Novel gadolinium-based compound, preparation method thereof and MRI contrast agent containing same

Abstract

The present invention relates to a novel gadolinium-based compound having a structure in which a gadolinium complex and ferulic acid are connected via a connecting group, a preparation method thereof and an MRI contrast agent containing the same.

Description

Technical Field

[0001] The present invention relates to a novel gadolinium-based compound, a preparation method thereof, and an MRI contrast agent containing the same. Specifically, the present invention relates to a novel gadolinium-based compound having a structure in which a gadolinium complex and ferulic acid are linked via a linking group, a preparation method thereof, and an MRI contrast agent containing the same. Background Art

[0002] Nowadays, due to the aging of the population, the number of patients suffering from degenerative brain diseases is increasing, and therefore, early detection of diseases is urgently needed. Degenerative brain diseases include Parkinson's disease, vascular dementia, Alzheimer's disease, etc., and one of the causes of these diseases is neurotoxicity caused by excessive accumulation of amyloid β polymers (oligomeric Aβ).

[0003] Amyloid β (Aβ) is the main component of amyloid plaques found in the brains of Alzheimer's patients. It refers to a 36 to 43 amino acid peptide that plays a decisive role in Alzheimer's disease. This peptide is derived from the amyloid precursor protein (APP).

[0004] The amyloid β molecules can aggregate to form soluble polymers that can exist in various forms, and it is well known that the amyloid β polymers (oligomeric Aβ) formed are toxic to nerve cells, and therefore, when excessively accumulated in the brain, are directly involved in the onset of Alzheimer's disease. It is therefore predicted that by detecting changes in the concentration of amyloid β polymers, early diagnosis of degenerative brain diseases will be possible.

[0005] On the other hand, magnetic resonance imaging (MRI) is a method of obtaining anatomical, physiological, and biochemical information images of the body by utilizing the phenomenon of hydrogen atom relaxation in a magnetic field and the different distribution of hydrogen atoms between body tissues. Unlike CT or PET, MRI does not use radiation that is harmful to the human body, but uses magnetic field gradients and radio waves under a strong magnetic field to produce images in the body, so it is non-invasive, has high resolution, and is very suitable for soft tissue examinations.

[0006] In order to use MRI equipment more precisely, contrast agents are injected into the subject to obtain MRI images. The contrast between tissues on the MRI image is caused by the different relaxation effects of the nuclear spins of water molecules in the tissues returning to the equilibrium state. Contrast agents use paramagnetic or superparamagnetic materials to affect the relaxation effect, thereby increasing the difference in relaxation between tissues, causing changes in MRI signals, and playing a role in making the contrast between tissues clearer.

[0007] At present, the most commonly used contrast agent in clinical practice is a contrast agent based on gadolinium (Gd) chelate. Currently, Gd-DTPA (Magnevist), Gd-DOTA (Dotaram), Gd (DTPA-BMA) (Omniscan), Gd (DO3A-HP) (ProHance), Gd (BOPTA) (MultiHance) etc. are used. However, most commercially available contrast agents are non-specific contrast agents distributed in the extracellular space (extracellular fluide, ECF). As a specific contrast agent, only liver-specific contrast agents are used in particular. Although recent studies have promoted the development of contrast agents that can show signal enhancement by having specific targeting or physiological activity (pH changes, enzyme activity), sufficient results have not yet been obtained for specific MRI contrast agents for degenerative encephalopathy. Summary of the invention

[0008] The object of the present invention is to provide a gadolinium-based compound, which can be used as an MRI contrast material, and in particular, has specificity for degenerative brain diseases.

[0009] Another object of the present invention is to provide an MRI contrast agent containing the compound.

[0010] Another object of the present invention is to provide a method for preparing the compound.

[0011] According to the present invention, a gadolinium-based compound is provided, wherein it is represented by the following Chemical Formula 1.

[0012] [Chemical formula 1]

[0013]

[0014] In the chemical formula 1,

[0015] A represents *-(CH2) n -A 1 -*,

[0016] n represents any integer from 0 to 5,

[0017] A 1 represents *-COO-*, *-CO-*, *-NH-*, *-CH2-*, *-CONH-* or *-O-*,

[0018] Linking group indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OROL2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NH-L 2 -*, or *-L 1 -COO-L 2 -*,

[0019] L 1 represents a straight chain or branched (C1-C30) alkylene group,

[0020] L 2 represents a single bond, or a straight or branched (C1-C30) alkylene group,

[0021] R represents a linear or branched (C1-C20) alkylene group,

[0022] Fa represents chemical formula 2:

[0023] [Chemical formula 2]

[0024]

[0025] * indicates the attachment site.

[0026] Furthermore, according to the present invention, an MRI contrast agent is provided, which contains the gadolinium-based compound represented by the Chemical Formula 1.

[0027] Furthermore, according to the present invention, there is provided a method for preparing the gadolinium-based compound represented by the chemical formula 1, comprising the following steps:

[0028] (a) reacting a compound of the following Chemical Formula 1-1 with a compound of the following Chemical Formula 1-2 to obtain a compound of the following Chemical Formula 1-3;

[0029] [Chemical formula 1-1]

[0030]

[0031] In the chemical formula 1-1,

[0032] L 2 As defined in the above chemical formula 1,

[0033] [Chemical formula 1-2]

[0034]

[0035] [Chemical formula 1-3]

[0036]

[0037] In the chemical formulas 1-2 and 1-3,

[0038] (PT) 3C represents a protecting group,

[0039] Linking group indicates *-L 1 -NH-L 2 -*,

[0040] L 1 , L 2 , A and Fa are as defined in the above chemical formula 1,

[0041] (b) a step of removing the protecting group (PT) 3C from the compound of Chemical Formula 1-3; and

[0042] (c) a step of reacting the compound obtained in step (b) with gadolinium hydrate to obtain a compound of Chemical Formula 1.

[0043] Effects of the Invention

[0044] The novel gadolinium-based compound according to the present invention can be used not only as an MRI contrast material due to its sufficient self-relaxation property, but also can be combined with β-amyloid protein polymer (oligomeric Aβ) to obtain an MRI contrast enhancement effect in the presence of β-amyloid protein polymer, thereby being used for the diagnosis of diseases related to β-amyloid protein polymer, especially the diagnosis of degenerative brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1a is the compound L prepared in the preparation example of the compound of the present invention 1 H NMR spectrum.

[0046] Figure 1b is the compound L prepared in the preparation example of the compound of the present invention 13 C NMR spectrum.

[0047] Figure 1c is the HR-MS spectrum of compound L prepared in the preparation example of the compound according to the present invention.

[0048] Figure 1d is a HPLC chromatogram of compound L prepared in the preparation example of the compound according to the present invention.

[0049] Figure 2a is the HR-MS spectrum of GdL prepared in the preparation example of the compound according to the present invention.

[0050] Figure 2b It is the HPLC chromatogram of GdL prepared in the preparation example of the compound according to the present invention.

[0051] Figure 3A graph showing the self-relaxation rate (r1, r2) analysis of GdL according to the present invention.

[0052] Figure 4 Graph showing the kinetic stability of GdL according to the invention and various commercially available contrast agents (Gadovist, Dotarem, Prohance, Magnevist, Primovist, Omniscan, Multihance).

[0053] Figure 5 It is a graph comparing the MR signal intensity of the MR T1 volume membrane of the MRI sample. DETAILED DESCRIPTION

[0054] Below, the terms used in this application are only used to illustrate specific embodiments and are not intended to limit the present invention. Unless otherwise defined, all terms used in this specification, including technical or scientific terms, have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as meanings consistent with those in the context of the relevant technology and should not be interpreted as ideal or overly formalized meanings unless clearly defined in this application.

[0055] The gadolinium-based compound according to the present invention can be represented by the following Chemical Formula 1.

[0056] [Chemical formula 1]

[0057]

[0058] In the chemical formula 1,

[0059] A represents *-(CH2) n -A 1 -*,

[0060] n represents any integer from 0 to 5, specifically, it can represent any integer from 1 to 5, more specifically, it can be any integer from 1 to 3,

[0061] A 1 represents *-COO-*, *-CO-*, *-NH-*, *-CH2-*, *-CONH-*, or *-O-*, and specifically, may represent *-CONH-*,

[0062] Linking group indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OROL 2 -*, *-L 1-CH2-L 2 -*, *-L 1 -NH-L 2 -*, or *-L 1 -COO-L 2 -*, specifically, can represent *-L 1 -NH-L 2 -*,

[0063] L 1 represents a linear or branched (C1-C30) alkylene group, specifically, L 1 It may be a linear or branched (C1-C10) alkylene group, more specifically, it may be a linear or branched (C1-C5) alkylene group,

[0064] L 2 represents a single bond, or a linear or branched (C1-C30) alkylene group, specifically, it can be a single bond, R represents a linear or branched (C1-C20) alkylene group, specifically, it can represent a linear or branched (C1-C10) alkylene group, more specifically, it can represent a linear or branched (C1-C5) alkylene group, and Fa represents Chemical Formula 2:

[0065] [Chemical formula 2]

[0066]

[0067] * indicates the attachment site.

[0068] According to an implementation example of the present invention, in the chemical formula 1, n can represent any integer from 1 to 5, A 1 It can represent *-CONH-*.

[0069] According to another implementation example of the present invention, L 1 It can represent a straight chain or branched (C1-C10) alkylene group, L 2 Can represent a single bond.

[0070] In the gadolinium-based compound of the Chemical Formula 1, gadolinium may be coordinated with at least one water molecule. For example, in the gadolinium-based compound of the Chemical Formula 1, gadolinium may be coordinated with one or two water molecules.

[0071] In the gadolinium-based compound of Chemical Formula 1 of the present invention, when A 1 In the case of *-COO-*, *-CO-*, or *-CONH-*, the oxygen atom can form a coordinate bond with gadolinium.

[0072] Chemical formula 2 of the present invention is a part derived from ferulic acid. It is reported that ferulic acid is a phenolic chemical substance existing in plant cell walls, has high antioxidant activity against various active oxygen species from oxygen molecules, and has a strong antioxidant effect against oxidative transition metals such as iron ions and copper ions. Therefore, ferulic acid has been used as a main component of anti-aging agents and disease therapeutic agents, but it has not been considered to be used as an MRI contrast agent with oligomeric Aβ targeting function.

[0073] As shown in the examples described below, the gadolinium-based compound of Chemical Formula 1 of the present invention can specifically bind to oligomeric Aβ in mammals.

[0074] In addition, since the compound of the present invention is water-soluble and has self-relaxation properties by coordinating with at least one water molecule, it can improve image contrast by increasing the relaxation rate of at least one water molecule and hydrogen atom in the body, and thus can be used as an MRI contrast material.

[0075] Therefore, according to the present invention, an MRI contrast agent is provided, which contains a gadolinium-based compound represented by the chemical formula 1. In addition, since the compound of the present invention can bind to the β-amyloid protein polymer (oligomeric Aβ), the MRI contrast agent of the present invention can be specifically used for diseases related to the β-amyloid protein polymer (oligomeric Aβ), and more specifically, can be used to diagnose degenerative brain diseases, such as Parkinson's disease, vascular dementia, Alzheimer's disease, and other degenerative diseases. Therefore, according to an implementation example of the present invention, a specific MRI contrast agent for diagnosing degenerative brain diseases can be provided, which contains the compound of the chemical formula 1. Furthermore, as described above, according to the latest research, it is confirmed that the amyloid β polymer (oligomeric Aβ) induced by the amyloid precursor protein is excessively accumulated in the brain and is involved in the onset of Alzheimer's disease. Therefore, the MRI contrast agent containing the compound of the present invention targeting the β-amyloid protein polymer can become a specific MRI contrast agent for early diagnosis of Alzheimer's disease.

[0076] The gadolinium-based compound of Chemical Formula 1 according to the present invention can be prepared by a method comprising the following steps:

[0077] (a) reacting a compound of the following Chemical Formula 1-1 with a compound of the following Chemical Formula 1-2 to obtain a compound of the following Chemical Formula 1-3;

[0078] (b) a step of removing the protecting group (PT) 3C from the compound of Chemical Formula 1-3; and

[0079] (c) a step of reacting the compound obtained in step (b) with gadolinium hydrate to obtain a compound of Chemical Formula 1.

[0080] [Chemical formula 1-1]

[0081]

[0082] In the chemical formula 1-1,

[0083] L 2 As defined in the above chemical formula 1,

[0084] [Chemical formula 1-2]

[0085]

[0086] [Chemical formula 1-3]

[0087]

[0088] In the chemical formulas 1-2 and 1-3,

[0089] (PT) 3C represents a protecting group,

[0090] Linking group indicates *-L 1 -NH-L 2 -*,

[0091] L 1 , L 2 , A and Fa are as defined in the above Chemical Formula 1.

[0092] According to an implementation example of the present invention, in step (a), the L 2 Can be a single bond.

[0093] If necessary, before carrying out the step (a), the -OH group directly connected to the benzene ring in the chemical formula 1-1 can be protected by a protecting group commonly used for -OH, such as acetyl, benzoyl, methoxymethyl ether, methylthiomethyl ether, etc., and the step (a) can be carried out after the -OH group is protected. In addition, if necessary, before carrying out the step (a), a halogen compound can be used to replace the halogen in the chemical formula 1-1 with L 2 The directly linked -OH group can be subjected to step (a) after the halogenation.

[0094] In the chemical formulae 1-2 and 1-3, (PT)3C represents a protecting group, and the protecting group generally used to protect the -OH group includes, for example, a methyl group, a benzyl group, a tert-butyl group, and the like.

[0095] The step (b) is a step of removing the protecting group. The protecting group, i.e. (PT)3C, can be removed by a commonly used method in the art, for example, using an aqueous base solution or an aqueous acid solution.

[0096] In order to understand the present invention in detail, representative compounds of the present invention are exemplified below to explain the compounds according to the present invention, the preparation methods thereof, and the characteristics of MRI contrast agents containing the compounds.

[0097] 1. Preparation Examples of Compounds According to the Present Invention

[0098]

[0099] 1) Tri-tert-butyl 2,2`,2``-(10-(2-((2-(3-(3-hydroxy-4-methoxyphenyl)acrylamide)ethyl) Synthesis of (amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (Compound 1)

[0100] Ferulic acid (0.2 g, 1.03 mmol), N,N-diisopropylethylamine (0.26 mL, 2.06 mmol) and hydroxybenzotriazole (HOBt) (0.24 g, 1.54 mmol) were dissolved in dimethylformamide (DMF) (10 mL) and stirred for 30 minutes.

[0101] Then, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (0.78 g, 2.06 mmol) and DO3A-NH2 (0.64 g, 1.03 mmol) were added to the mixture and stirred at room temperature for 20 hours.

[0102] The reaction was stopped by adding 10% sodium bicarbonate and extracted with ethyl acetate (3×50 mL). The obtained organic layer was washed with brine solution, sodium sulfate was added to remove the remaining moisture, and dried under vacuum to obtain an oily substance (Compound 1). (Yield: 0.72 g (89%))

[0103] The results of HR-FAB-MS analysis of the obtained compound 1 are as follows.

[0104] HR-FAB-MS calculated for C 40 H 66 N6NaO 10 (m / z):813.4738[M+Na] + ; found, 813.4743[M+Na] +

[0105] 2)(E)-2,2`,2``-(10-(2-((2-(3-(3-hydroxy-4-methoxyphenyl)acrylamide)ethyl)amino Synthesis of 2-( ...

[0106] The compound 1 (0.7 g, 0.89 mmol) was dissolved in a mixed solution of trifluoroacetic acid (TFA) and dichloromethane (CH2Cl2) in an ice bath, and then stirred at room temperature until completely dissolved and transparent. While stirring, TLC was used to confirm whether the reactant reacted. After the reaction was fully carried out, the solvent was removed, and ether was added to obtain a precipitate. Then, the precipitate was filtered, washed with ether several times, and then vacuum dried to obtain a pale yellowish white solid (compound L).

[0107] The obtained compound L 1 H NMR spectrum, 13 C NMR spectrum, HR-MS spectrum and HPLC chromatogram are shown in Figures 1a to 1d (Yield: 0.48 g (87%))

[0108] 1 H NMR (500MHz, MeOD); d=7.43 (d, J=15.7Hz, 1H), 7.19 (d, J=1.6Hz, 1H), 7.06 (dd, J=8.2, 1.7Hz, 1H), 6.80 (d, J =8.2Hz, 1H), 6.69 (d, J = 15.7Hz, 1H), 3.89 (s, 3H), 3.88-3.82 (m, 4H), 3.72-3.34 (m, 16H), 3.20-3.06 (m, 8H).

[0109] 13 C NMR (126MHz, MeOD); d=169.73, 163.16, 162.88, 149.92, 149.41, 142.04, 128.48, 1 23.57, 119.56, 116.59, 111.87, 56.64, 56.17, 51.92, 50.90, 50.31, 40.61, 39.63.

[0110] HR-MS calculated for C 28 H 43 N6O 10 (m / z):623.3041[M+H] + ; found, 623.3042[M+H] +

[0111] Purity analysis using analytical HPLC: 96.04%

[0112] 3) Synthesis of compound GdL (compound of the present invention)

[0113] Compound L (0.4 g, 0.64 mmol) was dissolved in distilled water (10 mL), and the pH was adjusted to 7 using a 1 M sodium hydroxide solution, followed by the addition of Gd(Cl)3·H2O (0.24 g, 0.64 mmol) dissolved in distilled water (5 mL). Thereafter, the mixture was stirred at room temperature for 1 day, and the pH during stirring was maintained at 7 to 7.3. After the stirring was completed, the filtered substance was further purified by high performance liquid chromatography (HPLC) to obtain compound GdL.

[0114] The HR-MS spectrum and HPLC chromatogram of the obtained compound GdL are shown in Figure 2a and 2b . (Yield: 0.15 g (31%))

[0115] HR-MS calculated for C 28 H 40 GdN6O 10 (m / z):778.2047[M+H] + ; found, 778.2044[M+H] +

[0116] Purity analysis using analytical HPLC: 96.69%

[0117] Next, the representative compounds of the present invention prepared can be used as MRI contrast agents, and at the same time, property evaluation is performed to determine whether the amyloid β polymer has targeting ability.

[0118] 2. Methods and results for evaluating the properties of the compounds of the present invention

[0119] 1) Measurement of Relaxivity

[0120] In triple distilled water, the GdL synthesized by the method was diluted to various concentrations (0.0625, 0.125, 0.25, 0.5, 1 mM) to prepare a phantom, and then the T1 and T2 relaxation times were measured in 3T MRI. R (relaxation rate = 1 / T) of each concentration was calculated, and the self-relaxation rate (r1, r2) of the synthesized GdL was obtained by linear regression analysis. (Refer to Figure 3 )

[0121] like Figure 3 As shown, the r1 value of GdL was measured to be 3.73±0.10, and the r2 value was 5.29±0.10, thereby confirming that the self-relaxation rate is a sufficient value for the contrast agent of the present invention to be used clinically, and the synthesized GDL can be used as an MRI contrast agent.

[0122] 2) Kinetic stability evaluation

[0123] After preparing 2.5 mM solutions of GdL and several commercially available contrast agents (Gadovist, Dotarem, Prohance, Magnevist, Primovist, Omniscan, Multihance) in PBS (pH 7.4), 250 mM zinc chloride (ZnCl2) was added each time to evaluate the ligand-metal binding stability of the gadolinium complex. The results are shown in Figure 4 This can be confirmed by measuring the transmetallation of zinc ions to gadolinium ions as a change in the self-relaxation rate.

[0124] It is known that linear gadolinium complexes are less stable than cyclic structures. Figure 4 , it could be confirmed that the synthesized GdL had higher stability than linear contrast agents and exhibited a similar rate of change as cyclic commercially available contrast agents with excellent stability.

[0125] 3) Phantom experimental methods for confirming the targeting of amyloid β aggregates

[0126] HFIP (221.5 μl) was added to β-amyloid protein (1 mg, 1 mM), and the mixture was shaken on a shaker at room temperature for 1 hour to remove preaggregation.

[0127] Next, the pre-aggregated amyloid β protein was dried in air, DMSO (221.5 μl) was added to adjust the concentration to 1 mM, and then a suspension was formed using a mixer and a sonicator, and then PBS (1X, pH 7.4, 878.5 μl) was added to adjust the concentration to 0.2 mM.

[0128] Then, after culturing for 4 days at 37°C using a shaker, 200 μl of the aggregated amyloid β polymer (oligomeric Aβ) was dispensed, and the compound GdL and a commercial contrast agent dissolved in PBS at a concentration of 2 mM were added. 20 μl of each was added, and then cultured at 37°C for 24 hours using a shaker.

[0129] After 24 hours of culture, the supernatant was removed by centrifugation, and the amyloid-β protein polymer was washed with PBS. 200 μl of a PBS:DMSO=9:1 solution was added to the pellet to prepare an MRI phantom sample.

[0130] 4) Experimental results

[0131] The β-amyloid polymer targeting experiment was conducted on a 9.4T MR device, and the results are shown in Figure 5 middle. Figure 5 A graph comparing the MR signal intensities of the MR T1 volume membrane of MRI sample specimens is shown.

[0132] refer to Figure 5 It can be seen that the phantoms in which only β-amyloid protein polymers were cultured (control group) and the phantoms in which commercial contrast agents were cultured together The phantom (comparative example) showed similar effects in terms of contrast enhancement effect. There is little targeting effect on amyloid-β aggregates.

[0133] On the other hand, the phantom of GDL treated with the compound of the present invention was cultured together with a control group and a commercially available contrast agent. Compared with the comparative example, the contrast enhancement effect was significantly higher. Therefore, it was confirmed that the compound of the present invention has a targeting effect on the β-amyloid polymer, and it was found that the GdL of the present invention is suitable as a β-amyloid polymer targeting contrast agent.

[0134] Although the present invention has been described above with reference to the preferred embodiments, it should be understood that various modifications and variations may be made by those skilled in the art without departing from the spirit and scope of the present invention as described in the claims.

Claims

1. A gadolinium-based compound, characterized in that It is represented by the following chemical formula 1, [Chemical formula 1] In the chemical formula 1, A represents *-(CH2) n -A 1 -*, n represents any integer from 0 to 5, A 1 represents *-CONH-*, Linker means *-L 1 -NH-L 2 -*, L 1 represents a straight chain or branched (C1-C30) alkylene group, L 2 represents a single bond, or a straight or branched (C1-C30) alkylene group, Fa represents chemical formula 2: [Chemical formula 2] * indicates the attachment site.

2. The gadolinium-based compound according to claim 1, characterized in that L 1 represents a straight chain or branched (C1-C10) alkylene group, L 2 Indicates a single bond.

3. An MRI contrast agent, characterized in that: It contains the gadolinium-based compound according to claim 1.

4. Use of the gadolinium-based compound according to claim 1 or 2 in the preparation of a medicament for diagnosing degenerative brain diseases.

5. The use according to claim 4, characterized in that The degenerative brain disease is Alzheimer's disease.

6. A method for preparing a gadolinium-based compound according to claim 1, characterized in that: include: (a) reacting a compound of the following Chemical Formula 1-1 with a compound of the following Chemical Formula 1-2 to obtain a compound of the following Chemical Formula 1-3; [Chemical formula 1-1] In the chemical formula 1-1, L 2 As defined in the chemical formula 1 of claim 1, [Chemical formula 1-2] [Chemical formula 1-3] In the chemical formulas 1-2 and 1-3, (PT) 3C represents a protecting group, Linker means *-L 1 -NH-L 2 -*, L 1 , L 2 , A and Fa are as defined in Chemical Formula 1 as claimed in claim 1, (b) a step of removing the protecting group (PT) 3C from the compound of Chemical Formula 1-3; and (c) a step of reacting the compound obtained in step (b) with gadolinium hydrate to obtain the compound of Chemical Formula 1 described in claim 1.

7. The method according to claim 6, characterized in that In the step (a), the L 2 For a single key.

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