC monitored for progress of the reaction, and the reaction was complete for 2.5 h; the HCOOH was removed by rotary evaporation and dried in a vacuum oven to give a pale yellow solid (7.95 g, yield: 95.6%).
And a third step of: synthesis of Compound 3-FPBA-CON 3 ：
The preparation method comprises the following steps: compound 3 (7.95 g,28.78 mmol) was dissolved in THF and Et was added 3 N (4.37 g,43.17 mmol); diphenyl azide phosphate DPPA (9.50 g,34.53 mmol) was added to the system under ice bath conditions, and the mixture was returned to room temperature and stirred for 8h; the organic solvent was removed by rotary evaporation, and the mixture was further separated and purified by silica gel column chromatography using PE/EA (10/1. Fwdarw.8:1, v/v) as eluent to give a white solid (6.80 g, yield: 78.5%).
Fourth step: synthesis of Compound 5:
the preparation method comprises the following steps: compound 3-FPBA-CON 3 (5.36 g,17.80 mmol) toluene was azeotropically dehydrated three times; heating at 85deg.C for 5 hr; DO3A (8.32 g,16.16 mmol) toluene azeotropic dehydration was repeatedAdding anhydrous triethylamine, adding anhydrous DCM, dissolving a DO3A system, transferring to a compound 4 system by adopting a double needle, and stirring at room temperature overnight; the organic solvent is removed by rotary evaporator, the mixture system is further separated and purified by silica gel column chromatography, CH is used 3 OH/DCM (1/20, v/v) as eluent gave a white solid (11.28 g, yield: 88.6%).
Fifth step: synthesis of Compound 6:
the preparation method comprises the following steps: compound 5 (11.28 g,14.32 mmol) was dispersed in 80mL THF and stirred; diethanolamine (7.53 g,71.61 mmol) was mixed with 30mL THF and the system was dropped; the reaction was carried out at room temperature for 12h, THF was removed by spinning, DCM was dissolved, washed with water, dried, and the organic solvent was removed by spinning with a rotary evaporator to give a colorless oil (9.33 g, yield: 92.3%).
Sixth step: synthesis of 3-FPBA-DOTA:
the preparation method comprises the following steps: compound 6 (2.00 g,2.83 mmol) was dissolved in 4M HCl/Dioxane and stirred overnight at room temperature, with complete reaction monitored by TLC; diethyl ether was precipitated, filtered and dried to give a white solid (1.44 g, yield: 94.6%).
Seventh step: synthesis of Compound 3-FPBA-DOTA-Gd:
the preparation method comprises the following steps: 3-FPBA-DOTA (386.88 mg,0.72 mmol) was dissolved in deionized water (10 mL) and GdCl was added dropwise 3 ·6H 2 O (0.40 g,1.10 mmol), aqueous NaOH (1.0M) was slowly added to adjust the pH of the solution to 6.5, stirred at room temperature for 24h, and free Gd was removed by ion exchange resin 3+ Lyophilization afforded the desired product (0.50 g, yield: 98.3%))。
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims (8)

1. A contrast agent having a structure according to any one of formulas a-H:
wherein M is Gd, ga, cu or Zr;
m is an integer of 0 to 400;
n is an integer of 1 to 11;
R 1 is NH or O;
R 2 is any one of the following structures:
R 3 is H or methyl;
indicating the connection location.
2. A protein-bound contrast agent, characterized in that it is obtained by reactive binding of the contrast agent of claim 1 to a protein in a physiological environment.
3. A method of preparing a protein-bound contrast agent as claimed in claim 2, comprising the steps of:
the method for preparing protein-bound contrast agent comprising mixing the contrast agent according to claim 1 with protein in a physiological environment, and reacting to obtain the protein-bound contrast agent.
4. The method of claim 3, wherein the physiological environment is PBS buffer or sodium chloride solution;
the pH value of the PBS buffer solution is 5.0-11.0.
5. A method according to claim 3, wherein the reaction temperature is 25 to 37.5 ℃ and the reaction time is 0.5 to 1h.
6. The method according to claim 3, wherein the molar ratio of the contrast agent to the protein is 1:1 to 100:1.
7. A protein-bound contrast agent composition comprising the contrast agent of claim 1 or the protein-bound contrast agent of claim 2.
8. Use of the contrast agent of claim 1, the protein-bound contrast agent of claim 2 or the protein-bound contrast agent composition of claim 7 for the preparation of a contrast agent for imaging diagnosis.
CN202311789988.XA 2023-12-22 2023-12-22 Contrast agent and application thereof Pending CN117777174A (en)

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