KR101836461B1 - Gd-complex of DO3A-ferrocene Conjugates as MRI Contrast Agent - Google Patents

Abstract

The present invention relates to the development of a new type of MR contrast agent based on ferrocene, and more particularly, to a novel MR contrast agent based on ferrocene, more specifically a DO3A-ferrocene compound, a gadolinium complex containing the compound as a ligand, an MRI contrast agent including the gadolinium complex, And a manufacturing method thereof.
The compound of the present invention has a characteristic that the structure of DO3A is bonded with ferrocene and when it forms a complex with gadolinium, it can have enhanced contrast effect, dynamics and thermodynamic stability as compared with a compound used as a ligand or a backbone of a conventional contrast agent It can be used very useful as an MRI contrast agent.

Description

Development of new type MR contrast agent based on ferrocene {Gd-complex of DO3A-ferrocene Conjugates as MRI Contrast Agent}

The present invention relates to the development of a new type of MR contrast agent based on ferrocene, and more particularly, to a novel MR contrast agent based on ferrocene, more specifically a DO3A-ferrocene compound, a gadolinium complex containing the compound as a ligand, an MRI contrast agent including the gadolinium complex, And a manufacturing method thereof.

Magnetic Resonance Imaging (MRI) is a method of obtaining anatomical, physiological, and biochemical information images of a body using the phenomenon of spin relaxation of hydrogen atoms in a magnetic field, It is one of the excellent imaging devices that can be invasive and can be imaged in real time.

In life sciences and medical fields, in order to make various and precise use of MRI, materials are injected from outside to increase image contrast, and the substance used at this time is called a contrast agent. The contrast between tissues on the MRI image is due to the different tissues' different relaxations of the water molecule nuclear spin in the tissue to equilibrium, and the contrast agent affects this relaxation And the difference in the degree of relaxation between the tissues is opened to cause a change in the MRI signal, thereby making the contrast between the tissues clearer. Contrast agents have a difference in utilization and precision depending on the features, functions, and targets to be injected. Enhanced contrasts using contrast agents enhance or brighten the image of the surrounding organs and tissues of specific organs and tissues. The contrast agent which makes the image signal of the body part which wants to obtain the MRI image relatively high is referred to as a 'positive contrast agent,' while the contrast agent which makes it relatively lower than the surrounding is called a 'negative contrast agent'. N, N ', N', N ', N'-tetramethylethylenediamine, which exhibits a magnetic relaxation rate of about 4.7 mM -1 s -1 (20 MHz, 298 K) (Ⅲ) complexes such as N, N'-pentaacetate, (N-Me-glucamine) 2 [Gd (DTPA) (H2O)] (Magnevist, Schering) and magnetic relaxation rates of 4.4 mM- (III) complexes such as [Gd (DTPA-bismethylamide) (H2O)] (Omniscan, Nycomed)

The necessary properties of this contrast imaging contrast agent are the multidentate structure, which causes thermodynamic stability, water solubility, and paramagnetic Gd (Ⅲ) ion, that is, at least one molecule of water combined with high water magnetic relaxation . In contrast, the contrast agent should be chemically inactive, have low cytotoxicity in vivo, and should be completely excreted out of the body after diagnosis. However, the contrast agents have low water solubility and self-relaxation rate, and have a relatively high cytotoxicity in vivo. Therefore, it is still necessary to develop an optimized MRI contrast agent.

The inventors of the present invention have made intensive studies to overcome the problems of the prior arts as a result. As a result, it has been found that when a paramagnetic gadolinium chelate having a ring structure including a ferrocene having a high aromaticity is prepared, A contrast agent exhibiting a contrast effect and having excellent thermodynamic and kinetic stability can be produced, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a DO3A-ferrocene or a derivative thereof containing a ferrocene having a structure represented by the following formula

[Chemical Formula 1]

Another object of the present invention is to provide a process for producing the DO3A-ferrocene or derivatives thereof according to claim 1, which comprises the following steps:

a) adding DO3A (tBu) 3 to ferrocene acetyl chloride and stirring to obtain a mixture;

b) filtering and evaporating the mixture to obtain a crude compound;

c) purifying the crude compound to remove the solvent to obtain a DO3ATBE-ferrocene derivative;

d) adding the DO3ATBE-ferrocene derivative of step c) to a mixture of TFA (Trifluoroacetic acid) and CH2Cl2 and stirring to obtain a mixture; And

e) evaporating the mixture of step d) to obtain a DO3A-ferrocene compound.

Another object of the present invention is to provide a complex comprising the DO3A-ferrocene or a derivative thereof as a ligand (L), and a metal atom coordinating to the ligand.

Another object of the present invention is to provide a magnetic resonance imaging (MRI) contrast agent comprising the complex as an active ingredient.

In order to achieve the above object, the present invention provides a ferric oxide-containing DO3A-ferrocene or a derivative thereof having the structure of Formula 1:

[Chemical Formula 1]

In order to achieve the above object, the present invention provides a process for producing the DO3A-ferrocene or a derivative thereof according to claim 1, comprising the steps of:

a) adding DO3A (tBu) 3 to ferrocene acetyl chloride and stirring to obtain a mixture;

b) filtering and evaporating the mixture to obtain a crude compound;

c) purifying the crude compound to remove the solvent to obtain a DO3ATBE-ferrocene derivative;

d) adding the DO3ATBE-ferrocene derivative of step c) to a mixture of TFA (Trifluoroacetic acid) and CH2Cl2 and stirring to obtain a mixture; And

e) evaporating the mixture of step d) to obtain a DO3A-ferrocene compound.

In order to achieve the above object, the present invention provides a complex comprising the DO3A-ferrocene or a derivative thereof as a ligand (L), and a metal atom coordinating to the ligand.

In order to achieve the above object, the present invention provides a magnetic resonance imaging (MRI) contrast agent comprising the complex as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a DO3A-ferrocene or a derivative thereof containing a ferrocene having a structure represented by the following Formula 1:

[Chemical Formula 1]

The term " DO3A " used in the present invention means a metal affinity chelate compound 1,4,7,10-tetraazacyclododecane and is used as a ligand capable of binding gadolinium. Gadolinium, which is a lanthanide metal, is capable of 9 bonds. Gadolinium ions are very toxic and can not be administered to human body. Therefore, ligands such as DO3A should be combined to reduce their toxicity. DO3A has four N (nitrogen) and three COOH (carboxyl) groups and is a ligand suitable for bonding with gadolinium.

In the present invention, the 'ferrocene' is a metallocene in which the divalent iron cation is bound to two cyclopentadienyl anions. Since the ferrocene is inherently more aromatic than benzene, the phenyl moiety can be replaced by a ferrocene derivative. It is also known that ferrocene is chemically stable in a variety of non-oxidizing media and toxic to the human body because it forms a metabolic ferricinium ion in situ.

The DO3A-ferrocene or derivative compound of the present invention forms a complex with gadolinium due to its structural characteristics, and can have excellent contrast effect and thermodynamically excellent stability as compared with a compound used as a ligand or a backbone of a conventional contrast agent.

The DO3A-ferrocene of the present invention can be prepared by a process comprising the following steps:

a) adding DO3A (tBu) 3 to ferrocene acetyl chloride and stirring to obtain a mixture;

b) filtering and evaporating the mixture to obtain a crude compound;

c) purifying the crude compound to remove the solvent to obtain a DO3ATBE-ferrocene derivative;

d) adding the DO3ATBE-ferrocene derivative of step c) to a mixture of TFA (Trifluoroacetic acid) and CH2Cl2 and stirring to obtain a mixture; And

e) evaporating the mixture of step d) to obtain a DO3A-ferrocene compound.

In the present invention, the ferrocene acetyl chloride in step a) can be produced according to a known method, for example, the method described in Organometallics 2009, 28, 5412-5423. According to one embodiment of the present invention, ferrocene acetyl chloride is added to CH3CN together with K2CO3, stirred, and the reaction is carried out by adding DO3A-tert-butyl ether (DO3ATBE), followed by evaporation under reduced pressure to obtain a crude compound. Purification by chromatography yielded a red solid DO3ATBE-ferrocene compound.

The DO3ATBE-ferrocene compound was added to a mixture of TFA (trifluoroacetic acid) and CH2Cl2, stirred, and the solvent was evaporated under reduced pressure. The mixture was dissolved in methanol and diluted with diethyl ether to obtain a red solid DO3A-ferrocene there was.

The present invention provides a complex comprising the DO3A-ferrocene or a derivative thereof as a ligand (L), and a metal atom coordinating to the ligand.

In the complex of the present invention, the metal atom may be selected from the group consisting of gadolinium (Gd), calcium (Ca), zinc (Zn) and copper (Cu), preferably gadolinium .

As used herein, the term " complex " means that several different atoms, ions, molecules or atomic groups centering on one or more atoms or ions are oriented and coordinately coordinated to form a single atom It is a group of people. The atomic ion molecule or atom group coordinated to the central atom or ion here is called a ligand. In other words, it means that the DO3A-ferrocene compound of Formula 1 acts as a ligand to form a complex with gadolinium.

In the present invention, the complex in which gadolinium is coordinated to the DO3A-ferrocene ligand is characterized by having the structure represented by the following formula (2)

(2)

The present invention also provides a magnetic resonance imaging (MRI) contrast agent comprising said complex as an active ingredient.

According to one embodiment of the present invention, the contrast agent containing the complex of DO3A-ferrocene ligand and gadolinium as the active ingredient showed equivalent or better stability as compared with the DOTA-based contrast agent as the cyclic ligand, and the non-ligand type ligand DTPA -bis (amide) -based contrast agent.

The DO3A-ferrocene ligand used in the contrast agent of the present invention is a cyclic ligand that is superior in terms of thermodynamic stability and kinetic stability as compared with the non-coordinating chelate, and at the same time, when forming a paramagnetic complex, the ligand is connected by a ferrocene The dynamics of the ring is considerably limited and the increase in the molecular weight causes synergism at the same time, so that the R1 contrast effect can be significantly increased.

The MRI contrast agent of the present invention can be used as an MRI contrast agent used for obtaining an MRI image for diagnosis of a disease including cancer in which a gadolinium contrast agent can be conventionally used. Gadolinium contrast agent.

The contrast agent according to the present invention may be formulated according to a conventional formulation method for preparing a contrast agent, which is known in the art, and may be used in the form of a composition, preferably as an intravenous composition. Contrast agents of the invention may contain physiologically compatible buffers (e.g., 0.08% NaCl salt solution, or tris (hydroxymethyl) aminomethane) and physiologically applicable additives (such as stabilizers such as parabens) have.

The compound of the present invention is characterized in that ferrocene is bonded to the structure of DO3A and when it forms a complex with gadolinium, it has enhanced contrast enhancement effect, dynamics and thermodynamic stability as compared with the compound used as a ligand or backbone of the conventional contrast agent Therefore, it can be very useful as an MRI contrast agent.

FIG. 1 is a schematic diagram of a process for synthesizing a compound of Formula 1 and a process for synthesizing a gadolinium complex according to the present invention.
FIG. 2 is a graph for evaluating the kinetic stability of contrast agents with time. FIG.
Fig. 3 is a diagram for evaluating the relaxation rate of the contrast agent of the present invention (A: R 1 and B of the contrast agent of the present invention: R 2 of the contrast agent of the present invention).
FIG. 4 is a graph showing an MRI contrast effect according to time after administering the contrast agent of the present invention to a tail vein of a normal mouse.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Experimental material

Specific synthetic procedures for preparing the ferrocene-DO3A compound are described in detail below. All halvers were performed under dinitrogen atmosphere using standard Schlenk technology. The solvent was purified using standard procedures and dried. DO3A (tBu) 3 (= 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert-butyl acetate) was prepared according to literature ( Gu, S. et al., J. Med. Chem 2011, 54 (1), 143-152 ). All industrial reagents were purchased from Aldrich and used unless otherwise specified. Deionized water was used in all experiments. 1H NMR experiments were performed using a Bruker Advance 400 Spectrometer at Kyungpook National University Instrumental Analysis Center. Chemical shifts were provided as delta values for tetramethylsilane (TMS) as an internal standard. The coupling constant is expressed in Hz. The FAB-mass spectra were obtained using the JMS-700 model (Jeol, Japan) mass spectrometer at Korea Basic Science Institute (KBSI).

≪ Example 2 >

Synthesis of ferrocene acetyl chloride

In FIG. 1 (2), the compound, ferrocene acetyl chloride, was prepared according to a known method (Organometallics 2009, 28, 5412-5423).

Briefly, ferrocene (10 g, 53.75 mmol) and chloroacetyl chloride (3.6 g, 32.25 mmol) were added to 50 mL of CH 2 Cl 2 at -15 ° C and stirred while AlCl ₃ (4.3 g, 32.25 mmol) . The reaction mixture was then stirred at ambient temperature for 24 hours. When the reaction was completed, the reaction mixture was deactivated by addition of ice, followed by extraction with CH2Cl2. The crude product obtained was further purified by column chromatography (silica, hexane / ethyl acdetate) to obtain a brown solid. Yield: 3.5 g (25%)

≪ Example 3 >

Synthesis of DO3ATBE-Ferrocene Derivatives

Of the CH ₃ CN 30 mL at room temperature, the ferrocene chloride (2) (3g, 11.43 mmmol ) and K ₂ CO ₃ while stirring a (4.75 g, 34.35 mmol), DO3ATBE (4.27 g, 9.16 mmol) CH 3 CN And the mixture was slowly added over 30 minutes. The reaction was then stirred at ambient temperature for 24 hours. When the reaction was complete, the mixture was filtered to remove the inorganic solid and the organic phase was evaporated under reduced pressure. The resulting crode product was further purified via column chromatography (silica, CH ₂ Cl ₂ / MeOH, 95: 5) to obtain a red solid (compound (3) in FIG.

Yield: 4.9 g (58%). ^{1 H NMR (CDCl3): δ} = 4.68 (br, 4H, Fc), 4.47-4.45 (Br, 5H, Fc), 2.82-1.72 (m, 28H, cyclen), 1.40-1.85 (m, 27H, - ( CH3) 3). Maldi-TOF (m / z): C38H61FeN4O7 Calcd. for: 741.389 ([MH < + >]). Found: 741.7553 ([MH < + >]).

<Example 4>

Synthesis of DO3A-Ferrocene Derivatives

To the mixture of TFA (Trifluoroacetic Acid) / CH ₂ Cl ₂ (1: 1, 30 mL) was added the DO3ATBE-ferrocene compound (3 g, 4.05 mmol) prepared in Example 2 and stirred at room temperature for 24 hours. The solution was evaporated under reduced pressure. The mixture was dissolved in a small amount of methanol (MeOH) and diluted with diethyl ether (100 ml) to precipitate a red solid. This was separated by filtration, washed several times with diethyl ether, and then dried under vacuum to obtain a red solid (compound (4) in FIG. 1).

Yield: 1.4 g (62%). ¹ H NMR (CDCl 3):? = 3.15 (br, 4H, Fc), 2.81 (br, 5H, Fc), 2.20-1.77 (m, 24H, cyclen). Maldi-TOF (m / z) C26H37FeN4O7 Calcd. for: 573.2012 ([MH &lt; + &gt;]). Found: 573.3457 ([MH &lt; + &gt;]).

&Lt; Example 5 >

Preparation of gadolinium complex

Gadolinium (III) chloride hexahydrate (2.3 g, 6.29 mmol) was added to the DO3A-ferrocene compound (3 g, 5.24 mmol) of Example 3 dissolved in deionized water (15 ml). The mixture was stirred for 18 hours and during this time the pH of the solution was adjusted to 7.0-7.5 with NaOH (1.0 M) periodically. The water was removed by evaporation and the remaining organic product was dissolved in the minimum amount of MeOH (~ 5 ml) and diethyl ether was added for precipitation. The resulting red precipitate was filtered off and the solvent was further dissolved in a small amount of MeOH (~ 5 ml) and further purified through Emberite to obtain a red solid (compound (5) in FIG. 1).

Yield: 2.0 g, (53%). FAB-Mass (m / z) C26H33FeGdN4O7 Calcd. for: 726.6578 ([M]). Found: 726.70 ([M]).

&Lt; Example 6 >

Stability of Gadolinium Complex

1) Transmetallation kinetics

This test was carried out according to the literature in which the water proton relaxation rates of the same molar amount of compound 5 (2.5 mM) and ZnCl ₂ (2.5 mM) in phosphate buffer (pH 7.4) , R ₁ ^P ) was measured. That is, a buffer solution (10 μl) of ZnCl ₂ was added to a buffer solution (1 ml) of the compound (5). The mixture was stirred vigorously and 300 μl of the solution was used for a relaxometric study. In addition, comparative studies were performed using Gadovist, Dotarem, Multihance, Magnevist, and Omniscan, which were commercialized for comparison purposes.

The R ₁ ^P relaxation rate was obtained by subtracting the diamagnetic contribution of the underwater proton relaxation at the observed relaxation rate R ₁ = (1 / T 1). Measurements were performed at room temperature with a 3-tesla (T) whole body system (Magnetom Tim Trio, Siemens, Germany).

An advantage of compound (5) is that its form as a chelate backbone better than DTPA introduces DOTA. Thus, a significant improvement in the kinetic stability of compound (5) was expected over DTPA-based compounds. Indeed, this prediction is given in Fig. 2, which shows a plot of R ₁ ^p (t) / R ₁ ^p (0) as a function of time for various MRI CAs.

Thus, the relative value of R ₁ ^P at any time t, R ₁ ^P (t) / R ₁ ^P (0), is a good estimator of the degree of metal exchange by zinc. Of the endogenous ions, Zn ²⁺ can replace a significant amount of Gd, because the concentration of the former in the blood is relatively high (Laurent, S. et al., Invest. Radiol. 2001, 36,115-122). A dramatic increase in kinetic stability has been observed in compound (5) compared to the plots exhibited by DTPA-based CAs, a new form of BPCA developed by the present inventors (Kim, H.-K. et al. Chem. Commum., 2010, 46, 8442-8444). The kinetic stability of the compound (5) was also confirmed to be equal to or higher than that of Dotarem.

&Lt; Example 7 >

The relaxivity of the gadolinium complex

T ₁ measurements were performed using the inversion recovery method with variable inversion time (TI) at 1.5 T (64 MHz, GE Healthcare, Milwaukee, Wis., USA). Magnetic resonance (MR) images were required at 35 different TI values ranging from 50 to 1750 milliseconds. T ₁ relaxation time was determined from the non-linear least squares fit of the signal intensity measured at each TI value. The Carr-Purcell-Meiboon-Gill (CPMG) pulse sequence was applied to multiple spin-echo measurements for T ₂ measurements. Thirty-four images were required with 34 different echo time (TE) values ranging from 10 to 1900 msec. T ₂ relaxation time was determined from the non-linear least squares method of mean pixel values for multiple spin-echo measurements of each echo time. The relaxivity (R1 and R2) was then calculated inversely with the relaxation time per mM. The final relaxation time (T ₁ and T ₂ ) and the relaxation (R ₁ and R ₂ ) were finally image-worked to create relaxation time maps and relaxation rate maps, respectively.

Figure 3 below shows the proton relaxation r ₁ and r ₂ of the compound (5) of the present invention. Measurements were performed in PBS (pH 7.4). The r ₁ value of the compound 5 of the present invention has a value similar to that of dotarem (3.7 ± 0.12 mM -1 s -1), which is a contrast agent that is used as (3.3 ± 0.35 mM -1 s -1).

&Lt; Example 8 >

Contrast effect of gadolinium complex

All animal experiments were conducted according to the rules of the Animal Research Council of Kyungpook National University. Six-week-old male ICR mice weighing 29-31 g were used for MRI. (N = 4) were anesthetized with 1.5% isoflurane contained in oxygen. Measurements were performed before and after injection of compound 2 through the tail vein. The amount of CA per injection was 0.1 mmol [Gd] / kg for the MR image. After each measurement, the mice were restored in anesthesia and placed in a cage in which food and water could be taken freely. During this measurement, the animals were maintained at approximately 37 [deg.] C using a warm water bath. Whole body MR images were obtained with a 1.5 T MR unit (GE Healthcare, Milwaukee, Wis., USA) equipped with a home-made small animal RF coil. The coil is a receiver type having an inner diameter of 50 mm. Imaging parameters for SE (Spin Echo) are as follows: repetition time (TR) 300 ms; Echo time (TE) = 13 ms; 8 mm field of view (FOV); 192 x 128 matrix size; 1.0 mm slice thickness; Number of acquisition (NEX) = 8. Images were taken for 120 minutes after injection.

Figure 4 shows in vivo coronal images of mice obtained by tail vein injection of compound (5).

The compound of the present invention is characterized in that ferrocene is bonded to the structure of DO3A and when it forms a complex with gadolinium, it has enhanced contrast enhancement effect, dynamics and thermodynamic stability as compared with the compound used as a ligand or backbone of the conventional contrast agent Therefore, it is very useful as an MRI contrast agent, and thus it is very likely to be used in an industrial field.

Claims (7)

(D3A-ferrocene) compound containing a 1,4,7,10-tetraazacyclododecane-ferrocene (DO3A-ferrocene) compound containing a ferrocene having a structure represented by the following Formula 1 as a ligand (L) ) Complexes containing metal atoms:
[Chemical Formula 1]

A process for preparing a complex according to claim 1 comprising the steps of:
a) adding DO3A-1,4,7-tris-tert-butyl acetate (DO3A (tBu) ₃ ) to ferrocene acetyl chloride and stirring to obtain a mixture;
b) filtering and evaporating the mixture to obtain a crude compound;
c) purifying the crude compound to remove the solvent to obtain DO3A-tert-butyl ether (DO3ATBE) -ferrocene derivative;
d) adding the DO3ATBE-ferrocene derivative of step c) to a mixture of TFA (Trifluoroacetic acid) and CH ₂ Cl ₂ and stirring to obtain a mixture; And
e) evaporating the mixture of step d) to obtain a DO3A-ferrocene compound.
delete delete The complex of claim 1, wherein the complex has the structure of Formula 2:
(2)

A magnetic resonance imaging (MRI) contrast agent comprising the complex according to claim 1 or 5 as an active ingredient.
7. The contrast agent of claim 6, wherein the contrast agent has improved thermodynamic and kinematic stability.

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