KR20230084040A - Gadolinium-based compound, mri contrast agent comprising the same - Google Patents

Abstract

A gadolinium-based compound and an MRI contrast agent including the same are disclosed. The gadolinium-based compound may be a DO3A-based gadolinium compound to which a diaza crown ether to which an alkylguanidine functional group is bonded. The MRI contrast agent may include the compound.

Description

Gadolinium-based compound, MRI contrast agent containing the same {GADOLINIUM-BASED COMPOUND, MRI CONTRAST AGENT COMPRISING THE SAME}

The present invention relates to a gadolinium-based compound and an MRI contrast agent containing the same.

Due to the aging of the population today, the number of patients with degenerative brain diseases is increasing, and accordingly, the need for early detection of the disease is emerging. Degenerative brain diseases include Parkinson's disease, vascular dementia, Alzheimer's disease, and the like, and neurotoxicity due to overexpression of glutamate is considered as one of the causes of the disease.

Glutamate, which is responsible for more than 70% of excitatory signaling in the human brain, is an important amino acid regulating learning, memory, motor skills, and emotions. Excitatory signal transduction by glutamate at the synapse is tightly regulated by several glutamate transporters and receptors. The vesicular glutamate transporter (VGLUT) is known to play a key role in regulating the storage and concentration of glutamate in neuronal cells. Normally, the concentration of glutamate in the synaptic cleft is 1-3 μM, but when glutamate stored in vesicles is released, the concentration rises to hundreds to thousands of μM or more.

Therefore, when abnormalities in VGLUT occur, glutamate stored in vesicles is abnormally secreted in excess, and according to a recent study, when the concentration of glutamate increases, reuptake by glial cells decreases, and glutamate accumulates at the neuronal synapse, resulting in toxicity. It is said to cause damage to nerve cells by inducing generation of reactive oxygen species and oxidative stress.

In addition, recently, studies have shown that the concentration of glutamate increases in the gyrus or cerebrospinal fluid in the early stages of the onset of Alzheimer's disease, but as Alzheimer's progresses, nerve cells are damaged and the number decreases. . Therefore, it was expected that detecting changes in glutamate concentration would enable early diagnosis of degenerative brain diseases.

On the other hand, Magnetic Resonance Image (MRI) is a method of obtaining anatomical, physiological, and biochemical information images of the body by using a phenomenon in which the distribution of hydrogen atoms is different between tissues in the body and the hydrogen atoms are relaxed in a magnetic field. . Unlike CT or PET, MRI does not use radiation that is harmful to the human body and uses radio waves and the gradient of a magnetic field under a strong magnetic field to create images of the inside of the body.

In order to use such MRI equipment more precisely, a contrast agent is injected into an object to obtain an MRI image. Contrast between tissues on an MRI image is a phenomenon that occurs because a relaxation action in which a nuclear spin of a water molecule returns to an equilibrium state in a tissue is different for each tissue. The contrast agent uses a paramagnetic or superparamagnetic material to affect the relaxation effect, thereby widening the difference in relaxation between tissues and causing a change in the MRI signal, thereby making the contrast between tissues clearer.

Currently, the most commonly used contrast agent clinically is a contrast agent based on a gadolinium (Gd) chelate. Currently, Gd-DTPA (Magnevist®), Gd-DOTA (Dotaram®), Gd(DTPA-BMA) (Omniscan®), Gd(DO3A-HP) (ProHance®), Gd(BOPTA) (MultiHance®), etc. It is being used. However, most commercially available contrast agents are non-specific contrast agents that are distributed in the extracellular space (ECF). As a specific contrast agent, only a liver-specific contrast agent is used.

Recent studies have been promoting the development of contrast agents that have specific targets or can exhibit signal enhancement by physiological activity (pH change, enzyme activity), but so far, MRI contrast agents with specific targets, especially for degenerative brain Sufficient results have not been obtained for disease-specific MRI contrast agents.

One object of the present invention is to provide a compound that specifically binds to cationic neurotransmitters.

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

In one aspect, the present invention provides a compound having Formula (1) below.

(1)

(One)

here,

L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*;

x, y and z are each independently selected as any integer from 0 to 5;

A ¹ and A ² are a single bond, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* one or more structures, each independently selected,

X is a structure having formula (2):

(2)

(2)

* is a bonding site.

In one embodiment, the A ¹ may be *-CO-*.

In one embodiment, the A ² may be a single bond.

In one embodiment, the x may be 1.

In one embodiment, the y may be 0.

In one embodiment, the z may be 0.

In one embodiment, the compound may have the following formula (3).

(3)

(3)

In one embodiment, the gadolinium (Gd) may coordinate with one or more water molecules.

In one embodiment, the compound may specifically bind to a mammalian cationic neurotransmitter.

In one embodiment, the compound is specifically for a cationic neurotransmitter containing at least one selected from glutamate, GABA, acetylcholine, and aspartic acid. can be combined

In one embodiment, the compound may have a relaxation rate of 3.4 to 10.6 s ^-1 .

In one embodiment, the compound can cross the blood-brain barrier (BBB) when injected intravenously.

In another aspect, the present invention provides an MRI contrast agent containing the compound.

In one embodiment, the MRI contrast agent can be used for diagnosis of degenerative brain disease.

In one embodiment, the MRI contrast agent may be used for diagnosis of Alzheimer's disease.

A compound according to an embodiment of the present invention is a cationic neurotransmitter, specifically, a cation containing at least one selected from glutamate, GABA, acetylcholine, and aspartic acid. It can specifically bind to sexual neurotransmitters, and therefore, an MRI contrast agent containing the compound can be used for diagnosis of degenerative brain diseases including Alzheimer's disease.

1 to 5 are views showing experimental results according to experimental examples of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention can have various changes and various forms, specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals have been used for like elements throughout the description of each figure. In the accompanying drawings, the dimensions of the structures are shown enlarged than actual for clarity of the present invention.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as "comprise" or "have" are intended to designate that there is a feature, number, step, operation, component, or combination thereof described in the specification, but one or more other features or numbers However, it should be understood that it does not preclude the presence or addition of steps, operations, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one 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 having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

A compound according to an embodiment of the present invention may have the following formula (1).

(1)

(One)

In the above formula (1), the gadolinium ion (Gd ³⁺ ) may coordinate with the carboxylate (COO ⁻ ) group of the above formula (1) to form a complex compound.

In Formula (1), L may be *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*, and x, y and z are any of 0 to 5 Each can be independently selected as an integer of, A ¹ and A ² are single bonds, *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* And *-O-* may be one or more structures each independently selected from the group containing. * is a bonding site.

The L may be a linker connecting nitrogen and the X in the cyclic structure of the compound. The A ¹ and A ² may determine a method in which the linker connects nitrogen and the X in the cyclic structure of the compound or a functional group determined by the method. The x, y and z may determine the length of the chain linking the A ¹ and A ² in the linker. In one embodiment, the A ¹ may be *-CO-*. In one embodiment, the A ² may be a single bond. In one embodiment, the x may be 1. In one embodiment, the y may be 0. In one embodiment, the z may be 0.

As long as the compounds according to the embodiments of the present invention substantially have the structure of Formula (1) above, binding or removal of binding acceptable to those skilled in the art is not excluded from the scope of the present invention. For example, in one embodiment, the gadolinium (Gd) in the compound may coordinate with one or more water molecules.

In Formula (1), X may have a structure having the following Formula (2).

(2)

(2)

* is a bonding site.

In one embodiment, the compound may have the following formula (3).

(3)

(3)

In one embodiment, the compound may specifically bind to a mammalian cationic neurotransmitter. In one embodiment, the compound is specifically for a cationic neurotransmitter containing at least one selected from glutamate, GABA, acetylcholine, and aspartic acid. can be combined

In one embodiment, the compound may have a relaxation rate of 3.4 to 10.6 s ^-1 . In one embodiment, the compound can cross the blood-brain barrier (BBB) when injected intravenously.

As described above, the compound according to the embodiment of the present invention is cationic neurotransmission including at least one selected from glutamate, GABA, acetylcholine, and aspartic acid. It can specifically bind to a substance.

Meanwhile, an MRI contrast agent according to an embodiment of the present invention may include the compound. In one embodiment, the MRI contrast agent can be used for diagnosis of degenerative brain disease. In one embodiment, the MRI contrast agent may be used for diagnosis of Alzheimer's disease.

It is known that concentrations of cationic neurotransmitters targeted by compounds according to embodiments of the present invention decrease with the progression of Alzheimer's disease. Therefore, an MRI contrast agent containing the compound can be used for diagnosis of degenerative brain diseases including Alzheimer's disease.

Hereinafter, embodiments of the present invention will be described in detail. However, the examples described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

<Synthesis of GdL (Gd-crown)>

A Gd-crown (GDL) compound was prepared by stepwise synthesis of material 1 to final material GdL .

① Synthesis of Tert-butyl-3-bromopropylcarbamate, 1

It was synthesized in 95% yield by referring to a known synthesis method.

② Synthesis of Benzyl (3-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)propyl)-carbamate, 2

4,13-Diaza-18-crown-6-ether (0.98 g, 3.7 mmol) was dissolved in dichloromethane (70 mL), and triethylamine (0.63 mL, 4.5 mmol) was added. Compound 1 (1.07 g, 3.9 mmol) was dissolved in dichloromethane (5 mL) and slowly added to the reaction mixture over 10 minutes, followed by stirring at room temperature for two days. Then, the reactant was concentrated under reduced pressure, and the precipitate formed by adding an excess of diethyl ether was filtered, and after precipitation, the solvent was removed from the precipitated solution under reduced pressure and silica gel chromatography was used (1-2% methanol / 1% triethyl dichloromethane containing amine) to obtain a pale yellow oil. Yield: 0.4 g (24%)

③ Tri-tert-butyl 2,2',2”-(10-(2-(16-(3-(((benzyloxy)carbonyl)amino)propyl)-1,4,10,13-tetraoxa-7, Synthesis of 16-diazacyclooctadecan-7-yl)2-oxoethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, 3

Compound 2-1 (0.65 g, 1.14 mmol), TBTU (0.46 g, 1.42 mmol) and HOBt (0.19 g, 1.42 mmol) were dissolved in dimethylformamide (7.5 mL) under nitrogen. Then add DIPEA (0.5 mL, 2.84 mmol) and stir the reaction for another 10 min. Next, compound 2 (0.43 g, 0.95 mmol) dissolved in dimethylformamide (3mL) was added to the reaction mixture, stirred at room temperature for an additional 18 hours, concentrated under reduced pressure, and dissolved in dichloromethane. Thereafter, the organic layer was washed three times with a saturated aqueous sodium carbonate solution, and then purified by fractionation using silica gel column chromatography (2-5% methanol/dichloromethane) to obtain Compound 3 as a white semi-solid. Yield: 0.75 g (76%)

④ Tri-tert-butyl 2,2'2”-(10-(2-(16-(3-aminopropyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)-2 Synthesis of -oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, 4

Pd/C (0.33 g, 0.15 mmol, 10% wt, wet type) was weighed into a well-dried reaction flask and nitrogen gas was purged. Compound 3 (0.52 g, 0.51 mmol) dissolved in methanol (20 mL) and ethyl acetate (10 mL) was added, and 2N ammonia (NH ₃ ) dissolved in methanol was added, followed by bubbling with hydrogen gas at room temperature for 5 hours. . The reactant was filtered under reduced pressure using a celite pad and concentrated to obtain a white foamy solid, which was used for the next step without further purification.

⑤ Tri-tert-butyl 2,2'2”-(10-(2-(16-(3-(2,3-bis(tert-butoxycarbonyl)-guanidino)-propyl)-1,4,10,13 Synthesis of -tetraoxa-7,16-diazacyclooctadecan-7-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(Z)-triacetate, 5

To N,N'-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamide (0.16 g, 0.54 mmol) dissolved in dichloromethane (10 mL), add triethylamine (76 μL, 0.54 mmol). . Then, Compound 4 (0.44 g, 0.5 mmol) dissolved in dichloromethane (5 mL) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 18 hours. Next, the reactant was concentrated under reduced pressure and separated and purified using silica gel column chromatography (2-3% methanol/dichloromethane) to obtain a white foamy compound 5 . Yield: 0.4 g (72%)

⑥ 2,2'2”-(10-(2-(16-(3-guanidinopropyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)-2-oxoethyl)-1 Synthesis of ,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, L

Compound 5 (0.1 g, 0.09 mmol) dissolved in trifluoroacetic acid (TFA, 10 mL) was stirred at room temperature overnight. Then, the mixture was concentrated under reduced pressure using C18 silica gel column chromatography (0-5% acetonitrile/water (0.1% TFA)) to obtain a white solid, L (0.1 g).

⑦ Synthesis of GdL (Gd-crown)

L (0.1 g, 0.13 mmol) and GdCl ₃ 6H ₂ O (50 mg, 0.13 mmol) were dissolved in tertiary distilled water (15 mL), and the pH of the solution was adjusted to 6.5-7.0 using 1N aqueous sodium hydroxide solution. . Next, the reactant was stirred at room temperature overnight and the reaction was confirmed by LC-MS. Thereafter, the reaction product was separated and purified using C18 silica gel column chromatography (0-5% acetonitrile/water) to obtain a white solid, GdL (Gd-crown). Yield: 85 mg (72%)

<Results of HR-MS and purity analysis of GdL (Gd-crown)>

Gd-crown was analyzed by HR-MS. 1 is a diagram showing the result. Referring to FIG. 1, it can be confirmed that a peak (903.3583 m/z) corresponding to the peak (903.3579 m/z) predicted for Gd-crown appears.

In addition, the results of analyzing the purity of Gd-crown using HPLC are shown in FIG. 2 . Referring to FIG. 2 , it can be confirmed that the Gd-crown has a purity of about 100%.

<GdL (Gd-crown) performance evaluation method and results>

1) Measurement of relaxation rate

The synthesized Gd-crown and Gd-DOTA, a commercially available cyclic contrast agent, were used as comparative groups to measure the self-relaxation rates ( r ₁ , r ₂ ) of each material, and the results are shown in Table 1 below.

Specifically, samples were prepared by diluting the gadolinium complex at 5 concentrations (0.0625, 0.125, 0.25, 0.5, 1 mM) in the solution (PBS, 25 mM HEPES buffer (pH 7.4), 0.67 mM HAS solution), and then 3T MRI was performed. In T ₁ , T ₂ relaxation time (Relaxation time) was measured. Here, T ₁ uses 35 inversion times (TI, 50 ~ 1750 ms), and T ₂ uses a Carr-Purcell-Meiboon-Gill (CPMG) pulse sequence and 32 echo times (echo time, TI). TE, 30 to 960 ms), and was calculated using a nonlinear least-squares fit of the signal intensity at each TI or TE value. The relaxation rate ( R ₁ , R ₂ ) was obtained as the reciprocal of the relaxation time, and the self-relaxation rates (relaxivities, r ₁ , r ₂ ) were obtained from the slope of the linear graph of the relaxation rate change according to the concentration of gadolinium (Gd). got

r ₁ (mM ^-1 s ^-1 ) r ₂ (mM ^-1 s ^-1 ) HEPES ^{a PBSb} HSA ^c HEPES ^{a PBSb} HSA ^c Gd-crown 3.44 4.55 4.80 7.50 7.98 10.59 Gd-DOTA 3.13 3.03 3.47 6.06 6.00 7.04 ^a [HEPES] = 25 mM in water, pH 7.4; ^b PBS: pH 7.4; ^c [HSA] = 0.67 mM in water.

Referring to Table 1, it can be confirmed that the self-relaxation rate of Gd-crwown was higher than that of Gd-DOTA in all tested solutions.

2) Confirmation of binding affinity between Gd-crown and glutamate

Glutamic acid was dissolved at various concentration ratios ([Glu]/[Gd compound]: 0 to 100) in a solution (HEPES buffer solution, pH 7.4) in which 2 mM of Gd-crown and the control Gd-DOTA compound were dissolved, respectively. The R ₁ relaxation rate in the solution was measured.

Referring to FIG. 3 showing the results, it can be seen that the relaxation rate ( R ₁ ) increases more than twice as high as the concentration of glutamic acid in the case of Gd-crown compared to that of Gd-DOTA, which is a control group. It is predicted that glutamic acid and Gd-crown form a ternary complex, which slows down the tumbling rate and increases the relaxation rate.

3) Confirmation of binding affinity between Gd-crown and neurotransmitters

Neurotransmitters were added at various concentration ratios ([neurotransmitter]/[Gd compound]: 0 to 100) in a solution (HEPES buffer, pH 7.4) in which 2 mM of Gd-crown and the control Gd-DOTA compound were dissolved, respectively. Phosphorus aspartic acid, GABA, acetylcholine, and glycine were dissolved, and the R ₁ relaxation rate in each solution was measured.

Referring to Figure 4 showing the results, in the case of Gd-crown, aspartic acid, GABA, and acetylcholine were compared to the control Gd-DOTA, as the concentration increased, the relaxation rate ( R ₁ ) can be seen as a result of which the change in .

4) Comparison of brain MR images of commercially available contrast agents (Gd-DOTA) and glutamic acid-targeted contrast agents (Gd-crown) in dementia animal models (APP/PS1) and normal rats

Using a 6-8-month-old dementia animal model (APP/PS1) and normal mice, brain MR images were obtained before contrast agent injection under respiratory anesthesia, and then 50 mM Gd compounds (Gd-DOTA, Gd-crown), 4 μL was injected into the intraventricular space (intracerebroventricular, icv injection) to obtain T ₁ -weighted MR images up to 5 hours. Parameters used to acquire the T ₁ -weighted image are as follows. (Repetition time (TR) = 700 ms, Echo time (TE) = 7 ms, (Number of acquisition (NEX) = 4, 20 mm field of view (FOV), 128*128 matrix size, 0.5 mm slice thickness)

Referring to FIG. 5 showing the results, in the case of Gd-crown , the CNR changes in the hippocampus (at 1 hour) and the cortex (at 1, 3, and 5 hours) were statistically significant in normal mice compared to the dementia model. It can be seen that it has a significantly higher value. On the other hand, in the case of the control group, Gd-DOTA, there was little difference between the two animal groups.

Several papers report that the concentrations of glutamic acid and aspartic acid present in the hippocampus and cortex areas of the APP/PS1 model of 6-8 months are lower than those of normal mice ( Biochimica et Biophysica Acta 2014, 1842 , 2395-2402 ; Proceedings of the National Academy of Sciences 2005, 102 (33), 11906-11910; Nature Communications 2014, 5 , 1-12). Through this, the possibility of diagnosing dementia using the Gd-crown of the present invention as a contrast agent was confirmed.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. You will understand that you can.

Claims (13)

Having the following formula (1),
compound:

(One)
here,
L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*;
x, y and z are each independently selected as any integer from 0 to 5;
A ¹ and A ² are a single bond, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* one or more structures, each independently selected,
X is a structure having formula (2):

(2)
* is a bonding site. According to claim 1,
Wherein A ¹ is *-CO-*,
compound. According to claim 2,
Wherein A ² is a single bond;
compound. According to claim 3,
wherein x is 1,
wherein y is 0,
wherein z is 0;
compound. According to claim 1,
Having the following formula (3),

(3)
compound. According to claim 1,
The gadolinium (Gd) coordinates with one or more water molecules,
compound. According to claim 1,
The compound specifically binds to mammalian cationic neurotransmitters,
compound. According to claim 7,
The compound specifically binds to a cationic neurotransmitter containing at least one selected from glutamate, GABA, acetylcholine and aspartic acid,
compound. According to claim 1,
The compound has a relaxation rate of 3.4 to 10.6 s ^-1 ,
compound. According to claim 1,
The compound, when injected via intravenous injection, crosses the blood-brain barrier (BBB),
compound. Comprising the compound according to any one of claims 1 to 10,
MRI contrast agents. According to claim 11,
The MRI contrast agent is used for diagnosis of degenerative brain disease,
MRI contrast agent. According to claim 12,
The MRI contrast agent is used for diagnosis of Alzheimer's disease,
MRI contrast agents.

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