WO2009145378A1

WO2009145378A1 - Gd complex comprising dtpa-bis-amide ligand and method for preparing the same

Info

Publication number: WO2009145378A1
Application number: PCT/KR2008/003144
Authority: WO
Inventors: Tae-Jeong Kim; Yong-Min Chang; Ji-Ae Park
Original assignee: Kyungpook National University Industry-Academic Cooperation Foundation
Priority date: 2008-05-27
Filing date: 2008-06-04
Publication date: 2009-12-03
Also published as: KR20090123171A

Abstract

A method of preparing a new DTPA-bis-amide ligand includes the following: first step of adding DTPA-bis-anhydride to N,N-dimethylformamide to with stirring; second step of adding 2-hydroxyethyl-trans-4-(aminomethyl)cyclohexyl carboxylate hydrochloride, 2-methoxyethyl-trans-4-(aminomethyl) cyclohexyl carboxylate hydrochloride, or allyl-trans-4-(aminomethyl)cyclohexyl carboxylate hydrochloride with stirring; third step of conducting silica gel chromatography after removing all of solvent under low pressure and adding methanol to dissolve; and fourth step of drying the product under vacuum state. Further, a method of preparing a new Gadolinium complex further includes fifth step of putting the obtained DTPA-bis-amide ligand to the distilled water, and adding Gd₂O₃ with stirring; sixth step of removing impurity and solvent; and seventh step of dissolving it in methanol and precipitating it with acetonitrile to obtain a solid.

Description

[DESCRIPTION] [Invention Title]

GD COMPLEX COMPRISING DTPA-BIS-AMIDE LIGAND AND METHOD FOR PREPARING THE SAME [Technical Field]

<i> The present invention relates to a new method of preparing a DTPA-bis- amide ligand having high molecular weight to increase magnetic relaxation ratio.

<2> Further, the present invention relates to a new method of preparing a gadolinium (Gd) complex having high water-solubility by using the ligand.

<3> Further, the present invention relates to an MR contrast medium for diagnosing cancer, which contains a gadolinium (Gd) complex synthesized by the above mentioned method, has a contrast enhancing effect, and has a reduced cell toxicity. [Background Art]

<4> In general, Magnetic Resonance Image (MRI) method has been widely used owing to the imaging of a diagnosis. Although magnetic resonance image method can support images of weak skin tissue, the quality of images may be determined by high shading contrast enhancing action of the contrast medium used in shading contrast reaction. Thus, growth of contrast medium field for efficient magnetic resonance image method has attracted the skilled experts' sufficient attention recently.

<5> The necessary Properties of a contrast medium in such a magnetic resonance image include thermodynamic stability, water-solubility, and a multidentate ligand structure being a cause of making paramagnetic Gd (III) ion. That is, it has to couple with at least one molecule of water to show high magnetic relaxation with water. Further, a magnetic resonance image contrast medium should not have a chemical activity, its cell toxicity in vivo should be low, and it should be exhausted completely after a diagnosis.

<6> An example of a contrast medium being approved for human body as a contrast medium in magnetic resonance image method includes an ionized Gd (III) complex such as diethylenetriamine-N,N,N' ,N' ' ,N¹ ' -pentaacetate, (N-Me- glucamine)2[Gd(DTPA)(H₂O)] (Magnevist, Schering) having magnetic relaxation

ratio of 4.7 mM^'V¹ (20MHz, 298K) and a neutral Gd(III) complex such as [Gd(DTPA-bismethylamide)(H₂O)] (Qmniscan , Nycomed) having magnetic

relaxation ratio of 4.4InMV¹ (20MHz, 298K).

<7> However, the above contrast medium has problems such as low water- solubility, low magnetic relaxation ratio, and relatively high cell toxicity in vivo. [Disclosure] [Technical Problem]

<8> The present invention has been made to solve the foregoing problems with the prior art, and embodiments of the present invention provide a gadolinium complex having high water-solubility by using the ligand prepared according to a method of preparing the ligand of the present invention. <9> Further, embodiments of the present invention provide an MR contrast medium capable of diagnosing cancer, which has higher contrast enhancing effect than the MR contrast medium of the prior art by using a complex prepared according to a method of preparing a gadolinium (Gd) complex of the present invention.

<io> Further, embodiments of the present invention provide a contrast medium capable of diagnosing cancer, which can reduce cell toxicity more than the MR contrast medium of the prior art.

[Technical Solution]

<π> According to an aspect of the present invention, there is provided a method of preparing a new DTPA-bis-amide ligand. The method includes: first step of adding DTPA-bis-anhydride to N,N-demethylformamide with stirring; second step of adding 2-hydroxyethy\-trans~A- (aminomethyl)cyclohexylcarboxylatehydrochloride to the mixture with stirring", third step of conducting silica gel chromatography after removing all of solvent under low pressure and dissolving the mixture in methanol; and fourth step of drying the product obtained in the third step under vacuum state to obtain a ligand L4. <12> In the second step, 2-methoxyethyWr,affs^4-(aminomethyl) cyclohexaneethyl carboxylatehydrochloride may be added instead of 2- hydroxyethyWra/7sH:-(aminomethyl) cyclohexyl carboxylatehydrochloride to obtain a ligand L5 in the fourth step. <i3> In the second step, a\\y\-trans-4-(minomethy\) cyclohexylethylcarboxylate hydrochloride may be added instead of 2- hydroxyethyl-^ra/2_?-4-(aminomethyl ) cyclohexylethylcarboxylate hydrochloride to obtain a ligand L6 in the fourth step. <14> The method may further include: fifth step of putting the DTPA-bis- amide ligand obtained in fourth step to the distilled water, and adding Gd₂O₃ with stirring to prepare a mixture solution; sixth step of removing impurity and solvent from the mixture solution obtained in the fifth step; and seventh step of dissolving the material obtained in the sixth step in methanol and precipitating it with acetonitrile to obtain a solid. <15> The ratio of molarity of the DTPA-bis-amide ligand to that of Gd₂O₃ in the fifth step may be 1 to 1. <i6> The stirring of the fifth step may be conducted at a temperature ranging from 90 °C to 100 °C for the time ranging from 5 hours to 7 hours. <17> The sixth step may include passing the mixture solution obtained in the fifth step through CeI ite to remove impurity and solvent. <i8> According to another aspect of the present invention, a DTPA-bis-amide ligand is prepared by the method as set forth above.

<19> The DTPA-bis-amide ligand may have an improved thermodynamic stability. <20> According to a further aspect of the present invention, a gadolinium

(Gd) complex is prepared by the method as set forth above. <2i> The gadolinium (Gd) complex may be expressed by a chemical formula (1) be1ow: <22> [Gd(L)(H₂O)] -H₂O (1),

<23> where L represents a ligand according to claim 8, and x represents 0 to

12. <24> The gadolinium (Gd) complex may have an improved solubility and magnetic relaxation ratio.

<25> The gadolinium (Gd) complex may have a low cell toxicity. <26> The MR contrast medium for diagnosing cancer may contain the gadolinium

(Gd) complex. [Advantageous Effects] <27> As set forth above, the ligand prepared by the method of preparing the present invention contains a transamic acid, a polar functional group, which makes the gadolinium complex have a high water-solubility. <28> Further, when the gadolinium complex prepared by the method of preparing the present invention is used as an MR contrast medium for diagnosing cancer, it makes a contrast enhancing effect higher than the MR contrast medium of the prior art. <29> Further, the MR contrast medium of the present invention for diagnosing cancer contains a gadolinium (Gd) complex of the present invention, and it makes cell toxicity lower than the MR contrast medium of the prior art.

[Description of Drawings] <30> FIG. 1 shows a process for synthesizing ligands (L4 to L6) and gadolinium complexes (Gd (L4) to Gd (L6)) of the present invention, wherein: <3i> When DTPA-bis-anhydride is added to DMF with stirring, and 2-hydroxy ethyWra/7.?-4-(aminomethyl) cyclohexylcarboxylate hydrochloride (R = (CH₂)₂0H) is added, a ligand L4 is formed, and when GdA is added hereto, a gadolinium complex Gd (L4) is formed;

<32> When DTPA-bis-anhydride is added to DMF with stirring, 2-methoxy ethyl-

cyclohexylcarboxylate hydrochloride (R = (CH₂)₂0Me) is added, a ligand L5 is formed, and when is added hereto, a gadolinium complex Gd (L5) is formed; and <33> When DTPA-bis-anhydride is added to DMF with stirring and a\\yl-trans- 4-(aminofflethyl) cyclohexylcarboxylatehydrochloride (R=CH₂CH=CH₂) is added, a ligand L6 is formed, and when Gd₂Os is added hereto, a gadolinium complex Gd

(L6) is formed. <34> FIG. 2 shows relaxation times (Ti and T₂) and corresponding relaxation ratios (Ri and R₂) of gadolinium complexes (Gd (L4) to Gd (L6)), Omniscan and pure water. <35> FIG. 3 shows a relaxation time map (T₁ map) and a corresponding relaxation ratio map (Ri map) of gadolinium complexes (Gd (L4) to Gd (L6)),

Omniscan and pure water.

<36> FIG. 4 compares cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by an MTT test. [Best Mode]

<37> The present invention relates to a new method of preparing a new DTPA- bis-amide ligand and a new gadolinium complex, wherein the DTPA-bis-amide ligand contains a transamic acid having polar functional groups and ester conjugates. This ligand contains polar functional groups and thus has a high water-solubility.

<38> Further, DTPA of the present invention is an abbreviated term of DiethyleneTriamine PentaAcetic acid, a metal-affinitive chelate compound, which is a chemical protective agent against radiation hazard. The protective agent functions to remove a radioactive material out of it and to reduce cell toxicity.

<39> The complex of the present invention contains one or more atom(s) or ion(s) as a core and several ion molecules of other atoms or atomic groups, which have aromaticity, with being sterically coordinated to the core. An atomic ion molecule or an atomic group, which coordinates a core atom or an ion, is called "a ligand." To specify a complex, its chemical formula is represented in a bracket "[ ]," the bond between a core atom and a ligand is an ion bond or a covalent bond, the number of a ligand is included even though the generated atomic group is not electrified.

<40>

<4i> Now, the following non-limiting examples are used to further describe or illustrate the invention. The examples are given for illustration of the invention and are not intended to be limiting thereof.

<42>

<43> EXAMPLES

<44>

<45> Preparation Example 1 (Ligand L4)

<46> 0.71g (2mmol) of DTPA-bis-anhydride was dissolved in 15mL N,N- dimethylformamide (DMF) with stirring, and 0.63g (4mmol) of 2-hydroxyethyl- £r,3/7S-4-(aminomethyl) eyelohexylcarboxylate hydrochloride was added. The reaction mixture was stirred at 65°C for 4 hours, all of solvent was removed under low pressure, and 1OmL of methanol was added to dissolve. Short silica gel (60 meshes) chromatography of the solution was conducted by passing methanol, and all of solvent was removed again. The obtained white solid was dried at 50°C for 8 hours and was kept under vacuum state.

<47> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L4.

<48> Yield: 3.93 g (87%)

<49> ¹H [J₆-DMSO, 400 MHz] : δ 8.25 (s , 2H, CH₂COM) , 4.17 (s , 2H, H2) , 3.54

(m, 8H, OCrøPD , 3.40 (m, 8H, H7, H5) , 3.07 (m, 4H, H3/H4) , 2.94 (m, 4H,

H3/H4) , 2.24 (m, 2H, H13) , 2.08 (m, 4H, H9) , 1.81 (m, 8H, H11/H12) , 1.37 (m, 2H, HlO) , 1.08 (m, 8H, H11/H12)

<50> ¹³C NMR (J₆-DMSO, 100 MHz) : δ 175.46 (C1/C8) , 175.09 (C1/C8) , 172.96

(C14) , 172.26 (C6) , 65.83 (0Oi₂CH₂OH) , 65.26 (OCH₂Ol₂OH) , 63.99 (C2) , 59.25

(C7) , 56.49 (C5) , 54.83 (C3/C4) , 54.54 (C3/C4) , 44.84 (C13) , 43.02 (C9) , 42.76 (ClO) , 29.59 (CIl) , 28.52 (C12) <5i> Calculated for C₃₄H₅₇N₅OuSH₂O: C: 45.17, H: 8.14, N: 7.75

<52> Measured for C₃₄H₅₇N₅O₁₄SH₂O: C: 45.33, H: 7.86, N: 7.66

<53> FAB-MS im/z) :

<54> Calculated for C₃₄H₅₈N₅Oi₄: 760.85 ( [MH] ⁺)

<55> Measured for C₃₄H₅₈N₅O₁₄: 760.65 ( [MH] ⁺)

<56> Calculated for C₃₄H₅₇N₅NaOi₄: 782.83 ( [MNa] ⁺)

<57> Measured for C₃₄H₅₇N₅NaOi₄: 782.60 ( [MNa] ⁺)

<58>

<59> Preparation Example 2 (Ligand L5)

<60> This preparation method was progressed as the same way in Preparation Example 1, except that 2-methoxyethyl-fra/7S^u-4-(aminomethyl) cyclohexylcarboxylatehydrochloride was used instead of

4-(aminomethyl) eyelohexylcarboxylate hydrochloride. The obtained white solid was dried at 50"C for 8 hours and was kept under vacuum state.

<6i> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L5.

<62> Yield: 3.87 g (83%)

<63> ¹H [ cfs-DMSO, 400 MHz] : δ 8.31 (s , 2H, CH₂CON/?) , 4.14 (s , 2H, H2) , 4.09

(m, 4H, OC^₂CH₂OCH₃) , 3.56 (m, 4H, OCH₂CTy₂OCH₃) , 3.48 (m, 8H, H7, H5) , 3.36 (m, 4H, H3/H4) , 3.23 (s , 6H, OCH₂CH₂OCyY₃) , 3.08 (m, 4H, H3/H4) , 2.93 (m, 4H, H9) ,

2.21 (m, 2H, H13) , 1.76 (m, 8H, H11/H12) , 1.37 Gn, 2H, HlO) , 1.08 (m, 8H, H11/H12)

<64> ¹³C U-DMSO, 100MHz]: δ 175.34 (C14), 172.88 (C1/C8), 172.05 (C1/C8), 170.09 (C6), 70.11 (OCH₂OI₂OCH₃), 63.20 (OaI₂CH₂OCH₃), 58.42 (OCH₂CH₂OOI₃), 56.45

(C2), 54.83 (C7), 54.55 (C5), 51.85 (C3/C4), 49.72 (C3/C4), 44.84 (C13), 42.66 (C9), 37.07 (ClO), 29.53 (CIl), 28.52 (C12) <65> Calculated for C₃₆H₆₁N₅Oi₄SH₂O: C: 46.39, H: 8.33, N: 7.51

<66> Measured for C₃₆H₆IN₅O₁₄SH₂O: C: 46.22 , H: 7.97 , N: 7.83

<67> FAB-MS {m/z) :

<68> Calculated for C₃₆H₆₂N₅Oi₄: 788.9 ( [MH] ⁺)

<69> Measured for C₃₆H₆₂N₅Oi₄: 788.67 ( [MH] ⁺)

+,

<70> Cal culated for C₃₆H₆₂N₅NaOi₄ : 810.88 ( [MNa] )

+.

<7i> Measured for C₃₆H₆₁N₅NaOi₄: 810.61 ( [MNa] )

<72>

<73> Preparation Example 3 (Ligand L6)

<74> This preparation method was progressed as the same way in Preparation Example 1, except that

cyclohexylcarboxylatehydrochloride was used instead of 2-hydroxyethyl-trans- 4-(aminomethyl) eyelohexylcarboxylate hydrochloride. The obtained white solid was dried at 50°C for 8 hours and was kept under vacuum state.

<75> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L6.

<76> Yield: 3.6Og (82%)

<77> ¹H NMR (oH)MS0, 400 MHz): 58.28 (s, 2H, CH₂OM, 5.89 (m, 2H, OCH₂C^=CH₂), 5.22 (m, 4H, OCH₂CH=CTy₂), 4.52 (d, /= 4.52, 4H, OC#₂CH=CH₂), 4.15

(s, 2H, H2), 3.55 (m, 4H, H7), 3.48 (m, 4H, H5), 3.36 (m, 4H, H9), 3.08 (m, 4H, H3/H4), 2.94 (m, 4H, H3/H4), 2.25 (m, 2H, H13), 1.81 (m, 8H, H11/H12), 1.38 (m, 2H, HlO), 1.12 (m, 8H, H11/H12)

<78> ¹³C NMR U-DMSO, 100 MHz) : 5 174.94 (C14) , 172.93 (C1/C8) , 172.13

(C1/C8) , 170.24 (C6) , 133. 14 (OCH₂Oi=CH₂) , 117.75 (OCH₂CH=OI₂) , 64.41

(00I₂CH=CH₂) , 56.46 (C2) , 54.81 (C7) , 54.55 (C5) , 53.05 (C3/C4) , 52.27 (C3/C4) , 44.85 (C13) , 42.70 (C9) , 37.09 (ClO) , 29.58 (CIl) , 28.55 (C12) <79> Calculated for C₃₆H₅₇N₅O₁₂TH₂O: C: 49.25 , H: 8.15, N: 7.98

<80> Measured for C₃₆H₅₇N₅O₁₂TH₂O: C: 49.13 , H: 7.80 , N: 8.17

<8i> FAB-MS Wz) :

<82> Calculated for C₃₆H₅₈N₅O₁₂: 752.41 ( [MH] ⁺)

<83> Measured for C₃₆H₅₇N₅O₁^H₂O: 752.55 ( [MH] ⁺)

+.

<84> Calculated for C₃₆H₅₇N₅NaO₁₂: 774.39 ( [MNa] )

+,

<85> Measured for C₃₆H₅₇N₅0₁₂7H₂0: 774.50 ( [MNa] )

<86>

<87> Preparation Example 4 (Gadolinium complex Gd (L4))

<88> 0.73 g (1 mmol) of a ligand L4 was dissolved in 10 mL of the third distilled water and 0.18 g (0.5 mmol) of Gd₂O₃ was added. Suspension type reaction mixture was stirred at 100°C for 6 hours. A pale yellow solution was obtained at the end of the reaction. Dissoluble impurity and all of solvent were removed by passing the solution through CeI ite. The remained material was dissolved sufficiently by adding 5mL of methanol and was reprecipitated with 100 mL of acetonitrile to obtain the white solid. The obtained solid was filtered and then was dried. FIG. 1 shows a method of preparing a gadolinium complex briefly.

<89> From the experimental data below, the resulting product of the Preparation Example was identified as a gadolinium complex Gd (L4).

<90> Yield: 1.89 g (88%)

<9i> Calculated for C₃₄H₅₆GdN₅0₁₅8H₂0: C: 37.94, H: 6.74, N, 6.51

<92> Measured: C: 37.92 , H: 6.48 , N: 6.88

<93> FABMS WzY

<94> Calculated for C₃₄H₅₇GdN₅O₁₅: 933.09 ( [MH] ⁺)

<95> Measured for C₃₄H₅₇GdN₅O₁₅: 932.77 <96> Cal culated for C₃₄H₅₅GdN₅Oi₄: 915.08 (MH - (H₂O) )⁺

<97> Measured for C₃₄H₅₅GdN₅O₁₄: 914.84

<98>

<99> Preparation Example 5 (Gadolinium complex Gd (L5))

<ioo> This preparation method was progressed as the same way in Preparation Example 4, except that a ligand L5 was used instead of a ligand L4. FIG. 1 shows a method of preparing a gadolinium complex briefly.

<ioi> From the experimental data below, the resulting product of the preparation Example was identified as a gadolinium complex Gd (L5).

<iO2> Yield: 1.98 g (90%)

<i03> Calculated for C₃₆H₆₀GdN₅O₁₅SH₂O: C: 39.16 , H: 6.94 , N: 6.34

<i04> Measured for C₃₆H₆₀GdN₅Oi₅SH₂O: C: 39.02, H: 6.70, N: 6.65

<iO5> FABMS Wz) :

+

<iO6> Calculated for C₃₆H₅₉GdN₅Oi₄: 943.13(MH - (H₂O) )

+

<iO7> Measured for C₃₆H₅₉GdN₅Oi₄: 942.78(MH - (H₂O))

<108>

<iO9> Preparation Example 6 (Gadolinium complex Gd (L6))

<iio> This preparation method was progressed as the same way in Preparation Example 4, except that a ligand L6 was used instead of a ligand L4. FIG. 1 shows a method of preparing a gadolinium complex briefly.

<iii> From the experimental data below, the resulting product of the preparation Example was identified as a gadolinium complex Gd (L6).

<ii2> Yield: 1.80 g (87%)

<ii3> Cal culated for C₃₆H₅₆GdN₅0i₃6H₂0: C: 41.89, H: 6.64, N: 6.78

<ii4> Measured for C₃₆H₅₆GdN₅0i₃6H₂0: C: 42.09, H: 6.47 , N: 6.97

<ii5> FABMS im/z) :

<ii6> Calculated for C₃₆H₅₅GdN₅Oi₂: 907.1 (MH - (H₂O) )⁺ <ii7> Measured for C₃₆H₅₅GdN₅O₁₂: 906.78

<118>

<ii9> The test in the Preparation Example was conducted by using standard Schlenk techniques under nitrogen atmosphere, and moisture was also previously removed from the required solvent. Reagents were purchased from Aldrich. The used water was third distilled water.

1 13

<i20> As H and C NMR measuring apparatus, Bruker Advance 400 or 500 in

Korea Basic Science Institute (RBSI) was used, and NMR measuring values were calculated on the basis of tetramethylsilane (TMS), which was preset to 0. Coupling constant (J) was measured by using Micromass QUATTRO II GC8000 series model, which has electronic energy ranging from 20 to 70 eV. The IR Spectrum was obtained by using Mattson FT-IR Galaxy 6030E in KBSI. Element analysis was conducted at the co-experimental laboratory in Kyungpook National University.

<i2i> In this experimental example below, in order to verify a remarkable effect of ligands (L4 to L6) and gadolinium complexes (Gd (L4) to Gd (L6)) of the present invention, they were compared with Omniscan, which is known as a paramagnetic contrast medium.

<122>

<123> Test Example 1 (Proton adding equilibrium constant, stabilization constant, selection constant, conditional stabilization constant, and pM value)

H

<124> Table 1 shows proton adding equilibrium constant(K_\ ) of ligands (L4 to L6) prepared in a Preparation Example, and stabilization constant (ik), selection

conditional stabilization constant(K s_ei) of gadolinium complex es (Gd(L4) to Gd(L6)), and pM values of gadolinium (Gd(III)), calcium (Ca(II)), zinc (Zn(II)), and copper (Cu(II)) at pH 7.4 (the values represent bonding strength of ligands and metals). <125> Proton adding equilibrium constant [K₁ ) can be obtained by the

H 4" formula, K_x = [HjL]/ [H_H1L] [H], wherein H_;L means a proton added ligand, and i = 1, 2

<126> Stabilization constant iK_&) can be obtained by the formula, 4L = [ML]/

[M] [L], wherein M is Gd, Ca, Zn, and Cu, L represents a ligand.

<i27> pM value can be obtained by the formula, pM = -log [M ], wherein pH = 7.4.

3 3

<i28> Further , the concentrat ions of [M]= l μ mol/dm and [L]= l . l u mol/dm were used. <129> [Table 1]

Constant log K (25t , μ, = 0.10 M (KCl))

L4 L5 L6 DTPABMA

[HL]Z[L][H] 9.94 9.95 9.81 9.37

[HzL]/[HLI[H] 5.15 5.18 4.81 4.38

[H₃L]Z[H₂L][H] 3.72 3.79 3.67 3.31

∑pAT, 18.81 18.92 18.29 17.06

[GdL]Z[Gd][L] 21.10 20.80 20.99 16.85

{logKc_dL(pH7.4)} 18.56 18.25 18.58 14.84

[CaL]/[Ca][Ll 7.85 7.86 7.17 7.17

{logffcuføHM)} 5.31 5.31 4.76 5.11

[ZnL]Z[Zn][L] 11.78 11.80 1 1.03 12.04

(1OgA_2nL(PH^)) 9.24 9.25 8.62 10.02

[CuL]ZlCu][L] 12.46 12.23 11.52 13.03

{logKc:_uL(pH7.4)} 9.92 9.68 9.11 11.06

JlogK^Gd/Ca)] 13.25 12.94 13.82 9.68

pGd 17.56 17.25 17.58 13.88 pCa 4.31 4.31 3.76 4.19 pZn 8.24 8.25 7.61 9.06 pCu 8.92 8.68 8.11 10.05

<130>

<i3i> When a gadolinium complex was used as a contrast medium, gadolinium could be separated from a ligand in human body, and cell toxicity might be caused by a toxic gadolinium. In order not to make this separation, the stronger the bond between a ligand and a gadolinium might be, the more stable it is. Thus, as can be seen at the Table 1, ligands (L4 to L6) use gadolinium (Gd) showing higher values than Omniscan (DTPABMA) in proton adding equilibrium constant (^ ), stabilization constant (4_L), selection constant conditional stabilization constant (K'sei), and pM values, and it means that the bonding state between the ligand and the gadolinium is more stable, and it can be used as a good contrast medium.

<132>

<i33> Test Example 2 (Relaxation time and relaxation ratio)

<134> Relaxation time (Ti, T₂) and relaxation ratio (Ri, R₂) of gadolinium complexes (Gd (L4) to Gd (L6)) prepared in Preparation Example were measured. Even though the relatively less amount of contrast medium having high relaxation ratio was administrated, it showed a high contrast enhancing effect, and thus it was very important in magnetic resonance image. As can be seen in FIG. 2, Gd (L4) to Gd (L6) having higher molecular weights showed 1.6 times to 1.7 times higher relaxation ratio than that of Omniscan. Thus, the complex of the present invention can be considered as a contrast medium that can give signals more effectively. <i35> Further, FIG. 3 shows relaxation time map (Ti map) and relaxation ratio map (Ri map). In relaxation time map (Ti map), as relaxation time (Ti) is showed as a signal strength, the brighter signal strength means the longer relaxation time on the map. Thus, as can be seen in Fig. 3, Gd (L4) to Gd (L6) shows brighter signal strength comparing with Omniscan. Since the longer relaxation time can be confirmed, the contrast medium of the present invention can keep its contrast effect longer, and thus it is proved to be more effective. <136> Measurement of relaxation time Ti was progressed by a reverse recovery method of variable conducting time (TI) at 1.5T (64MHz). Magnetic resonance (MR) image needs another 35 TIs ranging from 50 to 1750 msec region. Relaxation time Ti was obtained from non-linear, square strokes of signal strengths measured at each TI values.

<137> CPMG (Carr-Purcell-Meiboon-Gill) continuous wave for measuring relaxation time T₂ can be obtained by measuring multi spinal echo. 34 images can acquire 34 other echo time (TE) ranging 10 to 1900 msec. Relaxation time T2 can be obtained from non-linear regular square forms for measuring multi spin-echo at each echo time. <138> Relaxation ratio(Ri and R₂) was calculated as a reverse value of relaxation time(Ti and T₂) per mM.

<139>

<i40> Test Example 3 (Cell toxicity)

<i4i> In order to investigate cell toxicity, MTT (Tetrazolium-based colorimetric) searching method was used. MTT searching method is to use an ability of mitochondria to reduce yellow soluble MTT tetrazolium to celadon green non-soluble MTT formazan (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) by dehydrogenase function. If alive cells are treated with MTT tetrazolium, MTT tetrazolium is reduced to form MTT formazan (3- (4,5~dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) by reductase of mitochondria. That is, when the cell was treated with a certain compound depending on its concentrations for predetermined hours to induce cell death, and treated with MTT tetrazolium, MTT formazan was formed at low concentration not representing cell toxicity, and MTT formazan was not formed at high concentration representing cell toxicity. Cell viability can be determined by measuring the formation of MTT formazan depending on the concentration gradient of such compound.

<142> FIG. 4 shows the result of comparing cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by using MTT test. That is, the higher cell viability a gadolinium complex might have, the lower the cell toxicity is. Cells which was not added anything are regarded as a control group. Cell viability of a control group was determined as 100% by measuring formation of MTT formazan. When various concentrations (0.5mM to 1OmM) of gadolinium complexes (Gd(L4) to Gd(L6)) and Omniscan were added to cells, the cells showed an amount of 60% or more of cell viability. It was identified that gadolinium complexes (Gd(L4) to Gd(L6)) having low cell toxicities can be effectively used a contrast medium through a graph of FIG. 4.

Claims

[CLAIMS] [Claim 1]

A method of preparing a new DTPA-bis-amide ligand, comprising: first step of adding DTPA-bis-anhydride to N,N-demethylformamide with stirring; second step of adding 2-hydroxyethyl-trans-A- (aminomethyl)cyclohexylcarboxylatehydrochloride to the mixture with stirring; third step of conducting silica gel chromatography after removing all of solvent under low pressure and dissolving the mixture in methanol; and fourth step of drying the product obtained in the third step under vacuum state to obtain a ligand L4. [Claim 2]

The method according to claim 1, wherein 2-methoxyethy1-trans-A- (aminomethyl)cyclohexylcarboxylatehydrochloride in the second step is added instead of 2-hydroxyethyl-£r,

2/?s^4-(aminomethyl )eyelohexylcarboxylate hydrochloride to obtain a ligand L5 in the fourth step.

[Claim 3]

The method according to claim 1, wherein allyWraπs-4- (aminomethyl)cyclohexylcarboxylatehydrochloride in the second step may be added instead of 2-hydroxyethyWra/7s-4- (aminomethyl)cyclohexylcarboxylatehydrochloride to obtain a ligand L6 in the fourth step.

[Claim 4]

The method according to any one of claims 1 to 3, further comprising: fifth step of putting the DTPA-bis-amide ligand obtained in fourth step to the distilled water, and adding Gd₂C"3 with stirring to prepare a mixture solution; sixth step of removing impurity and solvent from the mixture solution obtained in the fifth step; and seventh step of dissolving the material obtained in the sixth step in methanol and precipitating it with acetonitrile to obtain a solid.

[Claim 5]

The method according to claim 4, wherein a ratio of molarity of the DTPA-bis-amide ligand to that of Gd₂O₃ in the fifth step is 1 to 1.

[Claim 6]

The method according to claim 4, wherein the stirring of the fifth step is conducted at a temperature ranging from 90 °C to 100 ^°C for the time ranging from 5 hours to 7 hours. [Claim 7]

The method according to claim 4, wherein the sixth step comprises passing the mixture solution obtained in the fifth step through CeI ite to remove impurity and solvent. [Claim 8]

A DTPA-bis-amide ligand prepared by the method according to any one of claims 1 to 3. [Claim 9]

The DTPA-bis-amide ligand according to claim 8, wherein the DTPA-bis- amide ligand has an improved thermodynamic stability. [Claim 10]

A gadolinium (Gd) complex prepared by the method according to any one of claims 4 to 7. [Claim 11]

The gadolinium (Gd) complex according to claim 10, wherein the gadolinium (Gd) complex is expressed by a chemical formula (1) below: [Gd(L)(H₂O)] -H₂O (1), where L represents a ligand according to claim 8, and x represents 0 to 12. [Claim 12]

The gadolinium (Gd) complex according to claim 11, wherein the gadolinium (Gd) complex has an improved solubility and magnetic relaxation ratio. [Claim 13]

The gadolinium (Gd) complex according to claim 11, wherein the gadolinium (Gd) complex has a low cell toxicity. [Claim 14]

An MR contrast medium for diagnosing cancer containing the gadolinium (Gd) complex according to any one of claims 10 to 13.